Monoclonal antibody against FLT4 RECEPTOR TYROSINE KINASE AND ITS USE IN DIAGNOSIS AND THERAPY

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DESCRIPTION

Monoclonal antibody against the FLT4 receptor tyrosine kinase and its use in diagnosis and therapy.

Field of the invention

The present invention relates generally to receptor tyrosine kinase, nucleic acid probes and antibodies that specifically recognize said receptors, and to the use of said probes and antibodies to identify lymphatic vessels and upper endothelial venules (VEAs) in animal and human tissues and endothelial lymphatic cells in culture. More specifically, the present invention is directed to antibodies specific for FLT4, a receptor tyrosine kinase, to methods to identify the expression of FLT4 in lymphatic vessels and fundamentally to diagnose and treat pathological conditions in animals and humans that involve changes in lymphatic tissue, such as inflammatory, infectious, and immune diseases, metastatic lymph nodes, and lymphangiomas.

Background of the invention

The vascular endothelium lining the blood vessels is involved in the physiology of the vascular system, embryonic vasculogenesis and angiogenesis, blood clotting, wound healing and reproduction, as well as in various diseases. The development of the vascular tree occurs through angiogenesis and, according to some theories, the formation of the lymphatic system begins shortly after arterial and venous development through the sprouting of veins (1, 2).

After the fetal period, endothelial cells proliferate only very slowly, except during angiogenesis associated with neovascularization. Growth factors that stimulate angiogenesis exert their effects through specific tyrosine kinase receptors on the surface of endothelial cells.

The protein product of the FLT4 receptor tyrosine kinase cDNA, cloned from a human erythroleukemic cell line, is N-glycosylated and contains seven immunoglobulin-like loops in its extracellular domain. The cytoplasmic domain of FLT4 tyrosine kinase is approximately 80% identical at the amino acid level with the corresponding domains of FLT1 and KDR, and approximately 60% identical with receptors for the platelet-derived growth factor, factor colony stimulant-1, stem cell factor, and the FLT3 receptor (3).

Although the biological function of FLT4 has not yet been clarified, its restricted expression pattern indicates that its functions may involve the vascular endothelium. Our previous results revealed that the expression of FLT4 mRNA in endothelial cells in the developing vessels of various fetal organs as described in Kaipainen et al., JExp Med 178: 2077-2088,1993. A comparison of the mRNA signals of the FLT4, FLT1 and KDR / FLK-1 receptors showed an overlap although with different expression patterns in the study tissue (4). These data suggest that the receptor tyrosine kinase encoded by this family of genes may have different functions in regulating the growth and / or differentiation of blood vessels.

A primary function of the lymphatic system is to provide a return for fluid from the tissues and to transport many extravascular substances back into the blood. Additionally, during the maturation process, the lymphocytes leave the blood, migrate through the lymphoid organs and other tissues, and enter the lymphatic vessels, and return to the blood through the thoracic duct. Specialized venules, called high endothelial venules (VEAs) bind to blood lymphocytes again, causing their extravasation into the tissues. Therefore, lymphatic vessels and especially lymph nodes play an important role in immunology and are also sites of development of metastases of different tumors.

Since the early twenty-first century, three different theories have emerged about the origin of the lymphatic system. However, prior to the present invention, it has been difficult to identify lymphatic vessels as there are no specific markers for them.

Lymphatic vessels are usually studied with the help of lymphography. In lymphography, the X-ray contrast medium is injected directly into a lymphatic vessel. The contrast medium is distributed along the efferent drainage vessels of the lymphatic system. The contrast medium is collected in the lymph nodes, where it remains for up to half a year, and during this period X-ray analyzes allow monitoring of the size of the lymph nodes and their structure. This diagnosis is especially important in cancer patients with lymph node metastases and in lymphatic malignancies, such as lymphoma.

Summary of the invention

The present application relates to FLT4 peptides and other constructs and to the use of FLT4 as a specific marker for lymphatic endothelial cells.

The invention relates to antibodies that specifically recognize FLT4, especially monoclonal antibodies, and to compositions containing such antibodies. Furthermore, the present application describes the use

ES 2 263 152 T3 of these monoclonal antibodies for diagnostic purposes, for the detection and measurement of the amount of FLT4 receptors in tissues, especially in lymphatic tissues and in lymphatic endothelial cells.

In a preferred embodiment, the invention provides monoclonal antibodies that specifically recognize the FLT4 receptor. More specifically, this invention provides a monoclonal antibody designated 9D9F9. The hybridoma cell line that produces the monoclonal antibody 9D9F9 has been deposited with the Deutsche Sammlong von Mikroorganismen und Zellkulturen GMBH (DSM) under the provisions of the Budapest Treaty (accession number DSM ACC2210).

Monoclonal antibodies labeled with a detectable marker are also provided. As used herein, the term "detectable marker" encompasses any detectable marker known to those of skill in the art. However, in a preferred embodiment of the present invention, the detectable label is selected from the group consisting of radioisotopes, fluorochromes, dyes, enzymes, and biotin. For the purposes of this invention, appropriate radioisotopes include, but are not limited to, ¹²⁵I and ¹³¹1.

The monoclonal antibodies of the present invention can also be used in a method for detecting the presence of FLT4 receptors in a cell sample, comprising the steps of exposing a cell sample to a monoclonal antibody of the present invention and detecting the binding of said monoclonal antibody to FLT4 receptors.

Therefore, another aspect of the present invention relates to a method for determining the presence of FLT4 receptors in the cell sample, comprising the steps of:

(a) exposing a sample of cells to an antibody of the present invention;

(b) detecting the binding of said monoclonal antibody to FLT4 receptors.

