FI111267B - Antibody against a Tie-receptor tyrosine kinase - used to diagnose and treat neoplastic diseases and for radiological imaging - Google Patents

Abstract

Antibody against a Tie-receptor tyrosine kinase is claimed. Also claimed are: (1) a hybridoma cell line producing the Ab; and (2) a pharmaceutical compsn. comprising the Ab (1-10 mg/ml) or a deriv. and a diluent, adjuvant or carrier.

Description

111267 ANTIBODIES RECOGNIZING THE ROAD RECEPTOR AND THEIR USE

The invention generally relates to antibodies that react with the Tie tyrosine kinase receptor found in various endothelial cells and in some tumor cell populations. Furthermore, the present invention relates to methods for producing and using such antibodies. In particular, the present invention relates to anti-Tie monoclonal antibodies as diagnostic tools for the identification of certain hematopoietic cells and hematological and angiogenic diseases, as well as for vascular imaging and as a therapeutic agent.

10 TECHNICAL BACKGROUND

Cardiovascular disease and cancer are very common in Western countries and these groups of diseases are economically important because patients with these diseases are usually incapacitated and need treatment for long periods. Blood vessels play an important role in the development of cardiovascular diseases, as well as in the pathogenesis of cancer. Endothelial cells in the blood vessels play a central role in the development of vascular diseases. Traumas and endothelial cell metabolic disorders cause so-called. atheroma plaques and finally arteriosclerosis. Through tumor cell growth factors, neovascularization, induced by stimulation of endothelial cells, is an important factor in various cancers. It is known from experimental studies that in order for cells to develop and grow cancerous colonies, they need neovascularization to ensure the supply of nutrients and oxygen to the growing tissue.

The cellular behavior responsible for the development, maintenance and repair of differentiated cells and tissues is mainly regulated by intracellular signals through growth factors and related ligands and their receptors. The receptors are located on the surface of the receiving cell and bind peptide or polypeptide growth factors and other hormone-like ligands. As a result of this interaction, the receiving cell undergoes rapid biochemical changes, leading to both rapid and long-term regulation of cellular gene expression. Many receptors on different cell surfaces can bind specific growth factors.

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Tyrosine phosphorylation forms a key form of signaling in plasma membranes. Several tyrosine kinase genes encoding transmembrane receptors for polypeptide growth factors and hormones are currently known, such as epidermal growth factor (EGF), insulin, insulin-like growth factor, (IGF-I), platelet-derived growth factors (PDGF-AA, ABb) and BB. See, e.g., Heidin and Westermark, Cell Reg., 1: 555-556 (1990); Ullrich and Schlessinger, Cell, 61: 2243-354, (1990). Growth factor receptors on endothelial cells are of particular interest due to the potential association of growth factors such as FGF with a number of physiologically and pathologically important events: angiogenesis, arteriosclerosis and inflammatory diseases (Folkman and Klagsbrun,

Science, 235: 442-447, 1987). A number of hematopoietic growth factor receptors are also tyrosine kinases. These include the colony stimulating factor 1 receptor j (Sherr et al., Cell, 41: 665-676, 1985) and the c-kit, 15 stem cell factor receptor (Huang et al., Cell, 63: 225-233, 1990).

Receptor tyrosine kinases can be subdivided into developmental biologics based on their structural similarities and differences. These proteins differ in their specificity and affinity (Ullrich and Schlessinger, supra). Generally speaking, receptor tyrosine kinases are glycoproteins composed of an extracellular portion capable of binding a growth factor, a transmembrane portion, which is generally an alpha helical portion of a protein, a membrane proximal portion that may regulate a receptor, e.g. the enzymatic moiety, as well as the carboxy-terminal end involved in the identification and binding of specific substrates at many receptors.

Recently, International Patent Publication No. WO 93/14124 describes a novel endothelial cell receptor tyrosine kinase called Tie. The pathway is an acronym derived from Tyrosine kinase containing Immunoglobulin and EGF-like domains (tyrosine kinase with immunoglobulin and EGF-like domains). The pathway is useful in the diagnosis and treatment of diseases associated with endothelial cells and their Tie receptors, such as neoplastic diseases associated with tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis, and inflammatory diseases.

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The inventors have now produced monoclonal antibodies to the outer part of the cell endothelial cell receptor tyrosine kinase. These antibodies have been used to identify Tie in cell cultures and in vivo immunohistological assays. The results show that antibodies that specifically recognize the extracellular portions of Tie can be used to monitor hematopoietic and endothelial cells in tissue samples and in the body, and in the diagnosis of various cancers.

BRIEF DESCRIPTION OF THE PICTURES

Figure 1 represents an analysis of Tie for MOLT-4 and HEL cells by immunofluorescence and flovv cytometry.

Figure 2 shows immunoperoxidase staining of Tie in human blood cells.

Figure 3 shows the distribution and binding of 125 I-labeled monoclonal antibody, 3C4C7G6, to selected target tissues in a mouse with an 8-day healing wound.

Figure 4. Immunohistochemical staining of Tie in brain tissue: normal, glioblastoma multiforme and metastatic melanoma. Measuring column: 0.05 mm.

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Figure 5. Immunohistochemical staining of hemangioblastoma and hemangioperytoma using Tie, vWF or CD44 as endothelial cell specific markers (40x).

Figure 6. Distribution (% ID / g) of anti-TIE antibody 10F11 to important organs after 48, 72, 96 and 120 hours (N = 3, 3, 3 and 4)

Figure 7. Distribution (% ID / g) of 3c4 anti-TIE antibody to important organs after 48, 72, 96 and 120 hours (N = 3, 3, 7, 2 and 4)

Figure 8. Radioactivity accumulation at 48, 96, and 120 hours with 10F11 antibody, and at 6, 24, 48, 96, and 120 hours with 3c4 antibody. 1

Figure 9. Standard curve for four different pairs of antibodies (fixed / labeled antibody) 111267 4

Figure 10. Individual Tie antigen concentrations (g / l) in a serum sample of patients with breast cancer (N = 2), ovarian cancer (N = 5), and small cell lung cancer (N = 6). Reference samples (N = 20) are from non-cancer patients.

DESCRIPTION OF THE INVENTION

One embodiment of the present invention describes diagnostic methods for monitoring hematopoietic cells and endothelial cells in tissue samples and in whole organisms. In another embodiment, the present invention relates to clinical detection methods which describe the condition of the endothelial cells (trauma, growth, etc.) and methods for detecting endothelial cells, and thus vascular growth, in the body. The present invention relates to antibodies that recognize Tie. In a preferred embodiment, the antibodies of the invention recognize extracellular portions of Tie.

In another preferred embodiment, the invention relates to monoclonal antibodies that specifically recognize various epitopes on the extracellular part of the Tie receptor. The present invention relates in particular to the monoclonal antibodies 3C4C7G6 and 10F11G6. The hybridoma cell line producing the monoclonal antibody 3C4C7G6 has been deposited with the Depository Deutsche Sammlung von 20 Mikroorganismen und Zellkulturen GmbH (DSM) (DSM Accession Number ACC2159) pursuant to the Budapest Treaty.

:: Antibodies labeled with a recognizable marker have also been described.

