MXPA00002422A - Tie receptor tyrosine kinase ligand homologues - Google Patents

Abstract

The present invention concerns isolated nucleic acid molecules encoding the novel TIE ligand homologues NL2, NL3 and NL6 (FLS139), the proteins encoded by such nucleic acid molecules, as well as methods and means for making and using such nucleic acid and protein molecules.

Description

LIGAND0S HOMOLOGOUS OF TIE Field of the Invention The present invention relates to isolated nucleic acid molecules encoding novel TIE homologous ligands, the TIE homologous ligand proteins encoded by such nucleic acid molecules, as well as methods and means for the manufacture and use of such protein and acid molecules. nucleic acids, and antibodies bound to the TIE homologous ligands disclosed.

Art Background The abbreviations "TIE" or "tie" are acronyms, which mean "tyrosine kinase containing homologous domains of Ig and EGF" and were coined to designate a new family of receptor tyrosine kinases which are expressed almost exclusively in vascular endothelial cells and in early hematopoietic cells, and are characterized by the presence of an EGF-like domain, and extracellular doubling units stabilized by interchain disulfide bridges, generally referred to as "immunoglobulin-like (IG) -like folds." A homologous cDNA fragment of the human leukemia cell tyrosine kinase (tie) was REF .: 32873 described by Partanen et al., Proc. Nati Acad. Sci. USA 87, 8913-8917 (1990). The mRNA of this human "tie" receptor has been detected in all embryonic tissues of mouse and human fetus, and has been reported to be located in vascular and cardiac endothelial cells. Coronen et al., Blood 80, 2548-2555 (1992); PCT Application Publication No. WO 93/14124 (published July 22, 1993). The homolog of the rat of the human tie, referred to as "tie-1", was identified by Maisonpierre et al., Oncogene 8_. 1631-1637 (1993). Another tie receptor, designated "tie-2" was originally identified in rats (Dumont et al., Oncogene 8, 1293-1301 (1993)), whereas the human homolog of tie-2, referred to as "ork" was described in the US Patent No. 5,447,860 (Ziegler). The urine counterpart of tie-2 was originally called "tek." The cloning of a mouse tie-2 receptor from a cDNA brain capillary library is disclosed in PCT Application Publication No. WO 95/13387 (published May 18, 1995). It is believed that TIE receptors are actively involved in angiogenesis, as well as they may play a role in hematopoiesis.

The cloning of expression of human TIE-2 ligands has been described in the PCT Application Publication No. WO 96/11269 (published on April 18, 1996) and in the U.S. Patent No. 5,521,073 (published May 28, 1996). A vector designated as? GtlO encoding a TIE-2 ligand called "htie-2 ligand 1" or "hTLl" has been deposited under ATCC Accession No. 75928. A plasmid encoding another TIE-2 ligand designated "htie" -2 2"or" hTL2"is available under ATCC Accession No. 75928. This second ligand has been described as an antagonist of the TAI-2 receptor. The identification of human and mouse ligands secreted for the TIE-2 receptor has been reported by Davis et al., Cell 7, 1161-1169 (1996). The human ligand designated "Angiopoietin-1", to reflect its role in angiogenesis and its action potential during hematopoiesis, is the same ligand as the ligand variably designated as "htie-2 1" or "hTL-1" in WO 96/11269. Angiopoietin-1 has been described to later play an angiogenic role and distinguish it from that of VEGF (Suri et al., Cell 87, 1171-1180 (1996)). Since TIE-2 is apparently positively regulated during pathological angiogenesis required for tumor growth (Kaipainen et al., Cancer Res. 54, 6571-6577 (1994)) it has been suggested that angiopoietin-1 is additionally useful to specifically target the tumor vasculature (Davis et al., supra).

BRIEF DESCRIPTION OF THE INVENTION The present invention deals with novel human TIE homologous ligands with powerful effects on the vasculature. The invention also provides isolated nucleic acid molecules that encode such homologous ligands or functional derivatives thereof, and vectors containing such nucleic acid molecules. The invention subsequently treats host cells transformed with such nucleic acid to produce the novel TIE homologous ligands or functional derivatives thereof. The novel homologous ligands may be agonists or antagonists of the TIE receptors, known or discovered hereafter. Its therapeutic or diagnostic use, including the delivery of other therapeutic or diagnostic agents to cells expressing the respective TIE receptors, is also within the scope of the present invention.

The present invention further provides agonist or antagonist antibodies that specifically bind to the TIE homolog ligands described herein, and the diagnostic or therapeutic use of said antibodies.

In another aspect, the invention relates to compositions comprising the novel homologous ligands or antibodies.

In a further aspect, the invention relates to conjugates of the novel TIE homologous ligands of the present invention with other therapeutic or cytotoxic agents, and of compositions comprising said conjugates. It has been reported that the TIE-2 receptor is positively regulated during pathological angiogenesis that is a requirement for tumor growth, and other TIE receptors may have similar properties. Accordingly, conjugates of the TIE homologous ligands of the present invention for cytotoxic or other antitumor agents can be useful targeting the tumor vasculature specifically.

In yet another aspect, the invention relates to a method for the identification of a cell that expresses a TIE receptor, which comprises contact with a cell with a detectably labeled TIE counterpart ligand of the present invention under conditions that allow the binding of such a ligand TIE homologue to the TIE receptor, and the determination of whether such binding has actually occurred.

In a different aspect, the invention relates to a method for measuring the amount of a TIE homologous ligand of the present invention in a biological sample by contacting the biological sample with at least one antibody that specifically binds to the homologous ligand of TIE, and the measurement of the amount of the homologous-complex ligand formed of TIE antibodies.

The invention further relates to a screening method for the identification of polypeptides or small molecule agonists or antagonists of a TIE receptor based on its ability to compete with a native TIE homolog ligand or variant of the present invention for receptor binding. Corresponding TIE.

The invention also relates to a method for representing the presence of angiogenesis in the healing of wounds, in inflammation or in tumors of human patients, which comprises the administration of detectably labeled TIE-homologous ligands or agonist antibodies of the present invention, and the detection of angiogenesis.

In another aspect, the invention is directed to a method of promoting or inhibiting neovascularization in a patient by administering an effective amount of a TIE homologous ligand of the present invention in a pharmaceutically acceptable carrier. In a preferred embodiment, the present invention relates to a method for the promotion of wound healing. In another embodiment, the invention relates to a method for promoting-angiogenic processes, such as the induction of collateral vascularization in a heart or ischemic limb. In a further preferred embodiment, the invention relates to a method for the inhibition of tumor growth.

In yet another aspect, the invention relates to a method for promoting the development and / or maturation and / or growth of bone in a patient, comprising administering to the patient an effective amount of a TIE homologous ligand of the present invention in a pharmaceutically acceptable vehicle.

In a further aspect, the invention relates to a method for promoting muscle growth and development, which comprises administering to a patient in need of an effective amount of a TIE homologous ligand of the present invention in a pharmaceutically acceptable carrier.

In yet another aspect, the invention relates to a method of inhibiting the growth of endothelial cells and / or inducing endothelial cell apoptosis by administering an effective amount of a TIE homologous ligand of the present invention. In addition, the invention relates to a method of inhibiting inflammation, which comprises administering to a patient an effective amount of an antagonist of a TIE-homologous ligand of the present invention, such as an antibody to a homologous ligand of TIE described herein, eg, an anti-NLβ antagonist antibody.

The TIE homologous ligands of the present invention can be administered alone, or in combination with each other and / or with other therapeutic or diagnostic agents, including members of the VEGF family. Combination therapies can lead to new approaches to promote or inhibit neovascularization, bone and / or muscle growth, development or differentiation, or treatment of conditions associated with unwanted endothelial cell growth, e.g. treatment of tumors.

Brief Description of the Figures Figure 1 is a graph depicting the relationship of the homologous ligands NL2, NL3 and FLS139 with the two known homologous ligands of the TIE2 receptor (h-TIE2L1 and h-TIE2L2) and with other TIE homologous ligands disclosed in the application No. Serial 08 / 933,821, filed on September 19, 1997.

Figure 2 is the nucleotide sequence of TIE ligand NL2 (SEQ ID NO: 1) (DNA 22780).

Figure 3 is the amino acid sequence of TIE ligand NL2 (SEQ ID NO: 2). Figure 4 is the nucleotide sequence of the TIE ligand NL3 (SEQ ID NO: 3) (DNA 33457).

Figure 5 is the amino acid sequence of the TIE NL3 ligand (SEQ ID NO: 4).

Figure 6 is the nucleotide sequence of the TIE ligand FLS139 (SEQ ID NO: 5) (DNA 16451).

Figure 7 is the amino acid sequence of the TIE ligand FLS139 (SEQ ID NO: 6).

Figures 8-9 - Northern blots showing the expression of the mRNAs of TIE NL2 and NL3 homologous ligands in various tissues.

Figure 10 shows the effect on the formation of the HUVEC tube of the NL6 polypeptide conjugated to poly-his at a dilution of 1% and of a control buffer (10 mM HEPES / 0.14 M NaCl / 4% mannitol, pH 6.8) at a dilution of 1%. Comparative results with another novel TIE homolog ligand (NL1) and two known TIE ligands TIE-1 and TIE-2, tested as IgG fusions, are also shown in Figure.

Detailed description of the invention A. TIE HOMOLOGOUS LIGANDS AND THE NUCLEIC ACID MOLECULES THAT CODE THEM The TIE homologous ligands of the present invention include the native human homologous ligands designated NL2 (SEQ ID NO: 2), NL3 (SEQ ID NO: 4), and FLS139 (subsequently renamed "NL6"; ID NO: 6), their counterparts in other, non-human mammalian species, including, but not limited to, higher mammals, such as the monkey; rodents, such as mice, rats, hamsters; pigs; equines; cattle; variants of naturally occurring alleles and splices, and biologically active (functional) derivatives, such as, variants in the amino acid sequence of such native molecules, as long as they differ in a native ligand TL-1 or TL-2. Native NL-2, as disclosed herein, possesses an amino acid sequence identity of 27% with hTL-1 (TIE2L1) and about 24% identity of the amino acid sequence with hTL-2 (TIE2L2). The amino acid sequence of native NL3, as disclosed herein, is about 30% identical to that of hTL-1 and about 29% identical to that of hTL-2. The identity of the amino acid sequence between native FLS139 (NL6), as disclosed herein, and hTL-1 and hTL-2 is about 21%. The native TIE homologous ligands of the present invention are substantially free of other proteins with which they are associated in their native environment. This definition is not limited in any way by the method (s) by which the TIE homologous ligands of the present obtaining are obtained, and includes all the homologous ligandss within the definition, whether purified from their natural source, obtained by recombinant DNA technology, synthesized or prepared by any combination of these and / or other techniques. The amino acid sequence variants of the native TIE homolog ligands of the present invention should have at least about 90%, preferably, at least about 95%, more preferably at least about 98%, and yet more preferably at least about 991 sequence identity with a full-length human native TIE homolog ligand of the present invention, or with the fibrinogen-like domain of a human native TIE homologous ligand of the present invention. Such variants in the amino acid sequence preferably exhibit or inhibit a qualitative biological activity of a homologous ligand of native TIE.

The term "fibrinogen domain" or "fibrinogen-like domain" is used to refer to amino acids near position 278 near position 498 in the amino acid sequence of known hTL-1; the amino acids near position 276 to near position 496 in the amino acid sequence of known hTL-2; amino acids from near position 180 to about 453 in the amino acid sequence of NL2; the amino acids near position 77 near position 288 in the amino acid sequence of NL3; and the amino acids near position 238 to near position 460 in the amino acid sequence of FLS139, and to homologous domains in other TIE homologous ligands. The fibrinogen-like domain of NL2 is about 37-38% identical to that of hTL-1 (TIE2L1) and hTL-2 (TIE2L2). The fibrinogen-like domain of NL3 is about 37% identical to the fibrinogen-like domains of hTL-1 and hTL-2, while the fibrinogen-like domain of FLS139 is about 32-33% identical to the domains similar to fibrinogen. fibrinogen of hTL-1 and hTL-2.

The term "nucleic acid molecule" includes RNA, DNA and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given TIE ligand can be produced. The present invention specifically contemplates each possible variation of the nucleotide sequences, which encode the TIE homologous ligands of the present invention, based on all possible codon options. Although the nucleic acid molecules which encode the TIE-homologous ligands described herein are preferably capable of hybridizing, under stringent conditions, to a naturally occurring TIE-homologous ligand gene, it may be advantageous to produce nucleotide sequences which encode TIE homologous ligands, which possess a substantially different codon usage. For example, the codons can be selected to increase the rate at which the expression of the polypeptide occurs in particular prokaryotic or eukaryotic host cells, according to the frequency with which a particular codon is used by the host. In addition, RNA transcripts with improved properties, e.g. the half-life can be produced by an appropriate selection of the nucleotide sequences encoding a given TIE homolog ligand.

The "sequence identity" must be determined by aligning two sequences to be compared following the Clustal method of multiple sequence alignment (Higgins et al., Co put., Appl. Biosci., 5, 151-153 (1989), and Higgins et al., Gene 73, 237-244 (1988)) which is incorporated in version 1.6 of the Lasergene biocomputation software (DNASTAR, Inc., Madison, Wisconsin), or any updated or equivalent version of this software.

The "severity" of the hybridization reactions is readily determined by someone of ordinary skill in the state of the art, and is generally an empirical calculation dependent on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper analysis, while shorter probes need lower temperatures. Hybridization generally depends on the ability of the denatured DNA to re-grow when the complementary strands are present in an environment below its melting temperature. The higher the desired degree of homology between the probe and the sequence to be hybridized, the higher the relative temperature that can be used. As a result, it is understood that the higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures would make them less. For additional details and explanation of the stringency of hybridization reactions, see Ausubel et al. , Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

The "stringent conditions" or "high stringency conditions", as defined herein, can be identified by those that: (1) employ low ionic strength and a high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C; (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% formamide (v / v) with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM buffer of sodium phosphate at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x sodium Denhardt, sonic salmon sperm DNA (50 μg / ml), 0.1% SDS, 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a high stringency wash consisting of 0.1 x SSC containing EDTA at 55 ° C.

"Moderately stringent conditions" can be identified as described by Sambrook et al. , Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of a washing solution and hybridization conditions (e.g., temperature, ionic strength and% SDS) less stringent than those described above. An example of moderately stringent conditions is incubation overnight at 37 ° C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg / mL denatured broken salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50 ° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. As necessary to accommodate factors such as the length of the probe and its like.

The term "labeled epitope" when used herein refers to a chimeric polypeptide comprising a TIE polypeptide homologous ligand fused to a "tag polypeptide". The tag polypeptide has sufficient residues to provide an epitope against which an antibody can be formed, and is still short enough such that it does not interfere with the activity of the polypeptide to which it is fused. The labeling polypeptide is also preferably exactly unique such that the antibody substantially does not cross-react with other epitopes. Appropriate labeling polypeptides generally have at least six amino acid residues and usually about 8 to 50 amino acid residues (preferably, about 10 to 20 amino acid residues).

The terms "biological activity" and "biologically active" taking into consideration the TIE homologous ligands of the present invention refer to the ability of a molecule to specifically bind to and signal through a native receptor of a TIE ligand, known or to be discovered, (hereinafter referred to as a "TIE receptor"), eg a native TIE-2 receptor, or block the activity of the native TIE receptor (e.g., TIE-2) to participate in the transduction of the signal. Thus, TIE ligands (native and variants) of the present invention include agonists and antagonists of a native TIE receptor, e.g. TIE-2. The preferred biological activities of the TIE ligands of the present invention include activity to induce or inhibit vascularization. The ability to induce vascularization will be useful for the treatment of biological conditions and diseases, where vascularization is desirable, such as wound healing, ischemia, and diabetes. On the other hand, the ability to inhibit or block vascularization may, for example, be useful in the prevention or attenuation of tumor growth. Another preferred biological activity is the ability to alter muscle growth or development. A preferred biological activity later is the ability to influence bone development, maturation or growth. Yet another preferred biological activity is the ability to inhibit the growth of endothelial cells and / or induce apoptosis.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and / or causes cell destruction. The term is intended to include radioactive isotopes (e.g. I131, I125, Y90 and Re186), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include adria icine, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxine, taxoids, e.g. paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Anthony, Rnace), toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone , vincristine, vinorelbine, carbolpatine, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, esperamycins (see US Pat No. 4,675,187), melphalan and other related nitrogen mustards. Also included in this definition are the hormonal agents - which act to regulate or inhibit hormonal action in tumors such as tamoxifen and onapristone.

A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits the growth of a cell, especially a cancer cell that over expresses any of the genes identified herein, either in vi tro or in vivo. Thus, the growth inhibitory agent is one which significantly reduces the percentage of cells that over express such genes in the S phase. Examples of growth inhibitory agents include agents that block the progression of the cell cycle (in a different place to the phase S), such as agents that induce the arrest of Gl and the arrest of phase M. Classical blockers of the M phase include vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents who arrest Gl also spread over the arrest of phase S, for example, agents that rent DNA such as tamoxifen, prednisone, dacarbazine, ecloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. More information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Regulation of the cell cycle, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13"Doxorubicin" is an atracycline antibiotic. The complete chemical name of doxorubicin is (8S-cis) -10- [(3-amino-2,3,6-trideoxy-aL-lixo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6 , 8, 11-trihydroxy-8- (hydroxyacetyl) -l-methoxy-5, 12-naphtacenedione.

The term "cytokine" is a generic term for proteins released by a cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monocins, and traditional polypeptide hormones. Included within the cytokines are growth hormones such as human growth hormone, homana N-methionyl growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and -β; Mullerian inhibitory substance; peptide associated with mouse gonadotropins; inhibin; activin; Vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, ß, and - ?; colony stimulating factors (CSFs) such as macrophage CSF (M-CSF); CSF of macrophages and granulocytes (GM-CSF); and granulocyte CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-a or TNF-β; and other polypeptide factors including LIF and the ligand kit (KL). As used herein, the term cytosine includes proteins from natural or recombinant cell culture sources and biologically active equivalents of the native sequence cytokines.

The "vascular endothelial growth factor 'Vascular Permeability Factor" (VEGF / VPF) is a specific mitogen of endothelial cells which has recently shown to be stimulated by hypoxia and is required for tumor angiogenesis (Senger et al. ., Cancer 46: 5629-5632 (1986), Kim et al., Nature 362: 841-844 (1993), Schweiki et al., Nature 359: 843-845 (1992), Piet et al., Nature 359: 845-848 (1992)). It is a disulfide-linked glycoprotein, dimeric of 34-43 kDa (with the predominant species of about 45 kDa) synthesized and secreted by a variety of normal and tumor cells. In addition, cultured human reticular cells such as pigmented epithelial cells and pericytes have been shown to secrete VEGF and increase expression of the VEGF gene in response to hypoxia (Adamis et al., Biochem. Biophys. Res. Commun. 193: 631-638 (1993), Plouet et al., Invest Ophthalmol, Vis. Sci. 34: 900 (1992), Adamis et al., Invest Ophthalmol, Vis. Sci. 34: 1440 (1993), Aiello et al. ., Invest Ophthalmol, Vis. Sci. 35: 1868 (1994); Si orre-pinatel et al., Invest Ophthalmol, Vis. Sci. 35: 3393-3400 (1994)). In contrast, VEGF in normal tissues is relatively low. Thus, VEGF seems to play a major role in many processes and pathological states related to neovascularization. The regulation of VEGF expression in tissues affected by the various conditions described above could then be key in the treatment or preventive therapies associated with hypoxia.

The term "agonist" is used to refer to peptide and non-peptide analogs of the native TIE homologous ligands of the present invention and to antibodies that specifically bind to such TIE homologous ligands, provided they have the ability to send a signal through the native TIE receiver (eg TIE-2). In other words, the term "agonist" is defined in the context of the biological role of the TAR receptor, and not in relation to the biological role of a native TIE homologous ligand, which, as stated above, can be an agonist or a antagonist of the biological function of the TIE receptor. Preferred agonists possess the preferred biological activities of the TIE homologues as listed above, and include vascularization promoters, molecules that play a role in growth, maturation or bone formation, and promote growth and / or muscle development. .

The term "antagonist" is used to refer to peptide and non-peptide analogs of the native TIE homologous ligands of the present invention and to antibodies that specifically bind to such native TIE homologous ligands, provided they possess the ability to inhibit function of a native TIE receptor (eg TIE-2). Once again, the term "antagonist" is defined in the context of the biological role of the TAR receptor, and not in relation to the biological activity of the homologous ligand of native TAR, which may be either an agonist or an antagonist of the biological function of the TAR receptor. Preferred antagonists are inhibitors of vasculogenesis, or of the pathological development or growth of bone or muscle.

"Tumor", as it is used here, to all the growth and proliferation of neoplastic cells, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, cancer of the vulva, thyroid cancer, hepatic carcinoma and various types of cancer of the head and neck.

The "treatment" is an intervention carried out with the intention of preventing the development or altering the pathology of a disease. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already have the disease as well as those in which the disease can be prevented. In the treatment of tumors (e.g. cancer), a therapeutic agent can directly decrease the pathology of the tumor cells, or look for tumor cells more susceptible to treatment with other therapeutic agents, e.g. radiation and / or chemotherapy.

