MX2008000919A - Plexin d1 as a target for tumor diagnosis and therapy - Google Patents

Abstract

The present invention relates to plexin Dl for use as a targetable protein in the treatment or diagnosis of disorders that involve expression of plexin Dl. Diagnosis is suitably effected by detecting the presence of plexin Dl in the body or a bodily tissue or fluid, whereas treatment is effected by targeting plexin Dl for delivery of therapeutics to the site where treatment is needed. The invention further relates to the use of molecules that bind plexin Dl, a nucleic acid encoding plexine Dl or a ligand of plexin Dl for the preparation of a therapeutical composition for the treatment or diagnosis of disorders that involve expression of plexin Dl. The disorders comprise disorders in which plexin Dl is expressed on tumor cells, tumor blood vessels or activated macrophages.

Description

PLEXIN D1 AS AN OBJECTIVE FOR DIAGNOSIS AND TUMOR THERAPY Field of the Invention The present invention relates to the identification of a novel airworthy protein that can be used in the treatment and diagnosis of tumors, in particular solid tumors, and to disorders involving inflammation, in particular rheumatoid arthritis, atherosclerosis and multiple sclerosis. Background of the Invention To grow to a size beyond 2-3 mm 3, tumors have to recruit a neovasculature by angiogenesis. Tumors achieve this by expressing Vascular Endothelial Growth Factor-A (VEGF-A), either induced by hypoxia at the tumor center or as a result of malfunction of the tumor suppressor or proto-oncogene gene products. activated. A number of compounds that direct the VEGF-A signaling pathway have been developed in order to inhibit angiogenesis, and consequently, tumor growth. Although such anti-angiogenic therapies have been effective in animal tumor models, translation at the clinical level has been proven to be less successful (Eichhorn, ME and associates, Drug Resist Update 7: 125-138 (2004)). Because of this, there are a number of possible explanations. In clinically relevant situations, tumors that have grown for months or even years at the time of diagnosis, and a significant proportion of the vasculature may be more or less mature and therefore insensitive to inhibition with angiogenesis. This situation is a difficult contrast with that of most animal models, in which, as a rule, fast-growing aggressive tumors are studied. In addition, patients who are candidates for anti-angiogenic therapy are usually patients with disseminated, uncontrolled cancer and the growth of metastases may not always be strictly dependent on angiogenesis. Because most of the metastases are born in the blood, they grow in organs with densities of intrinsically high vessels such as liver, lung and brain where they can grow in a way independent of angiogenesis through the joint option of previously existing vessels. In fact, an angiogenesis inhibitor that very effectively inhibits tumor growth in a number of subcutaneous tumor models (Wedge, SR and associates, Cancer Res 62: 4645-4655 (2002)) does not inhibit the growth of tumor tumors. infiltration in mouse brain. Furthermore, at the time of treatment of mice bearing highly angiogenic brain tumors, the inhibition of angiogenesis does not result in an obstacle to the further progress of the tumor, but rather in a progress after a phenotypic change towards the joint option and infiltration (Leenders, WP and associates, Clin Cancer Res 10: 6222-6230 (2004)). These results imply that anti-angiogenic therapy must be supplemented by vascular targeting therapies in which the vascular bed of the existing tumor is attacked, resulting in secondary tumor brain death due to the interruption of the tumor's blood supply. To achieve effective vascular targeting therapy, markers that have specificity for the tumor vasculature must be identified. Much effort has been put into this, but with mixed success. The direction of effective vascular tumor has been achieved using single chain antibodies, directed against the ED-B domain of fibronectin, which is expressed in an elective form and deposited in the extracellular matrix of vessels recently formed in angiogenic tumors (Santimaria, and Associates, Clin Cancer Res 9: 571-579 (2003)). The direction of av 3-integrin (whose expression is restricted to immature vessels) using RGD or Vitaxin peptides, produced disappointing results, whereas endoglin-expression was not specific for tumor blood vessels (Posey, JA and associates, Cancer Biother Radiopharm 16: 125-132 (2001), Balza, E and associates, Int J Cancer 94: 579-585 (2001)). In inflammatory diseases, such as arthritis Rheumatoid (RA) or atherosclerosis, angiogenesis and activation of the vasculature is also frequently part of the pathology. The vasculature here paves the way for the inflammatory cells to extravasate and exert their destructive action. Therefore, these diseases can benefit from the direction to blood vessels. BRIEF DESCRIPTION OF THE INVENTION Accordingly, the object of the present invention is to provide a new targetable protein that can be used in the treatment and diagnosis of cancer and inflammatory diseases or diseases involving inflammation. In the research that led to the present invention, it was discovered that plexin D1 is expressed in the luminal part of endothelial cells in tumor blood vessels, in the tumor cells themselves and in activated macrophages found in tumors, in inflammation and in atherosclerotic plaques. The present invention therefore relates to plexin D1 for use as a targeted protein in the diagnostic treatment of disorders involving plexin D1 expression.