As used herein, the term recognizable marker means any recognizable marker known to those skilled in the art. In a preferred embodiment of the invention, however, the detectable marker is selected from the group consisting of radioisotopes, fluorochromes, dyes, enzymes, and biotin. Suitable radioisotopes for the invention include, but are not limited to, 125I and 1311.

The present invention also relates to monoclonal antibodies conjugated to an imaging agent. In this context, the term imaginable substance encompasses radioisotopes, but is not limited to what I see. A preferred radioisotope is 99m technetium.

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The present invention further relates to a method of detecting and identifying human tissues undergoing neovascularization, comprising the steps of (a) obtaining a tissue and / or body fluid sample suspected of having neovascularization, and (b) contacting the sample with with a specific monoclonal antibody under conditions suitable for complexing the monoclonal antibody and antigen; and (c) detecting the resulting complex.

The tissue detectable by this method is normal tissue, a precursor of cancer, or a solid tumor containing Tie-containing endothelial cells or leukemia cells expressing the Tie receptor. In one embodiment of the invention, the monoclonal antibody is labeled with a recognizable marker as described above.

The methods of the invention are useful in the detection and identification of various types of cancer.

The present invention also relates to a method for diagnosing and monitoring the disease state of various types of cancers by determining a circulating Tie antigen in human serum.

The monoclonal antibodies of the invention may also be used to identify Tie receptors in a cell sample in which:. the method comprises contacting a cell sample with a monoclonal antibody and detecting the binding of the monoclonal antibody to the Tie receptor.

In a preferred embodiment of the invention, the monoclonal antibodies can be used to identify and track specific hematopoietic cells, especially B cell line cells.

Contacting the cell mixture with the monoclonal antibodies of the invention may be in solution, such as in fluorescence-activated cell screening, or may be accomplished using solid tissue samples such as biopsy material, or may be performed using solid support-bound monoclonal antibodies such as direct immunoadherence. The cell mixture that reacts with the monoclonal antibody may be any solution containing blood cells or tissue cells. Preferably, the cell mixture is derived from mammalian normal cells, mammalian bone marrow or circulation, or tissue suspected of being a tumor, especially normal cells, leukemia cells, and solid tumor cells. When the cell mixture is contacted with the monoclonal antibody, the cells having the Tie receptors bind to the monoclonal antibody to form the antibody-Tie receptor complex. Identification of this antibody-Tie receptor complex, and thus the Tie-10 receptor, can be accomplished using methods known in the art. These methods include e.g. ELISA, IRMA (sandwich-type immunochemical method), immunohistochemistry, 125 I-labeled RIA and autoradiography.

The present invention also relates to a method of imaging neovascularization in a human patient in wound healing, inflammatory condition, or tumor. This method involves the administration of labeled antibodies and imaging to identify targets that present endothelial cells involved in the formation of new vessels, or to detect leukemia cells in the blood, bone marrow, or tissue.

The humanized monoclonal antibodies of the invention are useful in treating neoplastic diseases involving Tie receptor endothelial cells by administering a therapeutically effective amount of an anti-neoplastic therapeutic agent coupled to such a monoclonal antibody to a patient with such a disease. A therapeutically effective amount of a therapeutic agent is an amount that inhibits tumor growth, preferably causing neoplastic cell death and a decrease in the total number of neoplastic cells in the body. Examples of such therapeutic agents are, for example, antibodies bound to a 90Y radioisotope or a toxin conjugate such as ricin or various microbitoxins.

Conjugation of a substance for the treatment of leukemia to a monoclonal antibody may be performed using methods known per se, such as, for example, in Press et al., J. Clin.Oncol. 7: 1027-1038 (1989). Preferably, the monoclonal antibody conjugation site 35 is located at a site which is not close to the monoclonal antibody Tie111267 7 receptor binding site. It is also preferred that the conjugation site of the therapeutic agent is a different functional group than the active site of the therapeutic agent. More preferably, the conjugation site is also at a point where structural changes in the monoclonal antibody or therapeutic agent are kept to a minimum.

The present invention also relates to a method of treating neoplastic diseases comprising administering a therapeutically effective amount of a therapeutic agent conjugated to a binding moiety of the monoclonal antibody of the invention having the specific portions of the antibody that bind the antibody. Suitable binding fragments are of sufficient size and structure to allow binding to the Tie receptor. Such fragments can be prepared using a variety of methods known per se. The resulting binding fragments can be screened for Tie receptor binding ability using the 15 binding assays described in Example 5.

Administration of the monoclonal antibodies of the present invention means the administration of an appropriate amount of a pharmaceutical composition containing the monoclonal antibody as an active agent. In addition to the active ingredient, the pharmaceutical composition may contain suitable buffers, diluents, and additives. Suitable buffers include e.g. Tris-HCl, acetate, glycine and phosphate, preferably a phosphate buffer having a pH of 6.5 to 7.5. Suitable diluents include sterile aqueous solutions of NaCl, lactose or mannitol, preferably NaCl. Suitable additives include albumin or helartin which inhibit surface absorption, detergents (such as TWEEN 20 ™, 25 TWEEN 80 ™), solubilizers (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite) and preservatives (e.g. , benzyl alcohol, paraben).

Administration may be by conventional means such as intravenous, subcutaneous or intramuscular administration. Intravenous administration is preferred.

The mode of administration may be a single dose or may be administered in appropriate amounts in divided doses.

Preferably, the pharmaceutical preparation is in unit dosage form. The preparation is then subdivided into unit doses containing a suitable amount of the active ingredient, e.g., an effective amount to achieve the desired effect.

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The actual dose used may vary depending on the needs of the patient and the severity of the condition being treated. Determination of the correct dosage for a given situation is within the skill of the art. Generally, treatment is initiated at a lower dose, less than the optimal dose. This dosage is gradually increased until optimum efficacy is achieved. In practice, the total daily dose may be divided and administered as desired, either continuously or in separate portions throughout the day. The dosage and frequency of administration are adjusted according to the judgment of the attending physician, taking into account factors such as age, general condition and weight of the patient, as well as the severity of the disease being treated.

The usual preferred dosage according to the present invention is about 0.1 to 10 mg of active ingredient per day.

The development and use of mouse antibodies as therapeutic agents suffers from the fact that their half-life in humans is low due to the formation of the human anti-mouse antibody response (HAMA). Therefore, the potency of mouse antibodies in patients is lower (Adair et al., 1990). Repeated administration of foreign proteins also causes adverse side effects. Many of these problems can be solved using human monoclonal antibodies. Currently, these antibodies can be produced from murine monoclonal antibodies using molecular biology methods, which associate the murine antibody binding interaction region (CDR) with a human monoclonal antibody. These humanized antibodies are suitable for human immunotherapy. One can also * 25 construct single chain antibodies (scFv). These are constructed using linker sequences of various lengths, such as Whitlow et al. in Protein Eng., 6 (8): 989-95, (1993), whereby the binding of the antibody to the antigen is optimized.

As stated above, the antibodies of the present invention are useful in the diagnosis and recognition of disease states (e.g., various types of cancers), in the detection and monitoring of wound healing, in the treatment and prevention of various neoplastic diseases. Other uses of the agent described herein will be apparent to those skilled in the art.