Exposure of a mixture of cells to the monoclonal antibodies of the invention can be carried out in solution, as is the case with fluorescence-activated analysis for cell sorting, or it can be on solid tissue samples, such as tissue material. biopsy, or it can be with the monoclonal antibody immobilized on a solid support, such as is the case with column chromatography or immunological direct adherence. The mixture of cells to be exposed to the monoclonal antibody can be any solution of blood cells or tissue cells. Preferably, the cell mixture is derived from normal or pathological tissue that contains or is suspected of containing endothelial lymphatic cells. After exposure of the mixture of cells to the monoclonal antibodies, those cells with the FLT4 receptors will bind to the monoclonal antibody to form an antibody-FLT4 receptor complex, and therefore, the FLT4 receptors can be detected by methods known in the art. state of the art. Such methods include standard immunohistochemical methods in the art, such as immunofluorescence, FACS analysis, ELISA, IRMA (an immunochemical sandwich assay), immunohistochemistry, RIA with labeling. ¹²⁵I and autoradiography.

The present invention also provides monoclonal antibodies conjugated to an agent that can be used for imaging. As used herein, the term that can be used for imaging includes, but is not limited to, radioisotopes. A preferred radioisotope is 99m-technetium.

In a specific embodiment, the invention relates to a method for monitoring lymphatic vessels and their endothelial cells in tissue samples and in organisms. The present invention also provides clinical detection methods to describe the state of lymphatic tissue, and especially lymphatic vessels (inflammation, infection, trauma, growth, neoplasms, etc.) and methods to detect lymphatic vessels and thus lymphatic vascularization in a organism.

More specifically, the present invention provides a method for detecting and identifying lymphatic changes characterized by the expression of FLT4 in relation to metastatic cancers, and inflammatory, infectious and immunological conditions, said method comprising the steps of (a) obtaining a tissue and / or body fluid suspected of lymphatic changes, and (b) exposing the sample to a monoclonal antibody specific for FLT4 under appropriate conditions to form a complex between the monoclonal antibody and the antigen, and (c) detecting the presence of any complex that forms.

A tissue that can be detected by this method is any solid precancerous or cancerous tumor tissue with lymphatic cells containing the FLT4 or cells expressing the FLT4 receptor. In one embodiment of the present invention, the monoclonal antibody is labeled with a detectable marker as described herein. The methods of the invention are useful for detecting and differentiating between various forms of cancer, especially lymph node metastases and other lymphatic neoplastic processes, such as lymphomas, as well as lymphangiomas.

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The present invention also provides a method for imaging lymphatic vessels, upper endothelial venules, or lymph nodes in human patients. This method comprises the administration of labeled antibodies and detection by imaging at sites where FLT4-expressing cells are present, in lymphatic vessels or lymph nodes.

The invention further relates to a method for stimulating or antagonizing the function of FTL4 in lymphatic vascularization and under inflammatory, infectious and immunological conditions, said method comprising the inhibition of FLT4-mediated lymphatic vascularization with sufficient amounts of a compound that is binds to FLT4 to block FLT4 sites on endothelial cells that participate in this reaction, especially where FLT4 function is associated with disease such as metastatic cancers, lymphomas, inflammation (chronic or acute), infections, and immune diseases.

Brief description of the figures

Figure 1. The expression of FLT4 mRNA in murine tissues. A. Hybridization of polyadenylated RNA isolated from indicated tissues of adult mice. The size of the FLT4 mRNA band is expressed in kilobases. B Analysis of RNAse protection of RNA isolated from murine embryos at various gestational ages (E8E18) and from a newborn mouse (1 day). Sample E8 + P also contains placenta. The size of the probe and the protected fragment are given in nt; ^ -actin was used as a control.

Figure 2. The expression of FLT4 mRNA in embryos of 7.5, 8.5 and 11.5 days pc. Darkfield and brightfield photomicrographs of in situ autoradiographs are presented. FLT4 mRNA expression could not be detected in a 7.5 day embryo (A). The expression of an 8.5 day pc murine embryo is presented in (B) and (C). Arrows point to FLT4-positive cells in the endothelium of the posterior cardinal vein (vc), in the allantoic (al) in (B), and in the angioblasts (ab) of the head mesenchyme in (C). In the 8.5-day pc placenta, FLT4 transcripts can be objected to in the endothelial cells of the venous lacunae (lv) (D). Panels E and F show a comparison of FLT4 and Tie hybridization signals in 11.5-day pc embryos The developing dorsal aorta region and the metanephros (mn) (20x) are shown. Note that the dorsal aorta is negative for FLT4, but positive for Tie mRNA, while both probes hybridize with the subendocardial vein endothelium (sv). Also, the FLT4 probe gives a signal from the ureteral vein (v), while the Tie generally hybridizes with the ureteral capillaries (c, arrows). Ad: dorsal aorta, sn; neural groove. Scale bar: 30 pm.

Figure 3. Expression of FLT4 mRNA in a 12.5-day embryo. A sagittal section through the axillary plane is shown. Note that FLT4 mRNA is prominent in the dilated vessels of the axilla (ax), in a plexus-like pattern in the periorbital region (po), in the paravertebral tissue (arrows), and in the subcutaneous tissue (sc). c: brain, hi: liver. Scale bar: 5 pm.

Figure 4. FLT4 in embryos of 14 and 16.5 days. Panels A and B show the light and dark field images of a mid-sagittal section. Po: periorbital, mi: lower jaw, cu: neck region, sc: subcutaneous, mt, mesentery, ao: aorta, ts: thoracic duct. (C) shows a cross section of an embryo 16.5 days after hematoxylin-eosin staining. Ti: thymus, tr: trachea, e: esophagus, ca: carotid artery, ba: brachiocephalic artery, ct: thoracic duct. (D) shows a higher magnification (40x) of the thoracic duct region; autoradiographic grains can be distinguished above endothelial cells. Also, the small glass (v) at the top of the photo is positive. Scale bar: 10 pm (AC), 1 pm (D).

Figure 5. A comparison of FLT4 and Tie mRNA expression in cultured endothelial cells. A Northern analysis of polyadenylated RNA from human foreskin (MV), femoral vein (VE), aortic (AO) and umbilical (HU) microvascular cells. For comparison, the hybridization signal of the Tie receptor tyrosine kinase mRNA is shown. Bands resulting from the specific binding of the probe to ribosomal RNA are indicated by asterisks.

