AU749312B2 - Novel modified tie-2 receptor ligands - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Regeneron Pharmaceuticals, Inc.

Actual Inventor(s): SAMUEL DAVIS, GEORGE D. YANCOPOULOS Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: NOVEL MODIFIED TIE-2 RECEPTOR LIGANDS Our Ref: 626913 POF Code: 1271/134671 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): NOVEL MODIFIED TIE-2 RECEPTOR LIGANDS

INTRODUCTION

The present application is a divisional application from Australian patent application number 724032 (39687/97) the entire disclosure of which is incorporated herein by reference.

The present invention relates generally to the field of genetic engineering and more particularly to genes for receptor tyrosine kinases and their cognate ligands, their insertion into recombinant DNA vectors, and the production of the encoded proteins in recipient strains of microorganisms and recipient eukaryotic cells. More specifically, the present invention is directed to a novel modified TIE-2 ligand that binds the TIE-2 receptor, as S: well as to methods of making and using the modified ligand. The invention 15 further provides a nucleic acid sequence encoding the modified ligand, and methods for the generation of nucleic acid encoding the modified ligand and the gene product. The modified TIE-2 ligand, as well as nucleic acid encoding it, may be useful in the diagnosis and treatment of certain diseases involving endothelial cells and associated TIE receptors, such as neoplastic 20 diseases involving tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis and inflammatory diseases. In addition, the modified ligand may be used to promote the proliferation and/or differentiation of hematopoietic stem cells.

More generally, the receptor activating modified TIE-2 ligands described herein may be used to promote the growth, survival, migration, and/or differentiation and/or stabilization or destabilization of cells expressing TIE receptor. Biologically active modified TIE-2 ligand may be used for the in vitro maintenance of TIE receptor expressing cells in culture. Cells and tissues expressing TIE receptor include, for example, cardiac and vascular endothelial cells, lens epithelium and heart epicardium and early hematopoietic cells.

Alternatively, such human ligand may be used to support cells which S1 o are engineered to express TIE receptor. Further, rodified TIE-2 ligand and its cognate receptor may be used in assay systems to identify further agonists or antagonists of the receptor.

BACKGROUND OF THE INVENTION The cellular behavior responsible for the development, maintenance, and repair of differentiated cells and tissues is regulated, in large part, by intercellular signals conveyed via growth factors and similar ligands and their receptors. The receptors are located on the ceil surface of responding cells and they bind peptides or polypeptides known as growth factors as well as other hormone-like ligands. The results of this interaction are rapid biochemical changes in the responding cells, as well as a rapid and a long-term readjustment of cellular gene expression. Several receptors associated with various cell surfaces may bind specific growth factors.

The phosphorylation of tyrosine residues in proteins by tyrosine kinases is one of the key modes by which signals are transduced across the plasma membrane. Several currently known protein tyrosine kinase genes encode transmembrane receptors for polypeptide growth factors and hormones such as epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), platelet derived growth factors (PDGF-A and and fibroblast growth factors (FGFs). (Heldin et al., Cell Regulation, 1: 555-566 (1990); Ullrich, et al., Cell, 61: 243-54 (1990)). In each instance, these growth factors exert their action by binding to the extracellular portion of their cognate receptors, which leads to activation of the intrinsic tyrosine kinase present on the 1 o cytoplasmic portion of the receptor. Growth factor receptors of endothelial cells are of particular interest due to the possible involvement of growth factors in several important physiological and pathological processes, such as vasculogenesis, angiogenesis, atherosclerosis, and inflammatory diseases. (Folkman, et al. Science, 15 235: 442-447 (1987)). Also, the receptors of several hematopoietic growth factors are tyrosine kinases; these include c-fms, which is the colony stimulating factor 1 receptor, Sherr, et al., Cell, 41: 665-676 (1985), and c-kit, a primitive hematopoietic growth factor receptor reported in Huang, et al., Cell, 63: 225-33 (1990).

20 The receptor tyrosine kinases have been divided into evolutionary subfamilies based on the characteristic structure of their ectodomains. (Ullrich, et al. Cell, 61: 243-54 (1990)). Such subfamilies include, EGF receptor-like kinase (subclass I) and insulin receptor-like kinase (subclass II), each of which contains repeated homologous cysteine-rich sequences in their extracellular domains. A single cysteine-rich region is also found in the extracellular domains of the eph-like kinases. Hirai, et al., Science, 238: 1717-1720 (1987); Lindberg, et al. Mol. Cell. Biol., 10: 6316-24 (1990); Lhotak, et al., Mol.

Cell. Biol. 11: 2496-2502 (1991). PDGF receptors as well as c-fms and c-kit receptor tyrosine kinases may be grouped into subclass IIl; while the FGF receptors form subclass IV. Typical for the members of both of these subclasses are extracellular folding units stabilized by intrachain disulfide bonds. These so-called immunoglobulin (Ig)-like folds are found in the proteins of the immunoglobulin superfamily which contains a wide variety of other cell surface receptors having either cell-bound or soluble ligands. Williams, et al., Ann. Rev.

Immunol., 6: 381-405 (1988).

10 Receptor tyrosine kinases differ in theirS specificity and affinity.

In. general, receptor tyrosine kinases are glycoproteins which consist of an extracellular domain capable of binding the specific growth factor(s); a transmembrane domain which usually is an alphahelical portion of the protein; a juxtamembrane domain where the 1 5 receptor may be regulated by, protein phosphorylation; a tyrosine kinase domain which is the enzymatic component of the receptor; and a carboxyterminal tail which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.

20 Processes such as alternative exon splicing and alternative choice of gene promoter or polyadenylation sites have been reported to be capable of producing several distinct polypeptides from the same gene. These polypeptides may or may not contain the various domains listed above. As a consequence, some extracellular domains may be expressed as separate, secreted proteins and some forms of the receptors may lack the tyrosine kinase domain and contain only the extracellular domain inserted in the plasma membrane via the transmembrane domain plus a short carboxyl terminal tail.

A gene encoding an endothelial cell transmembrane tyrosine kinase, originally identified by RT-PCR as an unknown tyrosine kinasehomologous cDNA fragment from human leukemia cells, was described by Partanen, et al., Proc. Natl. Acad. Sci. USA, 87: 8913-8917 (1990).

This gene and its encoded protein are called "TIE" which is an abbreviation for "tyrosine kinase with Ig and EGF homology domains." Partanen, et al. Mol. Cell. Biol. 12: 1698-1707 (1992).

It has been reported that tie mRNA is present in all human fetal and mouse embryonic tissues. Upon inspection, tie message has been 1 o localized to the cardiac and vascular endothelial cells. Specifically, tie mRNA has been localized to the endothelia of blood vessels and endocardium of 9.5 to 18.5 day old mouse embryos. Enhanced tie expression was shown during neovascularization associated with developing ovarian follicles and granulation tissue in skin wounds.

1 5 Korhonen, et al. Blood 80: 2548-2555 (1992). Thus the TIEs have been suggested to play a role in angiogenesis, which is important for developing treatments for solid tumors and several other S angiogenesis-dependent diseases such as diabetic retinopathy, psoriasis, atherosclerosis and arthritis.

20 Two structurally related rat TIE receptor proteins have been reported to be encoded by distinct genes with related profiles of expression. One gene, termed tie-1, is the rat homolog of human tie.

Maisonpierre, et al., Oncogene 8: 1631-1637 (1993). The other gene, tie-2, may be the rat homolog of the murine tek gene, which, like tie, has been reported to be expressed in the mouse exclusively in endothelial cells and their presumptive progenitors. Dumont, et al.

Oncogene 8: 1293-1301 (1993). The human homolog of tie-2 is described in Ziegler, U.S. Patent No. 5,447,860 which issued on September 5, 1995 (wherein it is referred to as which is incorporated in its entirety herein.

Both genes were found to be widely expressed in endothelial cells of embryonic and postnatal tissues. Significant levels of tie-2 transcripts were also present in other embryonic cell populations, including lens epithelium, heart epicardium and regions of mesenchyme. Maisonpierre, et al., Oncogene 8: 1631-1637 (1993).

The predominant expression of the TIE receptor in vascular endothelia suggests that TIE plays a role in the development and t lo maintenance of the vascular system. This coulq include roles in Sendothelial cell determination, proliferation, differentiation and cell migration and patterning into vascular elements. Analyses of mouse embryos deficient in TIE-2 illustrate its importance in angiogenesis, particularly for vascular network formation in endothelial cells. Sato, et al., Nature 376:70-74 (1995). In the mature vascular system, the TIEs could function in endothelial ceil survival, maintenance and response to pathogenic influences.

The TIE receptors are also expressed in primitive hematopoietic stem cells, B cells and a subset of megakaryocytic cells, thus 20 suggesting the role of ligands which bind these receptors in early hematopoiesis, in the differentiation and/or proliferation of B cells, and in the megakaryocytic differentiation pathway. Iwama, et al.

Biochem. Biophys. Research Communications 195:301-309 (1993); Hashiyama, et al. Blood 87:93-101 (1996), Batard, et al. Blood 87:2212-2220 (1996).

SUMMARY OF THE INVENTION The present invention provides for a composition comprising a modified T1E-2 ligand substantially free of other proteins. As used herein, modified TIE-2 ligand refers to a ligand of the TIE family of ligands, whose representatives comprise ligands TL1, TL2, TL3 and TL4 as described herein, which has been altered by addition, deletion or substitution of one or more amino acids, or by way of tagging, with for example, the Fc portion of human IgG-1, but which retains its ability to bind the TIE-2 receptor. Modified TIE-2 ligand also includes a chimeric TIE-2 ligand comprising at least a portion of a first TIE-2 ligand and a portion of a second TIE-2 ligand which is different from 1 o the first. By way of non-limiting example, the 4irst TIE-2 ligand is TL1, and the second TIE-2 ligand is TL2. The invention envisions other combinations using additional TIE-2 ligand family members. For example, other combinations for creating a chimeric TIE-2 ligand are possible, including but not limited to those combinations wherein the 15 first ligand is selected from the group consisting of TL1, TL2, TL3 and TL4, and the second ligand, different from the first ligand, is selected from the group consisting of TL1, TL2, TL3 and TL4.

The invention also provides for an isolated nucleic acid molecule 2o encoding a modified TIE-2 ligand. In one embodiment, the isolated i nucleic acid molecule encodes a TIE-2 ligand of the TIE family of ligands, whose representatives comprise ligands TL1, TL2, TL3 and TL4 as described herein, which has been altered by addition, deletion or substitution of one or more amino acids, or by way of tagging, with for example, the Fc portion of human IgG-1, but which retains its ability to bind the TIE-2 receptor. In another embodiment, the isolated nucleic acid molecule encodes a modified TIE-2 ligand which is a chimeric TIE-2 ligand comprising at least a portion of a first TIE-2 ligand and a portion of a second TIE-2 ligand which is different from the first. By way of non-limiting example, the first TIE-2 ligand is TL1 and the second TIE-2 ligand is TL2. The invention envisions other combinations using additional TIE-2 ligand family members. For example, other combinations are possible, including but not limited to those combinations wherein the isolated nucleic acid molecule encodes a modified TIE-2 ligand which is a chimeric TIE-2 ligand comprising a portion of a first ligand selected from the group consisting of TL1, TL2, TL3 and TL4, and a portion of a second ligand, different from the.

1 0 first ligand, selected from the group consisting of TL1, TL2, TL3 and TL4.

The isolated nucleic acid may be DNA, cDNA or RNA. The invention also provides for a vector comprising an isolated nucleic acid molecule 1 5 encoding a modified TIE-2 ligand. The invention further provides for a host-vector system for the production in a suitable host cell of a polypeptide having the biological activity of a modified TIE-2 ligand.

The suitable host cell may be bacterial, yeast, insect or mammalian.

The invention also provides for a method of producing a polypeptide having the biological activity of a modified TIE-2 ligand which comprises growing cells of the host-vector system under conditions permitting production of the polypeptide and. recovering the polypeptide so produced.

The invention herein described of an isolated nucleic acid molecule encoding a modified TIE-2 ligand further provides for the development of the ligand as a therapeutic for the treatment of patients suffering from disorders involving cells, tissues or organs which express the TIE-2 receptor. The present invention also provides for an antibody which specifically binds such a therapeutic molecule.

The antibody may be monoclonal or polyclonal. The invention also provides for a method of using such a monoclonal or polyclonal antibody to measure the amount of the therapeutic molecule in a sample taken from a patient for purposes of monitoring the course of therapy.

The present invention also provides for an antibody which specifically binds a modified TIE-2 ligand as described herein. The antibody may be monoclonal Opoyclonal. Thus the invention further i 10 provides for therapeutic compositions comprising an antibody which specifically binds a modified TIE-2 ligand, in a pharmaceutically acceptable vehicle. The invention also provides for a method of S• blocking blood vessel growth in a mammal by administering an effective amount of a therapeutic composition comprising an antibody 1 5 which specifically binds a receptor activating modified T1E-2 ligand as described herein, in a pharmaceutically acceptable vehicle.

The invention further provides for therapeutic compositions comprising a modified TIE-2 ligand as described herein, in a pharmaceutically acceptable vehicle. The invention also provides fcr 20 method of promoting neovascularization in a patient by adminiserinc an effective amount of a therapeutic composition comprising a receptor activating modified TIE-2 ligand as described herein, in a pharmaceutically acceptable vehicle. In one embodiment, the method may be used to promote wound healing. In another embodiment, the method may be used to treat ischemia. In yet another embodimen:. a receptor activating modified TIE-2 ligand as described herein is used.

alone or in combination with other hematopoietic factors, to promote the proliferation or differentiation of hematopoietic stem cells, B cells or megakaryocytic cells.

Alternatively, the invention provides that a modified TIE-2 ligand may be conjugated to a cytotoxic agent and a therapeutic composition prepared therefrom. The invention further provides for a receptorbody which specifically binds a modified TIE-2 ligand. The invention further provides for therapeutic compositions comprising a receptorbody which specifically binds a modified TIE-2 ligand in a pharmaceutically acceptable vehicle. The invention also provides for a method of blocking blood vessel growth in a mammal by administering 1 o an effective amount of a therapeutic composition comprising a receptorbody which specifically binds a modified TIE-2 ligand in a pharmaceutically acceptable vehicle.

The invention also provides for a TIE-2 receptor antagonist as well as a method of inhibiting TIE-2 biological activity in a mammal comprising administering to the mammal an effective amount of a TIE- 2 antagonist. According to the invention, the antagonist may be a modified TIE-2 ligand as described herein which binds to, but does not activate, the TIE-2 receptor.

i' BRIEF DESCRIPTION OF THE FIGURES FIGURES 1A and 1B TIE-2 receptorbody (TIE-2 RB) inhibits the development of blood vessels in the embryonic chicken chorioallantoic membrane (CAM). A single piece of resorbable gelatin foam (Gelfoam) soaked with 6 Iig of RB was inserted immediately under the CAM of 1day chick embryos. After 3 further days of incubation, 4 day old embryos and surrounding CAM were removed and examined. FIGURE 1A: embryos treated with EHK-1 RB (rEHK-1 ecto/hlgG1 Fc) were viable and possessed normally developed blood vessels in their surrounding CAM. FIGURE 1B all embryos treated with TIE-2 RB (r TIE-2 ecto h IgG1 Fc) were dead, diminished in size and were almost completely devoid of surrounding blood vessels.

FIGURE 2 Vector pJFE14.

FIGURE 3 Restriction map of Xgtl0.

FIGURE 4 Nucleic acid and deduced amino acid (single letter code) sequences of human TIE-2 ligand 1 from clone Xgt10 encoding htie-2 S. ligand 1.

:1s5 FIGURE 5 Nucleic acid and deduced amino acid (single letter code) sequences of human TIE-2 ligand 1 from T98G clone.

FIGURE 6 Nucleic acid and deduced amino acid (single letter code) Ssequences of human TIE-2 ligand 2 from clone pBluescript KS encoding human TIE 2 ligand 2.

o.

FIGURE 7 Western blot showing activation of TIE-2 receptor by TIE-2 ligand 1 (Lane L1) but not by TIE-2 ligand 2 (Lane L2) or control (Mock).

FIGURE 8 Western blot showing that prior treatment of HAEC cells with excess TIE-2 ligand 2 (Lane 2) antagonizes the subsequent ability of dilute TIE-2 ligand 1 to activate the T1E-2 receptor (TIE2-R) as compared with prior treatment of HAEC cells with MOCK medium (Lane FIGURE 9 Western blot demonstrating the ability of TL2 to competitively inhibit TL1 activation of the TIE-2 receptor using the human cell hybrid line, EA.hy926.

FIGURE 10 Histogram representation of binding to rat TIE-2 IgG immobilized surface by TIE-2 ligand in C2C12 ras, Rat2 ras, SHEP, and T98G concentrated (10x) conditioned medium. Rat TIE-2 (rTIE2) specific binding is demonstrated by the significant reduction in the binding activity in the presence of 25 tig/ml soluble rat TIE-2 RB as compared to a minor reduction in the presence of soluble trkB RB.

i FIGURE 11 Binding of recombinant human TIE-2 ligand 1 (hTL1) and 1 5 human TIE-2 ligand 2 (hTL2), in COS cell supernatants, to a human TIE- 2 receptorbody (RB) immobilized surface. Human TIE-2-specific binding was determined by incubating the samples with 25 pg/ml of either soluble human TIE-2 RB or trkB RB; significant reduction in the binding activity is observed only for the samples incubated with human 20 TIE-2 RB.

FIGURE 12 Western blot showing that TIE-2 receptorbody (denoted TIE-2 RB or, as here, TIE2-Fc) blocks the activation of TIE-2 receptors by TIE-2 ligand 1 (TL1) in HUVEC cells, whereas an unrelated receptorbody (TRKB-Fc) does not block this activation.

FIGURE 13 Agarose gels showing serial dilutions [undiluted to 10-4] of the TL1 and TL2 RT-PCR products obtained from E14.5 mouse fetal liver (Lanes 1- total, Lanes 3- stromal enriched, and Lanes 4- ckit+TER119 hematopoietic precursor cells) and E14.5 mouse fetal thymus (Lanes 2- total).

FIGURE 14 Agarose gels showing serial dilutions [undiluted to 10-3]of the TL1 and TL2 RT-PCR products obtained from E17.5 mouse fetal thymus cortical stromal cells (Lanes 1- CDR1+/A2B5-) and medullary stromal cells (Lane CDR1-/A2B5+).

0 FIGURE 15 A/schematic representation of the hypothesized role of the,,TIE-2/TIE ligands in angiogenesis. TL1 is represented by TL2 S. is represented by TIE-2 is represented by VEGF is represented by and flk-1 (a VEGF receptor) is represented by 5 FIGURE 16 In situ hybridization slides showing the temporal expression pattern of TIE-2, TL1, TL2, and VEGF during angiogenesis associated with follicular development and corpus luteum formation in the ovary of a rat that was treated with pregnant mare serum. Column .1 1: Early pre-ovulatory follicle; Column 2: pre-ovulatory follicle; Column 3: early corpus luteum; and Column 4: atretic follicle; Row A: bright field; Row B: VEGF; Row C: TL2; Row D: TL1 and Row E: TIE-2 receptor.

FIGURE 17 Comparison of amino acid sequences of mature TL1 protein and mature TL2 protein. The TL1 sequence is the same as that set forth in Figure 4, except that the putative leader sequence has been removed. Similarly, the TL2 sequence is the same as that set forth in Figure 6, except that the putative leader sequence has been removed.

Arrows indicate residues Arg49,. Cys245 and Arg264 of TL1, which correspond to the residues at amino acid positions 69, 265 and 284, respectively, of TL1 as set forth in Figure 4.

FIGURE 18 Western blot of the covalent multimeric structure of TL1 and TL2 (Panel A) and the interconversion of TL1 and TL2 by the mutation of one cysteine (Panel B).

FIGURE 19 A typical curve of TIE-2-lgG binding to immobilized TL1 1 0 in a quantitative cell-free binding assay.

FIGURE 20 A typical curve showing TIE-2 ligand 1 ligandbody comprising the fibrinogen-like domain of the ligand bound to the Fc S domain of IgG (TL1-fFc) binding to immobilized TIE-2 ectodomain in a 15 quantitative cell-free binding assay.

FIGURE 21 Nucleotide and deduced amino acid (single letter code) sequences of TIE ligand-3. The coding sequence starts at position 47.

;The fibrinogen-like domain starts at position 929.

FIGURE 22 Comparison of Amino Acid Sequences of TIE Ligand Family Members. mTL3 mouse TIE ligand-3; hTL1 human TIE-2 ligandl; chTL1 chicken TIE-2 ligandl; mTL1 mouse TIE-2 ligand 1; mTL2 mouse TIE-2 ligand 2; hTL2 human TIE-2 ligand 2. The boxed regions indicate conserved regions of homology among the family members.

FIGURE 23 Nucleotide and deduced amino acid (single letter code) sequences of TIE ligand-4. Arrow indicates nucleotide position 569.

FIGURE 24 Nucleotide and deduced amino acid (single letter code) sequences of chimeric TIE ligand designated 1N1C2F (chimera The putative leader sequence is encoded by nucleotides 1-60.

FIGURE 25 Nucleotide and deduced amino acid (single letter code) sequences of chimeric TIE ligand designated 2N2C1F (chimera The putative leader sequence is encoded by nucleotides 1-48.

0o FIGURE 26 Nycleotide and deduced amino acid (single letter code) sequences of chimeric TIE ligand designated 1N2C2F (chimera The putative leader sequence is encoded by nucleotides 1-60.

FIGURE 27 Nucleotide and deduced amino acid (single letter code) sequences of chimeric TIE ligand designated 2N1C1F (chimera The putative leader sequence is encoded by nucleotides 1-48.

DETAILED DESCRIPTION OF THE INVENTION As described in greater detail below, applicants have created novel modified TIE-2 ligands that bind the TIE-2 receptor. The present invention provides for a composition comprising a modified TIE-2 ligand substantially free of other proteins. As used herein, modified TIE-2 ligand refers to a ligand of the TIE family of ligands, whose representatives comprise ligands TL1, TL2, TL3 and TL4 as described herein, which has been altered by addition, deletion or substitution of one or more amino acids, or by way of tagging, with for example, the Fc portion of human IgG-1, but which retains its ability to bind the TIE-2 receptor. Modified TIE-2 ligand also includes a chimeric TIE-2 ligand comprising at least a portion of a first TIE-2 ligand and a portion of a second TIE-2 ligand which is different from the first. By way of non-limiting example, the first TIE-2 ligand is TL1 and the second TIE-2 ligand is TL2. The invention envisions other combinations using additional TIE-2 ligand family members. For example, other combinations for creating a chimeric TIE-2 ligand are possible, including but not limited to those combinations wherein the first ligand is selected from the group consisting of TL1, TL2, TL3 and TL4, and the second ligand, different from the first ligand, is selected from the group consisting of TL1, TL2, TL3 and TL4.

The invention also provides for an isolated nucleic acid molecule 1 5 encoding a modified TIE-2 ligand. In one embodiment, the isolated nucleic acid molecule encodes a TIE-2 ligand of the TIE family of ligands, whose representatives comprise ligands TL1, TL2, TL3 and TL4 as described herein, which has been altered by addition, deletion or substitution of one or more amino acids, or by way of tagging, with for 20 example, the Fc portion of human IgG-1, but which retains its ability to bind the TIE-2 receptor. In another embodiment, the isolated nucleic acid molecule encodes a modified TIE-2 ligand which is a chimeric TIE-2 ligand comprising at least a portion of a first TIE-2 ligand and a portion of a second TIE-2 ligand which is different from the first. By way of non-limiting example, the first TIE-2 ligand is TL1 and the second TIE-2 ligand is TL2. The invention envisions other combinations using additional TIE-2 ligand family members. For example, other combinations are possible, including but not limited to those combinations wherein the isolated nucleic acid molecule encodes a modified TIE-2 ligand which is a chimeric TIE-2 ligand comprising a portion of a first ligand selected from the group consisting of TL1, TL2, TL3 and TL4, and a portion of a second ligand, different from the first ligand, selected from the group consisting of TL1, TL2, TL3 and TL4.

The present invention comprises the modified TIE-2 ligands and their amino acid sequences, as well as functionally equivalent variants 0 thereof, as wellas proteins or peptides comprising substitutions, deletions or insertional mutants of the described sequences, which i bind TIE-2 receptor and act as agonists or antagonists thereof. Such variants include those in which amino acid residues are substituted for residues within the sequence resulting in a silent change. For 5 example, one or more amino acid residues within the sequence can be substituted by another amino acid(s) of a similar polarity which acts S as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members.of the class to which the amino acid belongs. For example, the class of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and Smethionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Also included within the scope of the invention are proteins or fragments or derivatives thereof which exhibit the same or similar biological activity as the modified TIE-2 ligands described herein, and derivatives which are differentially modified during or after translation, by glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Functionally equivalent molecules also include molecules that contain modifications, including N-terminal modifications, which result from expression in a particular recombinant host, such as, for .example, Nterminal methylation which occurs in certain bacterial E. coli) 1 o expression systems.

The present invention also encompasses the nucleotide sequences that encode the proteins described herein as modified TIE-2 ligands, as well as host cells, including yeast, bacteria, viruses, and mammalian cells, which are genetically engineered to produce the proteins, by e.g.

15 transfection, transduction, infection, electroporation, or microinjection of nucleic acid encoding the modified TIE-2 ligands described herein in a suitable expression vector. The present invention also encompasses introduction of the nucleic acid encoding modified TIE-2 ligands through gene therapy techniques such as is described, for 20 example, in Finkel and Epstein FASEB J. 9:843-851 (1995); Guzman, et al. PNAS (USA) 91:10732-10736 (1994).

One skilled in the art will also recognize that the present invention encompasses DNA and RNA sequences that hybridize to a modified TIE- 2 ligand encoding nucleotide sequence, under conditions of moderate stringency, as defined in, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 101-104, Cold Spring Harbor Laboratory Press (1989). Thus, a nucleic acid molecule contemplated by the invention includes one having a nucleotide sequence deduced from an amino acid sequence of a modified TIE-2 ligand prepared as described herein, as well as a molecule having a sequence of nucleotides that hybridizes to such a nucleotide sequence, and also a nucleotide sequence which is degenerate of the above sequences as a result of the genetic code, but which encodes a ligand that binds TIE-2 receptor and which has an amino acid sequence and other primary, secondary and tertiary characteristics that are sufficiently duplicative of a modified TIE-2 ligand described herein so o0 as to confer on,the molecule the same biological activity as the modified TIE-2 ligand described herein.

The present invention provides for an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds and activates TIE-2 5 receptor comprising a nucleotide sequence encoding TIE-2 ligand 1 wherein the portion of the nucleotide sequence that encodes the N- S terminal domain of TIE-2 ligand 1 is replaced by a nucleotide sequence that encodes the N-terminal domain of TIE-2 ligand 2. The invention also provides for such a nucleic acid molecule, with a further '020 modification such that the portion of the nucleotide sequence that encodes the coiled-coil domain of TIE-2 ligand 1 is replaced by a nucleotide sequence that encodes the coiled-coil domain of TIE-2 ligand 2.

The present invention also provides for an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds and activates TIE-2 receptor comprising a nucleotide sequence encoding TIE-2 ligand 1 wherein the portion of the nucleotide sequence that encodes the Nterminal domain of TIE-2 ligand 1 is replaced by a nucleotide sequence that encodes the N-terminal domain of TIE-2 ligand 2 and which is further modified to encode a different amino acid instead of the cysteine residue encoded by nucleotides 784-787 as set forth in Figure 27. A serine residue is preferably substituted for the cysteine residue. In another embodiment, the nucleic acid molecule is further modified to encode a different amino acid instead of the arginine residue encoded by nucleotides 199-201 as set forth in. Figure 27. A serine residue is preferably substituted for the arginine residue.

The present invention also provides for an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds and activates TIE-2 receptor comprising a nucleotide sequence encoding TIE-2 ligand 1 which is modified to encode a different amino acid instead of the 1 5 cysteine residue at amino acid position 245. A serine residue is preferably substituted for the cysteine residue.

The invention further provides for an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds but does not activate TIEo* 2 0 2 receptor comprising a nucleotide sequence encoding TIE-2 ligand 1 wherein the portion of the nucleotide sequence that encodes the Nterminal domain of TIE-2 ligand 1 is deleted.. The invention also provides for such a nucleic acid molecule further modified so that the portion of the nucleotide sequence that encodes the coiled-coil domain of TIE-2 ligand 1 is deleted and the portion encoding the fibrinogenlike domain is fused in-frame to a nucleotide sequence encoding a human immunoglobulin gamma-1 constant region (IgG1 Fc).

The invention further provides for an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds but does not activate TIE- 2 receptor comprising a nucleotide sequence encoding TIE-2 ligand 2 wherein the portion of the nucleotide sequence that encodes the Nterminal domain of TIE-2 ligand 2 is deleted. The invention also provides for such a nucleic acid molecule further modified so that the portion of the nucleotide sequence that encodes the coiled-coil domain of TIE-2 ligand 2 is deleted and the portion encoding the fibrinogenlike domain is fused in-frame to a nucleotide- sequence encoding a 0 human immunogiobulin gamma-1 constant region (IgG1 Fc).

The invention further provides for an isolated nucleic acid molecule encoding a modified TIE-2 ligand that binds but does not activate TIE- 2 receptor comprising a nucleotide sequence encoding TIE-2 ligand 1 .1 5 wherein the portion of the nucleotide sequence that encodes the fibrinogen-like domain of T1E-2 ligand 1 is replaced by a nucleotide sequence that encodes the fibrinogen-like domain of TIE-2 ligand 2.

The invention also provides for such a nucleic acid molecule further modified so that the portion of the nucleotide sequence that encodes the coiled-coil domain of TIE-2 ligand 1 is replaced by a nucleotide sequence that encodes the coiled-coil domain of TIE-2 ligand 2.

The invention further provides for a modified TIE-2 ligand encoded by any of nucleic acid molecules of the invention.

The present invention also provides for a chimeric TIE-2 ligand comprising at least a portion of a first TIE-2 ligand and a portion of a second TIE-2 ligand which is different from the first, wherein the first and second TIE-2 ligands are selected from the group consisting of T1E-2 Ligand-1, TIE-2 Ligand-2, TIE Ligand-3 and TIE Ligand-4.

Preferably, the chimeric TIE ligand comprises at least a portion of TIE-2 Ligand-1 and a portion of TIE-2 Ligand-2.

The invention also provides a nucleic acid molecule that encodes a chimeric TIE ligand as set forth in Figure 24, 25, 26, or 27. The invention also provides a chimeric TIE ligand as set forth in Figure 24, 26, or 27. The invention further provides a chimeric TIE ligand as 1 0 set forth in Figure 27, modified to have a different amino acid instead' of.the cysteine residue encoded by nucleotides 784-787.

Any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding a modified TIE-2 ligand using appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinations (genetic recombination). Expression of a nucleic acid sequence encoding a 20 modified TIE-2 ligand or peptide fragments thereof may be regulated by a second nucleic acid sequence which is operably linked to the a modified TIE-2 ligand encoding sequence such that the modified TIE-2 ligand protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a modified TIE- 2 ligand described herein may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control expression of the ligand include, but are not limited to the long terminal repeat as described in Squinto et al., (Cell 65:1-20 (1991)); the SV40 early promoter region (Bernoist and Chambon, Nature 290:304-310), the CMV promoter, the M-MuLV 5' terminal repeat, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:144- 1445 (1981)), the adenovirus promoter, the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the 3-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731 o (1978)), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. U.S.A.

80:21-25 (1983)), see also "Useful proteins from recombinant bacteria" in Scientific American, 242:74-94 (1980); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) 5 promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals; elastase I gene control region which is active in pancreatic acinar cells (Swift et ai., Cell 38:639- 646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399- 409 (1986); MacDonald, Hepatology 7:425-515 (1987); insulin gene control region which is active in pancreatic beta cells [Hanahan, Nature 315:115-122 (1985)]; immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel.

1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel.

1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic protein gene control region which is active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal mus9le (Shani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378). The invention further encompasses the production of antisense compounds which are capable of specifically hybridizing with a sequence of RNA 15 encoding a modified TIE-2 ligand to modulate its expression. Ecker, U.S. Patent No. 5,166,195, issued November 24, 1992.

Thus, according to the invention, expression vectors capable of being replicated in a bacterial or eukaryotic host comprising a nucleic acid encoding a modified TIE-2 ligand as described herein, are used to S 20 transfect a host and thereby direct expression of such nucleic acid to produce a modified TIE-2 ligand, which may then be recovered in a biologically active form. As used herein, a biologically active form includes a form capable of binding to TIE receptor and causing a biological response such as a differentiated function or influencing the phenotype of the cell expressing the receptor. Such biologically active forms could, for example, induce phosphorylation of the tyrosine kinase domain of TIE receptor. Alternatively, the biological activity may be an effect as an antagonist to the TIE receptor. In alternative embodiments, the active form of. a modified TIE-2 ligand is one that can recognize TIE receptor and thereby act as a targeting agent for the receptor for use in both diagnostics and therapeutics. In accordance with such embodiments, the active form need not confer upon any TIE expressing cell any change in phenotype.

Expression vectors containing the gene inserts can be identified by four general approaches: DNA-DNA hybridization, presence or absence of "marker" gene functions, expression of inserted sequences and PCR detection. In the first approach, the presence of: o0 a foreign gene ipserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are *sees: homologous to an inserted modified TIE-2 ligand encoding gene. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" 5 gene functions thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if a nucleic acid encoding a modified TIE-2 ligand .4 is inserted within the marker gene sequence of the vector, recombinants containing the insert can be identified by the absence of

V.

0 the marker gene function. In the third approach, recombinant S. expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of a modified TIE-2 ligand gene product, for example, by binding of the ligand to TIE receptor or a portion thereof which may be tagged with, for example, a detectable antibody or portion thereof or by binding to antibodies produced against the modified TIE-2 ligand protein or a portion thereof. Cells of the present invention may transiently or, preferably, constitutively and permanently express a modified TIE-2 ligand as described herein. In the fourth approach, DNA nucleotide primers can be prepared corresponding to a tie specific DNA sequence. These primers could then be used to PCR a tie gene fragment. (PCR Protocols: A Guide To Methods and Applications, Edited by Michael A. Innis et al., Academic Press (1990)).

The. recombinant ligand may be purified by any technique which allows for the subsequent formation of a stable, biologically active 1o protein. Preferably, the ligand is secreted into the culture medium from, which it is recovered. Alternatively, the ligand may be recovered from cells either as soluble proteins or as inclusion bodies, from i' which it may be extracted quantitatively by 8M guanidinium hydrochloride and dialysis in accordance with well known methodology.

1 5 In order to further purify the ligand, affinity chromatography, conventional ion exchange chromatography, hydrophobic interaction o chromatography, reverse phase chromatography or gel filtration may be used.

0.0 20 In additional embodiments of the invention, as described in greater detail in the Examples, a modified TIE-2 ligand encoding gene may be used to inactivate or "knock out" an endogenous gene by homologous recombination, and thereby create a TIE ligand deficient cell, tissue, or animal. For example, and not by way of limitation, the recombinant TIE ligand-4 encoding gene may be engineered to contain an insertional mutation, for example the neo gene, which would inactivate the native TIE ligand-4 encoding gene. Such a construct, under the control of a suitable promoter, may be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, transduction, or injection. Cells containing the construct may then be selected by G418 resistance. Cells which lack an intact TIE ligand-4 encoding gene may then be identified, by Southern blotting, PCR detection, Northern blotting or assay of expression. Cells lacking an intact TIE ligand-4 encoding gene may then be fused to early embryo cells to generate transgenic animals deficient in such ligand. Such an animal may be used to define specific in vivo processes, normally dependent upon the ligand.

0o The present invention also provides for antibodies to a modified TIE-2 ligand described herein which are useful for detection of the ligand in, for example, diagnostic applications. For preparation of monoclonal antibodies directed toward a modified TIE-2 ligand, any technique which provides for the production of antibody molecules by 15 continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human Bcell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp. 77-96) and the like are within the scope of the present invention.

The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.

Human monoclonal antibodies may be made by any of numerous techniques known in the art Teng et al., 1983, Proc. Natl. Acad.

Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72- 79; Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad.

Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).

Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of a modified TIE-2 ligand described herein. For the production of antibody, various host animals, including but not limited to rabbits, mice and rats can be immunized by injection with a modified TIE-2 ligand, or a fragment or derivative thereof. Various adjuvants may be used to increase the 1 o immunological response, depending on the host species, and including but,.not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, S°keyhole limpet hemocyanins, dinitrophenol, and potentially useful 1 5 human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

A molecular clone of an antibody to a selected a modified TIE-2 :ligand epitope can be prepared by known techniques. Recombinant DNA methodology (see Maniatis et al., 1982, Molecular Cloning, A 20 Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.

The present invention provides for antibody molecules as well as fragments of such antibody molecules. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Antibody molecules may be purified by known techniques, immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.

The present invention further encompasses an immunoassay for measuring the amount of a modified TIE-2 ligand in a biological sample 1o by a) contacting the biological sample with at least one antibody which specifically binds a modified TIE-2 ligand so that the antibody forms a complex with any modified TIE-2 ligand present in the sample; and 5 b) measuring the amount of the complex and thereby measuring the amount of the modified TIE-2 ligand in the biological sample.

The invention further encompasses an assay for measuring the amount of TIE receptor in a biological sample by 20 a) contacting the biological sample with at least one ligand of the invention so that the ligand forms a complex with the TIE receptor; and b) measuring the amount of the complex and thereby measuring the amount of the TIE receptor in the biological sample.

The present invention also provides for the utilization of a modified TIE-2 ligand which activates the TIE-2 receptor as described herein, to support the survival and/or growth and/or migration and/or differentiation of TIE-2 receptor expressing cells. Thus, the ligand may be used as a supplement to support, for example, endothelial cells in culture.

Further, the creation by applicants of a modified TIE-2 ligand for the TIE-2 receptor enables the utilization of assay systems useful for the identification of agonists or antagonists of the TIE-2 receptor.

Such assay systems would be useful in identifying molecules capable of promoting or inhibiting angiogenesis. For example, in one embodiment, antagonists of the TIE-2 receptor may be identified as 110 test molecules that are capable of interfering with the interaction of the TIE-2 receptor with a modified TIE-2 ligand that binds the TIE-2 receptor. Such antagonists are identified by their ability to 1) block the binding of a biologically active modified TIE-2 ligand to the receptor as measured, for example, using BIAcore biosensor technology 1 5 (BIAcore; Pharmacia Biosensor, Piscataway, NJ); or 2) block the ability of a biologically active modified TIE-2 ligand to cause a biological response. Such biological responses include, but are not limited to, phosphorylation of the TIE receptor or downstream components of the S"TIE signal transduction pathway, or survival, growth or differentiation S 20 of TIE receptor bearing cells.

In one embodiment, cells engineered to express the TIE receptor, may be dependent for growth on the addition of a modified TIE-2 ligand. Such cells provide useful assay systems for identifying additional agonists of the TIE receptor, or antagonists capable of interfering with the activity of the modified TIE-2 ligand on such cells. Alternatively, autocrine cells, engineered to be capable of coexpressing both a modified TIE-2 ligand and receptor, may provide useful systems for assaying potential agonists or antagonists.

