AL-2 NEUROTROPHIC FACTOR

Abstract: The present invention provides nucleic acids encoding AL-2 protein, as well as AL-2 protein produced by recombinant DNA methods. Such AL-2 protein and nucleic acid are useful in preparing antibodies and antagonists and in diagnosing and treating various neuronal disorders and disorders or conditions associated with angiogenesis.

Description: AL-2 NEUROTROPHIC FACTOR INTRODUCTION Technical Field This application relates to a receptor protein tyrosine kinase ligand and its uses. In particular this application relates to the production and use of purified forms of AL-2 and related proteins. Background Protein neurotrophic factors, or neurotrophins. which influence growth and development of the vertebrate nervous system, are believed to play an important role in promoting the differentiation, survival, and function of diverse groups of neurons in the brain and periphery. Neurotrophic factors are believed to have important signaling functions in neural tissues, based in part upon the precedent established with nerve growth factor (NGF). NGF supports the survival of sympathetic, sensory, and basal forebrain neurons both in vitro and in vivo. Administration of exogenous NGF rescues neurons from cell death during development. Conversely, removal or sequestration of endogenous NGF by administration of anti-NGF antibodies promotes such cell death (Heumann. J. Exp. Biol.. 132:133-150 (1987); Hefti, J. Neurosci., 6:2155-2162 (1986); Thoenen et ai., Annu. Rev. Physiol., 60:284-335 (1980)). Additional neurotrophic factors related to NGF have since been identified. These include brain-derived neurotrophic factor (BDNF) (Leibrock, et al., Nature. 341 :149-152 (1989)), neurotrophin-3 (NT-3) (Kaisho, et al., FEBS Lett., 266:187 (1990); Maisonpierre, et ai. Science, 247: 1446 (1990); Rosenthal, et ai, Neuron, 4:767 (1990)), and neurotrophin 4/5 (NT-4/5) (Berk eier, et al.. Neuron, 7:857-866 (1991)). Neurotrophins, similar to other polypeptide growth factors, affect their target cells through interactions with cell surface receptors. According to our current understanding, two kinds of transmembrane glycoproteins act as receptors for the known neurotrophins. Equilibrium binding studies have shown that neurotrophin- responsive neuronal cells possess a common low molecular weight (65,000 - 80,000 Daltons), a low affinity receptor typically referred to as p75 LNGFR or p75, and a high molecular weight (130,000-150,000 Dalton) receptor. The high and low affinity receptors are members ofthe trk family of receptor tyrosine kinases. Receptor tyrosine kinases are known to serve as receptors for a variety of protein factors that promote cellular proliferation, differentiation, and survival. In addition to the trk receptors, examples of other receptoi tyrosine kinases include the receptors for epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). Typically, these receptors span the cell membrane, with one portion of the receptor being intracellular and in contact with the cytoplasm, and another portion of the receptor being extracellular. Binding of a ligand to the extracellular portion of the receptor induces tyrosine kinase activity in the intracellular portion ofthe receptor, with ensuing phosphorylation of various intracellular proteins involved in cellular signaling pathways. Recently, a receptor tyrosine kinase subclass referred to as the Eph receptor subclass or family has been identified. Eph was the first member of this Eph subclass of receptor tyrosine kinases to be identified and characterized by molecular cloning (Hirai et ai. Science. 238 : 1717- 1720 ( 1987)). The name Eph is derived from the name of the cell line from which the Eph cDNA was first isolated, the erythropoietin-producing human hepatocellular carcinoma cell line. ETL-1. The general structure of Eph is similar to that of other receptor tyrosine kinases and consists of an extracellular domain, a single membrane spanning region, and a conserved tyrosme kinase catalytic domain However, the structure ofthe exσacellular domain of Eph which comprises an immunoglobulin (lg)-lιke domain at its amtno terminus, followed by a cysteine-nch region and two fibronectin type III repeats in close proximity to the transmembrane domain, is completely distinct from that of previously descπbed receptor tyrosine kmases The juxtamembrane domain and carboxy-termmus regions of Eph also are unrelated to the corresponding regions of other tyrosine kinase receptors Newly discovered members of the Eph receptor family include Elk. Cek5, Mek4. Cek4 Hek Hek4 (Sajjadi et al.. The New Biologist, 3 769-778 ( 1991 )), Cek6 through Cek 10 (Sajjadi et al . Oncogene 8 1807- 13 ( 1993). Sek, Hek2, and Ehk3 (Tuzi, et al , Br J Cancer, 69 417-421 (1994), Zhou, et al , J Neurosci Res . 37 129-143 (1994)) Other Eph-related receptor kinases that have been identified include Sek (Gilardi-Hebenstreit et al, Oncogene, 7 2499-2506 ( 1992)), Eck (Lindberg et al Mol Cell Biol , 106316-6324 (1990)), Elk (Lhotak e/ a/ , >/ Cell Biol , 1 1 2496-2502 (1991)), Eek (Chan et al , Oncogene, 6 1057-1061 (1991)), Rek7 (Wuιslow et al.. Neuron, 14 973-981 (1995) Rek7 is a rat homolog of chicken Cek7 and closely related to Ehk- 1 (Davis et al.. Science, 266 816-819 ( 1994)) and bsk (Maisonpierre et al., Oncogene 8 3277- 3288 (1993), Zhou et al , J Neurosci Res, 37 129-143 (1994)), the Rek7 cDNA coiTesponds to a splice vanant of Ehk- 1. lacking the first of two tandem fibronectin type-Ill domains Human homologs of the chicken Cek receptors are referred to as Hek receptors (See WO 95/28484, which is incoφorated herein by reference) For example, Hek5 (Fox et al Oncogene, 10(5) 897-905 (1995), WO 95/28484) is the human homolog of chicken Cek5 The amino acid sequence of Hek5 is very closely related (96% amino acid identity in the catalytic domain) to the chicken receptor Cek5 (Pasquale et al., J Neuroscience 12 3956-3967 ( 1992), Pasquale, Cell Regulation, 2 523-534 (1991)) A portion ofthe Hek5 sequence was previously disclosed as Erk, a human clone encoding about sixty ammo acids (Chan et al , Oncogene, 6 1057-1061 (1991)) Mature Erk showed high homology with Cek5 (92.5%) and mouse Nuk (99 1%) (Kiyokawa et al , Cancer Res , 54 (14) 3645-50 (1994)) Other human Eph-family receptors include Hek (Wicks et al., Proc Natl Acad Sci USA, 89(5) 1611-1615 ( 1992), also known as Hek4), Hek2 (Boh e et al Oncogene, 8.2857-2862 (1993)), Heks 7, 8 and 11 (WO 95/28484), Hek3, which is a homolog of rat Eek and murine Mdk-1, and Hekl2, which is a homolog of rat Ehk2 Many ofthe Eph-receptor family members are "orphan receptors " However, recently, ligands have been reported including B61, an Eck receptor ligand (Bartley et al . Nature, 368 558-560 (1994) and Pandey et ai. Science, (1995) 268-567-569), Elf-1, a Mek4 and Sek receptor ligand (Cheng et al.. Cell, (1995) 82 371-381 , Cheng et al , Cell, 79 157-168 (1994)), Htk-L (Bennett et al , Proc Natl Acad Set USA, 92(6).1866-70 (1995)), AL-1 (Winslow e/ α/ , Neuron, 14 973-981 (1995)) and RAGS (Drescher et al , Cell, (1995) 82 359-370), which are Rek7 ligands, Ehk- 1 -L (Davis et al , Science 266 816-819 (1994), see also efl-2 in WO 95/27060), Cek5-L, and Lerk2 (Beckmann et al . EMBO J 13 3757-3762 (1994)) Aberrant expression of receptor tyrosme kinases correlates with transforming ability This relationship includes members of the Eph subclass of receptor tyrosine kinases For example, carcinomas of the liver, lung, breast and colon show elevated expression of Eph Unlike many other tyrosine kmases. this elevated expression can occur in the absence of gene amplification or rearrangement Such involvement of Eph in carcinogenesis also has been shown by the formation of foci of NIH 3T3 cells in soft agar and of tumors in nude mice following overexpression of Eph Moreover, Hek has been identified as a leukemia-specific marker present on the surface of a pre-B cell leukemia cell line As with Eph. Hek also was overexpressed in the absence of gene amplification or rearrangements in for example, hemopoietic tumors and lymphoid tumor cell lines Over-expression of Myk-1 (a murine homolog of human Htk (Bennett et al J Biol Chem . 269(19) 1421 1-8 (1994)) was found in the undifferentiated and invasive mammary tumors of transgenic mice expressing the Ha-ras oncogene (Andres et al Oncogene 9(5) 1461-7 (1994) and Andres et al Oncogene 9(8) 2431 ( 1994)) In addition to their roles in carcmogenesis, a number of transmembrane tyrosine kinases have been reported to play key roles dunng development Some receptor tyrosine kinases are developmentallv regulated and predominantly expressed in embryonic tissues Examples include Cek 1 , which belongs to the FGF subclass and the Cek4 and Cek5 tyrosine kinases (Pasquale et al Proc Natl Acad Set USA 86 5449-5453 (1989), Sa j jadi et al , New Biol . 3(8) 769-78 ( 1991 ), and Pasquale, Cell Regulation 2 523-534 ( 1991 )) Eph family members are expressed in many different adult tissues, with several family members expressed in the nervous system or specifically in neurons (Maisoπpierre et al Oncogene, 8 3277-3288 ( 1993), Lai et al Neuron 6 691-704 (1991)) The aberrant expression or uncontrolled regulation of any one of these receptor tyrosine kinases can result ui different malignancies and pathological disorders Therefore, there exists a need to identify means to regulate, control and manipulate receptor tyrosme kmases and their ligands in order to provide new and additional means for the diagnosis and therapy of Eph-pathway related disorders and cellular processes The present application provides the clinician and researcher with such means by providing new molecules that are specific for interacting with Eph-famι!y receptors These compounds and their methods of use. as provided herein, allow exquisite therapeutic control and specificity Additional advantages are provided as well SUMMARY The present mvention provides a novel cytokine, an Eph-related tyrosine kinase receptor ligand referred to as AL-2 The present mvention provides nucleic acid encoding AL-2, particularly two forms referred to herein as AL-2s ("AL-2-short") and AL-21 ("AL-2-long"), and methods to use the nucleic acid to produce AL-2 in recombinant host cells for diagnostic or therapeutic purposes Also provided are uses of nucleic acids encoding AL-2. and portions thereof, to identify related nucleic acids in the cells or tissues of various animal species By pioviding the full nucleotide coding sequence for AL-2, the mvention enables the production of AL- 2 by means of recombmant DNA technology, thereby making available for the first time sufficient quantities of substantially pure AL-2 protem or AL-2 antagonists for diagnostic and therapeutic uses For example, method embodiments mclude treatment or prevention of a variety of neurological disorders and diseases as well as conditions that are angiogenesis-dependent such as solid tumors, diabetic retmopathy, rheumatoid arthritis, and wound healing Also provided are deπvatives and modified forms of AL-2. including ammo acid sequence variants and covalent derivatives thereof, as well as antagonists of AL-2, that are preferably biologically active (e g , antigenically active In one embodiment, the invention provides a soluble form ofthe gand with at least the transmembrane region deleted Usually, the cytoplasmic domain will also be absent Immunogens are provided for raising antibodies, as well as to obtain antibodies, capable of binding to, preferably neutralizing, AL-2 or derivatives or modified forms thereof In a preferred embodiment, the invention provides AL-2 that is free of other human proteins -i- AL-2 and modified and variant forms of AL-2 are produced by means ot chemical or enzymatic treatment or by means of recombmant DNA technology, including in vivo production Variant polypeptides can differ from native AL-2 for example, by virtue of one or more amino acid substitutions, deletions or insertions or in the extent or pattern of glycosylation, but will substantially retain a biological activity of native AL-2 Chimeras comprising AL-2 (or a portion thereof) fused to another polypeptide are provided An example of such a chimera is epitope-tagged AL-2 In another embodiment a soluble form of an AL-2 chimera is provided, for example, as an lmmunoadhesm, which is a fusion ofthe extracellular domain of AL-2 and an immunoglobulin sequence Antibodies to AL-2 are produced by immunizing an animal with AL-2 or a fragment thereof, optionally in conjunction with an immunogenic polypeptide. and thereafter recovering antibodies from the serum ofthe immunized animals Alternatively, monoclonal antibodies are prepared from cells ofthe immunized animal in conventional fashion Antibodies obtained by routine screening will bind to AL-2 but, preferably, will not substantially bind to (i e , cross react with) NGF, BDNF, NT-3, NT-4/5, GDNF, AL- 1 , Htk-L, Lerk-2, or other neurotrophic factors or cytokmes Immobilized antι-AL-2 antibodies are particularly useful in the detection of AL-2 clinical samples for diagnostic purposes, and in the purification of AL-2 AL-2, its denvatives, or its antibodies are formulated with physiologically acceptable earners, especially for therapeutic use Such carriers are used, for example, to provide sustained-release formulations of AL-2 In further aspects, the invention provides a method for determining the presence of a nucleic acid molecule encod g AL-2 in test samples prepared from cells, tissues, or biological fluids, compnsing contacting the test sample with isolated DNA compnsing all or a portion ofthe nucleotide coding sequence for AL-2 and determining whether the isolated DNA hybridizes to a nucleic acid molecule in the test sample DNA comprising all or a portion ofthe nucleotide cod g sequence for AL-2 is also used in hybridization assays to identify and to isolate nucleic acids sharing substantial sequence identity to the coding sequence for AL-2, such as nucleic acids that encode allelic variants of AL-2 Also provided is a method which involves contacting an AL-2 receptor with AL-2 in order to cause phosphorylation ofthe kinase domain of the receptor Also provided is a method for amplifying a nucleic acid molecule encoding AL-2 that is present in a test sample, comprising the use of an oligonucleotide having a portion ofthe nucleotide coding sequence for AL- 2 as a primer in a polymerase chain reaction BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-1B shows the AL-21-encodmg nucleotide sequence (SEQ ID NO 1 ), its complementary sequence, and the deduced ammo acid sequence (SEQ ID NO 2) of AL-2 ofthe isolated AL-21 ("AL-2-long") cDNA The deduced N-termmus of the mature AL-2 protem begins with glycme-27 as numbered from the initiation methionine The C-terminal hydrophobic transmembrane domain extends from amino acid Leu-220 to Ala-245 The deduced extracellular domain sequence includes amino acids Gly-27 to Pro-219 Figure 2A-2B shows the AL-2s-encodιng nucleotide sequence (SEQ ID NO 3). its complementary sequence, and the deduced amino acid sequence (SEQ ID NO 4) of AL-2 ofthe isolated AL-2s ("AL-2-short") cD A The deduced N-terminus ofthe mature AL-2 protein begins with glycιne-27 as numbered from the initiation methionine The C-terminal hydrophobic transmembrane domain extends from amino acid Leu-220 to Ala-245 The deduced extracellular domain sequence includes ammo acids Gly-27 to Pro-219 Figure 3A-3B depicts an alignment of the AL-21 nucleotide sequence with human EST sequence H10006 (SEQ ID NO 5) Figure 4 shows a comparison of the AL-21 and AL-2s ammo acid sequences with that of Lerk2 (Beckmann et al EMBO J 13 3757-3762 ( 1994)) and human Htk-L (Bennett et al Proc Natl Acad Sci USA 92 1866-70 (1995), WO 96/02645 published February 1 , 1996, both are incorporated by reference herein) Identical ammo acids are boxed, and gaps introduced for optimal alignment are indicated by dashes Conserved cysteine residues can be seen The deduced C-terminal amino acid for AL-2s is valine Figure 5 shows a comparison ofthe AL-21 ammo acid sequences with that of Lerk2 and human Htk-L Identical ammo acids are boxed, and gaps introduced for optimal alignment are dicated by dashes Conserved cysteine residues can be seen DETAILED DESCRIPTION "AL-2" or "AL-2 protem" refers to a polypeptide or protem encoded by the AL-2 nucleotide sequence set forth in Figures 1A-1B (showing AL-21) or 2 (showing AL-2s), a polypeptide that is the translated amino acid sequence set forth in Figures 1A-1B or 2A-2B, fragments thereof having greater than about 5 contiguous ammo acid residues and comprising an immune epitope or other biologically active site of AL-2, amino acid sequence vaπants ofthe ammo acid sequence set forth m Figures 1 A-1B or 2A-2B wherein one or more amino acid residues are added at the N- or C-terminus of, or within, said Figures 1 A- IB or 2A-2B sequences or its fragments as defined above, ammo acid sequence variants of said Figures 1 A- IB or 2A-2B sequences or its fragments as defined above wherein one or more ammo acid residues of said Figures 1 A-2B or 2 A-2B sequences or fragment thereof are deleted, and optionally substituted by one or more amino acid residues, and deπvatives ofthe above proteins, polypeptides, or fragments thereof, wherein an am o acid residue has been covalently modified so that the resulting product is a non-naturally occurring ammo acid Preferred embodiments retain a biological property of AL-2 AL-2 ammo acid sequence vaπants may be made synthetically, for example, by site-directed or PCR mutagenesis, or may exist naturally, as m the case of allelic forms and other naturally occurring variants of the translated am o acid sequence set forth m Figures 1 A- IB or 2A-2B that occur in human or other animal species Accordmgly, within the scope ofthe present mvention are AL-2 proteins deπved from other animal species, preferably mammalian, including but not limited to murine, rat, bovine, porcme, or vaπous primates As used here , the term " AL-2" includes membrane-bound proteins (compnsing a cytoplasmic domain, a transmembrane region, and an extracellular domain), including the long and short forms of AL-2, as well as truncated protems that retain Eph-family-receptor binding property Truncated AL-2 proteins include, for example, soluble AL-2 comprising only the extracellular (receptor binding) domain Such fragments, vanants, and deπvatives exclude any polypeptide heretofore identified, including any known neurotrophic factor, such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophιn-3 (NT-3), and neurotrophιn-4/5 (NT-4/5), Eph family receptor ligand such as Erk-L or Lerk-2, as well as statutoπly obvious variants thereof A preferred AL-2 is one having a contiguous amino acid sequence of or derived from mature AL-2 shown Figures 1A-1B or 2A-2B By "Eph-related protem tyrosme kinase" or "Eph-related kinase" or "Eph-family receptor" means herein a receptor tyrosine kinase having an extracellular ligand binding domain, a transmembrane domain, and a cytoplasmic catalytic domain, and belonging to the Eph subclass of receptor tyrosine kinases Eph-family receptors include, for example, human receptor tyrosine kmases, Eph, Erk/Nuk. Htk. Eck. and Heks (e g , Hek. Hek2. Hek3. Hek4, Hek5, Hek6, Hek7. Hek8. Hek9. Hekl l. Hekl2). and their non-human counterparts including chicken Ceks (e g , Cek5. Cek6. Cek7, Cek8. Cek9. Cek 10), murine Nuk. Seks (e g . Sekl, Sek2, Sek3. Sek4; Gilardi-Hebenstreit et al, Oncogene 7.2499-2506 (1992)), Myk-1 (Andres et al . Oncogene 9(5) 1461-7 ( 1994)), Mek4, Mdk- 1 , and rat Tyros (e g , Tyro 1 , Tyro4, Tyro5, Tyro6, Tyro 1 1 ), Rek7, Ehk 1. Ehk2, Ehk3, Bsk, Eek, and Elk Natural ligands for these receptors can be characterized by means of ligand attachment to a cell membrane-either by a GPI-anchor (e g , Lerk3 and Lerk4 (Kozlosky et al , Oncogene, 10(2) 299-306 ( 1995)) or by a transmembrane sequence. Preferred receptors for AL-2 are receptors that are recognized by transmembrane-sequence type ligands Preferred receptors include rat Elk, Tyro5 (Marcelle et al , Oncogene, 7.2479-87 (1992)), and Tyro6, murine Nuk/Sek3 (Henkemeyer et al , Oncogene, 9(4) 1001-14 (1994). Becker et al , Mech Dev , 47(1) 3-17 (1994)), Myk-1, and Sek4 (Becker et al.. Mechanisms of Development, 47 3-17 (1994)), chicken Cek5, Cek6 and CeklO, and their human homologs More preferred are human receptors Hek5. Hek6, Hek3, Hek2. Keh9. Hekl 1, Hek 12, Htk, Erk and Eph. Even more preferred are human receptors Htk (WO 96/02645 published February 1, 1996), Hek2 (Bohme et al .Oncogene, 8 2857-2862 (1993)), Hek5 Erk (Fox et al., Oncogene, 10(5):897-905 (1995), (Kiyokawa et al., Cancer Res., 54 (14):3645-50 (1994), and Hek6 Of particularly preferred interest as an AL-2 binding receptor candidate are "orphan receptors," including human Eph, Hek3, Hek9, Hekl 1 and Hekl2, and less preferably their non-human homologs, as well as Ehk3 for which a human homolog is not known Eph-family-receptor-bindmg-transmembrane-sequence-containing ligands mclude the human Erk-L or Lerk2 ligand (Fletcher et al , Oncogene. 9(11)3241-7 (1994)) and the human Htk-L or Lerk5 ligand (Cerretti et al , Mol Immunol , 32(16): 1 197-205 (1995)). Non-human ligands mclude Cek5-L, Elf-2 and Elk-L Biologically active or antigenically active AL-2 polypeptides embodiments of this invention include the polypeptide represented by the entire translated nucleotide sequence of AL-21 and AL-2s (including their signal sequence), mature AL-2, i e , AL-2 without the signal sequence, fragments consisting essentially of the intracellular domam or transmembrane domain of AL-2; fragments ofthe AL-2 having a contiguous sequence of at least 5, 10, 15, 20, 25, 30, or 40 consecutive ammo acid residues from AL-2; ammo acid sequence variants of AL-2 wherem an ammo acid residue has been inserted N- or C-termmal to, or within, AL-2 or its fragment as defined above, ammo acid sequence vanants of AL-2 or its fragment as defined above wherem an ammo acid residue of AL-2 or its fragment as defined above has been substituted by another residue, including predetermined mutations by, e. , site-directed or PCR mutagenesis, AL-2 of various animal species such as rabbit, rat, porcme, non-human primate, equ e. murine. and ovme AL-2 and alleles or other naturally occurring vaπants ofthe foregoing and human AL-2, deπvatives of AL-2 or its fragments as defined above wherein AL-2 or its fragments have been covalent modified, by substitution, chemical, enzymatic, or other appropriate means. with a moiety other than a naturally occurring ammo acid; and glycosylation variants of AL-2 (insertion of a glycosylation site or alteration of any glycosylation site by deletion, insertion, or substitution of suitable residues) The preferred AL-2 is human AL-2 especially native human AL-2 having the sequence shown in Figures lA- lB or 2A-2B One embodiment ofthe present invention provides soluble AL-2 By "soluble AL-2" is meant AL-2 which is essentially free of at least a transmembrane sequence and optionally the intracellular domain of native AL-2 Bv 'essentially free" is meant that the soluble AL-2 sequence has less than 2% ofthe transmembrane domain, preferably less than 1% of the transmembrane domain, and more preferably less than 0 5% of this domain The transmembrane domain ofthe native human mature ammo acid sequences are delineated in Figures 1 A- I B and 2A-2B (for AL-21 and AL-2s respectively), ; e , resides Gly-27 to Pro-219 Soluble AL-2s have therapeutic advantages because they are generally soluble m the patient's blood stream Similarly, soluble ligands may prove to be particularly useful as diagnostics s ce they are expected to have a reduced tendency to incorporate in the cell membrane Soluble AL-2 polypeptides comprise all or part ofthe extracellular domain of a native AL-2 but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane Soluble AL-2 polypeptides advantageously compnse the native (or a heterologous) signal peptide when initially synthesized to promote secretion, but the signal peptide is cleaved upon secretion In preferred embodiments, the soluble AL-2 polypeptides retain the ability to b d an Eph-family receptor with preferences as discussed herein Soluble AL-2 can also mclude part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble AL-2 prote is capable of being secreted or otherwise isolated in one embodmient a soluble AL-2 is an "immunoadhesin" The term "immunoadhesin" is used interchangeably with the expression "AL-2-ιmmunoglobuhn chimera" and refers to a chimeric molecule that combines the extracellular do am ("ECD") of AL-2 with an immunoglobulin sequence The immunoglobulin sequence preferably, but not necessaπly, is an immunoglobulin constant domain The immunoglobulin moiety tn the chimeras ofthe present mvention may be obtained from IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA, IgE, IgD or IgM. but preferably IgG-1 or IgG-3 The expression "extracellular domam" or "ECD" when used herem refers to any polypeptide sequence that shares a receptor bmding function ofthe extracellular domain ofthe naturally occurring AL-2 disclosed herem Receptor bmdmg function refers to the ability ofthe polypeptide to bmd the extracellular domain of a Eph-family receptor, with preferences as discussed herein, and. optionally, activate the receptor Accordmgly, it is not necessary to mclude the entire extracellular domain since smaller segments are commonly found to be adequate for receptor binding The term ECD encompasses polypeptide sequences in which the cytoplasmic domain and hydrophobic transmembrane sequence (and, optionally, 1-20 amino acids amino-termmal to the transmembrane domain) of the mature AL-2 have been deleted The extracellular domain sequence of AL-2 is provided in Figures 1 A- IB and 2A-2B The term "epitope tagged" when used herein refers to a chimeric polypeptide compnsing the entire AL- 2, or a portion thereof, fused to a "tag polypeptide " The tag polypeptide has sufficient amino acids to provide an antibody-binding epitope but not mterfere with activity of the AL-2 The tag poiypeptide preferably also is fairly unique so that an antibody against it does not substantially cross-react with other epitopes Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 ammo acid residues, preferably between about 9-30 residues 'Isolated", when used to describe the various proteins disclosed