Figure 6. FLT4 in lymphatic vessels of the mesentery (A, B), lung (C, D), and amygdala (E, F) of human adults. Note that only lymphatic vessels in A and C give rise to a FLT4 signal, whereas veins, capillaries, and arteries are negative for FLT4 mRNA. In the amygdala, a signal is found in the endothelium of some VEAs. Scale bar: 200 pm.

Figure 7. FLT4 mRNA in normal (A, B) and metastatic (C, D) lymph nodes and in a lymphangioma (E, G). Arrows indicate lymphatic sinuses and VEAs, which are positive for FLT4. A comparison of FLT4 and von Willebrand factor signals shows that both are present in lymphatic endothelium but only von Willebrand factor signal is present in capillary (c) and venous (v) endothelium. Scale bars: 10 pm (AD) and 100 pm (EG).

Figure 8. The expression of FLT4 in fetal mesenteric vessels detected by immunoperoxidase staining. Sections were stained with affinity-purified antibodies to FLT4 (A), with antigen-blocked serum (B) and with pre-immune serum (C), and with specific antiserum against factor VIII-related antigen (D). Note that staining is limited to some but not all vessels (v). Scale bar: 0.05mm.

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Detailed description of the invention

In recognition of the importance of identifying changes in lymphatic tissues, especially lymph nodes in relation to metastatic cancers and immunological diseases, the present inventors have shown that FLT4 is a specific marker that detects the endothelium of lymphatic vessels and therefore it is useful as a marker of lymphatic changes in disease states in humans.

The present inventors have previously shown that the expression pattern of FLT4 compared to FLT1 and KDR is very different in the tissues of 18-week-old human fetuses (4). In order to understand the role of FLT4 during development, the inventors cloned partial murine FLT4 cDNAs. Using these probes in in situ hybridization, the expression of FLT4 mRNA during mouse development was analyzed and FLT4 was found to be expressed during vasculogenesis and angiogenesis of the lymphatic system. The relevance of these findings was also confirmed in normal and pathological human tissues, since FLT4 was found in lymphatic endothelial cells of adult human tissues under both normal and pathological conditions, as well as in some high endothelial venules (VEAs).

Cloning of murine FLT cDNA fragments showed that the deduced amino acid sequence is almost identical to the corresponding human sequence (approximately 96% amino acid identity in the two segments studied). Further clues to the identity of the murine FLT4 cDNA were obtained from Northern hybridization where probes from both species provided the typical 5.8 kb mouse tissue mRNA signal. Analysis of RNA isolated from various tissues of adult mice showed FLT4 expression in liver, lung, heart, spleen, and kidney, with little or no hybridization in brain and testes. This pattern is similar to the pattern previously described by Galland et al (5). The RNase protection results suggest that the FLT4 gene is needed during mouse development, starting from 8.5 days pc embryos, and the relative expression levels appeared quite stable.

For in situ hybridization, two fragments of the FLT4 cDNA were selected, which encoded extracellular domain sequences. This allowed a clear distinction to be made between the FLK-1 and FLT-1 receptor hybridization patterns, which only show a low degree of sequence identity in the extracellular region (6, 7, 8, 9). FLT4, like the genes for FLK-1, FLT-1, Tie and Tek receptor endothelial tyrosine kinase, was not expressed in the 7.5 day pc embryos. In an 8.5 day pc embryo, the strongest signals are located in the allantois, the angioblasts of the head mesenchyme and the cardinal vein. In contrast, the dorsal aorta, the endocardium of the heart and the yolk sac angioblasts were negative, in exchange for Tie, Tek, FLK1 and FLT1, Tie and Tek (10, 8). The restriction of the expression of FLT4 to the venous system was even clearer in samples from the embryos of 11.5-day-old mice, in which the Tie mRNA was also expressed in the arteries. In the 12.5-day pc embryos, the FLT4 signal appeared in the venous and putatively lymphatic endotheliums, but unlike the Tie tyrosine kinase receptor, the arterial endotheliums were negative. During the later stages of development, FLT4 mRNA has been restricted to vascular plexuses that lacked blood cells, which represent developing lymphatic vessels. Only the lymphatic endothelium and some high endothelial venules expressed FLT4 mRNA in adult human tissue. The increased expression occurred in lymphatic sinuses and upper endothelial venules and in metastatic lymph nodes and in lymphangioma.

Due to difficulties in interpreting data from mouse embryos, human endotheliums were studied, as the lymphatic system is much better defined in humans. Furthermore, established cells from various endotheliums could be studied in cell culture in order to find out whether the specificity of FLT4 expression persists under in vitro conditions. Endothelial cell lines are known to lose differentiation characteristics when cultured in vitro. Therefore, one would expect that they were negative for FLT4. Aortic endothelial cells also lacked the FLT4 mRNA. However, signals were obtained from cultured human endothelial cells from the microvasculature and from the femoral and umbilical veins. Thus, at least some of the specificity of FLT4 expression was maintained in cell culture.

Analysis of in situ hybridization of human adult tissues confirmed the restriction of FLT4 to the lymphatic system observed in developing mouse embryos. FLT4 expression was observed in lymphatic endothelial cells and human lymph node sinuses. Interestingly, some of the VEAs that have a cuboid endothelium and that have been shown to be involved in the transport of leukocytes to the lymph nodes were also positive for FLT4. Furthermore, a parallel analysis of hybridization showed that FLT4 mRNA levels were enhanced in these metastatic structures compared to normal lymph nodes. FLT4 was also very prominent in lymphangiomas, which are benign tumors composed of a connective tissue stroma and growing lymphatic channels with an endothelial lining. FLT4 mRNA was limited to the lymphatic endothelium of these tumors and was not observed in their arteries, veins, and capillaries. In the human lung, we were able to identify lymphatic structures, which were the only FLT4-positive vessels in this tissue.

The results presented suggest that FLT4 is a novel marker for lymphatic vessels and some high endothelial venules in adult human tissues. They also support the theory about the venous origin of the lymphatic vessels. FLT4, as a growth factor receptor, may be involved in the differentiation and function of these vessels.