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EXAMPLES

The following examples are provided to illustrate specific uses of the present invention without limiting its scope. The obvious value of other uses and applications 5 will be fully understood by those skilled in the art.

EXAMPLE 1

Production of the extracellular part of the Tie receptor by the baculovirus

The cDNA sequence of the Tie protein is described in Partanen J. et al., Mol. Cell Biol. 12: 1698-1707, (1992), which is hereby incorporated herein by reference. The cDNA sequence encoding the outer Tie portion of the cell ηο was amplified by PCR and cloned into the BamHI site of the PVT-Bac vector (Tessier et al., Gene, 98: 177-183, 1991) using 5'-CGTAGATCTGGCGGTGGACCTGAC as PCR primers. and 5'-GGCCATGATCACTAGTGATGGTGATGGTGATGCTGCTGATCCAGGCC CTCTTCAGC-3 '.

The sequence encoding the Factor X cleavage site (IEGR) followed by six consecutive histidine residues was inserted at the 3 'end of the cDNA. The vector thus obtained, PVT-Tie, was transfected into insect cells to express the outer portion of the Tie cell.

20 PVT-Road was transfected with Baculo Gold baculovirus DNA (Pharmingen Cat. 21100D) into SF-9 insect cells. Virus isolates were purified from the culture broth (TNMFH + 5% FCS) of cells transfected with agarose and screened for the best expression of recombinant protein in High Five insect cells (Invitrogen). One isolate 25 (BG-3 virus) was selected for large-scale protein production.

High Five cells were infected with BG-3 virus and the infected cells broth (EX-CELL 400, HRH Scientific) was harvested after two days. The recombinant BG-3 protein was purified from the culture broth by ConA affinity chromatography.

EXAMPLE 2 111267 1 o

Production of Anti-Tie Monoclonal Antibodies in Balb / C Mouse

Three-month-old Balb / C female mice were immunized by intraperitoneal injection of BG-3 (50 pg / mouse) emulsified in Freund's 5 complete adjuvant solution. Every three to four weeks, 50 µg of supplemental injections and a final booster injection (20 µg of BG-3 in PBS intravenously) were given after three weeks. Mice were sacrificed four days after the last dose and mouse spleen lymphoid cells were attached to SP 2/0 plasma cytoma cells in a ratio of 2: 1. Fusion cells were harvested in a 96-10 well culture plate (Nunc) in Ex-Cell 320 growth broth (Seralab) containing fetal calf serum (FCS, 20%) and HAT supplement (hypoxanthine-aminopterin-thymidine, Gibco, 043-01060H, 50x diluted). Cells were cultured at + 37 ° C, 5% CO 2. After 10 days, HAT-supplemented broth was switched to HT-supplemented cell culture broth 15 (Gibco, 043-0106H, 50x diluted). The HT broth was identical to the HAT broth except that aminopterin was absent.

Two to three weeks after the fusion, antibody production was checked by the antigen-specific immunofluorometric method described in Example 5, IFMA. The main clones were cloned by limiting dilution (Staszewski, 1984). Positive clones were expanded into a 24-well tissue culture plate (Nunc), cloned again and retested by the same method. Positive clones were further tested by fluorescence-activated cell isolation (FACS). Stable clones secrete IgG immunoglobulins.

One clone, 3C4C7G6, stably secreted the monoclonal antibody found to be of IgG 1 immunoglobulin class by IFMA. Hybridoma 3C4C7G6 was deposited in the German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Mascheroder Weg 1b. , Germany, December 30, 1993, and was assigned the accession number ACC2159.

Other clones were produced by similar methods which produced Tie-specific monoclonal antibodies against the same or another epitope. One such clone, 10F11G6, was selected for further experiments in combination with the 3C4C7G6 antibody described above.

11126711 Balb / C mice were used to produce monoclonal antibodies in ascites fluid. The hybridomas were injected intraperitoneally (i.p.) into mice after pristane treatment (98%, 2,6,10,14-tetramethylpentadecan, Aldrich-Chemie D-7924 Steinheim, cat.no T 2,280-2). 0.5 ml of pristane (i.p.) was injected 5 approximately two weeks before the hybridoma cells were injected. The number of cells injected was 7.5-9 x 10® per mouse. The resulting ascites were harvested 10-14 days after the hybrid injection and contained an average of 0.3 mg / ml antibody as determined by the antigen-specific IFMA assay described in Example 6.

10 EXAMPLE 3

Production of Anti-Tie Monoclonal Antibodies in Hollow Fiber Bioreactor

Monoclonal antibodies were produced against Know in vitro using a Technomouse System (Cellex Inc.) bioreactor. Korkkeineen the roller bottles with filter 15 and the first autoclaved at 121 ° C and 1.1 bar pressure for half an hour. They were then filled with 1L Dulbeccon MEM (Gibco, 042-02501, glucose 6.4 g / L, glutamine 2 mmol / L 066-1051 H, Na-pyruvate 1 mmol / L 066-1840E). The bioreactor holder was aseptically transferred to the Techomouse tray. The pump was charged and the culture broth hoses and empty waste bottles 20 (outflow hose) were aseptically connected.

The filling and rinsing program was performed as recommended by the manufacturer to remove any residual material from the bioreactor's intracapillary space (IC). The program was started at a flow rate of 150 ml / h for 4 hours. Thereafter, washing was carried out at a flow rate of 50 ml / h for 20 hours, during which the extracapillary (EC) state was also washed with 5% FCS-enriched Dulbecco MEM (DMEM). The EC broth was replaced aseptically with a new broth. One day after that, the bioreactor was ready to be charged with hybridoma cells.

Hybridoma cells were grown in cell culture flasks in 10% FCS-DMEM and 30 72 x 10 6 cells were harvested and loaded into a reactor in 5 µl of DMEM with 5% FCS. The flow rate of the intracapillary space was 100 ml / h. The recycling method was used to recover the monoclonal antibodies as follows: the culture broth tube was connected to the culture flask 1 2 111267 "out" to remove the culture broth from the bottle into the intracapillary space of the bioreactor; The outflow hose was connected to a growth bottle "in", whereupon the broth was returned to the bottle. Monoclonal antibodies were harvested three times a week, on Mondays, Wednesdays, and Fridays, and each time 10 ml of new 5% FCS-DMEM was added.

The anti-BG-3 cell line, 3C4C7G6, produced an average antibody of 152 pg / ml in the cell culture broth. After cell inoculation into the Technomouse System Bioreactor (72 x 10 6 cells), antibodies were harvested in 2-3 day intervals. The average production was 4.5 mg / week and the cumulative production over the two months was 37 mg.

Antibodies produced in ascites fluid or Technomouse system were purified using Affigel ™ Protein A MAPS II Kit (BioRad) according to the manufacturer's instructions. The column was equilibrated for purification with binding buffer (pH 9.0). Antibody bound to protein A matrix in binding buffer and the column was washed with binding buffer until baseline (detected at 280 nm by UV spectrophotometry). The specifically bound material was eluted from the protein A in elution buffer pH 3.0 and the fractions were collected in tubes containing 1 mol / L Tris-HCl, pH 9.0, which was required to immediately neutralize the fractions. The column was regenerated with regeneration buffer 20 and stored for subsequent use in 50 mM Na phosphate buffer containing 0.05% NaN 3 as a preservative.