The family of plexin receptors consists of four classes (PLXNA-D) and nine members in mammals. The plexins comprise a family of single pass, large membrane proteins with homology to disperse receptors of factor, encoded by the MET gene family. Members of the plexin family share Sema domains, Met-related sequences (MRS), a transmembrane region, and intracellular motifs that are predictive of Rac / Rho-GTPase signaling (figure 1). Since GTPase signaling results in cytoskeletal readjustments, events that are important in the formation of filopodia and lamelipodia and cell migration, plexins can be considered as regulators of migration. The plexins are receptors of the semaphorins, a family of transmembrane proteins or anchored-GPI, segregated which are subdivided into seven subclasses. Each plexin has its own (established) part of semaphorin binding, and each combination of plexin-semaphorin results in a specific response. Semaphorins class 3 are powerful axon repulsors and are therefore involved in morphogenesis of the nervous system (for a review, consult the publications of (Pasterkamp, RJ and associates, Curr Opin Neurobiol 13: 79-89 (2003); Fujisawa, H, J Neurobiol 59: 24-33 (2004).) For the activation of plexins by semaphorins, additional plexin binding portions may be required.These binding sites, neuropilin-1 and -2 (NP-1 and NP- 2) have no signaling motifs in the intracellular domain and are considered as passive co-receptors, allowing the interaction between semaphorins and plexins. Some plexins form even larger membrane complexes with, and Off Track active signaling receptors (Otk) and the dispersion factor receptors Met and Ron. A direct interaction between plexinAl and the receptor-2 of angiogenic Vascular Endothelial Growth Factor (VEGFR2) have also been demonstrated (Toyofuku, T and associates, E-plublication in Genes Dev 18: 435-447 (2004)). Because NP-1 binds to the members of the plexin family but also to VEGFR2, it is conceivable that there are multiple component membrane protein complexes comprising VEGFR2, NP-1 and plexins, establishing a link between plexins and angiogenesis (see also the publication of Weinstein, BM, Cell, 120: 299-302 (2005)). Neuropilins are also co-receptors for the potent angiogenic Factor-A Vascular Endothelial Growth Factor (VEGF-A165) and increase their affinity for VEGFR2. Importantly, the VEGF-A165 binding site in NP-1 overlaps with that of semaphorin 3A (Miao, H Q and associates, J Cell Biol 164: 233-242 (1999)). It has been postulated that the binding of VEGF-A to NP-1 promotes the migration of endothelial cells competing in the binding of semaphorins class 3, which is generally followed by a depolymerization of F-actin and repulsion of cell extensions (Bachelder, RE , Cancer Res 63: 5230-5233 (2003)). The antagonistic behavior Similar to VEGF-A and semaphorins class 3 has been described in a neuronal progenitor cell line (Bagnard, D and associates, J Neurosci 21: 3332-3341 (2001)) and tumor cells (Bachelder (2003), supra). Since antagonistic effects were observed in tumor cells that are devoid of VEGF receptors, it is conceivable that the underlying mechanism involves members of the plexin family, establishing an additional link between plexins and VEGF-A signaling. The inventors of the present invention have previously found that the plexin D1 family member (plxnD1) is not only expressed in neuronal cells but also in endothelial cells of the vasculature during early stages of development (van der Zwaag, B and associates, Dev Dyn 225: 336-343 (2002)), and it was an observation that was confirmed by two other groups (Gitler, AD and associates, Dev Cell 7: 107-116 (2004); Torres-Vázquez, J and associates, Dev Cell 7: 117-123 (2004)). In adult vasculature, plxnD1 is absent. The elimination mice-plxndl and zebrafish that carry mutations in the plxndl gene, are characterized by a poor development of the cardiovascular system (Gitler, Ad, and associates, (2004), supra: Torres-Vázquez, J and associates, (2004 ), supra). Mice with double elimination of Neuropilin-1 (NP-1) and NP-1 / Neuropilin-2 (NP-2) also suffer from lethal defects in vascularization malformation and aortic arch during embryonic development (Kawasaki, T and Associates, Development 126: 4895-4902 (1999); Takashima, S and associates, Proc Nati Acad Sci USA 99: 3657-3662 (2002); Gu, C and associates, Dev Cell 5: 45-57 (2003)). Furthermore, the morpholine-borne elimination of NP-1 in zebrafish leads to poor development of intersegmental vessels, and in this model system a clear link between NP-1 and VEGF-A165 has been established (Lee, P and associates, Proc Nati Acad Sci USA 99: 10470-10475 (2002)). The resemblance of phenotypes of elimination mice plxndl, neuropilin-1 and semaphorin 3C (Feiner, L and associates, Development 128: 3061-3070 (2001)) is consistent with the discovery that Plexin D1 is a neuropilin-1 dependent receptor. of semaphorin 3C (Gitler, AD and associates (2004), supra). However, PIxnDI is also a semaphorin 3E receptor, and this interaction does not require signaling neuropilins transmitted by Semaforin 3E (Gu, C and associates, Science 307: 265-268 (2005)). In accordance with the present invention, it was discovered that plexin D1 is also involved in angiogenesis during tumor growth and is expressed in the luminal part of endothelial cells in tumor blood vessels. It was also discovered that plexin D1 is expressed by activated macrophages. Plexin D1 was also found to be expressed in tumor cells in a wide variety of tumor types. The present invention therefore relates to plexin D1 to be used as a targeted protein in the treatment or diagnosis of disorders involving plexin D1 expression.

The diagnosis is carried out by detecting the presence of plexin D1 or a nucleic acid encoding plexin D1 in the body or a tissue or body fluid. The treatment is carried out by directing plexin D1 for the therapeutic delivery to the site, when the treatment is necessary, interfering in the interaction between plexin D1 and its ligands, interfering in expression of the plexin D1 gene or capturing the plexin D1 ligands to inhibit the interaction with plexin D1. The present invention therefore further relates, to the use of plexin D1 binding molecules, a plexin D1 encoding nucleic acid or a plexin D1 ligand for the preparation of a therapeutic composition for the treatment or diagnosis of disorders involving the expression of plexin D1. All these molecules will be identified in the present invention as "binding molecules" or "linking entities". The disorders comprise in particular disorders in which plexin D1 are expressed as tumor cells, tumor blood vessels or activated macrophages. The tumor cells in which plexin D1 is expressed comprise brain tumors, in particular astrocytomas, oligodendrogliomas and hemangioblastomas, colon carcinomas, in particular ductal carcinomas of the colon, prostate carcinomas, renal cell carcinomas, in particular renal clear cell carcinomas, breast carcinomas, in particular ductal sinus carcinomas, ovarian carcinomas, squamous cell carcinomas, melanomas, lung carcinomas, in particular small cell lung carcinomas and non-small cell lung carcinomas, soft tissue sarcomas, etc. When the disorders that are treated according to the present invention are inflammatory diseases, they are in particular autoimmune diseases, more in particular rheumatoid arthritis, or they are atherosclerosis or multiple sclerosis. The molecules that bind plexin D1 are for example, selected from antibodies, fragments of antibodies, proteins, protein domains, peptides, small molecules. These molecules can be used to direct plexin. Molecules that bind to the nucleic acid encoding plexin D1 are for example, oligonucleotides, such as RNA or DNA aptamers, for example, selected from siRNA, antisense RNA, phosphothio-antisense oligonucleotides. These molecules can be used to interfere with the expression of plexin D1. The molecules that bind to the ligand plexin D1 are, for example, selected from antibodies against ligands, soluble plexin D1 electodomain or small molecules, such as peptides, that bind plexin D1 ligands. These molecules can be used to capture the plexin D1 ligand in the circulation, prevent binding of the ligand to plexin D1 in cells of tumor vessels, tumor cells or activated macrophages and interfere