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These results, in combination with the FLT4 binding compounds according to the present invention, allow a selective labeling of the lymphatic endothelium, especially through the use of antibodies of the present invention coupled to radioactive or high electron density substances or other indicator substances that can be viewed. It may be possible to inject into the lymphatic system substances containing the FLT4 receptor according to the invention for the detection of high endothelial venules, especially activated VEAs, which express enhanced levels of the FLT4 receptor. To our knowledge, no such specific markers are available for the lymphatic endothelium at present.

The following examples are presented only to illustrate the present invention and do not limit the scope thereof in any way.

Examples

Example 1

Cloning mouse FLT4 cDNA probes

Approximately 10⁶ Plates from a 129SV mouse iFIX®II genomic library (Stratagene) with the human FLT4 receptor cDNA fragment corresponding to the extracellular domain (3). A 2.5 kb Bam HI fragment was subcloned from a positive plate and sequenced from both ends. From this subclone, the polymerase chain reaction was used to amplify and clone an exon fragment corresponding to nucleotides 1745-2049 of the mouse FLT4 cDNA sequence into a pBluescript KSII +/- vector (Stratagene) (9) .

A second fragment corresponding to nucleotides 1-192 was identically cloned.

Example 2

FLT4 mRNA analysis in murine tissues

Total RNA was isolated from developing embryos (8-18 days pc and in day old mice) according to Chomczynski et al. (eleven). The 8-day embryo sample also included the placenta.

For the analysis of protection by RNase, the RNA probe was generated from the linear plasmid of FLT4 obtained according to example 1 with [³²P] -UTP and T7 polymerase for antisense orientation. The jd-actin probe used corresponds to nucleotides 1188-1279 of the published murine jd-actin sequence (12). After gel purification with 6% polyacrylamide / 7M urea, the labeled transcripts were hybridized to 30 pg of total RNA overnight at 52 ° C. Non-hybridized RNA was digested with RNase A (10 U / ml) and T1 (1 pg / ml) at 37 ° C, pH 7.5 for 1 hour. RNases were inactivated by digestion with proteinase K at 37 ° C for 15 minutes and samples were analyzed on a 6% polyacrylamide / 7M urea gel.

The expression pattern of FLT4 analyzed in this experiment showed that very weak mRNA signals were obtained from the lung, liver, heart, kidney, skeletal muscle and spleen, samples of which no specific signal was detected in the tests and the brain. (figure 1A). Analysis of the RNA series collected during the different phases of mouse development in the RNA protection assay showed that FLT4 mRNA was expressed throughout embryogenesis from day 8 pc to newborn mice without any large variations in signal intensity (Figure 1B).

Example 3

In situ hybridization of FLT4 in mouse embryos

To better assign FLT4 transcripts to cells and tissues, sections of 7.5 and 8.5 day pc mouse embryos were hybridized with FLT4 RNA molecules. The embryos were derived from crossing CBA mice and NMRI mice. Pregnant mice were euthanized by cervical dislocation and embryos were immediately frozen or transferred via phosphate buffer to 4% paraformaldehyde. Isolated embryos and organs were fixed for 18 hours at 4 ° C, dehydrated, embedded in paraffin, and cut into 6 pm sections.

RNA probes (antisense and sense) of nucleotides 192 and 349 (see Example 1) were generated from linear plasmids with [³⁵S] -UTP. In situ hybridization of the sections was carried out as described by Wilkinson et al (13,14), with the following modifications: 1) instead of toluene, xylene was used before including in the paraffin wax, 2) 6 pm sections were cut, placed in a layer of water treated with diethyl pyrocarbonate on the surface of pre-treated glass slides with 2% 3-triethoxysilylpropylamine, 3) hydrolysis of the probes was omitted, and 4) high stringency washing was performed for 80 minutes at 65 ° C in a solution containing 30 mM DTT and 1 x SSC. The sections were covered with an NTB-2 emulsion (Kodak) and stored at 4 ° C. The slides were exposed for 14 days, developed and stained with hematoxylin. Control hybridizations to the sense strand and RNase A treated sections did not exhibit a specific signal above background.

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As shown in Figures 2A and B, FLT4 mRNA was not expressed in 7.5 day old mouse embryos, but clear signals were detected in the posterior cardinal vein (cv) on day 8.5 of development. . In contrast, the developing heart and dorsal aorta (ad) were negative for FLT4 (data not shown). In extra-embryonic tissues, FLT4 was prominently expressed in the allantois ("al" in Table B), while the developing yolk sac blood islets were negative (data not shown). On the other hand, the angioblasts (ab) of the head mesenchyme were highly positive for FLT4 (C). In the developing placenta, the FLT4 signal was first observed in the peripheral sinusoidal veins (data not shown). In the 9.5 day pc placenta, the venous lacunae endothelium (lv in D) and giant cells partially fused to Reichert's membrane (data not shown) expressed FLT4 mRNA.

Therefore, although the expression of FLT4 was very prominent in the earliest precursors of endothelial cells, angioblasts, said expression appears to be limited to certain vessels of the 8.5-day pc embryos. The Tie receptor is known to be expressed in all the endothelial cells of developing mouse embryos and thus provides a marker for these cells. Notably, in exchange for the Tie probe, the FLT4 probe hybridized very weakly or not at all with the arterial endothelium of the day 11.5 pc embryos, for example, with the developing dorsal aortic endothelium ( ad in figure 2 E, F) or carotid arteries (data not shown). Instead, the FLT4 signal was much more prominent in developing veins. For example, the FLT4 signal was detected in veins around developing metanephros (v, sv in E), while the Tie probe primarily recognized capillaries (c) within the metanephros (F).