EXAMPLE 4

Antibodies against the pathway expressed by bacteria

The Bam HI fragment of the Tie cDNA (nucleotides 520-1087) was subcloned into the Bam HI site of 25 pGEXIT vector (Pharmacia) to give an open reading sequence encoding glutathione S-transferase fused to the amino acid 162-350 coding region of Tie (GST-Tie2). ). The construct was transformed into E.coli strain DH5a and expression of the fusion protein was induced by IPTG. The resulting 40 kD fusion protein was purified on 30 denaturing agarose gels (FMC) and used for immunizations.

Monoclonal antibodies were produced as described above against BG-3 Tie protein. After two subcloning, eight clones were obtained which produced antibodies against the GST-Tie2 protein. These 13111267 reacted approximately equally to Tie in Western immunoblotting. The ascites of the anti-GST-Tie2 clones were purified using a protein A column and used for Western blotting.

EXAMPLE 5 5 Europium Labeling of Tie Protein

The extracellular portion of Tie produced in Example 1, BG-3, was labeled for test use. Labeling was performed as described in Mukkala et al., Anal.Biochem. 176 (2): 319-325, (1989), with the following modifications: To a BG-3 solution (0.5 mg / ml in 50 mmol / L borate buffer, pH 8.6) was added 125 ul molar excess of isothiocyanate-DTTA-Eu (N chelate, Wallac, Finland) and the pH was adjusted to 9.8 by the addition of a tenth of 0.5 mol / L sodium carbonate buffer (Merck), pH 9.8. The labeling was performed overnight at + 4 ° C. Unbound label was removed with PD-10 (Pharmacia, Sweden) using TSA buffer (50 mmol / L Tris-HCl, pH 7.8, 0.15 mol / L NaCl) as the elution buffer.

After purification, 1 mg / ml bovine serum albumin (BSA) was added to labeled BG-3 and the label was stored at + 4 ° C.

The amount of bound europium per BG-3 molecule was 2.9 as determined by fluorescence measurement and compared to known EUCI3-20 standards (Hemmilä et al., Anal.Biochem. 137: 335-343, 1984).

Example 6

Immunofluorometric screening method (IFMA1

Antibodies raised against the Tie receptor were screened using a sandwich type immunofluorometric assay in '25 wells of a microtiter plate (Nunc, polysorb) coated with rabbit anti-mouse Ig (Z 259,

Dakopatts, Lövgren et al., Talanta 1984; 31 (10B): 909-916). The pre-coated wells were washed once with Platewash (1296-024, Wallac) using DELFIA wash. The DELFIA assay buffer was used as a dilution buffer for cell culture supernatants and serum from mouse splenectomy 30 (dilutions 1: 1000-1: 100,000) used as a positive control: in preliminary screening experiments.

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Anti-BG-3 / 3C4C7G6 antibody produced in ascites fluid and purified with Affigel ™ Protein A-MAPS was used as a standard in subsequent experiments at concentrations of 0.25 ng / ml to 60 ng / ml in assay buffer (1 µmΙ, DELFIA).

Incubation was started for 2 hours at room temperature (or alternatively overnight incubation at + 4 ° C) by shaking on a Plateshaker (1296-001, Wallac) for 5 minutes, followed by four washes with wash solution as described above.

Eu-labeled BG-3 produced as described in Example 4 was added at 10 ng / well in 100 silicon test buffer. After five minutes

In a Plateshaker and after incubation for 1 hour at room temperature, the plates were washed as described above.

DELFIA enhancement solution was added at 200 μΙ / well. Plates were shaken for 5 min on a Plateshaker and fluorescence intensity was measured on an ARCUS-15 1230 (Wallac) for 10-15 min ((Lövgren et al., In: Collins WP (ed), Alternative Immunoassays. John Wiley & Sons Ltd, 1985; 203-216).

The sandwich type DELFIA method is very sensitive, with a theoretical sensitivity of less than 0.25 ng / ml for this anti-Tie monoclonal antibody. Although the sensitivity was appropriate for quantification of Mabs produced in cell culture, the method was also practical for quantifying in vitro produced Mabs. The linear range was 0.25 ng / ml to 60 ng / ml (Figure 2). The intra-test variability was found to be very low.

EXAMPLE 7

Radiolabeled monoclonal antibodies for in vivo detection of Tie receptor

Monoclonal antibody 3C4C7G6 was labeled with 125 I using the chloramine T method (Greenwood et al., Biochem J. 89: 114-123, 1963). 1 mCi of Na125I was used to label 40 pg of antibody. Labeled antibodies were purified by elution with Sephadex-G25 ™ to give the bulk of the 30 labeled antibodies in 1.8ml.

1112671-125-labeled anti-BG-3 (3C4C7G6) was administered i.v. in two different doses, 1.2 l-125-labeled anti-BG-3 (3C4C7G6) was administered i.v. at two different dose levels, 1.2 pg and 2.4 pg Lewis lung carcinoma mice. The biodistribution of labeled antibody in the mouse was measured at five 5 time points: 1) 6h (N = 4), 2) 24h (N = 7), 3) 47h (N = 5), 4) 70h (N = 3) and 5) 117h. (n = 2).

Table 1. Biodistribution to tissues at time points 1 - 5 ίο _1. 2. 3. 4._5.

% ID / g% ID / g% ID / g% ID / g% ID / g 6h 24h 47h 70h 117h blood 36,22871 11,26086 9,343041 7,87285 2,749225 hearth 7,828641 2.842418 1.932395 1.391288 0.548053 aortha 16.07039 5.553582 3.707159 3.166192 0.798207 lung 9.091084 3.823566 3.087934 2.555693 0.987308 liver 6,940821 2, 355467 1.770392 1.568976 0.685282 kidney 9.331627 3,490624 2,556933 1,829169 0.802883 brain 0.548253 0.2228239 0.149502 0.146163 0.042287 blood Vess 22.64432 4.712374 2,435758 2,65011 0,485038 tumour 7,151142 3,620,555 3,155919 2,179194 0,901169 spleen 5,840843 2,004754 1,396837 1,195612 0,622342 bladder 6,889924 4,36051 3, 628196 3,523912 0,899099 ovarias 8,445497 3,617,958 2,622101 2,387713 0,662774

The results indicate that anti-BG-3 activity was predominantly in the blood, tumor and blood vessels, and to some extent in the lungs and ovaries. Blood activity was high at 48 and 70 hour points: 9.3% ID / g and 7.9% ID / g (expressed as a percentage of the injected dose per gram of tissue and normalized to 20 g in the mouse). Anti-Bg-3 activity in the blood was 11.3% at the 24-hour and 36.2% at the 6-hour measuring points. 11 L-labeled anti-BG-3 (3C4C7G6) was also administered to mice with 8 to 20 days of healing wound in the epithelium of the skin. The dose given to these mice was 0.03 µg / animal. The biodistribution of the active antibody is shown in Figure 3. In this figure, the Y-axis represents the dose rate in% (% ID / g) and the X-axis represents injection time points: 4h (N = 2); 24h (N = 6); 48h (N = 6); 72h (N = 4) and 120h (N = 4). There is a steady state between target tissue and plasma, which is also shown in Figure 3.