herein, means protein that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are matenals that would mterfere with diagnostic or therapeutic uses for the protein, and may include enzymes, hormones, and other protemaceous or non-protemaceous solutes "Essentially pure" protein means a composition compπs g at least about 90% by weight of the protem based on total weight ofthe composition, preferably at least about 95% by weight "Essentially homogeneous" protem means a composition comprising at least about 99% by weight of protein, based on total weight ofthe composition An AL-2 ammo acid sequence vaπant is mcluded within the scope ofthe invention provided that it is functionally active As used herem, "functionally active" and "functional activity" in reference to AL-2 for the purposes herein means an tn vivo effector or antigenic function or activity that is performed by AL-2 of the sequences Figures 1 A-1B or 2A-2B (whether in its native or denatured conformation) A principal effector function is the ability of AL-2 to bmd to, and/or activate, a receptor from the Eph-receptor family, preferably a receptor for the transmembrane-ligand family that is also, more preferably, a human receptor Less preferred are their non-human homologs Generally, the ligand will bmd to the extracellular domain of the receptor and thereby activate its intracellular tyrosme kinase domain Consequently, bmding of the ligand to the receptor can result in enhancement or inhibition of proliferation and or differentiation and or activation of cells having a receptor for AL-2 in vrvo, ex vivo, or in vitro Other effector functions mclude signal transduction, any enzyme activity or enzyme modulatory activity (e g , tyrosine kinase activity), or any structural role, for example An antigenic function means possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised agamst the polypeptide sequence of a naturally occurring polypeptide compns g the polypeptide sequences of Figures 1A-1B and 2A-2B In preferred embodiments, antigenically active AL-2 is a polypeptide that binds with an affinity of at least about 10 6 l/mole to an antibody capable of b dmg AL-2 Ordmanly, the polypepnde bmds with an affmity of at least about 10 l/mole In particular, an AL-2 is able to promote or enhance the growth, survival, function, activation, and/or differentiation of neurons and glia, whether the neurons be central, peripheral, motoneuio s, or sensory neurons, e g , photoreceptors, vestibular ganglia, spinal ganglia, auditory hair cells, and the AL-2 is immunologically cross-reactive with an antibody directed agamst an epitope of naturally occurring AL-2 Therefore, AL-2 ammo acid sequence vaπants generally will share at least about 75% (preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, with increasing preference to at least 99%, and finally 100%) sequence identity with the translated ammo acid sequence set forth in Figures 1 A- IB and 2A-2B, after aligning the sequences and introducing gaps, if necessary, to achieve maximal percent identity This is typically determmed. for example, by the Fitch, et al , Proc Nat Acad Set USA, 80 1382-1386 (1983), version of the algorithm described by Needleman, et al , J Mol Biol , 48 443-453 (1970) None of N-terminal, C- terminal. or internal extensions, deletions, or insertions into the AL-2 sequence shall be construed as affecting sequence identity or homology Preferably, the AL-2 nucleic acid molecule that hybridizes to nucleic acid sequence encoding AL-2 contains at least 20, more preferably 40, even more preferably 70, and most preferably 90 bases For fragments the percent identity is calculated for that portion of a native sequence that is present in the fragment In one embodiment an isolated AL-2 protein induces phosphorylation of an Eph-family receptor and contains an ammo acid sequence selected from the group consisting of (a) the am o acid sequence for mature AL-21 (b) the amino acid sequence for mature AL-2s, (c) the naturally occurring amino acid sequence for mature AL-2 from a non-human animal species, (d) allelic variants of the sequences of (a), (b), or (c) and (e) the sequences of (a), (b), (c), or (d) having a single preferred conservative amino acid substitution as defined in Table 1 In a preferred embodmient the phosphorylation-inducing AL-2 has the ammo acid sequence for mature human AL-2 shown m Figures 1 A- IB or 2A-2B Generally the AL-2 will be a chimera, membrane or liposome bound, or epitope tagged and "clustered" (see WO 95/27060, which is incorporated herein by reference), thus mimicking its membrane-bound state and ability to induce receptor phosphorylation In another embodiment an isolated AL-2 protem bmds to the Eph-family receptor and contains an ammo acid sequence selected from the group consisting of (a) the ammo acid sequence for mature AL-21, (b) the amino acid sequence for mature AL-2s, (c) the naturally occurring ammo acid sequence for mature AL-2 from a non-human animal species, (d) allelic vaπants ofthe sequences of (a), (b), or (c). and (e) the sequences of (a), (b), (c), or (d) having a single preferred conservative ammo acid substitution as defined in Table 1 in a preferred embodiment the AL-2 has the ammo acid sequence for mature human AL-2 shown in Figures 1 A- 1 B or 2A-2B In another embodiment isolated soluble AL-2 binds to a Eph-family receptor and contams an amino acid sequence selected from the group consisting of (a) the ammo acid sequence for mature soluble AL-21, (b) the ammo acid sequence for mature soluble AL-2s, (c) the naturally occurring ammo acid sequence for mature soluble AL-2 from a non¬ human animal species, (d) allelic variants ofthe sequences of (a), (b), or (c), and (e) the sequences of (a), (b), (c), or (d) havmg a smgle preferred conservative ammo acid substitution as defined m Table 1 In a preferred embodiment the soluble AL-2 has the ammo acid sequence for mature soluble human AL-2 shown in Figures 1 A- 1 B or 2 A-2B In another preferred embodiment, the soluble AL-2 is a chimeric polypeptide containing an ammo acid sequence encodmg mature soluble AL-2 fused to an immunoglobulm sequence In a more preferred embodiment the chimeric polypeptide contains a fusion of an AL-2 extracellular domain sequence to an immunoglobulm constant domam sequence Preferably the constant domam sequence is that of an immunoglobulm heavy cha Also preferred are chimeπc polypeptides containing a mature, soluble Al-2 ammo acid sequence fused to an epitope tag polypeptide sequence AL-2 can be recovered from culture of cells expressing AL-2, preferably from the culture medium as a secreted polypeptide, although, AL-2 can be recovered from host cell lysates when directly produced without a secretory signal When AL-2 is membrane-bound, it can be released from the membrane using a suitable detergent solution (e g , Tnton-X 100™) When AL-2 is produced m a recombmant cell other than one of human origin, AL-2 is completely free of protems or polypeptides of human oπgin However, it is necessary to purify AL-2 from recombmant cell protems or polypeptides to obta preparations that are substantially homogeneous in AL-2 As a first step, the culture medium or lysate is centrifuged to remove particulate cell debns Then AL-2 is puπfied from contaminant soluble proteins and polypeptides, with the following procedures being exemplary of suitable purification procedures by fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC. chromatography on silica or on a cation-exchange resin such as DEAE. chromatofocusing, SDS- PAGE ammonium sulfate precipitation, gel filtration using, for example. Sephadex™ G-75 and protein A Sepharose™ columns to remove contaminants such as IgG In a preferred embodiment, an AL-2- receptor-Fc fusion, using the preferred AL-2-receptors in Fc constructs, for example as disclosed by Bennett et al J Biol Chem , 269(19) 1421 1-8 ( 1994) is immobilized on a protein A Sepharose™ column and AL-2 can be isolated by affinity purification using this column AL-2 variants in which residues have been deleted, inserted, or substituted are recovered in the same fashion as native AL-2, taking account of any substantial changes in properties resulting from the variation For example preparation of aπ AL-2 fusion with another protein or polypeptide. e g . a bacterial or viral antigen facilitates purification, an immunoaffinity column containing antibody to the antigen can be used to adsorb the fusion polypeptide Immunoaffmity columns such as a rabbit polyclonal antι-AL-2 column can be employed to absorb the AL-2 vaπant by bmdmg it to at least one remaming immune AL-2 epitope A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may be useful to inhibit proteolytic degradation during purification, and antibiotics may be mcluded to prevent the growth of adventitious contaminants One skilled m the an will appreciate that purification methods suitable for native AL-2 can require modification to account for changes in the character of AL-2 or its vanants upon expression in recombinant cell culture Ammo acid sequence vanants of AL-2 are prepared by introducing appropπate nucleotide changes mto AL-2 DNA and thereafter expressmg the resulting modified DNA in a host cell, or by in vitro synthesis Such vaπants mclude, for example, deletions from, or insertions or substitutions of, ammo acid residues within the AL- 2 amino acid sequence set forth in Figures 1A-1B and 2A-2B Any combmation of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of AL-2, provided that such variant possesses the desired characteristics descnbed herein Changes that are made in the ammo acid sequence set forth in Figures 1 A- IB and 2A-2B to arrive at an ammo acid sequence variant of AL-2 also may result m further modifications of AL-2 upon its expression in host cells, for example, by virtue of such changes introducing or movmg sites of glycosylation, or introducing membrane anchor sequences as descπbed, for example, in PCT Pat Pub No WO 89/01041 (published February 9, 1989) There are two prmcipal variables in the construction of amino acid sequence vaπants of AL-2 the location ofthe mutation site and the nature of the mutation These are variants from the am o acid sequence set forth in Figures 1A-1B and 2A-2B, and may represent naturally occurring allelic forms of AL-2, or predetermined mutant forms of AL-2 made by mutating AL-2 DNA, either to amve at an allele or a variant not found in nature In general, the location and nature of the mutation chosen will depend upon the AL-2 characteristic to be modified For example, due to the degeneracy of nucleotide coding sequences, mutations can be made in the AL-2 nucleotide sequence set forth in Figures 1 A- 1 B and 2A-2B without affecting the ammo acid sequence ofthe AL- 2 encoded thereby Other mutations can be made that will result in a AL-2 that has an am o acid sequence different from that set forth in Figures 1 A-l B and 2A-2B but which is functionally active Such functionally active ammo acid sequence vaπants of AL-2 are selected, for example, by substituting one or more amino acid residues in the ammo acid sequence set forth in Figures 1 A- 1 B and 2A-2B with other am o acid residues of a similar or different polarity or charge One useful approach is called "alanine scanning mutagenesis ' Here, a an ammo acid residue or group of target residues are identified (e , charged residues such as arginine, aspartic acid, histidine. lvsine, and glutamic acid) and. by means of recombinant DNA technology, replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell (Cunningham, et al . Science. 244 1081-1085 ( 1989)) Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at or for the sites of substitution Obviously, such variations that for example convert the ammo acid sequence set forth in Figures 1 A- IB and 2A-2B to the ammo acid sequence of a known neurotrophic factor, such as NGF. BDNF, NT-3, NT-4/5, Eph-family receptor ligand (e g , see Figures 4 and 5). or another known polypeptide or protem are not mcluded within the scope of this invention, nor are any other fragments, vaπants, and derivatives ofthe ammo acid AL-2 that are not novel and unobvious over the pnor art Thus, while the site for introducing an ammo acid sequence vaπation is predetermined, the nature ofthe mutation per se need not be predetermined For example, to optimize the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed AL-2 variants are screened for functional activity Ammo acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues, and typically are contiguous Deletions from regions of substantial homology with other tyrosme kmase receptor ligands. for example, are more likely to affect the functional activity of AL-2 Generally, the number of consecutive deletions will be selected so as to preserve the tertiary structure of AL-2 in the affected domain, e g , beta-pleated sheet or alpha helix Ammo acid sequence insertions mclude ammo- and/or carboxyl-terminal fusions ranging in length from one ammo acid residue to polypeptides containing a hundred or more residues, as well as lntrasequence insertions of smgle or multiple ammo acid residues lntrasequence insertions, i e , insertions made within the ammo acid sequence set forth in Figures 1 A- 1 B or 2 A-2B, may range generally from about 1 to 10 residues, more preferably 1 to 5, even more preferably 1 to 3, and most preferably 1 to 2 Examples of terminal insertions mclude AL-2 with an N-terminal methionyl residue (such as may result from the direct expression of AL-2 m recombmant cell culture), and AL-2 with a heterologous N-terminal signal sequence to improve the secretion of AL-2 fi om recombmant host cells Such signal sequences generally will be homologous to the host cell used for expression of AL-2. and include STII or lpp for E coli, alpha factor for yeast, and viral signals such as heφes gD for mammalian cells Other insertions include the fusion to the N- or C-terminus of AL-2 of immunogenic polypeptides (for example, bacteπal polypeptides such as beta-lactamase or an enzyme encoded by the E coli tφ locus, or yeast protem), and C-termmal fusions with protems having a long half-life such as immunoglobulin constant regions, albumin, or femtin, as descnbed in PCT Pat Pub No WO 89/02922 published April 6, 1989 The third group of variants are those in which at least one ammo acid residue in the am o acid sequence set forth in Figures 1 A- 1 B or 2A-2B, preferably one to four, more preferably one to three, even more preferably one to two, and most preferably only one, has been removed and a different residue inserted in its place The sites of greatest mterest for making such substitutions are in the regions ofthe amino acid sequence set forth in Figures 1 A-1B or 2A-2B that have the greatest homology with other tyrosme kinase receptor ligands (for non-limiting examples, see comparisons in Figures 4 and 5) Those sites are likely to be important to the functional activity ofthe AL-2 Accordingly, to retain functional activity, those sites, especiallv those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner Such conservative substitutions are shown in Table 1 under the headmg of preferred substitutions if such substitutions do not result in a change in functional activity, then more substantial changes, denominated exemplary substitutions in Table 1. or as further described below in reference to amino acid classes may be introduced and the resulting variant AL-2 analyzed for functional activity TABLE 1 Original Residue Exemplary Substitutions Preferred Substitutions Ala (A) val, leu, lie val Arg (R) lys; gin, asn lys Asn (N) gin; his, lys, arg gin Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro pro His (H) asn, gin, lys, arg arg He (1) leu, val, met, ala. phe, norleucine leu Leu (L) norleucine, lie, val, met, ala, phe lie Lys (K) arg, gin; asn arg Met (M) leu, phe, ile leu Phe (F) leu, val; lie, ala leu Pro (P) giy giy Ser (S) thr thr Thr (T) ser ser Tφ (W) tyr tyr Tyr (Y) trp; phe; thr, ser phe Val (V) lie, leu, met, phe, ala. norleucine leu Insertional, deletional, and substitutional changes in the ammo acid sequence set forth in Figures 1 A- 1 B and 2A-2B may be made to unprove the stability of AL-2 For example, trypsm or other protease cleavage sites are identified by inspection ofthe encoded ammo acid sequence for an arginyl or lysinyl residue These are rendered inactive to protease by substituting the residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine. by deleting the residue, or by inserting a prolyl residue immediately after the residue Also, any cysteine residues not involved in maintaining the proper conformation of AL-2 for functional activity may be substituted, generallv with serine, to unprove the oxidative stability ofthe molecule and prevent aberrant crosslmkmg Additional sites for mutation are those sites that are conserved in AL-2 amongst species vaπants of AL- 2 but are not conserved between AL-2 and another ligand in the Eph ligand family, preferably between AL-2 and at least two ligands, and more preferably at least three ligands Such sites, which are not conserved between AL- 2 and another transmembrane-ligand, are candidates sites for modulating receptor specificity and selectivity Sites that are conserved between AL-2 and other transmembrane- gands are candidate sites for modulating activities shared by transmembrane-hgands. such as stability, folding, tertiary conformation, protease susceptibility, and amount of ligand specific activity A comparison of AL-2 am o acid sequences with other Eph-family receptor ligand sequences (see Figures 4 and 5) reveals AL-2 as a new Eph-family receptor ligand AL-2, having a transmembrane sequence, is more closely related to other transmembrane-containing ligands than to the GPI-anchored ligands of which AL-1 is an example Transmembrane-containing ligands mclude Lerk-2, a ligand for the Eph-related receptor Hek5, and Htk-L, a ligand for the Htk receptor Percent identities of ligand comparisons are provided in Table 2, in which "ECD" indicates extracellular domain TABLE 2 % IDENTITY Ligand Full Length ECD Cytoplasmic Domam Lerk2 vs HtkL 560% 49 3% 74 7% AL-2 vs Lerk2 41 5% 42 1% 48 2% AL-2 vs HtkL 40 8% 39 5% 56 6% AL-2 vs AL-l 28 0% Covalent modifications of AL-2 molecules also are included within the scope of this invention For example, covalent modifications are introduced into AL-2 by reacting targeted ammo acid residues ofthe AL-2 with an organic deπvatizing agent that is capable of reacting with selected ammo acid side chains or the N- or C-termmal residues Cysteinyl residues most commonly are reacted with α-haioacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives Cysteinyl residues also are deπvatized by reaction with bromotπfluoroacetone, α-bromo-β-(5-ιmιdozoyl)propιonιc acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nιtro-2-pyπdyl disulfide, methyl 2-pyπdyl disulfide, p- chloromercuπbenzoate, 2-chloromercuπ-4-nιtrophenol. or chloro-7-nιtrobenzo-2-oxa-l,3-dιazole Histidyl residues are deπvatized by reaction with diethylpyrocarbonate at pH 5 5-7 0 because this agent is relatively specific for the histidyl side chain Para-bromophenacyl bromide also is useful, the reaction is preferabl y performed in 0 1 M sodium cacodylate at pH 6 0 Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides Denvatization with these agents has the effect of reversing the charge ofthe lysinyl residues Other suitable reagents for derivatizing α-amino-containing residues include lmidoesters such as methyl picolinimidate, pyridoxal phosphate; pyridoxal; chloroborohydride; tπnitrobenzenesulfonic acid: O-methylisourea: 2.4- pentanedione; and transaminase-catalyzed reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione. and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because ofthe high pK a ofthe guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosyt residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly. N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125 I or , 31 I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'- N=C=N-R'), where R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-moφholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Derivatization with bifunctional agents is useful for crosslinking AL-2 to a water-insoluble support matrix or surface for use in the method for purifying anti-AL-2 antibodies, or for therapeutic use. Commonly used cross! inking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy- succinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters. including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N- maieimido-l,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691.016; 4,195,128; 4.247,642; 4,229,537; and 4.330,440 are employed for protein immobilization. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Altematively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention. Other modifications mclude hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation ofthe α -amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. (Creighton, Proteins: Structure and Molecular Properties, pp.79-86 (W.H. Freeman & Co., 1983)). AL-2 also is covalently linked to nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol or polyoxyalkylenes. in the manner set forth in U.S. Pat. Nos. 4, 179,337; 4,301,144; 4,496,689; 4,640,835; 4,670,417; or 4.791,192. "AL-2 antagonist" or "antagonist" refers to a substance that opposes or interferes with a functional activity of AL-2. "Cell," "host cell." "cell line," and "cell culture" are used interchangeably and all such terms should be understood to include progeny. Thus, the words "transformants" and "transformed cells" include the primary sub j ect cell and cultures derived theretrom without regard for the number of times the cultures have been passaged It should also be understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations 'Piasmids" are DNA molecules that are capable of repiicatmg within a host cell either extrachromosomally or as part ofthe host cell chromosome(s), and are designated bv a lower case "p' preceded and/or followed by capital letters and or numbers The starting piasmids herem are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available piasmids as disclosed herem and/or m accordance with published procedures In certain instances, as will be apparent to the ordinarily skilled artisan, other piasmids known m the art may be used interchangeably with piasmids described herein "Control sequences" refers to DNA sequences necessary for the expression of an operably linked nucleotide coding sequence in a particular host cell The control sequences that are suitable for expression in prokaryotes, for example, include oπgms of replication, promoters, ribosome bmdmg sites, and transcription termination sites The control sequences that are suitable for expression in eukaryotes, for example, mclude origins of replication, promoters, ribosome bindmg sites, polyadenylation signals, and enhancers An "exogenous" element is one that is foreign to the host cell, or homologous to the host cell but in a position within the host cell in which the element is ordinarily not found "Digestion" of DNA refers to the catalytic cleavage of DNA with an enzyme that acts only at certain locations in the DNA Such enzymes are called restriction enzymes or restriction endonucleases, and the sites within DNA where such enzymes cleave are called restnction sites If there are multiple restriction sites withm the DNA, digestion will produce two or more linearized DNA fragments (restriction fragments) The various restnction enzymes used herein are commercially available and their reaction conditions, cofactors. and other requirements as established by the enzyme manufacturers are used Restnction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters representing the microorganism from which each restnction enzyme originally was obtained and then a number designating the particular enzyme In general, about 1 μg of DNA is digested with about 1-2 units of enzyme m about 20 μl of buffer solution Appropπate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer, and or are well known in the art "Recovery" or "isolation" of a given fragment of DNA from a restnction digest typically is accomplished by separating the digestion products, which are referred to as "restriction fragments," on a polyacrylamide or agarose gel by electrophoresis, identify mg the fragment of mterest on the basis of its mobility relative to that of marker DNA fragments of known molecular weight, excising the portion ofthe gel that contains the desired fragment, and separating the DNA from the gel, for example by electroelution 'Ligation" refers to the process of forming phosphodiester bonds between two double-stranded DNA fragments Unless otherwise specified, ligation is accomplished using known buffers and conditions with 10 units of T4 DNA ligase per 0 5 μg of approximately equimolar amounts ofthe DNA fragments to be ligated Oligonucleotides" are short-length, single- or double-stranded poiydeoxynucleotides that are chemically synthesized by known methods (involving, for example, triester, phosphoramidite, or phosphonate chemistrv). such as described by Engels, et al , Agnew Chem Int Ed Engl. 28 716-734 (1989) They are then purified for example, by polyacrylamide gel electrophoresis 'Polymerase chain reaction,' or "PCR," as used herein generally refers to a method for amplification of a desired nucleotide sequence tn vitro as descπbed in ϋ S Pat No 4 683.195 In general, the PCR method involves repeated cycles of pπmer extension synthesis usmg two oligonucleotide primers capable of hybridizing preferentially to a template nucleic acid Typically, the primers used in the PCR method will be complementary to nucleotide sequences within the template at both ends of or flanking the nucleotide sequence to be amplified, although primers complementary to the nucleotide sequence to be amplified also may be used (Wang, et al . in PCR Protocols, pp 70-75 (Academic Press 1990) Ochman. et al . in PCR Protocols, pp 219-227 Tπglia et al Nuc Acids Res . 