Therefore, the present invention provides for introduction of a TIE-2 receptor into cells that do not normally express this receptor, thus allowing these cells to exhibit profound and easily distinguishable responses to a ligand which binds this receptor. The s type of response elicited depends on the cell utilized, and not the specific receptor introduced into the cell. Appropriate cell lines can be chosen to yield a response of the greatest utility for assaying, as well as discovering, molecules that can act on tyrosine kinase receptors. The molecules may be any type of molecule, including but S o not limited to peptide and non-peptide molecules, that will act in systems to be described in a receptor specific manner.

One of the more useful systems to be exploited involves the introduction of a TIE receptor (or a chimeric receptor comprising the extracellular domain of another receptor tyrosine kinase such as, for 1 5 example, trkC and the intracellular domain of a TIE receptor) into a fibroblast cell line NIH3T3 cells) thus such a receptor which does not normally mediate proliferative or other responses can, following introduction into fibroblasts, nonetheless be assayed by a variety of well established methods to quantitate effects of fibroblast growth 20 factors thymidine incorporation or other types of proliferation assays; see van Zoelen, 1990, "The Use of Biological Assays For Detection Of Polypeptide Growth Factors" in Progress Factor Research, Vol. 2, pp. 131-152; Zhan and M. Goldfarb, 1986, Mol. Cell. Biol., Vol. 6, pp. 3541-3544). These assays have the added advantage that any preparation can be assayed both on the cell line having the introduced receptor as well as the parental cell line lacking the receptor; only specific effects on the cell line with the receptor would be judged as being mediated through the introduced receptor. Such cells may be further engineered to express a modified TIE-2 ligand, thus creating an autocrine system useful for assaying for molecules that act as antagonists/agonists of this interaction. Thus, the present invention provides for host cells comprising nucleic acid encoding a modified TIE-2 ligand and nucleic acid encoding TIE receptor.

The TIE receptor/modified TIE-2 ligand interaction also provides a useful system for identifying small molecule agonists or antagonists of the TIE. receptor. For example, fragments, mutants or derivatives of a modified TIE-2 ligand may be identified that bind TIE receptor but do 1 o not induce any.,other biological activity. Alternatively, the/ characterization of a modified TIE-2 ligand enables the further Scharacterization of active portions of the molecule. Further, the identification of a ligand enables the determination of the X-ray oo crystal structure of the receptor/ligand complex, thus enabling 1 5 identification of the binding site on the receptor. Knowledge of the binding site will provide useful insight into the rational design of novel agonists and antagonists.

The specific binding of a test molecule to TIE receptor may be measured in a number of ways. For example, the actual binding of test molecule to cells expressing TIE may be detected or measured, by detecting or measuring test molecule bound to the surface of intact cells; (ii) test molecule cross-linked to TIE protein in cell lysates; or (iii) test molecule bound to TIE in vitro. The specific interaction between test molecule and TIE may be evaluated by using reagents that demonstrate the unique properties of that interaction.

As a specific, nonlimiting example, the methods of the invention may be used as follows. Consider a case in which a modified TIE-2 ligand in a sample is to be measured. Varying dilutions of the sample i (the test molecule), in parallel with a negative control (NC) containing no modified TIE-2 ligand activity, and a positive control (PC) containing a known amount of a modified TIE-2 ligand, may be exposed to cells that express TIE in the presence of a detectably labeled modified TIE-2 ligand (in this example, radioiodinated ligand). The amount of modified TIE-2 ligand in the test sample may be evaluated by determining the amount of 1 2 5 1-labeled modified TIE-2 ligand that binds to the controls and in each of the dilutions, and then comparing the sample values to a standard curve. The more modified TIE-2 ligand.

4 o in the sample, the less 1 2 5 1-ligand that will bind to TIE.

The amount of 12 5 1-ligand bound may be determined by measuring the amount of radioactivity per cell, or by cross-linking a modified TIE-2 ligand to cell surface proteins using DSS, as described in Meakin and Shooter, 1991, Neuron 6:153-163, and detecting the amount of 1 s labeled protein in cell extracts using, for example, SDS polyacrylamide gel electrophoresis, which may reveal a labeled protein having a size corresponding to TIE receptor/modified TIE-2 ligand. The specific test molecule/TIE interaction may further be tested by adding to the assays various dilutions of an unlabeled control ligand that does not bind the TIE receptor and therefore should have no substantial effect on the competition between labeled modified TIE-2 ligand and test molecule for TIE binding. Alternatively, a molecule known to be able to disrupt TIE receptor/modified TIE-2 ligand binding, such as, but not limited to, anti-TIE antibody, or TIE receptorbody as described herein, may be expected to interfere with the competition between 12 5 1-modified TIE- 2 ligand and test molecule for TIE receptor binding.

Detectably labeled modified TIE-2 ligand includes, but is not limited to, a modified TIE-2 ligand linked covalently or noncovalently to a radioactive substance, a fluorescent substance, a substance that has enzymatic activity, a substance that may serve as a substrate for an enzyme (enzymes and substrates associated with colorimetrically detectable reactions are preferred) or to a substance that can be recognized by an antibody molecule that is preferably a detectably labeled antibody molecule.

Alternatively, the specific binding of test molecule to TIE may be measured by evaluating the secondary biological effects of a modified TIE-2 ligand/TIE receptor binding, including, but not limited to, cell growth /and/or differentiation or immediate early gene expression or phosphorylation of TIE. For example, the ability of the test molecule to induce differentiation can be tested in cells that lack tie and in comparable cells that express tie; differentiation in tieexpressing cells but not in comparable cells that lack tie would be indicative of a specific test molecule/TIE interaction. A similar analysis could be performed by detecting immediate early gene (e.g.

fos and jun) induction in tie-minus and tie-plus cells, or by detecting phosphorylation of TIE using standard phosphorylation assays known in the art. Such analysis might be useful in identifying agonists or 20 antagonists that do not competitively bind to TIE.

Similarly, the present invention provides for a method of identifying a molecule that has the biological activity of a modified TIE-2 ligand comprising exposing a cell that expresses tie to a test molecule and (ii) detecting the specific binding of the test molecule to TIE receptor, in which specific binding to TIE positively correlates with TIE-like activity. Specific binding may be detected by either assaying for direct binding or the secondary biological effects of binding, as discussed supra. Such a method may be particularly useful in identifying new members of the TIE ligand family or, in the pharmaceutical industry, in screening a large array of peptide and nonpeptide molecules peptidomimetics) for TIE associated biological activity. In a preferred, specific, nonlimiting embodiment of the invention, a large grid of culture wells may be prepared that contain, in alternate rows, PC12 (or fibroblasts, see infra) cells that are either tie-minus or engineered to be tie-plus. A variety of test molecules may then be added such that each column of the grid, or a portion thereof, contains a different test molecule. Each well could then be So scored for the presence or absence of growth and/or differentiation. An .extremely large number of test molecules could be screened for such activity in this manner.

In additional embodiments, the invention provides for methods of detecting or measuring TIE' ligand-like activity or identifying a 1 5 molecule as having such activity comprising exposing a test molecule to a TIE receptor protein in vitro under conditions that permit binding to occur and (ii) detecting binding of the test molecule to the TIE receptor protein, in which binding of test molecule to TIE receptor correlates with TIE ligand-like activity. According to such methods, the TIE receptor may or may not be substantially purified, may be affixed to a solid support as an affinity column or as an ELISA assay), or may be incorporated into an artificial membrane.

Binding of test molecule to TIE receptor may be evaluated by any method known in the art. In preferred embodiments, the binding of test molecule may be detected or measured by evaluating its ability to compete with detectably labeled known TIE ligands for TIE receptor binding.

The present invention also provides for a method of detecting the aoiiity OT a test molecule to Tunction as an antagonist oT I It tiganalike activity comprising detecting the ability of the molecule to inhibit an effect of TIE ligand binding to TIE receptor on a cell that expresses the receptor. Such an antagonist may or may not interfere with TIE receptor/modified TIE-2 ligand binding. Effects of a modified TIE-2 ligand binding to TIE receptor are preferably biological or biochemical effects, including, but not limited to, cell survival or proliferation, cell transformation, immediate early gene induction, or TIE phosphorylation.

1 The invention further provides for both a method of identifying antibodies or other molecules capable of neutralizing the ligand or blocking binding to the receptor, as well as the molecules identified by the method. By way of nonlimiting example, the method may be performed via an assay which is conceptually similar to an ELISA 5 assay. For example, TIE receptorbody may be bound to a solid support, such as a plastic multiwell plate. As a control, a known amount of a modified TIE-2 ligand which has been Myc-tagged may then be introduced to the well and any tagged modified TIE-2 ligand which binds the receptorbody may then be identified by means of a reporter antibody directed against the Myc-tag. This assay system may then be used to screen test samples for molecules which are capable of i) binding to the tagged ligand or ii) binding to the receptorbody and thereby blocking binding to the receptorbody by the tagged ligand. For example, a test sample containing a putative molecule of interest together with a known amount of tagged ligand may be introduced to the well and the amount of tagged ligand which binds to the receptorbody may be measured. By comparing the amount of bound tagged ligand in the test sample to the amount in the control, samples containing molecules which are capable of blocking ligand binding to the receptor may be identified. The molecules of interest thus identified may be isolated using methods well known to one of skill in the art.

Once a blocker of ligand binding is found, one of skill in the art would know to perform secondary assays to determine whether the blocker is binding to the receptor or to the ligand, as well as assays to determine if the blocker molecule can neutralize the biological activity of the ligand. For example, by using a binding assay which *o employs BIAcore biosensor technology (or the equivalent), in which either TIE receptorbody or a modified TIE-2 ligand or ligandbody is covalently attached to a solid support carboxymethyl dextran on a gold surface), one of skill in the art would be able to determine if the blocker molecule is binding specifically to the ligand, ligandbody or to 1 5 the receptorbody. To determine if the blocker molecule can neutralize the biological activity of the ligand, one of skill in the art could perform a phosphorylation assay (see Example 5) or alternatively, a functional bioassay, such as a survival assay, by using primary cultures of, for example, endothelial cells. Alternatively, a blocker o molecule which binds to the receptorbody could be an agonist and one of skill in the art would know to how to determine this by performing an appropriate assay for identifying additional agonists of the TIE receptor.

In addition, the invention further contemplates compositions wherein the TIE ligand is the receptor binding domain of a TIE-2 ligand described herein. For example, TIE-2 ligand 1 contains a "coiled coil" domain (beginning at the 5' end and extending to the nucleotide at about position 1160 of Figure 4 and about position 1157 of Figure and a fibrinogen-like domain (which is encoded by the nucleotide sequence of Figure 4 beginning at about position 1161 and about position 1158 of Figure The fibrinogen-like domain of TIE-2 ligand 2 is believed to begin on or around the same amino acid sequence as in ligand 1 (FRDCA) which is encoded by nucleotides beginning around 1197 of Figure 6. The fibrinogen-like domain of TIE ligand-3 is believed to begin on or around the amino acid sequence which is encoded by nucleotides beginning around position 929 as set forth in Figure 21. Multimerization of the coiled coil domains during 1 0 production of the ligand hampers purification. As described in Example 19,, Applicants have discovered, however, that the fibrinogen-like domain comprises the TIE-2 receptor binding domain. The monomeric forms of the fibrinogen-like domain do not, however, appear to bind the receptor. Studies utilizing myc-tagged fibrinogen-like domain, 15 which has been "clustered" using anti-myc antibodies, do bind the TIE- 2 receptor. [Methods of production of "clustered ligands and ligandbodies are described in Davis, et al. Science 266:816-819 (1994)]. Based on these finding, applicants produced "ligandbodies" which comprise the fibrinogen-like domain of the TIE-2 ligands 20 coupled to the Fc domain of IgG These ligandbodies, which form dimers, efficiently bind the TIE-2 receptor. Accordingly, the present invention contemplates the production of modified TIE ligandbodies which may be used as targeting agents, in diagnostics or in therapeutic applications, such as targeting agents for tumors and/or associated vasculature wherein a TIE antagonist is indicated.

The invention herein further provides for the development of the ligand, a fragment or derivative thereof, or another molecule which is a receptor agonist or antagonist, as a therapeutic for the treatment of patients suffering from disorders involving cells, tissues or organs which express the TIE receptor. Such molecules may be used in a method of treatment of the human or animal body, or in a method of diagnosis.

Because TIE receptor has been identified in association with endothelial cells and, as demonstrated herein, blocking of TIE-2 ligand 1 appears to prevent vascularization, applicants expect that a modified TIE-2 ligand described herein may be useful for the induction of vascularization in diseases or disorders where such vascularization Oo is indicated. Such diseases or disorders would include wound healing, ischaemia and diabetes. The ligands may be tested in animal models' and used therapeutically as described for other agents, such as vascular endothelial growth factor (VEGF), another endothelial cell- S: specific factor that is angiogenic. Ferrara, et al. U.S. Patent No.

5,332,671 issued July 26, 1994. The Ferrara reference, as well as other studies, describe in vitro and in vivo studies that may be used to demonstrate the effect of an angiogenic factor in enhancing blood flow to ischemic myocardium, enhancing wound healing, and in other therapeutic settings wherein neoangiogenesis is desired. [see Sudo, et al. European Patent Application 0 550 296 A2 published July 7, 1993; .i Banai, et al. Circulation 89:2183-2189 (1994); Unger, et al. Am. J.

Physiol. 266:H1588-H1595 (1994); Lazarous, et al. Circulation 91:145- 153 (1995)]. According to the invention, a modified TIE-2 ligand may be used alone or in combination with one or more additional pharmaceutically active compounds such as, for example, VEGF or basic fibroblast growth factor (bFGF), as well as cytokines, neurotrophins, etc.

Conversely, antagonists of the TIE receptor, such as modified TIE-2 ligands which bind but do not activate the receptor as described herein, receptorbodies as described herein in Examples 2 and 3, and TIE-2 ligand 2 as described in Example 9, would be useful to prevent or attenuate vascularization, thus preventing or attenuating, for example, tumor growth. These agents may be used alone or in combination with other compositions, such as anti-VEGF antibodies, that have been shown to be useful in treating conditions in which the therapeutic intent is to block angiogenesis. Applicants expect that a modified TIE-2 ligand described herein may also be used in combination with 1 o agents, such as cytokine antagonists such as IL-6 antagonists, that are.

J

0 known to block inflammation.

For example, applicants have determined that TIE ligands are expressed in cells within, or closely associated with, tumors. For example, TIE-2 ligand 2 appears to be tightly associated with tumor 1 5 endothelial cells. Accordingly, it and other TIE antagonists may also be useful in preventing or attenuating, for example, tumor growth. In addition, TIE ligands or ligandbodies may be useful for the delivery of toxins to a receptor bearing cell. Alternatively, other molecules, such as growth factors, cytokines or nutrients, may be delivered to a TIE receptor bearing cell via TIE ligands or ligandbodies. TIE ligands or ligandbodies such as modified TIE-2 ligand described herein may also Sbe used as diagnostic reagents for TIE receptor, to detect the receptor in vivo or in vitro. Where the TIE receptor is associated with a disease state, TIE ligands or ligandbodies such as a modified TIE-2 ligand may be useful as diagnostic reagents for detecting the disease by, for example, tissue staining or whole body imaging. Such reagents include radioisotopes, flurochromes, dyes, enzymes and biotin. Such diagnostics or targeting agents may be prepared as described in Alitalo, et al. WO 95/26364 published October 5, 1995 and Burrows, F.

and P. Thorpe, PNAS (USA) 90:8996-9000 (1993) which is incorporated herein in its entirety.

In other embodiments, the TIE ligands, a receptor activating modified TIE-2 ligand described herein are used as hematopoietic factors. A variety of hematopoietic factors and their receptors are involved in the proliferation and/or differentiation and/or migration of the various cells types contained within blood. Because the TIE receptors are expressed in early hematopoietic cells, the TIE ligands 1 o are expected to play a comparable role in the proliferation or S differentiation or migration of these cells. Thus, for example, TIE containing compositions may be prepared, assayed, examined in in S vitro and in vivo biological systems and used therapeutically as described in any of the following: Sousa, U.S. Patent No. 4,810,643, 1 5 Lee, et al., Proc. Natl. Acad. Sci. USA 82:4360-4364 (1985) Wong, et al.

S Science, 228:810-814 (1985); Yokota, et al. Proc. Natl. Acad. Sci (USA) o .o 81:1070 (1984); Bosselman, et al. WO 9105795 published May 2, 1991 entitled "Stem Cell Factor" and Kirkness, et al. WO 95/19985 published July 27, 1995 entitled "Haemopoietic Maturation Factor".

.20 Accordingly, receptor activating modified TIE-2 ligand may be used to diagnose or treat conditions in which normal hematopoiesis is suppressed, including, but not limited to anemia, thrombocytopenia, leukopenia and granulocytopenia. In a preferred embodiment, receptor activating modified TIE-2 ligand may be used to stimulate differentiation of blood cell precursors in situations where a patient has a disease, such as acquired immune deficiency syndrome (AIDS) which has caused a reduction in normal blood cell levels, or in clinical settings in which enhancement of hematopoietic populations is desired, such as in conjunction with bone marrow transplant, or in the treatment of aplasia or myelosuppression caused by radiation, chemical treatment or chemotherapy.

The receptor activating modified TIE-2 ligands of the present invention may be used alone, or in combination with another pharmaceutically active agent such as, for example, ctyokines, neurotrophins, interleukins, etc. In a preferred embodiment, the ligands may be used in conjunction with any of a number of the above referenced factors which are known to induce stem cell or other hematopoietic precursor proliferation, or factors acting on later cells in. the hematopoietic pathway, including, but not limited to, hemopoietic maturation factor, thrombopoietin, stem cell factor, erythropoietin, G-CSF, GM-CSF, etc.

In an alternative embodiment, TIE receptor antagonists are used 1 5 to diagnose or treat patients in which the desired result is inhibition of a hematopoietic pathway, such as for the treatment of myeloproliferative or other proliferative disorders of blood forming organs such as thrombocythemias, polycythemias and leukemias. In such embodiments, treatment may comprise use of a therapeutically effective amount of the a modified TIE-2 ligand, TIE antibody, TIE receptorbody, a conjugate of a modified TIE-2 ligand, or a ligandbody or fFC as described herein.

The present invention also provides for pharmaceutical compositions comprising a modified TIE-2 ligand or ligandbodies described herein, peptide fragments thereof, or derivatives in a pharmacologically acceptable vehicle. The modified TIE-2 ligand proteins, peptide fragments, or derivatives may be administered systemically or locally. Any appropriate mode of administration known in the art may be used, including, but not limited to, intravenous, intrathecal, intraarterial, intranasal, oral, subcutaneous, intraperitoneal, or by local injection or surgical implant. Sustained release formulations are also provided for.

The present invention also provides for an antibody which specifically binds such a therapeutic molecule. The antibody may be monoclonal or polyclonal. The invention also provides for a method of using such a monoclonal or polyclonal antibody to measure the amount of the therapeutic molecule in a sample taken from a patient for j0 purposes of monitoring the course of therapy.

The invention further provides for a therapeutic composition comprising a modified TIE-2 ligand or ligandbody and a cytotoxic agent conjugated thereto. In one embodiment, the cytotoxic agent may be a radioisotope or toxin.

1 5 The invention also provides for an antibody which specifically binds a modified TIE-2 ligand. The antibody may be monoclonal or polyclonal.

The invention further provides for a method of purifying a modified -T1E-2 ligand comprising: o a) coupling at least one TIE binding substrate to a solid matrix; b) incubating the substrate of a) with a cell lysate so that the substrate forms a complex with any modified TIE-2 ligand in the cell lysate; c) washing the solid matrix; and d) eluting the modified TIE-2 ligand from the coupled substrate.

i ne buustrate may oe any suosiance nat speciTicaiiy oinas tne modified T1E-2 ligand. In one embodiment, the substrate is selected from the group consisting of anti-modified TIE-2 ligand antibody, TIE receptor and TIE receptorbody. The invention further provides for a receptorbody which specifically binds a modified TIE-2 ligand, as well as a therapeutic composition comprising the receptorbody in a pharmaceutically acceptable vehicle, and a method of blocking blood vessel growth in a human comprising administering an effective amount of the therapeutic composition.

1 The invention also provides for a therapeutic composition S comprising a receptor activating modified TIE-2 ligand or ligandbody in a pharmaceutically acceptable vehicle, as well as a method of promoting neovascularization in a patient comprising administering to the patient an effective amount of the therapeutic composition.

1 5 In addition, the present invention provides for a method for identifying a cell which expresses TIE receptor which comprises contacting a cell with a detectably labeled modified TIE-2 ligand or ligandbody, under conditions permitting binding of the detectably labeled ligand to the TIE receptor and determining whether the detectably labeled ligand is bound to the TIE receptor, thereby identifying the cell as one which expresses TIE receptor. The present invention also provides for a therapeutic composition comprising a modified TIE-2 ligand or ligandbody and a cytotoxic agent- conjugated thereto. The cytotoxic agent may be a radioisotope or toxin.

The invention also provides a method of detecting expression of a modified TIE-2 ligand by a cell which comprises obtaining mRNA from the cell, contacting the mRNA so obtained with a labeled nucleic acid molecule encoding a modified TIE-2 ligand, under hybridizing conditions, determining the presence of mRNA hybridized to the labeled molecule, and thereby detecting the expression of a modified TIE-2 ligand in the cell.

The invention further provides a method of detecting expression of a modified TIE-2 ligand in tissue sections which comprises contacting the tissue sections with a labeled nucleic acid molecule encoding a modified TIE-2 ligand, under hybridizing conditions, determining the presence of mRNA hybridized to the labelled molecule, and thereby detecting the expression of a modified TIE-2 ligand in So tissue sections.

EXAMPLE 1 IDENTIFICATION OF THE ABAE CELL LINE AS REPORTER CELLS FOR THE TIE-2 RECEPTOR Adult BAE cells are registered in the European Cell Culture Repository, under ECACC#92010601. (See PNAS 75:2621 (1978)).

Northern (RNA) analyses revealed moderate levels of tie-2 transcripts in the ABAE (Adult Bovine Arterial Endothelial) cell line, consistent with in situ hybridization results that demonstrated almost exclusive localization of tie-2 RNAs to vascular endothelial cells. We therefore :.02 0 examined ABAE cell lysates for the presence of TIE-2 protein, as well as the extent to which this TIE-2 protein is tyrosine-phosphorylated under normal versus serum-deprived growth conditions. ABAE cell lysates were harvested and subjected to immunoprecipitation, followed by Western blot analyses of immunoprecipitated proteins with TIE-2 specific and phosphotyrosine-specific antisera. Omission or inclusion of TIE-2 peptides as specific blocking molecules during TIE-2 immunoprecipitation allowed unambiguous identification of TIE- 2 as a moderately detectable protein of -150 kD whose steady-state phosphotyrosine levels diminish to near undetectable levels by prior serum-starvation of the cells.

Culture of ABAE cells and harvest of cell lysates was done as follows. Low-passage-number ABAE cells were plated as a monolayer at a density of 2 x 106 cells/150mm plastic petri plate (Falcon) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing bovine calf serum (10 BCS), 2 mM L-glutamine and 1% each of penicillin and streptomycin in an atmosphere of 5% CO 2 Prior to harvest of cell lysates, cells were serum-starved for 24 hours in 1 o DMEM/Q/P-S, followed by aspiration of the medium and rinsing of the, plates with ice-cold phosphate buffered saline (PBS) supplemented with sodium orthovanadate, sodium fluoride and sodium benzamidine.

Cells were lysed in a small volume of this rinse buffer that had been supplemented with 1% NP40 detergent and the protease inhibitors 1 5 PMSF and aprotinin. Insoluble debris was removed from the cell lysates by centrifugation at 14,000 xG for 10 minutes, at 4 0 C and the supernatants were subjected to immunoprecipitation with antisera specific for TIE-2 receptor, with or without the presence of blocking peptides added to -20 pig/ml lysate. Immunoprecipitated proteins were resolved by PAGE Laemmli gel), and then electrotransferred to PVDF membrane and incubated either with various TIE-, 2- or phosphotyrosine-specific antisera. TIE-2 protein was visualized by incubation of the membrane with HRP-linked secondary antisera followed by treatment with ECL reagent (Amersham).

EXAMPLE 2 CLONING AND EXPRESSION OF TIE-2 RECEPTORBODY FOR AFFINITY-BASED STUDY OF TIE-2 LIGAND

INTERACTIONS

An expression construct was created that would yield a secreted protein consisting of the entire extracellular portion of the rat TIE-2 receptor fused to the human immunoglobulin gamma-1 constant region (IgG1 Fc). This fusion protein is called a TIE-2 "receptorbody" (RB), and would be normally expected to exist as a dimer in solution based on formation of disulfide linkages between individual IgG1 Fc tails.

The Fc portion of the TIE-2 RB was prepared as follows. A DNA Qo fragment encoding the Fc portion of human IgG1 that spans from the hinge region to the carboxy-terminus of the protein, was amplified from human placental cDNA by PCR with oligonucleotides corresponding to the published sequence of human IgG1; the resulting DNA fragment was cloned in a plasmid vector. Appropriate DNA 15 restriction fragments from a plasmid encoding the full-length TIE-2 receptor and from the human IgG1 Fc plasmid were ligated on either side of a short PCR-derived fragment that was designed so as to fuse, S in-frame, the TIE-2 and human IgG1 Fc protein-coding sequences.

Thus, the resulting TIE-2 ectodomain-Fc fusion protein precisely 20 substituted the IgG1 Fc in place of the region spanning the TIE-2 S transmembrane and cytoplasmic domains. An alternative method of preparing RBs is described in Goodwin, et. al. Cell 73:447-456 (1993).

Milligram quantities of TIE-2 RB were obtained by cloning the TIE-2 RB DNA fragment into the pVL1393 baculovirus vector and subsequently infecting the Spodoptera frugiperda SF-21AE insect cell line. Alternatively, the cell line SF-9 (ATCC Accession No. CRL-1711) or the cell line BTI-TN-5bl-4 may be used. DNA encoding the TIE-2 RB was cloned as an Eco RI-Notl fragment into the baculovirus transfer plasmid pVL1393. Plasmid DNA purified by cesium chloride density gradient centrifugation was recombined into viral DNA by mixing 3 Iig of plasmid DNA with 0.5 tg of Baculo-Gold DNA (Pharminigen), followed by introduction into liposomes using 30.g Lipofectin (GIBCO- BRL). DNA-liposome mixtures were added to SF-21AE cells (2x 106 dish) in TMN-FH medium (Modified Grace's Insect Cell Medium (GIBCO-BRL) for 5 hours at 2TC, followed by incubation at 27C for 5 days in TMN-FH medium supplemented with 5% fetal calf serum. Tissue culture medium was harvested for plaque purification 1 0 of recombinant, viruses, which was carried out using methods p;r:ei previously described (O'Reilly, L.K. Miller, and V.A. Luckow, Baculovirus Expression Vectors A Laboratory Manual. 1992, New York: W.H. Freeman) except that the agarose overlay contained 125 ig/mL Xgal (5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside;

GIBCO-BRL).

1 5 After 5 days of incubation at 27C, non-recombinant plaques were scored by positive chromogenic reaction to the X-gal substrate, and their positions marked. Recombinant plaques were then visualized by addition of a second overlay containing 100 tg/mL MTT dimethylthiazol-2-yl]2,5,diphenyltetrazolium bromide; Sigma).

20 Putative recombinant virus plaques were picked by plug aspiration, and purified by multiple rounds of plaque isolation to assure homogeneity.

Virus stocks were generated by serial, low-multiplicity passage of plaque-purified virus. Low passage stocks of one virus clone (vTIE-2 receptorbody) were produced.

SF-21AE cells were cultured in serum free medium (SF-900 II, Gibco BRL) containing 1X antibiotic/antimycotic solution (Gibco BRL) and 25 mg/L Gentamycin (Gibco BRL). Pluronic F-68 was added as a surfactant to a final concentration of 1g/L. Cultures (4L) were raised in a bioreactor (Artisan Cell Station System) for at least three days prior to infection. Cells were grown at 27'C, with gassing to 50 dissolved oxygen, at a gas flow rate of 80 mL/min (aeration at a sparge ring). Agitation was by means of a marine impeller at a rate of 100 rpm. Cells were harvested in mid-logarithmic growth phase (-2 X106 cells/mL), concentrated by centrifugation, and infected with plaque forming units of vTIE-2 receptorbody per cell. Cells and inoculum were brought to 400mL with fresh medium, and virus was o adsorbed for 2 hours at 27.C in a spinner flask. The culture was then resuspended in a final volume of 8L with fresh serum-free medium, Zoo: and the cells incubated in the bioreactor using the previously described conditions.

Culture medium from vTIE-2 receptorbody-infected SF21AE cells 5 were collected by centrifugation (500x g, 10 minutes) at 72 hours post-infection. Cell supernatants were brought to pH -8 with NaOH.

EDTA was added to a final concentration of 10 mM and the supernatant pH was readjusted to 8. Supernatants were filtered (0.45 tm, 9 Millipore) and loaded on a protein A column (protein A sepharose 4 fast 20 flow or HiTrap protein A, both from Pharmacia). The column was washed with PBS containing 0.5 M NaCI until the absorbance at 280 nm decreased to baseline. The column was washed in PBS and eluted with M acetic acid. Column fractions were immediately neutralized by eluting into tubes containing 1 M Tris pH 9. The peak fractions containing the TIE-2 receptorbody were pooled and dialyzed versus

PBS.

EXAMPLE 3 DEMONSTRATION THAT TIE-2 HAS A CRITICAL ROLE IN DEVELOPMENT OF THE VASCULATURE Insight into the function of TIE-2 was gained by introduction of "excess" soluble TIE-2 receptorbody (TIE-2 RB) into a developing system. The potential ability of TIE-2 RB to bind, and thereby neutralize, available TIE-2 ligand could result in an observable disruption of normal vascular development and characterization of the ligand. To examine whether TIE-2 RB could be used to disrupt vascular 1 o development in,early chick embryos, small pieces of a biologically resorbable foam were soaked with TIE-2 RB and inserted immediately beneath the chorioallantoic membrane at positions just lateral to the primitive embryo.

Early chicken embryos develop atop the yolk from a small disk of cells that is covered by the chorioallantoic membrane (CAM). The endothelial cells that will come to line the vasculature in the embryo arise from both extra- and intra-embryonic cell sources. Extraembryonically-derived endothelial cells, which provide the major source of endothelial cells in the embryo, originate from accretions of 9 20 mesenchyme that are situated laterally around the embryo-proper, just underneath the CAM. As these mesenchyme cells mature, they give rise to a common progenitor of both the endotheljal and hematopoietic cell lineages, termed the hemangioblast. In turn, the hemangioblast gives rise to a mixed population of angioblasts (the endothelial cell progenitor) and hematoblasts (the pluripotential hematopoietic precursor). Formation of rudiments of the circulatory system begins when endothelial cell progeny segregate to form a one-cell-thick vesicle that surrounds the primitive blood cells. Proliferation and migration of these cellular components eventually produces a vast network of blood-filled microvessels under the CAM that will ultimately invade the embryo to join with limited, intraembryonically-derived vascular elements.

Newly fertilized chicken eggs obtained from Spafas, Inc. (Boston, MA) were incubated at 99.5 0 F, 55 relative humidity. At about 24 hrs.

of development, the egg shell was wiped down with 70% ethanol and a dentist's drill was used to make a 1.5 cm. hole in the blunt apex of each egg. The shell membrane was removed to reveal an air space 110 directly above the embryo. Small rectangular pieces of sterile GelfQam (Upjohn) were cut with a scalpel and soaked in equal concentrations of either TIE-2- or EHK-1 receptorbody. EHK-1 receptorbody was made as set forth in Example 2 using the EHK-1 extracellular domain instead of the TIE-2 extracellular domain 15 (Maisonpierre et al., Oncogene 8:3277-3288 (1993). Each Gelfoam piece absorbed approximately 6 (ig of protein in 30 [il. Sterile watchmakers forceps were used to make a small tear in the CAM at a position several millimeters lateral to the primitive embryo. The majority of the piece of RB-soaked Gelfoam was inserted under the CAM and the 20 egg shell was sealed over with a piece of adhesive tape. Other similarly-staged eggs were treated in parallel with RB of the unrelated, neuronally expressed receptor tyrosine kinase, EHK-1 (Maisonpierre et al., Oncogene 8:3277-3288 (1993). Development was allowed to proceed for 4 days and then the embryos were examined by visual inspection. Embryos were removed by carefully breaking the shells in dishes of warmed PBS and carefully cutting away the embryo with surrounding CAM. Of 12 eggs treated with each RB, 6 TIE-2 RB and 5 EHK-1 RB treated embryos had developed beyond the stage observed at the start of the experiment. A dramatic difference was seen between these developed embryos, as shown in Figures 1A and 1B.

Those treated with EHK-1 RB appeared to have developed relatively normally. Four out of five EHK-1 embryos were viable as judged by the presence of a beating heart. Furthermore, the extra-embryonic vasculature, which is visually obvious due to the presence of red blood cells, was profuse and extended several centimeters laterally under the CAM. By contrast, those treated with TIE-2 RB were severely stunted, ranging from 2-5 mm. in diameter, as compared with more S1 0 than 10 mm in ,iameter for the EHK-1 RB embryos. All of the TIE-2 RB treated embryos were dead and their CAMs were devoid of blood vessels. The ability of TIE-2 RB to block vascular development in the chicken demonstrates that TIE-2 ligand is necessary for development •of the vasculature.

EXAMPLE 4 IDENTIFICATION OF A TIE-2-SPECIFIC BINDING SACTIVITY IN CONDITIONED MEDIUM FROM THE ras ONCOGENE-TRANSFORMED C2C12 MOUSE MYOBLAST CELL LINE 9 Screening of ten-fold-concentrated cell-conditioned media CCM) from various cell lines for the presence of soluble, TIE-2specific binding activity (BIAcore; Pharmacia Biosensor, P'iscataway, NJ) revealed binding activity in serum-free medium from oncogenicras-transformed C2C12 cells (C2C12-ras), RAT 2-ras (which is a ras transformed fibroblast cell line), human glioblastoma T98G and the human neuroblastoma cell line known as SHEP-1.

The C2C12-ras 10X CCM originated from a stably transfected line of C2C12 myoblasts that was oncogenically transformed by transfection with the T-24 mutant of H-ras by standard calcium phosphate-based methods. An SV40 based neomycin-resistance expression plasmid was physically linked with the ras expression plasmid in order to permit selection of transfected clones. Resulting G418-resistant ras-C2C12 cells were routinely maintained as a monolayer on plastic dishes in DMEM/glutamine/penicillinstreptomycin supplemented with 10 fetal calf serum (FCS). Serumfree C2C12-ras 10X CCM was made by plating the cells at 0 confluence in a serum free defined media for 12 hours. [Zhan and Goldfarb, Mol. Cell. Biol. 6: 3541-3544 (1986)); Zhan, et al. Oncogene 1: 369-376 (1987)]. The medium was discarded and replaced with fresh DMEM/Q/P-S for 24 hours. This medium was harvested and cells were re-fed fresh DMEM/Q/P-S, which was also harvested after a further 24 1 5 hours. These CCM were supplemented with the protease inhibitors PMSF (1mM) and aprotinin (10pg/ml), and ten-fold concentrated on sterile size-exclusion membranes (Amicon). TIE-2-binding activity could be neutralized by incubation of the medium with an excess of TIE-2 RB, but not by incubation with EHK-1 RB, prior to BIAcore S* 2 0 analysis.

Binding activity of the 10x CCM was measured using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ) which monitors biomolecular interactions in real-time via surface plasmon resonance. Purified TIE-2 RB was covalently coupled through primary amines to the carboxymethyl dextran layer of a CM5 research grade sensor chip (Pharmacia Biosensor; Piscataway, NJ). The sensor chip surface was activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC), followed by immobilization of TIE-2 RB (25 .g/mL, pH 4.5) and deactivation of unreacted sites with 1.0 M ethanolamine (pH A negative control surface of the EHK-1 receptorbody was prepared in a similar manner.

The running buffer used in the system was HBS (10 mM Hepes, 3.4 mM EDTA, 150 mM NaCI, 0.005% P20 surfactant, pH The 10x CCM samples were centrifuged for 15 min at 4° C and further clarified using a sterile, low protein-binding 0.45 gm filter (Millipore; Bedford, MA). Dextran (2mg/ml) and P20 surfactant (0.005%) were added to each CCM sample. Aliquots of 40 i.L were injected across the immobilized surface (either TIE-2 or EHK-1) at a flow rate of 5 pL/rpin and the receptor binding was monitored for 8 min. The binding activity (resonance units, RU) was measured as the difference between a baseline value determined 30 s prior to the sample injection and a o measurement taken at 30 s post-injection. Regeneration of the 1 5 surface was accomplished with one 12-pL pulse of 3 M MgCl 2 The instrument noise level is 20 RU; therefore, any binding activity with a signal above 20 RU may be interpreted as a real interaction with the receptor. For C2C12-ras conditioned media, the

S

binding activities were in the range 60-90 RU for the TIE-2 RB 20 immobilized surface. For the same samples assayed on a EHK-1 RB immobilized surface, the measured activities were less than 35 RU.

Specific binding to the TIE-2 receptorbody was evaluated by incubating the samples with an excess of either soluble TIE-2 or EHK-1 RB prior to assaying the binding activity. The addition of soluble EHK-1 RB had no effect on the TIE-2 binding activity of any of the samples, while in the presence of soluble TIE-2 binding to the surface is two-thirds less than that measured in the absence of TIE-2. A repeat assay using concentrated C2C12-ras CCM resulted in a four-fold enhancement over background of the TIE-2 specific binding signal.

EXAMPLE 5 C2C12-ras CCM CONTAINS AN ACTIVITY THAT INDUCES TYROSINE PHOSPHORYLATION OF TIE-2

RECEPTOR

C2C12-ras 10X CCM was examined for its ability to induce tyrosine phosphorylation of TIE-2 in ABAE cells. Serum-starved ABAE *o cells were briefly incubated with C2C12-ras CCM, lysed and subjected, to immunoprecipitation and Western analyses as described above.

Stimulation of serum-starved ABAE cells with serum-free C2C12-ras 10X CCM was done as follows. The medium of ABAE cells starved as described above was removed and replaced with either defined medium 1 5 or 10X CCM that had been pre-warmed to 37"C. After 10 minutes, the media were removed and the cells were twice rinsed on ice with. an excess of chilled PBS supplemented with orthovanadate/NaF/benzamidine. Cell lysis and TIE-2-specific immunoprecipitation was done as described above.

ABAE cells incubated for 10 minutes with defined medium S showed no induction of TIE-2 tyrosine phosphorylation, whereas incubation with C2C12-ras CCM stimulated at least a 100 X increase in TIE-2 phosphorylation. This activity was almost totally depleted by pre-incubation of the C2C12-ras 10X CCM for 90 minutes at room temperature with 13 jg of TIE-2 RB coupled to protein G-Sepharose beads. Medium incubated with protein G Sepharose alone was not depleted of this phosphorylating activity.