As can be seen in Figure 3, FLT4 mRNA is distributed in various regions of a 12.5-day-old mouse embryo, being especially prominent in the dilated vessel of the axillary region (ax). A FLT4 positive vessel with a similar structure was observed in the midsagittal section in the jugular area (data not shown). A plexus-like pattern of vessels expressing FLT4 appeared in the periorbital region (po) and developing vertebrae (vb). Furthermore, a positive vascular network for FLT4 (sc) could be seen just below the developing skin. Weaker capillary signals were obtained from several regions, including the developing brain (c) FLT4 mRNA could also be detected in small vessels in the neck region, the developing muzzle, and the base of the tongue. developing as well as in the tail region (data not shown). Furthermore, the liver (hi) was highly positive for FLT4 mRNA with a stippling pattern.

During further development, it appeared that FLT4 RNA was more restricted to certain vessels in the embryo. A 14.5-day embryo well illustrates this restricted pattern of expression (Figure 4A, B). In the midsagittal section of Figure 4, the most prominent signal of FLT4 can be seen along the developing vertebral column in its anterior part. The signal appeared to originate from endothelial cells in the thoracic duct (ct), which is the largest lymphatic vessel that forms at this point in development. In contrast, the dorsal aorta (ad) and the inferior vena cava (vc) were negative. Dilated vessels in the mesenteric region were also highly positive for FLT4. Furthermore, as in 12.5-day pc embryos, the networks of vessels near the anatomical borders in the mandible (ma) periorbital (po) as well as in the neck region (cu) contained positive endotheliums for FLT4. . Similar structures were present in the pericardial space and throughout the subcutaneous (sc) tissue. Notably, in exchange for the FLT4-negative vessels, all the FLT4-positive vessels lacked blood cells in their lumen. These expression patterns suggest that FLT4 is confined to endothelial lymphatic vessels at this time in development. An additional site where FLT4 expression was observed was in developing bone marrow sinusoids (mo).

Photographs of a cross section of the upper thorax of a 16.5 day pc embryo hybridized with a FLT4 probe are presented in Tables C and D of Figure 4. The section shown in C has been stained with hematoxylin- eosin to visualize the different types of vessels in this area. These include the carotid and brachycephalic arteries (ac, ab), the vena cava (vc), and the thoracic duct, which is smaller in size and lacks the surrounding muscle and connective tissue (arrow). A magnification of the thoracic duct region is shown in panel D, where autoradiographic FLT4 grains can be seen. The endothelial cells of the thoracic duct as well as a proximal small vessel (v) hybridize with the FLT4 probe.

Example 4

FLT4 mRNA analysis in cultured endothelial cells

The in situ hybridization results described in Example 3 showed that FLT4 is expressed in venous endothelial cells and later in lymphatic vessels and some venous endothelial cells but not in arterial endothelial cells. In order to find out if this regulation was maintained in vitro, we studied endothelial cells in culture using a Northern analysis and a hybridization analysis.

Human endothelial cells from the aorta, femoral vein, umbilical vein, and foreskin microvessels were isolated, cultured, and characterized as previously described by Van Hinsberg (15, 16). They were then used at confluent density after five to eight steps (1: 3 split ratio) for the isolation of polyadenylated RNA.

The endothelial cell lines EA-hy926, BCE and LEII did not express FLT4 (data not shown). However, cultured human umbilical, venous and microvascular cells tested positive for the 5.8 and 4.5 kb specific RNAs for FLT4, while aortic endothelial cells were negative (Figure 5). In exchange, another

ES 2 263 152 T3 endothelial receptor tyrosine kinase gene, Tie, was expressed as a 4.4 kb mRNA in all endothelial cell types studied.

Example 5

FLT4 mRNA in human adult tissues

The results obtained in Example 3 indicated that FLT4 mRNA is more confined to the endothelium of lymphatic vessels during development. In view of the potential significance of this finding in humans, we also studied FLT4 in human adult tissues with the human FLT probe. The human FLT probe used was an EcoRI-Sph1 fragment corresponding to base pairs 1-595 of the cDNA (3). The von Willebrand factor probe was an EcoRI-HindIII fragment corresponding to base pairs 1-2334 (17).

We routinely fix material to send for histopathological diagnosis. Normal lung tissue was obtained from a resection of the lower lobe of the left lung affected by squamous cancer. The mesentery and mesenteric lymph nodes were obtained from a patient with an adenocarcinoma of the colon. A normal lymph node adjacent to the salivary gland nucleated because it was abnormal in size. The tonsils of two patients and two appendages did not show diagnostic changes. Two lymphangiomas and three cystic lymphangiomas were studied with similar results.

For human tissues, which were routine 10% formalin-fixed samples for histopathological diagnosis, the normal in situ protocol provided only a background signal, whereas treatment with microwaves instead of proteinase K allowed specific hybridization (18 , 19).

In the mesentery, the lung and lymphatic endotheliums of the appendix (lv) provided FLT4 signals, while veins (v), arteries (a), and capillaries (c) were negative (Figure 6A-D and data not shown). To study whether FLT4 is expressed in VEAs, the tonsils were studied. In fact, in the tonsils, autoradiographic grains specific for FLT4 were detected in some VEAs (E, F).

Example 6

FLT4 mRNA analysis in normal and metastatic lymph nodes and in lymphangioma

A portion of a human mesenteric lymph node (see Example 5) was analyzed for expression of FLT4. The results are presented in Figure 7.

FLT4 is expressed in the lymphatic sinuses (sl) and the afferent and efferent lymphatic vessels (data not shown). The same pattern is seen in a lymph node containing adenocardinoma metastases (C, D). Some VEAs in both normal and metastatic lymph nodes were positive as well. In panel E, FLT4 expression is shown in a cystic lymphangioma (see comparison with hematoxylin-eosin stained section in F). It was notable that the specificity of FLT4 towards lymphatic endothelium was evident in a comparison with in situ signals of von Willenbrandt factor in all blood vessels (F).

Example 7

Location of FLT4 in fetal endothelial cells

A fragment of the FLT4 cDNA encoding the 40 amino acids with the carboxy terminus of the short form was cloned as a 657 bp EcoRI fragment into the bacterial expression vector pGEX-1IT (Pharmacia) in frame with the coding region of the glutathione S transferase. The resulting fusion protein, GST-FLT4, was produced in E. coli and purified by affinity chromatography on a glutathione-sepharose 4B column. The purified protein was lyophilized, dissolved in PBS, mixed with Freund's adjuvant, and used for immunization of rabbits. Antisera were used after the fourth booster immunization.