16 8186 ( 1988)) "PCR cloning" refers to the use ofthe PCR method to amplify a specific desired nucleotide sequence that is present amongst the nucleic acids from a suitable cell or tissue source, mcludmg total genomic DNA and cDNA transcnbed from total cellular RNA ( Frohman, et al , Proc Nat Acad Sci USA, 85 8998-9002 ( 1988), Saiki, et al . Science, 239 487-492 (1988), Mullis, et al , Meth Enzymol 155 335-350 (1987)) "Stringent conditions" for hybπdization or annealing of nucleic acid molecules are those that ( 1 ) employ low ionic strength and high temperature for washing, for example, 0015 M NaCl/00015 M sodium cιtrate/0 1 % sodium dodecyl sulfate (SDS) at 50° C, or (2) employ dunng hybndization a denaturing agent such as formamide, for example. 50% (vol vol) formamide with 0 1% bovine serum albumιn/0 1% Fιcoll 0 1% polyvmylpyrrolιdone/50 mM sodium phosphate buffer at pH 6 5 with 750 mM NaCl, 75 mM sodium citrate at 42° C Another example is use of 50% formamide. 5 x SSC (0 75 M NaCl. 0 075 M sodium citrate), 50 mM sodium phosphate (pH 6 8), 0 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/mL), 0 1% SDS, and 10% dextran sulfate at 42° C, with washes at 42°C in 0 2 x SSC and 0 1% SDS "AL-2 nucleic acid" is RNA or DNA that encodes AL-2 "AL-2 DNA" is DNA that encodes AL-2 AL-2 DNA is obtained from cDNA or genomic DNA libraπes, or by in vitro synthesis Identification of AL-2 DNA within a cDNA or a genomic DNA library, or in some other mixture of vaπous DNAs, is conveniently accomplished by the use of an oligonucleotide hybndization probe that is labeled with a detectable moiety, such as a radioisotope (Keller, et al , DNA Probes, pp 149-213 (Stockton Press, 1989)) To identify DNA encoding AL-2, the nucleotide sequence ofthe hybndization probe preferably is selected so that the hybridization probe is capable of hybndizmg preferentially to DNA encodmg the AL-2 ammo acid sequence set forth in Figures 1 A- 1B or 2A-2B, or a vaπant or denvative thereof as described herein, under the hybridization conditions chosen Another method for obtaining AL-2 nucleic acid is to chemically synthesize it using one of the methods described, for example, by Engels, et al , Agnew Chem Int Ed Engl , 28 716-734 (1989) A preferred embodiment is an isolated nucleic acid molecule that includes a nucleotide sequence encoding the amino acid sequence shown m Figures 1 A-1B or 2A-2B for mature AL-2, and in which, more preferably, the AL-2 codons are contiguous A preferred nucleotide sequence encoding the amino acid sequence for mature AL-2 can be found in Figures 1 A- 1 B or 2 A-2B Also included are AL-2-encodmg nucleic acid sequences based on the codon degeneracy ofthe genetic code If the entire nucleotide coding sequence tor AL-2 is not obtained in a single cDNA, genomic DNA or other DNA, as determined, for example, by DNA sequencing or restriction endonuclease analysis, then appropπate DNA fragments (e g , restriction fragments or PCR amplification products) may be recovered from several DNAs and covalently jomed to one another to construct the entri e codmg sequence The preferred means of covalently joining D A fragments is by ligation using a DNA ligase enzyme, such as T4 DNA ligase "Isolated" AL-2 nucleic acid is AL-2 nucleic acid that is identified and separated from (or otherwise substantially free from) contaminant nucleic acid encodmg another polypeptide or from nucleic acid with which it is normally associated the natural source of AL-2 nucleic acid Isolated AL-2 nucleic acid molecules therefore are distinguished from the AL-2 nucleic acid molecule as it occurs naturally in cells However, an isolated AL-2 nucleic acid molecule includes AL-2 nucleic acid molecules contained in cells that ordinarily express AL-2 where, for example, the nucleic acid molecule is a chromosomal location different from that of natural cells The isolated AL-2 nucleic acid can be incoφorated mto a plasmid or expression vector for in vitro, ex vivo or in vivo use, or can be labeled for diagnostic and probe puφoses, using a label as described further herein in the discussion of diagnostic assays and nucleic acid hybridization methods For example, isolated AL-2 DNA, or a fragment thereof comprising at least about 15 nucleotides. is used as a hybndization probe to detect, diagnose, or monitor disorders or diseases that mvolve changes in AL-2 expression, such as may result from neuron damage In one embodiment ofthe mvention, total RNA in a tissue sample from a patient (that is, a human or other mammal) can be assayed for the presence of AL-2 messenger RNA, wherem the decrease m the amount of AL-2 messenger RNA is indicative of neuronal degeneration The present mvention further provides antisense or sense oligonucleotides compπs g a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target AL-2 mRNA (sense) or AL-2 DNA (antisense) sequences Antisense or sense oligonucleotides, accordmg to the present mvention, comprise a fragment of the coding region of AL-2 cDNA Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides The ability to create an antisense or a sense oligonucleotide, based upon a cDNA sequence for a given prote is described for example, in Stein et al , Cancer Res , 48 2659 (1988) and van der Krol et al , BioTechniques, 6 958, 1988 Although not to be restncted by the following working model, it is generally believed that binding of antisense or sense oligonucleotides to target nucleic acid sequences results m the formation of duplexes that block translation (RNA) or transcription (DNA) by one of several means, mcludmg enhanced degradation of the duplexes, premature termination of transcπpnon or translation, or by other means Trie antisense oligonucleotides can be used to block expression of AL-2 proteins Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar Imkages. such as those descπbed W091/06629) and wherem such sugar Imkages are resistant to endogenous nucleases Such oligonucleotides with resistant sugar Imkages are stable in vivo (i e . capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those descπbed in WO 90/10448, and other moieties that increase affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lvsιne) Further still, mtercalatmg agents, such as ellipticine, and alkylatmg agents or metal complexes can be attached to sense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotide sequence Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including for example, CaP04-medιated DNA transfection. electroporation, or other gene transfer vectors such as Epstein-Barr virus or adenovirus Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the ceil with the retrovirus vector containing the inserted sequence, either m vivo or ex vivo Suitable retroviral vectors include but are not limited to, the muπne retrovirus M-MuLV N2 (a retrovirus derived from M-MuLV) or the double copy vectors designated DCT5A, DCT5B and DCT5C (see PCT U S application Ser No 90/02656) Alternatively, other promotor sequences may be used to express the oligonucleotide Most preferably target- tissue specific promoters (either constitutive or inducible) are used Sense or antisense oligonucleotides are also introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91 /04753 Suitable ligand bmdmg molecules mclude, but are not limited to, cell surface receptors, growth factors, other cytokmes or other ligands that bmd to specific cell surface receptors Alternatively, a sense or an antisense oligonucleotide is introduced mto a cell contaming the target nucleic acid sequence by formation of an oligonucleotide-hpid complex, as descπbed in WO 90/10448 The sense or antisense oligonucleotide-hpid complex is preferably dissociated withm the cell by all endogenous hpase Isolated AL-2 nucleic acid also is used to produce AL-2 by recombinant DNA and recombmant cell culture methods In various embodiments of the invention, host cells are transformed or transfected with recombmant DNA molecules comprising an isolated AL-2 DNA, to obtam expression ofthe AL-2 DNA and thus the production of AL-2 in large quantities DNA encoding ammo acid sequence variants of AL-2 is prepared by a vaπety of methods known m the art These methods mclude, but are not lunited to, isolation from a natural source (m the case of naturally occurring ammo acid sequence vanants of AL-2) or preparation by site-directed (or o gonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encodmg a variant or a non-variant form of AL-2 Site-directed mutagenesis is a preferred method for preparing substitution, deletion, and insertion vanants of AL-2 DNA This technique is well known in the art (Zoller et al , Meth Enz , 100 4668-500 ( 1983 ), Zoller et al , Meth Em 154 329-350 ( 1987), Carter, Meth Enz . 154 382-403 ( 1987), Horwitz et al , Meth En: 185 599- 11 (1990)), and has been used, for example, to produce amino acid sequence variants of trypsm and T4 lysozyme, which vaπants have certain desired functional properties (Perry et al , Science 226 555-557 (1984), Craik et al , Science 228.291-297 (1985)) Briefly, in carrying out site-directed mutagenesis of AL-2 DNA, the AL-2 DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such AL-2 DNA After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and us g the single strand of AL-2 DNA as a template Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA Oligonucleotides for use as hybridization probes or primers may be prepared by any suitable method such as by puπfication of a naturally occurring DNA or by in vitro synthesis For example, oligonucleotides are readily synthesized using various techniques in organic chemistry, such as described by Narang et al , Meth Enzvmol 68 90-98 (1979), Brown et al , Meth Enzymol , 68 109-151 (1979), and Caruther et al Meth Enzymol . 154 287-313 (1985) The general approach to selecting a suitable hybridization probe or primer is well known (Keller et al DNA Probes, pp 1 1-18 (Stockton Press, 1989)) Typically, the hybridization probe or primer will contain 10-25 or more nucleotides. and will include at least 5 nucleotides on either side of the sequence encodmg the desired mutation so as to ensure that the oligonucleotide will hybridize preferentially to the single-stranded DNA template molecule Multiple mutations are introduced into AL-2 DNA to produce amino acid sequence variants of AL-2 compnsing several or a combination of insertions deletions or substitutions of ammo acid residues as compared to the ammo acid sequence set forth Figures 1A-1 B or 2A-2B If the sites to be mutated are located close together, the mutations may be introduced simultaneously using a smgle oligonucleotide that encodes all of the desired mutations If, however, the sites to be mutated are located some distance from each other (separated by more than about ten nucleotides), it is more difficult to generate a single oligonucleotide that encodes all ofthe desired changes Instead, one of two alternative methods may be employed In the first method, a separate oligonucleotide is generated for each desired mutation The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all ofthe desired ammo acid substitutions The alternative method m vol ves two or more rounds of mutagenesis to produce the desired mutant The first round is as descnbed for introducing a smgle mutation a smgle strand of a previously prepared AL-2 DNA is used as a template, an oligonucleotide encodmg the first desired mutation is annealed to this template, and a heteroduplex DNA molecule is then generated The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template Thus, this template already contains one or more mutations The oligonucleotide encoding the additional desired ammo acid substιtutιon(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis This resultant DNA can be used as a template in a third round of mutagenesis, and so on PCR mutagenesis is also suitable for making ammo acid sequence variants of AL-2 (Higuchi, in PCR Protocols, pp 177-183 (Academic Press, 1990), Vallette et al , Nuc Acids Res 17 723-733 (1989)) Briefly, when small amounts of template DNA are used as startmg material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the pnmers differ from the template For introduction of a mutation into a plasmid DNA, for example, one of the pnmers is designed to overlap the position ofthe mutation and to conta the mutation, the sequence ofthe other pnmer must be identical to a nucleotide sequence within the opposite strand ofthe plasmid DNA, but this sequence can be located anywhere along the plasmid DNA It is preferred, however, that the sequence ofthe second primer is located within 200 nucleotides from that ofthe first, such that in the end the entire amplified region of DNA bounded by the primers can be easily sequenced PCR amplification using a primer pair like the one just descπbed results in a population of DNA fragments that differ at the position ofthe mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone (Wagner et al , in PCR Topics, pp 69-71 (Spπnger-Verlag, 1991)) If the ratio of template to product amplified DNA is extremely low. the ma j onty of product DNA fragments incoφorate the desired mutatιon(s) This product DNA is used to replace the corresponding region in the plasmid that served as PCR template using standard recombinant DNA methods Mutations at separate positions can be introduced simultaneously by either using a mutant second primer, or performing a second PCR with different mutant primers and ligating the two resulting PCR fragments simultaneously to the plasmid fragment in a three (or more)-paπ ligation Another method for preparing variants cassette mutagenesis. is based on the technique described by Wells et al Gene 34 315-323 (1985) The starting material is the plasmid (or other vector) compnsing the AL- 2 DNA to be mutated The codon(s) in the AL-2 DNA to be mutated are identified There must be a unique restnction endonuclease site on each side ofthe identified mutation sιte(s) If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the AL-2 DNA The plasmid DNA is cut at these sites to linearize it A double-stranded oligonucleotide encodmg the sequence ofthe DNA between the restnction sites but containing the desired mutatιon(s) is synthesized using standard procedures, wherem the two strands ofthe oligonucleotide are synthesized separately and then hybridized together using standard techniques This double-stranded oligonucleotide is referred to as the cassette This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid This plasmid now contains the mutated AL-2 DNA sequence In another embodiment, AL-2 suitable for therapy is AL-2 covalently joined to another protem, such as an immunoglobulm domam (for example, to produce an AL2-IgG fusion protem) Immunoglobulm fusions, immunoadhesins, are chimeric antibody-like molecules that combine the functional domaιn(s) of a binding protem (m this case AL-2 or its receptor) with the an immunoglobulm sequence The immunoglobulm sequence preferably (but not necessarily) is an immunoglobulm constant domain Immunoglobulins (lg) and certain variants thereof are known and many have been prepared in recombinant cell culture For example, see U S Patent 4,745,055, EP 256,654, Faulkner et al , Nature, 298 286 (1982), EP 120,694, EP 125,023, Momson, J Immun , 123 793 (1979), Kohler et al , Proc Nat'l Acad Sci USA, 77 2197 (1980), Raso et al Cancer Res 41.2073 (1981), Momson et al , Ann Rev Immunol , 2 239 (1984), Morrison, Science, 229 1202 (1985), Momson et al Proc Nat'l Acad Sci USA, 81 6851 ( 1984), EP 255,694; EP 266.663, and WO 88/03559 Reasserted immunoglobulm chains also are known See for example U S patent 4.444,878, WO 88/03565, and EP 68,763 and references cited therein The immunoglobul moiety in the chimeras ofthe present invention may be obtained from IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA, IgE, IgD or IgM, but preferably lgG-1 or IgG-3 Chimeras constructed from a receptor sequence linked to an appropπate immunoglobulm constant domain sequence (immunoadhesins) are known in the art Immunoadhesins reported in the literature include fusions of the T cell receptor* (Gascoigne et al , Proc Natl Acad Set USA, 84 2936-2940 (1987)), CD4* (Capon et al , Nature, 337 525-531 (1989), Traunecker et al , Nature 339 68-70 (1989), Zettmeissl et al . DNA Cell Biol USA 9 347-353 ( 1990), Bym et al , Nature, 344 667-670 (1990)), L-selectin (homing receptor) (Watson et al , J Cell Biol , 1 10 2221-2229 (1990), Watson et al . Nature 349 164-167 ( 1991)), CD44* (Aruffo et al , Cell 61 1303-1313 (1990)) CD28* and B7* (Linsley et al , J Exp Med 173721-730 ( 1991 )) CTLA-4 * (Lisley et al , J Exp Med , 174 61-569 ( 1991)), CD22* (Stamenkovic et al . Cell 66 1 133- 1 144 (1991)). where the asterisk (*) indicates that the receptor is member ofthe immunoglobul superfamily The simplest and most straightforward immunoadhesin design combined the binding regιon(s) of the adhesin' protein (in this case AL-2) with the hinge and Fc regions of an immunoglobul heavy chain Ordmaπiy. when preparing chimeras ofthe present invention, nucleic acid encoding the extracellular domain or a fragment thereof of AL-2 will be fused C-termmally to nucleic acid encoding the N-terminus of an immunoglobulm constant domam sequence, however N-termmal fusions are aiso possible Typically, in such fusions the encoded chimeric polypeptide will retain at least functionally active hinge. CH2 and CH3 domains ofthe constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus ofthe Fc portion of a constant domain, or immediately N-terminal to the CHI ofthe heavy chain or the corresponding region of the light chain. The precise site at which the fusion is made is not critical, particular sites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics of AL-2-ιmmunoglobulin chimeras. In some embodiments, chimeras are assembled as monomers, or hetero- or homo-multimers, and particularly as dimers or tetramers, essentially as illustrated in WO 91/08298. In a preferred embodiment, the AL-2 extracellular domain sequence is fused to the N-terminus of the C-terminal portion of an antibody (in particular the Fc domain), containing the effector functions of an immunoglobulin, e g , immunoglobulin G j (IgG- 1 ). It is possible to fuse the entire heavy chain constant region to the AL-2 extracellular domam sequence Preferably a sequence beginning in the hinge region just upstream ofthe papain cleavage site (which defines IgG Fc chemically; residue 216, taking the first residue of heavy chain constant region to be 1 14. or analogous sites of other immunoglobulins) is used in the fusion. In one embodiment, an AL-2 ammo acid sequence is fused to the hinge region and CH2 and CH3 or CH 1 , hinge, CH2 and CH3 domains of an IgG- 1 , IgG-2. or IgG-3 heavy chain. The precise site at which the fusion is made is not critical, and the optimal site can be determmed by routine experimentation. The immunoglobulin portion can genetically engineered or chemically modified to inactivate a biological activity ofthe immunoglobulm portion, such as T-cell binding, while retaming desirable properties such as its scaffolding property for presenting AL-2 function to an axon or target cell. Chimeras can be assembled as multimers, particularly as homo-dimers or -tetramers. Generally, these assembled immunoglobulins will have known unit structures. A basic four chain structural unit is the form in which IgG, IgD, and IgE exist. A four unit is repeated in die highei molecular weight immunoglobulins; IgM generally exists as a pentamer of basic four units held together by disulfide bonds. IgA globulin, and occasionally IgG globulin, may also exist in multimeric form in serum. In the case of multimer, each four unit may be the same or different. Alternatively, the AL-2 extracellular domain sequences can be inserted between immunoglobulin heavy chain and light cham sequences such that an immunoglobulin compnsing a chimeric heavy chain is obtained. In this embodiment, the sequences are fused to the 3' end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the CH2 domain, or between the CH2 and CH3 domains (see Hoogenboom et al.. Mol. Immunol., 28: 1027-1037 ( 1991)). The presence of an immunoglobulin light chain is not required in the immunoadhesins ofthe present invention; an immunoglobulin light chain might be present either covalently associated to a immunoglobulin heavy chain fusion polypeptide, or directly fused to the AL-2 extracellular domam. In the former case, DNA encodmg an immunoglobulin light chain is typically coexpressed with the DNA encodmg the AL-2-immunoglobulin heavy cham fusion protem. Upon secretion, the hybnd heavy cha and the light cham will be covalently associated to provide an immunoglobulin-like structure comprising two disulfide-l ked immunoglobulm heavy chain-light chain pairs Preparation of such structures are, for example, disclosed m U S Patent No 4,816.567 issued 28 March 1989 The immunoglobulin sequences used in the construction ofthe immunoadhesins ofthe present invention can be from an IgG immunoglobulin heavy chain constant domain For human immunoadhesins, the use of human IgGl and lgG3 immunoglobulm sequences is preferred A major advantage of using IgG l is that IgG l immunoadhesins can be purified efficiently on immobilized protein A In contrast, purification of IgG3 requires protein G a significantly less versatile medium However, other structural and functional properties of immunoglobulins should be considered when choosing the lg fusion partner for a particular immunoadhesm construction For example, the lgG3 hinge is longer and more flexible so it can accommodate larger 'adhes ' domains that may not fold or function properly when fused to IgGl Another consideration may be valency, IgG immunoadhesins are bivalent homodimers, whereas lg subtypes like IgA and IgM may give rise to dimertc or pentameπc structures respectively, ofthe basic lg homodimer unit For AL-2-Ig immunoadhesins designed for in vivo application, the pharmacokinetic properties and the effector functions specified by the Fc region are important as well Although IgGl , IgG2 and IgG4 all have in vivo half-lives of 21 days, their relative potencies at activating the complement system are different IgG4 does not activate complement, and IgG2 is significantly weaker at complement activation than IgGl Moreover, unlike IgG 1, IgG2 does not bmd to Fc receptors on mononuclear cells or neutrophils While IgG3 is optimal for complement activation, its in vivo half-life l approximately one third ofthe other IgG isotypes Another important consideration for immunoadhesins designed to be used as human therapeutics is the number of allotypic variants ofthe particular isotype In general, IgG isotypes with fewer serologically-defined allotypes are preferred For example, IgGl has only four serologically-defined allotypic sites, two of which (Glml and 2) are located in the Fc region, and one of these sites G lml, is non¬ immunogenic In contrast, there are 12 serologically-defined allotypes IgG3, all of which are m the Fc region, only three of these sites (G3m5, 11 and 21) have one allotype which is nonimmunogenic Thus, the potential immunogenicity of a γ3 immunoadhesin is greater than that of a γ I immunoadhesin In designing the chimeras of the present invention domams that are not required for neurotrophin bmdmg and/or biological activity may be deleted In such structures, it is important to place the fusion junction at residues that are located between domams to avoid misfoldtng With respect to the parental immunoglobulm a useful joining point is just upstream ofthe cyste es ofthe h ge that form the disulfide bonds between the two heavy chams In a frequently used design, the codon for the C-termmal residue of the 'adhesm' part of the molecule is placed directly upstream ofthe codons for the sequence DKTHTCPPCP (SEQ ID NO 6) of the IgGl hmge region The general methods suitable for the construction and expression of immunoadhesins are the same those disclosed here above with regard to (native or variant) AL-2 For example, AL-2-Ig immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding the AL-2 portion in-frame to an lg cDNA sequence However, fusion to genomic lg fragments can also be used (see. e g Gascoigne et al Proc Natl Acad Sci USA 84 2936-2940 (1987), Aruffo et al . Cell 61 1303-1313 (1990), Stamenkovic et al , Cell 66 1 133- 1 144 ( 1991 )) The latter type of fusion requires the presence of lg regulatory sequences for expression cDNAs encodmg IgG heavy-chain constant regions can be isolated based on published sequence from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybndization or by polymerase cham reaction (PCR) techniques The cDNAs encoding the 'adhesm and the lg parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in the chosen host cells For expression in mammalian cells pRK5-based vectors (Schall et al , Cell 61 361-370 (1990)) and CDM8-based vectors (Seed, Nature 329 840 (1989)) The exact junction can be created by removing the extra sequences between the designed junction codons using o gonucleotide-directed delenonal mutagenesis (Zoller et al Nucleic Acids Res 10 6487 (1982), Capon et al . Nature 337 525-531 (1989)) Synthetic oligonucleotides can be used in which each half is complementary to the sequence on either side ofthe desired junction, ideally these are 36 to 48- mers Alternatively, PCR techniques can be used to join the two parts of the molecule m-frame with an appropriate vector The choice of host cell line for the expression of AL-2-Ig immunoadhesins depends amly on the expression vector Another consideration is the amount of protein that is required Milligram quantities often can be produced by transient transfections For example, the adenovirus EIA-transfoimed 293 human embryonic kidney cell line can be transfected transiently with pRK5-based vectors by a modification of the calcium phosphate method to allow efficient immunoadhesin expression CDM8-based vectors can be used to transfect COS cells by the DEAE-dextran method (Aruffo et al , Cell, 61 1303-1313 (1990), Zettmeissl et al , DNA Cell Biol , (US) 9 347-353 (1990)) If larger amounts of protem are desired, the immunoadhesin can be expressed after stable transfection of a host cell line For example, a pRK5-based vector can be introduced into Chinese hamster ovary (CHO) cells m the presence of an additional plasmid encoding dihydrofolate reductase (DHFR) and conferring resistance to G418 Clones resistant to G418 can be selected in culture, these clones are grown the presence of mcreasmg levels of DHFR inhibitor methotrexate, clones are selected, in which the number of gene copies encoding the DHFR and immunoadhesin sequences is