EXAMPLE 6 EXPRESSION CLONING OF TIE-2 LIGAND COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% each of penicillin and streptomycin and 2 mM glutamine in an atmosphere of CO2. The mouse myoblast C2C12 ras cell line was cultured in Eagle's minimal essential medium (EMEM) with 10% FBS, and 2 mM glutamine. Full length mouse TIE-2 ligand cDNA clones were obtained by screening a C2C12 ras cDNA library in the pJFE14 vector expressed 1 o in COS cells. This vector, as shown in Figure 2, is a modified version of the vector pSR, (Takebe, et al. 1988, Mol. Cell. Biol. 8:466-472). The library was created using the two BSTX1 restriction sites in the .pJFE14 vector.

COS-7 cells were transiently transfected with either the pJFE14 15 library or control vector by the DEAE-dextran transfection protocol.

Briefly, COS-7 cells were plated at a density of 1.0 x 10 6 cells/100 mm plate 24 hours prior to transfection. For transfection, the cells were cultured in serum-free DMEM containing 400 tg/ml of DEAEdextran, 1 iM chloroquine, and 2 mM glutamine, and 1 pg of the appropriate DNA for 3-4 hours at 37°C in an atmosphere of 5% CO2.

The transfection media was aspirated and replaced with PBS with DMSO for 2-3 min. Following this DMSO "shock", the COS-7 cells were placed into DMEM with 10% FBS, 1% each of penicillin and streptomycin, and 2 mM glutamine for 48 hours.

Because the TIE-2 ligand is secreted it was necessary to permeabilize the cells to detect binding of the receptorbody probe to the ligand. Two days after transfection the cells were rinsed with PBS and then incubated with PBS containing 1.8% formaldehyde for min. at room temperature. Cells were then washed with PBS and incubated for 15 min. with PBS containing 0.1% Triton X-100 and Bovine Calf Serum to permeabilize the cells and block non-specific binding sites.

The screening was conducted by direct localization of staining using a TIE-2 receptorbody which consisted of the extracellular domain of TIE-2 fused to the IgG1 constant region. This receptorbody was prepared as set forth in Example 2. A 100 mm dish of transfected, fixed and permeabilized COS cells was probed by incubating them for 0o 30 min with TIE;2 RB. The cells were then washed twice with PBS anl incubated for an additional 30 min with PBS/10% Bovine Calf S Serum/anti-human IgG-alkaline phosphatase conjugate. After three PBS washes, cells were incubated in alkaline-phosphatase substrate for 30-60 min. The dish was then inspected microscopically for the 1 5 presence of stained cells. For each stained cell, a small area of cells including the stained cell was scraped from the dish using a plastic pipette tip and plasmid DNA was then rescued and used to electroporate bacterial cells. Single bacterial colonies resulting from the electroporation were picked and plasmid DNA prepared from these colonies was used. to transfect COS-7 cells which were probed for TIE- 2 ligand expression as evidenced by binding to TIE-2 receptorbodies.

This allowed identification of single clones coding for TIE-2 ligand.

Confirmation of TIE-2 ligand expression was obtained by phosphorylation of the TIE-2 receptor using the method set forth in Example 5. A plasmid clone encoding the TIE-2 ligand was deposited with the ATCC on October 7, 1994 and designated as "pJFE14 encoding TIE-2 ligand" under ATCC Accession No. 75910.

EXAMPLE 7 ISOLATION AND SEQUENCING OF FULL LENGTH cDNA CLONE ENCODING HUMAN TIE-2 LIGAND A human fetal lung cDNA library in lambda gt-10 (see Figure 3) was obtained from Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were plated at a density of 1.25 x 10 6 /20x20 cm plate, and replica filters taken following standard procedures (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed;, page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

1 o Isolation of human tie-2 ligand clones was carried out as follows. A 2.2 kb Xhol fragment from the deposited tie-2 ligand clone (ATCC NO. 75910 see Example 6 above) was labeled by random .priming to a specific activity of approximately 5xl08cpm/ng.

Hybridization was carried out at 65 0 C in hybridization solution 1 5 containing 0.5 mg/ml salmon sperm DNA. The filters were washed at in 2 x SSC, 0.1 SDS and exposed to Kodak XAR-5 film overnight at -70°C. Positive phage were plaque purified. High titre phage lysates of pure phage were used for isolation of DNA via a Qiagen S.column using standard techniques (Qiagen, Inc., Chatsworth, CA, 1995 catalog, page 36). Phage DNA was digested with EcoRI to release the cloned cDNA fragment for subsequent subcloning. A lambda phage vector containing human tie-2 ligand DNA was deposited with the ATCC on October 26, 1994 under the designation Xgt10 encoding htie-2 ligand 1 (ATCC Accession No. 75928). Phage DNA may be subjected directly to DNA sequence analysis by the dideoxy chain termination method (Sanger, et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 5463- 5467).

Subcloning of the human tie-2 ligand DNA into a mammalian expression vector may be accomplished as follows. The clone Xgtl0 encoding htie-2 ligand 1 contains an EcoRI site located 490 base pairs downstream from the start of the coding sequence for the human TIE-2 ligand. The coding region may be excised using unique restriction sites upstream and downstream of the initiator and stop codons respectively. For example, an Spel site, located 70 bp 5' to the initiator codon, and a Bpu1102i (also known as Blpl) site, located 265 bp 3' to the stop codon, may be used to excise the complete coding region. This may then be subcloned into the pJFE14 cloning vector, O0 using the Xbal (compatible to the Spel overhang) and the Psti sites (the Pstl .and Bpu1102i sites are both made blunt ended).

The coding region from the clone Xgt10 encoding htie-2 ligand 1 was sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster 1 5 City, CA). The nucleotide and deduced amino acid sequence of human TIE-2 ligand from the clone Xgtl0 encoding htie-2 ligand 1 is shown in Figure 4.

In addition, full length human tie-2 ligand cDNA clones were obtained by screening a human glioblastoma T98G cDNA library in the 20 pJFE14 vector. Clones encoding human TIE-2 ligand were identified by DNA hybridization using a 2.2 kb Xhol fragment from the deposited tie- 2 ligand clone (ATCC NO. 75910) as a probe (see Example 6 above). The coding region was sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). This sequence was nearly identical to that of clone Xgtl0 encoding htie-2 ligand 1. As shown in Figure 4, the clone encoding htie-2 ligand 1 contains an additional glycine residue which is encoded by nucleotides 1114-1116. The coding sequence of the T98G clone does not contain this glycine residue but otherwise is identical to the coding sequence of the clone Xgt10 encoding htie-2 ligand 1. Figure 5 sets forth the nucleotide and deduced amino acid sequence of human TIE-2 ligand from the T98G clone.

EXAMPLE 8 ISOLATION AND SEQUENCING OF SECOND FULL LENGTH cDNA CLONE A ENCODING HUMAN TIE-2 LIGAND r r r r r A human/fetal lung cDNA library in lambda gt-10 (see Figure 3) was obtained from Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were plated at a density of 1.25 x 10 6 /20x20 cm plate, and replica filters taken following standard procedures (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Duplicate filters were screened at low stringency (2 x SSC, 550 C) with probes made to the human TIE-2 ligand 1 sequence. One of the duplicate filters was probed with a 5' probe, encoding amino acids 25 265 of human TIE-2 ligand 1 as set forth in Figure 4.

probed with a 3' probe, encoding ligand 1 sequence (see Figure 4).

in hybridization solution containin Filters were washed in 2 x SSC a film. In addition, duplicate filter stringency (2 x SSC, 650 C) to th TIE-2 ligand 1 (F3-15, Xhol inser that fulfilled the following criteria The second duplicate filter was amino acids 282 498 of human TIE-2 Both probes were hybridized at 550 C g 0.5 mg/ml- salmon sperm DNA.

it 550 C and exposed overnight to X-ray s were also hybridized at normal e full length coding probe of mouse rt). Three positive clones were picked i. hybridization had not been seen to the full length (mouse) probe at normal stringency, and ii.

hybridization was seen at low stringency to both 5' and 3' probes.

EcoRI digestion of phage DNA obtained from these clones indicated two independent clones with insert sizes of approximately 2.2kb and approximately 1.8 kb. The 2.2kb EcoRI insert was subcloned into the EcoRI sites of both pBluescript KS (Stratagene) and a mammalian expression vector suitable for use in COS cells. Two orientations were identified for the mammalian expression vector. The 2.2kb insert in pBluescript KS was deposited with the ATCC on December 9, 1994 and designated, as pBluescript KS encoding human TIE 2 ligand 2. The start o site of the TIE-2/ligand 2 coding sequence is approximately 355 base S pairs downstream of the pBluescript EcoRI site.

COS-7 cells were transiently transfected with either the expression vector or control vector by the DEAE-dextran transfection protocol. Briefly, COS-7 cells were plated at a density of 1.0 x 106 cells/100 mm plate 24 hours prior to transfection. For transfection, the cells were cultured in serum-free DMEM containing 400 p.g/ml of DEAE-dextran, 1 IpM chloroquine, and 2 mM glutamine, and 1 pg of the appropriate DNA for 3-4 hours at 370C in an atmosphere of 5% CO2.

The transfection media was aspirated and replaced with phosphatebuffered saline with 10% DMSO for 2-3 min. Following this DMSO "shock", the COS-7 cells were placed into DMEM with 10% FBS, 1% each of penicillin and streptomycin, and 2 mM glutamine for 48 hours.

Because the TIE-2 ligand is secreted it was necessary to permeabilize the cells to detect binding of the receptorbody probe to the ligand. Transfected COS-7 cells were plated at a density of 1.0 x 6 cells/100 mm plate. The cells were rinsed with PBS and then incubated with PBS containing 1.8% formaldehyde for 15-30 min. at room temperature. Cells were then washed with PBS and incubated for min. with PBS containing 0.1% Triton X-100 and 10% Bovine Calf Serum to permeabilize the cells and block non-specific binding sites.

The screening was conducted by direct localization of staining using a TIE-2 receptorbody, which consisted of the extracellular domain of TIE-2 fused to the IgG1 constant region. This receptorbody was prepared as set forth in Example 2. Transfected COS cells were probed by incubating them for 30 min with TIE-2 receptorbody. The cells were then washed twice with PBS, fixed with methanol, and then incubated for an additional 30 min with PBS/10% Bovine Calf 1 0 Serum/anti-human IgG-alkaline phosphatase conjugate. After three PBS washes, cells were incubated in alkaline-phosphatase substrate for 30-60 min. The dish was then inspected microscopically for the presence of stained cells. Cells expressing one orientation of the clone, but not the other orientation, were seen to bind the TIE-2 1 5 receptorbody.

One of skill in the art will readily see that the described methods may be used to further identify other related members of the TIE ligand family.

SThe coding region from the clone pBluescript KS encoding human TIE-2 ligand 2 was sequenced using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The nucleotide and deduced amino acid sequence of human TIE-2 ligand from the clone pBluescript KS. encoding human TIE-2 ligand 2 is shown in Figure 6.

EXAMPLE 9 TIE-2 LIGAND 2 IS A RECEPTOR ANTAGONIST T ^IJV-r I Conditioned media from COS cells expressing either TIE-2 ligand 2 (TL2) or TIE-2 ligand 1 (TL1) were compared for their ability to activate TIE-2 receptors naturally present in human endothelial cell lines.

Lipofectamine reagent (GIBCO-BRL, Inc.) and recommended protocols were used to transfect COS-7 cells with either the pJFE14 expression vector alone, pJFE14 vector containing the human TIE-2 ligand 1 cDNA, or with a pMT21 expression vector (Kaufman, 1985, Proc. Natl. Acad. Sci. USA 82: 689-693) containing the human TIE-2 0 ligand 2 cDNA. /COS media containing secreted ligands were harvested.

S after three days and concentrated 20-fold by diafiltration (DIAFLO ultrafiltration membranes, Amicon, Inc.). The quantity of active TIE-2 S ligand 1 and TIE-2 ligand 2 present in these media was determined and expressed as the amount (in resonance units, of TIE-2 receptor 1 5 specific binding activity measured by a BIAcore binding assay.

S. Northern (RNA) analyses revealed significant levels of TIE-2 transcripts in HAEC (Human Aortic Endothelial Cell) human primary endothelial cells (Clonetics, Inc.). Therefore, these cells were used to examine whether TIE-2 receptor is tyrosine-phosphorylated when exposed to COS media containing the TIE-2 ligands. HAEC cells were maintained in a complete endothelial cell growth medium (Clonetics, Inc.) that contained 5% fetal bovine serum, soluble bovine brain extract, 10 ng/ml human EGF, 1 mg/ml hydrocortisone, 50 mg/ml gentamicin and 50 ng/ml amphotericin-B. Assessment of whether TL1 and TL2 could activate TIE-2 receptor in the HAEC cells was done as follows. Semi-confluent HAEC cells were serum-starved for two hours in high-glucose Dulbecco's MEM with added L-glutamine and penicillin-streptomycin at 37 0 C followed by replacement of the starvation medium with ligand-containing conditioned COS media for 7 minutes at 37°C in a 5% C02 incubator. The cells were subsequently lysed and TIE-2 receptor protein was recovered by immunoprecipitation of the lysates with TIE-2 peptide antiserum, followed by Western blotting with antiphosphotyrosine antiserum, exactly as described in example 1. The results are shown in Figure 7.

Phosphotyrosine levels on the TIE-2 receptor (TIE-2-R) were induced by treatment of HEAC cells with TIE-2 ligand 1 (Lane L1) but not by TIE-2 ligand 2 (Lane L2) conditioned COS media. MOCK is conditioned media from COS transfected with JFE14 empty vector.

Evidence that both TL1 and TL2 specifically bind to the TIE-2 receptor was demonstrated by using a BIAcore to assay the TIE-2 i receptor specific binding activities in transfected COS media and by immunostaining of TL1- and TL2-expressing COS cells with TIE-2 1 5 receptorbodies.

Because TL2 did not activate the TIE-2 receptor, applicants set out to determine whether TL2 might be capable of serving as an antagonist of TL1 activity. HAEC phosphorylation assays were performed in which cells were first incubated with an "excess" of TL2, "4 20 followed by addition of dilute TL1. It was reasoned that prior occupancy of TIE-2 receptor due to high levels of TL2 might prevent subsequent stimulation of the receptor following exposure to TL1 present at a limiting concentration.

Semi-confluent HAEC cells were serum-starved as described above and then incubated for 3 min., at 370C with 1-2 ml. of COS/JFE14-TL2 conditioned medium. Control plates were treated with COS/JFE14-only medium (MOCK). The plates were removed from the incubator and various dilutions of COS/JFE14-TL1 medium were then added, followed by further incubation of the plates for 5-7 min. at 37°C. Cells were subsequently rinsed, lysed and TIE-2-specific tyrosine phosphorylation in the lysates was examined by receptor immunoprecipitation and Western blotting, as described above. TL1 dilutions were made using 20X COS/JFE14-TL1 medium diluted to 2X, 0.1X, or 0.02X by addition of 20X COS/JFE14-alone medium. An assay of the initial 20X TL1 and 20X TL2 COS media using BIAcore biosensor technology indicated that they contained similar amounts of TIE-2-specific binding activities, 445 R.U. and 511 R.U. for TL1 and STL2, respectively, The results of the antiphosphotyrosine Western blot, .shown in Figure 8, indicate that when compared to prior treatment of HAEC cells with -MOCK medium (lane prior treatment of HAEC cells with excess TIE-2 ligand 2 (lane 2) antagonizes the subsequent ability of dilute TIE-2 ligand 1 to activate the TIE-2 "1s receptor (TIE-2-R).

The ability of TL2 to competitively inhibit TL1 activation of the TIE-2-R was further demonstrated using the human cell hybrid line, EA.hy926 (see Example 21 for detailed description of this cell line and its maintenance). Experiments were performed in which 0o unconcentrated COS cell media containing TL1 were mixed at varying dilutions with either MOCK- or TL2- conditioned media and placed on serum-starved EA.hy926 cell monolayers for 5 .minutes at 370C. The media were then removed, the cells were harvested-by lysis and TIE-2specific tyrosine phosphorylation was examined by Western blots, as described above. Figure 9 shows an experiment which contains three groups of treatments, as viewed from left to right. As shown in the four lanes at the left, treatment of the EA.hy926 cells with lx COS- TL1 alone robustly activated the endogenous T1E-2-R in these cells, whereas lx TL2 COS medium was inactive. However, mixture of TL1 with either MOCK or TL2 demonstrated that TL2 can block the activity of TL1 in a dose-dependent fashion. In the central three pairs of lanes the ratio of TL2 (or MOCK) was decreased while the amount of TL1 in the mixture was correspondingly increased from 0.1x to 0.3x. At any of these mixture ratios the TL1:TL2 lanes showed a reduced level of TIE-2-R phosphorylation compared to that of the corresponding TL1:MOCK lanes. When the amount TL1 was held steady and the amount of TL2 (or MOCK) was decreased, however (shown in the three pairs of 1 0 lanes at the righ), a point was reached at which the TL2 in the sample: was too dilute to effectively inhibit TL1 activity. The relative amount of each ligand present in these conditioned COS media could be estimated from their binding units as measured by the BIAcore assay and from Western blots of the COS media with ligand-specific 15 antibodies. Consequently, we can infer that only a few-fold molar excess of TL2 is required to effectively block the activity of TL1 in vitro. This is significant because we have observed distinct examples in vivo (see Example 17 and Figure 16) where TL2 mRNAs achieve considerable abundance relative to those of TL1. Thus, TL2 may be "4 20 serving an important physiological role in effectively blocking signaling by the TIE-2-R at these sites.

Taken together these data confirm that, -unlike TL1, TL2 is unable to stimulate endogenously expressed TIE-2-R on endothelial cells.

Furthermore, at a few fold molar excess TL2 can block TL1 stimulation of the TIE-2 receptor, indicating that TL2 is a naturally occurring TIE- 2 receptor antagonist.

EXAMPLE 10 IDENTIFICATION OF TIE-2-SPECIFIC BINDING ACTIVITY IN CONDITIONED MEDIUM AND COS CELL

SUPERNATANTS

Binding activity of 10x CCM from the cell lines C2C12-ras, Rat2 ras, SHEP, and T98G, or COS cell supernatants after transfection with either human TIE-2 ligand 1 (hTL1) or human TIE-2 ligand 2 (hTL2) was measured using biosensor technology (BIAcore; Pharmacia Biosensor, Piscataway, NJ) which monitors biomolecular interactions in real- @o time via surface ,plasmon resonance (SPR). Purified rat or human TIE-2' RB. was covalently coupled through primary amines to the carboxymethyl dextran layer of a CM5 research grade sensor chip (Pharmacia Biosensor; Piscataway, NJ). The sensor chip surface was activated using a mixture of N-hydroxysuccinimide (NHS) and N-ethyl- '1 5 dimethylaminopropyl)carbodiimide (EDC), followed by immobilization of TIE-2 RB (25 p.g/mL, pH 4.5) and deactivation of unreacted sites with 1.0 M ethanolamine (pH In general, 9000- 10000 RU of each receptorbody was coupled to the sensor chip.

The running buffer used in the system was HBS (10 mM Hepes, 150 mM NaCI, 0.005% P20 surfactant, pH The samples were centrifuged for 15 min at 4°C and further clarified using a sterile, low, protein-binding 0.45 (pm filter (Millipore; Bedford, MA). Dextran (2mg/ml) and P20 surfactant (0.005%) were added to each sample.

Aliquots of 40 .L were injected across the immobilized surface (either rat or human TIE-2) at a flow rate of 5 p.L/min and the receptor binding was monitored for 8 min. The binding activity (resonance units, RU) was measured as the difference between a baseline value determined 30 s prior to the sample injection and a measurement taken at 30 s post-injection. Regeneration of the surface was accomplished with one 15-pL pulse of 3 M MgCl 2 The CCM samples (C2C12-ras, Rat2-ras, SHEP, T98G) were tested on the rat TIE-2 RB immobilized surface, while the recombinant hTL1 and hTL2 were tested on the human TIE-2 RB immobilized surface. In each case, specific binding to the TIE-2 receptorbody was evaluated by incubating the samples with 25 .g/ml of either soluble TIE-2 (rat or human) RB or trkB RB prior to assaying the binding activity. As shown in Figures 10 and 11, the addition of soluble trkB RB causes a slight 1 o decrease in the, TIE-2 binding activity, while the addition of soluble TIE-2 RB significantly reduces the binding activity as compared to that measured in the absence of TIE-2 RB.

EXAMPLE 11 TIE-2 RB SPECIFICALLY BLOCKS ACTIVATION OF THE 1 5 TIE-2 RECEPTOR BY TIE-2 LIGAND 1 S****The applicants sought to determine whether soluble TIE-2 RB can serve as a competitive inhibitor to block activation of TIE-2 receptor by TIE-2 ligand 1 (TL1). To do this, TL1-containing COS media were preincubated with either TIE-2- or TrkB-RB and then compared for S their ability to activate TIE-2 receptors naturally present in a human endothelial cell line.

Conditioned COS media were generated from COS-7 cells transfected with either the pJFE14 expression vector alone (MOCK), or pJFE14 vector containing the human TIE-2 ligand 1 cDNA (TL1) and harvested as described in Example 9 hereinabove, with the exception that the media were sterile filtered but not concentrated. The quantity of TL1 was determined and expressed as the amount (in resonance units, of TIE-2 receptor-specific binding activity measured by BIAcore binding assay.

Northern (RNA) analyses revealed significant levels of tie-2 transcripts in HUVEC (Human Umbilical Vein Endothelial Cell) human primary endothelial cells (Clonetics, Inc.). Therefore, these cells were used to examine whether TIE-2 receptor can be tyrosinephosphorylated when exposed in the presence of TIE-2- or TrkB-RBs to COS media containing TL1. HUVEC cells were maintained at 370C, CO2 in a complete endothelial cell growth medium (Clonetics, Inc.) that Oo contained 5% fetal bovine serum, soluble bovine brain extract with p.g/ml heparin, 10 ng/ml human EGF, 1 ug/ml hydrocortisone, 50 ptg/ml gentamicin and 50 ng/ml amphotericin-B. Assessment of whether TL1 could activate TIE-2 receptor in the HUVEC cells was done as follows.

Confluent dishes of HUVEC cells were serum-starved for two-to-four 1 5 hours in low-glucose Dulbecco's MEM at 370C, 5% CO2, followed by minute incubation in-starvation medium that included 0.1 mM sodium orthovanadate, a potent inhibitor of phosphotyrosine phosphatases.

Meanwhile, conditioned COS media were preincubated 30 min. at room I temperature with either TIE-2- or TrkB-RB added to 50 tg/ml. The starvation medium was then removed from the HUVEC dishes and incubated with the RB-containing COS media for 7 minutes at 370C.

HUVEC cells were subsequently lysed and TIE-2 receptor protein was recovered by immunoprecipitation with TIE-2 peptide antiserum, followed by Western blotting with an anti-phosphotyrosine antibody, as described in Example 1. The results are shown in Figure 12.

Phosphotyrosine levels on the TIE-2 receptor were induced by treatment of HUVEC cells with TIE-2 ligand 1 (TL1) relative to that seen with control medium (MOCK) and this induction is specifically blocked by prior incubation with TIE-2-RB (TIE-2-Fc) but not by incubation with TrkB-RB (TrkB-Fc). These data indicate that soluble TIE-2 RB can serve as a selective inhibitor to block activation of TIE-2 receptor by TIE-2 ligand 1.

EXAMPLE 12 CONSTRUCTION OF TIE-2 LIGANDBODIES An expression construct was created that would yield a secreted protein consisting of the entire coding sequence of human TIE-2 ligand 1 (TL1) or TIE-2,ligand 2 (TL2) fused to the human immunoglobulin gamma-1 constant region (IgG1 Fc). These fusion proteins are called TIE-2 "ligandbodies" (TL1-Fc or TL2-Fc). The Fc portion of TL1-Fc and TL2-Fc was prepared as follows. A DNA fragment encoding the Fc portion of human IgG1 that spans from the hinge region to the carboxy- 1 5 terminus of the protein, was amplified from human placental cDNA by PCR with oligonucleotides corresponding to the published sequence of human IgG1; the resulting DNA fragment was cloned in a plasmid vector. Appropriate DNA restriction fragments from a plasmid encoding full-length TL1 or TL2 and from the human IgG1 Fc plasmid 20 were ligated on either side of a short PCR-derived fragment that was designed so as to fuse, in-frame, TL1 or TL2 with human IgG1 Fc protein-coding sequences.

Milligram quantities of TL2-Fc were obtained by cloning the TL2- Fc DNA fragment into the pVL1393 baculovirus vector and subsequently infecting the Spodoptera frugiperda SF-21AE insect cell line.

Alternatively, the cell line SF-9 (ATCC Accession No. CRL-1711) or the cell line BTI-TN-5bl-4 may be used. DNA encoding the TL2-Fc was cloned as an Eco RI-Notl fragment into the baculovirus transfer plasmid pVL1393. Plasmid DNA was recombined into viral DNA by mixing 3 lig of plasmid DNA with 0.5 p.g of Baculo-Gold DNA (Pharminigen), followed by introduction into liposomes using Lipofectin (GIBCO-BRL). DNA-liposome mixtures were added to SF- 21AE cells (2x 106 cells/60mm dish) in TMN-FH medium (Modified Grace's Insect Cell Medium (GIBCO-BRL) for 5 hours at 270C, followed by incubation at 270C for 5 days in TMN-FH medium supplemented with fetal calf serum. Tissue culture medium was harvested for plaque purification of recombinant viruses, which was carried out using methods previously described (O'Reilly, L.K. Miller, and V.A.

Luckow, Baculovirus Expression Vectors A Laboratory Manual. 1992, New York: W.H. Freeman) except that the agarose overlay contained 125 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-b- D-galactopyranoside; GIBCO-BRL). After 5 days of incubation at 270C, non-recombinant 0 1 5 plaques were scored by positive chromogenic reaction to the X-gal a* substrate, and their positions marked. Recombinant plaques were then visualized by addition of a second overlay containing 100 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]2,5,diphenyltetrazolium bromide; Sigma).

Putative recombinant virus plaques were picked by plug aspiration, and purified by multiple rounds of plaque isolation to assure homogeneity.

Virus stocks were generated by serial, low-multiplicity passage of plaque-purified virus. Low passage stocks of one virus clone (vTL2-Fc Clone were produced.

SF-21AE cells were cultured in serum-free medium (SF-900 II, Gibco BRL) containing 1X antibiotic/antimycotic solution (Gibco BRL) and 25 mg/L Gentamycin (Gibco BRL). Pluronic F-68 was added as a surfactant to a final concentration of 1g/L. Cultures (4L) were raised in a bioreactor (Artisan Cell Station System) for at least three days prior to infection. Cells were grown at 270C, with gassing to 50 dissolved oxygen, at a gas flow rate of 80 mL/min (aeration at a sparge ring). Agitation was by means of a marine impeller at a rate of 100 rpm. Cells were harvested in mid-logarithmic growth phase (-2 X10 6 cells/mL), concentrated by centrifugation, and infected with plaque forming units of vTL2-Fc per cell. Cells and inoculum were brought to 400mL with fresh medium, and virus was adsorbed for 2 hours at 27°C in a spinner flask. The culture was then resuspended in a final volume of 8L with fresh serum-free medium, and the cells incubated in the, bioreactor using the previously described conditions..

Culture medium from vTL2-Fc-infected SF21AE cells were collected by centrifugation (500x g, 10 minutes) at 72 hours postinfection. Cell supernatants were brought to pH 8 with NaOH. EDTA was added to a final concentration of 10 mM and the supernatant pH 1 5 was readjusted to 8. Supernatants were filtered (0.45 .tm, Millipore) and loaded on a protein A column (protein A sepharose 4 fast flow or HiTrap protein A, both from Pharmacia). The column was washed with SPBS containing 0.5 M NaCI until the absorbance at 280 nm decreased to baseline. The column was washed in PBS and eluted with 0.5 M acetic 20 acid. Column fractions were immediately neutralized by eluting into tubes containing 1 M Tris pH 9. The peak fractions containing the TL2- Fc were pooled and dialyzed versus PBS.

EXAMPLE 13- EXPRESSION OF TIE-1, TIE-2, TL1, AND TL2 IN RENAL CELL CARCINOMA In situ hybridization experiments were performed on human renal cell carcinoma tumor tissue using TIE-1, TIE-2, TL1, and TL2 cDNA probes. TIE-2, TIE-1, TL1, and TL2 expression were all up-regulated in the tumor vasculature. Ligand expression appeared to be localized to either the vascular endothelial cells (TL2) or very near the vascular endothelial cells in the mesenchyme (TL1). VEGF has been shown to be dramatically up-regulated in this tumor tissue. Brown, et al. Am. J.

Pathol. 143:1255-1262 (1993).

EXAMPLE 14 EXPRESSION OF TIE-1, TIE-2, TL1, AND TL2 IN WOUND

.:HEALING

In situ hybridization experiments were performed on crosssectional tissue slices obtained from a rat cutaneous wound model using TIE-1, TIE-2, TL1, and TL2 cDNA probes. The wound healing model involves pressing a small cork bore against the skin of a rat and removing a small, cylindrical plug of skin. As healing begins at the base of the wound, a vertical slice of tissue is taken and used for in situ hybridization. In the tested tissue sample, TL1 and TL2 appeared o to be slightly up-regulated by four days post-injury. In contrast to the slightly up-regulated expression of TL1 and TL2 in this tissue, VEGF expression, which may precede TL1 and TL2 expression, is dramatically up-regulated.

EXAMPLE 15 EXPRESSION OF TIE LIGANDS IN FETAL LIVER AND

THYMUS

Reverse transcription-PCR (RT-PCR) was performed on mouse E14.5 fetal liver and mouse E17.5 fetal thymus. Agarose gel electrophoresis of the RT-PCR products revealed that in the mouse fetal liver, TIE-2 ligand 1 (TL1) RNA is enriched in the stromal region, but is absent in c-kit+TER119 hematopoietic precursor cells. In this same tissue, TIE-2 ligand 2 (TL2) RNA is enriched in the stromal cells, but absent in the hematopoietic precursor cells (Figure 13). In the mouse fetal thymus, TL2 is enriched in the stromal cells (Figure 14).

1 0 EXAMPLE 16 TE TIE RECEPTOR/LIGAND SYSTEM IN ANGIOGENESIS Although the TIE-2/TIE ligand system appears to play an important role in endothelial cell biology, it has not been shown to play a significant, active role in the early to intermediate stages of vascularization angioblast or endothelial cell proliferation and migration, tubule formation, and other early stage events in vascular modeling). In contrast to the receptors and factors known to mediate these aspects of vascular development, the temporally late pattern of expression of TIE-2 and TL1 in the course of vascularization suggests 20 that this system plays a distinct role in the latter stages vascular development, including the structural and functional differentiation and stabilization of new blood vessels. The pattern of expression of TIE-2/TL1 also is consistent with a continuing role in the maintenance of the structural integrity and/or physiological characteristics of an established vasculature.

TIE Ligand .2 (TL2) appears to be a competitive inhibitor of TL1.

The spatiotemporal characteristics of TL2 expression suggest that this single inhibitory molecule may play multiple, context-dependent roles essential to appropriate vascular development or remodeling (e.g.

de-stabilization/de-differentiation of mature endothelial cells allowing the formation of new vessels from existing vasculature, inhibition of inappropriate blood vessel formation, and regression/involution of mature blood vessels). Figure 15 is a schematic representation of the hypothesized role of the TIE-2/TIE ligands in angiogenesis. In this figure TL1 is represented by TL2 is represented by TIE-2 is represented by VEGF is represented by and flk-1 (a VEGF receptor) is represented by EXAMPLE 17 EXPRESSION OF TIE LIGANDS IN THE FEMALE REPRODUCTIVE SYSTEM: EXPRESSION IN THE

OVARY

5 Preliminary observations made in experiments examining the expression of the TIE receptors and ligands in the female reproductive system are consistent with the hypothesis the TL1 plays a role in neovascularization which temporally follows that of VEGF. The pattern of TL2 expression is also consistent with an antagonism of the 0 action of TL1, and a specific role in vascular regression. To verify this, expression of relevant mRNAs can be examined following experimental induction of follicular and luteal development so that their temporal relation to various aspects of neovascularization/vascular regression can be more clearly defined in conjunction with endothelial cell staining, vascular fills).

Angiogenesis associated with follicular development and corpus luteum formation in staged ovaries of mature, female rats or following induced ovulation in pre-pubertal animals was followed using in situ hybridization. Figure 16 contains photographs of in situ hybridization slides showing the temporal expression pattern of TIE-2, TL1, TL2, and VEGF during the ovarian cycle [Column 1: Early preovulatory follicle; Column 2: pre-ovulatory follicle; Column 3: early corpus luteum; and Column 4: atretic follicle; Row A:bright field; Row B:VEGF; Row C: TL2; Row D: TL1 and Row E: TIE-2 receptor]. These studies revealed that VEGF, TL1 and TL2 are expressed in a temporally and spatially coordinate fashion with respect to the development and regression of vasculature in the ovary, specifically with respect to the establishment of, the vascular system which is generated in the course, of the conversion of an ovarian follicle to a corpus luteum (CL).

Briefly, VEGF expression increases in the follicular granule layer Sprior to its vascularization during the process of luteinization. During the process of CL formation, highest levels of VEGF expression are 1 5 apparent in the center of the developing CL in the vicinity of luteinizing cells which are not yet vascularized. VEGF levels remain moderately high and are diffusely distributed in the developed CL. In contrast, noticeably enhanced expression of TIE-2 ligand 1 occurs only late in process of CL formation, after a primary vascular plexus has been established. Later, TL1 expression is apparent throughout the CL at which time the definitive capillary network of the CL has been established.

TL2 exhibits a more complex pattern of expression than either VEGF or TL1. In the developing CL, TL2 is expressed at highest levels at the front of the developing capillary plexus- between the central avascular region of the CL where VEGF expression is highest, and the most peripheral portion of the CL where TL1 expression is dominant and where the luteinization process is complete and the vascular system is most mature. TL2 also appears to be expressed at high levels in the follicular layer of large follicles which are undergoing atresia. While TL1 is also apparent in atretic follicles, VEGF is not expressed.

The pattern of expression described above is most consistent with a role for VEGF in the initiation of angiogenesis, with TL1 acting late in this process-for example in modeling and/or stabilization of the definitive vascular network. In contrast, TL2 is present both in areas of active expansion of a newly forming vascular network (during Lo CL formation), and in regions which fail to establish a new vasculature' and vascular regression is in progress (atretic follicles). This suggests a more dynamic and complex role for TL2, possibly involving destabilization of existing vasculature (necessary for regression) or developing vasculature (necessary for the dynamic modeling of newly 15 forming vessels).

S. EXAMPLE 18 A RECEPTORBODY BINDING ASSAY AND A LIGAND BINDING AND COMPETITION ASSAY 0 A quantitative cell-free binding assay with two alternate formats has been developed for detecting either TIE-2 receptorbody binding or ligand binding and competition. In the receptorbody binding version of the assay, TIE-2 ligands (purified or partially purified; either TL1 or TL2) are coated onto an ELISA plate. Receptorbody at varying concentrations is then added, which binds to the immobilized ligand in a dose-dependent manner. At the end of 2 hours, excess receptorbody is washed away, then the amount bound to the plate is reported using a specific anti-human Fc antibody which is alkaline phosphatase tagged. Excess reporter antibody is washed away, then the AP reaction is developed using a colored substrate. The assay is quantitated using a spectrophotometer. Figure 19 shows a typical TIE- 2-lgG binding curve. This assay has been used to evaluate the integrity of TIE-2-IgG after injection into rats and mice. The assay can also be used in this format as a ligand competition assay, in which purified or partially-purified TIE ligands compete with immobilized ligand for receptorbody. In the ligand binding and competition version of the 1 0 binding assay, TIE-2 ectodomain is coated onto the ELISA plate. The 0 Fc-tagged fibrinogen-like domain fragments of the TIE ligands (TL1fFc and TL2-fFc) then bind to the ectodomain, and can be detected using the same anti-human Fc antibody as described above. Figure shows an example of TL1-fFc binding to TIE-2 ectodomain. This 1 5 version of the assay can also be used to quantitate levels of TL1-fFc in serum or other samples. If untagged ligand (again, either purified or unpurified) is added at the same time as the TL1-fFc, then a competition is set up between tagged ligand fragment and full-length ligand. The full-length ligand can displace the Fc-tagged fragment, 20 and a competition curve is generated.

EXAMPLE 19 EA.hy926 CELL LINE CAN BE USED AS A REPORTER CELL LINE FOR TIE LIGAND ACTIVITY EA.hy926 is a cell hybrid line that was established by fusion of HUVEC with the human lung carcinoma-derived line, A549 [Edgell, et al.

Proc. Natl. Acad. Sci. (USA) 80, 3734-3737 (1983). EA.hy926 cells have been found to express significant levels of TIE-2 receptor protein with low basal phosphotyrosine levels. The density at which EA.hy926 cells are passaged prior to their use for receptor assays, as well as 0 their degree of confluency at the time of assay, can affect TIE-2 receptor abundance and relative inducibility in response to treatment with ligand. By adopting the following regimen for growing these cells the EA.hy926 cell line can be used as a dependable system for assay of TIE-2 ligand activities.

1 EA.hy926 cells are seeded at 1.5 x 106 cells in T-75 flasks (Falconware) and re-fed every other day with high-glucose Dulbecco's MEM, 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, o and lx hypoxanthine-aminopterin-thymidine (HAT, Gibco/BRL). After three to four days of growth, the cells are passaged once again at 1.5 x 6 cells per T-75 flask and cultured an additional three to four days.

For phosphorylation assays, cells prepared as described above were serum-starved by replacement of the culture medium with highglucose DMEM and incubation for 2-3 hours at 370C. This medium was aspirated from the flask and samples of conditioned media or purified ligand were added to the flask in a total volume of 1.5 ml followed by incubation at 37 0 C for 5 minutes. Flasks were removed from the incubator and placed on a bed of ice. The medium was removed and replaced with 1.25 ml Lysis Buffer containing 1% nonidet P-40, sodium deoxycholate, 0.1% SDS in 20 mM Tris, pH 7.6, 150 mM NaCI, mM NaF, 1mM sodium orthovanadate, 5 mM benzamidine, and 1mM EDTA containing the protease inhibitors PMSF, aprotinin, and leupeptin.

After 10 minutes on ice to allow membrane solubilization, plates were scraped and cell lysates were clarified by microcentrifugation at top speed for 10 minutes at 4°C. TIE-2 receptor was immunoprecipitated from the clarified supernatant by incubation in the cold with an anti- 1 0 TIE-2 polyclonal, antiserum and Protein G-conjugated Sepharose beads..

The, beads were washed three times with cold cell lysis buffer and boiled 5 minutes in Laemmli sample buffer, which was then loaded on 7.5% SDS-polyacrylamide gels. Resolved proteins were electrotransferred to PVDF (Lamblia-P) membrane and then subjected 1 5 to Western blot analysis using anti-phosphotyrosine antibody and the ECL reagent. Subsequent comparison of total TIE-2 protein levels on the same blots was done by stripping the anti-phosphotyrosine antibody and reincubating with a polyclonal antiserum specific to the ectodomain of TIE-2.