Tissues from 17- and 20-week-old human fetuses were obtained from prostaglandin-induced legal abortions. The study was approved by the Ethics Committee of the Helsinki University Central Hospital. Gestational age was estimated by measuring the foot length of the fetus. The tissues were soaked in Tissue-Tek (Miles) and immediately frozen and stored at -70 ° C.

Anti-FLT4 antiserum was cross-absorbed onto a GST-sepharose column to remove anti-GST antibodies and purified by GST-FLT4 affinity chromatography. Several cryostatic sections of the tissues with a thickness of 6 pm were fixed with acetone and treated with H₂OR₂ 0.3% in methanol for 30 minutes to block endogenous peroxidase activity. After washing, the sections were incubated with 5% normal pig serum. The sections were then incubated with anti-FLT4 antibodies, washed, and bound antibodies detected with peroxidase-conjugated pig anti-rabbit IgG followed by staining for peroxidase activity with 0.2 3,3-diaminobenzidine. % (Amersham) as a substrate. The sections were counterstained in Meyer's hematoxylin.

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Anti-FLT4 immunoperoxidase staining of the mesentery of human fetuses showed an FLT4 protein in the endothelium of various vessels (Figure 4A), while control stains with antigen-blocked anti-FLT4 antibodies (B) and preimmune sera (C) were negatives. For comparison, Figure 4 shows the results of staining with an antiserum against factor VIII-related antigen, which is specific for vascular endothelial cells.

Example 8

The production of monoclonal antibodies against FLT4

Fusion I

Four month old male Balb / c mice were immunized by intraperitoneal injection of the FLT4 protein produced by recombination techniques (see example 7) in a concentrated medium (150 jug / mouse), emulsified with Freund's complete adjuvant. Booster injections of 150 pg were given at three to four week intervals and a final booster injection (10 pg of FLT4 in PBS administered intraperitoneally) was given after another three week interval. Four days after the final dose of the booster injection, the mice were sacrificed and the murine splenic lymphoid cells were fused with SP 2/0 plasmacytoma cells at a ratio of 2: 1, respectively.

The fused cells were harvested in 96-well culture plates (NUNC) in Ex-Cell 320 medium (SERALAB) containing 20% ​​fetal bovine serum and the supplement HAT (hypoxanthine-aminopterin-thymidine; GIBCO, 04301060H; diluted 50 times ). The cells were cultured at + 37 ° C in an atmosphere of 5% CO₂. After 10 days, the HAT supplemented medium was changed to cell culture medium with the HT supplement (GIBCO; 043-01065H, diluted 50 times). The HT medium is identical to the HAT medium, except that it does not contain the aminopterin.

After three weeks, the production of specific antibodies was determined by the antigen-specific immunofluorometric assay (IFMA), described in Example 10. Master clones were cloned by dilution as described in Staszewski et al., Yale Journal of Biology and Medicine, 57: 865-868 (1984). Positive clones were expanded into 24-well tissue culture plates (NUNC), cloned again, and retested again following the same method. Positive clones were tested for cell sorting by fluorescence activated analysis for cell sorting (FACS).

The stable clones secreted immunoglobulins belonging to the IgF1 class, except for one, which produced Ig that probably belonged to the IgA class. The monoclonal antibody subclass was determined by a rat monoclonal antibody to the murine subclass as a biotin conjugate (SEROTEC) in IFMA.

Balb / c mice were used to produce monoclonal antibodies in ascites fluid. The hybridomas described above were injected intraperitoneally into mice after pre-treatment of the animals with pristane (2,6,10,14-tetramethyl pentadecane 98%, ALDRICH-CHEMIE D7924 Steinheim, Cat. No. T 2 , 280-2). 0.5 ml of pristane (iv) was injected approximately two weeks before the hybridoma cells. The number of cells injected was approximately 7.5 to 9 x 10⁶ per mouse. Ascites was harvested 10-14 days after injection of the hybridomas.

Fusion II

Two month old Balb / c mice (female) were immunized by intraperitoneal injection of a recombinantly produced FLT4 protein (see example 7) (20 jug / mouse), they were emulsified with Freund's complete adjuvant. Booster injections of 20 pg were given at three to four week intervals and a final booster injection (10 pg FLT4 in PBS administered iv) was given after another three week interval. Four days after the final dose of the booster injection, the mice were sacrificed and the murine splenic lymphoid cells were fused with SP 2/0 plasmacytoma cells at a ratio of 2: 1, respectively.

The fused cells were harvested in 96-well plates (FALCON) in OptiMEM 1 medium (with Glutamax 1, 51985-026, GIBCO BRL) containing 20% ​​fetal calf serum and the supplement HAT (hypoxanthine-aminopterinathimidine; GIBCO BRL 21060- 017; diluted 50 times). The cells were grown at + 37 ° C in a 5% CO2 atmosphere. After 10 days, the HAT supplemented medium was changed to HT supplemented cell culture medium (GIBCO BRL; 41065-012, diluted 50 times). HT medium is identical to HAT medium, but does not contain aminopterin.

After three weeks, the production of specific antibodies was determined by the antigen-specific immunofluorometric assay (IFMA), described in Example 9. Master clones were cloned by limited dilutions as described in Staszewski et al., Yale Journal of Biology and Medicine, 57: 865-868 (1984). Positive clones were expanded into 24-well tissue culture plates (FALCON), reclined, and retested with the same method. Positive clones were tested by cell sorting by activated fluorescence (FACS).

IS 2 263 152 T3

Clones 2E11 and 6B2 secreted immunoglobulins belonging to the IgF class₁, clones 2B12 produced Ig belonging to the IgM subclass. The IgG subclass₁ The mouse was determined with the rat monoclonal antibody against the murine long-chain subclass as a biotin conjugate (SEROTEC) in IFMA and the murine IgM subclass was determined with the equipment for measuring monoclonal antibody isotopes (dipstick format) (19663 -012, Life Technologies Inc.)