1 6 111267 EXAMPLE 8

Tc-99m Labeling of Anti-Tie Monoclonal Antibodies for Imaging Assay

The antibody was labeled with 99m technetium as described in Schwarz et al., 5 J.NucI.Med., 1987, 28: 721, and Mather et al., J.NucI.Med., 1990, 31: 692-697. method. 2-Mercaptoethanol (ME) was used to open the disulfide bonds of the immunoglobulin heavy chain junction. The antibody was concentrated to about 10 mg / L and enough ME was added to the solution to give a molar ratio of 1000: 1 (0.47 μΙ ME / 1 mg of antibody). The mixture was incubated at room temperature for 30 min and the reduced antibody was purified by gel filtration on a 20 mL Sephadex-G-50 column and eluted using phosphate buffered saline as the mobile phase. Antibody fractions were pooled after optical density measurement (280 nm) and stored at -20 ° C in 0.5 mg aliquots for 99m-Tc 15 labeling.

For 99m Tc labeling, batches of antibody were thawed and reconstituted using methylene diphosphonate (MDP) in a bone imaging kit (Amerscan Medronate II Technetium Bone Agent, N.165) with 5 ml of 0.9% sterile saline according to the manufacturer's instructions. To the antibody batch was added 35 μΙ MDP solution containing 35 pg MDP and 2.4 pg SnF2 and mixed well. 99m-Tc pertechnetate was added to the mixture and shaken gently. The reaction was completed in 10 min. Radiochemical purity was measured by HPLC.

Labeling of the reduced antibody produces a stable 99m-Tc-labeled immunoglobulin because of the minimal non-specific binding of the label.

Labeling efficiency is determined by thin layer chromatography in 0.9% saline. Immunoreactivity remains above 85%. In vivo stability is analyzed by an in vitro cysteine assay.

99mTc-labeled anti-Tie antibodies can be detected with a * conventional gamma camera or SPECT (Single Photon 30 Emission Computerized Tomography) to provide a picture of antibody turnover in the human body.

1 7 111267 EXAMPLE 9

Detection of path positive cell lines by FACS

The anti-Tie antibodies prepared in Examples 2, 3 and 4 were used to detect Tie protein from human leukemia cell lines.

Hematopoietic cell lines HEL (human erythroleukemia) and MOLT-4 (T-lymphoblastic leukemia) were obtained from ATCC. HEL cells also expressing erythroid and megakaryocyte markers were used for indirect immuno-fluorescence staining of Tie using monoclonal antibodies and fluorescence activated cell (sorting) assay. Cells were counted, washed and incubated with antibodies in multiple dilutions (1: 1 to 1: 200), washed and incubated with FITC-conjugated mouse anti-immunoglobulin antibodies (secondary antibodies). Analyzes were performed using FACS IV equipment. As a negative control, cells stained with non-specific mouse immunoglobulin were used, followed by the same secondary antibodies. As a negative cell control, we used the MOLT-4 T cell leukemia cell line, which does not express Tie mRNA.

The results show that on average 85% of HEL cells stained anti-Tie antibodies, whereas less than 1% of MOLT cells stained 20 positive for Tie, respectively. When the cells of these two cell lines are mixed, the antibodies separate positive HEL cells from negative MOLT cells (Figure 1). In a human bone marrow specimen, less than 1% of the cells also show positive staining with these antibodies.

Cells of the human leukemia cell line MOLT4 (malignant T cell line that is Tie mRNA negative) and HEL cells (human erythroleukemia cell line which are Tie mRNA positive) were mixed in a ratio of about 1: 1.

The cells were then stained in suspension using the monoclonal Tie antibodies of Example 2 diluted 1:10 and FITC-conjugated anti-mouse IgG secondary antibody. As a negative control, the antibody was replaced with normal mouse serum. Analysis was performed on FACS IV. The result is two separate cell populations, one Tie-positive and one Tie-negative, each representing approximately 50% of the total cell population analyzed (Figure 1).

1 8 111267

Some Tie positive bone marrow cells were also positive for the CD19B cell marker and all were CD38 negative. This indicates that the Tie is expressed in the B cell line.

EXAMPLE 10 5 Analyzing Monoclonal Antibodies

The ability of monoclonal cell culture supernatants to recognize the Tie receptor on the surface of cells was tested using FACS analysis. NIH3T3 cells transfected with the Tie expression vector (full length Tie cDNA in the pLTRpoly vector, Mäkelä et al., 1991), NIH3T3 cells transfected with the control vector, and both HEL cells io (human erythroleukemia cell line, which endogenously expresses TOL). non-expressing cell line) was incubated in culture broths of different cell clones followed by FITC-labeled rabbit anti-mouse antibodies (Dako). Labeled cells were analyzed with a fluorescence-activated cell counter (Becton Dickinson).

Ascites were also produced from the following clones: 1H3H7, 3C4C7, 5C12H9. The 3C4D7 clone was subcloned and 3C4C7G6, 3C4C7B11, 3C4C7E7, 3C4C7F9 subclones were obtained which were similar.

1 9 111267

Table 2.

Results of anti-Tie monoclonal antibodies recognizing the outer part of the cell

Clone dolphin slot blot Western FACS

5 BG-3 _media denat._ 1H3F10 +++ +++ +++ (+) H6 +++ +++ +++ (+) ίο H7 +++ +++ +++ (+) 3C4C7 + ++ +++ + - ++ E4 +++ ++ (+) - + G4 +++ ++ (+) - + 5C12G11 +++ +++ +++ 15 H9 +++ +++ + ++ (+) 6A11A11 ++ ++ + + H6 ++ ++ (+) - + H9 ++ ++ + - + 9B10E6 + ++ +++ 20 G7 + ++ +++ 9E7E9 +++ + ++ +++ (+) E10 +++ ++ ++ (+) _H6 _ +++ _ ++ _ ++ _ (+) _ ^ _ 1 2 3 4 5 6 7 8 9 10 11 EXAMPLE 11 2

Tie's immunoperoxidase staining in human blood cells

The patient was given cyclophosphamide to hematopoietic stem cells 3:; mobilized to peripheral blood, and after 7 days, 4 x 5 peripheral blood auditory layer cells were harvested. The red cells were hemolysed from 6 cell suspensions and cytocentrifuge strips were made. These were used in 7 immunostained purified Tie monoclonal antibody 8 by the 3C4C7G6 immunoperoxidase method. By double immunofluorescence staining of bone marrow cells 9, Tie positive cells (less than 10 '· 1%) could not be clearly identified as belonging to any hematopoietic lineage.

11

5 positively stained cells were identified from about 70,000 cells per strip. In immunoperoxidase staining, Tie-positive cells were small and round and had a large nucleus and scarce cytoplasm (Figure 2). (The dark color of one positive cell in the image is due to peroxidase staining and indicates a Tie positive cell). Thus, anti-Tie monoclonal antibodies recognize specific hematopoietic cells from the bone marrow and can be used to identify specific subsets of hematopoietic cells.