co-amplified If the immunoadhesin contains a hydrophobic leader sequence at its N-terminus, it is likely to be processed and secreted by the transfected cells The expression of immunoadhesins with more complex structures may require uniquely suited host cells, for example, components such as light cham or J chain may be provided by certain myeloma or hybπdoma cell hosts (Gascoigne ef α/ , Proc Natl Acad Set USA, 84-2936-2940 (1987), Mait etal .J Virol , 67 3561-3568 (1993)) Immunoadhesins can be conveniently purified by affinity chromatography The suitability of protein A as an affinity ligand depends on the species and isotype ofthe immunoglobulin Fc domain that is used in the chimera Protem A can be used to puπfy immunoadhesins that are based on human γ 1 , γ2, or γ4 heavy chams (Lmdmark etal , J Immunol Meth , 62 1-13 (1983)) Protem G is recommended for all mouse isotypes and for human γ3 (Guss et al , EMBOJ 5- 15671575 (1986)) The matrix to which the affinity ligand is attached is most often agarose. but other matrices are available Mechanically stable matrices such as controlled pore glass or poly(styrenedιvιnyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose The conditions for bmdmg an immunoadhesin to the protein A or G affinity column are dictated entirely by the characteristics ofthe Fc domain, that is, its species and isotype Generally, when the proper ligand is chosen efficient binding occurs directly from unconditioned culture fluid One distinguishing feature of immunoadhesins is that, for human γ I molecules, the binding capacity for protein A is somewhat diminished relative to an antibody ofthe same Fc type Bound immunoadhesin can be efficiently eluted either at acidic pH (at or above 3 0). or in a neutral pH buffer containing a mildly chaotropic salt This affinity chromatography step can result in an immunoadhesin preparation that is >95% pure Other methods known in the art can be used m place of, or m addition to. affinity chromatography on protein A or G to purify immunoadhesins Immunoadhesins behave similarly to antibodies in thiophilic gel chromatography (Hutchens et al . Anal Biochem 159 217-226 ( 1986)) and immobilized metal chelate chromatography (Al-Mashikhi et al . J Dairy Sci 1 1756- 1763 ( 1988)) In contrast to antibodies however, their behavior on ion exchange columns is dictated not only by their lsoelectnc points, but aiso by a charge dipole that may exist in the molecules due to their chimeric nature If desired, the immunoadhesins can be made bispecific, that is, directed against two distinct ligands Thus, the immunoadhesins ofthe present mvention can have binding specificities for AL-2, or can specifically bind to a AL-2 and to an other determinant, for example one specifically expressed on the cells expressing a receptor to which the AL-2 portion ofthe immunoadhesin structure binds For bispecific molecules, tπmeπc molecules, composed of a chimenc antibody heavy cham in one arm and a chimeπc antibody heavy chain-light chain pair in the other arm of their antibody-like structure are advantageous, due to ease of purification In contrast to antibody-producing quadromas traditionally used for the production of bispecific immunoadhesins which produce a mixture of ten tetramers, cells transfected with nucleic acid encodmg the three chains of a trimeπc immunoadhesin structure produce a mixture of only three molecules, and purification ofthe desired product from this mixture is correspondingly easier This application encompasses chimenc polypeptides compnsing AL-2 fused to another polypeptide (such as the immunoadhesins mentioned above) In one preferred embodiment, the chimenc polypeptide contains a fusion ofthe AL-2 (or a fragment thereof, e g , the ECD ofthe AL-2) with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind The epitope tag is generally provided at the amino- or carboxyl- terminus ofthe AL-2 Such epitope-tagged- AL-2 can be detected using a labelled antibody agamst the tag polypeptide Also, the epitope tag allows AL-2 to be readily purified by anti-tag antibody affinity purification Affinity purification techniques and diagnostic assays involving antibodies are well-known Tag polypeptides and their respective antibodies are well known m the art Examples include the flu HA tag polypeptide and its antibody 12CA5, (Field et al . Mol Cell Biol , 8 2159-2165 ( 1988)), the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al Molecular and Cellular Biology 5(12) 3610-3616 (1985)), and the Heφes Sunplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al , Protein Engineering, 3(6) 547-553 (1990)) Other tag polypeptides have been disclosed Examples mclude the Flag-peptide (Hopp et al , BioTechnology, 6 1204-1210 (1988)), the KT3 epitope peptide (Martm et al , Science, 255 192-194 (1992)), an α-tubuhn epitope peptide (Skinner et al . J Biol Chem 266 15163-15166 (1991)). and the T7 gene 10 protem peptide tag (Lutz-Freyermuth et al Proc Natl Acad Sci USA 87 6393- 6397 ( 1990) Once the tag polypeptide has been selected, an antibody thereto can be generated using the techniques disclosed herein The general methods suitable for the construction and production of epitope tagged AL-2 are the same as those disclosed herein with regard to (native or variant) AL-2 AL-2-tag polypeptide fusions are most conveniently constructed by fusing the cDNA sequence encoding the AL-2 portion in-frame to the tag polypeptide DNA sequence and expressing the resultant DNA fusion construct in appropriate host cells Ordinarily, nucleic acid encoding the AL-2 (or a fragment thereof) will be fused at its 3 end to nucleic acid encoding the N-terminus of the tag polypeptide. however 5' fusions are also possible Epitope tagged AL-2 can be conveniently purified by affinity chromatography using the anti-tag antibody The matrix to which the affinity antibody is attached is most often agarose, but other matrices are available (e g controlled pore glass or poly(styrenedιvιnyl)benzene) The epitope tagged AL-2 can be eluted from the affinity column by varying the buffer pH or ionic strength or adding chaotropic agents, for example In another embodiment of the invention, multimeπc soluble ligands are prepared by expression as chimenc molecules utilizing flexible linker loops A DNA construct encoding the chimenc protein is designed such that it expresses two or more soluble or extracellular domams fused together in tandem (e g , "head-to- head") by a flexible loop This loop may be entirely artificial (e g polyglyc e repeats interrupted by serine or threonine at a certain interval) or "borrowed" from naturally occurring proteins (e g the hinge region of hlgG) Molecules can be engineered in which the length and composition ofthe loop is varied, to allow for selection of molecules with desired characteristics Although not wishing to be limited by theory, it is believed that membrane attachment of the ligands can facilitate ligand clustermg, which in turn can promote receptor multimenzation and activation Thus, one means of obtaining biological activity of the soluble AL-2 is mimicking, in solution, membrane associated ligand clustering Thus, a biologically active, clustered soluble Eph-family ligand compnses (soluble AL-2) n , wherem the soluble AL-2 is the receptor-bmding AL-2 extracellular domain and n is 2 or greater For example, despite the fact that receptor phosphorylation is markedly induced by stimulating receptor expressing reporter cells with mammalian cells overexpressing membrane-linked forms of the ligands AL-1 or B61 , there is little or no observable phosphorylation using soluble forms of these ligands. However, when secreted forms of B61 are myc-tagged and antibodies are used to cluster the ligands, or when AL- 1 -IgG chimera is used, the previously mactive soluble ligands strongly induce receptor tyrosme phosphorylation in reporter cells express g Ehk- 1 or Rek7 receptors, respectively Dimenzation of the soluble ligand, e g , utilizing Fc, can be sufficient for achiev g a biological response, however, further clustermg ofthe ligand accordmg to the mvention, for example usmg anti-Fc antibodies, may achieve an increase in biological activity Cells of the present mvention may transiently or, preferably, constitutively and permanently express AL-2 m native form, or in soluble form as chimenc tagged AL-2. AL-2 immunoadhesin, or clustered AL-2 as described herein Accordmgly, a method of enhancing the biological activity ofthe soluble AL-2 or its ECD is provided that cludes the steps of (a) expressing the soluble domain of AL-2 with an epitope tag and (b) exposing the tagged soluble domam to anti-tag antibodies The position ofthe tag with respect to AL-2 is not important so long as the tag does not interfere with AL-2 function and. in turn, AL-2 does not mterfere with tag function The tag is preferably located at either termini of AL-2, more preferably at the C-termmus of AL-2 However, the tag may be attached by covalent means, including with oxime linkages as taught for example in WO 9425071 published November 1 1. 1994 In additional embodiments are compounds of the formula (AL-2) n X, where n is an mteger greater than or equai to 2 and X is an organic linker covalently binding each AL-2 For example. AL-2 dimers and multimers can be made by attaching AL-2 peptides to an organic linker or baseplate (designated as X in the formula) us g methods and l kers (e g , baseplates or templates) described in the art. for example in WO 94/25071 WO 95/19567 or WO 95/04543 Accordingly, a biologically active, soluble AL-2 is provided that contains 2 or greater number of soluble AL-2 peptides where the soluble AL-2 is the AL-2 extracellular domam that binds an Eph-family receptor Preferably n is 2 to 20. more preterably 2 to 10, even more preferably 2 to 4 In one preferred embodiment n is 2 For example, multiple AL-2 are covalently attached to the same baseplate, e g , an organic molecule such as a penta-lysine where each AL-2 is attached site-specifically via a covalent linkage e g , an oxime linkage, which can be formed by reaction of a reactive group on AL-2 with its complementary reactive group on the baseplate Oxime linkages have supenor hydrolysis stability over a range of physiological conditions compared to hydrazones, etc Oxime linkages are not commonly subject to enzymatic hydrolysis Polyoxunes are therefore suited to applications where mtegnty and stability ofthe complex is desu-ed The Imker (or baseplate) should not interfere with AL-2 activity Baseplates can be designed to unprove solubility of peptides. as well as to present peptides to receptors A chemically reactive group suitable for oxime linkage formation can be site-specifically added to AL-2 through methods known in the art (see for example WO 90/02135. WO 94/25071, or EP 243929 B l issued September 27, 1995) It is apparent that AL-2 antagonists can be prepared or used applymg the above guidelines appropriately For example, a AL-2-bιndιng Eph-family-receptor-IgG chimera fusion (see Winslow et al , Neuron, 14 973-981 (1995)), for a method of making receptor-IgG fusions by recombmant means that is suitable for use with other Eph-family receptors and their extracellular domams) or antι-AL-2 antibody can be adsorbed onto a membrane, such as a silastic membrane, which can be implanted in proximity to tumors or arthπtic tissue, or can be incoφorated into liposomes (see for example WO 91/04014 published Apnl 4, 1991) It will be appreciated that some screening ofthe recovered vaπant will be needed to select one havmg the desu-ed activity A change m the immunological character ofthe AL-2 molecule, such as affinity for a given antibody, can be measured by a competitive-type immunoassay The variant is assayed for changes in the suppression or enhancement of its enzymatic activity by comparison to the activity observed for native AL-2 in the same assay For example, one can screen for the ability of the variant AL-2 to stimulate protein kinase activity of an Eph-family receptor using the techniques set forth, for example, in Lokker et al , EMBO J 1 1 2503-2510 (1992), Winslow et al , Neuron, 14 973-981 (1995), or Bennett et al J Biol Chem , 269( 19) 1421 1-8 (1994) Other potential modifications of protein or polypeptide properties such as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation, or the tendency to aggregate with carriers or into multimers are assayed by methods well known in the art AL-2 DNA, whether cDNA or genomic DNA or a product of in vitro synthesis, is ligated into a replicable vector for further cloning or for expression "Vectors" are piasmids and other DNAs that are capable of replicating autonomously within a host cell, and as such, are useful for performing two functions in conjunction with compatible host cells (a vector-host system) One function is to facilitate the cloning of the nucleic acid that encodes the AL-2, / e , to produce usable quantities ofthe nucleic acid The other function is to direct the expression of AL-2 One or both of these functions are performed by the vector-host svstem The vectors will contam different components depending upon the function they are to perform as well as the host cell with which they are to be used for cloning or expression To produce AL-2. an expression vector will contain nucleic acid that encodes AL-2 as descπbed above The AL-2 of this mvention is expressed directly in recombinant cell culture, or as a fusion with a heterologous polypeptide preferably a signal sequence or other polypeptide having a specific cleavage site at the junction between the heterologous polypeptide and the AL-2 In one example of recombinant host cell expression, mammalian cells are transfected with an expression vector comprising AL-2 DNA and the AL-2 encoded thereby is recovered from the culture, preferably cell culture medium m which the recombinant host cells are grown But the expression vectors and methods disclosed herein are suitable for use over a wide range of prokaryotic and eukaryotic organisms Prokaryotes may be used for the initial cloning of DNAs and the construction of the vectors useful in the mvennon However, prokaryotes may also be used for expression of DNA encoding AL-2 Polypeptides that are produced in prokaryotic host cells typically will be non-glycosylated Plasmid or viral vectors containing replication oπgms and other control sequences that are derived from species compatible with the host cell are used m connection with prokaryotic host cells, for clonmg or expression of an isolated DNA For example, E coli typically is transformed usmg pBR322, a plasmid derived from an £ coli species (Bolivar, et al , Gene 2 95-1 13 (1987)) PBR322 contains genes for ampicillin and tetracycline resistance so that cells transformed by the plasmid can easily be identified or selected For it to serve as an expression vector, the pBR322 plasmid, or other plasmid or viral vector, must also contain, or be modified to contain, a promoter that functions the host cell to provide messenger RNA (mRNA) transcripts of a DNA inserted downstream of the promoter (Rangagwala, et al , Bio/Technology, 9 477-479 (1991)) in addition to prokaryotes, eukaryotic microbes, such as yeast, may also be used as hosts for the clonmg or expression of DNAs useful in the mvention Yeast, for example, Saccharomyces cerevisiae, is a commonly used eukaryotic microorganism Piasmids useful for clonmg or expression in yeast cells of a desu-ed DNA are well known, as are various promoters that function in yeast cells to produce mRNA transcripts Furthermore, cells derived from multicellular organisms also may be used as hosts for the clonmg or expression of DNAs useful in the mvention Mammalian cells are most commonly used, and the procedures for maintaining or propagatmg such cells tn vitro, which procedures are commonly referred to as tissue culture, are well known Kruse and Patteison, eds , Tissue Culture (Academic Press, 1977) Examples of useful mammalian cells are human cell lines such as 293, HeLa, and WI-38, monkey cell lines such as COS-7 and VERO, and hamster cell lmes such as BHK-21 and CHO, all of which are publicly available from the Ameπcan Type Culture Collection (ATCC), Rockville, Maryland 20852 USA Expression vectors, unlike cloning vectors, should contain a promoter that is recognized by the host organism and is operably linked to the AL-2 nucleic acid Promoters are untranslated sequences that are located upstream from the start codon of a gene and that control transcription of the gene (that is, the synthesis of mRNA) Promoters typically fall mto two classes, inducible and constitutive Inducible promoters are promoters that initiate high level transcription of the DNA under their control in response to some change in culture conditions for example, the presence or absence of a nurnent or a change in temperature A large number of promoters are known, that may be operably linked to AL-2 DNA to achieve expression of AL-2 in a host cell This is not to say that the promoter associated with naturally occurring AL-2 DNA is not usable. However, heterologous promoters generally will result in greater transcription and higher yields of expressed AL-2. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoters. (Goeddel et al., Nature, 281 :544-548 ( 1979)), tryptophan (ttp) promoter, (Goeddel et al.. Nuc Acids Res.. 8:4057-4074 ( 1980)), and hybrid promoters such as the tac promoter. (deBoer et ai. Proc. Natl. Acad. Sci. USA. 80:21-25 (1983)). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, (Siebenlist et ai. Cell, 20:269-281 (1980)), thereby enabling a skilled worker operably to ligate them to DNA encoding AL-2 using linkers or adaptors to supply any required restriction sites (Wu et ai, Meth. Enz., 152:343-349 (1987)). Suitable promoters for use with yeast hosts include the promoters for 3 -phosphogly cerate kinase. (Hitzeman e/ α/., ./. Biol. Chem., 255:12073-12080 (1980); Kings an et al., Meth. Enz.. 185:329-341 (1990)), or other glycolytic enzymes such as enolase, giyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase (Dodson et ai, Nuc Acids Res., 10:2625-2637 ( 1982); Emr, Meth. E a., 185:231-279 (1990)). Expression vectors useful in mammalian cells typically include a promoter derived from a virus. For example, promoters derived from polyoma virus, adenovirus, cytomegalovirus (CMV), and simian virus 40 (SV40) are commonly used. Further, it is also possible, and often desirable, to utilize promoter or other control sequences associated with a naturally occurring DNA that encodes AL-2, provided that such control sequences are functional in the particular host cell used for recombinant DNA expression. Other control sequences that are desirable in an expression vector in addition to a promoter are a ribosome binding site, and in the case of an expression vector used with eukaryotic host cells, an enhancer. Enhancers are cis-acting elements of DNA, usually about from 10-300 bp, that act on a promoter to increase the level of transcription. Many enhancer sequences are now known from mammalian genes (for example, the genes for globin. elastase, albumm, α-fetoprotein and insulin). Typically, however, the enhancer used will be one from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers (Kriegler. Meth. Enz., 185:512-527 (1990)). Expression vectors may also contain sequences necessary for the termination of transcription and for stabilizing the messenger RNA (mRNA) (Balbas et ai, Meth. Era., 185:14-37 (1990)); Levinson. Meth. Enz.. 185:485-51 1 (1990)). In the case of expression vectors used with eukaryotic host cells, such transcription termination sequences may be obtained from the untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain polyadenylation sites as well as transcription termination sites (Birnsteil et ai. Cell, 41 :349-359 (1985)). In general, control sequences are DNA sequences necessary for the expression of an operably liked coding sequence in a particular host cell. "Expression" refers to transcription and/or translation. "Operably linked" refers to the covalent joining of two or more DNA sequences, by means of enzymatic ligation or otherwise, in a configuration relative to one another such that the normal function of the sequences can be performed. For example. DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotem that participates in the secretion ofthe polypeptide a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence or a ribosome bmdmg site is operably I ked to a codmg sequence if it is positioned so as to facilitate translation Generally 'operablv linked" means that the DNA sequences being linked are contiguous and, in the case of a secretor y leader contiguous and in reading phase Linking is accomplished by ligation at convenient restriction sites If such sites do not exist then synthetic oligonucleotide adaptors or linkers are used, in conjunction with standard recombinant DNA methods Expression and clonmg vectors also will contain a sequence that enables the vector to replicate in one or more selected host cells Generally, in clonmg vectors this sequence is one that enables the vector to replicate independently of the host chromosome(s), and includes origins of replication or autonomously replicating sequences Such sequences are well known for a vaπety of bactena, yeast, and viruses The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2μ plasmid oπgin is suitable for yeast, and vaπous viral oπgins (for example, from SV40, polyoma, or adenovirus) are useful for cloning vectors in mammalian cells Most expression vectors are "shuttle" vectors, i e they are capable of replication in at least one class of organisms but can be transfected to another organism for expression For example a vector may be cloned in E coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently ofthe host cell chromosome The expression vector may also include an amplifiable gene, such as that comprising the codmg sequence for dihydrofolate reductase (DHFR) Cells containing an expression vector that mcludes a DHFR gene may be cultured m the presence of methotrexate, a competitive antagonist of DHFR This leads to the synthesis of multiple copies ofthe DHFR gene and, concomitantly, multiple copies of other DNA sequences comprising the expression vector (Ringold et al , J Mol Apl Genet 1 165-175 (1981)), such as a DNA sequence encodmg AL-2 In that manner, the level of AL-2 produced by the cells may be increased DHFR protem encoded by the expression vector also may be used as a selectable marker of successful transfection For example, if the host cell prior to transformation is lacking in DHFR activity, successful transformation by an expression vector comprising DNA sequences encoding AL-2 and DHFR protein can be determmed by cell growth in medium containing methotrexate Also, mammalian cells transformed by an expression vector comprising DNA sequences encoding AL-2, DHFR prote , and aminogiycoside 3' phosphotransferase (APH) can be determmed by cell growth in medium containing an aminogiycoside antibiotic such as kanamycin or neomycm Because eukaryotic cells do not normally express an endogenous APH activity, genes encodmg APH protem, commonly referred to as neo r genes, may be used as dominant selectable markers in a wide range of eukaryotic host cells, by which cells transfected by the vector can easily be identified or selected ( mmez et al . Nature, 287 869-871 (1980), Colbere-Garapin et al , J Mol Biol . 