Example 9

The specificity of monoclonal antibodies against FLT4

Fusion Antibodies I

The extracellular domain of FLT4 described in Example 7 was labeled according to that described in Mukkala et al. in Anal. Biochem. 176 (2): 319-325, 1989, with the following modification: a 250-fold molar excess of DTTA-Eu isothiocyanate (N1 chelate, WALLAC, Finland) was added to the FLT4 solution (0.5 mg / ml in PBS) and the pH was adjusted to approximately 9 by adding 0.5 mol / L sodium carbonate buffer, pH 9.8. Labeling was carried out overnight at + 4 ° C. Unbound label molecules were removed using PD10 (PHARMACIA, Sweden) with TSA buffer (50 mmol / L, Tris-HCl, pH 7.8 containing 0.15 mol / L NaCl) as eluent.

After purification, 1 mg / ml bovine serum albumin (BSA) was added to the labeled FLT4 and stored at + 4 ° C. The number of europium ions that had been incorporated by each FLT4 molecule was 1.9, a figure that was determined by measuring fluorescence at a ratio corresponding to that of known EuCl3 standards (Hemmilaetal., Anal. Biochem ., 137: 335-343, 1984).

The antibodies produced in Example 8 were screened by a sandwich immunofluorometric assay with microwell strips (NUNC, polysorb) coated with rabbit anti-murine Ig (Z 259, DAKOPATTS). The pre-coated wells were washed once with the DELFIA wash solution on the Platewash 1296024 kit (WALLAC). DELFIA Assay Buffer was used as a dilution buffer for cell culture supernatants and for serum from splenectomized mice (at dilutions of 1: 1000 to 1: 100,000) used as positive control in the screening assay. preliminary.

An overnight incubation at + 4 ° C (or alternatively, for 2 hours at room temperature) was started with shaking on a Plateshake shaker (1296-001, WALLAC) for 5 minutes followed by four washes with such washing solution. as described above.

Europium-labeled FLT4 was added at a dilution of 1: 500 in 100 µl of the assay buffer. After 5 minutes on a Plateshake shaker and an hour incubation at room temperature, the strips were washed as described above.

Enhancement solution (DELFIA) was added at 200 µl / well. Plates were shaken for 5 minutes on a Plateshake shaker and fluorescence intensity was measured with ARCUS-1230 (WALLAC) for 10-15 minutes (Lovgren et al., In: Collins WP (Ed) Alternative Microassays, John Wiley & Sons Ltd, 1985, pp. 203-216).

The resulting monoclonal antibodies against FLT4 and the corresponding FACS results are summarized in Table 2.

TABLE 2

<td>MAb clones</td><td>LTR%^(to)</td><td>NEO%^(b)</td><td>DELFIA counts</td>

<td>1B1</td><td> 67,3</td><td> 1</td><td> 20625</td>

<td>1B1D11</td><td> 75</td><td> 1,2</td><td> 19694</td>

<td>1B1F8</td><td> 76,1</td><td> 1,4</td><td> 18580</td>

<td>4F6</td><td> 69,9</td><td> 1,2</td><td> 23229</td>

<td>4F6B8G12</td><td> 75</td><td> 0,3</td><td> 24374</td>

<td>4F6B8H11</td><td> 75,9</td><td> 0,3</td><td> 28281</td>

<td>4F6B8E12</td><td> 74,8</td><td> 0,4</td><td> 27097</td>

<td>4F6B8G10</td><td> 75,3</td><td> 0,4</td><td> 26063</td>

IS 2 263 152 T3

TABLE 2 (continued)

<td>MAb clones</td><td>LTR%^(to)</td><td>NEO%^(b)</td><td>DELFIA counts</td>

<td>9D9</td><td> 45,1</td><td> 0,75</td><td> 17316</td>

<td>9D9D10</td><td> 71,7</td><td> 2,3</td><td> 18230</td>

<td>9D9F9</td><td> 73</td><td> 1,8</td><td> 11904</td>

<td>9D9G6</td><td> 74,3</td><td> 2,9</td><td> 16743</td>

<td>9D9G7</td><td> 70,7</td><td> 1,3</td><td> 17009</td>

<td>10E4</td><td> 24,2</td><td> 1,4</td><td> 39202</td>

<td>10E4B10E12</td><td> 32,3</td><td> 0,3</td><td> 42490</td>

<td>10E4B10G10</td><td> 36,5</td><td> 0,3</td><td> 54815</td>

<td>10E4B10F12</td><td> 45,6</td><td> 0,4</td><td> 43909</td>

<td>10E4B10G12</td><td> 45,7</td><td> 0,5</td><td> 35576</td>

<td>11G2</td><td> 30,2</td><td> 1,6</td><td> 11304</td>

<td>11G2D12</td><td> 74,4</td><td> 1,5</td><td> 14660</td>

<td>11G2G9</td><td> 74,2</td><td> 0,9</td><td> 10283</td>

<td>11G2H7</td><td> 74,4</td><td> 2,1</td><td> 25382</td>

^(to) FACS Results with LTR-Transfected Cells ^(b) FACS results with NEO cells (control)

One clone, designated anti-FLT4 9D9F9, was observed to stably secrete the monoclonal antibody which was determined to belong to the IgG1 immunoglobulin class by IFMA. Hybridoma 9D9F9 was deposited with the German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell and Virus Cultures, Mascheroder Weg 1b, 3300 Braunschweig, Germany, on March 23, 1995, and was provided with the number ACC2210.

Fusion antibodies

The extracellular domain of FLT4 described in Example 7 was labeled as described by Mukkala et al., In Anal. Biochem. 176 (2): 319-325, 1989, with the following modification: A 250-fold molar excess of DTTA-Eu isothiocyanate (N1 chelate, Wallac, Finland) was added to the FLT4 solution (0.5 mg / ml in PBS) and the pH was adjusted to 9 by adding a sodium carbonate buffer at 0.5 mol / L and a pH of 9.8. Labeling was carried out overnight at + 4 ° C. Unbound label was removed using PD-10 (PHARMACIA) with TSA buffer (50 mmol / L Tris-HCl, pH 7.8 containing 0.15 mol / L NaCl) as eluent.