EXAMPLE 20 ISOLATION AND SEQUENCING OF FULL LENGTH cDNA CLONE ENCODING MAMMALIAN TIE LIGAND-3 TIE ligand-3 (TL3) was cloned from a mouse BAC genomic library (Research Genetics) by hybridizing library duplicates, with either mouse TL1 or mouse TL2 probes corresponding to the entire coding sequence of those genes. Each copy of the library was hybridized using phosphate buffer at 55 0 C overnight. After hybridization, the filters were washed using 2xSSC, 0.1% SDS at 60°C, followed by exposure of X ray film to the filters. Strong hybridization signals were identified corresponding to mouse TL1 and mouse TL2. In addition, signals were s identified which weakly hybridized to both mouse TL1 and mouse TL2.

DNA corresponding to these clones was purified, then digested with restriction enzymes, and two fragments which hybridized to the original probes were subcloned into a bacterial plasmid and sequenced.

The sequence of the fragments contained two exons with homology to both mouse TL1 and mouse TL2. Primers specific for these sequences were used as PCR primers to identify tissues containing transcripts corresponding to TL3. A PCR band corresponding to TL3 was identified in a mouse uterus cDNA library in lambda gt-11. (Clontech "0..9 Laboratories, Inc., Palo Alto, CA).

Plaques were plated at a density of 1.25 x 10 6 /20x20 cm plate and replica filters taken following standard procedures (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., page 8.46, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Duplicate filters were screened at "normal" stringency (2 x SSC, 650C) with a 200 bp PCR radioactive probe made to the mouse TL3 sequence. Hybridization was at 65°C in a solution containing 0.5 mg/mi salmon sperm DNA.

Filters were washed in 2 x SSC at 65 0 C and exposed for 6 hours to Xray film. Two positive clones that hybridized in duplicate were picked. EcoRI digestion of phage DNA obtained from these clones indicated two independent clones with insert sizes of approximately 1.2 kb and approximately 2.2 kb. The 2.2kb EcoRI insert was subcloned into the EcoRI site of pBluescript KS (Stratagene). Sequence analysis showed that the longer clone was lacking an initiator methionine and signal peptide but otherwise encoded a probe homologous to both mouse TL1 and mouse TL2.

Two TL3-specific PCR primers were then synthesised as follows: US2: cctctgggctcgccagtttgttagg US1: ccagctggcagatatcagg The following PCR reactions were performed using expression libraries derived from the mouse cell lines C2C12ras and MG87. In t-)e primary PCR reaction, the specific primer US2 was used in conjunction with vector-specific oligos to allow amplification in either orientation. PCR was in a total volume of 100ml using 35 cycles of 94° C, 1 min; 420C or 480 C for 1 min; 72° C, 1 min. The secondary PCR reaction included the second specific primer, US1, which is contained o within the primary PCR product, in conjunction with the same vector oligos. The secondary reactions were for 30 cycles, using the same temperatures and times as previous. PCR products were gel isolated and submitted for sequence analysis. On the basis of sequences 20 obtained from a total of four independent PCR reactions using two different cDNA libraries, the 5' end of the TL3 sequence was deduced.

Northern analysis revealed moderate to low levels of mouse TL3 transcript in mouse placenta. The expression of mouse TL3 consisted of a transcript of approximately 3 kb. The full length TL3 coding sequence is set forth in Figure 21.

The mouse TL3 sequence may then be used to obtain a human clone containing the coding sequence of human TL3 by hybridizing either a human genomic or cDNA library with a probe corresponding to mouse TL3 as has been described previously, for example, in Example 8 supra.

EXAMPLE 21 ISOLATION OF FULL LENGTH GENOMIC CLONE ENCODING HUMAN TIE LIGAND-4 TIE ligand-4 (TL4) was cloned from a mouse BAC genomic library (BAC HUMAN Genome Systems Inc.) by hybridizing library duplicates, with either a human TL1 radioactive probe corresponding to the entire fibrinogen coding sequence of TL1 (nucleotides 1153 to 1806 of Figure, 4) or a mouse TL3 radioactive probe corresponding to a segment of186 nucleotides from the fibrinogen region of mouse TL3 (nucleotides 1307 to 1492 of Figure 21). Each probe was labeled by PCR using exact oligonucleotides and standard PCR conditions, except that dCTP was 1 5 replaced by P 32 dCTP. The PCR mixture was then passed through a gel filtration column to separate the probe from free P32 dCTP. Each copy of the library was hybridized using phosphate buffer, and radiactive probe at 55 0 C overnight using standard hybridization conditions. After hybridization, the filters were washed using 2xSSC, 0.1% SDS at 55 0

C,

i* 0o followed by exposure of X ray film. Strong hybridization signals were observed corresponding to human TL1. In addition, signals were identified which weakly hybridized to both human TL1 and mouse TL3.

DNA corresponding to these clones was purified using standard procedures, then digested with restriction enzymes, and one fragment which hybridized to the original probes was subcloned into a bacterial plasmid and sequenced. The sequence of the fragments contained one exon with homology to both human TL1 and mouse TL3 and other members of the TIE ligand family. Primers specific for these sequences may be used as PCR primers to identify tissues containing transcripts corresponding to TL4.

The complete sequence of human TL4 may be obtained by sequencing the full BAC clone contained in the deposited bacterial cells. Exons may be identified by homology to known members of the TIE-ligand family such as TL1, TL2 and TL3. The full coding sequence of TL4 may then be determined by splicing together the exons from the TL4 genomic clone which, in turn, may be used to produce the TL4 protein.

1 o Alternatively, the exons may be used as probes to obtain a full length cDNA clone, which may then be used to produce the TL4 protein. Exons may also be identified from the BAC clone sequence by homology to S protein domains such as fibrinogen domains, coiled coil domains, or protein signals such as signal peptide sequences. Missing exons from 1 5 the BAC clone may be obtained by identification of contiguous BAC clones, for example, by using the ends of the deposited BAC clone as probes to screen a human genomic library such as the one used herein, by using the exon sequence contained in the BAC clone to screen a cDNA library, or by performing either 5' or 3' RACE procedure using oligonucleotide primers based on the TL4 exon sequences.

Identification of Additional TIE Ligand Family Members The novel TIE ligand-4 sequence may be used in a rational search for additional members of the TIE ligand family using an approach that takes advantage of the existence of conserved segments of strong homology between the known family members. For example, an alignment of the amino acid sequences of the TIE ligands shows several regions of conserved sequence (see boxed regions of Figure 22).

Degenerate oligonucleotides essentially based on these boxes in combination with either previously known or novel TIE ligand homology segments may be used to identify new TIE ligands.

The highly conserved regions among TL1, TL2 and TL3 may be used in designing degenerate oligonucleotide primers with which to prime PCR reactions using cDNAs. cDNA templates may be generated by reverse transcription of tissue RNAs using oligo d(T) or other appropriate Sprimers. Aliquots of the PCR reactions may then be subjected to electrophoresis on an agarose gel. Resulting amplified DNA fragments may be cloned by insertion into plasmids, sequenced and the DNA sequences compared with those of all known TIE ligands.

5 Size-selected amplified DNA fragments from these PCR reactions may S be cloned into plasmids, introduced into E. coli by electroporation, and transformants plated on selective agar. Bacterial colonies from PCR transformation may be analyzed by sequencing of plasmid DNAs that are purified by standard plasmid procedures.

Cloned fragments containing a segment of a novel TIE ligand may be used as hybridization probes to obtain full length cDNA clones from a cDNA library. For example, the human TL4 genomic sequence may be used to obtain a human cDNA clone containing the complete coding sequence of human TL4 by hybridizing a human cDNA library with a probe corresponding to human TL4 as has been described previously.

rCAMvirt'l_- z ULUNINvI Ut- I 1t t-ULL (;UUING SEQUENCE OF hTL4 Both 5' and 3' coding sequence from the genomic human TL-4 clone encoding human TIE ligand-4 (hTL-4 ATCC Accession No. 98095) was obtained by restriction enzyme digestion, Southern blotting and hybridization of the hTL-4 clone to coding sequences from mouse TL3, followed by subcloning and sequencing the hybridizing fragments.

Coding sequences corresponding to the N-terminal and C-terminal amino acids of hTL4 were used to design PCR primers (shown below), which in turn were used for PCR amplification of TL4 from human 0 ovary cDNA. A PCR band was identified as corresponding to human TL4 by DNA sequencing using the ABI 373A DNA sequencer and Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA). The PCR band was then subcloned into vector pCR-script and several plasmid clones were analyzed by sequencing. The complete human TL4 coding sequence was then compiled and is shown in Figure o 23. In another embodiment of the invention, the nucleotide at position 569 is changed from A to G, resulting in an amino acid change from 0 to R.

The PCR primers used as described above were designed as follows: hTL4atg 3' hTL4not gtgtcgacgcggccgctctagatcagacTTAGATGTCCAAAGGCCGTATCATCAT-3' Lowercase letters indicate "tail" sequences added to the. .CR primers to facilitate cloning of the amplified PCR fragments.

EXAMPLE 23 CONSTRUCTION AND CHARACTERIZATION OF MODIFIED TIE LIGANDS A genetic analysis of TIE-2 ligand-1 and TIE-2 ligand-2 (TL1 and TL2) was undertaken to gain insight into a number of their observed properties. Although TL1 and TL2 share similar structural homology, they exhibit different physical and biological properties. The most prominent feature that distinguishes the two ligands is that although A0 they both bind to the TIE-2 receptor, TL1 is an agonist while TL2 is an, *fee*: 0. o* antagonist. Under non-reducing electrophoretic conditions both proteins exhibit covalent, multimeric structures. TL1 is produced as a mixture of disulfide cross-linked multimers, primarily trimers and higher order species, without any dimeric species. But TL2 is produced 5 almost exclusively as a dimeric species. Also, while TL2 is produced well in most expression systems, TL1 is expressed poorly and is difficult to produce in large quantities. Finally, production and purification conditions also appear to predispose TL1 to inactivation by proteolytic cleavage at a site near the amino terminus.

0 *o To study these differences, several modified ligands were constructed as follows.

23.1. Cysteine substitution Investigations into what factors might be contributing to the different physical and biological properties of the two molecules revealed the presence in TL1 of a cysteine residue (CYS 265 in Figure 4; CYS 245 in Figure 17) preceding the fibrinogenlike domain in TL1 but absent in TL2 there was no corresponding cysteine residue in TL2. The CYS265 residue in TL1 is encoded by TGC and is located at about nucleotides 1102-1104 (see Figure 4) at the approximate junction between the coiled-coil and fibrinogen-like domains. Because cysteine residues are generally involved in disulfide bond formation, the presence of which can contribute to both the tertiary structure and biological properties of a molecule, it was thought that perhaps the presence of the CYS265 residue in TL1 might be at least partially responsible for the different properties of the two molecules.

To test this hypothesis, an expression plasmid was constructed which contained a mutation in TL1 in which the CYS (residue 265 in Figure 4; residue 245 in Figure 17) was replaced with an amino acid (serine) which does not form disulfide bonds. In addition to this TL1/CYS- 1 5 mutant, a second expression plasmid was constructed which mutated the approximately corresponding position in TL2 (Met247 in Figure 17) so that this residue was now a cysteine. Both non-mutated and mutated expression plasmids of TL1 and TL2 were transiently transfected into COS7 cells, cell supernatants containing the 20 recombinant proteins were harvested, and samples were subjected to both reducing and non-reducing SDS/PAGE electrophoresis ard subsequent Western blotting.

Figure 18 shows the Western blots under non-reducing conditions of both non-mutated and mutated TL1 and TL2 proteins, revealing that the TL1/CYS- mutant runs as a dimer much like TL2 and that the TL2/CYS+ mutant is able to form a trimer, as well as higher-order multimers, more like TL1. When the two mutant proteins were tested for their 88 ability to induce phosphorylation in TIE-2 expressing cells, the TL1/CYS- mutant was able to activate the TIE-2 receptor, whereas the TL2/CYS+ mutant was not.

Thus, when the cysteine residue (residue 265 in Figure 4; residue 245 in Figure 17) of TL1 was genetically altered to a serine, it was found that the covalent structure of TL1 became similar to that of TL2, i.e., primarily dimeric. The modified TL1 molecule still behaved as an agonist, thus the trimeric and/or higher order multimeric structure b was not the determining factor giving TL1 the ability to activate.

Although the removal of the cysteine did make a molecule with more desirable properties, it did not improve the production level of TL1.

23.2. Domain deletions The nucleotide sequences encoding TL1 and TL2 share a genetic structure that can be divided into three domains, based on the amino acid sequences of the mature proteins. The last approximately 215 amino acid residues of each mature protein contains six cysteines and bears strong resemblance to a domain of fibrinogen. This region was thus denoted the "fibrinogen-like" domain o or "F-domain." A central region of the mature protein containing approximately 205 residues had a high probability of assuming a "coiled-coil" structure and was denoted the "coiled-coil" domain or "Cdomain." The amino-terminal approximately 55 residues of the mature protein contained two cysteines and had a low probability of having a coiled-coil structure. This region was designated the "Nterminal" domain or "N-domain." The modified ligands described herein are designated using a terminology wherein N N-terminal domain, C coiled-coil domain, F fibrinogen-like domain and the numbers 1 and 2 refer to TL1 and TL2 respectively. Thus 1N indicates the N-terminal domain from TL1, 2F indicates the fibrinogen-like domain of TL2, and so forth.

In order to test whether the fibrinogen-like domain (F-domain) of the TIE-2 ligands contained TIE-2 activating activity, expression plasmids were constructed which deleted the coiled-coil and N-terminal domains, leaving only that portion of the DNA sequence encoding the Fdomain (for TL1, beginning in Figure 4 at about nucleotide 1159, amino.

acid residue ARG284; for TL2, corresponding to about nucleotide 1200, in Fgure 6, amino acid residue 282). This mutant construct was then .transiently transfected into COS cells. The supernatant containing the S' recombinant protein was harvested. The TL1/F-domain mutant was tested for its ability to bind the TIE-2 receptor. The results showed.

that, as a monomer, the TL1/F-domain mutant was not able to bind TIE-2 at a detectable level.

But when the TL1/F-domain monomer was myc-tagged and subsequently clustered with an antibody directed against the myc tag, 20 it exhibited detectable binding to TIE-2. However, the antibodyclustered TL1/F-domain mutant was not able to induce phosphorylation in a TIE-2 expressing cell line.

Thus it was determined that the F-domain of the TIE-2 ligands is involved in binding the receptor but that a truncation consisting of just the F-domain alone is not sufficient for receptor binding. This raised the possibility that the coiled-coil domain was responsible for holding together several fibrinogen-like domains, which might be essential for receptor binding. In an attempt to confirm this hypothesis, the F-domain was fused with the Fc section of human antibody IgG1. Because Fc sections dimerize upon expression by mammalian cells, these recombinant proteins mimicked the theoretical configuration of the F-domains were the native ligands to dimerize. This F-domain-Fc construct bound but failed to activate the receptor. Apparently, multimerization caused by other regions of the ligands is necessary to enable the ligands to bind the TIE-2 receptor.

In addition, some other factor outside of the F-domain must contribute b to phosphorylation of the receptor.

Mutants were then constructed which were missing the fibrinogen-like domain, and therefore contained only the N-terminal and coiled-coil domains. They were not capable of binding to the receptor. To assess the role of the N-terminal domain in receptor binding and activation, the ligands were truncated to just their C- and F-domains and tagged with a FLAG tag at the N-terminus, creating constructs termed FLAG- 1C1F and FLAG-2C2F. Although these molecules stained robustly in COS7 cells transfected transiently to express the TIE-2 receptor, they :o failed to respond in a phosphorylation assay. Thus the N-domain does contain an essential factor for receptor activation although, as disclosed infra, the ability of chimeric molecule 2N2C1F to activate the receptor shows that even the N-domain of an inactive ligand can fill that role.

The differences in behavior between the myc-tagged F-domain truncation and the Fc-tagged F-domain truncation described previously suggested that the TIE ligands can only bind in dimeric or higher multimeric forms. Indeed, non-reducing SDS-PAGE showed that the TIE ligands exist naturally in dimeric, trimeric, and multimeric forms.

That the FLAG-1C1F and FLAG-2C2F truncations can bind to the TIE-2 receptor without dimerization by a synthetic tag (such as Fc), whereas the F truncations cannot, suggests that the C-region is at least partly responsible for the aggregation of the F-domains.

r 23.3. Swapping constructs (chimeras): Applicants had noted that the level of production of TL1 in COS7 cells.

was approxima;ely tenfold lower than production of TL2. Therefore, chimeras of TL1 and TL2 were constructed in an attempt to explain this difference and also to further characterize the agonist activity of TL1 as compared to the antagonist activity of TL2.

Four chimeras were constructed in which either the N-terminal domain or the fibrinogen domain was exchanged between TL1 and TL2 and were designated using the terminology described previously such that, for example, 1N1C2F refers to a chimera having the N-terminal and coiledcoil domains of TL1, together with the fibrinogen-like domain from 20 TL2. The four chimeras were constructed as follows: chimera 1 1N1C2F chimera 2 2N2C1 F chimera 3 1N2C2F chimera 4 2N1C1F The nucleotide and amino acid sequences of chimeras 1-4 are shown in Figures 24-27 respectively.

Each chimera was inserted into a separate expression vector pJFE14.

The chimeras were then transfected into COS7 cells, along with the empty pJFE14 vector, native TL1, and native TL2 as controls, and the culture supernatants were. collected.

In order to determine how the swapping affected the level of expression of the ligands, a 1:5 dilution and a 1:50 dilution of the COS7 supernatants were dot-blotted onto nitrocellulose. Three ligands that contained the TL1 N-domain native TL1, 1N2C2F and 1N1C2F) were then probed with a rabbit antibody specific to the N-terminus of TL1.

*o Three ligands containing the TL2 N-domain, native TL2, 2N1C1F and 2N2C1F) were probed with a rabbit antibody specific for the Nterminus of TL2. The results demonstrated that the COS7 cells were expressing any molecule containing the N-domain of TL2 at roughly ten times the level of any molecule containing the TL1 N-domain, 5 regardless of the makeup of the rest of the protein. The conclusion was that the N-domain must principally control the level of expression of the ligand.

The next question addressed was the chimeras' ability or inability to o activate the TIE-2 receptor. EAhy926 cells were challenged with the four chimeras, as well as TL1 as a positive control for phosphorylation and TL2 or an empty pJFE14-transfected COS7 cell supernatant as negative controls for phosphorylation. The cells were lysed, and the TIE-2 receptor was immunoprecipitated out of the cell lysate and run on an SDS-PAGE. The samples were Western blotted and probed with an anti-phosphotyrosine antibody to detect any receptors that had been phosphorylated. Surprisingly, only the constructs containing the TL1 fibrinogen-like domain (2N1C1F and 2N2C1F) could phosphorylate the TIE-2 receptor. Thus, although the N-terminal region of TL1 is essential for activation, it can be replaced by the N-terminal region of TL2, the information that determines whether the ligand is an agonist or an antagonist is actually contained in the fibrinogen-like Sdomain.

Thus it was determined that the F-domain, in addition to binding the TIE-2 receptor, is responsible for the phosphorylation activity of TL1.

Further, when TL2, an otherwise inactive molecule, was altered by replacing its F-domain with the TL1 F-domain, the altered TL2 acted as an agonist.

The 2N1C1F construct was somewhat more potent, however. The signal caused by chimera 2N1C1F appeared slightly stronger than that of chimera 2N2C1F, leading to speculation that the C-domain of TL1, though not crucial for phosphorylation, might enhance the potency of TL1. However, since the samples used for the phosphorylation assay were not normalized in terms of the concentration of ligand, it was possible that a stronger phosphorylation signal only indicated the i* 20 presence of more ligand. The phosphorylation assay was therefore repeated with varying amounts of ligand to determine whether the active chimeras displayed different potencies. The concentration of ligand in the COS7 supernatants of ligand transfections was determined through BIAcore biosenser technology according to methods previously described (Stitt, et al. (1995) Cell 80: 661-670).

BIAcore measured the binding activity of a supernatant to the TIE-2 receptor in arbitrary units called resonance units Fairly good correlation between RU's and ligand concentration has been generally observed, with 400 RU of activity corresponding to about 1 pg of protein per mL of supernatant. Samples were diluted to concentrations of 100 RU, 20 RU, and 5 RU each and the phosphorylation assay was repeated. The results demonstrated that chimera 2N2C1F was clearly more potent than either the native TL1 or chimera 1N1C2F at the same concentrations.

Another interesting aspect of these exchange constructs is in their levels of expression. Each of the four chimeras was tested for its 0o level of production in COS cells, its ability to bind to TIE2, and its ability to phosphorylate TIE2. The results of these experiments showed that chimeras 1 and 3 were produced at levels comparable to TL1, whereas chimeras 2 and 4 were produced at levels comparable to TL2. Thus a high level of protein production was correlated with the 1 5 TL2 N-terminal domain. Additionally, when tested on endothelial EAhy926 cells, chimeras 2 and 4 were active, whereas 1 and 3 were not. Thus activity (phosphorylation of the receptor) correlates with the TL1 fibrinogen-like domain. Chimeras 2 and 4 therefore each had the desirable properties of high production levels as well as agonist i o activity.

23.4. Proteolytic resistant constructs Based on the observation that a large fraction of TL1 preparations was often proteolytically cleaved near the N-terminus, it was proposed that an arginine residue located at position 49 of the mature protein (see Figure 17) was a candidate cleavage site that might be involved in the regulation of the protein's activity in vivo, and that replacing the arginine with a serine (R49might increase the stability of the protein without necessarily affecting its activity. Such a mutant of TL1 was constructed and was found to be about as active as the native TL1 but did not exhibit resistance to proteolytic cleavage.

23.5. Combination mutants The most potent of the chimeric constructs, 2N1C1F, was additionally altered so that the cysteine encoded by nucleotides 784-787 as shown in Figure 27 was converted to a serine. This molecule (denoted 2N1C1F (C246S)) was expressed well, potently activated the receptor, was-resistant to proteolytic 1 cleavage and was primarily dimeric, rather than higher-order multimeric. Thus the 2N domain appeared to confer protease resistance on the molecule. Finally, this molecule was further altered to eliminate the potentially protease sensitive site encoded by nucleotides 199-201 as shown in Figure 27, to give a molecule (denoted 2N1C1F (R51->S,C246->S)) which was expected to be activating, well expressed, dimeric, and protease resistant.

'.Table 1 summarizes the modified TIE-2 ligand constructs that were made and characterizes each of them in terms of ability to bind the i. 20 TIE-2 receptor, ability to activate the TIE-2 receptor, the type of structure formed (monomer, dimer, etc.) and their relative production levels. Unmodified TL1 (plain) and TL2 (striped) are shown with the three domains as boxes. Thus striped boxes indicate domains from TL2.

The cysteine located at position 245 of the mature TL1 protein is indicated by a An through the indicates that that cysteine residue was substituted for by another amino acid as in, for example, the TL1 CYS- mutant. Similarly, an through the in the last construct indicates the substitution for an Arg residue at position 49 of the mature TL1 protein. The is present in one modified TL2 construct showing the TL2 CYS mutant. Constructs having Fc tails or flag tagging are also indicated.

Based upon the teachings herein, one of skill in the art can readily see that further constructs may be made in order to create additional modified and chimeric TIE-2 ligands which have altered properties.

For example, one may create a construct comprised of the N-terminal domain of TL2 and the F-domain of TL1 fused with the Fc section of Shuman antibody )gG1. This construct would be expected to bind and activate the TIE-2 receptor. Similarly, other constructs may be created using the teachings herein and are therefore considered to be S within the scope of this invention.

23.6.Materials and Methods Construction of Chimeras Swapping constructs were inserted into a pJFE14 vector in which the Xbal site was changed to an Ascl site. This vector was then digested with Ascl and Notl yielding an Ascl-Notl backbone. DNA fragments for the chimeras were generated by PCR using appropriate oligonucleotides.

The FLAG-1C1F and FLAG-2C2F inserts were subcloned into a pMT21 vector backbone that had been digested with EcoRI and Notl. The "CF" 2 5 truncations were obtained through PCR, and the FLAG tag and a preceding trypsin signalling sequence were constructed by annealing synthetic oligonucleotides.

I ranstections All constructs were transfected transiently into COS7 cells using either DEAE-Dextran or LipofectAMINE according to standard protocols.

Cell cultures were harvested 3 days after the transfection and spun down at 1000 rpm for 1 minute, and the supernatants were transferred to fresh tubes and stored at -20 0

C.

Staining of FLAG-1C1 F-Transfected and FLAG-2C2F-Transfected Cells 6-well dishes of COS7 cells were transfected transiently with the 1 0 TIE-2 receptor. The COS7 supernatant from various ligand tansfections was incubated on the cells for 30 minutes, followed by two washes with Phosphate Buffered Saline (PBS) without magnesium or calcium. The cells were fixed in -20 0 C methanol for 3 minutes, washed once with PBS, and incubated with anti-FLAG M2 antibody (IBI;1:3000 dilution) in PBS/10% Bovine Calf Serum (BCS) for minutes. The cells were washed once with PBS and incubated with goat anti-mouse IgG Alkaline Phosphatase (AP) conjugated antibody (Promega;1:1000) in PBS/10% BCS. The cells were washed twice with PBS and incubated with the phosphate substrate, BCIP/NBT, with 1mM levamisole.

Phosphorylation Assays Dilution of COS7 supernatants for the dose response study was done in the supernatants of COS7 cells transfected with the empty vector pJFE14. EA cells that naturally express the TIE-2 receptor were starved for >2 hours in serum-free medium, followed by challenge with the appropriate COS7 supernatant for 10 minutes at 37 0 C in an atmosphere of 5% C02. The cells were then rinsed in ice-cold PBS and lysed with 1% NP40 lysis buffer containing protease inhibitors p.g/ml leupeptin, 10 ilg/ml aprotinin, 1mM PMSF) followed by immunoprecipitation with an antibody specific for the TIE-2 receptor.

Samples were then subjected to immunoblot analysis, using anti pTyr antibodies.

Dot Blots Samples were applied to a nitrocellulose membrane, which was blocked and probed with the appropriate antibodies.

SO

23.7 Production of Chimeric Tie-2 Ligand from CHO and Baculovirus Infected Insect Cells Virus Production 5 The gene for the chimeric ligand (denoted 2N1C1F (C246S)) was engineered into a baculovirus expression plasmid and recombined with viral DNA to generate recombinant baculovirus, amplified and S harvested using methods previously described (O'Reilly, L.K.

Miller, and V.A. Luckow, Baculovirus Expression Vectors A Laboratory i.0 o Manual 1992, New York: W.H. Freeman). SF21 insect cells (Spodoptera frugiperda) obtained from Invitrogen were adapted and expanded at 27 0 C in Gibco SF900 II serum-free medium. Uninfected cells were grown to a density of 1x106 cells/mL. Cell density was determined by counting viable cells using a hemacytometer. The virus stock for the ligand was added to the bioreactor at a low multiplicity 0.01-0.1 PFU/cell to begin the infection. The infection process was allowed to continue for 3-4 days allowing maximum virus replication without incurring substantial cell lysis. The cell suspension was aseptically aliquoted into sterile centrifuge bottles and the cells removed by centrifugation (1600 RPM, 30 min). The cell-free supernatant was collected in sterile bottles and stored at 4°C in the absence of light until further use.

.0 6*@OsS 6666 4@S* 66 66

C

6066

C

6 *9*C 966 96 66 C c..

6 The virus titer was determined by plaque assay as described -by O'Reilly, Miller and Luckow. The method is carried out in tissue-culture dishes which are seeded with 1.5x106 cells. Serial dilutions of the virus stock are added to the attached cells and the mixture incubated with rocking to allow the virus to adsorb to individual cells. An agar overlay is added and plates incubated for days at 27°C. Viable cells were stained with neutral red revealing circular plaques which were counted to give the virus titer expressed in plaque forming unit per milliliter (PFU/mL).

Infection of Cells for Protein Production Uninfected SF21 cells were grown in tissue culture plates, and virus containg the chimeric ligand gene was added at a multiplicity of 1-10 pfu/cell. The virus was allowed to adsorb for 90 minutes at 27C with gentle rocking, after which the cells were refed with fresh amounts of Sf-900 II serum-free medium. After 3 days of growth at 27C, tissue culture fluids were harvested, and the ligand detected by immunoblotting.

CHO expression of Tie-2 ligand chimeras Tie-2 ligand chimeras were cloned into any of several mammalian cell expression vectors, including (but not limited to) pJFE, pcDNA3, 100 pMT21, pED or others. Plasmids were transfected into CHO DG44 cells (Urlaub, G. and Chasin, L.A. 1980.. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad.

Sci. U.S.A. 77:4216-4220; Urlaub, Kas, Carothers, and Chasin, L.A. 1983. Deletion of the diploid dihydrofolate locus from cultured mammalian cells. Cell 33:405-412) by calcium phosphate preciptation or cationic liposomes. In the case of vectors lacking a dhfr selectable marker, the plasmid pSV2.dhfr was cotransfected at a molar ratio to the plasmid containing the TIE ligand chimera.

SDHFR+ cells were selected by growth in selection medium (a medium lacking nucleosides and nucleotides containing 10% dialyzed fetal calf serum), and clones screend for production of chimeric TIE ligands by immunoblotting with a TIE2 receptor body. Clones expressing the desired protein were subjected to several rounds of gene amplification "1.5 using graded concentrations of methotrexate in selection medium.

Highly expressing clones were identified after gene amplification by similar immunoblotting techniques.

Cell lines expressing chimeric TIE ligands were cultured in monolayers, suspension flasks, roller bottles, and bioreactors in selection medium or in medium lacking selection, and can be grown in serum-free medium formulations.

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

101 TABLE 1 MUJTATION ANALYSIS OF TIE LIG ANDS N cOILED-0OIL RIBRINOGEN4

LIKE

T~C

TL2 HKM LO

ORDER

DM1M IGH DMM31 LOW ORDER

HG

9 N.D. N.D. LOW N.D. N.D. IGH MONOE HIGH MONOE IGH DIE HIG DMAE I-UG HIGH-EST PRODUCTION OF RUJ MOST POTENTLY ACTIVATING N.D. NOT DETERMINED I ICl Fe Ey-zzzzzl,6 Fe flag- 1 c

ORDER

ORDER

LOW

N.D. LOW flagp C x111 N.D. IGH N.D. I-m- N H-IGH- N.D. LOW

C

N.D. HI-UI- N.D. LOW N.D. IUGH DOER IGH N.D. LOW Lx ~Cl

DEPOSITS

The following have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 in accordance with the Budapest Treaty. A plasmid clone encoding a TIE- 2 ligand was deposited with the ATCC on October 7, 1994 and designated as "pJFE14 encoding TIE-2 ligand" under ATCC Accession No. 75910. Recombinant Autographa californica baculovirus encoding TIE-2 receptorbody was deposited with the ATCC on October 7, 1994 Sand designated a§ "vTIE-2 receptorbody" under ATCC Accession No.

VR2484. A lambda phage vector containing human tie-2 ligand DNA was deposited with the ATCC on October 26, 1994 and designated as "lgt10 encoding htie-2 ligand 1" under ATCC Accession No. 75928. A S plasmid clone encoding a second TIE-2 ligand was deposited with the '1*5 ATCC on December 9, 1994 and designated as "pBluescript KS encoding human TIE 2 ligand 2" under ATCC Accession No. 75963. E. coli strain containing plasmid pBeLoBacll with a human TL-4 gene insert encoding human TIE ligand-4 was deposited with the ATCC on July 2, 1996 and designated as "hTL-4" under ATCC Accession No. 98095.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

103 SEQUENCE

LISTING

(1GENERAL

INFORMATION

APPLICANT: REGENERON PHARMACEUTICALS,

INC.

(LI) TITLE OF THE INVENTION: NOVEL MODIFIED

LIGANDS

(ILL) NUMBER OF SEQUENCES: 28 (iv) OORRESPONDENCE

ADDRESS:

A4DDRESSEE: Regeneron Pharmaceticals, Inc.

STREET: 777 Old Saw Kill Road CITY: Tarrytown STATE: NY COUNTRY I USA ZIPt 10591 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ Vers~ionl CURNT APPLICATION

DATA:

APPLICATION NUMBER: NOT YET KNOWN FILING DATE: FILED HEREWITH

CLASSIFICATION-:

(vii) PRIOR APPLICATION

DATA:

ka) -APPLICATION NUMBER: USSN 08/740,22-1 FILING DATE: 25-OCT-1996

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: USSR 60/022/999 FILING DATE: 02-AUG-1996 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Cobert, Robert J REGISTRATION NUMBERt 36,108 REFERENCE/DOCKET NUMBER: REG 333 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 914-345-7400 TELEFAX: 914-345-7721 INFORMATION FOR SEQ ID NO:lt SEQUENCE

CHARACTERISTICS:

LENGTH: 2149 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CodIng Sequence LOCATION: 3 .1803 OTHER INFORMATION: NAME/KEY: Human TIE-2 ligand 1 LOCATION: .2149 OTHER INFORMATION: from clone 1gtlO encoding htie-2 ligand 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CAGCTGACTC AGGCAGGCTC GCTACTATGC AATAAATATC AAAATTTTAA AATTTTAGAA AACGCTTTCT TTGAGGGGGA CTAGTTTTAG AGGTCAGAAG GGCAGTACA ATG ACA GTT Met Thr Val.

1 CATGCTGAAC GGTCACACAG AGAGGAAACA ATAAATCTCA TCAAGTTTTA ACGAAGAAAA ACATCATTGC AGTGAAATAA CAAAGCTAAC AAATGGCTAG TTTTCTATGA TTCTTCTTCA AAGAGTCAAA CAAACAAGCA GTTTTACCTG AAATAAAGAA AAAGGAGCAA GTTTTGCGAG AGGCACGGAA GGAGTGTGCT TTC CTT TCC TTT OCT TTC CTC OCT GCC ATT CTG Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu 5

ACT

Tkxr CAC ATA GO TGC His Ile Giy Cya

AGC

Ser 20 AAT CAG COC CGA Aen Gln Arg Arg

PLOT

Ser CCA GAA AAC AGT Pro Giu Aen Ser AGA AGA TAT AAC Arg Arg Tyr Asn

CGG

Arg ATT CAA CAT CG Ile Gin His Gly

CAA

Gin 40 TGT GCC TAC ACT Cys Ala Tyr Thr TTC ATT Phe Ile 120 180 240 300 351 399 447,- 495S 543 591 639 687 CTT CCA GAA Leu Pro Giu AAC ACA AAC Asn Th~r Asn

CAC

His s0 GAT GGC AAC TGT Asp Gly Aen Cys

CGT

Arg 55 GAG AGT ACG ACA Giu Ser Thr Thr GAC CAG TAC Asp Gin Tyr CCG GAT TTC Pro Asp Phe OCT CTG CAG AGA Ala Leu. Gin Arg

GAT

Asp 70 OCT CCA CAC OTO Ala Pro His Val

GPA

Giu TCT TCC Ser Ser CAG AAA CTT CAA Gin Lys Leu Gin

CAT

His 85 CTG GAA CAT GTG Leu Giu His Val

ATG

Met GAA AAT TAT ACT Giu Aen Tyr Thr

S

CAG

Gin TOG CTG CAA AAA CTT GAG AAT TAC ATT GTG OPLA AAC ATG PLAG TCG Trp Leu Gin Lys Leu Giu Aen Tyr Ile Val Giu Aen Met Lys Ser 100 105 110 GAG ATO GCC CAG Giu Met Ala Gin

ATA

Ile 115 CAG CAG PLAT GCA Gin Gin Asn Ala

OTT

Val 120 CAG AAC CAC ACO Gin Aen His Thr OCT ACC Ala Thr 125 ATG CTG GAG Met Leu Giu AGA AAG CTG Arg Lye Leu 145

ATA

Ile 130 GGA ACC AGC CTC Gly Thr Ser Leu

CTC

Leu 135 TCT CAG ACT OCA Ser Gin Thr Ala GAG CAG ACC.

Glu Gin Thr 140 ACT TCT CGA Thr Ser Arg ACA OAT OTT GAG Thr Asp Val Giu

ACC

Thr 150 CAG GTA CTA AAT Gin Val Leu Asn

CAA

Gin 155 CTT GAG Leu Giu 160 ATA CAG CTG CTG Ile Gin Leu Leu

GAG

Giu 165 AAT TCA TTA TCC Asn Ser Leu Ser

ACC

Thr 170 TAC AAG CTA GAG Tyr Lye Leu Oiu 735 783 831 879 9 27

AAG,

Lys 175 CAA CTT CTT CAA Gin Leu Leu Gin

CAG

Gin 1S0 ACA AAT GAA ATC Thr Asn Glu Ile

TTG

Leu 185 AAG ATC CAT OAA Lys Ile His Glu Lys 190 PLAC AGT TTA TTA Asn Ser Leu Leu CAT AALA ATC TTA His Lys Ile Leu GAA ATG GAA GGA AAA CAC AAG Oiu Met Giu Gly Lys His Lys 200 205 GAA GAG TTG Giu Glu Leu GTT ACT CGT Val Thr Arg 225 GAC ACC TTA AAG GAA GAG Asp Thr Leu Lys Giu Giu 210 215 AAA GAG AAC CTT Lys Giu Asn Leu CAA GGC TTG Gin Giy Leu 220 CAA TTA AAC Gin Leu Aen CAA ACA TAT ATA Gin Thr Tyr Ile

ATC

Ile 230 CAG GAG CTG GAA Gin Glu Leu Giu AGA GCT Arg Ala 240 ACC ACC AAC AAC Thr Thr Asn Asn

AGT

Ser 245 GTC CTT CAG AAG Val Leu Gin Lye

CAG

Gin 250 CAA CTG GAG CTG Gin Leu Glu Leu ATG GAC ACA GTC CAC AAC CTT GTC AAT CTT TGC ACT AAA GAA GOT GT Met Asp Thr Val Hie Aen Leu Val Aen Leu Cys Thr Lys Giu Gly Vai 255 260 265 270 TTA CTA AAG GGA Leu Leu Lys Gly

GGA

0 ly 275

CAA

Gin AAA AGA GAG GAA Lys Arg Oiu Oiu AAA CCA TTT AGA Lys Pro Phe Arg GAC TGT Asp Cys 285 975 1023 1071 1119 1167 1215 1263 1311 1359 GCA GAT GTA Ala Asp Val TATATT AAT Tyr Ile Asn 305

TAT

Tyr 290 OCT GOT TTT Ala Gly Phe

AAT

Aen 295 AAA AGT OGA ATC Lys Ser Gly Ile TAC ACT ATT Tyr Thr Ile 300 AAT ATG GAT Aen Met Asp AAT ATG CCA GAA Aen Met Pro Giu

CCC

Pro 310 A.AA AAG GTG TTT Lys Lye Vai Phe

TGC

Cys 315 GTC AAT Val Aen 320 GOO GGA GGT TG Gly Gly Gly Trp

ACT

Thr 325 OTA ATA CAA CAT Val Ile Gin His CGT GAA OAT GGA Arg Giu Asp Giy 330 ATG GOT TTT GGA Met Gly Phe Gly

AGT

Ser

AAT

Asn 350

CTA

Leu 335 OAT TTC CAA AGA Asp Phe Gin Arg

GGC

Oly 340 TGG AAG GAA TAT Trp Lys Giu Tyr

AAA

Lys 345 CCC TCC GOT GAA Pro Ser Gly Glu TAT TGG CTG 000 AAT GAG TTT ATT TTT GCC ATT ACC Tyr Trp Leu Gly Asn Giu Phe Ile Phe Ala Ile Thr 355 360 365 1407 AGT CAG AGO Ser Gin Arg AAC CGA GCC Asn Arg Ala 385 CAG TAC ATO CTA Gin Tyr Met Leu 370 TAT TCA CAG TAT Tyr Ser Gin Tyr

AGA

Arg

GAC

Asp 390

ATT

Ile 375 GAG TTA ATG GAC Oiu Leu Met Asp TGG GAA 000 Trp Oiu Gly 380 AAT GAA AAG Asn Giu Lys AGA TTC CAC ATA Arg Phe His Ile

GGA

Gly 395 CAA AAC Gin Asn 400 TAT AGO TTG TAT Tyr Arg Leu Tyr

TTA

Le u 405 AAA GOT CAC ACT Lys Gly His Thr 000 Gly 410 ACA GCA GGA AAA Thr Ala Giy Lye

CAG

Gin 415 AGC AGC CTG ATC Ser Ser Leu Ile

TTA

Leu 420 CAC GOT OCT OAT His Gly Ala Asp

TTC

P he 425 AGC ACT AAA OAT Ser Thr Lys Asp

OCT

Ala 430 1455 1503 1551 1599 1647 1695 1743 OAT AAT GAC AAC Asp Asn Asp Aen

TOT

Cys 435 ATO TGC AAA TOT Met Cys Lys Cys 0CC Ala 440 CTC ATO TTA ACA Leu Met Leu Thr OGA OGA Oly Oly 445 TOO TOO TTT Trp Trp Phe ACT 000 GGA Thr Ala Gly 465 OCT TOT GOC CCC TCC AAT CTA AAT Ala Cys Oly Pro Ser Asn Leu Asn GGA ATO TTC TAT Gly Met Phe Tyr 460 CAA AAC CAT OGA Gin Asn His Gly AAA CTO AAT 000 ATA A.AG TGO CAC TAC Lys Leu Aen Giy Ile Lye Trp His Tyr 470 475 TTC AAA GGG CCC AGT TAC TCC TTA CGT TCC ACAL ACT ATG ATG ATT CGA 1791.