The "pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, the abnormal or uncontrollable growth of cells, metastasis, interference with the normal functioning of contiguous cells, the release of cytokines or other secretory products to abnormal levels, the suppression or aggravation of the inflammatory or immunological response, etc.

"Mammal" for purposes of treatment refers to an animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

The administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. The term "functional derivative" is used to define the variants in the sequence of biologically active amino acids of the native TIE homologous ligands of the present invention, as well as to covalent modifications, including derivatives obtained by reaction with organic derived agents, post-modification modifications. translational, derivatives with non-proteinaceous polymers, and immunoadhesins.

The term "isolated" when used to describe the various polypeptides described herein means polypeptides that have been identified and separated and / or recovered from a component of their natural environment. The contaminating components of their natural environment are materials that w typically interfere with the diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary cup sequencer, or (2) to homogeneity by SDS -PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. The isolated polypeptides include polypeptides in themselves within the recombinant cells, since at least one component of the natural environment of the TIE ligand will not be present. Ordinarily, however, the isolated polypeptides will be prepared by at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid. An isolated nucleic acid molecule is different from the shape or framework in which it is found in nature. Therefore isolated nucleic acid molecules are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes nucleic acid molecules contained in cells that ordinarily express a TIE ligand of the present invention, where, for example, the nucleic acid molecule is in a chromosomal location different from that of the natural cells The term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences compared to a native amino acid sequence.

Substitutional variants are those that have at least one amino acid residue in a native sequence removed and a different amino acid inserted in its place in the same position. The substitutions can be simple, where only one amino acid in the molecule has been replaced, or they can be multiple, where two or more amino acids have been substituted in the same molecule.

Insertion variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid.

Deletion variants are those with one or more amino acids in the native amino acid sequence removed. Ordinarily, the deletion variants will have one or more amino acids deleted in a particular region of the molecule. Deletion variants include those that have C- and / or N-terminal deletions (truncations) as well as variants with internal deletions of one or more amino acids. Preferred deletion variants of the present invention contain deletions outside the fibrinogen-like domain of a native TIE homolog ligand of the present invention.

The variants in the amino acid sequence of the present invention may contain various combinations of amino acid substitutions, insertions and / or deletions, to produce molecules with optimum characteristics.

The amino acids can be classified according to the composition and chemical properties of their side chains. They are generally classified into two groups, loaded and unloaded. Each of these groups is divided into subgroups to classify amino acids more appropriately.

I. Amino Acids Charged Residues Acids: aspartic acid, glutamic acid Basic Waste: lysine, arginine, histidine II. Non-charged Amino Acids Hydrophilic Wastes: serine, threonine, asparagine, glutamine Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine Non-Polar Residues: cysteine, methionine, proline Aromatic Residues: phenylalanine, tyrosine, tryptophan Conservative substitutions involve the exchange of one member within a group by another member within the same group, while non-conservative substitutions will involve the exchange of one member of one of these classes by another. It is expected that the variants obtained by non-conservative substitutions result in significant changes in the properties / biological function of the variant obtained.

Deletions in the amino acid sequence are generally within the range of about 1 to 30 residues, more preferably about 1 to 10 residues, and are typically contiguous. Deletions can occur in regions not directly involved in the interaction with a native TIE receptor. Deletions are preferably carried out outside of the fibrinogen-like regions at the C-terminus of the TIE homologous ligands of the present invention.

The amino acid insertions include ino- and / or carboxyl-terminal fusions that are within the range in length of a residue to polypeptides containing one hundred or more residues, as well as intrasequential insertions of single or multiple amino acid residues. The intrasequential insertions (i.e. insertions within the amino acid sequence of the TIE homologous ligand) can generally be in the range of about 1 to 10 residues, more preferably 1 to 5 residues, more preferably 1 to 3 residues. Examples of terminal insertions include the TIE homologous ligands with an N-terminal methionine residue, an artifact of direct expression in the bacterial recombinant cell culture, and the fusion of a heterologous N-terminal signal sequence with the N-terminal of the TIE homologous ligand molecule to facilitate secretion of the mature TIE homolog ligand from the recombinant host cells. Such signal sequences will generally be obtained from, and thus homologous to, the targeted host cell species. Appropriate sequences include, for example, STII or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for mammalian cells.

Other insertion variants of the native TIE ligand molecules include γ- or C-terminal fusion of the TIE homologous ligand molecule with immunogenic polypeptides, e.g. bacterial polypeptides such as beta-lactamase or an enzyme encoded by the trp locus of E. coli, or the yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin regions (preferably constant regions * of the immunoglobulin), albumin, or ferritin, as described in WO 89/02922 published April 6, 1989.

Since it is often difficult to predict in advance the characteristics of a variant TIE homologous ligand, it will be appreciated that some protection will be needed to select the optimal variant.

The amino acid sequence variants of native TIE homologous ligands of the present invention are prepared by methods known in the art by introducing appropriate nucleotide changes into the DNA of the native TIE homologue or variant ligand, or by Synthesis in vi tro of the desired polypeptide. There are two main variables in the construction of variants in the amino acid sequence: the location of the mature site and the nature of the mutation. With the exception of naturally occurring alleles, which do not require manipulation of the ADN sequence encoding the TIE homologous ligand, variants in the amino acid sequence of the TIE homologous ligands are preferably constructed by mutation of the DNA , either to reach an allele or a variant in the amino acid sequence that does not occur in nature.

A group of the mutations will be created within the domain or domains of the TIE homologous ligands of the present invention and it has been identified that it is involved in the interaction with a TIE receptor, e.g.

TIE-1 or TIE-2, or a receiver still to be discovered.

In addition or alternatively, alterations of the amino acids can be performed at sites that differ in TIE homologous ligands of several species, or in highly conserved regions, depending on the goal to be achieved.

Sites with such locations will typically be modified in series, e.g. by (1) substituting first with conservative options and then with more radical selections depending on the results achieved (2) eliminating the white residue or residues, or (3) inserting residues of the same or different class adjacent to the localized site, or combinations of options 1-3.

One aid technique is called "alanine scan" (Cunningham and Wells, Science 244, 1081-1085 [1989]). Here, a residue or group of white residues is identified and replaced by alanine or polyalanine. Those domains that demonstrate functional sensitivity to alanine substitutions are then refined by the subsequent introduction of other substituents at or for the alanine substitution sites.

After identifying the desired mutation (s), the gene encoding the amino acid sequence variant of a TIE homolog ligand can, for example, be obtained by chemical synthesis as described above.

More preferably, DNA encoding a variant in the amino acid sequence of a TIE homologous ligand is prepared by site-directed mutagenesis of DNA encoding a previously prepared variant or a non-variant version of the ligand. Site-directed mutagenesis (site-specific) allows the production of ligand variants by the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient sequence size and complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, site-specific mutagenesis techniques are well known in the state of the art, as exemplified by publications such as, Edelman et al. , DNA 2_, 183 (1983). As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both the form of a single and double stranded chain. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage, for example, as disclosed in Messing et al. , Third Cleveland Symposium on Macro olecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam (1981). This and other phage vectors are commercially available and their use is well known to those skilled in the art. A versatile and efficient method for the construction of site-specific mutations directed to oligodeoxyribonucleotide in DNA fragments using vectors derived from M13 was published by Zoller, M.J. and Smith, M., Nucleic Acids Res. 10, 6487-6500 [1982]). In addition, plasmid vectors containing a single chain phage originating from replication (Veira et al., Meth. Enzymol 153, 3 [1987]) can be used to obtain single chain DNA. Alternatively, nucleotide substitutions are introduced by synthesis of the appropriate DNA fragment in vi tro, and amplified by PCR procedures known in the art.

In general, site-specific mutagenesis is carried out by first obtaining a single chain vector that includes within its sequence a DNA sequence encoding the relevant protein. An oligonucleotide primer carrying the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al. , Proc. Nati Acad. Sci. USA 75, 5765 (1978). This primer is then annealed with the container vector of the single chain protein sequence, and subjected to DNA polymerase enzymes such as, Klenow fragment I E. coli polymerase, to complete the synthesis of the mutation carrier chain. Thus, a heteroduplex is formed where one strand encodes the original non-mutated sequence and the second strand carries the desired mutation. This heteroduplex vector is then used to transform the appropriate host cells such as JP101 cells, and clones are selected that include the recombinant vectors carrying the array of the mutated sequence. Accordingly, the mutated region can be removed in an appropriate expression vector for protein production.

The PCR technique can also be used in the creation of variants in the amino acid sequence of a TIE ligand. When small amounts of hardened DNA are used as the starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a hardened DNA can be used to generate relatively large amounts of a specific DNA fragment that differs from the sequence hardened only in the positions where the primers differ from the hardened one. For the introduction of a mutation into a DNA plasmid, one of the primers is designed to cover the position of the mutation and to contain the mutation; the sequence of the other primer must be identical for a sequence narrowing of the opposite strand of the plasmid, but this sequence can be located anywhere along the DNA plasmid. It is preferred, however, that the sequence of the second primer be located within 200 nucleotides of that of the first, such that in the end the entire amplified region of DNA surrounded by the primers can be easily sequenced. PCR amplification using a pair primer such as the one just described results in a population of DNA fragments that differ in the position of the mutation specified by the primer, and possibly in other positions, such as the copying of the annealing is from some way prone error.

If the rate of tempering to product material is extremely low, the vast majority of the product DNA fragments incorporate the desired mutation (s). This product material is used to replace the corresponding region in the plasmid that served as a PCR tempering using standard DNA technology. Mutations at separate positions can be introduced simultaneously either by using a second mutant primer or by carrying out a second PCR with different mutant primers and by ligating the two resulting PCR fragments simultaneously to the vector fragment in a ligation of three (or more) parts .

In a specific example of PCR mutagenesis, the tempered DNA plasmid (1 μg) is linearized by digestion with a restriction endonuclease that has a unique recognition site in the DNA plasmid outside of the region to be amplified. From this material, 100 ng is added to the PCR mixture containing the PCR buffer, which contains the four deoxynucleotide triphosphates and is included in the GeneAmpR equipment (obtained from Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA), and 25 pmol of each oligonucleotide primer, to a final volume of 50 μl. The reaction mixture is covered with 35 μl of mineral oil. The reaction is denatured for 5 minutes at 100 ° C, placed briefly on ice, and then 1 μl of Thermus aquaticus (Taq) DNA polymerase (5 units / 1, purchased from Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA ) is added below the layer of mineral oil. The reaction mixture is then inserted into a DNA Thermal Cycler (purchased from Perkin-Elmer Cetus) programmed as follows: 2 min. 55 ° C, 30 sec. 72 ° C, then 19 cycles of the following: 30 sec. 94 ° C, 30 sec. 55 ° C, and 30 sec. 72 ° C.

At the end of the program, the reaction bottle is removed from the thermal cycler and the aqueous phase transferred to a new bottle, extracted with phenol / chloroform (50:50 vol), and precipitated ethanol, and the DNA is recovered by standard procedures. This material is subsequently subject to appropriate treatments for insertion into a vector.

Another method for the preparation of variants, cartridge mutagenesis, is based on the technique described by Wells et al. , [Gene 34, 315 (1985)]. The starting material is the plasmid (or vector) comprising the DNA of the TIE homologous ligand to be mutated. The codon (s) within the TIE homologous ligand to be mutated is' identified. There must be a unique restriction endonuclease site on each side of the identified mutation site (s). If such restriction sites do not exist, they can be generated using the oligonucleotide-mediated mutagenesis method described above to enter them at appropriate locations in the DNA encoding the TIE homologous ligand: After the restriction sites have been introduced into the plasmid , the plasmid is cut by these sites to linearize it. A double-stranded oligonucleotide that encodes the DNA sequence between the restriction site but that contains the desired mutation (s) is synthesized using standard procedures. The two chains are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cartridge. This cartridge is designed to have 3 'and 5' ends that are compatible with the ends of the linearized plasmid, so that they can be ligated directly to the plasmid. This plasmid now contains the DNA sequence of the homologous ligand of mutated TIE.

Additionally, the so-called phagemid display method may be useful in performing variants in the amino acid sequence of native TIE homologues or variants. This method involves (a) the construction of a replicable expression vector comprising a first gene encoding a receptor to be mutated, a second gene encoding at least a portion of a wild-type or wild-type phage coat protein where the first and second genes are heterologous, and a regulatory element of transcription operably linked to the first and second genes, thus forming a gene fusion that encodes a fusion protein; (b) mutation of the vector to one or more selected positions within the first gene and consequently forming a family of related plasmids; (c) transformation of appropriate host cells with the plasmids; (d) infection of the transformed host cells with an assist phage having a gene encoding the phage coat protein; (e) culturing the infected host cells and transformed under conditions suitable for the formation of recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the adjusted conditions such that no more than a smaller amount of phagemid particles exhibit more than one copy of the fusion protein on the surface of the particle; (f) contacting the phagemid particles with an appropriate antigen such that at least a portion of the phagemid particles binds to the antigen; and (g) the separation of the phagomid particles that are joined from those that do not. Steps (d) to (g) may be repeated one or more times. Preferably in this method the plasmid is under strict control of the transcriptional regulatory element, and the culture conditions are adjusted in such a way that the number of phagemid particle number exhibiting more than one copy of the fusion protein on the surface of the the particle is less than about 1%. As well, preferably, the amount of phagemid particles exhibiting more than one copy of the fusion protein is less than 10% of the amount of phagemid particles exhibiting a single copy of the fusion protein. More preferably, the amount is less than 20%. Typically in this method, the expression vector will later contain a secretory signal sequence linked to the DNA encoding each subunit of the polypeptide and the transcription regulatory element will be a promoter system. Preferred promoter systems are selected from lac Z,? PL, tac, T7 polymerase, tryptophan and alkaline phosphatase promoters and combinations thereof. Also, normally the method will employ a helper phage selected from M13K07, M13R408, M13-VCS, and Phi X174. The preferred helper phage is M13K07, and the preferred coat protein is the M13 coat protein Phage gene III. Preferred hosts are E. coli, and the E. coli protease deficient chains.

Further details of the future and similar mutagenesis techniques are found in general textbooks, such as, for example, Sambrook et al. , 'Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), and Current Protocols in Molecular Biology, Ausubel et al., Eds. , Wiley-Interscience, 1991.

The "immunoadhesins" are chimeras which are traditionally constructed from a receptor sequence linked to a constant domain sequence of an appropriate immunoglobulin (immunoadhesins). Such structures are well known in the state of the art. The immunoadhesins reported in the literature include fusions of the T cell receptor * [Gascoigne et al., Proc. Nati Acad. Sci. USA 84, 2936-2940 (1987)]; CD4 * [Capón et al., Nature 337, 525-531 (1989); Traunecker et al., Nature 339, 68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA 9, 347-353 (1990); Byrn et al., Nature 344, 667-670 (1990)]; L-selectin (host receptor) [Watson et al., J. Cell. Biol. 110, 2221-2229 (1990); Watson et al., Nature 349, 164-167 (1991)]; CD44 * [Aruffo et al., Cell 61, 1303-1313 (1990)]; CD28 * and B7 * [Linsley et al., J. Exp. Med. 173, 721-730 (1991)]; CTLA-4 * [Lisley et al., J. Exp. Med. 174, 561-569 (1991)]; CD22 * [Stamenkovic et al., Cell 66. 1133-1144 (1991)]; TNF receptor [Ashkenazi et al., Proc. Nati Acad. Sci. USA 88, 10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27, 2883-2886 (1991); Peppel et al., J. Exp. Med. 174, 1483-1489 (1991)]; NP receptors [Bennett et al. , J. Biol. Chem. 266, 23060-23067 (1991)]; IgE receptor chain-a * [Ridgway and Gorman, J. Cell. Biol. 115, abstr. 1448 (1991)]; HGF receptor [Mark, M. R. et al. , 1992, J. Biol. Chem. Proposed], where the asterisk (*) indicates that the receptor is a member of the immunoglobulin superfa ilia.

Chimeric immunoglobulins ligands are also known, and are disclosed, for example, in the U.S. Patents Nos. 5,304,640 (for ligands of L-selectin); 5,316,921 and 5,328,837 (for HGF variants). These chimeras can be made in a manner similar to the construction of the chimeric immunoglobulin receptor.

Covalent modifications of the TIE homologous ligands of the present invention are included here within the scope. Such modifications are traditionally introduced by reacting the white amino acid residues of the TIE ligand with an organic derivatized agent that is capable of reacting with selected sides or terminal residues, or by latching onto mechanisms of post-translational modifications that function in host cells selected recombinants. The resulting covalent derivatives are useful in programs directed to the identification of residues important for biological activity, for immunoassays, or for the preparation of TIE anti-ligand antibodies for the purification of the immunoaffinity of the recombinants. For example, the complete inactivation of the biological activity of the protein after reacting with ninhydrin would suggest that at least one arginyl or lysyl residue is critical to its activity, where the individual residues which were modified under the selected conditions are identified by isolation of a peptide fragment containing the modified amino acid residue. Such modifications are within the ordinary skill in the state of the art and are carried out without undue experimentation.

The cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give the carboxymethyl or carboxyamidomethyl derivatives. The cysteinyl residues are also derived from the reaction with bromotrifluoroacetone, a-bromo-β- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmamoimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercury-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l, 3-diazole.

The histidyl residues are derived by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful, the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

The amino terminal and lysinyl residues are reacted with succinic acid anhydrides or other carboxylic anhydrides. The derivation with these agents has the effect of reversing the charge of the lysinyl residues. Other reagents suitable for derivatizing residues containing a-amino include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0-methylisourea; 2, 4-pentanedione; and reaction catalyzed by transaminase with glyoxylate.

The arginyl residues are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. The derivation of arginine residues requires that the reaction be carried out under alkaline conditions by the high pKa of the guanidine functional group. In addition, these reagents can react with the lysine groups as well as with the epsilon-amino group of arginine.

The specific modification of the tyrosyl residues can be done, with particular interest in the introduction of the spectral labels in the tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. The tyrosyl residues are iodinated using 125I or 131I to prepare labeled proteins for use in radioiminusoassay.

The carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R '-N = C = NR') such as l-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or l-ethyl- 3- (4-azonia-4, 4-di ethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

The glutaminyl and asparaginyl residues are frequently unlinked to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Any form of these residues falls within the scope of this invention.

Other modifications include the hydroxylation of proline and lysine, the phosphorylation of hydroxyl groups of seryl, threonyl, or tyrosyl residues, the methylation of the a-amino groups of the side chains of lysine, arginine, and histidine (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman &Co., San Francisco, pp. 79-86 [1983]), the acetylation of the N-terminal amine, and the amidation of any C-terminal carboxyl group. Later the molecules can be covalently bound to non-protein-forming polymers, e.g. polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner prescribed in U.S. Patents 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Derivatization with bifunctional agents is useful for the preparation of intramolecular aggregates of the TIE ligand with polypeptides as well as for cross-linking the TIE ligand polypeptide to a water-insoluble support matrix or surface for use in assays or affinity purification. A study of crossed bridges between chains provides direct information in the conformational structure. Commonly used cross-linking agents include 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, homobifunctional imidoesters, and bifunctional maleimides. Derivatizing agents such as methyl-3- [(p-azidophenyl) dithio] propioimidate provide "photoactivatable intermediates which are capable of cross-linking in the presence of light." Alternatively, reactive water-insoluble matrices such as activated cyanogen bromide carbohydrates and reactive substrates of systems described in US Patent Nos. 3,959,642, 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, 4,055,635, and 4,330,440 are used for immobilization and cross-linking of proteins.

Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. The glutaminyl and aspariginyl residues are frequently deamidated post translationally to the corresponding residues glutamyl and aspartyl. Alternatively, these residues are deamidated under mildly acidic conditions. Any form of these residues falls within the scope of this invention.

Other post-translational modifications include the hydroxylation of proline and lysine, the phosphorylation of the hydroxyl groups of the seryl, threonyl or tyrosyl residues, the methylation of the a-amino groups of the side chains of lysine, arginine and histidine [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co. , San Francisco, pp. 79-86 (1983)] Other derivatives comprise the novel peptides of this invention covalently linked to the non-protein-rich polymer. The non-proteolyceous polymer is ordinarily a synthetic hydrophilic polymer, i.e. a polymer not otherwise found in nature. However, polymers which exist in nature and are produced by recombinant methods or in vi tro are useful, as are polymers which are isolated from nature. Polyvinyl hydrophilic polymers fall within the scope of this invention, e.g. polyvinyl alcohol and polyvinyl pyrrolidone. Particularly useful are polyvinyl alkylene ethers such as polyethylene glycol, polypropylene glycol.

TIE homologous ligands can be attached to several non-protein polymers, such as polyethylene glycol (PEG), polypropyleneglycol or polyoxyalkylenes, in the manner imposed in the U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. These variants, like the immunoadhesins of the present invention, are expected to have longer half-lives than the corresponding native TIE homologous ligands.

The TIE homologous ligands can be wrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (eg liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A., Ed. (1980).