EXAMPLE 12

Humanization of 3C4C7G6 Monoclonal Antibody 3C4C7G6 can be humanized using methods previously described (Kolbinger et al., 1993; Kettleborough et al., 1991). The humanization method involves incorporating the mouse kappa light chain (L) and heavy chain (H) determinant regions (CDRs) into human variable regions (V) and providing point mutations in the ίο peripheral regions of the human antibody, while maintaining the original CDR conformation.

The reconstituted VL and VH chains are inserted into DNA encoding the human kappa and gamma-1 constant regions, respectively, by means of suitable expression vectors.

scFv is produced as previously described in Whitlow et al., 15 supra, (1993).

The affinities and scFvs of the humanized monoclonal antibodies are tested using the methods previously described in this application.

EXAMPLE 13

Conjugation of Tie-Specific Monoclonal Antibodies to a Therapeutic Substance

Monoclonal antibody 3C4C7G6, or the humanized antibodies described in Example 12, can be coupled or conjugated to various agents for diagnostic use, as described in the other examples herein, or for therapeutic use of the conjugates produced. 1

For example, in tumor therapy or in the treatment of dispersion malignancy such as leukemia, antibodies can be coupled to radioisotopes such as, 32P, 1311, 125I, 90Y, 188Re, 212Pb, 212Bi or 10B (See, e.g., Scheinberg et al., Oncology, 1: 31-37, 1987) ). Conjugation of radioisotopes to the antibody is accomplished by directly attaching the isotope to the antibody using the methods described previously (See eg, Schwartz J., Nuclear Medicine 21111267 28: 721, 1987) or by chelating linkers that bind the radioisotope to the antibody, or with an antibody that binds to a specific antibody. Various other agents may also be linked to antibodies. Such agents include antitumor drugs and antibiotics that are toxic because they bind to DNA by intercalation (eg daunomycin, adriamycin, aclasinomycin) or cleave DNA (eg esperamycin, calikeamycin, neocasstatin) and other toxic cytostatic drugs such as see, e.g., Greenfield et al., Antibody, Immunoconjucates and Radiopharmaceuticals, 4: 107-119, 1991). These agents are covalently linked using appropriate derivatives of the agents.

Many proteins and glycoproteins can also be used as therapeutic conjugates of antibodies. These include, for example, bacterial toxins such as Diphtheria toxin, Shigelloxin, and Pseudomonan exotoxin; plant toxins such as ricin, abrin, modeckin, viscumin, antiviral protein isolated from sour cherry, saporin, momordine and gelonin. These toxins contain a catalytic fragment and, in some cases, fragments or portions which recognize cell surface structures or facilitate passage through the cell membrane. Suitably modified toxins are used that provide improved specificity without reduced efficacy. Conjugation of toxins to antibodies is accomplished by heterobifunctional linkers such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) or 2-iminothiolane.

Prior to therapeutic use, conjugated antibodies are tested for toxic activity, target specificity, in vitro and in vivo stability, and other properties (see, e.g., Immunotoxins, Ed. Frankel, Kluwer Academic Publishers, Boston, 1988). It is desirable that the toxicity of the conjugated agent and the binding affinity and specificity of the antibody be minimized with the binding method used. Therefore, the binding of conjugates to the Tie receptor is tested (see Example 10). In vitro toxicity in target cells, such as the Dami leukemia cell line, is assayed by measuring bound labeled compound relative to control conjugate treated cell culture, and more directly, by assaying cultures capable of growing in cloning assays and extrapolation of 35 cell growth rate in the assay. In vivo stability, elimination rate and specific toxicity are determined by administering conjugates 22111267 to a suitable target animal such as a mouse, rat, rabbit or monkey. Other targets include normal mice and in vivo tumor and leukemia models, such as human cancer cells administered to immuno-deficient mouse strains, such as a nude mouse or an SCID mouse.

5 EXAMPLE 14

Preparation of a pharmaceutical composition containing the monoclonal antibody 3C4C7G6

The pharmaceutical compositions of the present invention contain an effective amount of an active agent, a Tie-specific monoclonal antibody, in particular monoclonal antibody 3C4C7G6, alone or in combination with a suitable buffer, diluent and / or excipient. Such compositions are prepared as sterile aqueous solutions or as lyophilized or otherwise dried formulations. Preferably, the antibodies are formulated in such a carrier at a concentration of 1 mg / ml to 10 mg / ml.

One example of an injectable pharmaceutical composition contains 3C4C7G6 monoclonal antibody (1 mg / ml) in an aqueous buffered solution of monobasic sodium phosphate solution (0.45 mg / ml; pH 7.0 ± 0.5) and Tween 80 ™ (0.2 mg / ml). The compositions of the invention are administered in dosages determined by one of ordinary skill in the art, taking into account the disease, the severity of the symptoms and the characteristics of the patient. As an example, the pharmaceutical compositions of the invention may be administered topically to achieve maximal effect in arresting tumor growth and neovascularization or wound healing. However, different doses are required for systemic use, depending on the characteristics of the patient, such as weight, age, disease progression, metabolism, etc.

Other uses may be apparent to one skilled in the art upon consideration of this description. Thus, the present invention is limited only by the requirements below.

23 111267 EXAMPLE 15

Immunohistochemical staining of Tie in normal brain, glioblastoma multifore, and melanoma metastases

Recent untreated cerebellar specimens and intracranial meningeal specimens were obtained from an open-air neurological surgery unit at Helsinki University Hospital. All tumor patients received corticosteroid treatment. A non-neoplastic control brain tissue sample was obtained from surgery for severe epilepsy. Several tumor specimens also contained the surrounding brain tissue. All samples were frozen in liquid nitrogen as soon as possible after surgery and stored at -70 ° C. The histopathological diagnosis was based on hematoxylin / eosin-stained ice cubes and routinely stained paraffin sections of formaldehyde-fixed parallel tumor specimens. Table 3 provides a complete list of samples.

15 Table 3. Human tissue samples

Well kudosnäyte__Age (y) Sex_ 1 kontrolliako__66__M_ 2 kontrolliako__Y \ __ F_ 3 grade II oligoastrosytoma__34__F_ 4 grade II oligoastrosytoma__66__M_ 5 grade III astrosytoma__36__M_ 6 _ GBM__53__M_ _7_ GBM__63__M_ 8 _ GBM__56_JM_ 9 GBM adjacent kudos__69__F_ 10 grade I meningeoma__52__M_ 11 grade II meningeoma__35__F_ 12 grade II meningeoma__55__M_ 13 melanoma metastasis__54__M_ 14 me surrounding tissue__67__M_ 15 Stage III glioma__44__M_ 16 | gbm 65 | m GBM = glioblastoma multiforme 24 111267

Sterilized plates were dipped in 2% aminopropyltriethoxysilane (TESPA) in acetone to ensure tissue adhesion. 5-μιτι ice cutters were cut on pre-treated plates at -20 ° C, fixed in 4% paraformaldehyde and stored at -70 ° C.

5-pm ice sections of the samples used in the in situ hybridization assay and two additional malignant glioma samples were cut on TESPA plates as described above. The sections were dried in vacuo at 37 ° C overnight and stained immunohistochemically using murine monoclonal antibodies against human Tie (a mixture of a few β1 monoclonal antibodies recognizing the extracellular portion of Tie) and human von Willebrand factor (vWF). rabbit antibodies (Dakopatts) as an endothelial cell-specific marker.