Phe Lys Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg 480 485 490 CCT TTA GAT TTT TGA AAG CGCA ATGTCAGAAG CGATTATGAA AGCAACAAAG AAATC 1848 Pro Leu Asp Phe 495 CGGAGAAGCT GCCAGGTGAG AAACTGTTTG AAAACTTCAG AAGCAAACAA TATTGTCTCC 1908 CTTCCAGCAA TAAGTGGTAG TTATGTGAAG TCACCAAGGT TCTTGACCGT GAATCTGGAG 1968 CCGTTTGAGT TCRCAAGAGT CTCTACTTGG GGTGACAGTG CTCACGTGGC TCGACTATAG 2028 AAAACTCCAC TGACTGTCGG, GCTTTAAAAA GGGAAGAAAC TGCTGAGCTT GCTGTGCTTC 2088 ARACTACTAC TGGACCTTAT TTTGGAACTA TGGTAGCCAG ATGATAAATA TGGTTAATTT 2148 C 2149 4 4* INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 498 amino acids TYPE./amino acid STRANDEDNESS: single (D3) TOPOLOGYt linear (ii) MOLECULE TYPE: protein '-v)FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Human TIE-2 ligand 1 LOCATION: 498 OTHER INFORMATION: f rom clone XgtlO encoding htie-2 ligand 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Thr Val Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His M~ t- 1 Ile G Tyr A Glu t Asn Gin Leu4 Ala Giu Leu 145 Ile Leu Leu Leu Arg 225 Thr Thr 5 10i ly ~sn is kl a Lays Gin Ile 130 Thr Cys Arg Asp Leu Leu Lys Ile 115 Gly *Asp Ser Ile G ly Gin GI .n Leu 100 Gin Thr Val ken Gin Aen Arg His 85 Glu Gin Ser Giu G in His Cys Asp 70 Leu Asn Asn Leu Thr Arg G ly Arg 55 Ala Giu Tyr Ala *Leu 135 *Gin Arg Ser Pro Glu ken Ser Gin 40 Giu Pro His Ile Val.

120 Ser Val Cys Ala Ser Thr His Val.

Val Met 90 Val Giu 1.05 Gin ken Gin Thr Leu ken Tyr Thr Glu 75 Giu ken His Ala Gir 155 Thr Asp 60 Pro ken Met Thr Giu 140 Thr.

Phe 3P Tyr Lys Ala 125S Gin Ser Ile Tyr Phe Thr Scr 110 Thr Thi Ar Arg Arg Leu Pro ken Thr Ser Scr Gin Trp Giu Met Met Leu Arg Lye ;Leu Glu 160 a Lye Gin 175 s ken Ser 0 s Giu Giu u Val Thr .n Arg Ala 240 !u Met Asp 255 il Leu Leu 150 Gin Leu Leu Asp 210 Gin Thr Val Leu Gin G iu 195 Thr Thr ken His Leu Gin 180 His Leu Tyr As n Aen Glu 165 Thr Lye Lye Ile Ser 245 Leu Asn Aen lie G iu Ile 230 Val1 Val Ser G iu Leu Giu 215 Gin Leu Aen Leu Ile Giu 200 Lys G iu Gin Leu Ser Leu 185 Met Giu Leu Lys Cy e 170 Lys Glu Gin 250 Thr Tyr Ile Gly Leu Lye 235 Gin Lye Lye Leu G1, His Giu Ly 19 Lye His Ly 205 Gin Gly Le 220 Gin Leu As Leu Giu Le Giu Gly Ve Lys GlyC Val Tyr 290 Asn Asn 305 Gly Gly Phe Gin Gly Glu Arg Gin 370 Ala Tyr 385 Tyr Arg Ser Leu Asp Aen Phe Asp .450 Gly Giln 465 Gly Pro Asp Phe 260 ;iy Lys Iln Ala M1et Pro Gly Trp Arg Gly 340 Tyr Trp 355 Tyr Met Ser Gin Leu Tyr Ile Leu 420 Cys Met 435 Ala Cys Asn His 265 Lys Arg Glu Giu Giu 280 Pro Phe Arg Asp 285 270 CyS Ala Asp Ile Tyr Ile Gly G lu Thr 325 Trp Leu Leu Tyr Leu 405 His Cys G ly Gly Phe Pro 310 Val Lys Gly Arg Asp 390 Lys G ly Lys Pro Lys 470 ksn 29 5 -'ye Ile Gliu Ile 37S Arg Giy Ala Cys Ser 455 Lys Ser Gly Ile Lys Gin Tyr G lu 360 G iu Phe His Asp Ala 440 Aen Aern Val Phe4 His Arg 330 Lye Met 345 Phe Ile Leu Met His Ile Thr Gly 410 Phe Ser 425 Leu Met Leu Aen Gly Ile Cys 315 Glu Gly P he Asp G ly 395 Thr Thr Leu LyE 471 Tyr '1 300 Aen Asp Phe4 Ala Trp 380 Aen Ala Lye Thr Met 460 Trp hr 4et Ily Gly Ile 365 Glu Giu G ly Asp Gly 445 Phe Hig Asp Ser Aen 350 Thr G ly Lye Lys Ala 430 Gly Tyr Tyr Val Leu 335 Pro Ser Aen Gin Gin 415 Asp Trp *Thr *Phe Pro- 495 320 Asp Ser G In Acrg Aen 400 Ser ken Trp Ala Lys 480 Leu 0 .0 0 0 V.00.

0 ser Tyr Ser 485 Leu Arg Ser Thr Thr 490 Met Met Ile Arq INFORMATION FOR SEQ ID NO:3: SEQUENCE CHAR~ACTERISTICS: LENGTH: 2146 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (Ui) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 310. 1800 OTHER INFORMATION: NAME/KEY: Human TIE-2 ligand 1 LOCATION: .2146 OTHER INFORMATION: from T98G clone (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CAGCTGACTC AGGCAGGCTC CATGCTGAAC GGTCACACAG AGAGGAAACA ATAAATCTCA GCTACTATGC AATAAATATC TCAAGTTTTA ACGAAGAAAA ACATCATTGC AGTGAAATPA AAAATTTTAA AATTTTAGAA CAAAGCTAAC AAATGGCTAG TTTTCTATGA TTCTTCTTCA AACGCTTTCT TTGAGGGGGA AAGAGTCAAA CAAACAAGCA GTTTTACCTG AAATAAAGAA CTAGTTTTAG AGGTCAGAAG AAAGGAGCAA GTTTTGCGAG AGGCACGGAA GGAGTGTGCT GGCAGTACA ATG ACA GTT TTC CTT TCC TTT GCT TTC CTC GCT GCC ATT CTG Met Thr Val Phe Leu. Ser Phe Ala Phe Leu Ala Ala Ile Leu 1 5 ACT CAC ATA GGG TGC AGC AAT CAG CGC CGA AGT CCA GAA AAC AGT GGG Thr His Ile Gly Cye Ser Asn Gin Arg Arg Ser Pro Glu Aen Ser Gly is 20 25 120 180 240 300 351 399 AGA AGA TAT ARC Arg Arg Tyr Ren

CGG

Arg ATT CAR CAT GGG Il.e Gin His Gly TGT GCC TAC ACT Cys Ala Tyr Thr TTC ATT Phe Ile CTT CCA GAR Leu Pro Giu ARC ACA AAC Asn Thr RAn

CAC

His s0 GAT GGC AAC TGT Asp Gly Aan Cys

CGT

Arg 55 GAG AGT ACG ACA Giu Ser Thr Thr GAC CAG TAC Asp Gin Tyr CCG GAT TTC Pro Asp Phe GCT CTG CAG AGA Ala Leu Gin Arg

GAT

Asp 70 GCT CCA CAC GTG Ala Pro His Val

GAA

Giu 447 495 543 591 639 TCT TCC Ser Ser CAG AAA CTT CAA Gli Lys Leu Gin CTG CAA AAA CTT Leu Gin Lys Leu 100

CAT

His 85 CTG GAA CAT GTG Leu Giu His Val

ATG,

Met GRA AAT TAT NCT- Giu Asn Tyr Thr

CAG

Gin

TG

Trp GAG RAT TAC ATT Giu'Asn Tyr Ile

GTG

Val 105 GAA ARC ATO, RAG Giu Asn Met Lys

TCG

Ser 110

C

GAG ATO GCC Giu Met Ala ATG CTG GAG Met xleu Giu AGA RAG CTG Arg Lys Leu 145 CAG ATA CAG Gin'Ile Gin 115 CR0 RAT GCA Gin An Ala

GTT

Val 120 CAG ARC CAC ACG Gin An His Thr OCT ACC Ala'Thr 125

ATAGGA

Ile Gly 130 ACC AGC CTC Thr Ser Leu

CTC

Leu 135 TCT CAG ACT GCA Ser Gin Thr Ala GAG CR0 ACC Giu Gin Thr 140 ACT TCT CGR Thr Ser Arg ACA GAT GTT GAG Thr Asp Val Giu

ACC

Thr 150 CR0 GTA CTA RAT Gin Val Leu Asn

CAA

Gin 155 CTT GAG Leu Olu 160 ATA CRC CTG CTG Ile Gin Leu Leu

GAG

0 iu 165 RAT TCA TTA TCC Asn Ser Leu Ser

ACC

Thr 170 TAC. RAG CTA GAG Tyr Lys Leu Giu 687 735,' 783 831 879 927 975 1023

RAG

Lys 175 CAA CTT CTT CAA Gin Leu Leu Gin

CAG

Gin 180 RCA RAT ORA ATC Thr An Giu Ile

TTG

Leu 185 RAG ATC CAT A Lye Ile His Olu RAC AGT TTA TTA An Ser Leu Leu

GAR

0 lu 195 CAT AAA ATC TTA His Lye Ile Leu

GAA

Giu 200 ATG OAR GGA AA Met Ciu Gly Lys CRC RAG His Lys 205 GRA GAG TTG Giu Giu Leu OTT ACT COT Vai Thr Arg 225

GAC

Asp 210 ACC TTA RAG OAR Thr Leu Lys Giu

GAG

0 iu 215 RAA GAG RAC CTT Lys Giu an Leu CAR GGC TTG Gin Gly Leu 220 CRA TTA AAC Gin Leu Rsn CRA RCA TAT ATA Gin Thr Tyr Ile

ATC

Ile 230 CAG GAG CTG GA Gin Giu Leu Giu

RAG

Lye 235 AGA OCT Arg Ala 240 ACC ACC ARC RAC Thr Thr an an

AGT

Ser 245 GTC CTT CAG RAG Val Leu Gin Lye

CAG

Gin 250 CAA. CTG. GAG .CTG Gin Leu Giu Leu 1071

ATG

Met 255 GAC ACA GTC CRC Rep Thr Val His

AC

An 260 CTT GTC RAT CTT Leu Val RAn Leu

TGC

Cys 265 ACT ARA GRA GTT Thr Lye Giu Val

TTA

Leu 270 CTA RAG GGA GOR Leu Lys Giy Gly

AA

Lys 275 AGA GAG GRA GAG Arg Giu Giu Giu Lys 280 CCA TTT AGA GAC Pro Phe Arg Asp TGT GCR Cye Aia 285 1119 1167 1215 OAT GTA TAT Asp Val Tyr

CA

Gin 290 OCT OCT TTT RAT Ala Gly Phe an

AA

Lys 295 ROT GGA ATC TAC ACT ATT TAT Ser Gly Ile Tyr Thr Ile Tyr 300 ATT AAT NAT ATG CCA GAA CCC AAA AAG GTG TTT Ile NAn NAn Met Pro Glu Pro Lys Lys Val Phe 305 310 TGC AAT Cys An 315 ATG GAT GTC Met Asp Val NAT GGG GGA GOT TOG ACT GTA ATA CAN CAT CGT GAA GAT GGA NOT CTA An Giy Gly Gly Trp Thr Val Ile Gin His Arg Giu Asp Gly Ser Leu 320 325 330

GAT

Asp 335 TTC CAN AGA GGC Phe Gin Arg Gly

TGO

Trp 340 AAG GAN TAT AAA Lye Giu Tyr Lys

ATG

Met 345 GGT TTT GGA ANT Giy Phe Giy NAn

CCC

Pro 350 TCC OCT GAN TAT Ser Gly Giu Tyr

TGG

Trp 355 CTG GGG ANT GAG Leu Gly hen Giu NTT TTT GCC ATT Ile Phe Ala I 1e ACC AGT Thr Mer 365 CAG AGO CAG Gin Arg Gin CGN GCC TAT Nrg Ala Tyr 385

TAC

Tyr 370 ATG CTA NOA ATT Met Leu Arg Ile

GAG

0 iu .375 TTA ATG GAC TGG Leu Met Asp Trp GAA GOG AAC Giu Oly Aen 380 GAA NAG CAA Giu Lys Gin 1263 1311 1359 1407 1455 1503 1551 1599 1647 TCN CAG TAT GAC Ser Gin Tyr Asp

NON

Nrg 390 TTC CAC ATA GGN Phe His Ile Gly

ANT

Asn 395 ANC TAT an'%Tyr 400 AGG TTG TAT TTA ANN GOT CAC ACT G00 ACA GCNA GGN ANN -CAG Arg Leu Tyr Leu Lye Gly*His Thr Gly Thr Ala Gly Lys Gin 405 410 NGC NOC CTG ATC TTA Ser Ser Leu Ile Leu CNC GOT GCT GAT His Gly Ala Asp 420 TOC ANN TGT GCC Cye Lys -Cys Ala

TTC

Phe

CTC

Leu 440

NOC

Ser 425 ACT AA OAT GCT Thr Lys Asp Ala

GAT

Asp 430 NAT GAC ANC TOT Asn Asp NAn Cys

ATO

Met 435 ATG TTN ACA GGA Met Leu Thr Gly CON TG Gly Trp 445 TOG TTT GAT Trp Phe Asp GCG GGA CAN Ala Gly Gin 465

OCT

Ala 450 TGT GGC CCC TCC Cys Gly Pro Ser

AT

An 455 CTN NAT GGA ATG Leu an Giy Met TTC TAT ACT Phe Tyr Thr 460 CAC TAC TTC His Tyr Phe ANC CAT CON AAN An His Arg Lys

CTG

Leu 470 ANT GGG ATA NAG an Oly Ile Lys

TG

Trp 475 1695 1743 1791 AA GGG Lye Oly 480 CCC ACT TAC TCC Pro Ser Tyr Ser

TTA

Leu 485 CGTV TCC ACA ACT Arg Ser Thr Thr ATG ATT CON CCT Met Ile Arg Pro TTA GAT TTT TON AAGCGCA NTGTCAGAAG CGATTATGNA AGCAACANAG AATCCGGA 1849 Leu Asp Phe 495 GAAGCTGCCA GGTGAGAAAC CACCANTAAG TGGTAGTTAT TTGAGTTCAC NAGAGTCTCT CTCCACTGNC TGTCGGGCTT TACTACTGGA CCTTATTTTG

TGTTTGAAAA

GTGANGTCAC

ACTTGGGGTG

TAAAGOGA

GAACTATGGT

CTTCAGAAGC

CAAGGTTCTT

NCAGTGCTCN

AGAAACTGCT

AGCCAGATGA

AAACAATATT

GACCGTGANT*

CGTGGCTCGA

GAGCTTGCTG

TAAATATGGT

GTCTCCCTTC

CTGGAGCCGT

CTNTAGNAA

TGCTTCAAAC

TAATTTC

1909 1969 2029 2089 2146 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 497 amino-acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal.

(ix) FEATURE: NAME/KEY: Human TIE-2 ligand 1 LOCATION: 1..-2146 OTHER INFORMATION: from T98G clone Met

I

IlIe (xi) SEQUENCE DESCRIPTION: SEQ ID Thr Val. Phe Leu Ser Phe Ala Phe Leu 5 10 Gly Cys Ser Aen GIn Arg Arg Ser Pro 25S NO: 4: Ala Ala Giu Asn Tyr Thr 0 Tyr Aen Ar 3S Giu His As so Aen Ala Le GIn Lys L( Leu Gin L~ Ala.Qin I 1 Giu. Ile G 130 Leu Thr A 14S Ile Gin L Leu Leu G Leu Leu G 1 Leu Asp Tx 210 Arg Gin I1 22S Thr Thr I Thr Val Giy Gly Tyr Gin 290 Aen Met 305 Gly Gly Gin Arg Giu Tyr Gin Tyr 370 Tyr Ser 385 Arg Leu Leu Ile Asn CyG Asp Ala 'p (a L e 15 ly sp eu ilu .95 !hr ~ht 271 P r Tr Gi1 Tr He G I 4:

C'

I

Gly A~ Gin A~ Gin H Leu G, 100; Gin G Thr S Val G Leu G

IS

His I Leu Tyr i Aen Asn 260 Arg a Gly o Giu p Thr y Trp 340 p Leu 5 .t Leu .n Tyr rr Leu ~u His 420 at Cys 35 (a Gly i-s 5 i n e r iu liu .65 .ys .ye EIle 24! Le~ G I: Phi P r Va 32 Ly G I Ar

AS

4C P2 Cys Asp 70 Leu Aen Asn Leu Thr

ISO

Asn Asr Gi% 234 *Va i V& jGi e As o Ly 31 1 I 5 s Gi y As g I] p A: 3C ~sG -v A Arg GJ 55 Ala P~ Giu H.

Tyr I Ala V Leu S 135 Gin V Ser L Glu I Leu C aGiu 1 215 3 Gin 1 Leu I Asn u Giu Lys 29S Lys 0 e Gin .u Tyr ;n Glu .e Giu 375 :g Phe Ly His La Asp Le Gin His Giy GI n Lu le 20 er 'al .eu le iu ~00 "ye G I~ Le~ Lyi 284 Se.

Va Hi1 Ly P h 36 Le H I P1 Ser His Val Vai 105 Gin Gin Leu Ser Let~ 18! Met Gi1 Le' i Ly iCy 26 s Pr r Gi 1 Ph S At 8 Me 34 ~eI ~0 ~u H~ .5 1 ir G 'Ie S Thr Th Val. GI 7E Met GJ 90 Glu Ai Asn H.

*Thr A Aen G Thr T 170 Lys I Giu G i Asn I u1 Giu I a Gin 250 s Thr o Phe y Ile Le Cys :g Giu 330 at Gly I5 le Phe et Asp le Gly ly Thr 410 er Thr ~r As .u Pr Lu AS sn M.

is TI la G .1 in T yr L le H liy I ael. C .ysC Z3S ;In Lys Nrg Tyr Aen 315 Asp Phe Ala Trp Asn 395 Ala Lys 'p 3n at iu hr ye Lis Dys 31 As 30 me

GJ

31

G

G

A

C1 a Ala Ile Lei Ser G 1 Phe 11 Gin Ty Asp P h Tyr Ti' Lye Sc Ala TI 125 Gin TI Ser ILeu G Giu L 1 His L 205 *Gly L 0 *x Leu *i Glu I u Vai p Cys 285 *r Ile 0O ~t Asp -y Ser Ly Asn le Thr 365 lu Gly s0 iu Lye ly Lys an Ala a1 y e r Le Lr

LO

ir 9

'YE

Ty 27 Al Ty

A

4 Thr HI: Arg Ar~ Leu Pri Aen Th Ser Se Gin Tr Giu He Met Le Arg LI Leu G~ Lys G.

175 Asn S 3Giu G i Val -T n Arg A 2 u Met P 255 u Leu I 0 a Asp Ile LI Asn ?u Asp 335 ro Ser 50 er Gin en Arg in Asn In Ser 415 ~sp Aen 0 r r p r lu hr.

Lsp lai 3iy 320 Phe Gly Arg Ala Tyr 400 Ser Asp :0 Cys Ala Ser Aen Leu 440 Leu Met Leu Asn Gly Thr Met Gly Gly Trp Trp 445 Phe Tyr Thr Ala Phe Gly Gin 465 Pro Phe 450 455 Aen Hie Arg Lys Leu Aen Gly Ile Lys Trp 470 475 Ser Tyr ser Leu Arg.Ser Thr Thr Met Met 485 490 460 Hie Tyr Phe Lys Gly 480 Ile Arg Pro Leu Asp 495 INFORMATION FOR SEQ ID SEQUENCE CHAR.ACTERISTICS: LENGTH: 2282 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 357 1844 OTHER INFORMATION: NAME/KEY: Human TIE-2 ligand 2 LOCATION: l...2282 OTHER INFORMATION: from clone p~luescript KS encoding human TIE 2 ligand 2 (xi) SEQUENCE DESCRIPTION: SEQ ID 9*

GAATTCCTGG

TCTGGGGAGA

CCAAGTGAGC

AACACAGCAG

ACGGACCCAG

TGGACGTGTG

GTTGGTGTTT

GAGGAACAAA

AGGACTGTTC

TAAAAACCAG

CCATGGCAGC

TTTGCCCTCA

ATCTCCTCCC

GGACCGTIGAA

TTCCCACTGC

GTTTGCTACT

GTAGCAGCCC

AGTTTGCTAA

AGCCTTGAGG

AGCTGCTCTG

AATCTGACAG

GGAAAAAGAG

TGCGTTTCAG

GCTGCTGGTT

GAGGGAACAA

TAAAAGCTGA

TTTACTGCAT

GAAAGAGAAG

ACGGCAGCAG

TATTACTGAA

CACTGTAGGA

CACAGCCCTC

GCCTGGAGAG

ACTTTCATTG

CTCGGGACTC

GAAAGA ATG Met 1 120 180 240 300 359 407 455 TOG CAG ATT OTT Trp Gin Ile Val GCC TAT AAC. AAC Aia Tyr Aen Aen TTC TTT ACT CTG Phe Phe Thr Leu

AGC

Ser 10 TGT GAT CTT Cys Asp Leu TTT COG AAG Phe Arg Lys

AGC

Ser 25 ATG GAC AGC ATA met Asp ser Ile GTC TTG GCC GCA Val Leu Ala Ala OGA AAG AAG CAA Gly Lys Lys Gln CTC CTG CCA GAG Leu Leu Pro Glu TAT CAG Tyr Gin OTC CAG CAT GG Val Gin HIS Gly

TCC

Ser TGC AGC TAC ACT Cys Ser Tyr Thr

ATO

Met

CAG

Gin GAC AAC TOO CGC Asp Asn Cys Arg AGG GAC GCG CCG Arg Asp Ala Pro

TOT

Ser 55 TOO TCC AGO CCt Ser Ser Ser Pro

TAO

Tyr GTG TCC AAT GCT Val Ser Aen Ala 503, 551 599 CTC GAA TAC GAT Leu Giu Tyr Asp

GAC

Asp 75 TCG GTG CAG AGO Ser Val Gin Arg CTG CAA Leu Gin GTG CTG GAG Val Leu Giu GAG AAT TAT Glu Aen Tyr 100

AAC

Asn ATC ATG GAA AAC Ile Met Giu Asn

AAC

Aen 90 ACT CAG TOG CTA Thr Gin Trp Leu ATG AAG CTT Met Lye Leu GAG ATA CAG Glu Ile Gin 647 695 ATC CAG GAC AAC Ile Gin Asp Asn

ATO

Met 105 AAG A.AA GAA ATO Lys Lys Glu Met

GTA

Val 110 CAG AAT Gin Asn 1 15 GCA GTA CAG AAC Ala Val Gin Asn CAG ACG Gin Tkir 120 OCT GTG ATG Ala Val Met NTA OAk ATA 000 Ile Giu Ile Gly 125 AAG TTA ACT GAT Lys Leu Thr Asp

ACA

Thr

GTG

Val 145

AAC

Aen 130 CTG TTG AAC CAA Leu Lou ken Gin

ACA

Thr 135 GCT GAG CAA ACG Ala Glu Gin Thr

CGG

Arg 140 GAA GCC. CAA GTA Glu Ala Gin Val

TTA

Leu 150 AAT CAG ACC ACG ken Gin Thr thr

AGA

Arg 155 CTT OAk CTT CAG Leu Giu Leu Gin CTC TTG Lou Leu 160 OAk CAC TCC Glu His Ser ACC AGT GAA Thr ser Oiu 180

CTC

Leu 165 TCG, ACA AAC AAA Ser Thr Aen Lys

TTG

Leu 170 OAk AAA CAG ATT Giu Lys Gin Ile TTG GAC CR0- Leu Asp dln 175 CTA GAk kAG Leu Oiu Lys ATA AAC AAA TTG Ile ken Lys Leu CAR OAT Gin Asp 185 AkO AAC AGT Lys Aen Ser

TTC

Phe 190

S

AAG GTG Lye Val 195 CTA GCT ATG OAk Leu Ala Met Giu GAC RAG CAC RTC ATC CAA CTA CAG TCA ATA Asp Lys His Ile Ile Gin Lou Gin Ser Ile 200

AA

Lys 210 GAA GAG AAA OAT Glu Giu Lys Asp

CAG

Gin 215 CTA CAG OTO TTA Lou Gin Val Lou

GTA

Val 220 TCC AAG CAA AAT Ser Lys Gin.Asn

TCC

Ser 225 ATC. ATT OAA GAA Ile Ile Glu Giu

CTA

Lou 230 OAk AA AAA ATA Oiu Lys Lye Ile

GTG

Val 235 ACT 0CC ACG GTG Tkir Ala Thr Val RAT AAT Aen Asn 240 TCA GTT CTT Ser Val Leu

CAA

Gin 245 AAG CAG CAA CAT Lye Gin Gin Hie

GAT

Asp 250 CTC ATG GAG ACA Lou Met Giu Thr OTT AAT AAC Vai Aen Aen 255 CCC ACT GTT Pro Thr Val 743 791 839 887 93S 983.

103 1' 1079 1127 1175 1223 1271 1319 1367 1415 1463 1511 TTA CTG ACT ATO Lou Lou Thr Met 260.

ATO TCC ACA Met Ser Thr

TCA

Ser 265 ARC TCA OCT RAG ken Ser Ala Lys

GAC

Asp 270 OCT AA Ala Lye 275 OAR OAk CAA ATC Oiu Oiu Gin Ile TTC AGA GAC TOT Phe krg Asp Cys

OCT

Ala 285 OAk GTA TTC AAA Giu Val Phe Lye

TCA

Ser 290 GGA CAC ACC ACA Gly His Thr Thr

AAT

Asn 295 GGC ATC TAC ACG Oly Ile Tyr Thr

TTA

Lou 300 ACA'TTC CCT RAT Thr Phe Pro ken

TCT

Ser 305 ACA OAk GAG ATC Thr Giu iu Ile

AAG

Lys 310 0CC TAC TGT GAC Ala Tyr Cys Asp GRA OCT OGA GGA Oiu Ala Gly Oly GOC 000 Giy Gly 320 TOG ACA ATT Trp Thr Ile ACT TOG AAA Thr Trp Lye 340

ATT

Ile 32S CAG CGA COT GAG Gln krg Arg Glu

OAT

Asp 330 GGC AOC OTT OAT Gly Ser Val Asp TTT CAG AGO Phe Gin krg 335 GGk OAk TAT Gly Oiu Tyr GAA TAT AAA OTG Giu Tyr Lye Val 00k Gly 345 TTT GOT AAC CCT Phe Gly ken Pro

TCA

Ser 350 TOG CTG Trp Lou OCR AAT GAG TTT Gly ken Glu Phe TCG CAA CTG ACT Ser Gin Lou Thr

RAT

ken 365 CAG CAA CG TAT Gin Gin Arg Tyr

GTG

Val 370 CTT AA ATA CAC Lou Lye Ile His CTT AAA GAC TG Lou Lye Asp Trp 375 Giu Gly 380 AAT GAG OCT TAC ken Oiu Ala Tyr

TCA

Ser 385 TTG TAT OAk CAT Lou Tyr Glu His

TTC

Phe 390 TAT CTC TCA AGT A Tyr Lou Ser Sdr Glu 39S GAA CTC AAT TAT Giu Lou Aen Tyr AGO ATT Arg Ile 400 CAC CTT AAA His Leu Lye cAA CCA GGk Gin Pro Oly 420

GGA

G ly 405 CTT ACA GGG ACA Leu Thr Gly Thr

GCC

Ala 410 GGC AAA ATA AGC Gly Lye Ile Ser AGC ATC AGC Ser Ile Ser 415 AAT GAT TTT AGC ken Asp Phe Ser ACA AAG Thr Lye 425 GAT GGA GAC AAC GAC AAA TOT Asp Gly Asp ken Asp Lye Cys 430 ATT TOC Ile Cys 435 AAA TOT TCA CAA Lys Cys Ser Gin

ATG

Met 440 CTA ACA GGA GGC Leu Thr Gly Gly

TGG

Trp 445 TOG TTT GAT GCA Trp Phe Asp Aila

TGT

Cys 450 GOT CCT TCC AAC Gly Pro Ser ken

TTG

Leu 455 AAC GOA ATG TAC ken Gly Met Tyr

TAT

Tyr 460 CCA CAG AGO CAG Pro Gin Arg Gin AAc Aen 465 i5s9 1607 1655 1703 1751 1799 1849' 1909 1969 2029 2089 2149 2209 2269 2282 ACA AAT AAO TTC Thr ken Lye Phe

AAC

ken 470 GOC ATT AAA TG Gly Ile Lys Trp TAC TOO AAA GGC Tyr Trp Lye Gly TCA GGC Ser Oly 480 TAT.TCG CTC Tyr Ser Lou

AAG

Lye 485 GCC ACA ACC Ala Thr Thr ATG ATO Met- Met 490 ATC CGA CCA Ile Arg Pro OCA OAT TTC TAAAC' Ala-Asp Phe 495

ATCCCAGTCC

GAAAGTCAG

CGGGACCCAC

AACGGACCAA

AGATGAACCC

AATGTTATGT

ACAGATCATC

ATGTCCTGAA

ACCTGAGGAA

GCTGCGCACT

ATGCTCCAGA

AGCAAGACCC

GAGGCTGAGA

GCAAGTTTAT

TTGGAACTGC

TTC

CTGTCTCGAA

GTGTCCTCTT

TTAGAGCCTG

TAAACATCCA

ATCAGACTGA

CAGTAAATAA

ATTCTTCTGA

CTATTTTCAA

CCACCACAGA

TAAACTTTAT

TAATTGTGAT

CAGTTTACAG

CTGGAAAACA

GCACTGTTTA

AGACTTAAGC

GGGCGTGTGC

CACTTAAACT

TAGACAGAAC

ACGCTGCTGT

GAACACTTAT

TACACTGTGT

CCAGTGCACT

TCGGTGCTGA

TGCATCACTT

ACCTATGCAA

CACAACCAAG

GTTATACAAT

AAATACCCAT

*9 a INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 496 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (1x) FEATURE: NAME/KEY: Human TIE-2 ligand 2 LOCATION: 496 OTHER INFORMATION: from clone pBluescript KS encoding human TIE 2 ligand 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met 1 Ala Gin O iu Val O in Trp Gin Ile Ala Tyr Aen Tyr Gin Vai Met Asp Asn Val 5 Phe Phe Thr Leu Ser 10 Ser Met Cys Asp Lou Asp Ser Ile Aen Phe Arg Lys 25 Cy 5 Gin His Gly Ser 40 Ser Ser Tyr Thr Phe Val Val Leu Ala Gly Lys Lye Lou Leu Pro Ser ken Ala Gin Arg Lou Lou Met Lye Cys Arg Gln Ser 55 Leu Ser Ser Pro Tyr krg Asp Ala Pro 70 Ile Glu Tyr Asp Asp 75 Thr Ser Val Gin Trp Val Lou Glu ken Met Giu ken ken Leu Giu ken Tyr 100 Gln Gin ken Ala 115 Ile Val Gin Asp ken Met 105 90 Lye Lye Glu Met VI~i Giu Ile

LO

Lu Ile Gly G

C.

C Thr A

I

Val G 145 Leu C Gin I] Lye I Ile Ser 225 ken Asn Val Lys Ser 305 Gly Arg Tyr Tyr Ser 385 Ile Ser Cys Ala Asn 465 Gly asn .30 ilu ilu chr .ys ys 210 Ile Ser Leu Ala Ser 290 Thr Trp Thr Trj Val 37C Let His Iii Cyi 45 Th: Ty Leu I Ala C His Ser Val 195 Giu I Ile Val Leu Lys 275 Gly Glu Thr Trp Leu 355 Leu a Tyr Leu i Pro a Cys 435 a Gly r Asn r Ser jeu 1n 3er 3iu 180 Leu Glu GLu Leu Thr 260 Glu His GlU Ile Lys 340 Gly Lye Glu Lye G13 42( Lyt Prc Lyi Let ken G Val I Leu 1 165 Ile I Ala I Lye Glu Gin 245 Met Giu Thr Ile Ile 325 Glu ken Ile His Gly 405 r ken 3 CYB Ser s Phe j Lye 485 In ;In aeu Lso jer ksn 4et kep Leu 230 Lye Met Gln Thr Lye 310 GIr Tyx Gl His Ph 39( Le As] Se As As 47 Al ken G 1 Thr A 135 Asn G Thr A Lye L Glu P Gin I 215 Glu I Gin C Ser Ile Asn I 295 Ala Arg Lye Phe 3 Leu 375 a Tyr a Thr p Phe r Gln n Leu 455 n Gly 0 a Thr in Thr .en L .eu G spp !00 aeu C 4s I ;in rhr Ser 280 ly Tyr Arg Val Val 360 Lye Leu Gly Ser Met 440 Asn Ile Thr 'ys ,In .85 'ys ;In .ys {ie ser 265 Phe Ile Cye Glu Gly 345 Sex

AE

Se2 Th~ Thl 42! Le' Gi Ly Me Thr Arg 155 Leu Glu 170 Asp Lye His Ile Val Leu Ile Val 235 Asp Leu 250 ken Ser Arg Asp Tyr Thr Asp Met 315 Asp Gly 330 Phe Gil Gin Les Trp Gt.

Ser Gli 39! Ala GI, 410 Lye Aei 5 u Thr GI, y Met Ty s Trp Ty 47 in Thr Ala Val 20 la Giu Gin Thr Met Ile G 125 Arg Lye Li 140 Leu Giu L Lye Gin I ken Ser P 1 Ile Gin L 205 Val Ser L 220 Thr Ala T Met Giu I] Ala Lye I Cys Ala 285 Leu Thr 300 Glu Ala 4 Ser Val ken Pro i Thr Asn 365 i Gly Asn 380 a Glu Leu Lye lie p Gly Asp y Gly Trp 445 r Tyr Pro 460 r Tyr Trp eu eu le he eu 'ye !hr hr 'ep "lu Phe Gly Asj Set 35C G1: Asi Se: As 43 Tr G1 Ly Thr Gin Leu 175 Leu Gin Gin Val Val 255 Pro Val Pro Gl Phe 335 )Gl i G1: a All n Ty: r Se 41 n As 0 p Ph n Ar se GI Asp Leu 160 Asp Glu Ser ken ken 240 ken Thr Phe en Gly 320 Gin t Glu i Arg Tyr Arg 400 r Ile s p Lye e Asp g GIn .y Ser 480 a

S

*5 t Met 490 Ile Arg Pro Ala Asp Phe 495 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 478 amino acids TYPE: amino acid STPANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Mature TL1 protein LOCATION: 1.-.478 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID 140:7t 0 *s @0@0 9 0 0099 0900 09 99 S 9 9 0 9 09 9 9 999* 99.9 *960 9 *900 9900 99 9. 9 9.99 9 *9 9 9* 9* Asn Gin GIn His Aen Cys Arg Asp His Leu Gin AR Ser Leu Giu Thi 13( Glu Asi 145 Thr Asi Lye G' Lye G I Ile 11 21 Ser Va 225 Leu Va Arg G I Gly Pk Giu P I Thr Vi 305 Trp L' Leu G Leu A Tyr A 3 Leu L 385 His G Cys L Gly P Gly I Ser I 465

I

Gly Gi Arg GI Ala Pi G1u H~ Tyr I Ala V.