The term "native TIE receptor" is used herein to refer to a TIE receptor of many animal species, including, but not limited to, humans, other higher primates, e.g. monkeys, and rodents, e.g. rats and mice, known or to be discovered. The definition specifically includes the TIE-2 receptor, disclosed, for example, in PCT Application Serial No. WO 95/13387 (published May 18, 1995), 'and the endothelial cell receptor tyrosine kinase called "TIE" in PCT Application Publication No. WO 93/14124 (published July 22, 1993), and preferably is TIE-2.

B. ANTIBODIES ANTI LIGANDO TIE HOMOLOGOUS The present invention covers agonist and antagonist antibodies, specifically binding to TIE homologous ligands. The antibodies can be monoclonal or polyclonal, and include, without limitation, mature antibodies, antibody fragments (e.g. Fab, F (ab ') 2, F v, etc.), single chain antibodies and various combinations of chains.

The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) that specifically bind to a TIE ligand of the present invention and antibody compositions with polyepitopic specificity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i. e. , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a simple antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen.

The monoclonal antibodies mentioned herein include hybrid and recombinant antibodies produced by the splicing of a variable domain (including hypervariable) of a TIE homologous anti-ligand antibody with a constant domain (eg "humanized" antibodies), or a light chain with a heavy chain , or a chain of one species with another chain of another species, or fusions with heterologous proteins, regardless of the species of origin or the class or subclass designation of immunoglobulins, as well as antibody fragments (eg Fab, F (ab ') 2, and Fv), until they exhibit the desired biological activity. See e.g. U.S. Pat. No. 4,816,567 and Mage et al., In Monoclonal Antibody Production Techniques and Applications, pp. 79-97 (Marcel Dekker, Inc .: New York, 1987).

Thus, the "monoclonal" modifier indicates the type of the antibody as it is obtained from a substantially homogeneous population of antibodies, and should not be analyzed as a required production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be made by the hybridoma method first described by Kohler and Milstein, Nature, 256: 495 (1975), or they can be made by recombinant DNA methods as described in US Pat. No. 4,816,567. The "monoclonal antibodies" can also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990), for example.

The "humanized" forms of non-human antibodies (eg murine) are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other subsequences of binding antibodies. antigen) which contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) whose residues of a complementary determining region (CDR) of the receptor are replaced by residues of a CDR of non-human species (donor antibody) such as those of the mouse, rat, or rabbit that has the affinity, specificity and desired capacity. In some cases, the residues of the framework region Fv (FR) of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues which are not found in the recipient antibody or in the CDR imported from the frame sequences. These modifications are made to refine and optimize antibody performance later on. In general, the humanized antibody will substantially comprise all or at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of a constant domain or region of an immunoglobulin (Fe), typically that of a human immunoglobulin.

Polyclonal antibodies of a TIE homologous ligand of the present invention are generally collected from animals by multiple subcutaneous (se) or intraperitoneal (ip) injections of the TIE ligand and an adjuvant. It may be useful to conjugate the TIE ligand or a fragment containing the target amino acid sequence with a protein that is immunogenic in the species to be immunized, e.g. eye limpet hemocyanin, serum albumin, bovine thyroglobulin, or trypsin inhibitor from soy beans using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, S0C12, or RXN = C = NR, where R and R1 are different alkyl groups.

The animals are immunized against the conjugates or immunogenic derivatives by the combination of 1 mg or 1 μg of conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freud's assistant and injecting the solution intradermally at multiple sites. One month later the animals are stimulated with 1/5 to 1/10 of the original amount of conjugate in the complete Freud's assistant via subcutaneous injection at multiple sites. From 7 to 14 days later the animals are bled and the serum is assayed for TIE anti-ligand antibody titre. The animals are stimulated until the title reaches a plateau. Preferably, the animal stimulated with the conjugate of the same TIE ligand, but conjugated to different protein and / or through different cross-linking reagents. The conjugates can also be made in recombinant cell culture as protein fusions. Also, adding agents such as alum are used to improve the immune response.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the "monoclonal" modifier indicates the type of antibody not being a mixture of discrete antibodies.

For example, the anti-TERT homologous monoclonal antibodies of the invention can be made using the hybridoma method first described by Kohler & Milstein, Nature 256: 495 (1975), or can be made by recombinant DNA methods [Cabilly, et al. , U.S. Pat. No. 4,816,567].

In the hybridoma method, a mouse or other appropriate animal host, such as the hamster, is immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that will bind specifically to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using an appropriate fusion agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)].

The thus prepared hybridoma cells are seeded and cultured in an appropriate culture medium which preferably contains one or more substances that inhibit the growth or survival of the parental, non-fused myeloma cells. For example, if the parental lymphoma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or PRET), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), the substances of which prevent cell growth. deficient of HGPRT.

Preferred myeloma cells are those that fuse efficiently, support a high stable level expression of antibody by the selected antibody producing cells, and are sensitive to a medium such as the HAT medium. Among these, the preferred myeloma cell lines are urine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-cells. 2 available from American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol. 133: 3001 (1984); Brodeur, et al. , Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].

The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies directed against the TIE homologous ligand. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. 107: 220 (1980) After the hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity are identified, the clones can be subcloned by limiting dilution methods and cultured by standard methods. Goding, Monoclqnal Antibodies: Principies and Practice, pp. 59-104 (Academic Press, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are appropriately separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or chromatography. of affinity.

The DNA encoding the monoclonal antibodies of the invention is rapidly isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as the preferred source of such DNA. Once isolated, the DNA can be placed in expression vectors, which are then transferred into host cells such as COS simian cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein , to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for the constant domains of the human heavy and light chains in place of the homologous murine sequences, Morrison, et al. , Proc. Nati Acad. Sci. 81, 6851 (1984), or by covalent attachment to the immunoglobulin coding sequence of all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, the "chimeric" or "hybrid" antibodies are prepared containing the binding specificity of a monoclonal anti-TIE ligand antibody described herein.

Typically such non-immunoglobulin polypeptides are substituted by the constant domains of an antibody of the invention, or are substituted by the variable domains of an antigen combining site of an antibody of the invention to create a chimeric bivalent antibody comprising a combination site of antigen having specificity for a TIE ligand of the present invention and another antigen combining site having specificity for a different antigen.

Chimeric or hybrid antibodies can also be prepared in vitro using methods known in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention will typically be labeled with a detectable portion. The detectable portion can be any which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable portion can be a radioisotope, such as 3H, s, or 125 I, a chemiluminescent fluorescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; biotin; radioactive isotopic labels, such as, e.g., 1 5I, 32P, 14C, or 3H, or an enzyme, such as alkaline phosphatase, beta galatosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody to the detectable portion can be employed, including those methods described by Hunter, et al. , Nature 14: 945 (1962); David, et al. , Biochemestry 13: 1014 (1974); Pain, et al. , J. Immunol. Meth. 40: 219 (1981); and Nygren, J. Histochem. and Cytochem. 30: 407 (1982).

The antibodies of the present invention can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard (which may be a TIE homologous ligand or an immunologically reactive portion thereof) to compete with the test sample analyte (TIE ligand) for binding with a limited amount of antibody. The amount of TIE homologous ligand in the test sample is inversely proportional to the amount of standard that happens to bind to the antibodies. To facilitate the determination of the amount of standard that comes together, the antibodies are generally insolubilized before or after the competition, so that the standard and the analyte that are bound to the antibodies can be conveniently separated from the standard and the analyte. which remain without joining.

Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected. In a sandwich assay the test sample analyte is bound by a first antibody which is immobilized on a solid support, and therefore a second antibody binds to the analyte, thus forming an insoluble three part complex. David & Greene, U.S. Pat. No. 4,376,110. The second antibody can label itself with a detectable portion (direct sandwich assays) or can be measured using an anti-immunoglobulin antibody that is labeled with a detectable portion (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable portion is an enzyme.

Methods for the humanization of non-human antibodies are well known in the state of the art; generally, a humanized antibody has one or more amino acid residues introduced therein from a source which is not human. These non-human amino acid residues are often referred to as "imported" residues, which are typically taken from an "imported" variable domain. The humanization can be carried out essentially following the method of Winter and his cotrabajadores [Jones et al. , Nature 321, 522-525 (1986); Riech ann et al. , Nature 332, 323-327 (1988); Verhoeyen et al. , Science 239, 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly, supra), where substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by the residues of analogous sites in rodent antibodies.

It is important that the antibodies are humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to the preferred method, the humanized antibodies are prepared by a process of analysis of the parental sequences and several conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available which illustrate and show probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The inspection of these samples allows the analysis of the probable role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, the FR residues can be selected and combined from the consensus and import sequence in such a way that the desired antibody characteristic, such as the increased affinity for the target antigen (s), is achieved. In general, CDR residues are directly and more substantially involved in the influence of antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the gene from the antibody heavy chain binding region (JH) in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene formation in such germline mutant mice will result in the production of human antibodies on the challenge of the antigen. See, e.g. Jakobovits et al. , Proc. Nati Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al. , Nature 362, 255-258 (1993).

Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities with at least two different antigens. In the present case, one of the binding specificities is one for a particular TIE ligand, the other is for any other antigen, and preferably another ligand. For example, bispecific antibodies that bind specifically to two different TIE homologous ligands are within the scope of the present invention.

The methods for making bispecific antibodies are known in the state of the art.

Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two heavy chain-immunoglobulin light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305, 537-539 (1983)). Because of the randomization of heavy and light chains of immunoglobulins, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually carried out by affinity chromatography steps, is somewhat uncomfortable, and the productions of the product are low. Similar procedures are disclosed in PCT application publication No. WO 93/08829 (published May 13, 1993), and in Traunecker et al. , EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, the variable domains of antibodies with the desired binding specificities (antigen-antibody combination sites) are fused with immunoglobulin constant domain sequences. The fusion preferably is with the constant domain of the heavy chain of the immunoglobulin, comprising at least part of the hinge, and the second and third constant regions of the heavy chain of an immunoglobulin (CH2 and CH3). It is preferable to have the first constant region of the heavy chain (CH1) containing the site necessary for the binding of the light chain present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and are cotransfected in an appropriate host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in modalities when different rates of the three polypeptide chains used in the construction provide the optimal products. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into an expression vector when the expression of at least two polypeptide chains at the same rates results in higher yields or when the rates are not particularly significant. In a preferred embodiment of this approach, bispecific antibodies are composed of the heavy chain of a hybrid immunoglobulin with a first binding specificity in one arm and a heavy chain-light chain pair of hybrid immunoglobulin (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations., since the presence of a light chain of an immunoglobulin in only one half of the bispecific molecule provides an easy way of separation.

For more details on the generation of bispecific antibodies see, for example, Suresh et al. , Methods in Enzymology 121, 210 (1986).

The term "diabodies" refers to small fragments of antibody with two antigen binding sites, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH) -V) By using a linker that is too short to allow pairing between the two domains of the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. The diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al. , Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993).

An "isolated" antibody is defined similarly to the definition provided above by the isolated polypeptides. Specifically, an "isolated" antibody is one of which has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of their natural environment are materials which would interfere with the diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other protein-free or non-protein solutes. In preferred embodiments, the antibody will be purified (1) at greater than 95% by weight of antibody as determined by the Lowry method, and more preferably by more than 99% by weight, (2) to a sufficient degree to obtain less 15 residues of an N-terminal or internal amino acid sequence by using a rotating cup sequencer, or (3) homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain . The isolated antibody includes the antibody itself within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, the isolated antibody will be prepared by at least one purification step.

The word "label" when used herein refers to a detectable compound or composition which is directly or indirectly conjugated to the antibody in such a manner as to generate a "labeled" antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze the chemical alteration of a compound or substrate composition which is detectable.

By "solid phase" is meant a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases collected here include those formed partially or entirely of glass (e.g., controlled porous glass), polysaccharides (e.g. agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase may comprise the well of a test dish; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U. S. Patent No. 4,275,149.

A "liposome" is a small vesicle composed of various types of lipids, phospholipids and / or surfactants which is useful for the delivery of a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal . The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of the biological membranes.

The "agonists" and "antagonists" of antibodies are as defined above.

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two antibodies covalently linked. It has been proposed that such antibodies tend, for example, to direct cells of the immune system to unwanted cells (US Patent No. 4,676,980), and for the treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92). / 200373; EP 03089). Heteroconjugate antibodies can be made using any of the convenient cross-linking methods. Suitable cross-linking agents are well known in the state of the art, and are disclosed in U. S. Patent No. 4,676,980, together with a number of cross-linking techniques.

The "single chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of the antibody, where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which allow the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

C. CLONING AND EXPRESSION OF THE TIE HOMOLOGOUS LIGANDS In the context of the present invention the terms "cell", "cell line", and "cell culture" are used interchangeably, and all of these designations include progeny. It is also understood that all progeny can not be exactly identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological property, as well as are protected in the originally transformed cell, are included.

The terms "replicable expression vector" and "expression vector" refers to a part of DNA, usually double stranded, in which a piece of foreign DNA may be inserted. Foreign DNA is defined as heterologous DNA, which is DNA not found naturally in the host cell. The vector is used to transport the foreign or heterologous DNA into an appropriate host cell. Once inside the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA can be generated. In addition, the vector contains the necessary elements that allow translation of the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by foreign DNA can thus be rapidly synthesized.

The expression and cloning vectors are well known in the state of the art and contain a nucleic acid sequence that allows the vector to be replicated in one or more selected host cells. The selection of the appropriate vector will depend on 1) whether it will be used for DNA amplification or for DNA expression, 2) the size of the DNA to be inserted in the vector, and 3) the host cell to be transformed with the vector. Each vector contains several components depending on its function (DNA amplification or DNA expression) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an increment element, a promoter, and a sequence termination sequence. transcription. (i) Signal Sequence Component In general, the signal sequence can be a component of the vector, or it can be a part of the TIE ligand molecule that is inserted into the vector. If the signal sequence is heterologous, it should be selected in such a way that it is recognized and processed (i.e. cut by a signal peptidase) by the host cell.

The heterologous signal sequences appropriate for prokaryotic host cells are preferably prokaryotic signal sequences, such as α-amylase, ompA, ompC, ompE, or pF, alkaline phosphatase, penicillinase, Ipp, or thermostable leader enterotoxins II. For the yeast secretion yeast invertase, amylase, alpha factor, or acid phosphatase leaders can, for example, be used. In mammalian cell expression the mammalian signal sequences are more appropriate. The signal sequences listed are for illustration only, and do not limit the scope of the present invention in any way. (ii) Replication Origin Component Both the expression vectors and the cloning vectors contain a nucleic acid sequence that allowed the vector to replicate in one or more selected host cells. Usually, in the cloning vectors this sequence is one that allows the vector to replicate independently of the host chromosomes, and includes origins of replication or sequences of autonomous replication. Such a sequence is well known for a variety of bacteria, yeasts and viruses. The origin of replication of the well-known plasmid pBR322 is appropriate for most gram negative bacteria, the origin of 2 μ of the plasmid for yeast and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for vectors of cloning in mammalian cells. Origins of replication are not needed by mammalian expression vectors (SV40 origin can typically be used only because it contains 1 early promoter). The majority of expression vectors are "transporter" vectors, i.e. they are capable of replication in at least one class of organisms but can be transferred in another organism for their expression. For example, a vector is cloned in E. coli and then the same vector is transferred in yeast or mammalian cells for expression even if it is not capable of replicating independently of the chromosome of the host cell.

The DNA is also cloned by insertion into the host genome. This is rapidly achieved by using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to the sequence found in Bacillus genomic DNA. Transfection of Bacillus with its vector results in homologous recombination with the genome and insertion of the DNA encoding the desired heterologous polypeptide. However, the recovery of genomic DNA is more complex than that of an exogenously replicated vector because restriction enzyme digestion is required to suppress the encoded polypeptide molecule. [iii) Selection Gen Component The expression and cloning vectors should contain a selection gene, also called a selectable marker. This is a gene that encodes a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures that no host cell which eliminates the vector will not obtain an advantage in the growth or reproduction on the transformed hosts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, (b) supplement auxotrophic deficiencies, or (c) provide critical nutrients not available in complex media, e.g. the gene that codes for D-alanine racemase for bacilli.

An example of a selection scheme uses a drug to arrest the growth of a host cell. Those cells that are successfully transformed with a heterologous gene express a protein that confers resistance to the drug and thus survive the selection regimen. Examples of such dominant selection use the neomycin drugs [Southern et al. , J. Molec. Appl. Genet 1, 327 (1982)], mycophenolic acid [Mulligan et al. , Science 209, 1422 (1980)], or hygromycin [Sudgen et al. , Mol. Cel. Biol. 5, 410-413 (1985)]. The three examples given here employ bacterial genes under eukaryotic control to transmit resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Other examples of selectable markers suitable for mammalian cells are dihydrofolate reductase (DHFR) or thymidine kinase. Such markers allow the identification of cells which were competent to take the desired nucleic acid. The transformants of mammalian cells are placed under selection pressure to which only the transformants have been adapted to survive by virtue of having taken the marker. The selection pressure is imposed by the culture of the transformants under conditions in which the concentration of the selection agent in the medium is successively changed, and therefore guiding the amplification of both the selection gene and the DNA encoding the desired polypeptide. Amplification is the process by which the genes in greatest demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. The increased amounts of the desired polypeptide are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are first identified by culturing all the transformants in a culture medium which suffers from hypoxanthine, glycine and thymidine. An appropriate host cell in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Nat'l. Acad. Sci. USA 77, 4216 (1980). A particularly useful DHFR is a mutant DHFR that is highly resistant to MTX (EP 117,060). This selection agent can be used with any guest in a different appropriate way, e.g. ATCC No. CCL61 CHO-Kl, despite the presence of endogenous DHFR. The DNA encoding the DHFR and the desired polypeptide, respectively, is then amplified by exposure to an agent (methotrexate, or MTX) that inactivates DHFR. One ensures that the cell requires more DHFR (and consequently amplifies all exogenous DNA) by selecting only for cells that can grow in successive series of even higher concentrations of MTX. Alternatively, hosts cotransformed with genes encoding the desired polypeptide, wild type DHFR and another selectable marker such as the neo gene can be identified using a selection agent for the selectable marker such as G418 and then selected and amplified using methotrexate in a host Wild type containing endogenous DHFR. (See also U. S. Patent No. 4,965,199).

A suitable selection gene for use in yeast is the trpl gene present in yeast plasmid YRp7 (Stinchcomb et al., 1919, Nature 282: 39; Kings an et al., 1979, Gene 1_: 141; or Tschemper et al. al., 1980, Gene 10: 157). The trpl gene provides a selection marker for a mutant strand of yeast deficient in the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85: 12). The presence of the trpl lesion in the genome of the yeast host cell then provides an effective environment for the detection of transformation by growth in the absence of tryptophan. Similarly, the yeast chains provide an effective environment for the detection of transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast chains (ATCC 20,622, or 38,626) are complemented by known plasmids carrying the Leu2 gene.

,? Promoter component The expression vectors, in contrast to the cloning vectors, should contain a promoter which is recognized by the host organism and is operably linked to the nucleic acid encoding the desired polypeptide. The promoters are untranslated sequences located at the upper end of the initial codon of a structural gene (generally within about 100 to 1000 bp) that controls the transcription and translation of the nucleic acid under its control. They typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate high levels of transcription of DNA under their control in response to some change in culture conditions, e.g. the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to the DNA encoding the desired polypeptide by removing it from its source gene by restriction enzyme digestion, followed by insertion 5 'to the initial codon for the polypeptide to be expressed. Not to mention that the genomic promoter of a TIE ligand is not usable. However, heterologous promoters will generally result in higher transcription and higher productions of expressed TIE homologous ligands compared to promoters of native TIE ligands.

Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al., Nature 275: 615 (1978); and Goeddel et al. , Nature 281: 544 (1979)), alkaline phosphatase, a tryptophan promoter system (trp) (Goeddel, Nucleic Acids Res. 8_: 4057 (1980) and EPO Appln Publ. No. 36,776) and hybrid promoters such as the tac promoter (H. De Boer et al., Proc. Nat'l. Acad. Sci. USA 80: 21-25 (1983)). However, other known bacterial promoters are appropriate. Their nucleotide sequences have been published, thus allowing a skilled worker to operably link them to the DNA encoding a TIE ligand (Siebenlist et al., Cell 20: 269 (1980)) using linkers or adapters to provide any site of required restriction. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operably linked to DNA encoding a TIE ligand.

Promoter sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. X. 149 (1978) and Holland, Biochemistry 17: 4900 (1978)). Such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other promoters of yeasts, which are inducible promoters that have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytor or C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metalotionain, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose. Vectors and promoters suitable for use in the expression of yeast are described later in R. Hitzeman et al., EP 73, 657A. Yeast enhancers are also advantageously used with yeast promoters.

The promoter sequences are known by eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases towards the upper end of the site from which transcription is initiated. Another sequence found 70 to 80 bases towards the upper end of the start of the transcription of many genes is a CXCAAT region where X can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence which may be the signal for the addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are inserted appropriately into the expression vectors of the mammal.