Staining was performed using the Vectastain ABC Elite Biotin / Avid system (Vector Laboratories, Burlingame, CA) for mouse IgG.

The endogenous peroxidase activity of the dried, non-fixed tissue section was attenuated by treatment with 0.5% H2O2 in methanol for 30 min. Washed with PBS followed by incubation of the sections with diluted Vectasta blocking serum for 20 min and then with undiluted Tie antibody (1: 1) overnight at 4 ° C or von Willebrand factor antibody (1:50) for 1 20 hours at room temperature. temperature. The biotinylated secondary antibody was added for 45 min, followed by the addition of Vectastain ABC reagent for 30 min. The reaction was visualized with 0.2 mg / ml 3-amino-9-ethylcarbazole (AEC), 0.03% H 2 O 2, 14 mM acetic acid and 33 mM sodium acetal. Normal serum and. antigen-blocked primary antibodies were used as controls. Slices 25 were counterstained lightly with hematoxylin and stained in Aquoamount.

The vasculature of normal cortex tissue was Tie protein-negative (Figure 4), whereas in the glioblastoma vasculature (B) and endothelial tissue close to GBM (B, indentation), strong staining was observed. Tie protein was also detected in melanoma:. 30 metastases in vascular endothelial cells (C) and at the border of the tumor (not shown). When the sections were incubated with antigen-blocking antibody or normal serum in place of Tie antibodies, no specific staining was observed. For further evaluation of the amount and specificity of the tie staining, close sections were stained with the VIII factor. Table 4 summarizes 35 Tie staining results.

25 111267

Table 4. Tie immunohistological staining

No Tissue__Tie ab 2 Normal Brain __: _ 15 Stage III glioma __ ++ _ 7_GBM * _ +++ 16 GBM_ I +++ 11 Stage I meningoma __ ++ _ 13 melanoma metastasis __ ++ _ These results indicate that TIE plays an important role in tumor progression-related angiogenesis.

5 EXAMPLE 16

Immunohistochemical Detection of Tie Protein in Hemanioblastoma and Hemorrhoidal Nonoperative In this study, we analyzed the expression of endothelial growth factors and ßο receptors in two highly vascular CNS tumors of controversial origin; capillary hemagioblasoma and hemangioperic cytoma.

Recent samples of hemangioblastoma and hemangioperoma cytoplasm were obtained from an open surgery at the Neurological Surgery Unit at University of Helsinki 15 Hospital. (All tumor patients received corticosteroid treatment). The samples were immediately frozen immediately after surgery and stored at -70 ° C. The histopathological diagnosis was based on hematoxylin / eosin-stained ice cubes and routinely stained paraffin sections of formaldehyde-fixed tumor specimens.

Sterilized plates were treated with 2% aminopropyltriethoxysilane (TESPA) in acetone to ensure tissue adhesion. 5-μιτι icicles were cut on the plates, fixed in 4% paraformaldehyde in PBS and stored at -70 ° C.

The 5-μητι ice sections of the samples used in the in situ hybridization assay: 25 were cut on TESPA plates as described above. The sections were dried in vacuo at 26111267 at 37 ° C overnight and stained immunohistochemically using a mouse monoclonal antibody against human Tie (monoclonal antibodies recognizing the extracellular portion of Tie were obtained from Dr. Juha Partanen) and using human von Willebrand. 5 rabbit antibodies (Dakopatts) against factor (vWF) and CD34 as an endothelial cell specific marker (36).

Staining was performed using the Vectastain ABC Elite Biotin / Avid system (Vector Laboratories, Burlingame, CA) for mouse IgG. The endogenous peroxidase-10 activity of the dried, non-fixed tissue section was blocked by treatment with 0.5% H2O2 in methanol for 30 min.

Washed with PBS followed by incubation with diluted Vectastai blocking serum for 20 min followed by undiluted Tie antibody (1: 1) overnight at 4 ° C or CD34 / von Willebrand factor (1:50) for 1 h. at room temperature. Biotinylated secondary antibody 15 was added for 45 min. followed by the addition of Vectastain ABC reagent for 30 min. The reaction was visualized with 0.2 mg / ml 3-amino-9-ethylcarbazole (AEC), 0.03% H 2 O 2, 14 mM acetic acid and 33 mM sodium acetate. Normal serum and antigen-blocked primary antibodies were used as controls. Sections were counterstained lightly with hematoxylin and stained in Aquoamount.

Heavy staining of Tie protein was found both on the inner surface of the hemagioblastoma (Figure 4, A, B) and to a lesser extent on the hemangioperic cytoma (C-F). The amount of Tie protein varied significantly in tumor tissues, for example, being higher in the less obvious hemangioperic cytoma area (E, F), and lower in the more common tumor specimen (C, D). Overall, however, expression was significantly higher than in normal cortex samples previously analyzed (23). Compared to vWF immunostaining (B, D, F), the pathway was not expressed in hemangiopericytoma tumor cells (C-F).

:. These results support the hypothesis that Tie may influence the angiogenesis of these two vascular tumors.

27 111267 EXAMPLE 17

Use of anti-Tie antibodies in vivo in a mouse Lewis pulmonary carcinoma model

We have investigated the binding of radiolabeled (I125) anti-TIE monoclonal antibodies (10F11G6 and 3C4C7G6) to major organs in the mouse as well as lung metastases to determine the possibility of using anti-TIE monoclonal antibodies to detect malignant growth.

Radiolabelling of the antibodies was performed as follows: 40 μ9 antibodies were labeled by the Chloramin-T method using 37 MBq Na-125 I as described in Greenwood et al., Supra, 1963. The labeled antibody was purified with Sephadex-G25 and brought to a main fraction of 1.8 ml. Prior to initiating in vivo biodistribution studies, the affinities of three radiolabeled antibodies (3c4, 10F11, 7E8) were compared in a cell assay (Lindmo, Nucl Med 21: 15807-810, 1984). All antibodies bound specifically to LE GD 14-2 cells, which were transfected mouse endothelial cells that absorbed the TIE receptor on the outer surface, but 10F11 bound three-fold compared to the others (21 pMol / 100,000 cells vs. 6-7 pMol / 100,000 cells). ).

For lung carcinoma experiments, we used 20 female mice 6-8 weeks old (C57BI / 6, Bomholtgaard, Denmark) with Lewis lung carcinoma (LLC1 / 2) tumors grown by injection of 1-10 M cells per animal into the right hindlimb.

The thyroid glands of mice were saturated with iodine by initiating administration of KI solution 25 days prior to injection of radiolabeled antibodies and continued throughout the experiment, giving KI solution 400 mg / 100ml ad libidum.

The distribution of I125-labeled anti-TIE antibodies in the body was examined at 24, 48, 72.96 and 120 hours after intravenous antibody injection (30 ng in 20 mM PBS). 14 Lewis 30 lung carcinoma test animals with lung metastases were detected with 10F11 antibody: one after 24 hours, three after 48 hours, three after 72 hours, three after 96 hours and four after 7 days (see Figure 6). Lung metastases were detected with 111267 with 28 antibodies 3C4 in three animals after 24, 48 and 120 hours (see Figure 7).