150 1-14 (1981 ), Okayama et al , Mol Cell Biol 3 280-289 (1983)) Many other selectable markers are known that may be used for identifying and isolating recombinant host cells that express AL-2 For example, a suitable selection marker for use in yeast is the tφ 1 gene present in the yeast plasmid YRp7 (Stmchcomb et al , Nature 282 39-43 (1979), Kings an et al , Gene 7 141 -152 (1979). Tschemper et al . Gene 10 157-166 (1980)) The t l gene provides a selection marker for a mutant strain of veast lacking the ability to grow tryptophan. for example. ATCC No 44076 or PEP4-1 (available from Λe American Type Culture Collection Rockville, Maryland 20852 USA) (Jones Genetics 85 12 ( 1977)) The presence of the trp 1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan Similarly. Leu2-deficιent yeast strains (ATCC Nos 20622 or 38626) are complemented by known piasmids bearing the Leu2 gene Accordingly a method for producing AL-2 is provided that includes the steps of transforming a cell contammg an endogenous AL-2 gene with a homologous DNA comprising an amplifiable gene and a flanking sequence of at least about 150 base pairs that is homologous with a DNA sequence within or in proximity to the endogenous AL-2 gene, whereby the homologous DNA integrates mto the cell genome by recombination, then culturing the cells under conditions that select for amplification ofthe amplifiable gene whereby the AL-2 gene is also amplified, and thereafter recovering AL-2 from the cells For diagnostic applications, antι-AL-2 antibodies typically will be labeled with a detectable moiety The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal For example, the detectable moiety may be a radioisotope, such as 3 H 14 C, 32 P, 35 S or 5 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or lucifeπn radioactive isotopic labels, such as, e g , 125 I, 32 P, 14 C, or 3 H, or an enzyme, such as alkaline phosphatase. beta- galactosidase or horseradish peroxidase Any method known the art for separately conjugating the antibody to the detectable moietv may be employed, mcludmg those methods descπbed by David et al , Biochemistry 13 1014-1021 ( 1974), Pain e/ α/ , J Immunol Meth , 40 219-231 (1981), and Bayer et al , Meth Era , 184 138-163 (1990) The antι-AL-2 antibodies may be employed any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies A Manual of Techniques, pp 147-158 (CRC Press, Inc , 1987)) The term "antibody" is used m the broadest sense and specifically covers smgle antι-AL-2 monoclonal antibodies (mcludmg agonist and antagonist antibodies) and antι-AL-2 antibody compositions with polyepitopic specificity The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i e , the individual antibodies comprising the population are identical except for possible natural ly-occumng mutations that may be present m minor amounts Monoclonal antibodies are highly specific, being directed agamst a single antigenic site Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed agamst different determinants (epitopes), each monoclonal antibody is directed agamst a single determinant on the antigen The monoclonal antibodies herem include hybrid and recombinant antibodies produced by splicing a vaπable (mcludmg hypervanable) domam of an antι-AL-2 antibody with a constant domain (e g "humanized" antibodies), or a light cham with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation as well as antibody fragments {e g Fab. F(ab') 2 , and Fv), so long as they exhibit the desired biological activity (See, e g U S 4,816 567 and Mage et al Monoclonal Antibodv Production Techniques and Applications, pp 79-97 (Marcel Dekker, Inc , New York (1987)) Thus, the modifier "monoclonal" indicates the character of the antibody as being obtamed from a substantially homogeneous population of antibodies, and is not to be construed as requiring production ofthe antibodv bv any particular method Monoclonal antibodies include hybnd and recombmant antibodies produced by splicing a variable (mcludmg hypervaπable) domain of an antι-AL-2 antibody with a constant domain (e g "humanized" antibodies), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous protems, regardless of species of oπgm or immunoglobulin class or subclass designation, as well as antibody fragments (e g . Fab, F(ab') 2 , and Fv) so long as they exhibit the desired biological activity (See, e g Mage et al Monoclonal Antibodv Production Tecnmques and Applications pp 79-97 (Marcel Dekker. Inc . New York ( 1987)) The monoclonal antibodies to be used in accordance with the present mvention can be made by hybπdoma method known in the art, or may be made by recombinant DNA methods (U S Patent No 4,816,567) The "monoclonal antibodies" may also be isolated from phage libraries generated usmg the techniques descπbed m McCafferty et al , Nature, 348 552-554 ( 1990), for example The individual antibodies compπs g the monoclonal antibody population are identical except for possible naturally-occurring mutations that may be present m mmor amounts "Humanized" forms of non-human (e g murine) antibodies are specific chimenc immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-bmding subsequences of antibodies) which contain minunal sequence derived from non-human immunoglobulm For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) ofthe recipient are replaced by residues from a CDR of a non¬ human species (donor antibody) such as mouse, rat or rabbit havmg the desired specificity, affinity and capacity In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, the humanized antibody may comprise residues which are found neither m the recipient antibody nor m the imported CDR or framework sequences These modifications are made to further refine and optimize antibody performance In general, the humanized antibody will compπse substantially all of at least one. and typically two, vanabie domams, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulm and all or substantially all ofthe FR regions are those of a human immunoglobulm consensus sequence The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin DNA encodmg the monoclonal antibodies of the mvention is readily isolated and sequenced using conventional procedures (e g , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) The hybridoma cells of the invention serve as a preferred source of such DNA Methods for humanizing non-human antibodies are well known m the art Generally, a humanized antibody has one or more ammo acid residues introduced mto it from a source which is non-human These non¬ human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" vanabie domain Humanization can be essentially performed following the method of Winter and co-workers (Jones et al , Nature, 321 -522-525 (1986), Riechmann et al , Nature 332 323-327 (1988), Verhoeyen et al , Science 239 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody Accordmgly, such "humanized" antibodies are chimenc antibodies (US Patent 4,816,567 by Cabillv et al ) wherein substantially less than an intact human variable domam has been substituted by the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues, and possibly some FR residues, are substituted bv residues from analogous sites in rodent antibodies It is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties To achieve this goal, according to a preferred method, humanized antibodies are prepared bv a process of analysis ofthe parental sequences and various conceptual humanized products usmg three dimensional models ofthe parental and humanized sequences Three dimensional immunoglobulm models are familiar to those skilled in the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues m the functioning of the candidate immunoglobulm sequence, / e , the analysis of residues that influence the ability ofthe candidate immunoglobulm to bind its antigen In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antιgen(s), is achieved In general, the CDR residues are directly and most substantially involved in influencing antigen binding For further details see WO 92/22653, published December 23, 1992 Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens In the present case, one ofthe bmdmg specificities is for a AL-2, the other one is for any other antigen, and preferably for a receptor or receptor subunit For example, bispecific antibodies specifically bmding a Htk receptor and AL-2 are within the scope ofthe present invention Methods for making bispecific antibodies are known in the art Traditionally, the recombmant production of bispecific antibodies is based on the coexpression of two immunoglobulm heavy chain-light cham pairs, where the two heavy chains have different specificities (Millstein et al , Nature 305 537-539 (1983)) Because ofthe random assortment of immunoglobulin heavy and light chains, these hybπdomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure The puπfication ofthe correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low Similar procedures are disclosed m WO 93/08829, published May 13. 1993, and in Traunecker et al , EMBO J, 10 3655-3659 (1991) According to a different and more preferred approach, antibody variable domains with the desired bmdmg specificities (antibody-antigen combmmg sites) are fused to immunoglobulm constant domam sequences The fusion preferably is with an immunoglobulin heavy chain constant domam, comprising at least part ofthe hmge, CH2 and CH3 regions It is preferred to have the first heavy cham constant region (CH 1 ) containing the site necessary for light cham bmdmg, present in at least one ofthe fusions DNAs encodmg the immunoglobulm heavy cham fusions and, if desu-ed, the immunoglobulin light cham, are inserted into separate expression vectors, and are cotransfected into a suitable host organism This provides for great flexibility m adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide the optimum yields It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains m equal ratios results in high yields or when the ratios are of no particular significance In a preferred embodmient of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first bmding specificity in one arm and a hybrid immunoglobulm heavy chain-light cham pair -35- (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulm chain combinations, as the presence of an immunoglobulm light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690 published March 3. 1994. For further details of generating bispecific antibodies see, for example. Suresh et ai. Methods in Enzymology, 121 :210 ( 1986). Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linkmg agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques. Competitive binding assays rely on the ability of a labeled standard (e.g, AL-2 or an immunologically reactive portion thereof) to compete with the test sample analyte (AL-2) for binding with a limited amount of antibody. The amount of AL-2 in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate determming the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected. In a sandwich assay, the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex (for example, see U.S. 4,376,110). The second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. AL-2 antibodies may be useful in certain therapeutic indications to block activity of the AL-2 (for example in carcinogenesis). Therapeutic AL-2 antibody formulations and modes for administration will be similar to those described herein for AL-2. A typical daily dosage ofthe antibody ranges from about 1 μg/kg to up to 5 mg/kg or more, depending on the factors mentioned herein for AL-2 administration. AL-2 antibodies may also be useful in diagnostic assays for AL-2, e.g., detecting its expression in specific cells, tissues, or serum. The antibodies are labeled in the same fashion as AL-2 described above and/or are immobilized on an insoluble matrix. AL-2 antibodies also are useful for the affinity purification of AL-2 from recombmant cell culture or natural sources. AL-2 antibodies that do not detectably cross-react with other protems can be used to purify AL-2 free from these other known proteins. Suitable diagnostic assays for AL-2 and its antibodies are described herein. The anti-AL-2 antibodies ofthe invention also are useful for in vivo imaging, wherein an antibody labeled with a detectable moiety is administered to a host, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is useful in the staging and treatment of various neurological disorders. The antibody may be labeled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art. Neutralizing anti-AL-2 antibodies are useful as antagonists of AL-2. The term "neutralizing anti-AL-2 antibody" as used herein refers to an antibody that is capable of specifically binding to AL-2, and which is capable of substantially inhibiting or eliminating the functional activity of AL-2 in vivo or in vitro. Typically a neutralizing antibody will inhibit the functional activity of AL-2 at least about 50%, preferably greater than 80%, and more preferably greater than 90% as determined, for example, by an in vitro receptor binding assay, or in vitro cell-based receptor activation assays (for example, see Winslow et ai. Neuron, 14:973-981 ( 1995)). Other AL-2 antagonists are prepared using AL-2 receptor proteins. One example of an AL-2 antagonist is an Eph-family-receptor-IgG chimeric protein that binds AL-2 as described herein. Another example of an AL- 2 antagonist is a soluble form of an AL-2 receptor, which comprises the extracellular domain or the receptor substantially free of the transmembrane domain. The soluble form of the receptor can be used directly as an antagonist, or it can be used to screen for small molecules that would antagonize AL-2 activity. As stated previously, receptor tyrosine kinases are involved in many signal transduction events that regulate important cellular processes. Such processes include, for example, cellular differentiation and proliferation. Abnormal regulation or expression ofthe signal transduction machinery can lead to aberrant and malignant growth ofthe abnormally regulated cells. Abnormal expression of Eph is known to be associated with carcinomas ofthe liver, lung, breast and colon, for example. Likewise, since some Eph-related tyrosine kinases are, at least, found within the same tissues as Eph, their abnormal expression may also lead to the development ofthe carcinomas described above as well as other types of cancers. Additionally, cancers ofthe neuronal linage are likely to be caused by the abnormal expression or regulation of Cek5, since this Eph-related kinase is found exclusively in neuronal tissues. Cek5 and the other Eph-related kinases expressed in the nervous system also are likely to be involved in nerve regeneration. A change in the amount or activity of an Eph-related kinase in a sample, compared to a normal sample, will be indicative of cancerous stages and of their level of malignancy. Depending on whether the normal state is caused by the presence or absence of an Eph-reiated kinase, the change can involve either an increase or decrease in the amount or activity ofthe Eph-related kinase. One skilled in the art can measure these parameters and compare them to those obtained from a normal sample. Methods for determining the levels or activity of Eph-related kinases are known to one skilled in the art and include, for example. RNA and protem blot analysis, ELISA using specific antibodies to each ofthe Eph-related kinases and direct measurement of catalytic activity such as tyrosine kinase activity. Such methods can be found in Harlow et ai. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory ( 1989), which is incoφorated herein by reference. The compositions and methods of receptor modulation as taught herein can then be effectively applied where AL-2-binding receptors are involved. AL-2 is believed to find therapeutic use for treating mammals via stimulation or inhibition of growth and or differentiation and or activation and/or metabolism of cells having a receptor for AL-2 as described herein, such as Htk, Hek2, or Hek5. The interaction of ligand and receptor may result in activation of the receptor and transduction of a signal which modulates the physiological state ofthe receptor-bearing cells. The ligand can act as a growth factor to stimulate the proliferation of target cells. Alternatively, ligand binding may not activate the receptor. In this instance, the ligand may act as an antagonist for other molecules which activate the receptor and induce signal transduction. The invention provides a method of modulating the endogenous enzymatic activity of an AL-2-binding Eph-family receptor. The method includes the step of administering to a mammal an effective amount of AL-2 to modulate the receptor enzymatic activity. In one embodiment is provided a method for stimulating the proliferation, differentiation, metabolism, regeneration, growth, process-out growth, or cell migration of AL-2- receptor expressing cells in a mammal by administering a therapeutically effective amount of receptor-activating AL-2. Receptor-activating forms of AL-2, such as AL-2-IgG, find use in alleviating cell damage or promoting neurogenesis following disease or injury, such as cytotoxicity, caused by chemotherapy. For example, a method for stimulating proliferation of neurons innervating the liver includes the step of administering a therapeutically effective amount of AL-2. Treatment with AL-2 is useful for repairing liver damage resulting from disease or injury. Soluble Eph-family-receptor polypeptides can be used to modulate the activation ofthe cell-associated receptors, typically by competing with the cell-bound receptor for unbound AL-2. Modulation of Eph-family receptor activation may in turn alter the proliferation and/or differentiation of receptor-bearing cells. Antibodies to Eph-like receptors are useful reagents for the detection of receptors in different cell types using immunoassays conventional to the art. Antibodies are also useful therapeutic agents for modulating receptor activation. Antibodies may bind to the receptor so as to directly or indirectly block ligand bmdmg and thereby act as an antagonist of receptor activation. Alternatively, antibodies may act as an agonist by binding to receptor so as to facilitate ligand binding and bring about receptor activation at lower ligand concentrations. In addition, antibodies can themselves act as a ligand by inducing receptor activation. In this context the present invention provides anti-idiotype antibodies. Le., anti-AL-2-antibodies, that recognize an AL-2-binding-Eph- family receptor. Accordmgly, a method for modulating the activation of an AL-2-binding-Eph-family receptor by administering a modulation-effective amount of AL-2 or soluble AL-2. The term "modulation-effective amount" is that amount which effects an increase or decrease in the activation of an AL-2-biπding-Eph-family receptor. Preferably the amount will range from about 0.01 μg to about 100 mg of polypeptide per kg body weight. In general, for therapeutic purposes, therapy will be appropriate for a patient having a condition in part related to the state of proliferation and/or differentiation of receptor-bearing cells. Based in part upon the tissue distribution of AL-2, and thus presumably its receptors in some embodiments, treatment with the pharmaceutical compositions ofthe mvention may be particularly indicated for disorders involving brain, heart, muscle, lung, kidney, pancreas, skeletal muscle, liver, and more preferably involving brain, pancreas, and skeletal muscle. AL-2 is also useful for selection of cell populations enriched for AL-2-receptor bearing cells. Such populations can be useful in cellular therapy regimens where it is necessary to treat patients that are depleted of certain cell types. The human AL-2 is clearly also useful insofar as it can be administered to a human having depressed levels of endogenous AL-2, preferably in the situation where such depressed levels lead to a pathological disorder. The prominent expression of AL-2 DNA in the cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe temporal lobe, putamen amygdala, caudate nucleus, coφus callosum. hippocampus, substantia nigra. subthalamic nucleus and thalamus (see Examples) is consistent with the use of AL-2 to treat neurodegenerative diseases or, other neuronal disorders or conditions in which these structures or neurons projecting to these structures, are affected AL-2 finds use m treating neurodegenerative disorders or nerve damage where nerve regeneration and (re)establιshment of neuronal pathways are desired outcomes, since it may play a role in neuronal cell migration and axonogenesis A cπtical stage in the development ofthe nervous system, and during nerve regeneration as might occur after injury, is the projection of axons to their targets Navigational decisions are made at the growth cones ofthe migrating axons As axons grow their growth cones extend and retract filopodia and lamellipodia processes which are implicated in the navigational decisions and pathfinding abilities of migrating axons Like peπpheral nervous system axons, the growth cones of neurons associated with the central nervous system follow stereotyped pathways and apparently can selectively choose from a number of possible routes (reviewed by Goodman et al , Cell 72 77-98 (1993)) For example, subcellular localization of a Hek5 homolog, the murine Nuk receptor tyrosme kinase, indicates that this receptor is concentrated at sites of cell-cell contact, often involving migrating neuronal cells or their extensions (Henkemeyer et al , Oncogene, 9 1001 - 14 ( 1994)) Most notably, high levels of Nuk protein are found within initial axon outgrowths and associated nerve fibers The axonal localization of Nuk was transient and not detected after migrations have ceased, which suggests a role for mis tyrosme k ase durmg the early pathfinding and or fasciculation stages of axonogenesis, which can be important processes during recovery from neuronal damage Accordmgly, AL-2 (and embodiments disclosed herem or identified by the methods presented herein) is believed to be useful in promoting the development, maintenance, regeneration, migration, or process- outgrowth of neurons in vivo, including central (brain and spinal chord), peripheral (sympathetic, parasympathetic, sensory, and enteric neurons), and motoneurons The ligands, agonists and antagonists may accordingly be used to stimulate or inhibit these activities associated with neurodegenerative conditions and conditions mvolvmg trauma and injury to the nervous system Consequently, AL-2 may be utilized in methods for the diagnosis and/or treatment of a variety of neurologic diseases and disorders In some embodiments of the invention, purified AL-2 can be administered to patients in whom the nervous system has been damaged by trauma, surgery, stroke, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, or toxic agents, to promote the survival or growth of neurons For example, AL-2 can be used to promote the survival or growth of motoneurons that are damaged by trauma or surgery Also, AL-2 can be used to treat motoneuron disorders, such as amyotrophic lateral sclerosis (Lou Gehπg's disease), Bell's palsy, and various conditions involving spmal muscular atrophy, or paralysis AL-2 can be used to treat human neurodegenerative disorders, such as Alzheimer's disease. Parkinson's disease, epilepsy, demye nating diseases, such as multiple sclerosis. Huntington's chorea, Down's Syndrome, nerve deafness. Meniere's disease, and other disorders ofthe cerebellum (Hefti Neurobiol , 25( 1 1 ) 1418-35 ( 1994), Marsden. Lancet 335 948-952 (1990), Agid, Lancet. 337 1321-1327 (1991 ), Wexler e/ α/ Ann Rev Neurosci 14 503-529 1991)) A L-2 can be used as cognitive enhancer, to enhance learning particularly in dementias or trauma, since they can promote axonal outgrowth and synaptic plasticity, particularly of hippocampal neurons that express AL-2-bιnd g Eph-family receptors and cortical neurons that express AL-2. AL-2 can be used in bacterial and viral infections of the nervous system, deficiency diseases, such as Wernicke's disease and nutritional polyneuropath . progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo ponto cerebellar atrophy, and peripheral nerve damage. For example, in Alzheimer's disease there is a critical loss of basal forebrain cholinergic neurons, cortical neurons, and hippocampal neurons. Although maximally effective treatment of this neurodegenerative condition may require protection of all vulnerable neuronal populations, treatment with AL-2 alone is expected to provide therapeutic benefit. Alzheimer's disease, which has been identified by the National Institutes of Aging as accounting for more than 50% of dementia in the elderly, is also the fourth or fifth leading cause of death in Americans over 65 years of age. Four million Americans, 40% of Americans over age 85 (the fastest growing segment ofthe U.S. population), have Alzheimer's disease. Twenty-five percent of all patients with Parkinson's disease also suffer from Alzheimer's disease-like dementia. And in about 15% of patients with dementia, Alzheimer's disease and multi-infarct dementia coexist. The third most common cause of dementia, after Alzheimer's disease and vascular dementia, is cognitive impairment due to organic brain disease related directly to alcoholism, which occurs in about 10% of alcoholics. However, the most consistent abnormality for Alzheimer's disease, as well as for vascular dementia and cognitive impairment due to organic brain disease related to alcoholism, is the degeneration ofthe cholinergic system arising from the basal forebrain (BF) to both the codex and hippocampus (Bigl et ai. in Brain Cholinergic Systems, M. Steriade and D. Biesold, eds., Oxford University Press, Oxford, pp.364-386 (1990)). And there are a number of other neurotransmitter systems affected by Alzheimer's disease (Davies, Med. Res. Rev., 3:221 (1983)). However, cognitive impairment, related for example to degeneration ofthe cholinergic neurotransmitter system, is not limited to individuals suffering from dementia. It has also been seen in otherwise healthy aged adults and rats. Studies that compare the degree of learning impairment with the degree of reduced cortical cerebral blood flow in aged rats show a good correlation (Berman et ai, Neurobiol. Aging, 9:691 (1988)). In chronic alcoholism the resultant organic brain disease, like Alzheimer's disease and normal aging, is also characterized by diffuse reductions in cortical cerebral blood flow in those brain regions where cholinergic neurons arise (basal forebrain) and to which they project (cerebral cortex) (Lofti et ai, Cerebrovasc.and Brain Metab. Rev. 1 :2 (1989)). The progressive nature of Parkinson's disease is due to a loss of nigral dopaminergic neurons ofthe substantia nigra (Studer et ai, Eur. J. Neuroscience, 7 :223-233 ( 1995)). ALS in vol ves progressive degeneration of motoneurons ofthe spinal cord, brain stem and cerebral cortex. Further, AL-2 can be used to treat neuropathy, and especially peripheral neuropathy. "Peripheral neuropathy" refers to a disorder affecting the peripheral nervous system, most often manifested as one or a combination of motor, sensory, sensorimotor. or autonomic neural dysfunction. The wide variety of moφhologies exhibited by peripheral neuropathies can each be attributed uniquely to an equally wide number of causes. For example, peripheral neuropathies can be genetically acquired, can result from a systemic disease, or can be induced by a toxic agent. Examples include but are not limited to distal sensorimotor neuropathy, or autonomic neuropathies such as reduced motility of the gastrointestinal tract or atony of the urinary bladder. Examples of neuropathies associated with systemic disease include post-polio syndrome; examples of hereditary neuropathies include Charcot-Marie-Tooth disease. Refsum's disease. Abetalipoproteinemia. Tangier disease. Krabbe's disease. Metachromatic leukodystrophy. Fabry's disease, and Dejeπne-Sottas syndrome, and examples of neuropathies caused by a toxic agent mclude those caused by treatment with a chemotherapeutic agent such as vincπstme. cisplatin. methotrexate. or 3'-azιdo-3'-deoxythymιdιne. AL-2 may play a role in neurogenesis. for example in axon bundling or process outgrowth The following mechanism is not meant to be limiting to the invention. Any role of AL-2 in axon fascicle formation may be indirect. ; e AL-2 and its receptor, may not themselves function as adhesion molecules but rather are involved in regulatmg the fasciculation process. Accordingly, activation of AL-2-receptor expressed on the neurons, by AL-2 expressed on astrocytes for example, might activate a signaling pathway that promotes fasciculation, possibly by up-regulating or activating adhesion molecules. Activation of AL-2-receptor may cause growth cone repulsion and collapse, forcmg axons together for fasciculation. AL-2 expressed on astrocytes would serve as a repulsive cue to axons driving axons together. This model is compatible with the current view that astrocytes play an important role durmg development of the CNS, where they are thought to provide a substratum and trophic support for growing axons (Hatten et ai, Semtn. Neurosci , 2 455-465 (1990)). A tyrosine kinase is required for axon bundling. Neurons in the developing or regenerating nervous system presumably require two types of factors, those that promote growth and survival, and those that provide spatial or directional guidance m the establishment of neuronal pathways (Tessier-Lavigne, Curr Optn Genet Devel, 4:596-601 (1994)). Tyrosine kinases are known to play a well-established role in the former and can participate the latter. Accordingly, AL-2 can play role in the formation of neuronal pathways, a crucial feature of both development and regeneration in the nervous system. In still further embodiments ofthe invention, AL-2 antagonists, and especially anti-AL-2 antibodies, can be administered to patients suffering from neurologic diseases and disorders characterized by excessive production or activity of AL-2. AL-2 antagonists can be used in the prevention of aberrant regeneration of sensory neurons such as may occur post-operatively, or in the selective ablation of sensory neurons, for example, in the treatment of chronic pain syndromes. In yet another embodiment AL-2 stimulates hematopoiesis and thus find use m treating hematopoietic- related disorders. Htk, a candidate receptor for AL-2, has a wide tissue distribution including expression in several myeloid hematopoietic cell lines (Bennett et ai, J Biol Chem. 269:1421 1-8 (1994)). Hematopoietic expression of Htk m the monocytic lineage (myeloid but not lymphoid hematopoietic cells) mdicates that AL-2, upon Htk binding and activation, can activate differentiation and/or proliferation of these cells, finding use in treating conditions such as anemia, bone marrow transplant (autologous or otherwise) or as adjunct therapy in chemo- or radiation-therapies. Furthermore, AL-2 antagonists can reduce or prevent differentiation and or proliferation of these cells, a function that finds particular use in disease conditions involving malignant forms of these cells, for example, in treating acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or myelodysplastic syndrome (MDS). AL-2 antagonists can be administered in conjunction with other agents or therapies for AML or CML. The development of a vascular supply, angiogenesis. is essential for the growth, maturation, and mamtenance of normal tissues, including neuronal tissues. It is also required for wound healing and the rapid growth of solid tumors and is involved in a variety of other pathological conditions. Current concepts of angiogenesis, based m large part on studies on the vasculanzation of tumors, suggest that cells secrete angiogenic factors which induce endothelial cell migration, proliferation, and capillary formation. Numerous factors have been identified which induce vessel formation in vitro or in vivo in animal models. These include FGFα, FGFβ, TGF-α. TNF-α, VPF or VEGF, monobutyπn, angiotropin. angiogenin. hyaluronic acid degradation products, and more recently, B61 for TNF-α-induced angiogenesis (Pandey et ai. Science. 