Transcription of the TIE ligand of vectors in mammalian host cells can be controlled by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), adenovirus (such as Adenovirus 2). ), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and more preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, eg the actin promoter or an immunoglobulin promoter, the heat shock promoters, and the promoter normally associated with the sequence of the TIE ligand, such promoters provided are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained from an SV40 restriction fragment which also contains the viral origin of SV40 replication [Fiers et al. , Nature 273: 113 (1978), Mulligan and Berg, Science 209, 1422-1427 (1980); Pavlakis et al. , Proc. Nati Acad. Sci. USA 78, 7398-7402 (1981)]. The early immediate promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment [Greenaway et al. , Gene 18, 355-360 (1982)]. A system for the expression of DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in US 4,419,446. A modification of this system is described in US 4,601,978. See also, Gray et al. , Nature 295, 503-508 (1982) in the expression of cDNA encoding human immune interferon in monkey cells; Reyes et al. , Nature 297, 598-601 (1982) on the expression of human β-interferon cDNA in mouse cells under the control of a herpes simplex virus thymidine kinase promoter; Canaani and Berg, Proc. Nati Acad. Sci. USA 79, 5166-5170 (1982) in the expression of the human interferon-ßl gene in cultured mouse and rabbit cells; and Gorman et al. , Proc. Nati Acad. Sci. USA 79, 6777-6781 (1982) in the expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and HIN-3T3 cells using the long terminal repeat of the Rous sarcoma virus as a promoter. (v) Enhancing Element Component The transcription of the DNA encoding the TIE homologous ligands of the present invention by higher eukaryotes is often enhanced by the insertion of an enhancer sequence within the vector. Intensifiers are DNA elements that act like cis, usually about 10 to 300 bp, which act on a promoter to increase its transcription. The intensifiers are relatively independent of orientation and position and have been found 5 '[Laimins et al. , Proc. Nati Acad. Sci. USA 78./993 (1981)] and 3 '[Lasky et al. , Mol. Cel. Biol. 3, 1108 (1983)] to the transcription unit, inside an intron [Banerji et al. , Cell 33, 729 (1983)] as well as within the same coding sequence [Osborne et al. , Mol. Cel Biol. 4_, 1293 (1984)]. Many intensifying sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one will use an enhancer of a eukaryotic cell virus. Examples include the SV40 enhancer on the latter side of the origin of replication (bp 100-270), the cytomegalovirus promoter enhancer early, the polyoma enhancer on the latter side of the replication origin, and the adenovirus enhancers. See also Yaniv, Nature 297, 17-18 (1982) in enhancement elements for the activation of eukaryotic promoters. The enhancer can be spliced into the vector at a 5 'or 3' position of the TIE ligand DNA, but it is preferable located at a 5 'site of the promoter. vi Component of Termination of Transcription Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells of other multicellular organisms) will also contain sequences necessary for the termination of transcription and for the stabilization of MARN. Such sequences are commonly available from 5 'untranslated regions, and occasionally 3' of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the MARN encoding the TIE ligand. Untranslated-3 'regions also include transcription termination sites.

The construction of appropriate vectors containing one or more of the components listed above, the desired coding and control sequences, employs standard ligation techniques. The isolated plasmids or DNA fragments are cut, adapted, and religated in the desired manner to generate the required plasmids.

For confirmatory analysis of the correct sequences in constructed plasmids, ligation mixtures are used to transform the 294 chain of E. coli K12 (ATCC 31, 446) and successful transformants selected by ampicillin or tetracycline resistance where appropriate. The plasmids of the transformants are prepared, analyzed by digestion of the restriction endonuclease, and / or sequenced by the method of Messing et al. , Nucleic Acids Res. 9, 309 (1981) or by the method of Maxam et al. , Methods in Enzymology 65, 499 (1980).

Particularly useful in the practice of this invention are the expression vectors that provide for transient expression in mammalian DNA cells encoding a TIE ligand. In general, transient expression involves the use of an expression vector that is capable of efficiently replicating in a host cell, such as that the host cell accumulates many copies of the expression vector and, conversely, synthesizes high levels of a desired encoded polypeptide. by the expression vector. Passenger systems, comprising an appropriate expression vector and a host cell, allow convenient positive identification of polypeptides encoded by DNA clones, as well as for the rapid protection of such polypeptides for desired biological or physiological properties. Thus, transient expression systems are particularly useful in the invention for purposes of identifying analogs and variants of a TIE ligand.

Other methods, vectors, and host cells appropriate for adaptation to the synthesis of TIE polypeptides in recombinant vertebrate cell cultures are described in Getting et al. , Nature 293, 620-625 (1981); Mantel et al. , Nature 281, 40-46 (1979); Levinson et al.; EP 117,060 and EP 117,058. A particularly useful plasmid for the expression of TIE ligand polypeptides in mammalian cell culture is pRK5 (EP 307,247), together with its derivatives, such as, pRK5D having a sp6 transcription initiation site followed by a site of restriction enzyme Sfil preceding the cloning sites Xho / NotlI cDNA, and pRK5B, a precursor of pRK5D not containing the Sfil site; see, Holmes et al., Science 253, 1278-1280 (1991). (vii) Construction and vector analysis The construction of appropriate vectors containing one or more of the components listed above employs standard ligation techniques. The isolated plasmids or DNA fragments are cut, adapted, and religated in the desired form to generate the required plasmids.

For the analysis for the confirmation of the correct sequences in the constructed plasmids, the ligation mixtures are used to transform the 294 chain of E. coli K12 (ATCC 31, 446) and the successful transformants selected by resistance to ampicillin or tetracycline where it is appropriate. The plasmids of the transformants are prepared, analyzed by digestion of the restriction endonuclease, and / or sequenced by the method of Messing et al. , Nucleic Acids Res. 9, 309 (1981) or by the method of Maxam et al. , Methods in Enzymology 65, 499 (1980). (viii) Vectors of temporary expression Particularly useful in the practice of this invention are the expression vectors that provide for transient expression in mammalian DNA cells encoding a TIE ligand. In general, transient expression involves the use of an expression vector that is capable of efficiently replicating in a host cell, such as that the host cell accumulates many copies of the expression vector and, conversely, synthesizes high levels of a desired polypeptide. encoded by the expression vector. Sambrook et al. , supra, pp. 16.17-16.22. Passenger expression systems, comprising an appropriate expression vector and a host cell, allow convenient positive protection of such polypeptides for desired biological and physiological properties. Thus, transient expression systems are particularly useful in the invention for purposes of identifying analogs and variants of native TIE homologous ligands with the requisite biological activity. (ix) Appropriate Vectors of Exemplary Vertebrate Cells Other methods, vectors, and host cells appropriate for adaptation to the synthesis of a TIE ligand (including functional derivatives of native proteins) in recombinant vertebrate cell cultures are described in Getting et al. , Nature 293, 620-625 (1981); Mantel et al. , Nature 281, 40-46 (1979); Levinson et al.; EP 117,060 and EP 117,058. A particularly useful plasmid for the expression of a TIE ligand in mammalian cell culture is pRK5 (EP 307,247) or pSV16B (PCT Publication No. WO 91/08291).

Suitable host cells for the cloning or expression of the vectors described herein are the prokaryotic, yeast or higher eukaryotic cells described above. Appropriate prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. A preferred cloning host is E. coli 294 (ATCC 31, 446) despite other gram-positive or gram-positive prokaryotes such as E. coli B, E. coli X1776 (ATCC 31, 537), E. coli W3110 ( ATCC 27, 325), Pseudomonas species, or Serratia Marcesans are appropriate.

In addition to prokaryotes, eukaryotic microbes such as fungi or filamentous yeasts are appropriate hosts for the vectors described herein. Saccharomyces cerevisiae, or common baking yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and chains are commonly available and useful here, such as S. pombe [Beach and Nurse, Nature 290, 140 (1981)], Kluyveromyces lactis [Louvencourt et al. , J. Bacteriol. 737 (1983)]; yarrowia (EP 402,226); Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234), Neurospora crassa [Case et al. , Proc. Nati Acad. Sci. USA 76, 5259-5263 (1979)]; and Aspergillus hosts such as A. Nidulans [Ballance et al. , Biochem. Biophys. Res. Commun. 112, 284-289 (1983); Tilburn et al. , Gene 26, 205-221 (1983); Yelton et al. , Proc. Nati Acad. Sci. USA 81, 1470-1474 (1984)] and A. niger [Kelly and Hynes, EMBO J. 4, 475-479 (1985)].

Appropriate host cells can also be derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any culture of higher eukaryotic cells is workable, either from vertebrate or invertebrate cultures, although mammalian cells like humans are preferred. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral chains and variants and permissive insect host cells corresponding to hosts such as host cells of Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melangaster (fruit fly), and Bombyx mori have been identified. See, e.g. Luckow et al. , Bio / Technology 6, 47-55 (1988); Miller et al. , in Genetic Engineering, Setlow, J.K. et al. , eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al. , Nature 315, 592-594 (1985). A variety of such viral chains are available to the public, e.g. the L-1 variant of Autographa californica NPV, and such viruses can be used as the virus described herein according to the present invention, particularly for the transfection of Spodoptera frugiperda cells.

Generally, plant cells are transfected by incubation with certain chains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain the DNA of the TIE ligand: During the incubation of the plant cell culture with A. tumefaciens, the DNA encoding the TIE ligand is transferred to the host plant cell such that it is transfected, and, under appropriate conditions, will express the DNA of the TIE ligand. In addition, signal and regulatory sequences compatible with plant cells are available, such as the nopaline tapeza promoter and the polyadenylation signal sequences. Depicker et al. , J. Mol. Appl. Gen. 1, 561 (1982). In addition, segments of DNA isolated from the upper end region of the T-DNA 780 gene are capable of activating or increasing the levels of transcription of plant-expressible genes in plant tissue containing recombinant DNA. See EP 321,196 published June 21, 1989.

However, the interest has been higher in vertebrate cells, and the propagation of vertebrate cells in culture (tissue culture) is per se well known.

See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973). Examples of useful mammalian host cell lines are the monkey kidney line CV1 transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line [293 or 293 cells subcloned for growth in suspension culture, Graham et al. , J. Gen. Virol. 36, 59 (1977)]; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR [CHO, Urlaub and Chasin, Proc. Nati Acad. Sci. USA 77, 4216 (1980)]; mouse sertoli cells [TM4, Mather, Biol. Reprod. 23, 243-251 (1980)]; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3a, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells [Mather et al. , Annals N.Y. Acad. Sci. 383, 44068 (1982)]; MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). Preferred host cells are kidney cells from human embryo 293 and from Chinese hamster ovary.

Particularly preferred host cells for the purpose of the present invention are vertebrate cells that produce the TIE homologous ligands of 6. the present invention.

The host cells are transfected and preferably transformed with the cloning or expression vectors described above and cultured in conventional nutrient media modified as is appropriate to induce selected promoters or transformants containing amplified genes.

The prokaryotic cells used to produce the TIE homologous ligands of this invention are cultured in appropriate media as generally described in Sambrook et al. , supra.

Mammalian cells can be cultured in a variety of media. Commercially available media such as Ham 's FIO (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, some of the means described in Ham and Wallace, Meth. Enzimol 58, 44 (1979): Barnes and Sato, Anal. Biochem. 102 255 (1980), US 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195 or US Pat. Re. 30,985 can be used as culture media for host cells. Some of these media can be supplemented as a requirement with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as chloride, calcium, magnesium and phosphate), buffers (such as HEPES). ), nucleosides (such as adenosine and thymidine), antibiotics (such as the drug Gentamicin ™) transmitting elements (defined as inorganic elements usually present at final concentrations in the micromolar range), and glucose or an equivalent source of energy. Some other necessary supplement may also be included in appropriate concentrations as it should be known to those skilled in the art. The culture conditions, such as temperature, pH and others, are appropriately those previously used with the host cells selected for cloning or expression, as the case may be, and will be apparent to the ordinary artisan.

The host cells referred to in this disclosure encompass cells in cell culture in vi tro as well as cells that are within a plant or host animal.

It is further envisioned that the TIE homologous ligands of this invention can be produced by homologous recombination, or with recombinant production methods using control elements introduced into cells already containing DNA encoding the particular TIE ligand.

Amplification and / or gene expression can be limited in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Nati Acad. Sci. USA 77, 5201-5205 (1980)], dot blotting (DNA analysis), or hybridization in itself, using an appropriately labeled test, based on the sequences provided herein. Several markers can be used, most commonly radioisotopes, particularly 32t However, other techniques can also be employed, such as the use of biotin-modified nucleotides for introduction into a polynucleotide. Biotin then serves as a site for avidin binding or antibodies, which may be labeled with a wide variety of markers, such as radionuclides, fluorescers, enzymes and others. Alternatively, antibodies that can be employed can recognize specific duplicates, including duplicates of DNA, duplicates of RNA, and duplicates of DNA-RNA hybrids or duplicate DNA proteins. The antibodies can instead be labeled and the assay can be carried out where the duplicate is bound to the surface, so that through duplicate formation on the surface, the presence of the antibody bound to the duplicate can be detected.

The expression of the gene, alternatively, can be calculated by immunological methods, such as immunohistochemical staining of tissue sections and assay of cell culture or body fluids, which directly quantify the expression of product of the gene. With immunohistochemical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction with specific antibodies labeled for the matched gene product, where the markers are usually visibly detectable, such as enzymatic labels, fluorescent labels, markers luminescent, and others. A particularly sensitive staining technique suitable for use in the present invention is described by Hse et al. , Am. J. Clin. Pharm. 75, 734-738 (1980).

Antibodies useful for immunohistochemical staining and / or fluid sample testing can be both monoclonal and polyclonal, and can be prepared in any animal. Conveniently, the antibodies can be prepared against a native TIE ligand polypeptide of the present invention, or against a syngeneic peptide based on the DNA sequence provided herein as described hereinafter.

The TIE homologous ligand can be produced in host cells in the form of inclusion bodies or secreted within the periplasmic space or culture medium, and is typically recovered from the host cell used. Recombinant homologous ligands can be purified by any technique taken into account for the subsequent formation of a stable protein.

When the TIE homolog ligand is expressed in a recombinant cell other than one of human origin, it is completely free of proteins or polypeptides of human origin. However, this is necessary to purify the TIE homologous ligand of recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous with respect to the ligand. As a first step, the culture medium or lysate is centrifuged to remove the cell waste particles. The membrane and the soluble protein fractions are then separated. The TIE homologous ligand can then be purified from the soluble protein fraction. The following procedures are exemplary of appropriate purification procedures: fractionation on immunoaffinity columns or ion exchange; ethanol precipitation; Reverse phase HPLC; chromatography on silica or cation exchange resin such as DEAE; chromatofocus SDS-PAGE; precipitation in ammonium sulfate; gel filtration using, for example, 'Sephadex G-75; and Protein A Sepharose columns to remove contaminants such as IgG.

Functional derivatives of the TIE homologous ligands in whose residues have been, deleted, inserted and / or substituted are recovered in the same manner as the native ligands, taking into account any of the substantial changes in the properties caused by the alteration. For example, fusion of the TIE homolog ligand with another protein or polypeptide, e.g. a viral or bacterial antigen, facilitates purification; an immunoaffinity column containing antibody to the antigen can be used to absorb the fusion. Immunoaffinity columns such as a polyclonal rabbit anti-ligand homologous TIE column can be employed to absorb variants of the TIE homologous ligand by ligation to at least one surplus immune epitope. A protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may also be useful for inhibiting proteolytic degradation during purification, and antibiotics may be included to prevent the growth of foreign contaminants. The TIE homologous ligands of the present invention are conveniently purified by chromatographic affinity, based on their ability to bind to a TIE receptor, e.g. TIE-2.

A skilled in the art will appreciate that appropriate purification methods for the native TIE homologous ligand may require modification to account for changes in the character of a native TIE homologous ligand or these variants in expression in cell culture recombinant.

D. USE OF TIE HOMOLOGOUS LIGANDS, NUCLEIC ACID MOLECULES AND ANTIBODIES The TIE homologous ligands of the present invention were expected to be useful in promoting the survival and / or growth and / or differentiation of cells expressing TIE receptor in cell culture.

The TIE homologous ligands can be used additionally to identify cells which express native TIE receptors. For this purpose, a detectably labeled ligand is contacted with a target cell under conditions that allow this binding to these receptors (TIE receptors), and the ligation is monitored.

The TIE homologous ligands herein can also be used to identify molecules exhibiting a biological activity of a TIE homologous ligand, for example, by exposing a cell expressing a TIE homologous ligand here for a test molecule, and detecting the specific ligation. of the test molecule for a TIE receptor, either by direct detection, or based on secondary biological effects. This approach is particularly suitable for identifying new members of the TIE ligand family, or for projection of small molecule libraries of peptides or non-peptides.

The TIE homologous ligands reported herein are also useful in the screening of assays designed to identify agonists and antagonists of a native TIE homologous ligand receptor that plays an important role in bone development, maturation or growth, or growth of the muscle or development and / or promotion or inhibition of angiogenesis. For example, antagonists of a TIE receptor can be identified based on their ability to block ligation of a TIE homologous ligand of the present invention to a native TIE receptor, as calculated, for example, by use of TIE receptor technology. BiAcore biosensor (BIAcore, Pharmacia Biosensor, Midscataway, NJ); or by monitoring their abilities to block the biological response caused by an even biologically active TIE homologous ligand. Biological responses that can be monitored include, for example, the phosphorylation of a TIE receptor or components felt down the path of "TIE signal transduction, or survival, growth or differentiation of cells expressing the TIE receptor. Cell-based, using cells that do not normally produce the TIE receptor, manufactured to express this receptor, or to co-express the TIE receptor and TIE homologous ligand of the present invention, are particularly convenient for use.

In a particular embodiment, the small agonist molecules and antagonists of a native TIE receptor can be identified, based on their ability to interfere with the TIE / TIE receptor ligand interaction. There are numerous ways to measure the specific binding of a test molecule to a TIE receptor, including, but not limited to detecting or measuring the amount of test molecules bound to the surface of the intact cell by expressing the TIE receptor, crosslinked to the TIE receptor. TIE receptor in cells Used, or bound to the TIE receptor in vi tro.

The detectably labeled TIE homologous ligands include, for example, TIE homologous ligands covalently or non-covalently bound to a radioactive substance, eg125I, a fluorescent substance, a substance having enzymatic activity (preferably suitable for colorimetric detection), a substrate for an enzyme (preferably suitable for colorimetric detection), or a substance that can be recognized by an antibody molecule (detectably labeled).

The assays of the present invention can be performed in a manner similar to those described in PCT publication WO 96/11269, published April 18, 1996.

The TIE homologous ligands of the present invention are also useful for purification of TIE receptors, optionally used in the form of immunoadhesins, in which the TIE ligand or the TIE receptor bound to the portion thereof is fused to a heavy chain or light of the immunoglobulin constant region.

In addition, new TIE homologous ligands can even be used to promote neovascularization, and may be useful for inhibiting tumor growth.

The nucleic acid molecules of the present invention are useful for detecting the expression of TIE homologous ligands in cells or tissue sections. The cells or tissue sections may be in contact with a detectable labeled nucleic acid molecule encoding a TIE ligand of the present invention under hybridization conditions, and the presence of hybridized mRNA for the determined nucleic acid molecule, thereby detecting the expression of the TIE ligand.

The antibodies of the present invention can, for example, be used in immunological assays to measure the amount of a TIE ligand in a biological sample. The biological sample is contacted with an antibody or antibody mixture specifically bound to the TIE ligand of the present invention, and the amount of the complex formed with a ligand present in the test sample is calculated.

Antibodies to the TIE homologous ligands here can be used additionally for the transfer of cytotoxic molecules, e.g. radioisotopes or toxins, or therapeutic agents for cells expressing a corresponding TAR receptor. The therapeutic agents may, for example, be other TIE homologous ligands, including the TIE-2 ligand, members of a vascular endothelial growth factor (VEGF) family, or known anti-tumor agents, and agents known to be associated with the growth or development of the muscle, or bone development, maturation, or growth.

TIE-homologous anti-ligand antibodies are also suitable as diagnostic agents for detecting disease states associated with the expression of a TIE receptor (e.g., TIE-2). Thus, detectably labeled TIE homologous ligands and agonist antibodies of a TAR receptor can be used to reflect the presence of angiogenesis.

TIE homologous anti-ligand antibodies specifically anti-NL6 antibodies can also find utility as anti-inflammatory agents.

For therapeutic use, the TIE homologous ligands or TIE anti-ligand antibodies of the present invention are formulated as a therapeutic composition comprising the active ingredient (s) and mixed with a pharmacologically acceptable carrier., suitable for topical or systemic application. The pharmaceutical compositions of the present invention are prepared for storage by mixing the active ingredient having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition Osol, A. Ed. (1980)), form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to patients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid, low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone, amino acids such as glycine, glutamine, aspargin, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt formers such as sodium; and / or nonionic surfactants such as Tween, Pluronics or PEG.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation or interfacial polymerization techniques, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmetacylate) microcapsules respectively; in colloidal drug transfer systems (for example liposomes, albumin microspheres, microemulsion, nanoparticles and nanocapsules) or in macroeulsion. Such techniques are stated in Remington's Pharmaceutical Sciences, supra.

Formulations to be used for in vi ve administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes, prior to or followed by lyophilization and reconstitution.