with the function of plexin D1 in these cells. For diagnosis, the binding molecule is suitably labeled with a detectable label. Said detectable label is, for example, selected from an opening tag, paramagnetic tag, a fluorescent tag, a chemiluminescent tag. The diagnosis can be carried out on a sample of a body fluid or tissue in vivo, in situ or ex vivo. Examples of diagnostic techniques are in situ hybridization of, for example, plexin D1 mRNA or immunohistochemistry in biopsies or tumor cells. For treatment, the binding molecule is for example, supplied with an entity that damages or kills the tumor cell and / or the tumor endothelial cell, in particular a cytotoxic entity, such as a radionuclide, a toxin, a boron Therapy of Boron Neutron Capture (BNCT), or a prodrug that is coupled to the binding entity through a dissociable linker, which is activated in response to dissociation of said linker, or peptides that induce apoptosis, an example of which is the sequence (KLAKLAK) 2. Said peptides are added to the binding entity by molecular genetic engineering techniques. The entities described above may be conjugated directly to the linking entity, or they may be presented in nanoavices, such as liposomes or polymersomes, which are conjugated to the linking entity. Boron Neutron Capture Therapy (BNCT) comprises irradiation of an area in shape, such as a tumor or inflammation, where boron has accumulated after intravenous injection of the liposomal conjugate, with neutrons, after which Boron atoms will decay to lithium under the emission of alpha destructive particles. Alternatively, therapy can be carried out by inducing local thrombosis in the tumor vessels to block the blood supply to the tumor, and induce brain death. An example of said molecule is the Tissue Factor (TF). Conveniently, plexin D1 can be targeted with specific binding molecules in intravenous administration, since plexin D1 is expressed in the luminal part of the endothelial cells in tumor blood vessels. Therapeutic compounds for damaging or killing tumor cells that attach to the binding molecule can reach the tumor from within and the compounds that induce thrombosis are easily delivered to their site of action. The interference with the plexin function D1 represents a way to inhibit angiogenesis, to inhibit tumor cell migration, and inhibit the migration of macrophages. Therefore, the present invention provides methods for treating or suppressing disorders in which plexin D1 is involved, using the specific presence of plexin D1 to deliver (typically therapeutic) to diseased tissues and / or by interfering with plexin function D1 or in interaction between plexin D1 and its ligands Detailed Description of the Invention The present invention is therefore based on the fact that plexin D1 can be used as a targetable marker in tumor blood vessels, as a targetable protein involved in angiogenesis of tumor, as a targeting marker in tumor cells and a targeting protein involved in cell migration The present invention also relates therefore to the use of molecules binding to plexin D1, its gene or mRNA or its ligands in diagnostics and therapy. All types of specific binding molecules and derivatives thereof can be used in the present invention. n, in particular, proteinaceous compounds, such as, but not limited to, antibodies, antibody fragments, single-domain antibody fragments, other protein-binding domains, such as, but not limited to, lipocalins and small molecules that bind in a specific plexin D1 or its ligands. To bind the plexin D1 gene or mRNA transcribed from the plexin D1 gene, nucleic acid molecules, such as DNA or RNA aptamers, can be used. In a first embodiment of the present invention, plexin D1 or plexin ligand binding molecules D1 are antibodies, in particular monoclonal antibodies, or more particularly particular human or humanized antibodies in which the constant regions of the original antibody are substituted with the regions human antibody constants, or fragments thereof which still bind to plexin D1 or its ligand. The antibody is preferably a human IgG1 antibody. However, other isotypes of human antibodies are also included in the present invention, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD and IgE. Also all antibodies derived from animals of various isotypes can be used in the present invention. The antibodies may be full-length antibodies or antigen binding fragments of antibodies, including Fab, F (ab ') 2, single chain Fv or single domain VHH fragments, VH or single VL domains. Preferably, antibodies against plexin D1 are human monoclonal antibodies produced by a hybridoma cell which includes a B cell obtained from an immunized transgenic animal having a genome comprising a transgene of human heavy chain and a human light chain transgene, fused to an immortalized cell, or an antibody derived from animal or an antibody fragment produced by a hybridoma cell that includes a B cell obtained from an immunized animal, fused to a immortalized cell, or human and animal antibodies, produced by a eukaryotic cell transfected with the cDNA or genomic DNA encoding the antibody or antibody fragment. In a preferred embodiment of the present invention, the single domain Flame (VHH) antibodies with affinity for plexin D1 are provided, more specifically, the single domain antibodies A12 Flame (SEQ ID NO: 1) and F8 (SEQ ID NO: 2), deployed or not in M13 bacteriophages, also known to those skilled in the art, such as VHH pause display antibodies. A preferred single chain antibody is derived from the antibody 11F5H6 and 17E9C12. The sequence of the single chain antibody is shown in SEQ ID NO: 3 and SEQ ID NO: 4. The antibodies to be used according to the present invention may be high affinity antibodies which are evoked in non-transgenic laboratory animals, or in a transgenic animal in which the endogenous globulin locus has been replaced by the human globulin locus, thus allowing the production of human antibodies in said animals (Jakobovits, A. Curr Opin Biotechnol 6: 561-566 (1995)). The present invention further relates to a method for producing the antibodies of the present invention, wherein the method comprises immunizing an animal with a plexin D1, or a cell expressing plexin D1, or a nucleic acid encoding plexin D1 , or parts of the extracellular domain of plexin D1, so that antibodies against plexin D1 are produced by the B cells of the animal, isolating B cells from the animal and fusing B cells with a myeloma cell line to obtain immortalized cells that they secrete in antibody. The animal is preferably a transgenic animal having a genome comprising a human heavy chain transgene and a human light chain transgene, so that the resulting antibody is humanized. In one embodiment, the method includes immunizing a laboratory animal with a synthetic peptide, chosen from the plexin D1 extracellular domain, for example, peptide 47-63 which corresponds to the amino terminus of the mature plexin D1 amino acid sequence. However, immunizations are preferably performed with extracellular recombinant domains, preferably a region with low similarity to another member of the plexin family, for example, a region comprising amino acids 47-546, which lacks the Related Sequences. Met. The domains Recombinant extracellular plexin D1 can be produced in E. coli cells by inserting the nucleic acid coding into a suitable prokaryotic expression vector, for example, under the control of the β-galactosidase promoter, transforming E. coli cells with the vector, and the isolation of the recombinant proteins from the purified inclusion bodies. However it is preferred that the antibodies be evoked by immunization with the extracellular domain of recombinant plexin D1 which is produced by ecucariotic cells, thus containing post-translational modifications, which are as similar as those found in native plexin D1 , for example, by Chinese hamster ovary cells (CHO) after transfection with a vector, which contains the nucleic acids encoding said extracellular domains under the control of a cytomegalovirus promoter. The recombinant extracellular plexin D1 fragments may or may not be fused to labels facilitating purification, for example, a VSV tag or a heavy chain constant region of an immunoglobulin. The method for producing the antibody can also comprise cloning regions