Leu S' 115 Gin V Ser L Glu I Leu G 1 i Giu L 195 B Gin C 0 1 Leu C 1 Asn u Giu e Asn 2'75 o Lys 0 1i Ile se Glu -y Aen :g Ile 355 :p Arg 70 y s Gly ly Ala Ys Cys ro Ser 435 ye Leu so .eu Arg .n C .u S -o H~ le I al 00 er al eu ie' iu .80 In eu ilu 260 Lys L~ys Girl Tyr Giu 340 Giu Phe His Asr A16 42( As: As: Se~

Z

Li

S

L

1 Ala *r Thr *5 Val LI Met 710 3.1 Gilu Ln Aen in Thr eu Aen er. Thr 150 eu Lys 65 Tyr Thr G iu 55 G iu an His Ala G In 135 Tyr Ilie Thr Asp 40 Pro Ronf Met Thr Giu 120 *Thr *Lye His Phe 25 GIn Asp Tyr Lys Ala 105 Gin Ser Leu Giv Ile Tyr Phe Thr Ser 90 Thr Thr Arg Giu Lyf 17 Leu F Ron IJ Ser Gin Glu Met Arg Leu Lys 155 Ron 'ro Lhr Ser Trp Met Leu Lye Giu 140 Gin Se: G iu Aon Gin Leu Ala G iu Leu 125 Ile Leu Let A.ra Arci Ser Pro Giu Asn Ser Gly Arg Arg Tyr Met Giu Giy L Glu Asn Leu G 2 Leu Giu Lye G 215 Lye Gin Gin I 230 Cys Thr Lys C 245 Lye Pro Phe Ser Giy Ile Val Phe Cys 295 His Arg Giu 310 Lye Met Gly 325 Phe Ile Phe Leu* Met Asp His Ile Gly" 375 Thr Giy Thr 390 Phe Ser Thr 405 Leu Met Lau i Leu Aon Gly 'x Gly Ile Lye 455 r Thr Thr Met 470 ye His L~ 185 In Gly L4 ,00 Iln Lau A .eu Giu L ;iu Giy V *2 krg AspC 265 ryr Thr :I 280 Ron Met I Asp Gly Phe Gly Ala Ile 345 Trp Giu 360 Asn Giu Ala Giy Lys Asp Thr Gly 425 met Phe 440 Trp His Met Ile (s en eu.

al 50 le er ksr 33C Thi Ly Ly Al 41 G I Ty Ty Ax Giu GI Val T~ Arg A: 2: Met A 235 Leu L Ala A Tyr I Val A 315 Pro Ser e Gin a Asp 0 y Trp *r Thr *r Phe -g Pro 475 -u Lc 21 la TI 20 ep T eu L ep V le A~ 2 ~sn G 100 Lap 3er 31n Arn 380 Ser Aen Trp Ala Lys 460 Leu y a 6

A

3

T

S

A

P

Aen Arg Ile His Asp Gly Ala Leu GIr Lye Leu GIr Gin LTye Let Gin Ile al Ile Gly Th 110 Thr Asp Va Gin Leu Le Lau Gin GI Leu Glu HJ 175 1Asp Thr L4 190 g Gin Thr T~ r Thr Aon A r Val His A 2 a Gly Gly L 255 .1 Tyr Gin A 270 n Asn Met P -y Gly Gly IJ ie Gin Arg ly Glu Tyr 333 rg Gin Tyr 350 la Tyr Ser yr Arg Leu er Leu Ile sep Aen Cys 415 ~he Asp Ala 430 fly Gin Aen 145 ;1y Pro Ser Ap Phe n r

I

sn ye ~la 'ro 'rp ;ly 320 rrp Met Gin Tyr Leu 400 Met Cys His Tyr INFORMATION FOR SEQ ID 140:8: SEQUENCE CHARACTERISTICS: LENGTH: 480 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: Linear (1i) MOLECULE TYPE: protein (1x) FEATUREz NAME/KEY: Mature TL2 protein LOCATION: 480 OTHER. INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ala Ala Tyr Aen Gin T Giu M Val G Gin V Leu G Gin G Thr Val C Leu C 145 Gin Lys Ile Ser Asn 225 Aen Val Lys Ser Gly 305 Arg Tyr Tyr Ser Ile 385 Ser yr et in 0 ~lu In ~lu iu rhr [Uys Lys Ile 210C Ser Let Ali Sei Th2 29C Trj Th Trj Va Le 37 Hi Gin V 2 Asp A Arg A Leu G Asn T Asn A Leu I Ala C HisI Ser Val Giu 195 Ile Val Leu LLye Gly 275 SThr r Trp p Leu 1 Leu 355 u Tyr 0 s Leu al 0 an ,ep iu yr Ia.

.00 4 eu ;ln er ;iu eu Glu Leu Thz Gli 26C His Gl~ 11i Lyi Gi 34' Ly Gi Ly Asn Phe A Gin His G Cys Arg S Ala Pro L "Asn Ile 11 70 Ile Gin P Val Gin P Asn Gin I Vai Leu Leu Ser I50 Ile Asn 165 Ala Met Lye Asp Glu Leu Gin Lye 230 Met Met 245' zGiu Gin 3 Thr Thr a Ile Lye Ile Gin 310 Giu Tyr 325 y Asn Giu 0 s Ile His u His Phe s Gly Leu .390 iy er eu Let ~hr ksn 135 LChr G iu Gin Q iu 215 Gin Ser I Ilr Ast Al~ 29! Ar~ Lys Phi Le' Ty 37 Th Ser Cys 25 Ser Ser 40 Giu Tyr Giu Asn Amn Met Gin Thr 105 Ala Glu 120 Gin. .Thr Asn Lye Leu Gin Asp Lye 185 Leu Gin 200 Lys Lys Gin HIE Thr Sei Ser Ph 26! Gly 11i 280.

3. Tyr Cyi SArg Gil a Val Gi Val Se 34 Lye As 360 r Leu Se r Gly Tli rg Lye Ser met Asp Ser II 1 '0 Ser Tyr Thr Pt~ Ser Pro Tyr V: 4! Asp Asp Ser V~ Asn Thr Gin T: 75 Lye Lye Glu Mi 90 Ala Val Met I Gin Thr Arg L Thr Arg LeuG Led Giu Lye G 155 Asp Lye Aen S 170 His Ile Ile G *Val Leu Val S Ile Val Thr 220 IAsp Leu Met 235 *Asn Ser Ala 250 a Arg Asp cys e Tyr Thr Leu a Asp Met Gi u 300 ui Asp Gly Ser 315 y Phe Gly Aen 330 r Gin Leu Thr 5 p Trp Giu Giy r Ser Giu Giu 380 ~r Ala Gly Lye Ie ys 2S lu 'In Al Th 28 Al Va Pr

AS

Al L4 Gly Leu Ser Gin Leu Val Giu 110 Leu Leu Ile Phe Leu 190C Lys 5. Th~ u Th: s As) a GI 27 r Ph a GI .1 As Se *n GI 35 in G3 au A~ le SE Lye Lye Leti Pro Asn Ala Arg Leu Met Lye Giu Ile Ile Gly Tkir Asp Gin Leu Leu Asp 160 Leu Giu 175 Gin Ser Gin Aen Val Aen r Val Asn 240 p Pro Thr u Val Phe 0 e Pro Asn y Gly Giy p-Phe Gin 320 r Gly Giu 335 .n Gin Arg ~0 .u Ala Tyr in Tyr Arg ar Ser Ile 400 sn Asp Lye 415 395 Gin Pro Gly Aen 405 Asp Phe Ser Thr Lys 410 Asp Gly Asp As cys Ile Cys Ala Cys Gly 435 Asn Thr Aen 450 Giy Tyr Ser 465 Lys 420 Pro Cys Ser Gin Met Leu 425 Gly Thr Gly Gly Trp Trp Phe Asp 430 Gin Arg Gin Ser Asn Leu Asn 440 Ile Met Tyr Tyr Pro 445 Lys Phe Asn Gly 455 Thr Lys Trp Tyr Tyr 460 Arg Trp Lys Gly Ser Pro Ala Asp Phe Leu Lys Ala 470 Thr Met Met Ile 475 480 a INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1849 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (1i) MOLECULE TYPE: DNA (ix) FEATURE: NAM E/KEY: Coding Sequence LOCATION: 47...1573 OTHER INFORMATION: (A)NAME/KEY: TIE ligand-3 LOCATION: 1849 OTHER INFORMATION: The fibrinogen-lke domain starts at position 929., (xi) SEQUENCE DESCRIPTION: SEQ ID NQ:9: CTGTCCTGGT ACCTGACAAG ACCACCTCAC CACCACTTGG TCTCAO ATO CTC TOC Met Leu Cys

I

CAG CCA Gin Pro OCT ATG CTA CTA Ala Met Lou Lou

GAT

Asp 10 GGC CTC CTC CTG Gly Lou Leu Lou

CTG

Lou GCC ACC ATG OCT Ala Thr Met Ala

OCA

Ala 0CC CAG CAC AGA Ala Gin His Arg 000 O ly 25 CCA GAA GCC GOT Pro Giu Ala Gly

GG

Gly 30 CAC CGC CAG ATT His Arg Gin Ile

CAC

His 10 3 151 199 247 CAG GTC OG COT Gin Val Arg Arg

GOC

Gly CAG TGC AGC TAC Gin Cys Ser Tyr

ACC

Thr 45 TTT OTG GTG CCG Phe Val Val Pro GAG CCT Glu Pro 150 GAT ATC TGC CAG CTO GCG CCG ACA GCG GCG CCT GAG GCT TTG 000 GGC Asp Ile Cys Gin Lou Ala Pro Thr Ala Ala Pro Glu Ala Leu Gly Gly 60 TCC AAT AGC Ser Aen Ser CTC CAG AGG GAC Lou Gin Arg Asp

TTG

Leu 75 CCT GCC TCG AGG Pro Ala Ser Arg

CTG

Leu CAC CTA- ACA His Lou Thr GAC TG Asp Trp CGA GCC CAG AGG Arg Ala Gin Arg

GCC

Ala 90 CAG COG 0CC CAG Gin Arg Ala Gin

COT

Arg GTG AGC CAG CTO Val Ser Gin Lou 295 343 391 439

GAG

Giu 100 AAG ATA CTA GAG Lys Ile Lou Giu

AAT

Asn 105 AAC ACT CAG TG Asn Thr Gin Trp

CTG

Lou 110 CTG AAG CTG GAG Lou Lys Lou Glu

CAG

Gin 115 TCC ATC AAG GTG Ser Ile Lys Val

AAC

ken 120 TTG AGO TCA CAC Lou Arg Ser His CTG OTO CAG GCC CAG CAG GAC Lou Val Gin Ala Gin Gin Asp 125 130 118 ACA ATC CAG Thr Ile Gin ATG AAC CAG Met Asn Gin iso

AAC

135 cAG ACA ACT ACC Gin Thr Thr Thr

ATG

Met 140 CTG GCA CTG, GGT Leu Ala Leu Gly GCC AAC CTC Ala Asn Leu 145 GTG GAG GCA Vai Glu Ala ACC AAA GCT CAG Thr Lye Ala Gin

ACC

Thr 155 CAC AAG CTG ACT His Lye Leu Thr

GCT

Ala 160 CAG GTC Gin Val 165 CTA AAC CAG ACA Leu Asn Gin Thr

TTG

Leu 170 CAC: ATG AAG ACC His Met Lys Tkxr CAI ATO CTG GAG Gin Met Leu Glu 17 CTG ATG CAG AGC Leu Met Gin Ser AAc Asn

CGA.

Krg 195 487 535 583 631 679

TCA

Ser 180 CTG TCC ACC AAC Leu Ser Thr Asn PsAG Lys 185 CTG GAG COG CAG Leu Giu Arg Gin

ATG

Met 190 GAG CTG CAG CG Giu Leu Gin Arg

CTG

Leu 200 CAG GOT CGC AAC Gin Gly Arg An

AGG

Arg 205 0CC CTG GAG ACC Ala Leu Giu Thr AGG, CTG Arg Leu 210 CAG OCA CTG Gin Ala Leu GAA GCA Glu Ala 215 CAA CAT CAG Gln His Gin

GCC

Ala 220 CAG CTT AAC AGC Gin Leu Aen Ser CTC CAA GAG Leu Gin Giu 225 000 ACC CTG Gly Thr Leu AAG AGO GAA CAA CTG CAC AGT CTC Lye Arg Giu Gin Leu His Ser Leu 230 235 CTG GGC CAT CAG Leu Gly His Gin

ACC

Tkir 240 OCT AAC Ala Aen 245 CTG AAG CAC AAT Leu Lys His Asn

CTG

Leu 2S0 CAC GCT CTC AGC His Ala Leu Ser

AGC

Ser 255 AAT TCC. AGC TCC Asn Ser Ser Ser

CTG

Leu 260 CAG CAG CAG CAG Gin Gin Gin Gin

CAG

Gin 265 CAA CTG ACG GAG Gin Leu Th~r Giu

TTT

Phe 270 GTA CAG CGC CTG Val Gin Arg Leu

GTA

Val 275 CGO ATT GTA GCC Arg Ile Val Ala GAC CAG CAT CCG Asp Gin His Pro

GT

Val 28S TCC TTA AAG ACA Ser Leu Lys Thr CCT AAG, Pro Lys 290 CCA GTG TTC Pro Val Phe

CAG

Gin 295 GAC TOT GCA GAG Asp Cys Ala Giu

ATC

Ile 300 AAG COC TCC 000 Lys Arg.Ser Gly GTT AAT ACC Val Asn Thr 305 OCT CTC AAG Pro Leu Lys AGC GOT GTC TAT ACC ATC TAT Ser Gly Val Tyr Thr Ile Tyr 310

GAG

Giu 315 ACC AAC Thr Asn ATG ACA AAG Met Thr Lye 320 775' 823 871 919 967 1015.

1063 1111 1159 1207 1255 OTO TTC TOT GAC ATG GAG ACT GAT GGA GOT G TOG ACC CTC ATC CAG Vai Phe Cys Asp Met Glu Thr Asp Oly Gly Gly Trp Thr Leu Ile Gin 325 330 335

CAC

His 340 CGO GAG OAT GGA Arg Giu Asp Gly

AGC

Ser 345 OTA AAT TTC CAG, Val Asn Phe Gin

AGO

Arg 350 ACC TOG GAA GAA Thr Trp Giu Giu

TAC

Tyr 355 AAA GAG GOT TTT Lys Giu Oly Phe

GOT

Gly 360 AAT GTG 0CC AGA Asn Val Ala Ara

GAG

G2lu 365 CAC TGO CTG GGC His Trp Leu Gly AAT GAG Asn Giu 370 GCT OTO CAC Ala Val His CTC ACC AGC AGA Leu Thr Ser Arg

ACG

Thr 380 0CC TAC TTG CTA Ala Tyr Leu Leu CGC GTG GAA Arg Val Glu 385 CTG CAT GAC TGG GAA GOC CGC CAG ACC TCC ATC CAG TAT GAG AAC TTC Leu His Asp Trp Glu Oly Arg Gin Thr Ser Ile Gin Tyr Giu Ann Phe 390 395 400 119 CAG CTQ GGC AGC GAG AGG CAG CGG TAC AGC CTC TCT GTG AAT GAC AGC Gin Lou Gly Ser Giu Arg Gin Arg Tyr Ser Lou Ser Val Asn Asp Ser 405 410 415

AGC

Ser 420 AGT TCA GCA GG Ser Ser Ala Gly AAG AAC AGC CTG Lys Asn Ser Leu

GCT

Ala 430, CCT CAG GGC ACC Pro Gin Gly Thr

AAG

Lys 435 TTC AGC ACC AAA Phe Ser Thr Lys

GAC

Asp 440 ATG GAC AAT GAT Met Asp Aen Asp

AAC

Asn 445 TGC ATG TGT AAA Cys Met Cys Lye TGT GCT Cys Ala 450 1303 1351 1399 1447 1495 CAG ATG CTG Gin Met Leu CTC AAT GGC Lou Aen Gly 470

TCT

Ser 455 GGA COG TGG TG Gly Gly.Trp Trp

TTT

P he 460 GAT GCC TGT GCC Asp Ala Cys Gly CTC TCC AAC Leu Ser Asn 465 AAG, ATC AAT Lys Ile Asn ATC TAC TAT TCA Ile Tyr Tyr Ser CAT CAG CAC TTG His Gin His Leu GGC ATC CC Gly Ile Arj 485 ACA CGC AT( Thr"Arg Met 500

GATGCCGTAG

TCAGTGCCCA

AATTACPAAGA

AAGGCACCTG

CCTGCCATGA

TOO CAC TAC Trp His Tyr ATG CTG AGG tMet Lau Arg 505

TTC

Phe 490

CCA

Pro CGA GGC CCC AGC Arg Gly Pro Ser ATG GGT GCC TCA Met'Gly Ala

TAC

Tyr 495 TCA CTG CAC.GGC Ser Lau His Gly CACACAG CCCTGCAGAG ACT 1543 1596 1656 1716 1776 1836 1849 GAGGATTCTC AACCCAGOTG ACTCTGTGCA.

GGGCTCATCT TGACATTCTG GAACATCGGA ATTCACCTGC CTCCCTGTTG CCCTCTAATT CCTCTGTTGG AACCATACTC TTTCCCCCTC

ACT

CGCTGGGCCC

ACCAGCTTAC

GTGAAATTGC

CTGCTGCATG

TGCCCAGAAA

CTTGCCCCTG

TGGGTGCTTG

CCCGGGAATC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 509 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (iU) MOLECULE TYPE: protein FRAGMENT TYPE. internal (ix) FEATURE: NAHE/KEY: TIE iigand- 3 LOCATION: 1 509 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1O: Met 1 Thr Lou Cys Gin Met Ala Ala Pro 5 Ala Met Lau Lou Asp 10 Cly Pro Gly Lou Lou Lou Lou Ala His Arg Ala Gin His Arg Giu Ala Cly Gly 25 Gin Phe Val Val Gin Ile His Pro Glu Pro Gin Val Arg Arg Asp Ile Cys Gin 55 Ser Aen Ser Lou Cly 40 Cys Ser Tyr Thr Lou Gly Lou Ala Pro Gi1n Arg Asp Thr Ala Lou Pro Ala Pro Clu Ala G ly Ala Ser Arg His 70 Arg 75 Gin Lou Val Lou Thr ASP Trp as Ala Gin Arg Ala 90 Arg Ala Gin Arg Set Gin Lou Giu Lys Ile Lou Giu Asn Asn Thr Gin Trp Lou Lou Lys 120 Leu Gin Ala 145 Val LeU Gin Thr Leu 225 Giy Ser Giu Gin 115 Gin Asp 130 Aen Leu Giu Ala Glu Ken Ser Arg 195 Arg Leu 210 Gin Glu Thr Leu Ser Ser Leu Va3 271 100 Ser Ile Lys Vai P Thr Ile Gin Aen C 135 Met Aen Gin Thr 150 Gin Val Leu Aen 165 ser Leu Ser Thr 180 Glu Leu GIn Arg Gin Ala Leu Giu 215 Lys Arg Glu GIn 230 Ala Aen Leu Lys 245 Leu Gin Gin Gin 260' -Arg Ile Val Ala LBn .20 .In y a Asn Leu 200 Ala Lev Hi: G1i Gi: 281 As 105 Leu P Thr I] Ala Thr Lys 185 Gin Gin His Aen ,i Gin 265 ni Asp 0 p Cys Lrg S hr T ;In 'I Leu 170 Lieu Gly

HIS

Ser Leu 250 Gin Gin Ala er 'hr 'hr L55S Us 3 iu Arg Gin Leu 235 His Leu Hit G1~ Thr Pro Lye 290 Val Aen Thr 305 Pro Val Phe Gin 295 Pro Leu Giu Gly Arg 385 Giu Asn Gly Lye Leu 465 Lye Leu Ile

GLL

Asn 370 Vai Asn Asp Thr Cys 450 Lys Gin Tyr 355 G iu Giu Phe Ser Lys 435 Ala Ser Vai His 340 Lys Ala Leu Gin Ser 420 Phe Gin Gly Phe 325 Arg G iu Val His Leu 405 Ser Ser Met Ile 485 Val Tyr *1 310 Cys Asp Glu Asp Giy Phe His Arg 375 Asp Trp 390 Giy Ser Ser Ala Thr Lye Leu Ser 455 Giy Ile 470 Arg Trp ~hr 4et 31y 3 ly 360 Leu Giu G iu G ly Asp 440 G iy Tyx Hi~ His Lei 12 Met Le 140 His Ly Met Ly Arg G1 Aen Az 2( Ala GJ 220 Leu G Ala Li Thr G 3Pro V 2 1 Ile L 300 u Thr P 5 p Gly C n Phe( a Arg g Thr 380 .n Thr .g Tyr 3nl Ser sn Asp rp Phe 460 al His 75 rg Gly .le

GILL

Ser 345 As n Thr Gly Arg Arg 425 Met Gl) Ty~ 3Ty a1

B

Ln 31

S

A

4

I

Tyr G 3 Thr P 330 Val I Val2 Ser Arg Gin 410 Lys Asp Trp Se5r r Phe 490 39 Ar

A:

V

4

A

110 Val Gin Ala Ala Leu Gly Leu Thr Ala 160 .Thr Gin Met.

175 Met Leu Met 190 Ala Leu Giu Leux Asn Ser His Gin Thr 240 1 Ser Ser Aen 255 ar Phe Val Gin 270 I Ser Leu Lye s Arg 5cr Gly n Met Thr Lys 320 .y Giy Trp Thr 335 *n Arg Thr Trp 350 *u His Trp Leu La Tyr Leu. Leu er Ile Gin Tyr 400 er Leu Scr Val 415 eu Ala Pro Gin 430 en Cys Met Cys ~sp Ala Cys GlIy ;In His Leu His 480 ~ro Ser Tyr Ser 495 Ser Aen Leu Ile Asn Gl) Leu His Gly Thr S00 Arg Met Met Leu Arg 505 Pro Met Gly Ala INFORMATION FOR SEQ ID NO:ii: SEQUENCE CHARACTERISTICS: LENGTH-. 503 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: mTL3 LOCATION: 503 OTHER INFORMATION: mouse TIE iigand-3 9 Met His Arg Gin Leu Ala Leu Val.

Aan Thr 145 Aenr Thr Arg Giv Gin 225 LY.0 Gir All Gil Ty: As' As Ph Ar Tr 38 Se Al Se Ii Tz (xi) SEQUENCE Leu Leu Asp Gly Arg Gly Pro Giu DESCRIPTION: SEQ ID Leu Leu Leu Leu Ala 10 Ala Gly Gly His Arg Gly Gin Leu Ala Gin Arg Gin Arg Giu Asil Asn Leu 115 Gin Thi 130 Lys All Olin Thi Aean Lyi Leu GlI 19 Ala Gi 210 Leu Hi His As Gin Gi a Gin As 27 n Asp CY 290 r Thr I I 5 p Met GI p Gly SE e Gly Au 3! g Leu TI 370 p Giu G~ 5 r Giu &1 a Gly A 'a Asp M 4 ~r Gly G 450 .e Tyr T ~p His T n n a r r h

'I

3 Cys Pro Asp Ala Asn 100 Krg Thr Gin Leu Leu 180 Gly His ISer Let; GIr 2 6( GIl 3- Al: e Ty u Th: r Va 34, n Va 5 r Se y Ar 'g GI g Ly 42 ~t As 15 .y Tr rr Sc rr Pt Ser Tyr TI Thr Ala A 5 Leu Pro A; 70 Gin Arg A as Thr Gin T Ser His L Thr Met L 1 ,thr His L 150 His Met L 165 Giu Arg C Arg Asn Gin Ala C Leu Leu( 230 His Ala 245 Leu Thr4 i His Pro i Giu Ile r Giu Thr 310 r Asp Gly 325 1 Aen. Phe 0 1 Ala Arg r Arg Thr g Gin Thr 390 n Arg Tyr 405 a Asn Ser ,0 p Aen Asp p Trp Phe ~r Val His 470 ie Arg Gly 485 la 5 la rp eu .eu ,ys ~In ~rg In ;ly ['eu 3;iu Val Lys 295 Asra Gir Git Al~ 37 Se~ Se~ Lei As: As 45

GI

Pr Ph 40 Pr Se Li

VI

e *0 Ln Val Giu Arg Arg Leu 105 Gin Val Ala Leu Val 90 Lys Ala Pro Leu His 75 Ser Leu Gin Giu Gly Leu Gin Giu GIn NO: 11: Thr Met Gin Ile Ala His Pro Gly Thr Leu Gin Asp 125.

AIla Gin Asp Ser Asp Giu Ser 110 Thr Ala Val Ile Asn Trp Lye Ile Ile 120 Ala L Leu T Thr G Met L Ala L 200 Leu P, His C Ser Phe Ser 280 Arg Met -Gly Arg aHis 360 k Tyr Ile r Leu ui Ala n Cys 440 p Ala 5 n His 0 Ser eu hr in eu .85 ,eu Len ~In er la I Z65 L eu Ser Thr Trj Thz 34! Tr Lei Se Pr 42 Me Cy L~e Gly A] Ala Vi 1I Met Li 170 Met G Giu T Ser L Thr G 2 Asn S 250 Gin A~ Lys T *Gly N Lye ThrI 330 Trp ,Leu u Leu n Tyr r Val 410 o Gin

S

t Cys a Gly u His -r Sen 490 ~a Asn Leu Met Aen Gin 1i 55 In hr eu 35 er ~hr Tal ?ro 315 3 it Ar

GI:

39 As

GI

Ly Gin Arg Cys Ser Ile Lys Gin Giti A Ser A Arg 1 Gin C 220 Thr Sen Leu Pro Asn 300 Leu Sle Giu g Va-i 380 ui Asn 5 n Asp y Thr a Cys la an ~rg 4 eu ~0s 1u jeu Ser 1al Lys 285 Thr Lys GIr Tyx G I 36! Gl1 Phi Se Ly Al Gin Sen G iu 190 Gin Lys Ala Leu Ang 270 Pro Sex Val H!i Lyu 351 I Al i Le e Gi r Se a Ph 43 a GI Val Leu 175 Leu Ala Arg Asn Gin 255 Ile Val

GII

-PhE 3Arc 33~ a GI' a Va.

ui Hi n Le r Se 41 e Se 0 .n Me Leu 160 Ser G In Leu Giu Leu 240 *Gin Val *Phe Val B Cys 320 g Glu Li Gly 1 His 0 Asp u Gly 400 r Ser ~r Thr ~t Leu 445 Leu Lys 475 Ile Ser 460 Ile His Aen Aen Gly Leu Gly Thr Aen Ile Arg 49S G ly Ang 480 Met Met Leu Ang Pro Met Gly Ala 500 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 490 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (iU) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: hTLl LOCATION: .490 OTHER INFORMATION: human TIE-2 ligand 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO%12: Ala Phe 1 Arg Ser Gin'. Gys Glu Ser Pro His His Val Ile Val Val Gin Ser Gin 130 Val Leu 145 Leu Ser Ile Leu Glu Met Lys Glu 210 Glu Leu 225 Ieu Ala- Ala Ile Leu Thr His Ile G Pro Alia Thr Val Met Gbu Aen 115 Tkir Aen Thr Lys Glu 195 Aen Glu Gin Thr Phe 275 Ilbe Cye Gil Gl) Ph~ Asj .5 Glu ksn S Tyr Thr I Thr Asp C Giu Pro3 Glu An Asn Met 100 His Thr Ala Glu Gin Thr Tyr Lys 165 Ile His 180 Gly Lys Leo Gin Lys Gin Gin Leu 245 Lys Glu 260 Arg Asp Tyr Thr Asn Met aAsp Gly 325 rPhe Gly 340 a Ala Ile 5 Trp Glu ~er ~he In wep 70 ryr ys klia Ser 150 Leu G iu His G ly Leu 230 Glu Val Cys Ilbe Asr 31iC Sez As: Thi G1~ Gly I le Tyr 55 Phe Thr Ser Thr Thr 135 Arg G bo Lye Lye Leu 215 Asn Let~ Let Al: ~Ty2 29! Va ~Lei i Pr4 Se.

~As Arg Leu 40 Asn Ser Gin Glu Met 120 Arg Leu Lys Asn Giu 200 *Val Arg aMet aLet I Asi 2 8 -Iii I Asi *1 As~ Se Gi 36 n Ar Arg 7 25 Pro G Thr A Ser G Ttp L 9 Met A 105 Leu G Lys I Gb I Ser 185 *Glu *Thr Ala Asp Lye 265 Val a. Aen ni Gly p Phe r Gly 345 n Arg 0 g Ala rr A lu IH en In 1 eu C 0 la( Ilu ~eu ~le .eu 170 ,eu Leou Arg Tbhr Thr 250 Gly Tyr An Gly Gin 330 Glu ly s n ~Is lia.

ye, T5 In ;In Ilie rhr Leu Asi Th2 23! Va.

G1 Gi He 31 Ar Ty Cys SE Arg I2 Asp G.

4' Leu G Leu G Lys L Ile G Gly TI 1 Asp V~ 140 Leu I Gin C Glu Thr i Thr 220 c Asn I His y Lye n Ala t Pro 300 y Trp 5 g Gly 'r Trp le ly in In eu In hr al1 ~eu 1r iii Let 20! As Ar

GI.

28 Gi Ti, Tr Asn Gin Arg Gin His Gly Asn Cys Arg Arg Asp Ala His Leu Giu so Giu Asn Tyr Gin Ann Ala 110 Ser Leu Leu Glu Thr Gin GluAsnSer GluAen160 Thr Asn Glu 175 Lye Ile Leu 190 i Lye Glu Glu c Ile Ile Gin ni Ser Val Leu 240 n Leu Val An 255 g Gbu Glu.Giu 270' y Phe Aen Lye u Pro Lye Lys Lr Val Ile Gin 320 .p Lye Glu Tyr 335 :u Gly Ann Glu 350 diln Leu Lye Ser Val 305 His Lye Phe Leo Lye Cys Pro Gly 290 Phe Arg met Ile Met Gin Tyr Tyr Ser Met Leu Arg Ile Giu 365 Gin Tyr Asp Arg Phe His 385 Thr Phe Leu Leu Gly 465 Thr 370 375 380 Ile Gly Asfl Giu Lys Gin ken Tyr Arg Leu Tyr Leu Lys 390 395 Gly Thr Al.a Gly Lye Gin Ser Ser Leu Ile Leu His Gly 405 410 Ser Thr LYe Asp Ala Asp Aen Asp Aen Cys Met Cys Lye 420 425 430 Met Leu Thr Gly. Gly Trp Trp Phe Asp Ala Cys Gly Pro 435 440 445 ken Gly Met Phe Tyr Thr Ala Giy Gi .n ken His Giy Lys 450 455 460 Ile Lye Trp His Tyr Phe Lys Giy Pro Ser Tyr Ser Ile 470 475 Thr Met Met Ile Arg Pro Leu Asp Phe 485 490 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 491 amino acids TYPZ9: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: chTLi LOCATION: l...491 OTHER INFORMATION: chicken TIE-2 ligand 1 Gly Ala 415 Cys Ser Leu Arg His 400 Asp Ala ken ken Ser 480* 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Ala Phe Leu Ala Ala Ile Leu Ala His I 0. 0 Arg Gin Giu Pro His I le Val Ser Val 145 Leu Ile Giu Lye G lu 225 Gin Ser CYe ser His Val Vai Gin Gin 130 Leu Ser Leu Met Giu 210 Leu Lys pro Thr Thr Val Met Giu Aen 115 Thr ken Thr Lye Giu 195 ken Giu Gin Glu Tyr Thr G lu G lu ken 100 His Ala Gin Tyr Ile 180 Giu Leu Lys Gin ken Thr Asp Gin ken Met Thr Glu Thr Lye 165 His Arg Gin Gin Leu Ser Phe Gin Asp 70 Tyr LYe Ala Gin Ser 150 Leu Glu His Gly Leu 230 Glu Gly Ile Tyr 55 Phe Thr Ser Thr Thr 135 Arg Giu Lye Lye Leu 215 Asn Leu Arg Leu 40 ken Ser Gin Glu Met 120 Arg Leu Lye ken Glu 200 Vai Lye met k.rg as Pro rhr Phe Trp Met 105 Leu Lys G lu Gin Ser 185 G lu Thr Ala Asp le

LO

?he flu !Aen Gin Leu 90 Al a Giu Leu Ile Leu 170 Leu Met Arc Th, Th, 2 5 ken Gin Ala Lye 75 Gin Gin Ile Thr Gin 155 Leu Leu Asp Gin 7Thr 235 r Val 3 ~rg eu Leu Lyes Leu Gly Asp 140 Leu Gin Glu Thr Ser 220 ken His C;iy Cys Thr Thr Gin Arg :le iy ;ln In Leu Gin Thr 125 Val Leu Gin His Leu 205 Ty: As: Th Gin His Gly ken Cys Arg Arg Asp Ala His Leu Giu Giu Ser Tyr Gin ken Ala 110 Ser Leu Leu Giu Thr Gin Glu ken.Ser 160 Thr ken Giu 175 Lye Ile Leu 190 Lye Giu Giu Ile Ile Gin Ser Val Leu 240 Leu Ile Thr 255 B Arg Giu Glu 245' Leu Cys Ser Lye Glu Gly Val Leu Leu Lye ken Ala Ly~ Giu Lye P 2 Lys Ser G 290 Lys Val 1 305 Gin His Tyr Lyes Giu Phe Giu Leu 370 Phe His 385 His Ser Giu Phe Ala Leu Asn Leu Ae'450 Gln-dy 465 Ser Thr 'ro :75 liy ~he krg M1et Ile 355 Met Ile G ly Ser Met 435E Aer Ilie Thi 2 60 he 265 Arg Asp Cys Ala Asp 280 Val Tyr Gin Ala 285 Ile Tyr Cys Asn Giu Asp 325 Gly Phe 340 Phe Ala Asp Trp Gly An Thr Ala 405 Thr Lys 420.

*Leu Thr Gly'"Met Lye Trp -Met Met 485 Irhr met 310 G1y Giy I le G lu Giu 390 Gly Asp G ly Phe His 470 I le Ile *1 295 Asp Ser Ser Thr Giy 375 Lye Lys Ala Gly Tyr 455 Tyr Arg Lyr la 1 Leu Pro Ser 360 Asni Gin Gin Asp Trj 44C Tku Phs Pic Ile Aen Asp Ser 345 Gin Arg Aen Ser An 425 Trp -Ala a Lye Le~ Aen Aon N Gly Giy C .315 Phe Gin 330 Giy Giu Arg Gin Aia Tyr Tyr Arg 395 Ser Leu 410 Asp An Phe Asp Gly Gin Gly Pro 475 x Asp Phe 490 Tal I ~00 iy ys Tyr Tyr Ser 380 Leu Ile Cys Ala As n 460 Arg er Lrrp "iy Trp Ser 365 Gin Tyr Let; Mel Cyl 44! Hi Ty 270 Gly E Asp I Thr Trp Leu 350 Leu.

Tyr Leu His -Cys.

430 s Gly S Giy r Ser ~he ~ro lai.

Lys 335 Gly Arg Asp Lys G ly 415 Zye Pro Lys Ilie An Lys Ile 320 G iu Aen Ile lirg G ly 400 Ala, Cys Ser Leu Arg 480 INFORMATION FOR SEQ ID 14:14: SEQUENCE CHARACTERISTICS: LENGTH: 497 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (IX) FEATURE: NAME/KEY: mTL1 LOCATION: .497 OTHER INFORMATION: mouse TIE-2 ligand 2.

(xi) SEQUENCE DESCRIPTION: SEQ ID NOi4: Met Thr Vai Phe Leu Ser Phe Ala Phe Phe Aia Ala Ile Leu Ile Tyr Giu Asn Gin Leu Ala Giu Leu Gly Aen His so Al a Lys Gin Gin Ile 130 Thr cys Arg Asp Leu Leu Lys Ile 115 Gly Ser Ile G ly Gin Gin Leu 100 Gin Thr Aen Gin Gin His Aen Cys Arg Asp 70 His Leu.

Giu An Gin An Ser Leu.

Arg Arg Gly Gin 40 Arg Giu 55 Ala Pro Glu His Tyr Ile Ala Val 120 Leu Ser Asn 25 Cys Ser His Val Val 105 Gin Gin Pro Ala Thr Val Met 90 Giu Asn Thr Glu Tyr Thr Giu 75 Glu An His Al a Asn Thr As3p Pro Asn Met Thr Giu 140 Thr Ser Phe Gin Asp Tyr Lys Ala 125 Gin Gly Ile Tyr Phe Thr Ser 110 Thr Thr Thr Arg Leu An Ser Gin Giu Met Arg Hlis Arg Pro Thr Ser Trp Met Leu Lys 135 Asp Vai Giu Thr Gin Val Leu Aen Gin Ser Arg Leu Giu 125 145 Ilie 150 Aen Gin Leu Leu Giu Ser Leu Ser 155 Thr Tyr 170 165 Leu Leu Gin Thr Aen Giu Ile Leu Asp Gin 225 Asn Val Gly Tyr Aen 305 Gly Glu Thr 210 ser As n His Gly Gin 290 Val Gly His 195 Leu Phe Asn Aen Lys 275 Ala Pro Trp 180

L

Lys Ile Lys Giu Ile Ile Ser Ile 245 Leu Ile 260 Arg Giu Gly Phe Glu Pro pThr Val.

,325 *Trp'Lys 340 Leu Gly *Leu Arg Tyr Asp *Leu Lye 405 aHis Gly 420 -Cys Lye Gly Pro Gly Lye ~eu Glu too.

Gin Lys Giu Tyr Gin Tyr 370 Tyr Ser 385 Arg Leu Leu Ile Asn Cys Asp Ala 450 Gin Aen 465 Pro Arg Gly Trp 355 Met Gir TYz Le~ Mel 43! Cyi Hi' ilu .In 130 Leu Ser Giu Asn Lys 310 Ile Glu Asrn Ile Arg 390 G i) Al~ Cy' Se~ Le' 474 Lye G 215 Glu I Gin Leu Giu Lys 295 Lys Gin Tyr Giu Glu 375 Phe His xAsp Ala Aen 455 u Aen 0 eu Let 00 liu deu.

.dys ys Lys 280 Ser Val

HIE

Lys Ph 36( Le~ Hi Th Ph Le 44 Le Gi Lys I Giu G Aesn L Giu L Gin C 'Thr 1 265.

Pro Gly.

Phe Arg *met 345 met r Gly e Ser 425 u Met 0 u Asn v Ile is iy ,eu 'ye In ~so .ys ?'he Ile Cys His Lys Gin Gin 235 Leu G iu Arg Tyr Asri Glu 330 G iy Phe Asp Gly Thr 410 Thr Leu Gly Lys Met 490 315 Asp Giy 'Ser Leu Phe Ala Trp Asn 395 Ala Lye Thr Met Trp 475 Gly Ile Giu 380 Giu Giy Asp Gly Phe 460 His Ser Thr 365 Gly Lye Lys Ala G ly 445 Tyr Tyr Pro 350 Ser Aen Gin Gin Asp 430 Trp Thr Phe Asp 335 Ser Gin Arg Asri Ser 4-15 Trp Ali Lyi Gly Arg Ala Tyr 400 Ser Asp Phe Gly 3 Gly 480 Lye Leu C Giu Lyes His Lye 205 Giy Leu 220 Leu Ser Giu Leu Gly Val Asp Cys 285 Thr Ile 300 Met Asp iu ke n 190 1lu Arg Met Leu 270 Ala Tyr Val 160 Lye Gin 175 Ser Leu Glu Met Ser Arg Ala Thr 240 Asp Thr 255, Leu Lye Asp Vai Phe Aen Aen Gly 320 Tyr Ser Ile 485 Arg Ser Thr Thr Met Ile Arg Pro Leu Asp 495 Phe INFORMATION FOR SEQ -ID NO:1S: SEQUENCE CHARACTERISTICS: LENGTH: 496 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (1x) FEATURE: NAME/KEY: mTL2 LOCATION: .496 (D)-OTHER INFORI@.TION: mouse TIE-2 lI gand 2 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Trp Gin Ile Ile Phe Leu Thr Phe Gly Trp Asp Ala Val Leu Thr 1510 ser Ala Tyr Ser Asn Phe Arg Lye Ser Val Asp Ser Thr Gly Nrg Arg Arg Tyr Arg Ile Gin Asn Giy Pro Cys Ala Tyr Thr Phe 40 Leu Leu Pro Glu Thr Val Gin Gin Leu Leu Glu Gin Gin Thr Ser 130 Val Glu Asp Arg Leu Asn Asn 115 Leu Thr Leu Gin His **see: 0 0 1 ego.