268:567-569 ( 1995)). Inhibitors of angiogenesis include a cartilage-derived inhibitor identified as TIMP. PF-4. thrombospondin. laminin peptides, heparin cortisone, minocycline. fumagillin. difluoromethyl omithine. sulfated chitm deπvatives. and B61 antibody. The major development ofthe vascular supply occurs during embryonic development, at ovulation during formation ofthe coφus luteum, and during wound and fracture healing. Many pathological disease states are characterized by augmented angiogenesis including tumor growth, diabetic retinopathy. neovascular glaucoma, psoriasis, and rheumatoid arthritis. During these processes normally quiescent endothelial cells which line the blood vessels sprout from sites along the vessel, degrade extracellular matrix barriers, proliferate, and migrate to form new vessels. Angiogenic factors, secreted from surrounding tissue, direct the endotheliai cells to degrade stromal collagens, undergo directed migration (chemotaxis), proliferate, and reorganize into capillaries. AL-2 may stimulate either the growth or differentiation of cells expressing an AL-2 receptor. AL-2 that induces differentiation of AL-2-receptor bearing may be useful in the treatment of certain types of cancers. AL-2 may be used alone or in combination with standard chemotherapy or radiation therapy for cancer treatment. Where an AL-2-receptor is shown to be involved in the development of a cancerous state, either through stimulation of cell growth or through promotion of metastasis by stimulating cell mobility and adhesion, AL-2 antagonists as taught herein will find use. Fragments or analogs of AL-2 that bmd to but do not activate the receptor are useful antagonists. Administration of an antagonist having affinity for the receptor will block receptor binding and activation by endogenous activators. Administration of soluble AL-2 receptor may also be used to counteract the biological effects of receptor activation. Soluble AL-2 receptor will compete with endogenous cell surface receptors for binding to activators, including AL-2, and thereby reduce the extent of AL- 2 receptor activation. In addition, monoclonal antibodies directed either to AL-2 or to the receptor may be useful in blocking the interactions of AL-2, or other activator, with AL-2 receptors on cell surfaces. Accordingly, AL-2 can find further use in promoting or enhancing angiogenesis by receptor activation on endothelial or stromal cells. The induction of vascularization is a critical component ofthe wound healing process. Neovascularization, also known as angiogenesis, is a complex process involving several sequential steps including basement membrane degradation, endothelial cell mobilization and proliferation, vessel canalization, and new basement membrane formation (Mantovani, Int. J. Cancer, 25:617 ( 1980)). Vascularization ensures that proliferating and differentiating fibroblasts are supplied with nutrients and oxygen, and that elements of humoral and cellular immunity are delivered to sites of potential bacterial infection. It is desirable to induce neovascularization as early as possible in the course of wound healing, particularly in the case of patients having conditions that tend to retard wound healing, e.g.. burns, decubitus ulcers, diabetes, obesity and malignancies. Even normal post-surgical patients will be benefited if they can be released from hospital care at any earlier date because of accelerated wound healing. This invention provides novel compositions and methods for modulating angiogenesis. A patient bearing a wound can be treated by applying an angiogenically active dose of an AL-2 compound to the wound. This facilitates the neovascularization of surgical incisions, bums, traumatized tissue, skin grafts, ulcers and other wounds or injuries where accelerated healing is desired In individuals who have substantially impari ed wound healmg capacity, thereby lack the ability to provide to the wound site endogenous factors necessary for the process of wound healmg, the addition of exogenous AL-2 and compositions ofthe invention enable wound healmg to proceed m a normal manner The protems ofthe present invention are expected to accelerate the healmg process in a broad spectrum of wound conditions Novel topical compositions contammg an AI-2 compound are provided for use in the inventive method, as are novel articles such as sutures, grafts and dressings contammg an AL-2 compound The term "wound" is defined herein as an opening in the skm, mucosa or epithelial linings, most such openings generally being associated with exposed raw or abraded tissue There are no limitations as to the type of wound or other traumata that can be treated in accordance with this invention, such wounds including (but are not limited to) first, second and third degree bums (especially second and third degree), surgical incisions, including those of cosmetic surgery, wounds, mcludmg lacerations, incisions, and penetrations, and ulcers, e g , chronic non-healing dermal ulcers, including decubital ulcers (bed-sores) and ulcers or wounds associated with diabetic, dental, hemophihc, malignant and obese patients Furthermore, normal wound-healmg may be retarded by a number of factors, mcludmg advanced age, diabetes, cancer, and treatment with anti-inflammatory drugs or anticoagulants, and the protems descnbed herein may be used to offset the delayed wound-healing effects of such treatments Although the primary concern is the healmg of major wounds by neovascularization. it is contemplated that an AL-2 compound may also be useful for mmor wounds, and for cosmetic regeneration of epithelial cells Preferably, the wounds to be treated are bums and surgical incisions, whether or not associated with viral mfections or tumors In most cases wounds are not the result of a tumor or a viral infection and ordinarily they do not include tumor cells AL-2 is preferably delivered to wounds by topical application, "topical" m this context meaning topical to the wound, and does not necessarily refer to epidermal application. When applied topically, the AL-2 compound is usually combined with other mgredients, such as earners and/or adjuvants There are no limitations on the nature of such other mgredients, except that they must be pharmaceutically acceptable, efficacious for their intended administration, and cannot degrade or mactivate AL-2 AL-2 is applied to bums in the form of an imgant or salve, and if so then in an isotonic solution such as physiological salme solution or D5W AL-2 is particularly useful m accelerating the growth and survival of skin grafts applied to bu s Ordinarily, an AL-2- containing composition is impregnated into the grafts or adherently coated onto the face ofthe graft, either on the side ofthe graft to be applied to the bum or on the exterior side ofthe graft AL-2 also is mcluded in bum debπdement salves which contain proteases so long as the debπdement enzyme does not proteolytically inactivate the AL-2 AL-2 is impregnated to surgical articles in accordance with this invention, such articles bemg defined as items to be contacted with wounds which articles are typically water adsorbent or hydratable and which have a therapeutic utility in treating wounds Examples of surgical articles are dressings, sutures, pledgets, skin grafting films (including living skin grafts as well as collagen-containing membranes or synthetic skm substitutes) and the like as will be known to the clinician Dressings for use herein generally comprise water adsorbent laminates contammg AL-2 to be adherently placed into contact with wounds Improved dressings for use with AL-2 as described herein preferably will have a membrane such as a dialysis membrane teφosed between the wound surface and the adsorbent substance in the dressing, the membrane containing pores sufficiently small for AL-2 to diffuse into the wound but not sufficiently large for epithelial cells to penetrate into the adsorbent. The degree of adsorbency will vary considerably and in fact dressings are included herein which are nonadsorbent. i.e.. the AL-2 is deposited or stored in an aqueous reservoir which is used to irrigate the wound on a continuous or intermittent basis. AL-2 also is formulated into ointments or suspensions, preferably in combination with purified collagen, in order to produce semisolid or suspension vehicles. Conventional oleaginous formulations containing AL-2 are useful as salves. Such AL-2 carriers and formulations release AL-2 on a sustained basis at the wound, thereby serving to create a chemotactic gradient that directionally orients neovascularization. e.g.. into a skin graft. Sustained release formulations for AL-2 mclude semipermeable polymer matrices in the form of shaped articles, e.g.. films, or microcapsules. Implantable sustained release matrices include copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22(l):547-556 (1983)), poly (2-hydroxyethyl- methacrylate) (Langer et ai, J. Biomed. Mater Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et ai. Id.), or poly-D-(-)-3-Hydroxybutyric acid (EP 133.988A). These formulations may function as bioerodible matrices or as stable sources for the passive diffusion of AL-2. Sustained release AL-2 compositions for contact with wounds also include liposomally entrapped AL-2. Liposomes containing AL-2 are prepared by methods known per se: DE 3,218,121A; Epstein et ai. Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang et ai, Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52322A; EP 36676A; EP 88046A; EP 143949A; EP 142641A; Japanese patent application 83-1 18008; U.S. patents 4,485,045 and 4,544,545; and EP 102,324A. Ordinarily the liposomes are ofthe small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal rate of AL-2 leakage. AL-2 is formulated with other ingredients such as carriers and/or adjuvants, e.g., albumm, nonionic surfactants and other emulsifiers. There are no limitations on the nature of such other ingredients, except that they must be pharmaceutically acceptable, efficacious for their intended administration, and cannot degrade the activity ofthe active ingredients ofthe compositions. Suitable adjuvants mclude collagen or hyaluronic acid preparations, fibronectin, factor XIII, or other proteins or substances designed to stabilize or otherwise enhance the active therapeutic ingredient(s). AL-2 optionally is supplied wiΛ other known angiogenic agents such as heparin, which has been shown to accelerate the healing of thermal bums, TNF. TGF-α, TGF-β, fibroblast growth factor, epidermal growth factor, B61. angiogenin, platelet factor 4. insulin, PDGF, and angiogenesis factor and the angiogenic activity of the combinations observed for synergistic effects. AL-2 optionally also is combined with an with an IFN. e.g., IFN-γ, and other cytokmes, or may be free of interferons such as IFN-γ. Where such cytokines or known angiogenic agents are species-specific, the appropriate cytokine or agent will be selected for the species to be treated. Animals or humans are treated in accordance with this invention. It is possible but not preferred to treat an animal of one species with AL-2 of another species. A preferred AL-2 for use herein is soluble AL-2-IgG. The amount of AL-2 to be contacted with the wound depends upon a great number of variables that will be taken into account by the clinician, including the presence of other angiogenic agents in the AL-2 formulations, the nature ofthe wound to be treated the condition ofthe patient, the AL-2 formulation selected the neovasculanzing activity of the molecular species of AL-2 chosen and the route of administration Lesser amounts of AL-2 typically are administered when the AL-2 is formulated into a sustained release vehicle, e g dressing or ointment, and when the AL-2 is administered by direct topical contact rather than impregnated into a bandage or dressmg Concentrations in the range of 0 10 ng/ml - 100 ug ml mav be used The typical topical formulation will be capable of de venng a concentration of AL-2-IgG. or equivalent, at the neovascularization target site tfor example, a skm graft) in a range of about from 0 10 ng/ml to 10000 ng/ml, more preferably 0 20 ng ml to 1000 ng/mi, and even more preferably 0 25 ng/ml to 350 ng/ml, although this therapeutic dose range is subject to considerable variation as noted above Delivery of concentrations outside of this range may offer certain ofthe benefits of AL-2 neovascularization, but the clinician will be expected to monitor dosages in order to optimize performance of AL-2 wound healmg It also should be noted that the weight amount will vary for other AL-2 vaπants and forms if their molecular weight and/or angiogenic potency differ from that of AL-2-IgG Potency differences are easily determined by comparing the degree of neovasculanzation achieved with the candidate AL-2 and AL-2-IgG m any ofthe assays set forth the Examples herem Accordmgly, a method for accelerating the neovasculanzation of a wound is provided that mcludes the step of applymg to the wound an angiogenically effective dose of a composition comprising tumor necrosis factor The composition can be applied topically by direct contact with the wound, particularly when the wound is a fresh surgical incision In a preferred embodiment the composition further compnses collagen or a synthetic skm substitute The caregiver may further administer to the wound a growth factor, an antibiotic, a debndement agent and/or angiogenm A preferred composition for the debndement of bums contains an AL-2 compound and a proteolytic enzyme which does not inactivate the neovasculanzing activity of AL-2 The therapeutic compositions can be reapplied at one-to-several-day-intervals until healmg is complete Therapeutic options for patients with vascular disease, particularly vascular obstructive disease, are sometimes lunited Such patients are often refractory to conservative measures and typically unresponsive to drug therapy (Takeshita et al , J Clin Invest 93 662-670 (1994)) When vascular obstruction is lengthy and/or widespread, nonsurgical revasculaπzation may not be feasible Surgical therapy, consisting of arterial bypass and or amputation, may be complicated by a variable morbidity and mortality, and is often dependent for its efficacy upon short- and long-term patency of the conduit used Therapeutic angiogenesis constitutes an alternative treatment strategy for such patients The present invention provides methods for enhancing angiogenesis m a mammal comprising administering to the mammal an effective amount of AL-2 The AL-2 alone may be administered to the mammal, or altematively, may be administered to the mammal m combination with other therapies and/or pharmacologic agents In particular AL-2 finds use m patients suffering from vascular insufficiency or limb ischemia secondary to arteπal occlusive disease The effects of AL-2 proteins of the invention on angiogenesis can be tested, for example, in a rabbit model of hindltmb ischemia This rabbit model was designed to simulate ischemia characteristics of patients with severe lower extremity arteπal occlusive disease and is performed essentially as described in Takeshita et al J Clin Invest 93 662-670 ( 1994) Measurements of calf blood pressure (BP) index angiographic score of collateral formation lntravascular Doppler-wire analvsis of blood flow, and microsphere-based analysis of muscle perfusion at rest and during stress are performed AL-2 antagonists can find use in inhibiting, preventing or treating pathological angiogenesis. such as dunng tumor vasculaπzation Tumor neovasculanzation is a vital stage in the growth of solid tumors (Polvermi et al , Lab Invest , 51 635-642 (1985)) The progressive growth of solid tumors is strictly dependent on their ability to attract new blood vessels that will supply them with oxygen and essential nutrients (Bouck Cancer Cells 2(6) 179-185 ( 1990)) Angiogenesis has been shown to precede or accompany malignancy In the absence of neovasculanzation the size of tumor grafts becomes limited When angiogenesis is absent, tumors tend to remam dormant Therefore, angiogenic activity has been directly correlated with tumor growth AL-2 antagonist compositions and methods of the invention can modulate (e g , prevent or reduce) new capillary growth into tumors A variety of non-neoplastic diseases, previously thought to be unrelated, can be considered "angiogenic diseases" because they are dominated by the pathological growth of capillary blood vessels These diseases mclude diabetic retinopathy, arthntis, hemangiomas, psoriasis, and ocular neovasculanzation AL-2 antagonist compositions and methods ofthe invention can be used to treat these conditions Vascularization also plays a cπtical role in chronic inflammatory conditions such as rheumatoid arthntis (Koch ec al , Arthr Rheum , 29 471-479 (1986)) Rheumatoid arthntis ("RA") is a chronic heterogeneous disorder m which a variety of etiological agents may be responsible for initiating a series of events leadmg to mflammation multiple joints. The cause ofthe disease remams unknown, although by analogy with other forms of arthntis such as that accompanying Lyme disease, it has been postulated that infection with as yet unidentified bacteria or viruses in a genetically susceptible host is an initiating event Persistence could result from the presence of viral or bactenal antigens that generate an immune response or cross-react with host tissues together with amplification effects of cellular products ofthe host While many patients have systemic manifestations m RA, many ofthe most seπous consequences of RA stem from its effects on articular connective tissues, which are characterized by alterations ofthe synovial membrane with proliferation of lmmg cells and infiltration by chronic inflammatory cells Erosions of bone occur m areas contiguous with the inflammatory cell mass as well as m regions adjacent to bone marrow distant from the mflammation The bone erosions are probably produced through induction of differentiation and activation of osteoclast progenitors The erosion of soft connective tissues, e g , cartilage, joint capsules, tendons, and ligaments, results from direct release of proteolytic enzymes from cells ofthe inflammatory cell mass or from polymoφhonuclear leukocytes that are typically abundant in rheumatoid synovial fluids, although rare m the synovial membrane See, for example, Harris, W N Kelley et al , eds , Textbook of Rheumatology, W B Saunders, Philadelphia, pp 886-915 (1985), Dayer et al Clin Rheum Dis , 4 517-537 (1978), Krane, Arthritis and Allied Conditions A textbook of Rheumatology, ed by D McCarty, pp 593-604. Lea and Febiger, Philadelphia (1985), and Krane et al , Lymphokines 7 75- 136 ( 1982) Therapy for RA depends on the stage of the disease Stage 1 , where a postulated antigen is presented to T-cells with no obvious arthritic symptoms, is not treated Stage 2 mvolves T-cell and B-cell proliferation and angiogenesis in synovial membrane, resulting in malaise, mild joint stiffness, and swelling During Stage 3 neutrophils accumulate in synovial fluid and synovial cells proliferate without polarization or invasion of cartilage, resulting in joint pain and swelling, morning stiffness, malaise, and weakness Current therapy for Stages 2 and 3 includes bed rest, application of heat, supplemental eicosapentaenoic and docosahexanoic acid and drugs Nonsteroidal anti-inflammatory drugs, including aspirin, continue to be the foundation of drug therapy m treating Stages 2 and 3 of the disease Those anti-inflammatory drugs other than aspirin include mdomethacm, phenylbutazone, phenylacetic acid derivatives such as lbuprofen and fenoprofen naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometm) indoleacetic acids (sulindac) halogenated anthraniiic acid (meclofenamate sodium), piroxicam zomepirac and diflunisal Second-line drugs for RA Stages 2 and 3 mclude anti-malaπal drugs such as hydroxychloroqume, sulfasalazine, gold salts and penicillamine and low- dose methotrexate These alternatives frequently produce severe side effects, mcludmg retinal lesions and kidney and bone marrow toxicity The irreversible destruction of cartilage occurs m Stage 4 of the disease Currently available drugs and treatments include total lymphoid irradiation, high-dose intravenous methylpredmsolone, and cyclospoπne Cyclospoπne is nephrotoxic and the other treatments exert substantial toxicity as well As a result, such immunosuppressive agents heretofore have been used only in the treatment of severe and unremitting RA Other possible therapeutic drugs for Stage 4 of RA include cyclic oligosaccharides (cyclodextrins), which, when combined with a noninflammatory steroid (cortexolone), inhibit angiogenesis in vrvo Folkman et al , Science 243 1490-1493 (1989) Antibodies agamst crucial components ofthe early phase ofthe immune response mclude anti-Class II MHC antibodies (Gaston et al , Arthritis Rheum , 31 21-30 (1988), Sany et al . Arthritis Rheum 25 17-24 (1982)), antι-ιnterleukιn-2 receptor antibodies (Kyle et al . Ann Rheum Dts , 48 428-429 (1989)), antι-CD4 antibodies (Herzog et al , J Autotmmun , 2 627-642 (1989), Walker et al , J Autotmmun , 2 643-649 (1989)), and antithymocyte globulin (Shmerling et al , Arthritis Rheum , 32 1495- 1496 ( 1989)) The last three of these drugs have been used patients with RA The present mvention provides compositions and methods that down-regulate inflammatory and proliferative pathways RA by modulatmg the associated angiogenesis The compositions contain an angiogenesis-modulatmg effective amount of an AL-2 antagonist The compositions can further comprise other angiogenesis-modulatmg agents, particularly agents for treating RA as discussed above or agents for treating tumors The methods involve the step of administering an angiogenesis-modulatmg effective amount of an AL-2 antagonist to a mammal m need of such treatment By "modulating" in the context of conditions in which angiogenesis is undesirable is meant blockmg, inhibiting, preventing, reversing, or reducing angiogenesis, or preventing further progression of angiogenesis There seems to be little or no biochemical difference between angiogenic peptides expressed by tumors and those expressed by normal tissues Nor are theie any moφhological differences between the new capillanes that respond to a malignancy and the capillary growth that occurs during physiological neovasculanzation (Folkman et al , Science, 235 442-447 (1987)) As there is no qualitative difference between the angiogenic capabilities of nonmalignant and malignant diseases, results from normal and malignant vascularization assays can be easily compared, and progress can be made in either area independently ofthe system of investigation used (Paweletz et al . Critical Reviews in Oncology! Hematology, 9(3) 197-198 ( 1989)) Thus the mvention relates to a method comprising inhibiting angiogenesis by administering an effective amount of AL-2 antagonist The invention comprises a method for the treatment of angiogenesis-dependent diseases by administering an effective amount of AL-2 antagonist to a mammal Angiogenesis dependent diseases mclude, but are not lunited to diabetic retmopathy, arthritis, tumor growth and metastasis, neovascular glaucoma, retmopathy of prematurity, senile macular degeneration, and hypergeneration of scars after wound healmg The pharmaceutical composition ofthe present invention exhibit