encoding the antibody from the plexin D1-specific B cells into the expression vector and expressing the coding sequence. In a preferred embodiment, the expression vector is pHENIXHISVSV, allowing the expression by antibody E. coli host cells, flanked at the carboxy-terminal end through a Vesicular Stomatitis Virus (VSV-tag) and a His * 8 tag. The VSV label means that it facilitates the immunohistochemical detection, used in specific antibodies. The His * 8 tag means that purification is facilitated based on Nickel affinity chromatography. Other expression vectors can be used in the same way. More specifically, the present invention provides an isolated single domain A12 antibody, which has a dissociation constant less than 2x10"8 M, which binds to the amino terminus of plexin D1, and which detects plexin D1 in immunohistochemical stains and it hosts blood vessels of plexin D1 expressing tumors, and also with an isolated single domain antibody F8, which has a dissociation constant of less than 3x10"8 M, which binds to the amino terminus of plexin D1 and which detects plexin D1 in stained immunohistochemical and lodges tumor blood vessels expressing plexin D1. Both isolated single domain antibodies can be fused to the constant region of a human IgG1 heavy chain or the constant region of a mouse IgG1 heavy chain. Preferably, fully human antibodies are used within the scope of the present invention. In other modality, antibodies derived from laboratory or humanized animals can be used. The present invention further provides biospecific antibodies having a binding specificity for plexin D1, and a binding specificity for a cell presenting human antigen, or for a Fe receptor, wherein the Fe receptor is a Fe (gamma) R1 or a human Fc (alpha) receptor. The present invention also provides nucleic acid molecules that encode the preferred antibodies, or part of antigen binding. Recombinant expression vectors that include nucleic acids encoding the antibodies of the present invention, as well as host cells transfected with said vectors, are also encompassed by the present invention. Other binding molecules for use in the present invention are small molecules that specifically bind to Plexin D1. The term "small molecule" often refers to molecules with molecular weights of 500 or greater. The term is currently commonly used and therefore is clear to those skilled in the art. In addition, small molecule libraries are readily available or developed. An example of such a library is the Replenishment of Small Molecules from NIH Molecular Libraries (MLSMR). These libraries are subject to High Performance Classification (HTS) to identify molecules that bind to plexin D1. The present invention also relates to small molecules resulting from a classification of said libraries. Other compounds that can be used in accordance with the present invention comprise peptides or aptamers (Ulrich, Med. Chem, 1 (2): 199-208 (2005)) that bind to extracellular domains of plexin D1 and therefore interfere with the binding of ligands plexin D1 to plexin D1. Conversely, said peptides or aptamers can also bind to the plexin D1 binding sites of the plexin ligands D1, and therefore interfere with the ligand binding to plexin D1. To interfere with the expression of the plexin D1 gene, another type of binding molecule is used, in particular siRNA, antisense RNA or phosphothio-antisense nucleotides. The small interfering RNA (siRNA) comprises all strands of RNA that interfere with the translation of the messenger RNA. SiRNA binds the complementary part of the target messenger RNA and labels it for degradation, thereby inhibiting gene expression. This is commonly known as a genetic "silencing". SiRNA is usually 21 to 32 nucleotides in length. The antisense RNA has an RNA molecule transcribed by the coding, instead of the template, strand of DNA, so that it is complementary to the sense mRNA. The formation of a duplex between sense and antisense RNA molecules blocks translation and it can also subject both molecules to specific double-stranded nucleases, thereby inhibiting the expression of the gene. Inhibition of gene expression can be used to block angiogenesis and migration of tumor cells and macrophages. Preferably, the linker molecules described above bind to plexin D1, its gene or its ligand in eukaryotic cells. These molecules accumulate specifically in tumors at the time of intravenous injection, or accumulate specifically in tumor blood vessels at the time of intravenous injection. Antibodies, fragments thereof, small molecules and other proteinaceous compounds that bind plexin D1 can be used in various ways. In one embodiment, the present invention relates to compounds that bind to the extracellular part of plexin D1, and which result in interference from the plexin D1 function. Alternatively, the present invention relates to compounds that bind to the intracellular domain of plexin D1 and which prevent signaling by plexin D1. In a specific embodiment, said binding molecules bind plexin D1 to interfere with the formation of the multi-component membrane complex by inhibiting the binding of plexin D1 ligands, in particular neuropilin-1, neuropilin-2, semaphorin 3C, semaphorin 3E, receptor 1-VEGF, receptor 2-VEGF or VEGF-A, to plexin D1. Such binding molecules lead to the inhibition of GTPase signaling induced by ligand by plexin D1 or to the inhibition of migration of cells expressing plexin D1, in particular endothelial cells associated with tumor, tumor cells or macrophages. According to another aspect thereof, the present invention relates to a method for inducing the lysis of a cell expressing plexin D1, wherein the method comprises contacting the cell expressing plexin D1 with the linker molecules, in particular antibodies, of the present invention in the presence of human effected cells, so that lysis of cells expressing plexin D1 occurs. In a still further embodiment, the binding molecule is combined with, or coupled with, an effector compound which can detect the presence of plexin D1 for diagnostic purposes, or which can carry out an effect on the cell expressing plexin D1. The diagnostic or therapeutic effector compound can be coupled directly to the linker molecule or it can be present in a transport vehicle, such as a nanoaparate, in particular a liposome or polymersome, which is coupled to the linker molecule. Alternatively, the linker molecule can be a bispecific antibody that binds both plexin D1 and the effector compound, thereby directing the effector compound to a site or cell where plexin D1 is expressed. The present invention therefore provides in a particular embodiment thereof, the use of said binding molecules in a diagnosis method or disease transmitted by the expression of plexin D1, wherein the method comprises the intravenous administration of the molecules that bind plexin D1 protein, aptameric or small cell, conjugated with an effector compound that allows in vivo detection of the binding molecules. The effective diagnostic compounds are for example radioisotopes or contrast agents for Magnetic Resonance Imaging (MRI), such as gadolinium-DTPA, or fluorescent inks. Examples of radioactive substances include, but are not limited to, tecnetium 99m (99mTc), iodine-123 (123l), iodine-131 (131l), rhenium-186 or -188 (186 188Re), gallium-67 (67Ga) beta-90 (90Y) or lutetium-177 (177Lu) radiation emission substances, the positron emission isotopes Fluoro-18 (18F) and Carbon-11 (11C). The radioisotopes can be used either to detect or damage or kill cells expressing plexin D1. Normally different isotopes are used for diagnosis and therapy. Those skilled in the art will be aware of which isotope to use for which tissue and for which type of use. In another modality, molecular bond molecules Proteinaceous, aptameric and small for use in the present invention can be combined with, or coupled with, a toxic agent, such as a chemotherapeutic agent either directly or in a transport vehicle, in particular a nanoamate, such as a liposome or polymersome. In another embodiment, the plexin binding entity D1 of the present invention is coupled to one or more chemotherapeutic agents selected from the group consisting of nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines (e.g., thiotepa), sulfonates alkyl (for example busulfan), nitrosoureas (for example, carmustine and streptozocin), complexes of platinum (for example carboplatin and cisplatin), non-classical alkylating agent (for example, dacarbazine and temozolamide), folate analogs (for example, methotrexate) ), purine analogs (eg, fludarabine and mercaptopurine), adenosine analogs (eg, cladribine and pentostatin), pyrimidine analogues (eg, fluorouracil (alone or in combination with leucovorin) and gemcitabine), substituted ureas (eg hydroxyurea example), antitumor antibiotics (e.g., bleomycin and doxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide), microtubule agent (e.g. ocetaxel and paclitaxel), camptothecin analogues (eg, irinotecan and topotecan), enzymes (eg asparaginase), cytokines (eg, interleukin-2 and interferon-phalanx)), monoclonal antibodies (e.g., trastuzumab and bevacizumab), recombinant toxins and immunotoxins (e.g., recombinant cholera toxin B and TP-38), cancer gene therapies, and cancer vaccines (e.g., telomerase vaccine). The chemotherapeutic agents are preferably selected from the group consisting of doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil and hydroxyurea of cyclophosphamide. Other compounds are known to those skilled in the art. A tumor can also be treated by blocking its blood supply by inducing local thrombosis in the tumor vasculature. The binding molecules of the present invention can, in this embodiment, be used to target thrombosis inducing molecules, such as the coagulation factor TF of blood coagulation (Tissue Factor), a radioactive entity or a toxin, such as ricin to the site of the tumor. The effector compounds can be coupled to the linker molecule, in particular to molecules that bind plexin D1, or can be found in a nanoareate, such as a liposome or polymersome, which is coupled to molecules that bind plexin D1. An alternative method to treat cancer or an inflammatory disorder according to the present invention is with boron. The binding molecules of the present invention can be conjugated to transport vehicles, in particular nanoavices, such as Mposomas or polymersomes, which are filled with boron to obtain a therapeutic composition. After delivery and accumulation of this composition in the diseased area, this area is irradiated with neutrons, resulting in the emission of radioactive and cytotoxic alpha particles that damage or kill tumor endothelial cells, tumor cells and / or activated macrophages. It is desirable that for antibodies that target tumoral blood vessels have high affinities towards plexin D1, for example, greater than 10"8, preferably greater than 10" 9, more preferably greater than 10"10 M. High affinity and The high molecular weight of the antibodies, however, will restrict the penetration into the tumor tissue, Therefore, the nucleic acids encoding the monoclonal antibodies, obtained by RT-PCR cloning, can be used for antibody derivatives, for example, antibodies that they lack the constant region and are monovalent, or fragments of antibodies that adapt to optimal affinities for blood vessel targeting or tumor penetration by mutagenic methods.These antibody derivatives will have lower affinities and lower molecular weight and will have improved targeting properties. Tumor cells The different binding molecules of the present invention can be combined n a mixture In a specific modality, the members of the mixture have affinity diverse An example of such a combination is a mixture of monoclonal antibodies and / or antibody fragments or a mixture of antibodies with small molecules. Monoclonal antibodies that have high affinity can be used to direct vessels, whereas smaller fragments that have lower affinity are better in their ability to penetrate and reach tumor cells. Alternatively, a mixture of plexin D1 binding molecules can be used together with plexin D1 ligand binding molecules and / or with molecules that bind plexin D1 encoding nucleic acids. Or the plexin D1 ligand binding molecules can be combined with molecules that bind nucleic acids encoding plexin D1. The plexin D1 binding molecules of the present invention can be used in a method for treating a disease transmitted by the expression of plexin D1, which comprises the intravenous delivery of the binding molecules of the present invention in a dose, effective to treat said disease. Binding molecules can be used in a method for diagnosing a disease transmitted by plexin D1 expression, comprising the intravenous administration of conjugates of plexin D1 binding molecules with a paramagnetic, fluorescent or radioactive tracer followed by imaging of magnetic resonance, generation of optical image, SPECT or PET. The linker molecules can be used additionally in a method for treating or suppressing a disease transmitted by the expression of plexin D1, comprising the intravenous administration of the small protein and molecular binding molecules of the present invention or a composition of the molecules of small molecular binding proteins. The disease that will be treated or diagnosed may be cancer, an inflammatory disease, in particular an autoimmune disease, such as rheumatoid arthritis, or atherosclerosis, or multiple sclerosis. The diagnosis can be carried out in vivo and in vitro. An in vivo method is described above and can be carried out with magnetic resonance imaging (MRI) or with SPECT or PET cameras after accumulation of the radioactively labeled binding molecule in diseased tissue. Another diagnostic method comprises detecting the presence of plexin D1 in an in vitro or ex vivo sample. Said method comprises contacting the sample with plexin D1 binding molecules, or nucleic acids that bind the plexin D1 gene or its mRNA or a DNA copy derived from its mRNA, all linked to a detectable label, under conditions that form a complex between the antibody and plexin D1, and detect the formation of the complex. The complex can be detected by visualization of the detectable marker. The samples can be bodily fluids, such as blood, serum, plasma, saliva, urine, semen, feces or tissues, such as tumor cell biopsies. The present invention further relates to an expression vector, comprising the coding sequence of the antibody Flame F8 or A12, or of the single chain antibody derived from the 11F5H6 antibody and suitable regulatory sequences. The present invention also relates to a cell transfected with the expression vector. The present invention also relates to the recombinant protein obtainable by expressing the expression vector. Another aspect of the present invention relates to the expression vector, which comprises the coding sequence of the extracellular domain of plexin D1, optionally fused to a constant region of a human heavy chain and suitable regulatory sequences. The present invention also relates to a cell transfected with said expression vector. The present invention also relates to the recombinant protein obtained by expression of the expression vector. The recombinant protein comprises the extracellular domain of plexin D1, which binds to plexin ligands D1 and thus prevents the binding of ligands to plexin D1 associated with the cell. Preferably, the coding sequence encodes a recombinant protein comprising amino acids 47-506 of the extracellular domain of plexin D1, which binds to plexin ligands D1, and therefore prevents binding of ligands to plexin D1 with the cell, or amino acids 507-1274 of the extracellular domain of plexin D1, which binds plexin ligands D1, thus preventing the binding of ligands to plexin D1 associated with the cell. The recombinant protein can carry mutations that increase the integrity of plexin D1 ligands, and therefore have increased potency as decoy receptors. Said mutations are normally induced by making changes in the coding sequence used to produce the recombinant protein. The present invention further relates to the use of binding molecules of the present invention in a method for treating or suppressing a disease transmitted by plexin D1, comprising the intravenous or intratumoral delivery of extracellular dimeric plexin D1 domains as described above, or in a method for treating or suppressing a plexin D1-transmitted disease, comprising the intravenous administration of adenoviruses or lentiviruses, which contain the coding nucleotides of the recombinant extracellular domains of plexin D1 or part thereof, as described above .