The therapeutic compositions herein are generally placed within a container having a sterile access port, for example an intravenous solution bag or bottle having a plug penetrable by an injection of hypodermic needle.

The route of administration is according to known methods, e.g. by injection or intravenous infusion, intraperitoneal, intracerebral, intramuscular, intraocular, intrarterial or intralesional routes, topical administration, or by sustained release systems.

Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of adapted articles e.g. films, or microcapsules. Sustained-release matrices include polyester, hydrogels, polylactides (US Patent 3,773,919, EP 58,881), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al., 1983, "Biopolymers" 22 (1) : 547-556), poly (2-hydroxyethyl-methacrylate) (R. Langer, et al., 1981, "J. Biomed, Mater. Res." 15: 167-277 and R. Langer, 1982, Chem. Tech. "12: 98-105), ethylene vinyl acetate (R. Langer et al., Id) or poly-D- (-) -3-hydroxybutyric acid (EP 133, 988A)." Sustained-release compositions also include liposomes. Liposomes containing a molecule within the scope of the present invention are prepared by methods known per se: DE 3,218,121A; Epstein et al., 1985, "Proc. Nati. Acad. Sci. USA" 82: 3688-3692; Hwang et al., 1980, "Proc. Nati. Acad. Sci. USA" 77: 4030-4034; EP 52322A; EP 36676A; EP88046A; EP143949A; EP 142641A; Japanese patent application 83-118008; US Patents 4,485,045 and 4,544,545; and EP 102,324A. Ordinarily the liposomes are of the small unilamellar type (about 200-800 Angstroms) in which the lipid content is larger than about 30 mol. % cholesterol, with the selected proportion adjusted for optimal NT-4 therapy.

An effective amount of the molecule of the present invention to be employed therapeutically will depend, for example, on the therapeutic objectives, the route of administration and the condition of the patient. Therefore, it will be necessary for the therapist to titrate the dose and modify the route of administration as required to obtain the optimal therapeutic effect. A typical mid-range daily dose of around lμg / kg to more than 100 μg / kg or more, depending on the factors mentioned above. Typically the clinician will administer a molecule of the present invention until a dose is achieved that provides the required biological effect. The progress of this therapy is easily monitored by conventional tests.

If the therapeutic field is to prevent or treat tumors, the compounds here can be combined with other therapies. For example, the patient to be treated with such anti-cancer agents can also receive radiation therapy. Alternatively, or in addition, a chemotherapeutic agent can be administered to the patient. Dosage preparations and schedules for such chemotherapeutic agents may be used according to the manufacturer's instructions or as determined empirically by licensed practitioners. Dosage preparations and schedules for such chemotherapeutics are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992). The chemotherapeutic agent may precede or follow the administration of the anti-tumor agent, or may be concurrently with it.

This may also be desirable for the administration of antibodies against other associated tumor antigens, such as antibodies which bind to ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor (VEGF). Alternatively, or in addition, two or more antibodies binding the same or two or more different antigens stated herein may be coadministered to the patient. Sometimes, this may be beneficial to also administer one or more cytokines to the patient. In a preferred embodiment, the anti-tumor compounds are even administered with a growth inhibitory agent below.

For the prevention or treatment of disease, the appropriate dose of an anti-tumor agent, e.g. an antibody here will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, prior therapy, patient's medical history and response to the agent, and desire of the therapist. The agent is appropriately administered to the patient at a time or under a series of treatments.

Details of the invention will be apparent below from the following non-limiting examples.

Example 1 Identification of the FLS139 ligand FLS139 was identified in a cDNA library prepared from human fetal liver mRNA obtained from Clontech Laboratories, Inc. Palo Alto, CA USA, catalog no. 64018-1, following the protocol described in "Instruction Manual: Superscript® Lambda System for cDNA Synthesis and? Cloning," cat. No. 19643-014, Life Technologies, Gaithersburg, MD, USA which is incorporated herein by reference. Unless otherwise noted, all reagents were also obtained from Life Technologies. The complete procedure can be summarized in the following stages: (1) Synthesis of the first chain; (2) Synthesis of the second chain; (3) Adding the adapter; (4) Enzymatic digestion; (5) Isolation of gel cDNA; (6) Ligation within the vector; and (7) Transformation.

Synthesis of the first chain or strand: The Not-1 adapter (Life Tech., 2 μl, 0.5 μg / μl) was added to a sterile 1.5 ml microcentrifuge tube to which poly A + MARN (7μl, 5μg) was added. The reaction tube was heated at 70 ° C for 5 minutes or enough time to denature the secondary structure of the mRNA. The reaction was then cooled in ice and 5X first-chain buffer (Life Tech., 4 μl), 0.1 M DTT (2 μl) and 10 mM dNTP Mix were added.

(Life Tech., 1 μl) and then heated at 37 ° C for 2 minutes to equilibrate the temperature. The Superscript II® reverse transcriptase (Life Tech 5 μl) was then added, the reaction of the tube well mixed and incubated at 37 ° C for one hour, and terminated by placing on ice. The final concentration of the reagents was as follows: 50 mM Trist-HCL (pH 8.3); 75 piM KCl; 3mM MgCl 2; 10 mM DTT; 500 μM to each dATP, dCTP, dGTP and dTTP; 50 μg / ml Not 1 adapter-primer; 5 μg (250 μg / ml) of mRNA; 50,000 U / ml of Superscript II® reverse transcriptase.

Synthesis of the second chain or strand: In ice, the following reagents were added for the reaction in the first chain synthesis tube, the reaction well mixed and left to react at 16 ° C for 2 hours, taking care not to allow the temperature to fall below 16 ° C: distilled water (93 μl); second chain buffer 5X (30 μl); dNTP mix (3μl); 10 U / μl of E. Coli DNA ligase (1 μl); 10 U / μl of DNA polymerase I from E. Coli (4 μl); 2 U / μl of RN asa H from E. Coli (1 μl). 10 U T4 of DNA polymerase (2 μl) and the continued reaction to incubate at 16 ° C for another 5 minutes were added. The final concentration of the reaction was as follows: 25 mM Tris-HCl (pH 7.5); 100 mM KCl; 5 mM MgCl2; 10 mM (NH4) 2S04; 0.15 M ß-NAD +; 250 μM of each dATP, dCTP, dGTP, dTTP; 1.2 mM DTT; 65 U / ml of DNA ligase; 250 U / ml of DNA polymerase I; 13 U / ml of Rnasa H. The reaction has stopped by placing on ice and by adding 0.5 M EDTA (10 μl), then extracted through phenol: chloroform: isoaminoalcohol (25: 24: 1, 150 μl). The aqueous phase was removed, collected and diluted in 5 M NaCl (15 μl) and absolute ethanol (-20 ° C, 400 μl) and centrifuged for 2 minutes at 14,000 x g. The supernatant was carefully removed from the resulting DNA conglomerate, the conglomerate resuspended in 70% ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 x g. The supernatant was again removed and the conglomerate dried in a dryer.

Adding the adapter The following reagents were added to the cDNA conglomerate of the second chain synthesis from before, and the reaction was gently mixed and incubated at 16 ° C for 16 hours: distilled water (25 μl); 5X T4 DNA ligase buffer (10 μl); Adapters Sal I (10 μl); T4 DNA ligase (5 μl). The final composition of the reaction was as follows: 50 mM Tris-HCl (pH 7.6); 10 M mgCl2; IMM of ATP; 5% (w / v) of PEG 800; 1 mM DTT; 200 μg / ml Adapters Salt I; 100 U / ml of T4 DNA ligase. The reaction was extracted through phenol: chloroform: isoaminoglycol (25: 24: 1, 50 μl), the aqueous phase removed, collected and diluted in 5 M NaCl (8 μl) and absolute ethanol (-20 ° C, 250 μl ). This was centrifuged for 20 minutes at 14,000 x g, the supernatant removed and the conglomerate was resuspended in 0.5 ml of 70% ethanol, and centrifuged again for 2 minutes at 14,000 x g. Subsequently, the supernatant was removed and the resulting conglomerate dried in a dryer and used in the following procedure.

Enzymatic digestion; The following reagents were added to the cDNA prepared with the adapter Sal I of the previous paragraph and the mixture was incubated at 37 ° C for 2 hours: treated water-DEPC (41 μl); Not 1 restriction buffer (REACT, Life Tech., 5 μl), Not 1 (4 μl). The final composition of this reaction was as follows: 50mM Tris-HCl (pH 8.0); 10 mM MgCl2; 100 mM MaCl; 1,200 U / ml of Not 1.

Isolation of gel cDNA: The cDNA in size fractionated by acrylamide gel electrophoresis on a 5% acrylamide gel, and some fragments which were longer than 1 Kb, as determined by comparison with a molecular weight marker, were extracted from the gel. The cDNA was then electroextracted from the gel in 0.1 of buffer x TBE (200 μl) and extracted with phenol: chloroform: isoamylalcohol (25: 24: 1, 200 μl). The aqueous phase was removed, collected and centrifuged for 20 minutes at 14,000 x g. The supernatant was removed from the DNA conglomerate which was resuspended in 70% ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 x g. The supernatant was again discarded, the conglomerate dried in a dryer and resuspended in distilled water (15 μl).

Ligation of the cDNA within the vector pRK5: The following reagents were added together and incubated at 16 ° C for 16 hours; 5X T4 ligase buffer (3 μl); digested vector pRK5, Xhol, Not 1 (0.5 μg, 1 μl); the cDNA prepared from the previous paragraphs (5 μl) and distilled water (6 μl). Subsequently, additional distilled water (70 μl) and 10 mg / ml of tRNA (0.1 μl) were added and the entire reaction was extracted through phenol: chloroform: isoamylalcohol (25: 24: 1). The aqueous phase was removed, collected and diluted in 5 M NaCl (10 μl) and absolute ethanol (-20 ° C, 250 μl). This was then centrifuged for 20 minutes at 14,000 x g, decanted, and the conglomerate resuspended in 70% ethanol (0.5 ml) and centrifuged again for 2 minutes at 14,000 x g. The DNA conglomerate was then dried in (...) and washed in distilled water (3 μl) for use in the subsequent procedure.

Transformation of the ligation library into bacteria: The previously prepared ligated cDNA / pRK5 DNA vector was cooled in ice, for which an additional 1 was added. elctrocompetent DH10B bacteria (Life Tech., 20 μl). The mixture of the vector bacterium was then electrophoresed according to the recommendations of the manufacturers. Subsequently the SOC medium (1 ml) was added and the mixture was incubated at 37 ° C for 30 minutes: The transformants were then placed in 20 standard plates 150 mm LB containing ampicillin and incubated for 16 hours (37 ° C) to allow the growth of the colonies. The positive colonies were then removed by scraping and the DNA isolated from the conglomerate using standard CsCl gradient protocols. For example, Ausubel et al., 2.3.1.

Identification of FLS139 FLS139 can be identified in a human fetal liver library by any standard method known in the state of the art, including the methods reported by Klein R.D. et al. (1996), Proc. Nati Acad. Sci. 93, 7108-7113 and Jacobs (U.S. Patent No. 5,563,637 published July 16, 1996). According to Klein et al. And Jacobs, cDNAs encoding novel secreted proteins and mammalian membrane binding proteins are identified by detection of their leader secretory sequences using a yeast invertase gene as a reporter system. The reverse enzyme catalyzes the decomposition of sucrose to glucose and fructose as well as the decomposition of raffinose to sucrose and melibiose. The secreted form of invertase is required for the use of sucrose by yeast. { Saccharomyces cerevisiae) so that yeast cells that are unable to produce secreted invertase grow poorly in media containing sucrose as the sole source of energy and carbon. Both Klein R. D., supra and Jacobs, supra take advantage of the known ability of mammalian signal sequences to functionally replace the native sequence signal of the yeast invertase. A mammalian cDNA library is linked to a DNA encoding a non-secreted yeast invertase, the ligated DNA is isolated and transformed into yeast cells that do not contain an invertase gene. Recombinants containing the invertase gene of unseen yeast bound to a mammalian signal sequence are identified based on their ability to grow in a medium containing only sucrose or only raffinose as a carbon source. The identified mammalian signal sequences are then used to cover a second, full-length DNA library to isolate the full-length clones by coding the corresponding secreted proteins. The clone can, for example, be manufactured by expression of the clone or by any other technique known in the state of the art.

The primers used for the identification of FL139 are the following: OLÍ11 CCACGTTGGCTTGAAATTGA SEC. ID.NO: 13 OLÍ115 CCTCCAGAATTGATCAAGACAATTCATGATTTGATTCTCTATCTCCAGAG SEC. ID. O: 14 OLI116 TCGTCTAACATAGCAAATC SEC. ID.NO: 15 The nucleotide sequence of FLS139 is shown in Figure 6 (SEQ ID No. O), while this amino acid sequence is shown in Figure 7 (SEQ ID NO: 6). As illustrated in Figure 1, FLS139 contains a fibrinogen as a domain exhibiting a high degree of sequence homology with the two known human ligands of the TIE-2 receptor (h-TIE2L1 and h-TIE2L2). Consequently, FLS139 has been identified as a novel member of the TIE ligand family.

A clone of FLS139 was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, on September 18, 1997 under the terms of the Budapest Treaty, and has been assigned the deposit number ATCC 209281.

Example 2 Identification of NL2 and NL3 NL2 and NL3 were the GenBank database through the coverage using the BLAST computer program (Altshul et al., Methods in Enzymology 266: 460-480 (1996) .The NL2 sequence shows homology with the known EST sequences T08223, AA122061, and M62290. Similarly, NL3 shows homology with the known EST sequences T57280, and T50719. None of the known EST sequences have been identified as full length sequences, or described as ligands associated with the TIE receptors.

Following their identification, NL2 and NL3 were cloned from a human fetal lung library prepared from mRNA obtained from Clontech, Inc. (Palo Alto CA, USA), catalog # 6528-1, following the manufacturers' instructions. The library was covered by hybridization with synthetic oligonucleotide tests: For NL2: NL2, 5-1 ATGAGGTGGCCAAGCCTGCCCGAAGAAAGAGGC SEC. ID.NO: 7 NL2, 3-1 CAACTGGCTGGGCCATCTCGGGCAGCCTCTTTCTTCGGG SEC. ID.NO: 8 NL2,3-4 CCCAGCCAGAACTCGCCGTGGGGA SEC.ID.N0: 9 For NL3: NL3,5-1 TGGTTGGCAAAGGCAAGGTGGCTGACGATCCGG SEC.ID.NO:10 NL3,3-1GTGGCCCTTATCTCTCCTGTACAGCTTCCGGATCGTCAGCAC SEC.ID.NO.il NL3,3-2 TCCATTCCCACCTATGACGCTGACCCA SEC. ID. O: 12 Based on the ESTs found in the GenBank database. The cDNA sequences were sequenced in their entirety.

The nucleotide and amino acid sequences of NL2 are shown in Figure 2 (SEQ ID NO: 1) and Figure 3 (SEQ ID NO: 2), respectively. The nucleotide and amino acid sequences of NL3 are shown in Figure 4 (SEQ ID O: 3) and Figure 5 (SEQ ID NO: 4), respectively.

A clone of NL2 (NL2-DNA 22780-1078) was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, on September 18, 1997 under the terms of the Budapest Treaty, and has been assigned the ATCC deposit number 209284.

A clone of NL3 was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, on September 18, 1997 under the terms of the Budapest Treaty, and has been assigned the deposit number ATCC 209283.

Example 3 Northern blot analysis and in situ hybridization results The expression of FLS139, NL2 and NL3 mRNA in human tissues was examined by Northern Blot analysis. The spots of human mRNA were hybridized for a 32 P-labeled DNA test based on the total length of cDNAs; The tests were generated by digestion and by purifying the cDNA inserts, MTN human fetal RNA spots (Clontech) and adult human MTN-II RNA stains (Clontech) were incubated with DNA tests. The spots were incubated with the tests in hybridization buffer (5x SSPE, 2Denhardt solution, 100mg / mL Denatured cut salmon sperm DNA, 50% formamide, 2% SDS) for 60 hours at 42 ° C. spots were washed several times in 2x SSC; 0.05% SDS for one hour at room temperature, followed by a 30 minute wash in O.lx SSC; 0.1% SDS at 50 ° C. The spots were developed after exposure during the night by phosphoprojection (Fuji) analysis.

As shown in Figures 8 and 9, the transcribed mRNAs of NL2 and NL3 were detected.

The tissue expression model of NL3 was also determined by in situ hybridization (observing hybridization to cellular RNA), using an optimized protocol employing 33P-labeled PCR-generated riboprots. (Lu and Gillett, Cell Vision 1; 169-176 (1994)). Fetal tissues and human adults prepared in formalin, fixed in paraffin were sectioned, deparaffinized, deproteinized in proteinase K (20 g / ml) for 15 minutes at 37 ° C, and later processed by in situ hybridization as described by Lu and Gillet (1994). ). A marked [33-P] UTP counter-sense riboprobe was generated from a PCR product and hybridized at 55 ° C overnight. The slides were immersed in Kodak nuclear stamp emulsion NTB2 and exposed for 4 weeks.

Synthesis of Riboprueba? P 6. 0 μl (125 mCi) of 33P-UTP (Amersham BF 1002, SA <2000 Ci / mmol) were dried by centrifugation. For each tube containing dried 33P-UTP, the following ingredients were added: 2.0 μl 5x transcription buffer 1.0 μl DTT (100mM) 2.0 μl NTP mixture (2.5 mM: 10 μl, each 10 mM GTP, CTP and ATP + 10 μl H20) 1.0 μl of UTP (50 μM) 1.0 μl of Rnasin 1.0 μl of DNA template (1 μg) 1.0 μl of H20. 1.0 μl of RNA polymerase (for PCR products T3 = AS, T7 = S, usually) The tubes were incubated at 37 ° C for one hour. 1.0 μl of RQ1 Dnasa were added, followed by incubation at 37 ° C for 15 minutes. 90 μl of TE (10 mM Tris pH 7.6 / 1 mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The leftover solution was stored in a Microcon-50 ultrafiltration unit, and stirred using program 10 (6 minutes). The filtration unit was poured into a second tube and stirred using program 2 (3 minutes). After the final recovery lap, 100 μl of TE was added. 1 μl of the final product was pipetted on DE81 paper and contained in 6 ml of Biofluor II.

The test was run on a urea / TBE gel. 1-3 μl of the test or 5 μl of RNA Mrk III were added for 3 μl of transporter buffer. After heating in a heater at 95 ° C for three minutes, the gel was immediately placed on ice. The gel wells were washed, the sample stored, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifier projector in freezing at -70 ° C one hour overnight.

P hybridization Pretreatment of frozen sections The cuts were removed from the freezer, placed in aluminum trays and thawed at room temperature for five minutes. The trays were placed in an incubator at 55 ° C for five minutes to reduce condensation. The slices were fixed for ten minutes in 4% paraformaldehyde in ice on the steam cover, and washed in 0.5 x SSC for five minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ H20). After deproteinization in 0.5 μg / μl of proteinase K for ten minutes at 37 ° C (12.5 μl of 10 μg / μl base in 250 μl of RNase-free RNase buffer preheated), the sections were washed in 0.5 x SSC at ten minutes at room temperature. The sections were dehydrated in 70% ethanol, 95%, 100%, two minutes each.

Pretreatment of paraffin submerged sections The sections were deparaffinized, placed in SQ H20, and rinsed twice in 2 x SSC at room temperature for five minutes each time. Sections were deproteinized in 20 μg / μl of human embryo K proteinase (500 μl of 10 μg / μl in 250 μl of RNase-free RNase buffer at 37 ° C, 15 minutes), or 8 x proteinase (100 μl in 250 ml of Rnasa buffer, at 37 ° C, 30 minutes) -made of formalin. Subsequent rinsings in 0. 5 x SSC and dehydration were performed as described above.

Prehybridization The sections were extended in plastic boxes filled with box buffer (4 x SSC, 50% formamide) saturated filter paper. The tissue was covered with 50 μl of hybridization buffer (3.75 g of Dextran Sulfate + 6 ml SQ H20), vortexed and heated in the microwave for two minutes with the cover loose. After cooling on ice, 18.75 ml of formamide, 3.75 ml of 20 x SSC and 9 ml of SQ H20 were added, the tissues well vortexed, and incubated at 42 ° C for 1-4 hours.

Hybridization Test of 1.0 x 106 cpm and 1.0 μl of tRNA (50 g / μl base) per cut were heated at 95 ° C for three minutes. The slices were frozen in ice, and 48 μl of hybridization buffer per cut was added. After vortexing, 50 μl of 33P mixture was added to 50 μl of prehybridization in the cut. The sections were incubated overnight at 57 ° C.

Washes The washes were done 2x ten minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC + 16 ml 0.25 M EDTA, Vf = 4L), followed by a RNase treatment at 37 ° C for thirty minutes (500 μl) of 10 g / ml in 250 ml of RNase buffer = 20 μg / ml), the sections were washed 2 x 10 minutes with 2 x SSC, EDTA at room temperature, the strict washing conditions were as follows: two hours at 55 ° C , 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, Vf = 4L).