therapeutic properties and preventative or inhibitive properties against diseases associated with angiogenesis for example, inflammatory diseases (e , rheumatoid arthritis) diabetic retmopathy, tumors such as malignant tumors (e g , cancer such as mastocarcinoma, hepatoma. colic carcinoma, Kaposi's sarcoma, lung carcinomas and other epithelial carcinomas) Numerous methods, in vitro and in vivo, are available to screen candidate AL-2 or AL-2 antagonists compounds for angiogenic or angiogenesis- hibitmg properties Several tn vitro assays of endothelial cell growth migration and capillary tube formation are known and can be used with the compounds ofthe mvention, particularly as initial screenmg methods for angiogenic or angiostatic substances Further testing would typically use in vivo animal testing U S Patent 5,382,514, which is incoφorated herem, describes numerous models for angiogenesis in vivo For example, the comeal pocket assay involves the surgical implantation of polymer pellets contammg angiogenic factors m the cornea of larger animals such as rabbits S ce quantitation can be difficult the assay is usually used for preferred candidate compounds The rabbit ear chamber assay requires the surgical insertion of a glass or plastic viewing device and measurement of capillary migration by microscopy The rat dorsal air sac assay mvolves implants of stainless steel chambers containing angiogenic factors An algmate assay which generates an angiogenic response has been described which involves the injection of tumor cells encased algmate subcutaneously into mice The accumulation of hemoglobin in the injected gel is used to quantitate the angiogenic response A compound can be administered to the chorio-allantoic membranes of aged, typically three-day-aged, fertilized chicken eggs and the appearance of neovasculanzation after a lapse of time, typically two days is observed (CAM assay, Ausprunk et al , Am J Pathol , 97 597 ( 1975)) The neovasculanzation inhibitory rates are compared with an untreated control group A more recent assay method involves providing a liquid matrix matenal which forms a matrix gel when injected into a host, adding an angiogenic agent to the liquid matrix matenal, injecting the liquid matrix matenal containing the angiogenic agent mto a host to form a matrix gel, recovermg the matrix gel from the host, and quantitating angiogenesis of the recovered matrix gel A vaπation of this can be used to test for inhibitors of vascularization in a tissue by providing a liquid matπx material which forms a matrix gel when injected into a host, adding an angiogenic inhibiting agent to the liquid matπx matenal: and mjectmg the liquid matrix material containing the angiogenic inhibiting agent mto a tissue situs of a host to form a matrix gel This system can be used with compounds of the mvention when mducmg vasculanzation in a tissue is desu-ed by providmg a liquid matrix matenal which forms a matrix gel when injected mto a host, addmg an angiogenic mducmg agent to the liquid matrix material, and mjectmg the liquid matπx matenal contammg the angiogenic inducing agent into a tissue situs of a host to form a matπx gel In a preferred embodiment, a solution of basement membrane proteins supplemented with fibroblast growth factor and hepaπn is injected subcutaneously in a host, e g , a mouse, where it forms a gel Sprouts from vessels in the adjacent tissue penetrate into the gel within days connecting it with the external vasculature Angiogenesis is then quantitated by image analysis of vessels and by measuring the hemoglobin present in the vessels within the gel This assay method facilitates the testmg of both angiogenic and angiostatic agents in vivo In addition, the endothelial cells responding to the angiogenic factors can be recovered m vitro for further studies Preferred compounds have 50-70% inhibition rates, and more preferred compounds show 80- 100% inhibition rates As described herem the angiogenically active proteins ofthe invention provide use in in vitro and in vivo screens for compounds that inhibit angiogenesis by measuring inhibition of AL-2 stimulated angiogenesis in the presence and absence of the candidate inhibitor Therapeutic formulations of AL-2 and AL-2 antagonists for treating neurologic diseases and disorders and for modulat g angiogenesis and other disorders are prepared by mixing AL-2 or AL-2 antagonist, e g., anti- AL-2 antibody or a soluble AL-2-bιndmg-Eph-receptor, having the desired degree of purity, with optional physiologically acceptable carriers, excipients, or stabilizers which are well known Acceptable carriers. excipients or stabilizers are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants mcluding ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins: hydrophilic polymers such as polyvinylpyrrolidone; ammo acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextπns. chelatmg agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). It may be desirable to adsorb AL-2 onto a membrane, such as a silastic membrane, which can be implanted in proximity to damaged neural tissue, or to incoφorate AL-2 into liposomes (PCT Pat. Pub. No. WO 91/04014, published April 4, 1991 ). AL-2 optionally is combmed with or administered in concert with other neurotrophic factors to achieve a desired therapeutic effect. For example, AL-2 may be used together with NGF, NT-3, BDNF, NT-4/5, an insulin-like growth factor (e g., IGF-1, IGF-2, or IGF-3) or another neurotrophic factor to achieve a synergistic stimulatory effect on the growth of sensory neurons, wherein the term "synergistic" means that the effect ofthe combination of AL-2 with a second substance is greater than that achieved with either substance used individually. AL-2 and AL-2 antagonists to be used for in vivo administration must be sterile. This is readily accomplished by filtration of a solution of AL-2 or anti-AL-2 antibody through sterile filtration membranes. Thereafter, the filtered solution may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The filtered solution also may be lyophilized to produce sterile AL-2 or anti-AL-2 antibody in a powder form. Methods for administering AL-2 and AL-2 antagonists in vivo include injection or infusion by intravenous, intraperitoneal, intracerebral, intrathecal. intramuscular, intraocular, lntraartenal, or intralesional routes, and by means of sustained-release formulations. Sustained-release formulations generally consist of AL-2 or AL-2 antagonists and a matrix from which the AL-2 or AL-2 antagonists are released over some period of time. Suitable matrices include semipermeable polymer matrices m the form of shaped articles, for example, membranes, fibers, or microcapsules. Sustained release matrices may comprise polyesters, hydrogels, polylactides. U.S. Pat. No 3.773,919, copolymers of L- glutamic acid and gamma ethyl-L-glutamate, Sid an, et ai, Btopolymers. 22: 547-556 (1983), poly (2- hydroxyethy 1-methacry late), or ethylene vinyl acetate, Langer, et al . J Biomed Mater Res , 15: 167-277 (1981 ), Langer. Chem Tech . 12:98-105 ( 1982) In one embodiment ofthe invention, the therapeutic formulation comprises AL-2 or AL-2 antagonist entrapped within or complexed with liposomes For example. AL-2 covalently joined to a glycophosphatidyl- mositol moiety may be used to form a liposome comprising AL-2. In a further embodiment, the therapeutic formulation comprises cells actively producmg AL-2 or AL-2 antagonist. Such cells may be directly introduced into the tissue of a patient, or may be encapsulated within porous membranes which are then implanted in a patient in either case providmg for the delivery of AL-2 or antι-AL-2 antagonist into areas within the body of the patient in need of increased or decreased concentrations of AL-2 Altematively, an expression vector compnsing AL-2 DNA may be used for in vivo transformation of a patient's cells to accomplish the same result An effective amount of AL-2 or AL-2 antagonist, e , antι-AL-2 antibody, *o be employed therapeutically will depend for example upon the therapeutic objectives, the route of administration, and the condition of the patient Accordmgly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtam the optimal therapeutic effect A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kg or more, depending on the factors mentioned above Where possible, it is desirable to determme appropriate dosage ranges first in vitro, for example, using assays for neuronal cell survival or growth which are known m the art, and then in suitable animal models, from which dosage ranges for human patients may be extrapolated In a specific embodiment of the invention, a pharmaceutical composition effective in promoting the survival or growth of neurons will provide a local growth promotmg activity concentration m vivo of between about 0 1 and 10 ng/ml Typically, the clinician will administer AL-2 until a dosage is reached that achieves the desired effect Therapeutic progress is easily monitored by conventional assays In the treatment of tumors the compositions described herein can be administered subcutaneously or intramuscularly, for example, and the pharmacological activities of an AL-2 antagonist can be mamtained over a long penod of tune by the sustained-release effect of a composition ofthe present invention The number of administrations can therefore be reduced The composition can also be by directly injecting the composition mto a tumor-controlling artery In the case of treatment of an adult patient having a tumor, the dose ofthe AL-2 antagonist can be appropriately selected depending upon the kind of tumor, site, size, and kmd of AL-2 antagonist For example, the dose of a protem AL-2 antagonists, particularly an antibody, can be about 0 1 mg to about 500 mg, typically about 1 0 mg to about 300 mg, more typically about 25 mg to about 100 mg The administration frequency can be appropπately selected depending upon the kind of disease and dosage form In the case of injection into the tumor-controlled artery or tumor itself, frequently repeated injections are not required and a smgle injection once every one to 4 weeks may be sufficient for the desired therapeutic effects The nucleic acid encoding the AL-2 may be used as a diagnostic for tissue-specific typing For example, such procedures as in situ hybridization, Northern and Southern blotting, and PCR analysis, can be used to determine whether DNA and or RNA encoding AL-2 is present in the cell type(s) being evaluated AL-2 nucleic acid or polypeptide may also be used as diagnostic markers for such tissues For example, the AL-2 may be labeled using the techniques described herein, and expression of AL-2-receptor, including the preferred receptors disclosed herein, receptor can be quantified via its binding to labelled AL-2 AL-2 nucleic acid is also useful for the preparation of AL-2 polypeptide by recombinant techniques exemplified herein The invention also provides methods for studying the function ofthe AL-2 protein Cells, tissues, and non-human animals lackmg AL-2 expression, partially lackmg in AL-2 expression, or over-expressmg AL-2 can be developed using recombinant molecules ofthe invention having specific deletion or insertion mutations in the AL-2 gene For example the extracellular domain or parts thereof, the transmembrane region or parts thereof, and the cytoplasmic domain can be deleted A recombmant molecule may be used to macnvate or alter the endogenous gene by homologous recombination, and thereby create an AL-2 deficient (or over-expressing) cell, tissue or animal Null alleles can be generated in cells, such as embryonic stem cells by deletion mutation A recombmant AL-2 gene may also be engineered to contain an insertion mutation which inactivates AL-2 Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection, etc Cells lackmg an intact AL-2 gene can then be identified, for example by Southern blotting, Northern blotting or by assaying for expression of AL-2 protein using the methods described herein Such cells may then be fused to embryonic stem cells to generate transgenic non-human anunals deficient AL-2 Germme transmission ofthe mutation may be achieved, for example, by aggregatmg the embryonic stem cells with early stage embryos, such as 8 cell embryos, in vitro, transferring the resulting blastocysts mto recipient females and, generating germine transmission of the resulting aggregation chimeras Such a mutant animal may be used to define specific nerve cell populations, developmental patterns of axonogenesis, neural tube formation and nerve regeneration and in vrvo processes, normally dependent on AL-2 expression Methods for prepanng cells, tissues, and non-human animals lackmg AL-2 expression or partially lackmg in AL-2 expression, and deficient in the expression of other genes are provided In one embodiment an animal may be generated which is deficient m AL-2 and another tyrosme kinase receptor ligand Such animals could be used to determme how the members ofthe family cooperate m embryonic development, particularly development ofthe nervous system For example, an animal lacking or partially lacking AL-2 expression and Htk-L expression can be generated to determme how the receptor tyrosine kinases cooperate in neurogenesis, e g , the segmental patterning of the hindbrain Multiple deficient mice can also be generated to study the interaction of AL-2 and other protems such as the ligands ofthe Src-family of cytoplasmic tyrosme kmases For example, an animal may be generated which lacks or partially lacks AL-2 expression, and expression of one or more Src family tyrosine kmases including Src or Fyn and their ligands The bmdmg characteπstics of AL-2 (mcludmg vanants) can also be determmed usmg puπfied receptor e , conjugated, soluble receptor (for example, I-Htk-Fc or Hek5-IgG) in competition assays as descπbed herein For example, either intact cells expressmg AL-2 or soluble AL-2 bound to a solid substrate are used to measure the extent to which a sample contammg a putative AL-2 -receptor competes for bmdmg of a conjugated soluble receptor to AL-2 The AL-2 of the present invention can be used in a binding assay to detect cells expressmg an Eph- family receptor that bmds AL-2 For example, AL-2 or an extracellular domain or a fragment thereof can be conjugated to a detectable moiety such as I Radiolabeling with 1 can be performed by any of several standard methodologies that yield a functional 125 I-AL-2 molecule labeled to high specific activity Altematively, another detectable moiety such as an enzyme that can catalyze a colorimetric or fluorometric reaction biotin or avidin may be used Cells or samples to be tested for AL-2-receptor expression can be contacted with labeled AL-2 After incubation, unbound labeled AL-2 is removed and bmdmg is measured using the detectable moiety The AL-2 proteins disclosed herein can be employed to measure the biological activity of an AL-2 receptor terms of bmdmg affinity for AL-2 For example, AL-2 can be employed m a binding affinity study to measure the biological activity of a receptor that has been stored at different temperatures, or produced in different cell types Thus, AL-2 proteins find use as reagents in 'quality assurance" studies, e g to monitor shelf life and stability of receptor protein under different conditions Furthermore AL-2 can be used in determining whether biological activity is retained after modification of a receptor protein (e g , chemical modification, truncation, mutation, etc ) The bmdmg affinity ofthe modified receptor for an AL-2 is compared to that of an unmodified receptor to detect any adverse impact ofthe modifications on biological activity ofthe receptor Bmdmg of AL-2 to an Eph-family receptor can be determmed usmg conventional techniques, mcludmg competitive binding methods, such as RIAs, ELISAs, and other competitive bmding assays Ligand/receptor complexes can be identified usmg such separation methods as filtration, centπfugation. flow cytometry (see, e , Lyman e/ a/ , Ce// 75 1157-1 167 (1993), Urdal e/ o/ , J Biol Chem 263 2870-2877 (1988), and Gearing e/ α/ , EMBOJ 8 3667-3676 (1989)) Results from bmdmg studies can be analyzed usmg any conventional graphical representation ofthe bmdmg data, such as Scatchard analysis (Scatchard, Ann. NY Acad Sci 51 660-672 (1949), and Goodwin et al , Cell 73 447-456 (1993)), and the like Since the AL-2 mduces receptor phosphorylation, conventional tyrosine phosphorylation assays can also be used Isolated AL-2 polypeptide may be used m quantitative diagnostic assays as a standard or control agamst which samples containing unknown quantities of AL-2 may be prepared AL-2 preparations are also useful in generating antibodies, as standards in assays for AL-2 (e , by labeling AL-2 for use as a standard m a radioimmunoassay, or enzyme-linked immunoassay), for detecting the presence of an AL-2-receptor m a biological sample (e g , using a labelled AL-2), in affinity punfication techniques, and m competitive-type receptor bmdmg assays when labeled for example with radioiodine. enzymes, fluorophores, spin labels, or branched DNA AL-2 polypeptide can be produced in prokaryotic cells using the techniques taught herein, and the unglycosylated prote so produced can be used as a molecular weight marker, for example Preferably unglycosylated, soluble AL-2 is used AL-2 can be used as a molecular weight marker in gel filtration chromatography or SDS-PAGE, for example, either analytical or preparative modes, where it is desirable to determme molecular weιght(s) for separated peptides AL-2 is most preferably used in combination with other known molecular weight markers as standards to provide a range of molecular weights Other known molecular weight markers can be purchased commercially, e g , from Amersham Coφoration, Arlington Heights, IL, for example The molecular weight markers can be labelled to enable easy detection following separation Techniques for labelling antibodies and protems are discussed herem and are well known m the art For example, the molecular weight markers can be biotinylated and, following separation on SDS-PAGE for example, can be detected using streptavidin-horseradish peroxidase AL-2 is used for competitive screening of potential agonists or antagonists for binding to an AL-2 receptor AL-2 variants are useful as standards or controls assays for AL-2, provided that they are recognized by the analytical system employed, e g , an antι-AL-2 antibody One embodiment is a method for identify mg compounds that modulate the activity of an AL-2-bmdmg- Eph-family receptor The method cludes the steps of (a) exposmg cells exhibiting the receptor to ligand, / e AL-2 or modified or variant forms, for a time sufficient to allow formation of receptor-ligand complexes and induce signal transduction, (b) determining the extent of activity withm the cells, and (c) comparing the measured activity to the activity in cells not exposed to the gano Receptor activity may be detected bv changes in target cell proliferation, differentiation, metabolism, or other activity of interest (e g , axon-targeting neuronal outgrowth), preferably one predictive of success of a therapeutic method The receptor can be endogenous or can be present as a result of expression of a recombinant molecule AL-2 can be useful as a growth factor or differentiation for cells havmg an AL-2 receptor These cells. which can be grown ex vivo, can simultaneously be exposed to other known growth factors or cytokines Exemplary cytokmes include the interleukins (e , IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (GM-CSF), erythropoietin (Epo), lymphotoxin, steel factor (SLF), tumor necrosis factor (TNF) and gamma-interferon This results in proliferation and/or differentiation ofthe cells havmg a AL-2 receptor For example, human tumor cell lmes for which it is desired to isolate certam tumor associated factors (usually protems) therefrom can be grown ex vivo usmg AL-2 Also, antibodies agamst the tumor associated factors can be generated which may be useful for diagnostic puφoses Examples of such tumor cell lmes which are candidates for treatment with AL-2 mclude mammary cancer cells (e g MCF-7), liver cell lines, Colo 205, NCI 69, HM-1 and HeLa, for example A different use of an AL-2 is as a reagent m protem purification procedures AL-2 or AL-2-IgG fusion protems may be attached to a solid support matenal by conventional techniques and used to purify an AL-2- binding protem, e.g receptor, by affinity chromatography AL-2 can be used for affinity puπfication of an AL-2 receptor Bπefly, this technique involves covalently attach mg AL-2 to an inert and porous matπx (e g , agarose reacted with cyanogen bromide) A solution containing the AL-2 receptor can then be passed through the chromatographic matenal and can be subsequently released by changing the elution conditions (e g by changing pH or ionic strength) AL-2 polypeptides find use as carriers for delivering agents to cells bearmg an AL-2-bιndιng cell surface receptor Al-2 can be used to deliver diagnostic or therapeutic agents to these cells (or to other cell types found to express an AL-2 receptor on the cell surface) in in vitro ex vivo, or m vivo procedures One example of such use is to expose an AL-2 receptor expressmg neoplastic cell line to a therapeutic agent AL-2 conjugate to assess whether the conjugate exhibits a desired effect on the target cells A number of different therapeutic agents attached to AL-2 can be mcluded m an assay to detect and compare the effect ofthe agents on the target cells In a preferred embodiment the agent is a cytotoxin, however, the agent can be a viral protease inhibitor or the like In another embodiment, a diagnostic, l e detectable agent, is conjugated to AL-2 to detect the presence of AL-2-receptor-expressmg cells Diagnostic and therapeutic agents that may be attached to a AL-2 polypeptide include but are not lunited to, drugs, toxins, antiviral agents, radionuclides, chromophores, fluorescent compounds enzymes that catalyze a coloπmetnc or fluorometπc reaction, and the like, with the particular agent being chosen according to the intended application Examples of drugs include those used in treating vaπous forms of cancer, e g , nitrogen mustards such as L-phenylalanine nitrogen mustard or cyclophosphamide. intercalating agents such as cis-diaminodichloroplatinum. antmietabolites such as 5-fluorouracιl, vinca alkaloids such as vincπstine. and antibiotics such as bleomycin, doxorubicin. daunorubicm. and derivatives thereof Among the tox s are πcin, abrm. diphtheria tox , Pseudomonas aeruginosa exotoxm A. ribosomal inactivating protems. mycotoxins such as tπchothecenes. and derivatives and fragments (e , single chains) thereof Radionuclides suitable for diagnostic use include but are not limited to, I 1, m Tc, In. and Br Radionuclides suitable for therapeutic use include, but are not limited to, 131 1 2 H At. 77 Br. 186 Re 188 Re 2, 2 Pb 212 Bι, 109 Pd 64 Cu and 67 Cu Such agents may be attached to AL-2 by anv suitable conventional procedure AL-2 contains functional groups on ammo acid side chams that can be reacted with functional groups on a desired agent to form covalent bonds for example Alternatively, the protem or agent may be deπvatized to generate or attach a desued reactive functional group, preferably a site-specific reactive group The deπvatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Illinois) A number of techniques for radiolabelmg proteins are known Radionuclide metals may be attached to AL-2 by using a suitable bifunctional chelating agent for example Conjugates comprising AL-2 and a suitable diagnostic or therapeutic agent (preferably covalently linked) are administered or otherwise employed m an amount appropriate for the particular application In view of the sequence identity between AL-2 and Htk-L as shown in Figures 4 and 5. which is determmed for the first tune herem, and smce Htk-L is a ligand ofthe transmembrane-sequence type and binds an Eph-family receptor, namely Htk, the present application provides embodiments of methods of treatment wherem an effective amount of Htk-L is admmistered to a patient m need of such treatments as discussed for the first time herem for AL-2 Accordingly, WO 96/02645, published February 1 , 1996, is incoφorated by reference herem for its teachmgs regarding nucleic acid sequences encodmg Htk-L, Htk-L protems and vaπants, and methods for their production and formulation Consequently, it is the tent ofthe present inventors that new uses and methods of administration of AL-2. as taught for the first time herein, are to be applied to Htk-L For example, m one embodiment Htk-L will find use m methods of treatment of angiogenesis-related conditions as taught herein for AL-2 In summary, by providing nucleic acid molecules encodmg AL-2. the present invention enables for the first time the production of AL-2 by recombmant DNA methods, thus providing a reliable source of sufficient quantities of AL-2 for use m various diagnostic and therapeutic applications In view of its distinct biological properties, purified recombinant AL-2 will be especially useful in a variety of circumstances, such as in angiogenesis-related conditions and where it is necessary or desirable to assure neuronal function, growth, survival, or cell-cell contact but where other neurotrophic factors or angiogenic agents either cannot be used or are less effective The following examples are offered bv way of illustration only and are not intended to limit the invention in any manner All patent and literature references cited herein are expressly incoφorated EXAMPLES Example 1 Isolation of a full-length cDNA encoding AL-2 The Genbank EST database was screened with an AL- 1 sequence and with sequences from several other members ofthe Eph-receptor ligand family, nameiy B61, Lerk2 and Htk-L. From this search. EST sequence H10006 was identified (see Figure 3A-3B) and selected to provide a sequence from which a probe-based clonmg approach for a novel neurotrophic factor was devised Two 60-mer oligonucleotide probes were designed based on the sequence of the EST HI 0006. namely sense-probe-H 1006 (5'-GGA CAA AGT CCC GAG GAG GGG CTG TCC CCC GAA AAC CTG TGT CTG AAA TGC CCA TGG AAA-3' ) (SEQ ID NO 7) and antιsense-probe-H1006 (5'-CAG GTT CTC CTT CCC CAG GCT CCC AGG CTG TGG GCT GCC CCT CGG TCT CTT TCC ATG GGC-3') (SEQ ID NO' 8) The probes are altematively referred to as sense-probe-H 10006 and anti-sense-probe-H 10006, respectively The two synthetic probes were labeled and used to screen a human fetal brain cDNA library. Filters were hybridized in 50% formamide and washed m 0.2% SSC/0.1% SDS at 55°C. Six double-positive clones. / e , clones that hybπdized with both probes, were identified and selected. These clones were plaque purified, and their cDNA inserts were transferred mto a plasmid vector and sequenced. Two distmct sequences encoding identical proteins differing only at their C-termini were observed indicating a novel neurotrophic factor designated AL-2. The shorter form, which ends with the sequence "KV," was designated AL-2s (" AL-2-short"), and the longer form, which contains additional ammo acids at its C-terminal end, was designated AL-21 ("AL-2- long") Figure 1 A- IB depicts the AL-21 cDNA sequence and the deduced AL-21 amino acid sequence. Figure 2A-2B depicts the AL-2s cDNA sequence and the deduced AL-2s ammo acid sequence. Figure 3A-3B depicts alignment ofthe AL-21 nucleic acid sequence with the EST HI 0006 sequence. Example 2 Expression of AL-2 by Northern t>M Analysis A Northern blot of poly(A)+ RNA isolated from pancreas, kidney, skeletal muscle, liver, lung, placenta, brain and heart tissue was screened with an AL-2 probe. T e highest levels of AL-2 expression were m the brain, pancreas, and skeletal muscle. Lower levels were detectable in kidney, liver, placenta and heart Within the brain AL-2 was expressed in every brain region tested, mc ludmg cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe, putamen, amygdala, caudate nucleus, coφus callosum. hippocampus whole brain, substantia nigra, subthalamic nucleus and thalamus Interestingly, a second, shorter RNA transcript was observed in the brain samples. The shorter transcript may be the AL-2s mRNA. The ratio between the observed long and short transcripts differed amongst the brain regions screened Example 3 Construction and Production of an AL-2-IgG Fusion A soluble AL-2-IgG fusion protem was constructed by recombmant DNA techniques. DNA encoding a soluble AL-2-IgG chimera was constructed by joining the DNAs encoding the extracellular domain of AL-2 and the Fc domam of IgG t , similar to the construction of Rek7-IgG and AL-l-IgG (see Winslow et ai. Neuron. 