*0 Ce

C

0000 Gin I] Gin I Met i Ser 225 Asn Ser Ile Lys Ser 305 Gly Lys Tyr Tyr Ser 385 Ile Ser Cys Ala Aen 465 Gly Lhr I .ys 'ys 210 Val Ser Leu Arg Ala 290 Pro Trp Gly Trp Vai 370 Leu His Gin Ile Cys 450 Thr Tyr ier fal L95 ilu Ile Leu Leu Arg 275 Gl Glt Thi Tr Let Lei Ty: Le Pr Cy 43 Gi As Se Ser Asp Glu Tyr 100 Val Leu Gin Ser Glu 180 Leu Gin Asp Leu Thr 260 Giu Leu Gilu Val i Lye 34C j Gi) u Ly r As Th~ o Gi' 42 s Ly 5 y Pr n Ly r Il Gly Ala Asn Ile Val Ser Val lie 165 Ile Asp Lys Glu Gin 245 Met Git Thi Ile Sie 32! a Gil Asi a Iii 3 Hi r GI y Se 0 6 Cy o Se a Ph e Ly 48 Arg S

S

Pro P 70 Val M Gin A Gin A Gin T

I

Leu I 150 Ser I1 Asn I Met Asp Leu 230 Lye Met Gin Lye Lys 310 Gin i Tyr .I Glu a Gin s.Phe 390 y Leu 5 r Asp a Ser r Asn e Asn 470 s Ala '5 er 5 ro et sp ann 'hr .35 asn chr ys 'iu lu 215 Glu Gin Sex Thi Se2 29! Ali Hi Lyi Phi Le 37 Ty Th Ph Le Le 45 Gi Ser St Asp T] Glu A Aen H4 1 His T 120 Ala G Gin T Tyr L Ile F Giy I 200 *Leu C Lye I Gin I Sen Thr 280 Gly 3. Tyr a Arg 8 Met Ile 360 u Lye 5 r Ile Gly e Ser u Met 440 *u Kn 5 .y Ile rn Thr ar yr an.

et 05 hr lu hr .ys [is .8s ;In :,ys Iii Pro 265 Phe Ile Cy G1r Gi' 34! Se As Al Th Th 42 Le

GI

Ly He Ser Glu Tyr 90 Thr Asp 75 Thr Tyr Ser Gin Lye Lys G Ala Val M Gin Thr A 1 Thr Arg L 155 Leu Glu L 170 Asn Lys P His Ser C Val Leu Leu Val 235 Asp Leu I 250 A Ksn Ser Arg Asp Tyr Thr s Asn Met 315 i Asp Gly 330 y Phe Gly 5 r Gin Ile p Tnp Glu a Gly Glu 395 r Ala Ala 410 r Lys Asp iu et rg eeu 'ys

LB

lu tal a2C rhi Met Val Trp Met Ile 125 Lys Glu Gin Ser Gli.

Se Al 1 Thr I Gin Leu 1 Ala 4 110 Glu Leu Leu Ile Phe 190 Met Lys a Thr Glu Ile Thr Gin Leu 175 Leu Gin Gin Val sn Ala 3er Leu Aet Lys Ile Gly Aisp Leu 160 Asp Giu Thr Ser Asn 240 Met P Lye E Cys I Leu 300 Asp Ser Asn Thr Gly 380 G iu Lye Ser Gly Tyr 460 Tyr Arg sp ;er kla rhr lal Leu Pro Gly 365 Asn Sex Ile As TrI 44' Pr Tn Pr

C

C CC Thr Val A 255 Ser Leu A 270 Asp Val P Phe Pro dly Gly C Asp Phe 335 Leu Gly 4 350 Gin His Glu Ala Asn Tyr Ser Ser 415 A Ken Asp 430 3 Trp Phe o Gin Lys p Lys Gly o Ala Asp 495 an rla 1 he sn iy 320 ;In Glu Arg His Arg 400 Ile Lye Asp Gin Sen 480 Phe

S

'5 uu yy tt Thr Gin Trp Met 490 Gly Phe Tyr 475 Ile INFORMATION FOR SEQ ID NO:i6: SEQUENCE CHARACTERISTICS: LENGTH: 496 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 127 (ii) MOLECULE TYPE: protein (1x) FEATURE: NAME/KEY: hTL2 LOCATION; 496 OTHER INFORMATION: human TIE-2 ligand 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Trp Gin Ile Vai Phe Phe Thr Leu Ser Cyo Asp Ala Ala A Arg T Giu H Vai G Gin L Lou G Gin. C Thr S Vail 145 Lou Gin Lys Sor 225 An Asn O Vai Lys Ser 305 Gly Lye Tyr Tyr Ser 385 Ile Ser Cys Al a ia Tyr A~ 21 yr Arg I et Asp. A 0 in Arg A eu Lou G rlu Aen T 1 ,in Asn A 115 er LouL iu Thr G ;In His S rhr Ser C Lys Val 1 195 Lys Giu 210 Ile Ile Ser Vai Lou Lou Aia Arg 275 Ala Giy 290 Pro Glu Trp Thr Gly Trp Trp Lou 355 Val Leu 370 Leu Tyr His Lou Gin Pro Ile Cys 435 Cys Giy on Ai le G2 en G~ op A iu A yr1 00'i la V .eu S lIn V eor I 1u I iOu I flu 31u Leu Thr 260 Giu His G iu Ile Lys 340 Gly Lye Asp Lys Gly 420 Lys on en le or le -le 241 He1 Th 32

GI

As 1i Hi

GI

As Phe A, Hie G~ Arg S4 5! Pro P 70 Vai M Gin A Gin A Gin TI

IS

Sor I Asn I Met C Asp Lou 230 i Lye i Gin r Lye e Lye 310 e Gin u Tyr n Giu His *s Phe 390 .y Lou h5 in Asp a~ Ser Ly 5.

et op en hr .35 oen 'hr ~yo ;lu Se~ Ill Asi 29 Ai Ar Ly Ph Lo 37 Ty Tki Pt Le Lye Se 2! Ser C 40 Ser S4 Giu T' Glu A His T 120 Ala G Gin T Tyr I Ile F~ Asp 1 200 Lou Lyes i Gin :Thr a Ser 280 ri Giy 5 a Tyr g Arg a Vai 360 u Lye 'r Ile Lr Gly ie Ser au Met 440 ar Met 0' Ala Br Sor rr GIU en Tyr 90 Asp Tyr Thr Asp 75 Thr Ser Thr Tyr Ser Gin Ile Phe Val Val Trp Val Lou Gly Lye Lou Lou Thr An Gin Ser Lou Met Ala Giu 110 Giu Ile Lou Thr Thr Pro Ala Lou s0 Lye Ile Gly Asp et 05 hr.

lu 'hr 4 yo [is LB5 .Y13 ;In Lys His Ser 265 Phe Ilie Cy: Gi~ Gi' 34! Se As Se Th Th 42 Le Lye L Ala V Gin T ThrA Lou G 170 Asp I His I Val Ile Asp 250 An Arg Tyr 3 An Asp .330 y Phe 5 r Gin p Trp Gly r Ala 410 "r Lye .5 .u Thr ye al hr .rg iu 4 ys le 4 eu 235 Leu Sex Th: mel 31! Gl' Gi Ii 01 Gi 39 Al As

GI

Glu He met I~ Arg L: 140 Lou G Lye G Ann S Ile G 2 Val S 220 Thr A Met P Ala I SCys -Lou 300 t Asp 5 e yr Ser e Thr u Gly 380 u Giu 5 .a Lye ip Giy .y Giy iu In or iu or la Lep .ys klia 285 rhr Lei.

Prc As: 36! As Le 11 As Tr 44 Lou G Ile L Pho L 190 Met G Lys G Thr V Thr N Asp 270 *Asp *Phe Giy aAsp D Sor 350 ni Gin n Giu u Aen e Ser p An 430 p Trp In eu eu in in ral al1 er lal Pro Glj~ Phe 33! Gi'

GI~

Al Ty Se 41 As Ph Lou 160 Asp Glu Thr An An 240 Asn Thr Phe Asn Giy 320 aGin (Giu n& Arg a Tyr r Arg 400 r Ile p Lye ~e Asp at le Pro Ser Asn Lou Ann Giy Met Phe Tyr Pro Gin Arg Gin 450 455 hen Tkir Asn Lye Phe Aen Gly Ile Lys Trp Tyr 465 470 475 Gly Tyr Ser Ile Lye Ala Thr Thr Met Met Ile 485 490 INFORMATION FOR SEQ ID 00:17: ()SEQUENCE CHAR.ACTERISTICS: LENGTH: 1512 base pairs TYPE: nucleic acid STP.ANDEDNESS: single TOPOLOGY:* linear (ii) MOLECULE TYPE: DNA (1x) FEATUYRE: NAME/KEY: Coding Sequence LOCATION: 1509 OTHER INFORMATION: NAHN/KEY: TIE ligand-4 OTHER INFORMATION: 460 Tyr Arg Trp Lye Gly Ser 480 Pro Ala Asp Phe 495 a.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:i7: ATG CTC TCC CAG met Leu Ser Gin CTA GCC ATG CTG Ala Met Leu CAG GGC Gin Gly 10 AGC CTC CTC CTT Ser Leu. Leu Leu GTG GTT Val Val GCC ACC ATG Ala Thr Met TGC GAG ACA Cys Glu Thr 35

TCT

Ser GTG GCT CAA CAG Val Ala Gin Gin

ACA

Thr 2S AGG CAG Arg Gin GAG GCG GAT AGG GGC Giu Ala Asp Arg Giy CTT GTA GTC CAG Leu Val Val Gin "CAC GGC CAC TGT AGC TAC ACC TTC TTG His Gly His Cys Ser Tyr Thr Phe Leu 40 a 48 96 144 192 240 288 CTG CCC Leu Pro AAG TCT GAG CCC Lys Ser Glu Pro

TGC

Cys 55 CCT CCG GGG CCT Pro Pro Gly Pro

GAG

Giu GTC TCC AGG GAC Val Ser Arg Asp

TCC

Ser AAC ACC CTC CAG Aen Thr Leu Gin

AGA

Arg 70 GAA TCA CTG GCC Glu Ser Leu Ala

AAC

Asn 75 CCA CTG CAC CTG Pro Leu His Leu

CG

Gly so AAG TTG CCC ACC Lye Leu Pro Thr

CAG

Gin 85 CAG GTG AAA CAG Gin Val Lye Gin

CTG

Leu 90 GAG CAG GCA CTG Giu Gln Ala Leu CAG AAC Gin Aen AAC ACG CAG Aen Thr Gin AGG TCG AAG Arg Ser Lye 115

TGG

Trp 100 CTG AAG AAG CTA Leu Lye Lys Leu

GAG

G iu 105 AGG GCC ATC AAG Arg Ala Ile Lye ACG ATC TTG Thr Ile Leu 110 AAT CAG ACG Aen Gln Thr 336 384 CTG GAG CAG GTC Leu Glu Gin Val

CAG

Gin 120 CAG CAA ATG GCC Gin Gin Met Ala

CAG

Gin 125 GCC CCC Ala Pro 130 ATG CTA GAG CTG Met Leu Giu Leu

GC

Gly 135 ACC AGC CTC CTG Thr Ser Leu Leu

AAC

Asn 140 CAG ACC ACT GCC Gin Thr Thr Ala CAG ATC CCC AAG CTC ACC GAC ATG GAG GCT CAG CTC CTG A4AC CAG ACA Gin Ile Arg Lye Leu Thr Asp Met Glu Ala Gin Leu Leu Aen Gin Thr 480 150 155 TCA AGA ATG GAT Ser Arg Met Asp

GCC

Ala 165 CAG ATG CCA GAG Gin Met Pro Glu

ACC

Thr 170 TTT CTG TCC ACC Phe Leu Ser Thr AAC AAG Asn Lys 175 CTG GAG AA.C Leu Giu han

CAG

GIn 180 CTG CTG CTA CAG Leu Leu Leu Gin

AGG

Arg 185 CAG AAG CTC CAG Gin Lys Leu Gin CAG CTT CAG Gin Leu Gin 190 GAG ACC PAAG Giu Thr Lye GGC CAA AAC AGC Giy Gin Aen Ser 195 GCG CTC GAG Ala Leu Giu

AAG

Lys 200 CGG TTG CAG GCC Arg Leu Gin Ala 528 576 624 672 720 CAG CAG Gin Gin 210 GAG GAG CTG GCC Giu Glu Leu Ala

AGC

Ser 215 ATC CTC AGC AAG Ile Leu Ser Lys

AAG

Lys 220 GCG AAG CTG CTG Ala Lys Leu Leu

AAC

Asn 225 ACG CTG AGC CGC Thr Leu Ser Arg CAG AGC GCC GCC CTC ACC AAC ATC GAG CGC GGC Gin Ser Ala Ala Leu Thr Asn Ile Giu Arg Gly 230 235 .240 CTG COC GGT GTC AGG Leu Arg Giy Val Arg 245 CAC AAC TCC AGC.

His Asn Ser Ser

CTC

Leu 250 CTG CAG GAC Leu Gin Asp AGC CTG CGC Ser Leu Arg GCT AAC GCC Ala Aen Ala 275

CAG

Gin 260 CTG CTG GTG TTG Leu~ Leu Val Leu

TTG

Leu 265 CGG CAC CTG GTG Arg His Leu Val CAG CAG CAC -Gitw-On His 255 CAA GAA AGG Gin Glu Arg 270 CAG GTG TTC Gin Val Phe 768 816 864 TCG GCC CCG GCC Ser Ala Pro Ala

TTC

Phe 280 ATA ATG GCA GOT Ile Met Ala Gly

GAG

Giu 285 CAG GAC TGT GCA GAG ATC CAG CGC TOT G GCC ACT Gin Asp Cys Ala Glu Ile Gin Arg Ser Gly Ala Ser 290 295 300 GCC AGT GOT GTC Ala Ser Gly Val TAC ACC ATC CAG GTG TCC AAT GCA ACG AAG, CCC AGG AAG GTG TTC TGT Tyr Thr Ile Gin Val Ser Asn Aia Thr Lye Pro Arg Lys Val Phe Cys 305 310 315 320 GAC CTO CAG AGC Asp Leu Gin Ser

AGT

Ser 325 GGA GGC AGG TG Gly Oly Arg Trp

ACC

Thr 330 CTC ATC CAG CGC Leu Ile Gin Arg CGT GAG Arg Giu 335 AAT GGC ACC Asn Gly Thr TTC GGA GAO Phe Gly Asp 355

GTG

Val 340 AAT TTT CAG CG Asn Phe Gin Arg

AAC

Asn 345 TGG AAG CAT TAO Trp Lys Asp Tyr AAA CAG GGC Lye Gin Gly 350 GTG GTG CAC Val Val His CCA GCT COG GAG Pro Ala Gly Giu CAC TGG His Trp 360 CTG GGC, AAT Leu Gly Aen

GAA

0 iu 365 912 960 1008 1056 1104 1152 1200 1248 1296 CAG CTC Gin Leu 370 ACC AGA AGO GCA Thr Arg Arg Ala

GCC

Ala 375 TAC TOT CTG CGT Tyr Ser Leu Arg

OTG

Val 380 GAG CTO CAA GAC Giu Leu Gin Asp

TGG

Trp 385 GAA GGC CAC GAG Glu Gly His Giu G00 Ala 390 TAT GCC CAG TAC, Tyr Ala Gin Tyr

GAA

Glu 395 CAT TTC CAC CTG His Phe His Leu AGT GAG AAC, CAG CTA Ser Clu Asn Gin Leu .405 TAC AGO CTT TCT Tyr Arg Leu Ser

GTG

Val 410 GTC GGG TAC AGC Val Gly Tyr Ser GGC TCA Gly Ser 415 OCA GGG COC CAG AGC AGC CTO GTC CTO CAG AAC ACC AGC TTT AGC ACCO Ala Gly Arg Gin Ser Ser Leu Val Leu Gin Asn Thr Ser 420 425 Phe Ser Thr 430 CTT GAC TCA Leu Asp Ser 435 TCT GGA GG Ser Gly Gly

GAC

Asp AAC GAC CAC Asn Asp His

TOT

Cys 440

GCC

Ala CTC TGC AAG TGT Leu Cys Lye Cye

GCC

Ala 445

AAC

Asn CAG GTG ATG Gin Val Met CTC AAC GGC Leu Asn Gly TOG TOG TTT Trp Trp Phe

GTC

Val 465

TGG

Trp 450

TAC

Tyr

GAC

Asp 455

GAC

Asp TGT GGC .CTG Cys Gly Leu

TCA

Ser 460

ATG

Met 1344 1392 1440 1488 TAC CAC GCT Tyr His Ala

CCC

Pro 470 AAC AAG TAC Aen Lye Tyr

AAG

Lye 475

CTG

Leu GAC GGC Asp Gly ATC CGC Ile Arg CGC ATG Arg Met 495

CAC

Hie TAC TTC AAG GGC CCC AGC Tyr Phe Lys Gly Pro Ser 485 CG-CCT TTG GAC ATC TAA Arg Pro ,Leu Asp Ile 500, TAC TCA Tyr Ser .490 CGT GCC TCT Arg Ala Ser ATG ATA Met Ile 1512 INFOP1MATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 503 amino, acids TYPE: amino acid STRANDEDNESS:.single TOPOLOGY: linear (1i) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE:.

NAHE/KEY: TIE ligand-4 LOCATION: 1. 503

OTHER-.INFORMATION:

.4* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Leu Ser Gin Leu Ala Met Leu Gin Gly Ser Leu Leu Leu Val Val Met 1 Ala Cys Leu Ser Lys Asn Arg Ala Gin 145 Ser Leu Thr Giu Pro Asn Leu Thr Ser Pro 130 Ile Arg Giu Met Thr 35 Lye Thr Pro Gin Lys 115 Met Arg Met Asn Ser 20 Leu Ser Leu Thr Trp 100 Leu Leu Lye Asp Gin 180 5a Val Glu Gin Gin Leu O ii O iu Leu Ala 165 Leu Ala Val Pro Arg 70 Gin Lys Gin Leu Thr 150 Gin Leu Gin Gin Cys 55 GlU Val* Lys Val O ly 135 Asp Met Leu Gin Thr Arg 25 His Gly His 40 Pro Pro Gly Ser Leu Ala Lye Gin Leu 90 Leu Giu Arg 105 Gin Gin Gin 120 Thr Ser Leu Mgt Glu Ala Pro Giu Thr 170 Gin Arg Gin 185 Gin Cys Pro Asn 75 Glu Ala Met Leu Gin Phe Glu Ser Glu Pro Gin Ile Ala Aen 140 Leu Leu Ala Tyr Val Leu Ala.

Lys Gin 125 Gin Leu Ser Asp Thr Ser His Leu Thr 110 Asn Thr Aen Thr Arg Gly Phe Leu Arg Asp Leu Gly Gin Asn~ Ile Leu Gin Thr Thr Ala Gin Thr 160 *Aen Lye 175 Lys Leu Gin Gin Leu Gin 190 Gly Gin Gin GIn 210 Asnl Thr 225 Leu Arg4 Ser Leu Ala Asn Gln Asp 290 Tyr Tkir 305 Asp Lau Asn Gly Phe Gly Gin Leu 370 Trp. Giu 385 Ser Giu Ala Gly Leu Asp Ser Gly 450 Val Tyr 465 Trp His wsn ;lu Leu Giy Arg Ala 275 Cys Ile Gin Thr Asp 355 Thr G ly Aen Arg Ser 435 Gly Tyr Tyr I~er Ala Lau Giu Lys Arg 200 Leu Gin Ala Lau Glu Thr .205 Lys Giu Ser Val Gin 260 Ser Ala Gin Ser Val 340 Pro Arg His GIn Gln 420 Asp *Trp His *Phie Leu Arg C Arg 245 Lau Ala Giu Val Ser 325 Asn Ala Arg Giu Lau 405 Ser Asn Trp Ala Lys 485 ~la In Z30 Uls IUeu Pro Ie Ser 310 Gly Phe Gly Ala Ala 390 Tyr Ser Asp Phe Pro 470 Gly Ser 215 Ser Aen Val Ala Gin 295 Asn Gly Gin Giu Ala 375 Tyr Arg Leu His Asp 4S5 Asp Pro Ile kl.

Ser Lau Phe 280 Arg Ala Arg Arg His 360 Tyr Ala Let; Va] Cyc 44C Al: Asi Se~ Lau Ala Ser Leu 265 Ile Ser Thr Trp Asn 345 Trp *Ser Gill Ser *Let 42E aLet I Cys 'i Lyl r Ty~ Ser Leu Lau 250 Arg Met Gly Lye Thr 330 Trp *Lau Leu Tyr *Val 410 Glr aCyE 3 G1) a Ty~ r Se~ 49~ Lye Lj Thr Ai 235 Leu G His Li Ala G Ala S 3 Pro A 315 Leu I Lys Ai Gly A *Arg 1i *Giu 1 395 Val C Asn aLys SLeu :Lye 475.

a In ly er 00 rg le .sp onf ral lie L'hz Se2 46( H1e Plia Lye Lou Leu Ile G Asp C Val C Giu 285 Ala Lye GIn Tyr Glu 365 Giu.

Phe Tyr Ser aAla 445 Asn Asp liu lIn ~In In Ser VJal Arg Lye 350 Val Leu His Ser Plie 430 Gir Leau Glj Arg Gin 255 Glu Val Gly Phe Arg 335 Gin Val GIn Leu Gly 415 Ser Val Asr IiE Gly 240 His Arg Phe Val.

Cys 320 Glu G ly His Asp Gly 400 Ser *Thr *Met 1Gly BArg.

480 r Leu Arg Ala Ser Arg Met 495 Met Ile Arg Pro Leu Asp Ile 500 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 1497 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY.: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: .1494 OTHER INFORMATION: NAME/KEY: lN1C2F (chimera 1) LOCATION: .1497 OTHER INFORMATION: NAME/KEY: Other LOCATION: OTHER INFORMATION: Putative leader sequence is encoded by nucleotides 1-60 132 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: ATG ACA GTT TTC Met Thr Val. Phe

I.

TCC TTT GCT TTC Ser Phe Ala Phe GCT GCC ATT CTC Ala Ala Ile Leu ACT CAC Thr His ATA CGG TGC Ile Gly Cys TAT AAC CGG Tyr Asn Arg

AGC

Ser AAT CAG CGC CGA Asn Gin Arg Arg

AGT

Ser 25 CCA GAA AAC AGT Pro Glu Asn Ser GGG AGA AGA Gly Arg Arg ATT CTT CCA Ile Leu Pro ATT CAA CAT GGG Ile Gin His Gly TOT GCC TAC ACT Cys Ala Tyr Thr GAA CAC Glu His so GAT CCC AAC TGT Asp Giy Aen Cys

CGT

Arg 55 GAG ACT ACG ACA Giu Ser Thr Thr

GAC

Asp CAG TAG AAC ACA Gin Tyr Aen Thr

AAC

Aen GCT CTG CAG AGA Ala Leu Gin Arg GAT GCT CCA CAC GTC Asp Ala Pro His Val.

70 GAA CCC CAT TTC TCT TCC Giu Pro Asp Phe Ser Ser 75 GAA AAT TAT ACT CAG TGG.

Glu Aen Tyr Thr Gin Trp 96 144 192 240 288 336 384 GAG AAA CTT CAA Gin Lys Leu Gin

CAT

His CTG GAA CAT GTG Leu Giu His Val

ATG

Met 90 CTG CAA AAA Leu Gin.Lye CCC GAG ATA Ala Gin Ile 115

CTT

Leu 100 GAG AAT TAC ATT Giu ken Tyr Ile GAA AAC ATG AAG Giu Aen Met Lys TCG GAG ATG Ser Giu Met 110 ACC ATG CTG Thr Met Leu GAG GAG AAT GCA Gin Gin ken Ala GAG AAC CAC AG Gin Asn His Thr

OCT

Ala 125 GAG ATA GGA ACC AGC CTC CTC TCT GAG ACT GCA Glu Ile Giy Thr Ser Leu Leu Ser Gin Thr Ala 130 135 GAG GAG Glu, Gin 140 ACC AGA AAG Thr Arg Lys

CTG

Leu 145 ACA GAT GTT GAG Thr Asp Val Glu

ACC

Thr 150 GAG GTA CTA AAT Gin Val Leu Asfl

CAA

Gin 155 ACT TCT CGA CTT Thr Ser Arg Leu

GAG

Giu 160 p.

ATA CAG CTG CTG Ile Gin Leu Leu

GAG

C iu 165 AAT TCA TTA TCC Aen Ser Leu Ser ACC TAG AAG CTA GAG AAG CAA Thr Tyr Lye Leu Giu Lys Gin 170 -175 AAG ATC CAT OAk AAA AAC ACT Lye Ile His Giu Lye ken Ser 190 480 528 S76 624 CTT CTT CAA Leu Leu Gin TTA TTA GAA Leu Leu Oiu 195

CAG

Gin 180 ACA AAT GAA ATC Thr Aen Giu Ile

TTG

Lau 185 CAT AAA ATC TTA His Lye Ile Leu

GAA

C lu 200 ATO GAA GGA AAA Met Giu Gly Lye

CAC

His 205 AAG GAA GAG Lye Giu Giu TTG GAC ACC TTA AAG GAA GAG AAA GAG AAC CTT CAA CCC TTG GTT ACT Leu Asp Thr. .Leu Lye Ciu Giu Lye Glu ken Leu Gin Gly Leu Val Thr 210 215 220

CGT

Arg 225 CAA ACA TAT ATA Gin Thr Tyr Ile

ATC

Ile 230 GAG GAG CTG CAA Gin Giu Leu Glu

AAG

Lye 235 CAA TTA AAC AGA Gin Leu Asn Arg

GCT

Ala 240 ACC ACC AAC AAC Thr Thr Aen ken

ACT

Ser 245 GTC CTT CAG AAG Vai Leu Gin Lye

CAG

Gin 250 CAA CTG GAG CTG Gin Lau Giu Leu ATG GAG Met Asp 720 768 816 ACA GTC CAC AAC CTT CTC AAT CTT TGC ACT AAA GAA GGT GTT TTA CTA 1 33 Thr Val His hen Leu Va2l Aen Leu Cye Tkir Lys Glu Gly Val Lou Leu 260 265 270 AAG GGA GGA Lye Gly Gly 275 AAA AGA GAG GAA LYS Arg Giu Glu AAA CCA TTT AGA Lys Pro Phe Arg GAC TGT GCT OAA Asp Cys Ala Glu 285 GTA TTC AAA TCA GGA CAC, ACC ACA AAT GGCATC TAC ACO TTA ACA TTC Val Phe Lys Ser Gly His Thr Thr Aen Gly Ile Tyr Thr Leu Thr Phe 290 295 300

CCT

Pro 305 AAT TCT ACA GAA Aen Ser Thr Glu

GAG

Glu 310 ATC AAG GCC TAC Ile Lys Ala Tyr

TGT

Cys 315 GAC ATG GAA OCT Asp Met Glu Ala

GGA

Oiy 320 GGA GGC GG0 TG Gly Oly Oly.Trp ACA ATT Thr Ile 325.

ATT CAG CGA Ile Gin Arg

CGT

Arg 330 GAG GAT GGC AGC Glu Asp Giy Ser GTT GAT Val Asp 335 864 912 960 1008 1056 1104 .1152 1200 TTT CAG AGG ACT TOG AAA OAk TAT Phe Gin Arg Thr Trp Lye Glu Tyr 340 GGA GAA TAT TG CTO OGA AAT GAG oiy. Olu Tyr Trp Leu Gly ken Glu 355 360

AAA

Lye 345 GTG 00k, TTTGT Val Gly Phe Gly AAC, CCT TCA.

Ann Pro Ser 350 ACT AAT CAG Thr ken Gin TTT OTT TOG CAA Phe Val Ser Gin

CTG

Leu 365 CAA CGC Gin Arg 370 TAT GTG CTT AAA Tyr Val Leu Lye

ATA

Ile 375 CAC CTT AAA GAC His Leu Lye Asp

TG

Trp 380 GAA GGG AAT GAG Giu Gly Aen Giu OCT TAC TCA TTG, TAT GAA CAT TTC TAT CTC TCA AOT GAA OAk CTC AAT Ala Tyr Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Giu Lau Asn 385 390 395 400 TAT AGO ATT CAC Tyr Arg Ile His

CTT

Leu 405 AAA GGk CTT .ACA Lys Gly Leu Thr 000 G ly 410 ACA GCC GOC AAA Thr Ala Gly Lys ATA AGC Ile Ser 415 AGC ATC kOC Ser Ile Ser GAC AAA TOT Asp Lys Cys 435

CAA

Gin 420 CCA GGA AAT OAT Pro Gly ken Asp

TTT

Phe 425 AGC ACA AAG OAT Ser Thr Lye Asp GGA GAC AAC Oly Asp ken 430* GGC TOO TG Gly Trp Trp ATT TOC AAA TOT Ile Cys Lye Cys

TCA

Ser 440 CAA ATO CTA ACA Gin Met Leu Thr

GGA

O iy 445 1248 1296 1344 1392 1440 1488 TTT GAT Phe Asp 450 GCA TOT GGT CCT Ala CyB Gly Pro

TCC

Ser 455 AAC TTG AAC GGA Asn Leu Aen Gly

ATG

Met 460 TAC TAT CCA CAG Tyr Tyr Pro Gin

AGG

Arg 465 CAG AAC ACA AAT Gin ken Thr ken

AAG

Lye 470 TTC AAC GGC ATT Phe ken Gly Ile

AAA

Lye 475

ATG

Met TGG TAC TAC TGG AAA Trp Tyr Tyr Trp Lye 480 ATG ATC CGA CCA OCA Met Ile Arg Pro Ala 495 OGC TCA GGC TAT Gly Ser Oly Tyr

TCG

Ser 485 CTC AAG 0CC ACA Leu Lys Ala Thr

ACC

Thr 490 OAT TTC TAk 1497 Asp Phe INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 498 amino acids TYPE: amino acid STRANDEDNESSt single TOPOLOGY: linear (1i) 14OLECULE TYPE: protein FRAGMENT TYPE. internal (ix) FEATURE:I NAME/KEY: 3H1C2F (chimera 1) LOCATION: 1...498 OTHER INFOR.MATION: (xil SEQUE Met Thr Val Phe NCE DESCRIPTION: SEQ ID Leu Ser Phe Ala Phe Leu Ala Ala 5 *10 Aen Gin Arg Arg Ser Pro Glu Asn 25 Gin His Gly Gin cys Ala Tyr Thr Ile Tyr GlU **Aen Gly Asn H .is s0 Ala

C

I

I

Gin Lye Leu GIn Ala Gin Giu Ile 130 Lou Thr 145 Ile Gin Leu Leu Leu Leu Leu Asp 210 Arg Gin 225 Thr Thr ye S 2 rg 35 kepC LAeu Leu Lys Ile 115 Gly Asp Leu Gin GiU 195 Thr Thr Asn ;iy ;In G3n Leu 100 GIn Thr Val Leu Gin 180 His Leu Tyr Asr A~s.n C) krg Ai His Li 85 Gilu A Gin A Ser L Giu T Glu A 165 Thr A Lys I *Lye C 1Ser 245 Leu 3Arg Giy Giu p Thr 325 r Trp 0 p Leu .1 Leu u Tyr op A eu G on T an A eu L hr C 50 osn oanC lie ;iu Ile Z30 JFai V1al Q lu His G iu 310 Ile Lys Gly Lys G iu lu yr la 4 eu -35 In Ser 1lu Leu 215 Gir Gliu Ser Thr Thr Pro His Ile Val 120 Ser Val Leu Ile Giu 200 LYe 1Glu Hlis Val Val 105 Gin Gin Leu Ser Lell Val met 90 Giu Asn Thr Asn Thr 170 Lys Giu 75 Giu Aen His Aia *Gin 155 *Tyr Ile ;er l1e 60 Pro Asn Met Thr G iu 140 Thr Lys HiE Ile Ser Pfie Gin Asp Tyr Lye Ala 125 Gin Ser Leg Gi1A Met Gill Gly Gill Aen Leu Leu Gill Lys 235

I

Lye Gin 220 Gin ,eu Thr is ;iy Arg I ie Leu His 20I G I~ Le Phe Se Thr GI Ser GI 110 Thr Me Thr A~ Arg Li Glu L 1 Lye A 190 3 Lys G y Leu V~ u Aen u Leu y Val 270 .p Cys kr Leu :t Giu Ly Ser ly Asn 350 eu Thr 65 lu Gly iu Giu r :t 7! er as 4 e Al

A]

3:

A

A

L

His Arg Pro Thr Ser s0 .Trp Met Leu Lye 160 Gin Ser I Giu 1 Thr g Ala 240 t Asp u Leu .a Glu 1r Phe La Gly 320 al Asp ro Ser en Gin en Giu eu Asn 400 Leu Gin Lye Thr Lye Val Pro 305 Gly Phe G iy Gin Ala 385 Tyr Val Gly Phe 290 Asn Gly Gin Giu Arg 370 Tyr His G iy 27S Lys Ser Gly Arg Tyr 355 Tyr Ser Aer 26( LyE Se~ Th~ Tr: Th 34 Tr Va Le Aen Glu Thr 295 Ile Ile Giu Aen Ile 375 His Leu G lu 280 Thr Lye Gin Tyr G iu 360 His Phe Cya 265 Lye Aen Ala Arg Lys 345 Phe Leu Tyr 250 Thr Pro Gly Tyr Arg 330 Val Val Lys Let; Lye Phe Ile Cys 315 G iu Gly Ser Asp Ser 395 euC ;1u Ar4 Tyr 300 Asp Asp Phe Gin Trp 380 Ser As 28

MC

LI

3

G

G

Arg Ile His Leu Lye Gly Leu Thr Gly Thr Ala Gly Lye Ile Ser I 1r,; 405 Pro 410 Ser Ser Ile ser Asp Lys Cys 435 Phe Asp Ala Gin 420 IlIe Gly Asn Asp Phe 425 Gln Thr Lye Asp 415 Gly Asp Asn 430 Gly Trp Trp Tyr Pro Gin Cys Lys Cys Ser 440 Met Leu Thr Cys Giy Pro 450 Gin Ser 455 Phe Asn Leu Aen Gly Met 460 Aen Gly Ile Lys Trp O ly 445 Tyr Tyr hrg 465 Gly Asn Thr Asn Lys 470 Ser Gly Tyr Ser 485 Leu Lys Ala Thr Thr *Met 490 Met Ile Tyr Trp Arg Pro 495 Lys 480 Ala Asp Phe S S

S

S. INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 1491 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPO LOGY: linear (ii) MOLEC4IE TYPE: DNA (Ix) FEATUIRE: NAME/KEY: coding sequence LOCATION: 1...1488 OTHER INFORMATION: NAME/KEY: 2N2C1F (chimera 2) LOCATION: 1491 OTHER INFORMATION: NAME/KEY: Other LOCATION: I1.. .48 OTHER INFORMATION: Putative leader sequence Is encoded by nucleotides 1-48 (xi) SEQUENCE DESCRIPTION: SEQ-ID 140:21:

ATG

Met 1 TGG cAG ATT Trp Gin Ile TTC TTT ACT CTG Phe Phe Thr Leu AGC TGT GAT CTT GTC TTG GCC Ser Cys Asp Leu Val Leu Ala 10 GCA GCC TAT Ala Ala Tyr CAA TAT CAG Gin Tyr Gin

AAC

Asn AAC TTT CGG AAG Asn'Phe Arg Lys

AGC

Ser 25 ATG GAC AGC ATA Met Asp Ser Ile GGA AAG AAG Gly Lye Lye GTC CAG CAT GGG Val Gin His Gly TCC TGC AGC TAC ACT TTC CTC CTG CCA Ser Cys Ser Tyr Thr Phe Leu Leu;Pro 40 45 I GAG ATG Giu Met GAC AAC TGC CGC Asp Aen Cys Arg

TCT

Ser 55 TCC TCC AGC CCC Ser Ser Ser Pro

TAC

Tyr GTG TCC AAT OCT Val Ser Asn Ala 48 96 144 192 240 288 336

GTG

Val CAG AGG GAC GCG Gin Arg Asp Ala

CCG

Pro 70 CTC GAA TAC GAT Leu Glu Tyr Asp

GAC

Asp 75 TCG GTG CAG AGG Ser Val Gin Arg

CTG

Leu CAA GTG CTG GAG Gin Val Lau Glu

AAC

Asn ATC ATG GAA AAC Ile Met Glu Asn AAC ACT Asn Thr 90 CAG TGG CTA Gin Trp Leu ATG AAG Met LYe CTT GAG AAT TAT ATC CAG GAC AAC ATG AAG AAA GAA ATG GTA GAG ATA Leu Giu Aen Tyr Ile Gln Asp Aen Met Lye Lye Giu Met Val Glu Ile 100 105 110 CAG CAG AAT Gin Gin Asn 115 GCA GTA CAG AAC Ala Val Gin Aen CAG ACG GCT GTG ATG ATA Gin Thr Aia Val Met Ile 120 125 GAA ATA GGG Glu Ile Gly ACA AAC Thr Aen 130 CTG TTG AAC CAA Leu Leu Aen Gin

ACA

Thr 135 GCT GAG CMA ACG Ala Giu Gin Thr

CGG

Arg 140 AAG TTA ACT GAT Lye Leu Thr Asp

GTG

Val 145 GAA GCC CAA GTA Glu Ala Gin Val

TTA

Leu 150 AAT CAG ACC ACG Ann Gin Thr Thr

AGA

Arg 155 CTT GAA CTT CAG Leu Giu Leu Gln

CTC

Lau- 160 384 432 480 528 576 TTG GAA CAC TCC Leu Giu His Ser

CTC

Leu 165 TOG ACA AAC AAA TTG Ser Thr Ann Lye Lou 170 GAA AAA CAG ATT Glu Lye Gin Ile TTG GAC Leu Asp 175 CAG ACC AGT Gin Thr Ser

GAA

Glu 180 ATA AAC AAA TTG Ile Ann Lye Leu

CAA

Gin 185 GAT AAG AAC AGT Asp Lye Ann Ser TTC CTA GAA Phe Lou Glu 190

S

S

S.