Oligos: C-141F- NL3pl: 48mer GGA TTC TAA TAC GAC TCA CTA TAG GGC AAG TTG TCC TCC (SEQ ID NO: 16) C-141G- NL3p2: 47mer CTA TGA AAT TAA CCC TCA CTA AAG GGA CGT GGT CAG CGT (SEQ ID NO: 17) The adult tissues examined were: liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex, hippocampus, cerebellum, penis, eye, bladder, stomach, gastric carcinoma, colon, colon carcinoma and chondrosarcoma, injured liver induced by acetaminophen and liver cirrhosis. The - Fetal tissues examined were placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, large spleen, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and lower member. The expression was not observed in any of the normal or fetal tissues. The expression was detected in hepatic sinusoid cells (probably endothelial cells) in both acute liver injury (induced by acetabulum acetaminophen) and chronic (cirrhosis and adjacent metastasis to colorectal carcinoma). These results indicate that NL3 may play a role in the regulation of liver regeneration.

The expression of NL1 was also examined in a similar order of adult and fetal tissues but was not observed expressed under the conditions indicated above.

Example 4 Expression of FLS139, NL2 and NL3 in E. coli This example illustrates the preparation of a non-glycosylated form of the TIE homologous ligands of the present invention in E. coli. The DNA sequence encoding an NL-2, NL-3, or FLS139 ligand (SEC.ID.NOs: 1.3, and 5, respectively) is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites in the selected expression vector. A variety of expression vectors can be employed. The expression vector will preferably encode an antibiotic resistance gene, an origin of replication, a promoter, and a ribosome binding site. An example of a convenient vector is pBR322 (derived from E. coli; see Bolivar et al., Gene 2:95 (1977)) which contain genes for ampicillin and tetracycline resistance. The vector is degested with restriction enzyme and dephosphorylated the amplified PCR sequences are then ligated into the vector.

The ligation mixture is then used to transform a selected E. coli chain, using the methods described in Sambrook et al., Supra. Transformants are identified by their ability to grow in LB dishes and antibiotic resistant colonies are then selected. The plasmid DNA can be isolated and confirmed by restriction analysis.

The selected clones can be grown overnight in liquid culture medium-such as IB broth supplemented with antibiotics. The overnight culture can be used subsequently to inoculate a scale culture later. The cells are then grown to a desired optical density. An inductor, such as IPTG, can be added.

After culturing the cells for a few more hours, the cells can be harvested by centrifugation. The cell conglomerate, obtained by centrifugation, can be solubilized using various agents known in the state of the art, and the solubilized protein can then be purified using a metal chelating column under conditions that allow for close binding of the protein.

Truly, NL2 was expressed in E. coli in a poly-His labeled form, using the following procedure. The DNA encoding NL2 was initially amplified using selected PCR primers. The primers contained restriction enzyme sites which correspond to the restriction enzyme sites in the selected expression vector, and provide other profitable sequences for efficient and reliable initiation of translation, rapid purification on a metal chelation column, and Proteolytic removal with enterokinase. The amplified PCR, the labeled poly-His sequence was then ligated into an expression vector, which was used to transform a JE. chain-based host coli 52 (W3110fuhA (tonA) longalE rpoHts (htpRts) clpP (Iaclq) Transformants were first grown in LB containing 50 mg / ml carbenicillin at 30 ° C with shaking until an OD600 of 3-5 was The cultures were then diluted 50-100 covered in Crap medium (prepared by mixing 3.57 g (NH) 2S) 4.0.71 g of 2H20 sodium citrate, 1.07 g of KCl, 5.36 g of Difco yeast extract, 5.36 g Hykc Sheffield SF in 500 L of water, as well as 110 mM MPOS, pH 7.3, 0.55% (w / v) glucose and 7 M MgSO4) and grown for approximately 20-30 hours at 30 ° C with shaking. removed to verify expression by SDS-PAGE analysis, and the culture mass is centrifuged to settle the cells.The cell conglomerates were frozen until purification and refolding.

The mass of E. coli from 0.5 to 1 of fermentations L (6-10 g of conglomerates) was resuspended in 10 volumes (p / v) in 7 M guanidine, 20 mM Tris buffer, pH 8. Sodium sodium sulfite and tetrathionate sodium were added making final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4 ° C. This step results in a denatured protein with all the cysteine residues blocked by sulfitolization. The solution was centrifuged at 40,000 rpm in a Beckman ultracentrifuge for 30 minutes. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 M Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. Depending on the clarified extract it was stored on a Qiagen Ni-NTA chelating metal column balanced on the chelating metal column buffer. The column was washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was washed with buffer containing 250 mM of imidazole. The fractions containing the desired protein were combined and stored at 4 ° C. The protein concentration was estimated by absorbance at 280 nm using the extinction coefficient based on these amino acid sequences.

The protein was refolded by diluting the mixture slowly in freshly prepared refolded buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and lmM EDTA. Refolding volumes were chosen such that in the end the protein concentration was between 50 and 100 micrograms / ml. The refolding solution was gently shaken at 4 ° C for 12-36 hours. The refolding reaction was suppressed by adding TFA to a final concentration of 0.4% (pH of about 3). Before further purification of the protein, the solution was filtered through a 0.22 micron filter and added to acetonitrile for a final concentration of 2-10%. The refolded protein was chromatographed on a Poros Rl / H reversed phase column using a mobile shock absorber of 0.1% TFA with elution with an acetonitrile gradient of 10 to 80%. The aliquots of fractions with A280 absorbance were analyzed in SDS polyacrylamide gels and fractions containing homogeneous refolded protein were combined. Usually, -the adequately refolded species of more proteins are washed at the lowest concentration of acetonitrile than those species that are the most compact with their hydrophobic interiors protected from interaction with the reverse phase resin. The agreed species are usually washed at high concentrations of acetonitrile. In addition to transforming the unfolded forms of proteins of the desired form, the reverse phase step also removes endotoxin from the samples.

The fractions containing the desired NL2 folded protein were mixed and the acetonitrile removed using a moderate stream of nitrogen directed to the solution. Proteins were formulated in 20mM Hepes, pH 6.8 with 0.1M sodium chloride and 4% mannitol by dialysis or gel filtration using GF Superfine resins (pharmacy) balanced in the buffer formulation and sterile filtrate.

Example 5 Expression of FLS139, NL 2 and NL 3 in mammalian cells This example illustrates the preparation of a glycosylated form of the homologous ligands FLS139, NL2 and NL3 by recombinant expression in mammalian cells.

The vector, pRK5 (see EP307, 247, published March 15, 1989), is employed as the expression vector. Optionally, DNA FLS139, NL2 and NL3 using ligation methods such as described in Sambrook et al., Supra. The resulting vector is called pRK5-FLS 139, NL2 and NL3, respectively.

In one embodiment, the selected host cells can be 293 cells. Human 293 cells (ATCC CCL 1573) are developed by confluence in tissue culture dishes in media such as DMEM supplemented with fetal calf serum and optionally, nutritive components and / or antibiotics About 10 μg of pRK5-FLS139 DNA, NL2 and -NL3 is mixed with about 1 μg of DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31: 543 (1982)] and dissolved in 500 μl of Tris-HCl of 1 mM, 0.1 mM of EDTA, 0.227 M of CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 M NaP04, and a precipitate is left to form for 10 minutes at 25 ° C. The precipitate is suspended and added to the 293 cells and left to rest for about 4 hrs at 37 ° C. The culture medium is aspirated and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium. Fresh medium is added and the cells are incubated for about 5 days.

Approximately 24 hours after transfection, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi / ml 35S-cysteine and 200 μCi / ml of 35S-methionine. After 12 hours of incubation, the conditioned medium is collected, concentrated on a rotary filter, and stored in a 15% SDS gel. The processed gel can be dried and exposed to film for a selected period of time to reveal the presence of FLS139, NL2 and NL3 polypeptide. Cultures containing transfected cells can be incubated further (in serum-free medium) and the medium is tested in selected bioassays. In an alternative technique, FLS139, NL2 and NL3 can be introduced into 293 cells temporarily using the dextran sulfate method described by Somparyrac et al., Proc. Nati.Acad. Sci., 12: 7575 (1981) . The 293 cells are grown for maximum density in a rotating flask and 700 μg of DNA pRK5-FLS139, -NL2 and -NL3 are added. The cells are first concentrated from the spinning flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated in the cell conglomerate for 4 hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and reintroduced into the rotating flask containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After about four days, the conditioned medium is centrifuged and filtered to remove cells and debris. The sample containing the expressed FLS139, NL2 and NL3 can then be concentrated and purified by any selected method such as dialysis and / or column chromatography.

In another embodiment, FLS139, NL2 and NL3 can be expressed in CHO cells. PRK5-FLS139, -NL2 and -NL3 can be transfected into CHO cells using known reagents such as CaP04 or DEAE-dextran. As written before, cell cultures can be concealed, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 35 S-methionine. After determining the presence of FLS139, NL2 and NL3 polypeptide, the culture medium can be replaced with serum-free medium. Preferably, the cultures are incubated for about six days, and then the conditioned medium is harvested. The medium containing the expressed FLS139, NL2 and NL3 can then be concentrated by any selected method.

The epitope tagged FLS139, NL2 and NL3 can also be expressed in CHO host cells. FLS139, NL2 and NL3 can be subcloned out of the pRK5 vector. The inserted subclone can be subjected to PCR to fuse in shape with a selected epitope fragment such as a poly-his fragment within a Baculovirus expression vector. The poly-his labeled FLS139, NL2 and NL3 insert can then be subcloned into a boosted SB40 vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SB40 driven vector. The marking can be done, as described above to verify the expression. The culture medium containing the expressed poly-his labeled FLS139, NL2 and NL3 can then be concentrated and purified by any selection method, such as affinity of Ni2 + chromatography.

The glycocylated forms of NL2, NL3 and FLS139 (NL6) were effectively expressed in CHO cells in labeled poly-his forms. Following the PCR amplification, the DNA from NL2, NL3 or NL6 was subcloned into a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). The CHO expression vectors are constructed to have compatible restriction sites 5 'and 3' of the 7th DN of interest to allow convenient elongation of the cDNAs. The vector used for the expression of NL2, NL3 or NL6 in CHO cells is as described in Lucas et al. , Nucí Acids Res. 24: 9 (1774-1779 (1996), and using the SV40 early promoter-enhancer to drive the expression of the cDNA of interest and dihydrofolate reductase (DHFR) .The DHFR expression allows selection for stable maintenance of the plasmid following the transfiction Twelve micrograms of the DNA encoding plasmid of NL2, NL3 or NL6 were introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dossier® or Fugene® (Boehringer Mannheim). The cells were grown and described in Lucas et al. , supra. Approximately 3x10 ~ 7 cells were frozen in an ampoule for later growth and comp production was described below.

The ampoule containing the DNA encoding plasmid of NL2, NL3 or NL6 was dissolved by depositing in a tub of water and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mLc of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective medium (0.2 μm with 5% filtered PS20 0.2 μm diafiltered from fetal bovine serum). The cells were then aliquoted in a 100 mL warhead containing 90 mL of selective medium. After 1-2 days, the cells were transferred in a 250 mL warhead filled with 150 L of selective growth medium and incubated at 37 ° C. After another 2-3 days, warheads of 250 mL, 500 mL and 2000. L were seeded with 3x1O5 cells / mL. They were exchanged with fresh medium by centrifugation and resuspension in production medium. Any useful CHO medium can be employed e.g., as described in U.S.P.5, 122,469 recorded on June 16, 1992. A 3L production warhead is seeded at 1.2xl06 cells / mL. On day zero, the cell number and pH were determined. On day one, the warhead was sampled and irrigation was started with filtered air. Day two, the ogive was sampled, the temperature raised to 33 ° C, and 30 mL of 500g / L-glucose and 0.6 mL of 10% antifoam were added (eg, 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade emulsion ). Throughout the production, the pH was necessarily adjusted to keep it around 7.2. After 10 days, or until after the viability above 70%, the cell culture was harvested by centrifugation and filtered through a 0.22 μ filter. The filtrate was also stored at 4 ° C until storage in a purification column.

The labeled poly-His was purified using a Ni-NTA column (Qiagen). After the purification, imidazole was added to the selective medium for a concentration of 5 mM. The selective medium was placed in a column of 6 mL Ni-NTA equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 minimolar imidazole at a rate of 4-5 mL / min at 4 ° C after When stored, the column was washed with additional equilibration buffer and the purified protein with equilibrium buffer containing 0.25 M imidazole at a circulation percentage of 4-5 ml / min at 4 ° C. After storage, the column is washed with additional equilibration buffer and the protein washed with equilibrium buffer containing 0.26 M imidazole.

The purified protein was subsequently disrupted in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine column (Pharmacia) and stored at -80 ° C.

The homogeneity of the purified proteins was confirmed by SDS PEG and by sequencing N-terminal amino acid made by Edman degradation.

Example 6 Expression of FLS139, NL2 and NL3 in yeast First, the yeast expression vectors are 1 constructed by intracellular production or secretion of FLS139, NL2 and NL3 of the ADH2 / GAPDH promoter. The DNA encoding FLS139, NL2 and NL3, a selected signal peptide and the promoter is inserted into appropriate restriction enzyme sites in the selected plasmid to direct the intracellular expression of FLS139, NL2 and NL3. For secretion, the DNA encoding FLS139, NL2 and NL3 can be cloned into the selected plasmid, along with the DNA encoding the ADH2 / GAPDH promoter, the leader / secretory sequence of yeast alpha factor, and linked sequences (if needed) ) for the expression of FLS139, NL2 and NL3.

The yeast cells, such as yeast chain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue ink.

The recombinants FLS139, NL2 and NL3 can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing FLS139, NL2 and NL3 can later be purified using selected column chromatography resins.

Example 7 Expression of FLS139, NL2 and NL3 in Insect Cells transfected with Baculovirus The following method describes the recombinant expression of FLS139, NL2 and NL3 in Baculovirus transfected in insect cells.

The FLS139, NL2 and NL3 is fused upstream of an epitope tag contained with an expression vector of Baculovirus. Such an epitope tag includes poly-his tag and immunoglobulin tag (such as IgG Fe regions). A variety of plasmids can be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). In summary, the FLS139, NL2 and NL3 of the desired portion of FLS139, NL2 and NL3 (such as the sequence encoding the extracellular domain of a trans-embryonic protein) is amplified by PCR with complementary primers for the 5 'and 3' regions. The 5 'primer can incorporate flanking restriction enzyme sites (selected). The product is then digested with those selected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfection of the above plasmid and BaculoGold ™ virus DNA (Pharmigen) in Spodoptera frugiper a ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression is performed as described by O'Reilley et al., Baculovirus espression vectors: A laboratory Manual, Oxford: Oxford UniversitY Press (1994).

The labeled poly-his of expressed FLS139, NL2 and NL3 can be purified, for example by chelation affinity chromatography Ni2 + - as follows. The extracts are prepared from virus-infected recombinant Sf9 cells as described by Rupert et al., Nature 362: 175-179 (1993). In summary, the Sf9 cells were washed, resuspended in sonication buffer (25 mL of Hepes, pH 7.9, 12.5 mM MgCl2, 0.1 mM EDTA: 10% glycerol, 0.1% NP-40, 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are clarified by centrifugation, and the supernatant is diluted in 50 parts of buffered storage (50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni2 + -NTA agarose column (commercially available from Qiagen) is prepared with a base volume of 5 L, washed with 25 mL of water and equilibrated with 25 mL of storage buffer. The filtered cell extract is stored in the column at 0.5 mL per minute. The column is washed to basic line A280 with storage buffer, to which fraction point collection is started. The column is then washed with a secondary wash buffer (50 mM phosphate, 300 mM-NaCl, 10% Glycerol, pH 6.0), which washes non-specifically bound proteins. After extending the basic line 2so again, the column is developed with 0 to 500 mM gradient of imidazole in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or western blot with Ni-NTA conjugate to alkaline phosphatase (Qiagen). Fractions containing the Hisio-labeled FLS139, NL2 and NL3 are mixed and dialyzed against storage buffer.

Alternatively, the purification of labeled IgG (or labeled Fe) FLS139, NL2 and NL3 can be performed using known chromatography techniques, including for example, Protein A or Protein G chromatography column.

NL2 and FLS139 (NL6) were expressed in Baculovirus-infected High5 cells in poly-his labeled forms. While the expression was actually performed on a 0.5 L scale, it can easily be scaled up to longer preparations (e.g. 0.8 L).

Following the PCR amplification of the respective coding sequences, they were subcloned into the Baculovirus expression vector (pb.PH.His.c), and the Baculoogold® Baculovirus vector and DNA (Pharmigen) were co-transfected into High5 cells, using Lipofectin (Gibco BRL). Pb.pH.His is a modification of the commercially available Baculovirus expression vector pVL1393 (Pharmigen), with a modified polylinkel region to include the His sequences. The cells were grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone). The cells were incubated for 5 days at 28 ° C. The supernatant was subsequently cultured and used for the first viral amplification by infection of Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS at an approximate multiplicity of infection (MOI) of 10. The cells were incubated for 3 days at 28 days. ° C. The supernatant was cultured and the construction of the expression of NL2 and NL6 in the baculovirus expression vector was determined by binding of 1 ml of supernatant cluster to 25 mL of Ni-NTA drops (Qiagen) followed by SDS-PAGE analysis comparing a known concentration of standard protein per Coomassie Blue spot.

The first viral envelope supernatant was used to infect a warhead culture (500 ml) of High5 cells growing in medium ESF-921 (Expression Systems LLC) at an MOI of approximately 0.1. The cells were incubated for 3 days at 28 ° C. The supernatant was cultured and filtered. The cumulus junction and the SDS-PAGE analysis was repeated, as necessary, until the expression of the ogive culture was confirmed.

The conditioned medium of the transfected cells (0.5 to 3 L) harvested by centrifugation to remove the cells and filtered through a 0.22 micron filter. The labeled poly-his builder was purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the medium conditioned at a concentration of 5 mM. The conditioned medium was pumped in 6 ml of NI-NTA column equilibrated in 20 mM Hepes, pH 7.4, storage buffer containing 0.3 M NaCl and 5 mM imidazole at a fluid range of 4-5 ml / min. At 4 ° C. After storage, the column was washed with additional equilibration buffer and the protein washed with equilibrium buffer containing 0.25 M imidazole. The highly purified protein was subsequently deactivated in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with 25 ml of a Superfine G25 column (Pharmacia) and stored at -80 ° C.

The homogeneity of NL2 and NL6 proteins was verified by SDS acrylamide gel electrophoresis (PEG) and N-terminal amino acid sequencing by degradation Edman Example 8 Preparation of antibodies that bind FLS139, NL2 or NL3 This example illustrates the preparation of monoclonal antibodies which can specifically bind FLS139, NL2 and NL3.

Techniques for production of monoclonal antibodies are known in the state of the art and are described, for example in Goding, supra. Immunogens that can be employed include purified homologous ligands of the present invention, fusion proteins containing such homologous ligands, and cells expressing recombinant homologous ligands on the surface of the cell. The selection of the immunogen can be made by the skilled artisan without undue experimentation.

Mice, such as Balb / c, are immunized with the immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount of 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Reseca, Hamilton, MT) and injected into anterior plantar bearings of animals. The immunized mice are then redosed 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Serum samples can be obtained periodically from the mouse by retro-Orbital bleeding for ELISA assay test to detect the antibodies.

After an appropriate antibody titer has been detected, the animal "positive" for antibodies can be injected with a final intravenous injection of the given ligand. Three or four days later, the mice are sacrificed and the spleen cells are harvested. Spleen cells are then ligated (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU, available from ATCC, No. CRL 1597. The fusion generates cell hybridomas which can then be placed in 96-well tissue culture dishes containing HAT medium (hypoxanthine, aminopterin, and thymidine) to inhibit the proliferation of unfused cells, myeloma hybrids, and splenic cell hybrids.

Hybridoma cells will be protected in an ELISA for reactivity against the antigen. The determination of "positive" hybridoma cells by secreting the desired monoclonal antibodies against the TIE homologous ligands here is well known within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally in syngeneic Balb / c mice to produce ascites containing the monoclonal antibodies antigenic ligand of TIE. Alternatively, the hybridoma cells can be grown in cylindrical tissue culture bottles or jars. The purification of the monoclonal antibodies produced in the ascites can be accomplished by using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, the chromatographic affinity based on the binding of the antibody to protein A or protein G may be employed.