These antagonistic plexin D1 decoy receptors interfere with the interaction between plexin D1 and its ligands, in particular neuropilin-1, semaphorin 3C and semaphorin 3E, and consequently interfere with the plexin D1 function. Preferably, plexin D1 decoy receptors have increased affinity towards plexin D1 ligands, as compared to plexin D1. Said increased affinity can be obtained by creating a library of plexin D1 extracellular domains, leading to random introduced mutations, and selecting decoy receptors with the most potent antagonistic behavior in cell migration assays. In order to produce the proteinaceous molecules (including peptides, polypeptides and glycosylated polypeptides or polypeptides having other post- or peri-translation modifications) it is desirable to insert the recombinant nucleic acid encoding fragment of the extracellular domain of plexin D1 comprising the sites of link for semaphorin 3C, semaphorin 3E and NP-1 in expression vectors. Antagonists according to the present invention are preferably produced from a nucleic acid or an expression vector according to the present invention, preferably in a host cell. The molecules of the present invention can also be used in a combination of treatment methods as described above and / or with conventional therapies or anti-angiogenic therapy to additionally prevent the formation of a tumor neovasculature, or radiotherapy and / or adjuvant chemotherapy. The present invention also relates to a method for identifying molecules that have the ability to bind to plexin D1, wherein the method comprises contacting a collection of molecules with plexin D1 and selecting the molecules of the collection showing the linkage with plexin D1, such as plexin D1 binding molecules. The collection of molecules can be present, for example, in small molecule libraries, in a protein formation, etc. The techniques for classifying collections of molecules are known per se. The present invention resides in the identification of the target to be linked, which is plexin D1. In the present application, the term "binding molecule" is used for all types of binding molecules, ie, those that bind plexin D1, those that bind a nucleic acid encoding the plexin D1 gene and those that bind the ligands plexin D1. All these types of molecules can be coupled to the effector compounds as described above. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be further illustrated in the examples below, and which are not intended in any way to limit the present invention. invention. In the examples reference is made to the following figures: Figure 1: Structural domains of members of the plexin family: four subfamilies have been identified, namely plexin A-D. The boxes with horizontal stripes indicate Sema domains, the dashed boxes diagonally indicate Met-related sequence motifs (MRS), and the cleared chart indicates the atypical MRS motif of PLXND1. The members of the Plexin B subfamily have a potential furin-like proteolytic site, marked by a gray ribbon. The transmembrane region is marked through a shaded box and is followed by two conserved intracellular domains, together comprising the SP domain, marked through two ovals. Figure 2: A) Analysis of in situ hybridization of lesions I 57-VEGF-A-I65 brain using mouse specific plxndl RNA probe labeled with digoxigenin. The tumor vessels are strongly positive (arrow), while the brain capillaries, distant from the lesions, are negative (compared to the ISH profile with the CD34 spotting in Figure 2B). Figure 3: Specific ISH analysis of human PLXND1 of multiform brain metastasis of glioblastoma (A) of sarcoma (B), melanoma (C), and mammocarcinoma (D). The inserts show stained CD31 of sections in series. The ISH of control using sense probes were negative (not shown). It should be noted that in these tumors, the expression PLXND1 is not confined to blood vessels: also in tumor cells with high levels of PLXND1, transcripts were found. T = tumor, V = vessel. Figure 4: ISH analysis using an RNA probe labeled with human-specific digoxigenin (A) and immunohistochemical staining with CD31 (B) from normal brain. It should be noted that the vessels are found abundantly, but these do not express the plexin D1 transcript. Figure 5: Specificity of phages (A) and corresponding single domain antibodies (sdabs) (B) A12 and F8 for peptide H2N-ALEIQRRFPSPTPTNC-CONH2. In A, p1010 phages were allowed to bind to PLXND1-peptide, BSA, human IgG or irrelevant peptides as described in the text. After thorough washing, bound phages were detected using an anti-M13 antibody. In B, similar incubations were carried out but now with soluble sdabs. After washing, bound and semi-quantified sdabs were detected through the VSV-G label. Figure 6: The dissociation constants (kd's) of the link between the single domain antibodies A12 and F8 were determined using the Biacore 2000 biosensor (Uppsala, Sweden). The sensor chip and the protein coupling chemicals were purchased from Biacore AB. The conjugate PLXND1-peptide-KLH (27 μg / ml in Na-Acetate, pH 4.0) or BSA (1 μg / ml in Na-Acetate, pH 5.0) to activated CM5 surfaces using Ne \ -N '- (dimethylaminopropyl) carbodiimide , / V-hydroxysuccinimide, under conditions recommended by the manufacturer. Unreacted groups were deactivated through 1M ethanolamine, pH 8.5. Kinetic measurements were carried out at a temperature of 25 ° C with a flow range of 10 ml / min in an HBS-EP regulator (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) . Six concentrations of purified sdabs with Ni affinity (within the range of 1 mM to 50 μm) were used to determine the dissociation constants (Kds) of the interaction with the peptide-PLXNDI. After each experiment, the regeneration of the sensor surface was carried out with 10 mM NaOH. The specific binding, defined through the link to a surface-PLXNDI minus the link to a control-BSA surface, was analyzed using Bl Aevaluation 4.1 software and a 1: 1 Langmuir link model. The affinities of the single domain antibodies A12 and F8 were 2.1 x 10"8 M and 3.5 x 10" 8 M. Figure 7: Specificity evaluation of plexin D1 of single domain antibodies. Figure A shows immunohistochemical staining of the trabecular bone growth plate of a mouse embryo (E16.5) with antibody from A12 simple domain, using the VSV-G label for antibody detection. The box shows an in situ hybridization of a similar embryonic structure using a plexin D1 probe labeled with mouse-specific digoxigenin. Observe the plexin D1 overlap in in situ and immunoblot hybridization. Figure 7B is a representative example of a Me157-VEGF-A165 lesion in the brain of an unprotected mouse. The vasculature that was also positive in plexin D1 ISH (see also figure 2) is immunopositive with the single domain antibody F8. Figure 8: Immunoassays with A12 single domain antibody in a selection of human brain tumors. The brains shown are A) multiforme of glioblastoma, metastasis of B) melanoma C) mammocarcinoma and D) renal cell carcinoma. The inserts in A and B consist of control stains with only anti-VSV antibody, and show that the tumor staining is specific. It should be noted that the vessels and tumor cells are highly reactive with the antibody. Figure 9: Immunoassays with single domain antibody A12 in a melanoma progression series. The immunoblots were carried out in a nevus, nevus dysplastic and melanoma phases of horizontal and vertical growth. It should be noted that only the neoplastic cells express plexin D1.