AAG AAG GTG CTA OCT ATO GAA GAC AAG CAC ATC ATC CAA CTA CAG TCA Lye ye Val Leu Ala Met Giu Asp Lye His Ile Ile Gin Leu Gin Ser 195 200 205 ATA AAA Ile Lye 210 GA GAG AAA OAT Glu Giu Lye Asp

CAG

Gin 215 CTA CAG GTG TTA Leu Gin Val Leu

GTA

Val 220 TCC AAG CAA AAT Ser Lye Gin Aen 624" 672 720 768

TCC

Ser 225 ATC ATT GAA GA Ile Ile Giu Glu

CTA

Leu 230 GAA AAA AAA ATA Glu Lye Lye lie

GTG

Val 235 ACT GCC ACG GTG Thr Ala Thr Vai

AAT

Aen 240 AAT TCA GTT OTT Ann Ser Vai Leu

CAA

Gin 245 AAG CAG CAA CAT Lye Gin Gin His

GAT

Asp 250 CTC ATG GAG ACA Leu Met Giu Thr OTT AAT Val Aen 255 AAC TTA CTO Aen Leu Leu OTT OCT AAA Val Ala Lye 275

ACT

Thr 260 ATG ATO TCC ACA Met Met Ser Thr

TCA

Ser 265 AAC TCA GCT AAG Aen Ser Ala Lye GAO CCC ACT Asp Pro Thr 270 GAT GTA TAT Asp Vai Tyr GAA GAA CAA ATC Giu Giu Gin Ile

AGC

Ser 280 TTC AGA GAC TGT Phe Arg Asp Cys

GCA

Ala 285 CAA GCT Gin Ala 290 GGT TTT MAT AAA IOT OGA ATC TAC ACT ATT TAT ATT AAT AAT Gly Phe Ann Lys Ser Gly Ile Tyr Thr Ile Tyr Ile Ann Asn 295 300

ATG

Met 305 CCA GAA CCC AAA Pro Glu Pro Lye

AAG

Lye 310 GTG TTT TOC AAT Val Phe Cys Ann ATG OAT GTC AAT GGG GGA Met Asp Vai Ann Giy Giy 315 320 816 864 912 960 1008 1056 1104 GGT TOO ACT GTA Gly Trp Thr Val

ATA

Ile 325 CMA CAT COT GAA Gin His Arg Glu

GAT

Asp 330 OGA AGT CTA OAT Gly Ser Leu Asp TTC CAA Phe Gin 335 AGA 000 TOO Arg Giy Trp

AAG

Lye 340 GAA TAT AAAATG Glu Tyr Lye Met

GGT

O y 345 TTT OGA AAT CCC Phe Giy Aen Pro TCC GOT GAA Ser Oly Olu 350 TAT TGG CTG GGG AAT GAO TTT ATT TTT 0CC ATT ACC AGT CAG AGOG CAG Tyr Trp Leu Gly Asn Giu Phe Ile Phe Ala Ile Thr Ser Gin Krg Gin 355 360 365 TAC ATO Tyr Met 370 CTA AGA ATT GAG Leu Arg Ile Giu

TTA

Leu 375 ATG GAC TGG GAA Het Asp Trp Giu GGG AAC CGA GCC TAT Gly Aen Arg Ala Tyr 380

TCA

Ser 385 CAG TAT GAC AGA Gin Tyr Asp Arg

TTC

Phe 390 CAC ATA GGA AAT His Ile Gly Aen

GAA

G iu 395 AAG CAA AAC TAT Lys Gin Aen Tyr

AGG

Arg 400 TTG TAT TTA AAA Leu Tyr Leu Lye

GGT

Gly 405 CAC ACT GGG ACA His Thr Gly Thr GGA AAA CAG AGC Gly Lye Gin Ser AGC CTG Ser Leu 415 ATC TTA CAC Ile Leu His

GGT

Gly 420 GCT GAT TTC AGC Ala Asp Phe Ser ACT AAA GAT GCT GAT AAT GAC AAC Thr Lys Asp Ala Asp Asn Asp Asn 425 430 1152 1200 1248 1296 1344 1392 1446 TGT ATG TOC. AAA TGT GCC CTC ATG TTA ACA GGA OGA TGG TOG TTT OAT Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 GCT TOT Ala Cys 450 AAC, CAT Asn'His 465 GGC CCC TCC AAT Gly Pro Ser Aen GGA AAA CTG..AAT Gly Lye Leu Aen 470

CTA

Leu 455 AAT GGA ATG TTC Aen Gly Met Phe

TAT

Tyr 460 ACT GCG GGA CAA Thr Ala Gly Gin 000 ATA AAG TG Oly Ile Lye Trp

CAC

His 475 TAC TTC AAA 000 Tyr Phe Lys Gly

CCC

Pro 480 C AGT TAC TCC TTA Ser Tyr Ser Leu COT TCC Arg Ser 485 ACA ACT ATG Thr Thr Met ATT COA CCT TTA Ile Arg Pro Leu OAT TTT T 1489 Asp Phe 495 1491

C..

C. C a.

INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 496 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (1x) FEATURE: NAME/KEY: 2N2CiF (chimera 2) LOCATION: 1..-496 OTHER INFORMATION:' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met .1 Ala Trp Gin Ile Val Phe Phe

S

Asn Phe Arg Thr Lys Ala Gin Tyr Giu Met so Val Gin Tyr Gin Asp Val Gin Asn Cys Asn His Gly Ser 40 Arg Ser Ser Leu Ser Cys Asp 10 Ser Met Asp Ser Cys Ser Tyr Thr Ser Ser Pro Tyr Tyr Asp Asp Ser 75 Ile Phe Val Gly Lye Lys Leu Leu Pro Ser Asn Ala Leu Val Leu Ala is Arg Asp Ala Pro *70 Ile 55.

Leu Glu Val Gin Gin Arg Leu Met Lye Val Leu Giu Aen Met Giu Asn Aen 90 Thr Gin Trp Leu Leu Giu Aen Tyr Ile Gin Asp Aen Met Lye Lye Giu Met Val Giu Ile 100 105 110 Gin Gin Aen 115 Thr Val 145 Leu Gin Lys Ile Ser 225 Aen Aen Asn 130 Giu G iu Thr Lys Lys 210 Ile Ser Leu Leu Ala His Ser Val 195 Giu Ile Val.

Leu o .0.0 .0.4 Val Gin met 305 Giy Arg Tyr Tyr Ser 385 Leu Ile Cl's Ala Aen 465 Ser Ala Al.a4 290 Pro Trp Gly Trp met 370 Gin Tyr Leu Met Cys 450 His Tyr Lys 275 Gly Thi Tri Let Le: Ty: Le~

HL~

Cy 43 Gi Gi Se Ala Val Gin Aen Leu Asn Gin Thr 135 Gin Val Leu Aen 150 Ser Leu Ser Thr 165 Glu Ile Aen Lys 180 Leu Ala Met Giu Glu Lys Asp Gin 215 Giu Giu Leu Giu 230 Leu Gin Lys Gin 245 thr Met Met Ser 260 GiuGiu Gin Ile *Phe 4on Lye sex i 295 Pro Lye Lye Va.] 310 *Val Ile Gin Hi 325 Lys Giu Tyr Lyi 340 Gil' Aen Giu Ph4 a Arg Ile Giu Lei 37' Asp Arg Phe Hi 390 Lye Gly His Th 405 Gly Ala Asp Ph 420 Lys Cl's Ala Le 5 y Pro Ser Aen Le 45 y Lye Leu Aen G1 470 r Leu Arg'Ser Tkh 485 Gin 120 Ala Gin Asn Leu

ASP

200 Leu Lye Gin Thr Ser 280 Gly Phe Ar; Met Ile 36C Met G1' a Se: 44' ui As 5 y I r Th Thr T Lye L 1 Gin A 185 Lye I His I Ser 265 Phe Ile Cys Giu Gly 345 Phe

ASP

SGly ~Thr r Thr 425 t, Leu 0 n Gly e Lye r Met hr eu 70 ,ep al, le sep ~so Thr Ala Vai Met Ile 125 Giu Gin Thr Arg Lye 140 Arg 155 G iu Lys Ile Leu Val 235 Leu Ser Leu Lye Aen Ile Val 220 Thr Met Ala Giu Gin Ser Gin 205 Ser Ala Glu Lys krg Tyr Asp 330 P he Ala Trp Aen Ala 410 Lye Thr Met Trr Met 49( AspC Thr Met 315 Gly Gil' Ile Giu Giu 395 Gly Asp G ly Phe His 475 Ile .ys I le 300

FLOP

Ser Asn Thr G ly 380 Lys Lye Ala Gly Tyr 4 6C Tyi Arc Ala A 285 Tyr- I Vai I Leu Pro Ser4 365 Aen Gin Gin Asp Trp 445 Thr -Phe ;Pro ep Val Tyr Giu Leu Leu Ile Phe 190 Leu Lye Thr Thr Asp 270 Ile Gly rhr Asp Gin Leu 175 Leu Gin Gin Val Val 255 Pro le Oen kap Ser 350 Glm Arg Asn Ser As, 430 Trj Ai Lyi Le: Aen An Gly Giy 320 Phe Gin 335 Gly Giu Arg Gin Ala Tyr Tyr Arg 400 Ser Leu 415 Asp Aen Phe Asp Gly Gin a Gil' Pro 480 u Asp Phe 495 Leu 160 Asp Glu Ser Aen, Aen 240 Amn Thr INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 1500 base pairs TYPE: nucleic acid STP.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (1x) FEATURE: NAME/KEY: Coding Sequence LOCATION: .1497 OTHER INFORMATION: NAME/KEY: iN2C2F (chimera 3) LOCATION: 1500 OTHER INFORMATION: NAME/KEY: Other LOCATION: OTHER INFORMATION: Putative leader sequence is encoded by nucleotides 1-60 (xi) SEQUENCE DESCRIPTION: SEQ.ID NO:23: ATG ACA OTT TTC Met Thr Val Phe 1

CTT

Leu 5 TCC TTT GCT Ser Phe Ala TTC CTC Phe Lau 10 AGT CCA Ser.Pro, 25 GCT 0CC ATT CTG Ala Ala Ile Leu ACT CAC Thr His is ATA 000 TGC Ile Giy Cys TAT AAC CG Tyr Asn Arg

AGC

Ser ANT CAG CGC CGA An Gin Arg Arg GAN AAC AGT Glu Asn Ser GOG AGA AGA Gly Arg Arg ATT CTT OCA Ile Leu Pro 96 144 ATT CAN CAT 000, Ile Gin His Gly

CAA

Gin 40- TGT 0CC TAC ACT Cys Ala Tyr Thr

TTC

Phe GAA CAC Giu Hie GAT 000 AAC TOT Asp Gly Asn Cys

COT

Arg GAG AOT ACG ACA Glu Ser Thr Thr

GAC

Asp CAG TAC AAC ACA Gin Tyr Asn Thr 192

S

S

S.

S

S

*SSS**

S

S.

S

*SSS

55.5

S

*5S.

*@5G

S

S

SS S

S

S

0 5O AAC GCT CTG CAC AON OAT OCT CCA CAC GTG GAN CCO OAT'GAC TCG OTG Asn Ala Leu Gin Arg Asp Ala Pro His Val Glu Pro Asp Asp. Ser Val 65* 70 75 CAG AGO CTG CAN Gin Arg Leu Gin

OTG

Val 85 CTG GAO AAC ATC Leu Giu Asn Ile

ATG

Met 90 GAA AAC NAC ACT Oiu Asn Asn Thr CAG TG Gin Trp CTA ATO AAG Leu Met Lys G TA GAG ATA Val Oiu Ile 115

CTT

Lau 100 GAG ANT TAT ATC Giu Asn Tyr Ile GAC AAC ATO AAG Asp An Met Lys AAA GAA ATO Lys Giu Met 110 OTO ATO ATA Val Met Ile CAG CAG ANT OCA Gin Gin NAn Ala

GTA

Val 120 CAG AAC CAG ACO Gin Asn Gin Thr

OCT

Ala 125 240 288 336 384 432 480 528 GAA ATA Giu Ile 130 000 ACA NAC CTG Gly Thr Aen Leu

TTG

Leu 135 NAC CAA ACA OCT NAn Gin Thr Ala

GAG

Glu 140 CAN ACO COO AAG Gi.n Thr Arg Lys

TTA

Leu 145 ACT OAT OTO A Thr Asp Val Giu 0CC Ala I50 CAN OTA TTA AAT Gin Val Leu Asn

CAG

Gin 155 ACC ACG AGA CTT Thr Thr Arg Leu

GAA

Giu 160 CTT CAG CTC TTG Leu.Gin Leu Leu

GAN

Giu 165 CAC TCC CTC TCG His Ser Leu Ser

ACA

Thr 170 AAC ANA TTG GAN Aen Lye Leu Giu AA CAG Lye Gin 175 ATT TTG GAC Ile Leu Asp TTC CTA A Phe Leu Oiu 195

CAG

Gin 180 ACC NOT GAN ATA Thr Ser Oiu Ile

AAC

An 185 AA TTO CAN OAT Lye Leu Gin Asp AG ANC NOT Lye An Ser 190 ATC NTC CAN Ile Ile Gin 576 624 ANG ANG, GTG CTA Lye Lys Val Leu

OCT

Ala 200 ATG GAN GAC ANO Met Giu Asp Lys

CAC

His 205 CTA CAG Leu Gin 210 TCA ATA AAA GAN Ser Ile Lys Glu

GAG

Giu 215 NA OAT CAG CTA Lys Asp Gin Leu

CAG

Gin 220 OTO TTA OTA TCC Val Leu Val Ser ANG CAN ANT TCC ATC ATT GAN GAN CTA GAN AA ANA ATA OTG ACT 0CC 72 720 Lys Gin Aen Ser Ile Ile Giu Giu Leu Giu Lye Lye Ile Val Thr Ala.

225 230 235 240 ACG GTG AAT AAT Tkir Val Aen Asn

TCA

Ser 245 GTT CTT CAA hAG Val Leu Gin Lys

CAG

Gin 250 CAA CAT GAT CTC Gin His Asp Leu ATG GAG Met Giu 255 ACA GTT hAT Thr Val hen GAC CCC ACT Asp Pro Thr 275

AAC

hen 260 TTA CTG ACT ATG Leu Leu Thr Met

ATG

Met 265 TCC ACA TCA AAC Ser Thr Ser An TCA OCT AAG Ser Ala Lys* 270 GAC TOT GCT Asp Cys Ala- GTT OCT AAA Val Ala Lys

GAA'GAA

Oiu Giu 280 CAA ATC AGC TTC Gin Ile Ser Phe

AGA

hrg 285 816 864 912 GAA GTh Giu Val 290 TTC AAA TCA GGA Phe Lys Ser Gly

CAC

His 295 ACC ACA AAT GOC Thr Thr hen Gly

ATC

Ile 300 TAC ACG TTA ACA Tyr Thr Leu Thr

TTC

Phe 305 CCT AAT TCT ACA Pro Asn Ser Thr

GAA

Giu 310 GAG ATC AAG GCC Oiu Ile Lye Aia

TAC

Tyr 315 TGT GAC. ATG Cys Asp Met GAA GCT 01w Ala 320 AGC GTT Ser Val 335 960 GGA GGA GGC GG.' TOO ACA ATT ATT CAG CGA Giy. Gly Gly Giy Trp Thr Ile Ile Gin Arg 325 330 GAT TTT CAG AGG ACT TGG AAA GAA TAT AA Asp Phe Gin hrg Thr Trp Lye Giu Tyr Lye 340 345 CGT GAG GAT GGC Arg Oiu Asp Gly GTG GGA TTT Val Gly Phe GOT AAC CCT Gly Aen Pro 350 CTG ACT AAT Leu Thr hen TCA GGA GAA Ser Gly Glu 355 TAT TGG CTG GGA Tyr Trp Leu Oly hAT An 3.60 GAG TTT GTT TCG Giu Phe Val Ser

CAA

Gin 365 CAG CAA Gin Gin 370 fZGC TAT GTG CTT Arg Tyr Val Leu

AAA

Lys 375 ATA CAC CTT AAA Ile His LeU Lye

GAC

Asp 380 TGG GAA 000 AAT Trp Giu Gly Aen

GAG

Glu 385 GCT TAC TCA TTG Ala Tyr Ser Leu

TAT

Tyr 390 GAA CAT TTC TAT Glu His Phe Tyr

CTC

Leu 395 TCA AGT GAA GAA Ser Ser Giu Giu

CTC

Leu 400 hAT TAT AGO ATT hen Tyr Arg Ile CAC CTT His Leu 405.

AAA GGA CTT Lys Gly Leu

ACA

Thr 410 GGG ACA GCC GGC Gly Thr Ala Gly AAA ATA Lys Ile 415 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 AGC AGC ATC Ser Ser Ile AAC GAC AAA Aen Asp Lys 435

AGC

Ser 420 CAA CCA OGA AAT Gin Pro Gly Asn

GAT

Asp 425 TTT hOC ACA AAG Phe Ser Thr Lys GAT GGA GAC Asp Gly Asp 430 OGA GCC TGO.- Giy Oiy Trp TOT ATT TGC AAA Cys Ile Cys Lye TCA CAA ATO CTA Ser Gin Met Leu

ACA

Thr 445 TGG TTT Trp Phe 450 OAT GCA TGT GOT Asp Ala Cys Gly

CCT

Pro 455 TCC AAC TTG AAC Ser hen Leu Asn GGA ATG TAC TAT Gly Met Tyr Tyr 460 AAA TGG TAC TAC Lye Trp Tyr Tyr

CCA

Pro

TGG

Trp 480

CAG

Gin 465 AGO CAG hAd ACA Arg Gin hen Thr hAT hen 470 AAG TTC AAC GCC Lys Phe hen Gly

ATT

Ile 475 AAA GGd TCA OGC Lys Giy Ser Giy

TAT

Tyr 485 TCG CTC AAG GCC Ser Leu Lye Aia

ACA

Thr 490 ACC ATO ATG ATC Thr Met Met Ile CGA CCA Arg Pro 495 GCA GAT TTC TAA Ala Asp Phe INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 499 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: iN2C2F (chimera 3) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 1500 0 0 0 Met Thr Val Phe Leu I2. /5 Ile Gly Cys Ser' Aen Tyr Aen Arg Ile Gin Giu His Asp Gly Asn so Asn Ala Leu Gin Arg Gin Arg Leu GIn Val Leu Met Lys Leu Giu 100 Val Giu Ile Gin Gin 115 Glu Ile Gly Thr Aen 130 Leu Thr Asp Val Giu 145 Leu Gin Leu Leu Giu 165 Ile Leu Asp Gin Thr 180 Phe Leu Giu Lys Lys 195 Leu Gin Ser Ile Lyk 210 Lys Gin Aen Ser Ile 225 Thr Val Asn Asn Sei 24! Thr Val Asn Aen Let 260 Asp Pro Thr Val Ali 275 Giu Vai Phe Lye Sei 290 Phe Pro Asn Ser Th,, 305 Gly Gly Gly Gly Trj 32! Asp Phe Gin Arg Th 340 Ser Gly Giu Tyr Tr- 355 Gin A His C eye 7 Asp 70 Leu Aen Asn Leu Ala I50 His Ser Val iGlu 230 -Val iLeu 3. Lys r Gly r Giu 310 pThr r Trp p Leu brg ;ly ~rg is kia 1lu ryr Al a Leu 135 Gln Ser G lu Leu Giu 215 Git Let Th:

GI~

H!i 29

GI~

114 Ly Gi Arg S 2 Gin C 4C1 Giu z Pro I Asn Ile Val 120 Asn Val Leu Ile Ala 200 Lys Met i Giu 280 s Thr e Ile 8 Glu y Asn 360 er ~ys ier 'is Ile ;In 105 Leu Ser As n 185 Met Let Ly: Hel 26! GIa Th Ly Gi Ty 34 Gi Ser Phe Ala Phe Leu Aia 10 Pro Giu Ala Tyr Thr Thr Val Giu 75 Met Glu 90 Asp Aen Asn Gin Thr Ala Asn GIr Thr Asr 170 Lye Le~ Glu Asl Gin Le' 1Giu Ly 23 iGin Gi 250 Ser Th n Ile Se r Asn GI s Ala Ty 31 Ala Aen Thr Asp Pro Aen Met Thr C iu 140 Thr Lys Gir LyE IGi 22( 3Lyi *Hi Se Ph y Ii 30 r Cy 5 g GI 1 Gl .1 SEr Ile Leu TI 1' Ser Giy A Phe Ile L Gin Tyr A Asp Asp S Asn Thr G 9 Lys Lys G 110 Ala Val l 125 Gin Thr I Thr Arg Leu Ciu Asp Lye 190 His Ile 205 i Val Leu s Ile Val Asp Leu Asn Ser 270 e Arg Asp 285.

e Tyr Thr 0 a Asp Met *u Asp Gly .y Phe Gly 350 ~r Gin Leu 365 hr His rg Arg eu Pro en Thr er Vai in Trp liu Met ~et Ile .rg Lys .,eu Ciu 160 1,ys Gin 175 A~sn Ser Ile Gin Val Ser Thr Ala 240 Met Glu.

255 Aia Lye Cys Ala Leu Thr Giu Ala 320 Ser Val 335 Aen Pro Thr Asn n r 5 u Arg 330.

Lye Phe Ar Va Va 142 Gin Gin .370 Giu Ala Arg Tyr Val Leu Lye 375 Giu Ile His Leu Lye Asp 380 Ser Trp Glu Giy hen' Tyr Ser Leu 385 hen Tyr 390 Leu His Phe Tyr Leu 395 Gly ser Giu Glu Leu 400 Tyr Arg Ile His 405 Gin Lys Gly Leu Thr 410 Phe Thr Ala ser Ser Ile Ser 420 Cys Pro Gly Aen Asp 425 Ser Ser Thr Lye Giy Lys Ile 415 Asp Gly Asp 430 Gly Gly Trp hen Asp Trp Phe 450 Gin Arg Lys 435 Asp Ile Cys Lys Cys 440 Ser Gin Met Leu Ala Cye Giy Pro 455 Lys Aen Leu hen Gin hen Thr hen 470 465 Lys Phe hen Gly Ile 475 Lye Aia Thr Thr 490 Giy 460 Lye Met Thr 445 Met Trp Met Tyr Tyr Tyr Tyr Ile hrg 495 Pro Trp 480 Pro Gly Ser Giy Tyr Ser Leu 485 Ala Asp Phe INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LEN,1i: 1488 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: l..,;1485 OTHER INFORMATION: NAME/KEY: 2NlC1F (chimera 4) LOCATION: 1488 OTHER INFORMATION: NAME/KEY: Other LOCATION: 48 OTHER INFORMATION: Putative leader sequence (xi) SEQUENCE DESCRIPTION: SEQ ID

ATG

Met TGG CAG ATT Trp Gin Ile

GTT

Val 5 TTC.TTT ACT Phe Phe Thr CTG AGC Leu Ser 10 TGT GAT CTT GTC TTG GCC Cys Asp Leu Val Leu Ala is GCA GCC TAT Ala Ala Tyr CAA TAT CAG Gin Tyr Gin

AAC

hen AAC TTT CGG AAG hen Phe Arg Lye

AGC

Ser 25 ATG GAC AGc ATA Met Asp Ser Ile GGA hAG hAG Gly Lys- Lys CTC CTG CCA Leu Leu Pro GTC CAG CAT GGG Val Gin His Gly

TCC

Ser 40 TGC hOC TAC ACT Cys Ser Tyr Thr

TTC

Phe 96 144 192 240 GAG ATG Giu Met GAC A1AC TGC CG Asp hen Cys Arg

TCT

Ser 55 TCC TCC AGC CCC Ser Ser Ser Pro

TAC

Tyr GTG TCC hAT GCT Val Ser hen Ala

GTG

Val CAG AGG GAC GCG Gin Arg Asp Ala

CCG

Pro 70 CTC GAA TAC OAT Leu Glu Tyr Ap TTC TCT TCC GAG AAA CTT Phe Ser Ser Gin Lye Leu 75 CAA CAT CTG GAA Gin His Leu Giu

CAT

His GTG ATG GAA AAT Val Met Giu Aen

TAT

Tyr 90 ACT CAG TGG CTG Thr Gin Trp Leu CAA AAA Gin Lys 288 CTT GAG AAT Leu Giu Aen

TAC

Tyr 100 ATT GTG GAA AAC.

Ile Val Giu Aen AAG TCG GAG ATG Lys Ser Glu Met GCC CAG ATA Ala Gin lie 110 GAG ATA GGA Giu Ile Gly CAG CAG AAT Gin Gin Aen 115 GCA GTT CAG AAC Ala Val Gin Aen CAC ACG OCT ACC ATG His Thr Ala Thr Met 120,

CTG

Leu 125 ACC AGC Thr Ser 130 CTC CTC TCT CAG Leu Leu Ser Gin

ACT

Thr 135 GCA GAG CAG AC Ala Giu Gin Thr

AGA

Arg 140 AAG CTG ACA GAT Lys Leu Thr Xep

GTT

Val 145 GAG ACC CAG GTA Glu Thr Gin Val

CTA

Leu 150 AAT CAA ACT TCT Aen Gin Thr Ser

CGA

Arg 155 CTT GAG ATA CAG Leu Giu Ile Gin

CTG

Leu 160 CTG, GAG Leu Glu CAG ACA Gin \,Thr AAT TCA TTA Asn S er Leu ,165 AAT GAP. ATC Aen Giu Ile 180 TCC ACC TAC AAG Ser Thr Tyr Lys

CTA

Leu 170 GAG AAG, CAA CTT Giu. Lys Gin Leu CTT CAA Leu Gin 175 TTG AAG ATC Leu Lys Ile

CAT

His 185 GAA AAA AAC Giu Lye Asn AGT TTA TTA GAA Ser Leu Leu Giu 190 CAT AAA ATC His Lye Ile 195 TTA GAA ATO GAA Leu Giu Met Glu GGA AAA CAC AAG GAA GAG TTG GAC ACC Gly Lys His Lys Giu Giu Leu Asp Thr 200 205 TTA AAG Leu Lye 210 GAA GAG AAA GAG Giu Giu Lys Giu

AAC

Asn 215 CTT CAA GGC TTG Leu Gin Gly Leu

GTT

Val 220 ACT COT CAA ACA Thr Arg*Gin Thr

TAT

Tyr 225 ATA ATC CAG GAG Ile Ile Gin Giu

CTG

Leu 230 GAA AAG CAA TTA Giu Lys Gin Leu

AAC

Asn 235 AGA GCT ACC ACC Arg Ala Thr Thr

AAC

Aen 240 384 432 480 52.8 516 624 672 720 768 816 864 912 960 1008 1056 AAC AGT OTC CTT Aen Ser Vai Leu

CAG

Gin 245 AAG CAG CAA CTG Lys Gin Gin Leu

GAG

Glu 250 CTG ATG GAG ACA Leu Met Asp Thr GTC CAC Val His 255 AAC CTT GTC Asn Leu Vai AAA AGA GAG Lys A .rg Glu 275

AAT

Aen 260 CTT TGC ACT AAA Leu Cys Thr Lye

GAA

G iu 265 OCT GTT TTA CTA GlyVai. Leu Leu AAG, GGA GGA Lys Giy Gly 270 GAA GAG AAA CCA Giu Glu Lye Pro TTT AGA GAC TOT GCA GAT GTA TAT CAA Phe Arg Asp Cys Ala Asp Val Tyr Gin 280 285 OCT GGT Ala Gly 290 TTT AAT AAA AGT Phe Aen Lye Ser

GGA

Gly 295 ATC TAC ACT ATT Ile Tyr Thr Ile

TAT

Tyr 300 ATT AAT AAT ATG Ile Aen Asn Met

CCA

Pro 305 GAA CCC AAA AAG Giu Pro Lye Lye TTT TGC AAT ATG Phe Cys Asn Met

GAT

Asp 315 GTC AAT GOG GGA Val Asn Gly Gly

GOT

C ly 320 TGG ACT GTA ATA Trp Thr Val Ile GGC TGG ARG GAA Gly Trp Lye Glu 340

CAA

Gin 325 CAT CGT GAA GAT His Arg Glu Asp

GGA

Gly 330 AGT CTA CAT TTC Ser Leu Asp Phe CAA AGA Gin Arg 335 TAT AAA ATG GGT Tyr Lye Met Gly TTT GGA AAT CCC TCC GGT GAA TAT Phe Gly Asn Pro Ser Giy Giu Tyr 345 350 TGG CTG, GGG Trp Leu Giy 355 A.AT GAG TTT ATT Asn Glu Phe Ile

TTT

Ph~e 360 GCC ATT ACC AGT Ala Ile Thr Ser

CAG

Gin 365 AGG CAG TAC Arg Gin Tyr 1104 1152 ATG CTA AGA ATT GAG TTA Met Leu Arg Ile Glu Leu

ATG

Met CAC TGG GAA GG Asp Trp Glu Gly

AAC

An CGA GCC TAT TCA Ara Ala Tyr SAr QJ I %J J-1b 380

CAG

Gin 385 TAT GAC AGA TTC Tyr Asp Arg Phe

CAC

His 390 ATA GGA AAT GAA Ile Gly Asn Giu

AAG

Lys 395 CAA AAC TAT Gin Asn Tyr AGG TTG, Arg Leix 400 CTG ATC- Leu fle 415 TAT TTA AAA GGT Tyr Leu Lye Gly

CAC

His 405 ACT GGG ACA GCA Thr Giy Thr Ala

GGA

C ly 410 AAA CAG AGC AGC Lys Gin Ser Ser TTA CAC GGT Leu His Gly ATG TGC -AAA Met Cys Lys 435

GCT

Ala 420 GAT TTC AGC ACT Asp Phe Ser Thr AAA GAT OCT GAT AAT GAC AAC TGT Lys Asp Ala Asp Aen Asp Asn Cys 425 430 TOT GCC CTC ATG TTA ACA GGA GGA TGG TGG Cys Ala Leu Met Leu Thr Gly Giy Trp Trp 440 445 TTT GAT GCT Phe Asp Ala 1200 1248 1296 1344 1392 1440 1488 TOT GGC Cys.Gly 450 CCC TCC'AAT CTA Pro Ser Aen Leu

AAT

An 455 GGA ATG TTC TAT Gly Met Phe Tyr

ACT

Thr 460 GCG GGA CAA AAC Ala Gly Gin An

CAT

His 465

TAC

Tyr GGA AAA CTG AAT Gly Lys Leu An TCC TTA CGT TCC Ser Leu Arg Ser 485 GOG ATA AAG TGG CAC TAC TTC AAA GGG CCC AGT Gly Ile Lys Trp His Tyr Phe Lys Gly Pro Ser 470 475 480 ACA ACT ATG ATG Thr Thr Met Met

ATT

Ile 490 CGA CCT TTA GAT Arg Pro Leu Asp TTT TGA Phe 495 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 495 amino acids TYPE: amino acid STRANDEONESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: 2NlClF (chimera 4) LOCATION: 495 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Trp Gin Ile Ala Tyr An Val1Phe Phe Thr Leu Ser 10 Ser Met Cys Asp Leu Asp Ser Ile Gin Tyr G lu Met Val Gin Gin Val1 Ann Phe Arg Lys Gin His Gly Ser 40 Cys Arg Ser Ser Cys Ser Val Leu Ala Gly Lys Lys Leu Leu Pro Ser Asn Ala Asp An Arg Asp Ala Pro 70 Leu Glu His Val 55 Leu Ser Tyr Thr Ser Pro Tyr Asp Phe Ser 75 Tyr Thr Gin Phe Val Glu Tyr Gin Ser Gin Lye Leu Trp Leu Gin Lys His Met G i Aen 145 Leu Giu Asn Gin Thr Val 145 Leu Gin His Leu Tyr 225 Asn Aen Lye Ala Pro 305 Trp Gly Trp Met Gin 385 Tyr Leu Met Cys His 465 Tyr Gin Ser 130 Glu Glu Thr Lys Lys 210 Ile Ser Leu Arg Gly 290 G iu Thr Trp Leu Leu 370 Tyr Leu His Cys Giy 450 Gly Ser Aen Leu Thr Aen Aen Ile 195 Giu Ile Val Val Glu 275 Phe Pro Val Lys Gly 355 Arg Asp Lys Gly Lys 435 Pro Lys Leu Tyr Ile 100 Ala Val Leu Ser Gin Val Ser Lou 165 Glu Ile 180 Leu Giu Giu Lye Gin Giu Lou Gin 245 Asn Leu 260 Giu/ Glu Asen Lye Lys Lye Ile Gin 325 Glu Tyr 340 Aen Giu Ile Giu Arg Phe Gly His 405 Ala Asp 420 Cys Ala Ser Aen Lou Aen Arg Ser 485 Val Glu Aen Gin Gin Leu 150 Ser Leu Met Giu Lou 230 Lys Cys Lye Ser Val 310 His Lys Phe Lou His 390 Thr Phe Lou Leu G ly 470 Thr ken Thr 135 ken Thr Lye G iu Asn 215 Giu Gin Thr Pro Gly 295 Phe Arg Met Ile Met 375 Ile Gly Ser Met Aen 455 Ile Thr H is 120 Alia Gin Tyr Ile Gly 200 Lou Lye Gin Lye Phe 280 Ile Cya Giu Gly Phe 360 Asp Gly Thr Thr Lou 440 G iy Lye Met Met 105 Thr 0l Thr Lye His 185 Lye Gin Gin Lou G iu 265 Arg Tyr kAen Asp Phe 345 Ala Trp ken Ala Lys 425 Thr Met Trp Met Ala Thr Gin Thr Ser Arg 155 Lou Giu 170 Giu Lye His Lye Gly Lou Lou Aen 235.

Giu Lou 250 Gly Val Asp Cys Thr Ile Met Asp 315 Gly Ser 330 Gly ken Ile Thr Giu Gly Glu Lye 395 Gly Lye 410 Asp Ala Gly Giy Phe Tyr His Tyr 475 Ile Arg 490 NO 1.2 7: 4et I 4,rg I 140 Lou Lys Asen Giu Val 220 Arg Met Lou Ala Tyr 300 Val Lou Pro Sor ken 380 Gin Gin Asp Trp Thr 460 Phe Pro eOu .ye flu ;In Ser 'liu 205 Thr Ala Asp Lou Asp 285 Ile ken Ser Gir 3 Arc Asr 502 As: Trn 44! Al Lyl Le' ys Ser Giu Met Ala Gin Ile 110 Glu Ile Gly Lou Thr Asp Ile Gin Lou 160 Lou Lout Gin 175 Lou Lou Glu 190 Lou Asp Thr Arg Gin Thr Thr Thr ken 240 Thr Val His 255 Lye Gly Gly 270 Vai Tyr Gin Aen ken Met Gly Gly Gly 320 Phe Gin Arg 335 Gly Glu Tyr 350 Arg Gin Tyr Ala Tyr Ser Tyr Arg Lou 400 Ser Lou Ile 415 i Asp ken Cys 430 SPhe Asp Ala Gly Gin ken Gly Pro Ser 480 Asp Phe 495

S..

S

S

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRAliDEDNESS: single TOPOLOGY: linear (1i) MOLECULE TYPE: DNA (Ix) FEATURE: NAME/KEY: hTL4atg LOCATION: .47 OTHER INFORMATION: PCR primer 146 NAME/KEY: Other LOCATION: 1...20 OTHER INFORMATION: "tail" sequences added to PCR primer to facilitate cloning of the amplified PCR fragments (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GCATGCTATC TCGAGCCACC ATGCTCTCCC AGCTAGCCAT GCTGCAG 47 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 55 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: hTL4not S(B) LOCATION: 1...55 OTHER INFORMATION: PCR Primer NAME/KEY: Other LOCATION: 1...28 OTHER INFORMATION: "tail" sequence added to the S* PCR primers to facilitate cloning of the amplified PCR fragments (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GTGTCGACGC GGCCGCTCTA GATCAGACTT AGATGTCCAA AGGCCGTATC ATCAT 147

Claims (20)

1. A modified TIE-2 ligand that binds and activates TIE-2 receptor which is TIE-2 ligand 1 modified to comprise a different amino acid instead of the cysteine residue at amino acid position 265 set forth in Figure 4.

2. A ligand according to claim 1, which is modified such that the amino acid at position 265 is serine. 10 3. A modified TIE-2 ligand that binds but does not activate TIE-2 wherein the N-terminal domain of the TIE-2 ligand is deleted.

4. A ligand according to claim 3 which is TIE-2 ligand 1 or TIE-2 ligand 2 wherein the N-terminal domain is deleted.

5. A ligand according to claim 3 or 4, wherein the coiled-coil domain of the TIE-2 ligand is deleted and the fibrinogen-like domain is fused in- frame to a human immunoglobulin gamma-1 constant region. 20 6. A ligand according to claim 5 wherein the immunoglobulin constant region is IgG1-Fc.

7. A nucleic acid molecule which encodes a modified TIE-2 ligand of any one of the preceding claims.

8. A vector which comprises a nucleic acid molecule of claim 7.

9. A vector according to claim 8, wherein the nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a host cell. -149- A vector according to claim 8 or 9 which is a plasmid.

11. A host-vector system for the production of a modified ligand according to any one of claims 1 to 6 which comprises a host cell and a vector according to claim 9 or

12. A host-vector system according to claim 11 wherein the host cell is a bacterial, yeast, insect or mammalian cell. 10 13. A method of producing a ligand as defined in any one of claims 1 to 6 which comprises growing cells of a host-vector system according to claim 11 or 12, under conditions permitting production of the ligand and recovering the ligand so produced. 15 14. An antibody which specifically binds the ligand of any one of claims 1 to 6. An antibody according to claim 14 which is a monoclonal antibody. 20 16. A conjugate comprising a ligand according to any one of claims i to 6 and conjugated thereto, a cytotoxic agent.

17. A conjugate according to claim 16 wherein the cytotoxic agent is a radioisotope or toxin.

18. A pharmaceutical composition comprising a modified ligand according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising an antibody according to claim 14 or 15 and a pharmaceutically acceptable carrier. *.eee. .0 040 0000. 6 0 00 S *050 S S.. *500 S S. S 505S -150- A pharmaceutical composition comprising a conjugate according to claim 16 or 17 and a pharmaceutically acceptable carrier.

21. A ligand produced by the method of claim 13.

22. A modified TIE-2 ligand according to claim 1 or 3 substantially as hereinbefore described with reference to the Examples.

23. A nucleic acid molecule according to claim 7 substantially as 10 hereinbefore described with reference to the Examples.

24. A vector according to claim 8 substantially as hereinbefore described with reference to the Examples. 15 25. A host-vector system according to claim 11 substantially as hereinbefore described with reference to the Examples.

26. A method according to claim 13 substantially as hereinbefore described with reference to the Examples.

27. An antibody according to claim 14 substantially as hereinbefore described with reference to the Examples.

28. A conjugate according to claim 16 substantially as hereinbefore described with reference to the Examples.

29. A pharmaceutical composition according to claim 18, 19 or substantially as hereinbefore described with reference to the Examples. DATED: 26 September 2000 PHILLIPS ORMONDE FITZPATRICK ATTORNEYS FOR: REGENERON BHARMACEUTICALS, INC