Example 9 Inhibition of VEGF stimulated by endothelial cell proliferation Bovine adrenal cortical capillary endothelial cells (ACE) (from a primary culutive, maximum 12-14 passages) were placed in 96-well microtitre plates (Amersham Life Science) at a density of 500 cells / well per 100 μL in low DMEM in glucose, 10% calf serum, 2 mM glutamine, lx pen / strept and fungizone, supplemented with 3 ng / mL of VEGF. The controls were placed in the same manner but some did not include VEGF. Test samples of the NL8 polypeptide were added in a volume of 100 μl for a final volume of 200 mcl. The cells were incubated for 6-7 days at 37 ° C. The medium was aspirated and the cells washed Ix with PBS. An acid phosphatase reaction mixture (100 μL) was added., 0.1 M sodium acetate, pH 5.5, 0.1% Triton-100, 10 mM p-nitrophenyl phosphate). After incubation for 2 hours at 37 ° C, the reaction was stopped by adding lOmvL of IN NaOH, OD was calculated in a microtiter plate reader at 405 nm. The controls were non-cells, cells alone, cells + FGF (5ng / mL), cells + VEGF (3 ng / mL), cells + VEGF (3ng / mL) + TGF-β (1 ng / mL), and cells + VEGF (3 ng / mL) + LIF (5 ng / mL). (TGF-β at a concentration of 1 ng / ml is known to block 70-90% of cell proliferation stimulated by VEGF.) The results were evaluated by calculating the percentage of inhibition of cell proliferation stimulated by VEGF (3 ng / ml), determined by calculating the acid phosphatase activity a = D405 n, (1) relative to cells without stimulation, and (2) ) relative to the TGF-β inhibition reference of the activity stimulated by VEGF. The results are considered positive, if the inhibition is 30% or greater. The results shown in Table 1 below are indicative of the utility of NL5, and possibly related to polypeptides, in cancer therapy and specifically in inhibition of tumor angiogenesis. The numerical values (Relative inhibition) shown in Table 1 were determined by calculating the percentage inhibition of proliferation stimulated by VEGF by the TIE homolog ligands tested related to cells without stimulation and then dividing the percentage by the percentage of inhibition obtained by TGF-β to 2 ng / ml which is known to block 70-90% cell proliferation stimulated by VEGF.

Table 1 Example 10 Induction of Endothelial Cell Apoptosis The ability of NL2, NL3 and NL6 to induce apoptosis in endothelial cells was tested in venous umbilical vein endothelial cells in humans (HUVEC, Cell System), using a 96-well format, in 0% serum medium supplemented with lOOng / ml of VEGF. (As HUVEC cells are easily dislodged from the dish surface, all pipetting in the wells should be done as smoothly as possible). 15 The medium was aspirated and the cells washed once with PBS. 5ml of trypsin Ix was added to the cells in a T125 flask, and the cells were left to founder until they were removed from the dish (about 5-10 minutes). The trypsinization was stopped by the addition of 5 ml of growth medium. The cells were rotated at 100 rpm for 5 minutes at 4 ° C. The medium was aspirated and the cells were resuspended in 10 ml of medium supplemented with 10% serum (Cell Systems), lx penn / strep.

Cells were placed in 96-well microtiter plates (Amersham Life Science, cytostar-T scintillating microplate, RPNQ160, sterile tissue culture treated, individually wrapped), in 10% serum (CSG medium, Cell Systems), at a density of 2x104 cells per well in a total volume of 100 μl. The NL5 and NL8 polypeptides were added in triplicate at 1%, 0.33% and 0.11% dilutions. Wells without cells as a target and wells with cells only as a negative control. It was used as a positive control serial dilutions 1: 3 of 50 μl of a 3x base of staurosporine. Ability of NL5 polypeptide to induce apoptosis was detereminated using Annexin V, a member of phospholipid and calcium binding proteins, to detect apoptosis. 0. 2 ml of Annexin V-Biotin base solution (100 μg / ml) were diluted in 4.6 ml of 2 x Ca 2+ binding buffer and 2.5% of BSA (1:25 dilution). Fifty μl of the diluted Annexin V-Biotin solution was added to each well (except the controls) for a final concentration of 1.0 μg / ml. Samples were incubated for 10-15 minutes with the preceding Annexin-Biotin to direct the addition of 35S-Streptavidin. 35S-Stre? Tavidin was diluted in 2 x Ca2 + binding buffer, 2.5% BSA and was added to all wells at a final concentration of 3x104 cpm / well. The plates were then sealed, centrifuged at 1000 rpm per minute for 15 minutes and placed in an orbital shelter for two hours. The analysis was carried out in Microbeta Trilux 1450 (Wallac).

NL2, NL3 and NL6 were positive in this trial. This result further confirms the potential utility of the same, and potentially related, molecules in cancer therapy.

Example 11 Induction of c-fos in endothelial cells Human venous umbilical vein endothelial cells (HUVEC, Cell Systems) in growth medium (50% Ham fF12 w / oct: low in glucose, and 50 amp.; of DMEM without glycine: with NaHCO3, 1% glutamine, 10 mM Hepes, 10% FBS, 10 ng / ml bFGF) were placed in 96-well microtiter plates at a cell density of 1 x 104 cells / water well. The day after the placement, the cells were killed by removing the culture medium and treating the cells with 100 μl / well of test sample and controls (positive control: growth medium, negative control: 10 mM HEPES, 140 mM of NaCl, 4% mannitol (w / v), pH 6.8). The cells were incubated for 30 minutes at 37 ° C, in 5% C02. The samples were removed, and the first part of the bDNA kit protocol (Chiron Diagnostics, cat # 6005-037) was followed, where each buffer / reactive capitalized listed below was available from the kit.

In summary, the amounts of the TM Damper and Probes needed for the tests were calculated based on the information provided by the manufacturer. The appropriate amounts of dissolved probes were added to the Lysis TM absorber. The Hybridization Capture Buffer was heated to room temperature. The bDNA screwdrivers were placed in the metal screw brackets, and 100 μl of Hybridization Capture Buffer was added for each bDN well needed, followed by incubation for at least 30 minutes. The test plates with the cells were removed from the incubator, and the medium was gently removed using the vacuum manifold. 100 μl of Hybridization Lysis Buffer with Probe were quickly pipetted into each well of the microtiter plates. The dishes were then incubated at 55 ° C for 15 minutes. After removing from the incubator, the plates were placed in the vortex mixer with the microtiter head adapter and vortexed in frame # 2 for one minute. 80 μl of the lysate were removed and added to the bDNA wells containing the Hybridization Capture Buffer, and pipetted to mix. The plates were incubated at 53 ° C for at least 16 hours.

The next day, the second part of the bADN kit protocol was followed. Specifically, the plates were removed from the incubator and placed on the bench to cool for 10 minutes. The addition volumes needed were calculated based on the information provided by the manufacturer. A Working Amplifier Solution was prepared by making a 1: 100 dilution of the Concentrated Amplifier (20 fm / μl) in AL Hybridization Damper. The hybridization mixture was removed from the dishes and washed twice with Wash A. 50 μl of Working Amplifier Solution was added for each well and the wells were incubated at 53 ° C for 30 minutes. The plates were then removed from the incubator and left to cool for 10 minutes. The Marked Probe Solution was prepared by performing a 1: 100 dilution of labeled Concentrate (40 pmoles / μl) in an AL Hybridization Damper. After cooling under a period of 10 minutes, the Hybridization Amplifier Mix was removed and the dishes washed twice with Wash A. 50 μl of Probe Marker Solution was added for each well and the wells were incubated at 53 ° C. for 15 minutes. After cooling for 10 minutes, the substrate was heated to room temperature. Upon the addition of 3 μl of Substrate Intensifier to each ml of substrate needed for the assay, the dishes were left to cool for 10 minutes, the Brand Hybridization Mixture was removed, and the plates were washed twice with Wash A and 3 times with Wash D. 50 μl Solution of Substrate with Intensifier was added to each well. Plates were incubated for 30 minutes at 37 ° C and read RLU in an appropriate luminometer.

The replicates were averaged and the coefficient of variation was determined. The calculation of the fold activity increases over the negative control (HEPES buffer described above) the value was indicated by chemiluminescence units (RLU). The samples which showed a value of at least 2 folds on the value of the negative control were considered positive.

Table 2 Example 12 6 Endothelial Cell Ca Flow Test The flow of Ca is a well-documented reaction about the binding of certain ligands to their receptors. A test compound that results in a positive response in the present flow test of Ca can be said to bind a specific receptor and activate a pathway of biological signaling in human endothelial cells. This could eventually lead, for example, to cell division ^ inhibition of cell proliferation / Endothelial tube formation, cell migration, apoptosis, etc.

Human venous umbilical vein endothelial cells . { HUVEC, Cell Systems) in growth medium (50:50 without glycine, 1% glutamine, 10 mM Hepes, 10% FBS, 1 ng / ml bFGF), were placed in 96-well microtiter dishes ViewPlates-96 (Packard Instrument Company Part # 6005182) at a cell density of 2 x 104 cells / well. Cells were washed with buffer (HBSS + 10 mM Hepes) three times, leaving 100 μL per well. The NLß polypeptide test samples were prepared in a separate dish of 96 wells at 5x concentration in buffer. Positive control: 50 μM of iono icine (5x); Negative control: Protein 32. The cell dish and sample dish were run on a FLIPR 0 (Molecular Devices) machine. The FLIPR machine added 25 μl of test sample to the cells, and the readings were taken every second for one minute, then every 3 seconds for the next 3 minutes.

The fluorescence change from the baseline to the maximum elevation of the curve (? Change) was calculated, and the duplicates were averaged. The rate of fluorescence increase was onitored, and only those samples that have a? change greater than 0 1000 and elevated within 60 seconds, were considered positive. In the following Table 3 the results are expressed relative to the positive control.

Table 3 Example 13 Guinea Pig Biopsy Evaluation Hairless guinea pig weighing 350 grams or more were anesthetized with keta ina (75-80 mg / kg) and xylazine (5 mg / kg) intramuscularly. NL6 or test samples of conditioned medium were injected intradermally in the backs with 100 μl per injection site. There were approximately 16-24 injection sites per animal. One ml of Ebans blusher dye (1% in physiological saline buffer) was injected intracardially. The proinflammatory or vascular permeability response of the skin to the test compound was visually marked, by measuring the translucent blue color of the injection site at 1 and 6 hours after administration of the test material (NL6). The animals were sacrificed 6 hours after administration. Each skin site was biopsied and fixed in formalin. The skin was prepared for immunohistopathological evaluation. Each site was evaluated for inflammatory cell infiltration in the skin. Sites with visible inflammatory cell infiltration were marked as positive. Infiltrating inflammatory cells can be neutrophils, eosinophils, monocytes or lymphocytes. NL6 was identified as a potential proinflammatory substance in this assay.

Example 14 Stimulation of Endothelial Tube Formation This assay follows the assay described in Davis and Ca arillo, Experimental Cell Reseach, 224: 39-51 (1996), or a modification thereof as follows: Protocol: HUVE cells (number of passage less than 8 of primary) are mixed with rat tail type I collagen, final concentration of 2.6 mg / ml at a density of 6 x 10 5 cells / ml and placed 50 μl per well in a 96 well plate. The gel is left to solidify for 1 hour at 37 ° C, then 50 μl per well of Mil9 culture medium supplemented with 1% FBS and a sample of the NL6 polypeptide is added (at dilutions of 15, 0.1% and 0.01%, respectively) in parallel with 1 μM of 6-FAM-FITC dye to stain vacuoles while they are being formed. The cells are incubated at 37 ° C / 5% C02 for 48 hours, mixed with 3.7% formalin at a temperature of 4 for 10 minutes, washed with PBS 5 times, then stained with Rh-Phaloidin at 4 ° C followed by nuclear staining. with 4 μM of DAPI.

I. Apoptosis test This assay will identify the factors that facilitate the survival of the cell in a 3-dimensional matrix in the presence of exogenous growth factors (VEGF, bFGF without PMA).

A positive result is equal to or less than 1. 0 = no apoptosis, 1 = less than 20% of cells are apoptotic, 2 = less than 50% of cells are apoptotic, 3 = more than 50% of cells are apoptotic. The stimulators of apoptosis in this system are expected to be factors of apoptosis, and inhibitors are expected to prevent or diminish apoptosis. 2. Vacuolas test This assay will identify factors that stimulate endothelial vacuole formation in the presence of bFGF and VEGF (40 ng / ml).

A positive result is greater than or equal to 2. 1 -vacuoles present in less than 20% of cells, 2 = vacuoles present in 20-50% of cells, 3 = vacuoles present in more than 50% of cells. This assay is designed to identify factors that are involved in stimulation of pinocytosis, ion pump, permeability, and binding formation, 3. Tube Formation Test This test is to identify factors that stimulate the formation of the endothelial tube in a three-dimensional matrix. This assay will identify factors that stimulate endothelial cells to differentiate into a tube as a structure in a three-dimensional matrix in the presence of exogenous growth factors (VEGF, bFGF).

A positive result is equal to or greater than 2. 1 - the cells are around, 2 = the cells are elongated, 3 - the cells are fusing tubes with some connections, 4 = the cells are forming networks of tubular complex. This trial would identify factors that may be involved in trajectory stimulation, chemotaxis, or endothelial configuration change.

Figure 10 shows the effect on the HUVEC-forming tube of the NL6 polypeptide conjugated to poly-his at 1% dilution and of a control buffer (10 mM HEPES / 0.14 M NaCl / 4% anitol, pH 6.8) 1% dilution. Comparative results with another novel TIE homolog ligand (NL1) and two known TIE-1 and TIE-2 ligands 5, tested as IgG fusions, are also shown in Figure 10.

Deposit material 0 As noted above, the following materials have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (ATCC): These deposits were made under the provisions of the Budapest Treaty in the Deposit of Microorganisms of International Recognition for the Purposes of Regulations and Low Patent Procedure (Budapest Treaty). This ensures the maintenance of a viable crop or deposit for 30 years from the day of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genetech, Inc. and ATCC, which ensures the permanent and unrestricted availability of progeny from the deposit culture to the public upon the promulgation of the US patent. relevant or on the installation open to the public of any US or foreign patent application whichever comes first and ensures the availability of the progeny for that determined by the Commissioner of Patents and Trademarks to accredit it in accordance with 35USC § 122 and rules of the Commissioned accordingly (including 37 CFR § 1.14 with particular reference to 886 OG 683).

The assignment of the present application has agreed that if a crop of the materials on deposit should die or be destroyed when cultivated under appropriate conditions, the materials will be quickly replaced in notification with another or the same. The availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights guaranteed under the authority of any government under the patent laws.