3C4 shows no binding to lung metastases at 48 hours (tissue / blood ratio 0.07), but both antibodies bind to metastases at 96 hours 5 in the Lewis lung carcinoma model, with tissue-to-blood ratios of 2.4 (3C4C7G6) and 2.8 (10F11G6).

The application of antibodies 3C4 and 10F11 to lung metastases is similar, both antibodies bind to metastases in vivo, but 10F11 binds more strongly to serum, 20% ID / g at 10F11 compared to 10% ID / g at 3C4 after 48 hours.

EXAMPLE 18

Analysis of serum samples in the IRMA method

Serum samples from normal and cancer patients have been screened by sandwich-type immunoradiometry (IRMA) 15 (Miles L. et al., Nature 219: 186-189 (1968), in which anti-TIE capture antibodies are attached to polystyrene tubes and secondary anti-TIE -TIE antibodies are labeled with 125 I using the Cloramin-T method previously mentioned Recombinant TIE protein was used as a standard sample to determine antigen concentration in a serum sample The concentration of the 20 undiluted standard samples used was determined at 280 nm and diluted in a standard curve. , with tie antigen concentration of 0. 18, 36, 72, 144, 720 and 3600 pg / L. Figure 9 shows standard curves of four pairs of antibodies, paired and labeled antibody.

25 This method screened serum samples from patients with breast cancer, ovarian and various lung cancers, and from non-cancer patients. Patients with new growing metastases in their lungs showed elevated values in this test. Patients with stable disease showed values comparable to those of non-cancer patients. Figure 10 shows the determined antigen serum concentrations in samples of 20 non-cancer patients and three recent metastatic cancer patients. A small cell lung cancer patient (SCLC) donated 6 samples, with monthly intervals of 29 to 111267, and an ovarian cancer patient with abdominal metastases donated 5 samples. A breast cancer patient with new lung metastases donated two samples. The antibody pair used in Figure 10 was 3C4C7G6 / 10F11G6, which had a higher sensitivity (> 5 µg / L) according to the standard curve.

5 «> -.

30, 111267

Applicant * oragent * fiic International applies -raNo.

reference number .Ia-MAB / SN__PCT / F1 9 5/0 0 1 7 Π

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCTRule 13iii) A. The indication * made below relate to the microorganism referred to in the description on page 4, line 15 ~ 20.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | ] Name of Depositary institution

DSM-DEUTSCHE SAMMLUNG VON MICROORGANISMEN UND 2ELLKULTUREN GmbH

Address of Depositary institution (including postal code and country)

Mascheroder Weg lb D-38124 Braunschweig DEUTSCHLAND

Date Deposit Accession Number 1993-12-02 DSM ACC2159

C. ADDITIONAL INDICATIONS (leave blank if not applicable) JJ

As regards the corresponding Patent Offices of the respective designated States, the Applicant requests that a sample of the deposited microorganisms be made available to an expert. been refused or withdrawn or is withdrawn to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (tflhr irnlinuiimr nrr nrtfrrtll dnignastd Stein) E> SEPARA IF. FURNISHING OF INDICATIONS (Leone blank if not applicable)

The indications listed below will be submitted to tbe International Bureau isicr (specify dicgaurainaiurc of ti% eindicationse.g ^ 'Accession Humber of Deposit')

For receiving Office use only For International Bureau use only

□ This sheet was received by tbe International Bureau on: I

___ 21 APRIL 1995, UJJft.95)

Authorized Officer,, ΉΓ; υ. Authorized Officer e. MOyse \ y

Fonn PCT / RQ / 134 (July 1992)

Claims (21)

111267 A monoclonal antibody which recognizes the extracellular portion of Tie receptor tyrosine kinase, characterized in that said antibody is an anti-Tie monoclonal antibody 5C4C7G6 and is produced by a hybridoma cell line deposited with DSM accession number ACC2159. The hybridoma cell line producing the monoclonal antibody of claim 1, wherein said hybridoma cell line is deposited with DSM accession number ACC2159. The antibody of claim 1, wherein said antibody is detectably labeled. The detectably labeled antibody of claim 3, wherein said detectable label is a radioisotope, fluorochrome, dye, enzyme, or biotin. The humanized antibody of claim 1, characterized in that the complementary region of the murine light and heavy chain in said monoclonal antibody is linked to a human variable region, and wherein the conformation of said complementary region of the mouse is maintained. The monoclonal antibody of any one of claims 1 or 5, characterized in that it contains a conjugated cytotoxic agent, a cytostatic drug or a glycoprotein. A method for identifying Tie in a biological sample, comprising the steps of a) contacting a sample suspected of containing Tie with a detectably labeled antibody to the extracellular portion of Tie according to claim 3 or 4, b) washing the sample; and c) detecting the presence of said detectably labeled antibody in said sample. A method according to claim 7, characterized in that the biological sample is a solid tissue sample or a solution containing blood cells or tissue cells. A method according to claim 8, characterized in that the cells are leukemia cells or bone marrow cells. A method for diagnosing diseases characterized by endothelial cell proliferation characterized in that it comprises the steps of 111267 a) collecting a tissue sample from a patient suspected of having a characteristic endothelial cell proliferation b) contacting said tissue sample with a detectably labeled Tie os cell; c) washing said tissue sample; and d) detecting the presence of said detectably labeled anti-Tie antibody in said tissue sample. 11. A method according to claim 10, characterized in that the disease is a neoplastic disease such as leukemia or a disease associated with tumor angiogenesis, wound healing, thromboembolic disease, atherosclerosis or inflammatory disease. The method according to claim 11, characterized in that the disease is megakaryoplastic leukemia, glioma, meningioma, metastatic melanoma, hemangioblastoma or hemangioperic cytoma. Use of a detectable labeled antibody according to any one of claims 3 or 4 in the manufacture of a preparation for imaging neovascularization in an organism. Use according to claim 13, characterized in that said labeled antibody is labeled with 99m technetium. Use according to one of claims 13 or 14, characterized in that the detection is performed using a gamma camera or Single Photon Emission Computerized Tomography, SPECT. 16. A method for diagnosing and / or monitoring a cancerous disease state, characterized in that it * * comprises the steps of 25a) a serum sample from a patient suspected of suffering from a disease characterized by endothelial cell proliferation b) contacting said serum sample from a detectably labeled Tie os cell. c) detecting the presence of said detectably labeled anti-Tie antibody in said serum sample. A method according to claim 16, characterized in that metastasis is associated with the stage of the disease. 111267 Method according to one of Claims 16 or 17, characterized in that the detection step is carried out by a sandwich-type test. A pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of an extracellular Tie antibody according to claim 1 in a pharmaceutically acceptable diluent, adjuvant or carrier. The pharmaceutical composition of claim 19, wherein the effective amount of the anti-Tie antibody is from about 1 mg / ml to about 10 mg / ml. Use of an extracellular antibody recognizing Tie according to claim 1 in the manufacture of a medicament for the alleviation of symptoms of a disease characterized by abnormal growth of endothelial cells. 111267