14 973-981 ( 1995)), with the AL-2 sequence replacing the AL-1 sequence. The AL-2 coding region from its initiation methionine- 1 to glycme-218 was fused at its 3' end to the 5'-end (at the aspartic acid) of the 343 amino acid sequence of lgG2b AL-2-lgG fusion protein is generated by transfection of HEK 293 cells (Graham, et al . J Gen Virol 36:59 ( 1977)) with the plasmid pRKAL-2-IgG under conditions as described by Winslow et ai. ,\euron. 14 973- 981 ( 1995)) Conditioned media is collected after 3 days and AL-2-IgG is purified bv Protein A chromatography Example 4 Biological Activity. Activation of Eph-Related Receptors by AL-2 The ability of AL-2 or variant e g . AL-2-IgG. to activate a Eph-familv receptor can be determined by tyrosine autophosphorylation of the receptor in a receptor-expressing cell source as described herein Cells expressmg an Eph-family receptor, preferably Hek2. Hek5, Hek6/elk/Cek6 or Htk, are incubated with AL-2 and specific phosphorylation ofthe Eph-family receptor is monitored Specific phosphorylation dicates that AL-2 not only binds to the Eph-family receptor, but that it also activates the Eph-family receptor Cells expressmg an Eph-family receptor, e g , cultured primary cortical neurons, are detected and analyzed by in situ hybndization and/or immunoprecipitation and immunoblotting with anti-Eph-family-receptor antibodies preferably antι-Hek2, antι-Elk/Hek6/Cek6, antι-Hek5 or anti-Htk, and anti-phosphotyrosine antibodies Membrane-bound AL-2 is transiently expressed on the surface of transfected 293 cells and its activation ofthe endogenous Eph-family receptor in the receptor-expressmg cell is monitored Activation of endogenous Eph-family receptor is mdicated by autophosphorylation ofthe receptor Alternatively soluble AL-2 fusion, e g , AL-2-IgG, dimers, multimers, as taught herem, is provided to the receptor-expressmg cells and tested for activation of endogenous Eph-family receptor as descπbed, for example, by Wmslow et al , Neuron. 14 973- 981 ( 1995) Membrane-attachment has been reported as required or preferred for maximal receptor activation with other members of this ligand family (Davis et al , Science, 266 816-819 (1994)) HEK 293 cells are transfected with an AL-2 cDNA expression plasmid usmg the calcium phosphate coprecipitation method (Simonsen et al , Proc Natl Acad Set USA, 80 2495-2499 (1983)) Primary cortical neurons from E16 rats are plated at a density of 5 x 10 cells /15 cm dish and cultured for 4 days These cells are then treated with purified soluble AL-2 or soluble AL-2-IgG (0 1-1 μg/ml) or a number of 293 cells expressmg an equivalent number of membrane-bound AL-2 for 10 mm at 37 °C Immunoprecipitation of lysates with rabbit anti-Eph-family receptors and immunoblotting with mouse anti-phosphotyros e is essentially as described (Kaplan et al , Science 252 554-559 (1991), Kaplan et al , Nature, 350 158-160 (1991)) Immunoblotted bands are visualized usmg a horseradish peroxidase-conjugated sheep anti-mouse antibody and the ECL fluorescence detection system (Amersham) as described by the manufacturer An ability of AL-2-IgG to activate the autophosphorylation of receptor, preferably to a similar extent as membrane-bound AL-2, indicates that the soluble AL-2-IgG fusion protein and the like can be used as an agonist for Eph-family receptors in vitro, ex vivo and in vivo An inability of soluble AL-2 (e g , free ECD) to activate receptor autophosphorylation despite its ability to bind receptor indicates that certain soluble forms of AL-2 as taught herein can act as antagonists of Eph-family receptors SEQUENCE LISTING 11) GENERAL INFORMATION: (i) APPLICANT: Genentech, Inc. (ii) TITLE OF INVENTION: AL-2 Neurotrophic Factor (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Genentech, Inc. (B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA (F) ZIP: 94080 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: WinPatin (Genentech) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Torchia, PhD., Timothy E. (B) REGISTRATION NUMBER: 36,700 (C) REFERENCE/DOCKET NUMBER: PIOOIPCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415/225-8674 (B) TELEFAX: 415/952-9881 (C) TELEX: 910/371-7168 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1877 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Double (D) TOPOLOGY: Linear (ix) FEATURE: (A) NAME/KEY: Extra Cellular Domain (B) LOCATION: 244-899 (C) IDENTIFICATION METHOD: (D) OTHER INFORMATION: (i ) FEATURE: (A) NAME/KEY: Transmembrane Domain (B) LOCATION: 901-978 (C) IDENTIFICATION METHOD: (D) OTHER INFORMATION: (ix) FEATURE: (A) NAME/KEY: signal peptide (B) LOCATION: 244-321 (C) IDENTIFICATION METHOD: (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : GNTCTAGAAN TAGTGGATCC CCCCGGGCTG CAGGAATTCC GACGGCCCCT 50 GGAAGGGCTC TGGTGGGGCT GAGCGCTCTG CCGCGGGGGC GCGGGCACAG 100 CAGGAAGCAG GTCCGCGTGG GCGCTGGGGG CATCAGCTAC CGGGGTGGTC 150 CGGGCTGAAG AGCCAGGCAG CCAAGGCAGC CACCCCGGGG GGTGGGCGAC 200 TTTGGGGGAG TTGGTGCCCC GCCCCCCAGG CCTTGGCGGG GTC ATG 246 Met 1 GGG CCC CCC CAT TCT GGG CCG GGG GGC GTG CGA GTC GGG 285 Gly Pro Pro His Ser Gly Pro Gly Gly Val Arg Val Gly 5 10 GCC CTG CTG CTG CTG GGG GTT TTG GGG CTG GTG TCT GGG 324 Ala Leu Leu Leu Leu Gly Val Leu Gly Leu Val Ser Gly 15 20 25 CTC AGC CTG GAG CCT GTC TAC TGG AAC TCG GCG AAT AAG 363 Leu Ser Leu Glu Pro Val Tyr Trp Asn Ser Ala Asn Lys 30 35 40 AGG TTC CAG GCA GAG GGT GGT TAT GTG CTG TAC CCT CAG 402 Arg Phe Gin Ala Glu Gly Gly Tyr Val Leu Tyr Pro Gin 45 50 ATC GGG GAC CGG CTA GAC CTG CTC TGC CCC CGG GCC CGG 441 lie Gly Asp Arg Leu Asp Leu Leu Cys Pro Arg Ala Arg 55 60 65 CCT CCT GGC CCT CAC TCC TCT CCT AAT TAT GAG TTC TAC 480 Pro Pro Gly Pro His Ser Ser Pro Asn Tyr Glu Phe Tyr 70 75 AAG CTG TAC CTG GTA GGG GGT GCT CAG GGC CGG CGC TGT 519 Lys Leu Tyr Leu Val Gly Gly Ala Gin Gly Arg Arg Cys 80 85 90 GAG GCA CCC CCT GCC CCA AAC CTC CTT CTC ACT TGT GAT 558 Glu Ala Pro Pro Ala Pro Asn Leu Leu Leu Thr Cys Asp 95 100 105 CGC CCA GAC CTG GAT CTC CGC TTC ACC ATC AAG TTC CAG 597 Arg Pro Asp Leu Asp Leu Arg Phe Thr lie Lys Phe Gin 110 115 GAG TAT AGC CCT AAT CTC TGG GGC CAC GAG TTC CGC TCG 636 Glu Tyr Ser Pro Asn Leu Trp Gly His Glu Phe Arg Ser 120 125 130 CAC CAC GAT TAC TAC ATC ATT GCC ACA TCG GAT GGG ACC 675 His His Asp Tyr Tyr lie lie Ala Thr Ser Asp Gly Thr 135 140 CGG GAG GGC CTG GAG AGC CTG CAG GGA GGT GTG TGC CTA 714 Arg Glu Gly Leu Glu Ser Leu Gin Gly Gly Val Cys Leu 145 150 155 ACC AGA GGC ATG AAG GTG CTT CTC CGA GTG GGA CAA AGT 753 Thr Arg Gly Met Lys Val Leu Leu Arg Val Gly Gin Ser 160 165 170 CCC CGA GGA GGG GCT GTC CCC CGA AAA CCT GTG TCT GAA 792 Pro Arg Gly Gly Ala Val Pro Arg Lys Pro Val Ser Glu 175 180 ATG CCC ATG GAA AGA GAC CGA GGG GCA GCC CAC AGC CTG 831 Met Pro Met Glu Arg Asp Arg Gly Ala Ala His Ser Leu 185 190 195 GAG CCT GGG AAG GAG AAC CTG CCA GGT GAC CCC ACC AGC 870 Glu Pro Gly Lys Glu Asn Leu Pro Gly Asp Pro Thr Ser 200 205 AAT GCA ACC TCC CGG GGT GCT GAA GGC CCC CTG CCC CCT 909 Asn Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro 210 215 220 CCC AGC ATG CCT GCA GTG GCT GGG GCA GCA GGG GGG CTG 948 Pro Ser Met Pro Ala Val Ala Gly Ala Ala Gly Gly Leu 225 230 235 GCG CTG CTC TTG CTG GGC GTG GCA GGG GCT GGG GGT GCC 987 Ala Leu Leu Leu Leu Gly Val Ala Gly Ala Gly Gly Ala 240 245 ATG TGT TGG CGG AGA CGG CGG GCC AAG CCT TCG GAG AGT 1026 Met Cys Trp Arg Arg Arg Arg Ala Lys Pro Ser Glu Ser 250 255 260 CGC CAC CCT GGT CCT GGC TCC TTC GGG AGG GGA GGG TCT 1065 Arg His Pro Gly Pro Gly Ser Phe Gly Arg Gly Gly Ser 265 270 CTG GGC CTG GGG GGT GGA GGT GGG ATG GGA CCT CGG GAG 1104 Leu Gly Leu Gly Gly Gly Gly Gly Met Gly Pro Arg Glu 275 280 285 GCT GAG CCT GGG GAG CTA GGG ATA GCT CTG CGG GGT GGC 1143 Ala Glu Pro Gly Glu Leu Gly lie Ala Leu Arg Gly Gly 290 295 300 GGG GCT GCA GAT CCC CCC TTC TGC CCC CAC TAT GAG AAG 1182 Gly Ala Ala Asp Pro Pro Phe Cys Pro His Tyr Glu Lys 305 310 GTG AGT GGT GAC TAT GGG CAT CCT GTG TAT ATC GTG CAG 1221 Val Ser Gly Asp Tyr Gly His Pro Val Tyr lie Val Gin 315 320 325 GAT GGG CCC CCC CAG AGC CCT CCA AAC ATC TAC TAC ACA 1260 Asp Gly Pro Pro Gin Ser Pro Pro Asn He Tyr Tyr Thr 330 335 TCG ATT TCT GTG TTG GAG TGG CCC ATA TTG CAT ACG ATA 1299 Ser He Ser Val Leu Glu Trp Pro He Leu His Thr He 340 345 350 CAA CTG TTT TTC ATG CGA TCC AAG TGC TCC CGT GTC ACT 1338 Gin Leu Phe Phe Met Arg Ser Lys Cys Ser Arg Val Thr 355 360 365 ACA TTC TTA TTT CCT GTG CAA GTT ATT ACG ACA TCG ACT 1377 Thr Phe Leu Phe Pro Val Gin Val He Thr Thr Ser Thr 370 375 TGC CGG ATG ACT TCA TTT AGC TTT ACC ACC CTG AAC CCA 1416 Cys Arg Met Thr Ser Phe Ser Phe Thr Thr Leu Asn Pro 380 385 390 TCC ATG CAG GCC TGC AGA GCA CAG ATG GGG GAA TTC CGA 14S5 Ser Met Gin Ala Cys Arg Ala Gin Met Gly Glu Phe Arg 395 400 ATC AGA TGG TGT TTC TGG GGG GAC AGG ATC CTG GGT ACG 1494 He Arg Trp Cys Phe Trp Gly Asp Arg He Leu Gly Thr 405 410 415 GCT CTG TTT GTG CTT GTG CTT ATT CTT CTT CTT GGG AGG 1533 Ala Leu Phe Val Leu Val Leu He Leu Leu Leu Gly Arg 420 425 430 CTG AAT ATG CAT CAG ACG ACA CTG CTC CGG CAA CGG GCC 1572 Leu Asn Met His Gin Thr Thr Leu Leu Arg Gin Arg Ala 435 440 AGT GTG GAG GCG GAA GCC GGC CAG CAT GGT CCC CTG TG 1610 Ser Val Glu Ala Glu Ala Gly Gin His Gly Pro Leu 445 450 455 ATAGGATTGA AAGAGCTACT GAGAATAGGG GGCTTCTCAA TGAGAGAGCG 1660 GAGGCTGCTG TTATCATGGG AACCAGGCAG ATCAATCATC CCTGGCAGGT 1710 CAGGCAGGAA GTTACTTAGC TTCTCCTTCA CCTTCTTCCC ACAGAATTTA 1760 TTATAGGCTT GTTCCAAGTT GTAGTGTGTG ATCAGATTCG TGCTGCCTGT 1810 CAGCTCTGTG CTACCTGGCA GTTCCCCTCA TGGAATTCGA TATCAAGCTT 1860 ATCGATACCG TCGACCT 1877 (2) INFORMATION FOR SEQ ID NO:2 : (ι) SEQUENCE CHARACTERISTICS: (A) LENGTH: 455 ammo acids (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : Met Gly Pro Pro His Ser Gly Pro Gly Gly Val Arg Val Gly Ala 1 5 10 15 Leu Leu Leu Leu Gly Val Leu Gly Leu Val Ser Gly Leu Ser Leu 20 25 30 Glu Pro Val Tyr Trp Asn Ser Ala Asn Lys Arg Phe Gin Ala Glu 35 40 45 Gly Gly Tyr Val Leu Tyr Pro Gin He Gly Asp Arg Leu Asp Leu 50 55 60 Leu Cys Pro Arg Ala Arg Pro Pro Gly Pro His Ser Ser Pro Asn 65 70 75 Tyr Glu Phe Tyr Lys Leu Tyr Leu Val Gly Gly Ala Gin Gly Arg 80 85 90 Arg Cys Glu Ala Pro Pro Ala Pro Asn Leu Leu Leu Thr Cys Asp 95 100 105 Arg Pro Asp Leu Asp Leu Arg Phe Thr He Lys Phe Gin Glu Tyr 110 115 120 Ser Pro Asn Leu Trp Gly His Glu Phe Arg Ser His His Asp Tyr 125 130 135 Tyr He He Ala Thr Ser Asp Gly Thr Arg Glu Gly Leu Glu Ser 140 145 150 Leu Gin Gly Gly Val Cys Leu Thr Arg Gly Met Lys Val Leu Leu 155 160 165 Arg Val Gly Gin Ser Pro Arg Gly Gly Ala Val Pro Arg Lys Pro 170 175 180 Val Ser Glu Met Pro Met Glu Arg Asp Arg Gly Ala Ala His Ser 185 190 195 Leu Glu Pro Gly Lys Glu Asn Leu Pro Gly Asp Pro Thr Ser Asn 200 205 210 Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro Pro Ser Met 215 220 225 Pro Ala Val Ala Gly Ala Ala Gly Gly Leu Ala Leu Leu Leu Leu 230 235 240 Gly Val Ala Gly Ala Gly Gly Ala Met Cys Trp Arg Arg Arg Arg 245 250 255 Ala Lys Pro Ser Glu Ser Arg His Pro Gly Pro Gly Ser Phe Gly 260 265 270 Arg Gly Gly Ser Leu Gly Leu Gly Gly Gly Gly Gly Met Gly Pro 275 280 285 Arg Glu Ala Glu Pro Gly Glu Leu Gly He Ala Leu Arg Gly Gly 290 295 300 Gly Ala Ala Asp Pro Pro Phe Cys Pro His Tyr Glu Lys Val Ser 305 310 315 Gly Asp Tyr Gly His Pro Val Tyr He Val Gin Asp Gly Pro Pro 320 325 330 Gin Ser Pro Pro Asn He Tyr Tyr Thr Ser He Ser Val Leu Glu 335 340 345 Trp Pro He Leu His Thr He Gin Leu Phe Phe Met Arg Ser Lys 350 355 360 Cys Ser Arg Val Thr Thr Phe Leu Phe Pro Val Gin Val He Thr 365 370 375 Thr Ser Thr Cys Arg Met Thr Ser Phe Ser Phe Thr Thr Leu Asn 380 385 390 Pro Ser Met Gin Ala Cys Arg Ala Gin Met Gly Glu Phe Arg He 395 " 400 405 Arg Trp Cys Phe Trp Gly Asp Arg He Leu Gly Thr Ala Leu Phe 410 415 420 Val Leu Val Leu He Leu Leu Leu Gly Arg Leu Asn Met His Gin 425 430 435 Thr Thr Leu Leu Arg Gin Arg Ala Ser Val Glu Ala Glu Ala Gly 440 445 450 455 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2380 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 : GNTCTAGAAN TAGTGGATCC CCCCGGGCTG CAGGAATTCC GACGGCCCCT 50 GGAAGGGCTC TGGTGGGGCT GAGCGCTCTG CCGCGGGGGC GCGGGCACAG 100 CAGGAAGCAG GTCCGCGTGG GCGCTGGGGG CATCAGCTAC CGGGGTGGTC 150 CGGGCTGAAG AGCCAGGCAG CCAAGGCAGC CACCCCGGGG GGTGGGCGAC 200 TTTGGGGGAG TTGGTGCCCC GCCCCCCAGG CCTTGGCGGG GTCATGGGGC 250 CCCCCCATTC TGGGCCGGGG GGCGTGCGAG TCGGGGCCCT GCTGCTGCTG 300 GGGGTTTTGG GGCTGGTGTC TGGGCTCAGC CTGGAGCCTG TCTACTGGAA 350 CTCGGCGAAT AAGAGGTTCC AGGCAGAGGG TGGTTATGTG CTGTACCCTC 400 AGATCGGGGA CCGGCTAGAC CTGCTCTGCC CCCGGGCCCG GCCTCCTGGC 450 CCTCACTCCT CTCCTAATTA TGAGTTCTAC AAGCTGTACC TGGTAGGGGG 500 TGCTCAGGGC CGGCGCTGTG AGGCACCCCC TGCCCCAAAC CTCCTTCTCA 550 CTTGTGATCG CCCAGACCTG GATCTCCGCT TCACCATCAA GTTCCAGGAG 600 TATAGCCCTA ATCTCTGGGG CCACGAGTTC CGCTCGCACC ACGATTACTA 650 CATCATTGCC ACATCGGATG GGACCCGGGA GGGCCTGGAG AGCCTGCAGG 700 GAGGTGTGTG CCTAACCAGA GGCATGAAGG TGCTTCTCCG AGTGGGACAA 750 AGTCCCCGAG GAGGGGCTGT CCCCCGAAAA CCTGTGTCTG AAATGCCCAT 800 GGAAAGAGAC CGAGGGGCAG CCCACAGCCT GGAGCCTGGG AAGGAGAACC 850 TGCCAGGTGA CCCCACCAGC AATGCAACCT CCCGGGGTGC TGAAGGCCCC 900 CTGCCCCCTC CCAGCATGCC TGCAGTGGCT GGGGCAGCAG GGGGGCTGGC 950 GCTGCTCTTG CTGGGCGTGG CAGGGGCTGG GGGTGCCATG TGTTGGCGGA 1000 GACGGCGGGC CAAGCCTTCG GAGAGTCGCC ACCCTGGTCC TGGCTCCTTC 1050 GGGAGGGGAG GGTCTCTGGG CCTGGGGGGT GGAGGTGGGA TGGGACCTCG 1100 GGAGGCTGAG CCTGGGGAGC TAGGGATAGC TCTGCGGGGT GGCGGGGCTG 1150 CAGATCCCCC CTTCTGCCCC CACTATGAGA AGGTGAGTGG TGACTATGGG 1200 CATCCTGTGT ATATCGTGCA GGATGGGCCC CCCCAGAGCC CTCCAAACAT 1250 CTACTACAAG GTATGAGGGC TCCTCTCACG TGGCTATCCT GAATCCAGCC 1300 CTTCTTGGGG TGCTCCTCCA GTTTAATTCC TGGTTTGAGG GACACCTCTA 1350 ACATCTCGGC CCCCTGTGCC CCCCCAGCCC CTTCACTCCT CCCGGCTGCT 1400 GTCCTC3TCT CCACTTTTAG GATTC TTAG GATTCCCACT GCCCCACTTC 1450 CTGCCCTCCC G TTGGCCAT GGGTGCCCCC TCTGTCTCA GTGTCCCTGG 1500 ATCCT TTTC CTTGGGGAG G GGCACAGGCT CAGCCTCCTC TCTGACCATG 1550 ACCCAGGCAT CCTTGTCCC CTCACCCACC CAGAGCTAGG GGCGGGAACA 1600 GCCCACCTTT TGGTTGGCAC CGCCTTCTTT CTGCCTCTCA CTGGTTTTCT 1650 CTTCTCTATC TCTTATTCTT TCCCTCTCTT CCGTCTCTAG GTCTGTTCTT 1700 CTTCCCTAGC ATCCTCCTCC CCACATCTCC TTTCACCCTC TTGGCTTCTT 1750 ATCCTGTGCC TCTCCCATCT CCTGGGTGGG GGCATCAAAG CATTTCTCCC 1800 CTTAGCTTTC AGCCCCCCTT CTGACCTCTC ATACCAACCA CTCCCCTCAG 1850 TCTGCCAAAA ATGGGGGCCT TATGGGGAAG GCTCTGACAC TCCACCCCAG 1900 CTCAGGCCAT GGGCAGCAGG GCTCCATTCT CTGGCCTGGC CCAGGCCTCT 1950 ACATACTTAC TCCAGCCATT TGGGGTGGTT GGGTCATGAC AGCTACCATG 2000 AGAAGAAGTG TCCCGTTTTG TCCAGTGGCC AATAGCAAGA TATGAACCGG 2050 TCGGGACATG TATGGACTTG GTCTGATGCT GAATGGGCCA CTTGGGACCG 2100 GAAGTGACTT GCTCCAGACA AGAGGTGACC AGGCCCGGAC AGAAATGGCC 2150 TGGGAAGTAG CAGAAGCAGT GCAGCAGGAA CTGGAAGTGC CTTCATCCAG 2200 GACAGGAAGT AGCACTTCTG AAACAGGAAG TGGTCTGGCT GGAACTCCAA 2250 GTGGCTTAGT CTGGGGGATC AGGAGGTGGG AGGTGGATGG TTCTTATTCT 2300 GTGGAGAAGA AGGGCGGGAA GAACTTCCTT TCAGGAGGAA GCTGGAACTT 2350 ACTGACTGTA AGAGGTTAGA GGTGGACCGA 2380 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 340 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : Met Gly Pro Pro His Ser Gly Pro Gly Gly Val Arg Val Gly Ala 1 5 10 15 Leu Leu Leu Leu Gly Val Leu Gly Leu Val Ser Gly Leu Ser Leu 20 25 30 Glu Pro Val Tyr Trp Asn Ser Ala Asn Lys Arg Phe Gin Ala Glu 35 40 45 Gly Gly Tyr Val Leu Tyr Pro Gin He Gly Asp Arg Leu Asp Leu 50 55 60 Leu Cys Pro Arg Ala Arg Pro Pro Gly Pro His Ser Ser Pro Asn 65 70 75 Tyr Glu Phe Tyr Lys Leu Tyr Leu Val Gly Gly Ala Gin Gly Arg 80 85 90 Arg Cys Glu Ala Pro Pro Ala Pro Asn Leu Leu Leu Thr Cys Asp 95 100 105 Arg Pro Asp Leu Asp Leu Arg Phe Thr He Lys Phe Gin Glu Tyr 110 115 120 Ser Pro Asn Leu Trp Gly His Glu Phe Arg Ser His His Asp Tyr 125 130 135 Tyr He He Ala Thr Ser Asp Gly Thr Arg Glu Gly Leu Glu Ser 140 145 150 Leu Gin Gly Gly Val Cys Leu Thr Arg Gly Met Lys Val Leu Leu 155 160 165 Arg Val Gly Gin Ser Pro Arg Gly Gly Ala Val Pro Arg Lys Pro 170 175 180 Val Ser Glu Met Pro Met Glu Arg Asp Arg Gly Ala Ala His Ser 185 190 195 Leu Glu Pro Gly Lys Glu Asn Leu Pro Gly Asp Pro Thr Ser Asn 200 205 210 Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro Pro Ser Met 215 220 225 Pro Ala Val Ala Gly Ala Ala Gly Gly Leu Ala Leu Leu Leu Leu 230 235 240 Gly Val Ala Gly Ala Gly Gly Ala Met Cys Trp Arg Arg Arg Arg 245 250 255 Ala Lys Pro Ser Glu Ser Arg His Pro Gly Pro Gly Ser Phe Gly 260 265 270 Arg Gly Gly Ser Leu Gly Leu Gly Gly Gly Gly Gly Met Gly Pro 275 280 285 Arg Glu Ala Glu Pro Gly Glu Leu Gly He Ala Leu Arg Gly Gly 290 295 300 Gly Ala Ala Asp Pro Pro Phe Cys Pro His Tyr Glu Lys Val Ser 305 310 315 Gly Asp Tyr Gly His Pro Val Tyr He Val Gin Asp Gly Pro Pro 320 325 330 Gin Ser Pro Pro Asn He Tyr Tyr Lys Val 335 340 (2) INFORMATION FOR SEQ ID NO: 5 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 454 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCCGACGCTG TGAGGCACCC CCTGCCCCAA ACCTCCTTCT CACTTGTGAT 50 CGCCCAGACC TGGATCTCCG CTTCACCATC AAGTTCCAGG AGTATAGCCC 100 TAATCTCTGG GGCCACGAGT TCCGCTCGCA CCACGATTAC TACATCATTG 150 CCACATCGGA TGGGACCCGG GAGGCCTGGG AGAGCCTGCA GGGAAGTGTG 200 TGCCTAACCA GAGGCATGAA GGTGCTTCTC CGAGTNGGAC AAAGTCCCGA 250 GGAGGGGCTG TCCCCCGAAA ACCTGTGTCT GAAATGCCCA TGGAAAGAGA 300 CCGAGGGGCA GCCCACAGCC TGGGAGCCTG GGGAAGGAGA ACCTGCCAGG 350 TGACCCCACC AGCAATNCAA CCTTCCGGGG TTGCTTGAAG GGCCCCTTGA 400 CCCTTTCCCA GCATTGCNTG CANTTGGTTN GGGGCAGCAN GGGGGNGTTT 450 TGGC 454 (2) INFORMATION FOR SEQ ID NO:6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 : Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : GGACAAAGTC CCGAGGAGGG GCTGTCCCCC GAAAACCTGT GTCTGAAATG 50 CCCATGGAAA 60 (2) INFORMATION FOR SEQ ID NO:8 : li) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : CAGGTTCTCC TTCCCCAGGC TCCCAGGCTG TGGGCTGCCC CTCGGTCTCT 50 TTCCATGGGC 60

Claims: What is claimed is: 1 An isolated nucleic acid, which encodes a polypeptide havmg an amino acid sequence that is at least 75% identical to the ammo acid sequence for mature human AL-2 shown in Figure 1 A- 1 B or Figure 2 A- 2B 2 The isolated nucleic acid according to claim I. which encodes a polypeptide havmg an amino acid sequence that is at least 85% identical to the amino acid sequence for mature human AL-2 shown in Figure lA-lB or Fιgure 2A-2B 3 The isolated nucleic acid of claim 2, comprising a nucleotide sequence encodmg the ammo acid sequence shown in Figure 1 A- IB for mature AL-21 4 The isolated nucleic acid of claim 2, comprising a nucleotide sequence encodmg the ammo acid sequence shown in Figure 2A-2B for mature AL-2s 5 The isolated nucleic acid of claim 2, which encodes a polypeptide havmg an ammo acid sequence that is at least 75% homologous to the ammo acid sequence ofthe extracellular domam of AL-2 shown in Figure 1A-1 B 6 The isolated nucleic acid of claim 5. which encodes a polypeptide having the amino acid sequence ofthe extracellular domain shown in Figure 1A-1 B for AL-2 7 The isolated nucleic acid of claim 1, wherem AL-2 is joined to an immunoglobulin 8 The isolated nucleic acid of claim 7, which encodes AL-2-IgG 9 The isolated nucleic acid of claim 1 , wherem AL-2 is fused to a tag polypeptide 10 The isolated nucleic acid of claim 1 , which hybridizes to DNA encodmg mature human AL-21 of Figure 1 A- IB or mature human AL-2s of Figure 2A-2B under stringent conditions, and which encodes a polypeptide that is antigenically cross-reactive to mature human AL-2s or AL-21 1 1 An expression vector comprising the nucleotide sequence of claim 1 operably linked to a promoter 12 The expression vector of claim 1 1, wherein the nucleotide sequence encodes the ammo acid sequence for mature AL-2 shown in Figure 1 A- IB or Figure 2A-2B 13 The expression vector of claim 12, wherem the nucleotide sequence encoding the ammo acid sequence for mature AL-2 is that shown in Figure 1 A- I B or Figure 2A-2B 14 A host cell transformed with the expression vector of claim 1 1 15 The host cell of claim 14, wherem the nucleotide sequence encodes the ammo acid sequence for mature AL-2 shown in Figure 1 A- IB or Figure 2A-2B 16 A method of using the host cell of claim 14, which method comprises culturing the host cell under conditions that allow replication ofthe expression vector 17 A process which comprises transforming a host cell with an expression vector capable, in the host cell transformed with the vector of expressing a nucleotide sequence that encodes a polypeptide compπsmg the amino acid sequence shown in Figure I A- I B or Figure 2A-2B for mature AL-2 and culturing the transformed host cell under conditions such that the AL-2 polypeptide is synthesized 18 An isolated polypeptide having an amino acid sequence that is at least 75% homologous to the mature human AL-2 amino acid sequence shown in Figure 1 A- I B or Figure 2A-2B 19 The isolated polypeptide of claim 18 having an am o acid sequence that is at least 85% homologous to the mature human AL-2 ammo acid sequence shown in Figure 1 A- I B or Figure 2A-2B 20. The isolated polypeptide of claim 19 having the mature human AL-2 ammo acid sequence shown in Figure 1 A- IB or Figure 2A-2B 21. The isolated polypeptide of claim 18 having an amino acid sequence that is at least 75% homologous to the ammo acid sequence ofthe extracellular domain shown in Figure 1 A- 1 B for mature human AL-2 22. The isolated polypeptide of claim 21 having the ammo acid sequence of the extracellular domain shown in Figure 1 A- 1 B for AL-2 23. The isolated polypeptide of claim 18, wherein AL-2 is joined to an immunoglobulm 24 The polypeptide of claim 23, wherein the AL-2 extracellular domain is joined to an immunoglobulin constant domain. 25. The polypeptide of claim 24, wherem the constant domam is that of an immunoglobulin heavy chain. 26. The polypeptide of claim 23 that is AL-2-IgG. 27 The polypeptide of claim 18, wherein AL-2 is fused to a tag polypeptide. 28. A pharmaceutical composition comprising the polypeptide of claim 18 and a physiologically acceptable carrier. 29. An antibody that specifically binds to a polypeptide having the amino acid sequence shown in Figure 1 A- 1 B for mature AL-2. 30. The antibody of claim 29 that is a monoclonal antibody. 31. A method for activating a tyrosine kinase domain of an AL-2-binding Eph-family receptor, comprising contacting an extracellular domain ofthe receptor with the AL-2 of claim 1 32. A method of treating a neurologic disease or disorder in a mammal, compπs g administering to the mammal a therapeutically effective amount ofthe composition of claim 28. 33. The method of claim 32 wherein the neurologic disease or disorder is trauma-induced, surgery- induced, stroke-induced, ischemia-induced, infection-induced, metabolic disease-related, nutπtional deficiency- induced, malignancy-induced, neurotoxicity, Alzheimer's disease, amyotrophic lateral sclerosis. Bell's palsy, spinal muscular atrophy or paralysis, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's chorea, Down's Syndrome, nerve deafness, Meniere's disease, post-polio syndrome, Charcot-Marie-Tooth disease, Refsum's disease, Abetalipoproteinemia. Tangier disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry's disease, and Dejeπne-Sottas syndrome. 34. The method of claim 32 that further comprises administering a therapeutically effective amount of a second neurotrophic factor. 35 A method for accelerating the neovasculanzation of a wound, comprising applying to the wound an angiogenically effective amount ofthe composition of claim 28. 36 A method of modulating angiogenesis associated with a disease condition in a mammal, comprising administering to the mammal an angiogenically- odulating amount of an AL-2 antagonist 3 The method of claim 36. wherem the angiogenesis-associated disease condition is rheumatoid arthritis or tumor formation 38 A method of diagnosmg a neurologic disease or disorder, comprising contacting nucleic acid of a sample with a second nucleic acid comprising at least 10 nucleotides ofthe nucleotide sequence shown in Figures 1 A- 1 B or 2 A-2B under conditions that al low hybndization of complementary nucleotide sequences, and detecting any hybridization that occurs 39 The method of claim 38, further compnsing amplifying the sample nucleic acid to which the second nucleic acid hybridizes

Inventors: CARAS INGRID W, CARAS, INGRID, W.
Applicants: GENENTECH INC, GENENTECH, INC.
Application year: 1997
Main classification: C12N15
IPC codes: A61K38, C07K14, C07K16, C07K19, C12N5, C12N15, C12P21, C12Q1