The present specification is considered to be sufficient to enable the skillful in the state of the art to practice the invention. The present invention is not limited in scope by the deposited analysis, since the modality deposited is LIST OF SEQUENCE (1) GENERAL INFORMATION (ü APPLICANT GENETECH, INC. FONG, SHERMAN FERRARA, NAPOLEONE GODDARD, AUDREY GODOWSKI, PAUL J. GURNEY, AUSTIN L. HILLAND, KENNETH WILLIAMS, MICKEY (ii) TITLE OF THE INVENTION: TIE HOMOLOGOUS LIGANDS (iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE TO THE CONSIGNEE: (A) CONSIGNEE: Genetech, Inc. (B) STREET: 1 DNA Way (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA (F) ZIP: 94080 (v) LEGIBLE FORMAT IN COMPUTING: (A) MEDIA TYPE: DISKETTE 1.44 Mb, 3.5 ING (B) COMPUTER: IBM COMPATIBLE PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: WinPatin ( Genetech) (vi) CURRENT APPLICATION REFERENCE: (A) APPLICATION NUMBER: (B) REGISTRATION DATE: (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION (A) NAME: Dreger, Ginger R. (B) REGISTRATION NUMBER: 33,055 (C) REFERENCE / CELLULAR NUMBER: P1078P2PCT (ix) TELECOMMUNICATION INFORMATION - (A) TELEPHONE: 650 / 225-3216 (B) TELEFAX: 650 / 952-9881 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1869 base pairs (B) TYPE: acid nucie co (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. O: 1: GCCGAGCTGA GCGGATCCTC ACATGACTGT GATCCGATTC TTTCCAGCGG 50 C TCTGCAAC CAAGCGGGTC TTACCCCCGG TCCTCCGCGT CTCCAGTCCT 100 CGCACCTGGA ACCCCAACGT CCCCGAGAGT CCCCGAATCC CCGCTCCCAG 150 GCTACCTAAG AGGATGAGCG GTGCTCCGAC GGCCGGGGCA GCCCTGATGC 200 TCTGCGCCGC CACCGCCGTG CTACTGAGCG CTCAGGGCGG ACCCGTGCAG 250 TCCAAGTCGC CGCGCTTTGC GTCCTGGGAC GAGATGAATG TCCTGGCGCA 300 CGGACTCCTG CAGCTCGGCC AGGGGCTGCG CGAACACGCG GAGCGCACCC 350 GCAGTCAGCT GAGCGCGCTG GAGCGGCGCC TGAGCGCGTG CGGGTCCGCC 400 TGTCAGGGAA CCGAGGGGTC CACCGACCTC CCGTTAGCCC CTGAGAGCCG 450 GGTGGACCCT GAGGTCCTTC ACAGCCTGCA GACACAACTC AAGGCTCAGA 500 ACAGCAGGAT CCAGCAACTC TTCCACAAGG TGGCCCAGCA GCAGCGGCAC 550 CTGGAGAAGC AGCACCTGCG AATTCAGCAT CTGCAAAGCC AGTTTGGCCT 600 CCTGGACCAC AAGCACCTAG ACCATGAGGT GGCCAAGCCT GCCCGAAGAA 650 AGAGGCTGCC CGAGATGGCC CAGCCAGTTG ACCCGGCTCA CAATGTCAGC 700 CGC TGCACC GGCTGCCCAG GGATTGCCAG GAGCTGTTCC AGGTTGGGGA 750 GAGGCAGAGT GGACTATTTG AAATCCAGCC TCAGGGGTCT CCGCCATTTT 800 TGG GAACTG CAAGATGACC TCAGATGGAG GCTGGACAGT AATTCAG AGG 850 CGCCACGATG GCTCAGTGGA CTTCAACCGG CCCTGGGAAG CCTACAAGGC 900 GGGGTTTGGG GATCCCCACG GCGAGTTCTG GCTGGGTCTG GAGAAGGTGC 950 ATAGCATCAC GGGGGACCGC AACAGCCGCC TGGCCGTGCA GCTGCGGGAC 1000 TGGGATGGCA ACGCCGAGTT GCTGCAGTTC TCCGTGCACC TGGGTGGCGA 1050 GGACACGGCC TATAGCCTGC AGCTCACTGC ACCCGTGGCC GGCCAGCTGG 1100 GCGCCACCAC CGTCCCACCC AGCGGCCTCT CCGTACCCTT CTCCACTTGG 1150 GACCAGGATC ACGACCTCCG CAGGGACAAG AACTGCGCCA AGAGCCTCTC 1200 TGGAGGCTGG TGGTTTGGCA CCTGCAGCCA TTCCAACCTC AACGGCCAGT 1250 ACTTCCGCTC CATCCCACAG CAGCGGCAGA AGCTTAAGAA GGGAATCTTC 1300 TGGAAGACCT GGCGGGGCCG CTACTACCCG CTGCAGGCCA CCACCATGTT 1350 GATCCAGCCC ATGGCAGCAG AGGCAGCCTC CTAGCGTCCT GGCTGGGCCT 1400 GGTCCCAGGC CCACGAAAGA CGGTGACTCT TGGCTCTGCC CGAGGATGTG 1450 GCCGTTCCCT GCCTGGGCAG GGGCTCCAAG GAGGGGCCAT CTGGAAACTT 1500 GTGGACAGAG AAGAAGACCA CGACTGGAGA AGCCCCCTTT CTGAGTGCAG 1550 GGGGGCTGCA TGCGTTGCCT CCTGAGATCG AGGCTGCAGG ATATGCTCAG 1600 ACTCTAGAGG CGTGGACCAA GGGGCATGGA GCTTCACTCC TTGCTGGCCA 1650 GGGAGTTGGG GACTCAGAGG GACCACTTGG GGCCAGCCAG ACTGGCCTCA 1700 ATGGCGGACT CAGTCACATT GACTGACGGG GACCAGGGCT TGTGTGGGTC 1750 GAGAGCGCCC TCATGGTGCT GGTGCTGTTG TGTGTAGGTC CCCTGGGGAC 1800 ACAAGCAGGC GCCAATGGTA TCTGGGCGGA GCTCACAGAG TTCTTGGAAT 1850 AAAAGCAACC TCAGAACAC 1869 (2) INFORMATION FOR SEQ ID NO: 2: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 406 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Ser Gly Ala Pro Thr Ala Gly Ala Ala Leu Mee Leu Cys Ala 1 5 10 15 Wing Thr Wing Val Leu Leu Wing Wing Gln Gly Gly Pro Val Gln Ser 20 25 30 Lys Ser Pro Arg Phe Wing Ser Trp Asp Glu Met Asn Val Leu Wing 35 40 45 His Gly Leu Leu Gln Leu Gly Gln Gly Leu Arg Glu His Wing Glu 50 55 60 Arg Thr Arg Ser Gln Leu Ser Wing Leu Glu Arg Arg Leu Ser Wing 65 70 75 Cys Gly Be Ala Cys Gln Gly Thr Glu Gly Be Thr Asp Leu Pro 80 85 90 Leu Ala Pro Glu Ser Arg Val Asp Pro Glu Val Leu His Ser Leu 95 100 105 Gln Thr Gln Leu Lys Wing Gln Asn Being Arg lie Gln Gln Leu Phe 110 115 120 Has Lys Val Ala Gln Gln Gln Arg His Leu Glu Lys Gln His Leu 125 130 135 Arg lie Glp His Leu Gln Ser Gln Phe Gly Leu Leu Asp His Lys 140 145 150 Hie Leu Asp His Glu Val Wing Lys Pro Wing Arg Arg Lys Arg Leu 155 160 165 Pro Glu Met Wing Gln Pro Val Asp Pro Wing His Asn Val Ser Arg 170 175 180 Leu His Arg Leu Pro Arg Asp Cys Gln Glu Leu Phe Gln Val Gly 185 190 195 Glu Arg Gln Ser Gly Leu Phe Glu lie Gln Pro Gln Gly Ser Pro 200 205 210 Pro Phe Leu Val Asn Cys Lys Met Thr Ser Xaa Gly Gly Trp Thr 215 220 225 Val lie Gln Arg Arg His Asp Gly Ser Val Asp Phe Asn Arg Pro 230 235 240 Trp Glu Wing Tyr Lys Wing Gly Phe Gly Asp Pro Hxs Gly Glu Phe 245 250 255 Trp Leu Gly Leu Glu Lys Val His Ser lie Thr Gly Asp Arg Asn 260 265 270 Being Arg Leu Ala Val Gln Leu Arg Asp Trp Asp Gly Asn Ala Glu 275 280 285 Leu Leu Gln Phe Ser Val His Leu Gly Gly Glu Asp Thr Ala Tyr 290 295 300 Be Leu Gln Leu Thr Ala Pro Val Wing Gly Gln Leu Gly Ala Thr 305 310 315 Thr Val Pro Pro Ser Gly Leu Ser Val Pro Phe Ser Thr Trp Asp 320 325 330 Gln Asp Hie Asp Leu Arg Arg Asp Lys Asn Cys Ala Lys Ser Leu 335 340 345 Be Gly Gly Trp Trp Phe Gly Thr Cye Be His Ser Asn Leu Asn 350 355 360 Gly Gln Tyr Phe Arg Ser lie Pro Gln Gln Arg Gln Lys Leu Lys 365 370 .. 375 Lys Gly lie Phe Trp Lys Tnr Trp Arg Gly Arg Tyr Tyr Pro Leu 380 385 390 Gln Ala Thr Thr Met Leu He Gln Pro Met Ala Ala Glu Ala Wing 395 400 405 Ser 406 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1024 base pairs (B) TYPE: nucleic acids (C) CHAIN: simple (D) TOPOLOGY: linear (i) ) DESCRIPTION OF THE SEQUENCE: SE ID N?: 3: CGGACGCGTG GGCCCCTGGT GGGCCCAGCA AGATGGATCT ACTGTGGATC 50 CTGCCCTCCC TGTGGCTTCT CCTGCTTGGG GGGCCTGCCT GCCTGAAGAC 100 CCAGGAACAC CCCAGCTGCC CAGGACCCAG GGAACTGGAA GCCAGCAAAG 150 TTGTCCTCCT GCCCAGTTGT CCCGGAGCTC CAGGAAGTCC TGGGGAGAAG 200 GGAGCCCCAG GTCCTCAAGG GCCACCTGGA CCACCAGGCA AGATGGGCCC 250 CAAGGGTGAG CCAGGCCCCA GAAACTGCCG GGAGCTGTTG AGCCAGGGCG 300 CCACCTTGAG CGGCTGGTAC CATCTGTGCC TACCTGAGGG CAGGGCCCTC 350 CCAGTCTTTT GTGACATGGA CACCGAGGGG GGCGGCTGGC TGGTGTTTCA 400 GAGGCGCCAG GATGGTTCTG TGGATTTCTT CCGCTCTTGG TCCTCCTACA 450 GAGCAGGTTT TGGGAACCAA GAGTCTGAAT TCTGGCTGGG AAATGAGAAT 500 TTGCACCAGC TTACTCTCCA GGGTAACTGG GAGCTGCGGG TAGAGCTGGA 550 AGACTTTAAT GGTAACCGTA CTTTCGCCCA CTATGCGACC TTCCGCCTCC 600 TCGGTGAGGT AGACCACTAC CAGCTGGCAC TGGGCAAGTT CTCAGAGGGC 650 ACTGCAGGGG ATTCCCTGAG CCTCCACAGT GGGAGGCCCT TTACCACCTA 700 TGACGCTGAC CACGATTCAA GCAACAGCAA CTGTGCAGTG ATTGTCCACG 750 GTGCCTGGTG GTATGCATCC TGTTACCGAT CAAATCTCAA TGGTCGCTAT 800 GCAGTGTCTG AGGCTGCCGC CCACAAATAT GGCATTGACT GGGCCTCAGG 850 CCGTGGTGTG GGCCACCCCT ACCGCAGGGT TCGGATGATG CTTCGATAGG 900 GCACTCTGGC AGCCAGTGCC CTTATCTCTC CTGTACAGCT TCCGGATCGT 950 CAGCCACCTT GCCTTTGCCA ACCACCTCTG CTTGCCTGTC CACATTTAAA 1000 AATAAAATCA TTTTAGCCCT TTCA 1024 (2) INFORMATION FOR SEQ ID NO: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 288 amino acids < B) TYPE: amino acids (D) TOPOLOGY: linear (XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO:: Met Asp Leu Leu Trp He Leu Pro Ser Leu Trp Leu Leu Leu Leu 1 5 10 15 Gly Gly Pro Wing Cys Leu Lys Thr Gln Glu Hxe Pro Ser Cys Pro 20 25 30 Gly Pro Arg Glu Leu Glu Wing Ser Lys Val Val Leu Leu Pro Ser 35 40 45 Cys Pro Gly Pro Wing Gly Ser Pro Gly Glu Pro Lime Gly Wing 50 55 60 Pro Gln Gly Pro Pro Gly Pro Pro Gly Lys Met Gly Pro Lys Gly 65 70 75 Glu Pro Gly Pro Arg Asn Cys Arg Glu Leu Leu Ser Gln Gly Wing 80 85 90 Thr Leu Ser Gly Trp Tyr His Leu Cys Leu Pro Glu Gly Arg Wing 95 100 105 Leu Pro Val Phe Cys Asp Met Asp Thr Glu Gly Gly Gly Trp Leu 110 115 120 Val Phe Gln Arg Arg Gln Asp Gly Ser Val Asp Phe Phe Arg Ser 125 130 135 Trp Being Tyr Arg Wing Gly Phe Gly Asn Gln Glu Being Glu Phe 140 145 150 Trp Leu Gly Asn Glu Asn Leu His Gln Leu Thr Leu Gln Gly Asn 155 160 165 Trp Glu Leu Arg Val Glu Leu Glu Asp Phe Asn Gly Asn Arg Thr 170 175 180 Phe Ala His Tyr Ala Thr Phe Arg Leu Leu Gly Glu Val Asp His 185 190 195 Tyr Gln Leu Wing Leu Gly Lys Phe Ser Glu Gly Thr Wing Gly Asp 200 205 210 Ser Leu Ser Leu His Ser Gly Arg Pro Phe Thr Thr Tyr Asp Ala 215 220 225 Asp Hxs Asp Being Ser Asn Being Asn Cys Ala Val He Val His Gly 230 235 240 Wing Trp Trp Tyr Wing Ser Cys Tyr Arg Ser Asn Leu Asn Gly Arg 245 250 255 Tyr Ala Val Ser Glu Ala Ala Ala His Lys Tyr Gly He Asp Trp 260 265 270 Wing Being Gly Arg Gly Val Gly Hxs Pro Tyr Arg Arg Val Arg Met 275 280 285 Met Leu Arg 288 (2) INFORMATION FOR SEQ ID NO: 5 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 2042 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GCGGACGCGT GGOTGAAATT GAAAATCAAG ATAAAAATGT TCACAATTAA 50 GCTCCTTCTT TTTATTGTTC CTCTAGTTAT TTCCTCCAGA ATTGATCAAG 100 ACAATTCATC ATTTGATTCT CTATCTCCAG AGCCAAAATC AAGATTTGCT 150 ATGTTAGACG ATGTAAAAAT TTTAGCCAAT GGCCTCCTTC AGTTGGGACA 200 TGGTCTTAAA GACTTTGTCC ATAAGACGAA GGGCCAAATT AATGACATAT 250 TTCAAAAACT CAACATATTT GATCAGTCTT TTTATGATCT ATCGCTGCAA 300 ACCAGTGAAA TCAAAGAAGA AGAAAAGGAA CTGAGAAGAA CTACATATAA 350 ACTACAAGTC AAAAATGAAG AGGTAAAGAA TATGTCACTT GAACTCAACT 400 CAAAACTTGA AAGCCTCCTA GAAGAAAAAA TTCTACTTCA ACAAAAAGTG 450 AAATATTTAG AAGAGCAACT AACTAACTTA ATTCAAAATC AACCTGAAAC 500 TCCAGAACAC CCAGAAGTAA CTTCACTTAA AACTTTTGTA GAAAAACAAG 550 ATAATAGCAT CAAAGACCTT CTCCAGACCG TGGAAGACCA ATATAAACAA 600 TTAAACCAAC AGCATAGTCA AATAAAAGAA ATAGAAAATC AGCTCAGAAG 650 GACTAGTATT CAAGAACCCA CAGAAATTTC TCTATCTTCC AAGCCAAGAG 700 CACCAAGAAC TACTCCCTTT CTTCAGTTGA ATGAAATAAG AAATGTAAAA 750 CATGATGGCA TTCCTGCTGA ATGTACCACC ATTTATAACA GAGGTGAACA 800 TACAAGTGGC ATGTATGCCA TCAGACCCAG CAACTCTCAA GTTTTTCATG 850 TCTACTGTGA TGTTATATCA GGTAGTCCAT GGACATTAAT TCAACATCGA 9 00 ATAGATGGAT CACAAAACTT CAATGAAACG TGGGAGAACT ACAAATATGG 950 TTTTGGGAGG CTTGATGGAG AATTTTGGTT GGGCCTAGAG AAGATATACT 1000 CCATAGTGAA GCAATCTAAT TATGTTTTAC GAATTGAGTT GGAAGACTGG 1050 AAAGACAACA AACATTATAT TGAATATTCT TTTTACTTGG GAAATCACGA 1100 AACCAACTAT ACGCTACATC TAGTTGCGAT TACTGGCAAT GTCCCCAATG 1150 CAATCCCGGA AAACAAAGAT TTGGTGTTTT CTACTTGGGA TCACAAAGCA 1200 AAAGGACACT TCAACTGTCC AGAGGGTTAT TCAGGAGGCT GGTGGTGGCA 1250 TGATGAGTGT GGAGAAAACA ACCTAAATGG TAAATATAAC AAACCAAGAG 1300 CAAAATCTAA GCCAGAGAGG AGAAGAGGAT TATCTTGGAA GTCTCAAAAT 13SO GGAAGGTTAT ACTCTATAAA ATCAACCAAA ATGTTGATCC ATCCAACAGA January 00 TTCAGAAAGC TTTGAATGAA CTGAGGCAAT TTAAAGGCAT ATTTAACCAT 1450 TAACTCATTC CAAGTTAATG TGGTCTAATA ATCTGGTATA AATCCTTAAG 1500 AGAAAGCTTG AGAAATAGAT TTTTTTTATC TTAAAGTCAC TGTCTATTTA 1550 AGATTAAACA TACAATCACA TAACCTTAAA GAATACCGTT TACATTTCTC 1600 AATCAAAATT CTTATAATAC TATTTGTTTT AAATTTTGTG ATGTGGGAAT 1650 CAATTTTAGA TGGTCACAAT CTAGATTATA ATCAATAGGT GAACTTATTA 1700 AATAACTTTT CTAAATAAAA AATTTAGAGA CTTT TATTTT AAAAGGCATC 1750 ATATGAGCTA ATATCACAAC TTTCCCAGTT TAAAAAACTA GTACTCTTGT 1800 TAAAACTCTA AACTTGACTA AATACAGAGG ACTGGTAATT GTACAGTTCT 1850 TAAATGTTGT AGTATTAATT TCAAAACTAA AAATCGTCAG CACAGAGTAT 1900 GTGTAAAAAT CTGTAATACA AATTTTTAAA CTGATGCTTC ATTTTGCTAC 1950 AAAATAATTT GGAGTAAATG TTTGATATGA TTTATTTATG AAACCTAATG 2000 AAGCAGAATT AAATACTGTA TTAAAATAAG TTCGCTGTCT TT 2042 (2) INFORMATION FOR SEQ ID NO: 6: (1) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 460 amino acids (B) TYPE: apuno acids (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Met Phe Thr He Lys Leu Leu Leu Phe He Val Pro Leu Val He 1 5 10 15 Ser Ser Arg He Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser 20 25 30 Pro Glu Pro Lys Ser Arg Phe Wing Met Leu Asp Asp Val Lys He 35 40 45 Leu Ala Asn Gly Leu Leu Gln Leu Gly Hxs Gly Leu Lys Asp Phe 50 55 60 Val His Lys Thr Lys Gly Gln He Asn Asp He Phe Gln Lys Leu 65 70 75 Asn He Phe Asp Gln Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser 80 85 90 Glu He Lys Glu Glu Glu Lys Glu Leu Arg Arg Thr Thr Tyr Lys 95 OO 105 Leu Gln Val Lys Asn Glu Glu Val Lys Asn Met Ser Leu Glu Leu 110 115 120 Asn Ser Lys Leu Glu Ser Leu Leu Glu Glu Lys He Leu Leu Gln 125 130 135 Gln Lys Val Lys Tyr Leu Glu Glu Gln Leu Thr Asn Leu He Gln 140 '145 150 Asn Gln Pro Glu Thr Pro Glu His Pro Glu Val Thr Ser Leu Lys 155 160 165 Thr Phe Val Glu Lys Gln Asp Asn Ser He Lys Asp Leu Leu Gln 170 175 180 Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln His Ser Gln 185 190 195 He Lys Glu He Glu Asn Gln Leu Arg Arg Thr Ser He Gln Glu 200 205 210 Pro Thr Glu Be Ser Leu Ser Ser Lys Pro Arg Pro Pro Arg Thr 215 220 225 Thr Pro Phe Leu Gln Leu Asn Glu He Arg Asn Val Lys His Asp 230 235 240 Gly He Pro Wing Glu Cys Thr Thr He Tyr Asn Arg Gly Glu His 245 250 255 Thr Ser Gly Met Tyr Wing He Arg Pro Ser Asn Ser Gln Val Phe 260 265 270 His Val Tyr ys Asp Val He Ser Gly Ser Pro Trp Thr Leu He 275 280 285 Gln His Arg He Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu 290 295 300 Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu 305 310 315 Gly Leu Glu Lys He Tyr Ser He Val Lys Gln Ser Asn Tyr Val 320 325 330 Leu Arg He Glu Leu Glu Asp Trp Lys Asp Asn Lys His Tyr He 335 340 345 Glu Tyr Ser Phe Tyr Leu Gly Asn Hxs Glu Thr Asn Tyr Thr Leu 350 355 360 His Leu Val Wing He Thr Gly Asn Val Pro Asn Wing He Pro Glu 365 370 375 Asn Lys Asp Leu Val Phe Ser Thr Trp Asp His Lys Wing Lys Gly 380 385 390 His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp His 395 400 405 Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro 410 415 420 Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys 425 430 435 Ser Gln Asn Gly Arg Leu Tyr Ser He Lys Ser Thr Lys Met Leu 440 445 450 He Hie Pro Thr Asp Ser Glu Ser Phe Glu 455 460 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID N?: 7: ATGAGGTGGC CAAGCCTGCC CGAAGAAAGA GGC 33 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: CAACTGGCTG GGCCATCTCG GGCAGCCTCT TTCTTCGGG 39 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: CCCAGCCAGA ACTCGCCGTG GGGA 24 (2) INFORMATION FOR SEQ ID NO: 10: (x) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) Linear TOPOLOGY (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: TGGTTGGCAA AGGCAAGGTG GCTGACGATC CGG 33 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY : linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: GTGGCCCTTA TCTCTCCTGT ACAGCTTCCG GATCGTCAGC CAC 43 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY : linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: TCCATTCCCA CCTATGACGC TGACCCA 27 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: CCACGTTGGC TTGAAATTGA 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: CCTCCAGAAT TGATCAAGAC AATTCATGAT TTGATTCTCT ATCTCCAGAG 50 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: TCGTCTAACA TAGCAAATC 19 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: TTCTAATACG ACTCACTATA GGGCAAGTTG TCCTCC 36 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: TGAAATTAAC CCTCACTAAA GGGACGTGGT CAGCGT 36 intended as an isolated illustration of certain aspects of the invention and any analysis that is functionally equivalent is within the scope of the invention. The deposit of material here does not constitute an admission that the written description is inadequate to enable the practice of any aspect of the invention, including the best of it, nor is it constructed as limiting the scope of the claims for the specific illustrations that this It represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those qualified in the prior art of the preceding description and will fall within the scope of the appended claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (29)

CLAIMS.

1. An isolated nucleic acid molecule encoding a mammalian TIE homologous ligand polypeptide, (a) selected from a group consisting of human NL2 (SEQ ID NO: 2), human NL3 (SEQ ID NO: 4), human NL6 (SEQ ID NO: 6), and homologues thereof in a kind of non-human mammal; or (b) a biologically active functional derivative thereof, provided that if the functional derivative is an amino acid sequence variant, it has at least about 90% sequence identity with the fibrinogen-equivalent domain of a human ligand. NL2, human NL3 or human NL6.

2. The isolated nucleic acid molecule of claim 1 characterized in that it comprises the coding region of SEC. ID.NO: 1; SEC. ID.NO: 3; or SEC.ID.NO:5.

3. The isolated nucleic acid molecule of claim 1 characterized in that it comprises a fibrinogen-equivalent domain of SEC. ID.NO: 1; SEC.ID.NO:3; or SEC.ID.NO:5.

4. A vector characterized in that it comprises a nucleic acid of claim 1.

5. A recombinant host cell transformed with a nucleic acid molecule according to claim 1

6. The recombinant host cell of claim 5 characterized in that it is a prokaryotic cell.

7. The recombinant host cell of claim 5 characterized in that it is a eukaryotic cell.

8. A TIE ligand homologue polypeptide, isolated from a mammal, (a) selected from a group consisting of human NL2 (SEQ ID NO: 2), human NL3 (SEQ ID NO: 4), and human NL6 ( SEC.ID.NO: 6), and homologs thereof in a non-human mammal species; or (b) a biologically active functional derivative thereof, provided that if the functional derivative is an amino acid sequence variant, it has at least about 90% sequence identity with the fibrinogen-equivalent domain of a ligand. of human NL2, human NL3 or human NL6.

9. An antibody characterized in that it specifically binds the TIE ligand according to claim 8.

10. The antibody of claim 9 characterized in that it is a monoclonal antibody.

11. The antibody of claim 10, characterized in that it has residues of non-human complementarity determining region (CDR) and human structure residues (FR).

12. The antibody of claim 9 characterized in that it inhibits endothelial cell growth.

13. The antibody of claim 12 characterized in that said cell is a tumor cell.

14. The antibody of claim 9 characterized in that it induces apoptosis of the cell.

15. The antibody of claim 9 characterized in that it inhibits tumor cell vascularization.

16. The antibody of claim 9 characterized in that it is an anti-NL2, anti-NL3 or anti-NL6 antibody.

17. The antibody of claim 9, characterized in that it is labeled.

18. A composition characterized in that it comprises a polypeptide of claim 8 in association with a carrier or carrier.

19. A composition characterized in that it comprises an antibody of claim 9 in association with a carrier.

20. The composition of claim 19 characterized in that it comprises an amount of growth inhibitor of said antibody.

21. The composition of claim 20 characterized in that it comprises an anti-NL2, anti-NL3 or anti-NL6 antibody. fuS

22. The composition of claim 21, characterized in that it also comprises a second antibody of a cytotoxic or chemotherapeutic agent.

23. A conjugate characterized in that it comprises a polypeptide of claim 8 or antibody of claim 9, fused to a further therapeutic or cytotoxic agent.

24. The conjugate of claim 23 characterized in that the subsequent therapeutic agent is a toxin, a different TIE ligand, or a member of the vascular endothelial growth factor (VEGF) family.

25. A method for projecting the presence of angiogenesis, characterized in that it comprises. administering to a patient a detectably labeled TIE homologous ligand of claim 8, or an agonist antibody of claim 9, and monitoring angiogenesis.

26. A method for inhibiting vasculogenesis, characterized by comprising the administration to a patient of an effective amount of a TIE homologous ligand of claim 8 or an agonist antibody of claim 9.

27. A method for the inhibition of endothelial cell proliferation characterized in that said treatment comprises endothelial cells with an effective amount of a TIE homologous ligand polypeptide of claim 8.

28. A method for the induction of endothelial cell apoptosis characterized in that said treatment comprises endothelial cells with an effective amount of a TIE homologous ligand polypeptide of claim 8.

29. A method for inhibiting tumor growth, characterized in that it comprises administering to a patient an effective amount of an antagonist TIE ligand according to claim 8. LIGANDOS HOMOLOGOS DE TIE SUMMARY OF THE INVENTION The present invention concerns isolated nucleic acid molecules encoding the novel homologous ligands of TIE NL2, NL3, and NL6 (FSL139), the proteins encoded by such nucleic acid molecules, as well as methods and means for the realization and use of such molecules of protein and nucleic acid