After purification, 1 mg / ml bovine serum albumin (BSA) was added to the labeled FLT4 and stored at + 4 ° C. The number of europium ions that were incorporated by each FLT4 molecule was 1.9 as determined by measuring fluorescence at a ratio corresponding to that of EuCl standards.₃ known (Hemmil et al., Anal. Biochem., 137: 335-343, 1984).

Antibodies produced in Example 8 were screened with an IFMA specific for FLT4 using microwells (Nunc, Polysorb) coated with rabbit anti-mouse Ig (Z 259 DAKO). The precoated wells were washed once in the wash solution (Wallac) with the DELFIA Platewash kit.

DELFIA Assay Buffer was used as a dilution buffer for the culture of cell supernatants (1: 2 dilution in preliminary screening) and for the serum of splenectomized mice (1: 1000 and 1: 100,000 dilutions) which it was used as a positive control. As a standard, purified anti-FLT49 9D9F9 (murine IgG1 subclass) was used at concentrations between 1.0 ng / ml and 250 ng / ml. Samples were first shaken at room temperature for 5 minutes on Plateshake equipment (Wallac) and then incubated for approximately 18 hours at + 4 ° C. The frames were first washed four times, then the Eu-labeled FLT4 (1: 2000 in 100 µl assay buffer) was added, and finally the frames were incubated for one hour at room temperature. After

Wash as described, the enhancement solution (200 µl / well, Wallac) was added and the frames were shaken for 5 minutes on the Plateshake kit. Fluorescence intensity was measured with ArCUS-1230 (Wallac).

The resulting monoclonal antibodies against FLT4 and the corresponding results are summarized in the table

3.

A standard curve for the quantification of anti-FLT4 antibodies was constructed with affinity purified anti-FLT4 9D9F9. The linear range extended from 1.0 ng / ml to 250 ng / ml.

The lysate of NIH 3T3 cells co-transfected with the pLTRFLT4 construct expressing complete FLT4 on the surface was electrophoresed on 6.5% SDS-PAGE; the proteins were transferred to the nitrocellulose nitrate membrane (0.45 pm, SCHLEICHER &amp; SCHUELL) and an immunoblot was carried out with the supernatants of the MAb cells cultures (1:10, 50 mmol / L TRIS - buffer of 40 mmol / L glycine containing 4% methanol and 0.04% SDS). The specificity of the MAb was detected by incubation with conjugated rabbit anti-mouse Ig in HRP (P 161, DAKO, with a dilution of 1: 1000 in TRIS buffer of 20 mmol / L at pH 7.5 containing 150 mmol / L of solution saline, and 5% milk powder) and ECL (enhanced chemiluminescence, AMERSHAM).

TABLE 3

<td>MAb clones</td><td>LTR%^(to)</td><td>NEO^(b)</td><td>Prod. Approx. of MAb ng / ml / 10⁶cells</td><td>WB</td>

<td>2B12E10</td><td> 39,5</td><td> 6,0</td><td> 440</td><td> +</td>

<td></td><td></td><td></td><td></td><td></td>

<td>2E11D11</td><td> 44,6</td><td> 8,8</td><td> 110</td><td> +</td>

<td>2E11F9</td><td> 49,5</td><td> 4,5</td><td> 100</td><td> +</td>

<td>2E11F12</td><td> 46,0</td><td> 4,1</td><td> 180</td><td> +</td>

<td>2E11G8</td><td> 41,2</td><td> 7,8</td><td> 160</td><td> +</td>

<td></td><td></td><td></td><td></td><td></td>

<td>6B2E12</td><td>NF</td><td>NF</td><td> 1390</td><td> +</td>

<td>6B2F8</td><td>NF</td><td>NF</td><td> 470</td><td> +</td>

<td>6B2G6</td><td>NF</td><td>NF</td><td> 630</td><td> +</td>

<td>6B2H5</td><td>NF</td><td>NF</td><td> 740</td><td> +</td>

<td>6B2H8</td><td>NF</td><td>NF</td><td> 1800</td><td> +</td>

^(to) FACS results with transfected LTR cells ^(b) FACS results with NEO cells (control)

NF: Not functional in the FACS ^(c) Quantification of MAb production based on affinity purified anti-FLT 9D9F9 antibody as standard.

As follows from the foregoing, the antibodies according to the present invention are useful in the diagnosis and identification of lymphatic vessels, lymphatic endothelial cells, upper endothelial venules, lymphangiomas, metastatic lymph nodes, and other pathological conditions of the system. lymphatic, detection and monitoring of the extent of metastasis, in the stimulation and inhibition of the endothelial cells of the lymphatic vessels and the upper lymphatic venules, in the selective introduction of molecules in the endothelial cells and in the taking of images of the lymphatic vessels in diseased subjects. Other utilities than described in the present invention are apparent to one of skill in the art.

IS 2 263 152 T3

INDICATIONS REGARDING A DEPOSITED MICROORGANISM (PCT Rule 13b¡s)

A. The indications presented below refer to the microorganism referenced on page 3, lines 13 to 17

B. DEPOSIT IDENTIFICATION Additional deposits are identified on an additional sheet []

Name of the depository institution

DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GMBH

Address of the depository institution (with postal code and country) Mascheroder Weg 1 b D-38124 Braunschweig Germany

Deposit date 03-23-1995

Accession number DSM ACC2210

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Regarding the respective Patent Offices of the respective designated states, the applicant wishes that a sample of the deposited microorganisms only be made available to an expert appointed by the applicant until the date the patent is accepted or the date e in which the application has been rejected, withdrawn, or has been considered as withdrawn.

D. DESIGNATED STATES FOR WHICH THE INDICATIONS ARE MADE (if the indications are not for all the indicated states)

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The indications detailed below will be presented later in the International Bureau (specify the general nature of the indications eg “accession number of the deposit)

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IS 2 263 152 T3

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