Figure 10: Immunoassays with A12 single domain antibody in sections of brain tumors Me157-VEGF-A in mice, treated with ZD6474. In untreated or placebo treated mice, staining of tumor vessels was positive with this antibody. However, in mice treated with ZD6474, there is a dose-dependent decrease in plexin D1 expression. ZD6474 was administered orally, once a day, in the dose as indicated. Figure 11: Double immunoassays with the macrophage marker CD68 (blue spotting) and single domain A12 antibody (red spotting) or mammocarcinoma. A subpopulation of macrophages expresses plexin D1 as revealed by the purple spotting protein. Figure 12: In vivo housing of phage A12, F8 or an irrelevant phage for brain lesions Me157-VEGFi65- Mice containing tumors were injected with phage 1012 in the tail vein and after 5 minutes the mice were anesthetized and subjected to a cardiac perfusion with 15 ml of phosphate-regulated saline. The mice were sacrificed, the brains were eliminated and the frozen sections were analyzed with respect to content and distribution of phages. A) M13 spotting of frozen section of Me157-VEGF 6s brain lesions. The phages are clearly associated with vessels, as evidenced by the anti-CD34 immunoblot in a section in series, shown in B). The arrows point to a positive container-CD34, distant from the lesion, which is not signaled by the anti-M13 stain. The sub-frame in (A) shows a control experiment in which an irrelevant phage was injected. C) Distribution of F8 sdab after intravenous injection in mice containing tumor. Sdabs were visualized by immunohistochemistry using an anti-VSV antibody. It should be noted that sdab is detected in tumor vessels but not in capillaries of normal brain. The inset shows the control experiment in which an irrelevant sdab was injected. An interstitial location consistent with the pierced nature of the vessels in these tumors was observed. D) Quantification of phage housing. Tumor tissue was sectioned from 10 μ? P frozen sections using laser capture dissection microscopy. The number of phage forming colonies (cfp) was counted after the injection of TG1 cells. Twenty times more F8 phages were eluted from tumors than from comparable areas of unaffected brain tissues. Figure 13: Single-domain antibody housing for tumor vessels that have not formed by themselves. Unprotected mice were inoculated with a cell suspension of 1.5 x 105 cells from the human glioma semi-graft E98, which was obtained from subcutaneous E98 tumor. After 3 weeks, phages carrying a single F8 domain antibody were injected into the tail vein, and After 5 minutes the mice were anesthetized and subjected to cardiac perfusion using 15 ml of phosphate-buffered saline. The mice were subsequently sacrificed, the brains were removed and fixed in formalin. Sections in series were stained with antibodies against M13 protein p8 (A), the endothelial marker CD34 (B) and glut-1 (C, a marker for pre-existing brain capillaries). The comparison of A, B and C reveals that not only newly formed tumor vessels accumulate F8 phage, but also undilated cerebral vessels that express glut-1 and that therefore are considered pre-existing brain vessels that had been incorporated into the brain. the tumor Figure 14: Effects of extracellular domains of plexin D1 on the development of tumor vasculature. The double transfectants of the human melanoma cell line Me157, which expresses VEGF-A16s and the extracellular domain of plexin D1 comprising amino acids 1-850, were injected into the right internal carotid artery of unprotected mice. After three weeks, the mice were subjected to magnetic resonance image generation enhanced with Gadolinium-DTPA. Figure 14A shows MR images of two control mice carrying Me157 brain tumors expressing only VEGF-A165. Figure 14B shows brain MR images of two mice carrying the double transfectant. The filtration vascular, as tested by Gd-DTPA extravasation, tends to be lower in double transfectants, suggesting that vascular filtration induced by VEGF-A is counteracted by the plexin D1 ectodomain. More importantly, blood vessels in double transfected tumors are activated, as indicated by activation of CD34, which still express glut-1, strongly suggesting that these vessels are pre-existing vessels that are incorporated into the tumor. through the phenomenon of co-option. It should be noted that blood vessels in tumors that express only VEGF-A165, are negative for glut-1 and therefore can be considered as newly formed. Figure 15: Western blots were generated with recombinant plexin D1 ectodomains, expressed in E. coli and comprising amino acids 47-506 (column 1) or 225-358 (column 2). Mouse serum 25 was tested before (panel A) and after (panel B) immunization with plexin region D1 47-506. As shown in Figure 15B, the mouse immune serum specifically recognized the recombinant protein E. coli 47-506 (52 kDa, column 1) and the protein comprising residues 225-388 of plexin D1 (a protein 18 kDa resting completely within the sequence that was used for immunization, column 2). The pre-immune serum did not show reactivity (panel A). When it was tested in Immunohistochemical staining in a brain metastasis of a soft alveolar tissue sarcoma, the mouse immune serum (panel B), but not the pre-immune serum (panel C), showed positivity towards blood vessels and tumor cells, a pattern of spotting that was similar to that of the A12 simple domain antibody. Figure 16: Immunohistochemistry with monoclonal IgM antibodies, obtained from mouse B-lymphocytes 25. 11F5H6 and 17E9C12 antibodies were selected based on reactivity against protein 47-506 in ELISA, and analyzed for their potential to detect plexin D1 in sections frozen of human tumors. These antibodies showed strong positivity in sarcoma and melanoma brain metastases, as illustrated in the figure. It should be noted that the inserts in the C-F panels represent control stains in which the primary antibody was omitted. Panels A and B show that these antibodies do not noticeably recognize vessel structures in normal brain tissue. Figure 17: Tumor housing of antibody 11F5H6.

To further assess whether monoclonal antibody 11F5H6 has the ability to recognize tumor blood vessels, angiogenic Me157-VEGF-A tumors were grown in brains of unprotected mice, essentially as described in example 10. The 11F5H6 antibody was injected ( 1 mg) in a vein of the lateral tail and allowed to circulate for 15 minutes. After this period, the mice were anesthetized with 1.3% isoflurane and the chest was opened, at which time a cardiac perfusion was carried out with 20 ml of phosphate-regulated saline. After this procedure, the mice were decapitated, and the brains were removed and either pressurized or fixed in formalin. The frozen sections of 4 μp ?, were stained with anti-IgM antibody. In Figure 17A, antibody 11F5H6 is shown to house and accumulate in tumor vessels but not in normal vessels (compare anti-IgM staining of Figure 17A with anti-endothelial staining CD31 in Figure 17B). Such staining is not seen when anti-IgM staining is carried out in non-injected mice. Therefore, 11F5H6 is a promising antibody that allows the direction of the tumor. Figure 18: Expression of Plexin D1 in macrophages in a mouse model of rheumatoid arthritis. The smears were carried out with single domain antibody A12. Figure 19: Plexin D1 expression in atherosclerosis. A subgroup of macrophages in human atherosclerotic plaques expresses plexin D1. The smears were carried out with single domain antibody A12. A double spotting was carried out, which displays plexin D1 in red and the macrophage marker CD68 in blue. A purple color indicates co-expression. The table shows the following: Table I: Analysis of different pathologies of plexin D1 expression. Table II: Plexin D1 expression in melanocytic lesions increases from benign to malignant lesions. EXAMPLES EXAMPLE 1 Specific expression of plexin D1 in blood vessels associated with tumor Plexin D1 expresses neurons but also in endothelial cells in angiogenic vessels during embryogenesis. The present invention demonstrates that plexin D1 is expressed in blood vessels associated with tumor but not in normal blood vessels. This has been shown through in situ hybridization of mouse brain, which contains angiogenic human melanoma lesions (Figure 2). The animal tumor model is described in (Kusters, B and associates, Cancer Res 63: 5408-5413 (2003)). In summary, the tumor cells are injected through a microsurgical procedure in the right carotid artery, resulting in tumor growth in the parenchyma of the right hemisphere of the brain. After three weeks, in the generation of neurological symptoms, the mice were sacrificed and the brains were removed and fixed in formalin. Sections of 4 μ? T were subjected to in situ hybridization? with sense and antisense RNA fragments labeled with digoxigenin. RNA probes were generated by transcription using T3 and T7 RNA polymerase, respectively, of a PCR product, comprising 600 bases in the 3'-untranslated region, and which was flanked by T7 and T3 promoters (Van der Zwaag and associates . (2002), supra). In situ hybridizations using antisense RNA probes and RNA sense probes as negative controls were carried out using standard protocols. The sections were dewaxed by paraffin fusion at a temperature of 60 ° C and subsequent treatments with xylene and ethanol. After rehydration in phosphate buffered saline (PBS) proteinase K (10 μg / m \ PBS in 20 mM Tris-HCI pH 7.4 / 5 mM EDTA) was digested for 15 minutes at a temperature of 37 ° C. Sections were post-fixed in formaldehyde buffered at 4% for 10 minutes, and acetylated in 0.1 M acetic acid anhydride. Slides were subsequently washed in 2xSSC (Sodium Citrate / Sodium Chloride) and milliQ. After drying, slides were hybridized with RNA probes labeled with digoxigenin overnight at a temperature of 65 ° C in 50% formamide / 2xSSC. High levels of plexin D1 RNA were observed in vessels of angiogenic Me157 tumors (Figure 2) using a mouse-specific plexin D1 RNA probe. The tumor cells were also positive for transcription. The homology does not Perfect plexin between human mouse D1 resulted in a weaker signal in human tumor cells using the mouse probe. EXAMPLE 2 Expression of plexin D1 in tumors To investigate the expression of plexin D1 RNA in human tumor samples, we carried out in situ hybridizations with a human-specific plexin D1 RNA probe. High levels of plexin D1 RNA expression were found in a number of human tumors, of which (glioblastoma multiforme, brain sarcoma metastasis, renal brain carcinoma, colon and breast adenocarcinoma), both in tumor vasculature and in tumor cells. Table 1 provides a summary of tumor types expressing plexin D1. Figure 3 shows some examples of in situ hybridizations, for example, a glioblastoma, a metastasis of brain melanoma and a brain metastasis of colon carcinoma. The plexin D1 RNA was found not only in the tumor vasculature, but also excessively in the tumor cells themselves. Importantly as in Figure 4A, plexin D1 RNA expression was not observed in normal brain vasculature. In Figure 4B, a CD31 stain is shown, demonstrating that abundant vessels are present in these sections.

EXAMPLE 3 Preparation of plexin D1 antibodies To detect plexin D1 protein, antibodies with affinity towards plexin D1 were selected. For this purpose, a M13 pHENIX phage library expressing VH antibodies of the single domain Llama was constructed, constructed by RT-PCR of B-lymphocytes Flame as described in (van Koningsbruggen, S et al., J Immunol Methods 279: 149 -161 (2003)). The resulting population of cDNAs encoding single domain-VH antibody fragments (sdab) was ligated into the phagemid vector pHENIXHis8VSV (results not shown), resulting in a fusion product with a tag-8 * His and a tag- VS VG in the C-term. After electroporation in TG1 E. coli cells, ampicillin-resistant colonies were harvested and collected. The resulting library had complexity of 8 x 108 clones. Eighty percent of the plasmids contained the full length sdab insert determined by PCR analysis and spot spot immunological detection of VSV-G tags in sdab (see below). The phage library was propagated in phagemids in TG1 E. coli bacteria. The phage particles were rescued by injection with the trypsin-sensitive auxiliary phage M13K07 (50). The phages were purified and concentrated from the culture supernatant by precipitation with Polyethylene glycol / 2.5 M NaCl 20% by means of methodology standard. To select the phages, the display of antibodies with affinity to plexin D1, immunotubes (Nunc, Roskilde, Denmark) were coated overnight at a temperature of 4 ° C with conjugated peptide-KLH 5 g / ml (H2N-ALEIQRRFPSPTPTNC-CONH2 ) corresponding to amino acids 1-16 of mature human PLXND1 protein (access No. AY116661) in 50 mM NaHCO3 (pH 9.6). It should be noted, that the glutamic acid at position 3 in this peptide is a lysine in the mouse sequence, the remaining amino acids are homologs with mouse plxndl. After rigorous washing with PBS / 0.5% Tween 20 (PBST), non-specific binding sites were blocked with 5% marvel in PBST (MPBST, 1 hour at room temperature (RT)) and 1013 phage particles of the existence of libraries were incubated with the immobilized peptide for 90 minutes at room temperature. After thorough washing with PBST and PBS, bound phages were eluted by trypsin treatment (10 mg / ml, 30 minutes at room temperature). After deactivation of trypsin with 1% newborn calf serum, the eluate was used to infect phase-log TG1 cells to amplify the phage-binding PLXND1 and calculate the number of linkers. To enrich the binding phages, four selection rounds were carried out. As of the second round, the selections were carried out against unconjugated peptides, immobilized on DNA-binding plates (Costar) to avoid selection of linkers-KLH. The individual PLXND1 binding phages with full length sdab inserts confirmed with PCR were tested for plexin D1 specificity. the deposits of DNA-binding plates or immunoplates (Nunc) were coated overnight at a temperature of 4 ° C with PLXND1-peptide or a relevant peptide (1 μg / tank in PBS / 0.5 M NaCl pH 9.0), Bovine serum albumin (1 μg / reservoir in 50 mM NaHC03 pH 9.6) or human immunoglobulin G (1 μg / reservoir in 50 mM NaHC03 pH 9.6). After blocking non-specific binding sites with MPBST, the deposits were incubated with phage in MPBST for 1 hour at room temperature and the unbound phages were removed by thorough washing. Binding phages were detected using anti-M13 conjugate-HRP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and tetramethylbenzidine (TMB, bioMerieux B.V., The Netherlands). The reaction was terminated with 2M H2SO4 and the enzymatic activity was quantified by measuring the absorbance at 450 nm using an ELISA reader. Using this selection procedure, the phages displaying the single domain antibodies V-H A12 and F8 on their surfaces were identified as specific linkers. Figure 5A shows that the associated antibodies A12 and F8 with M13 phage, bind specifically to the plexin D1 peptide, but not to bovine serum albumin, immunoglobulins or an irrelevant peptide. The expression of soluble single domain antibodies was induced in phase-log E. coli TG1 cells by culturing at a temperature of 30 ° C in a 2xTYA / 1 mM IPTG medium. Sdabs were harvested by osmotic lysis using ice-cooled TES buffer (200 mM TrisHCI, 0.5 mM EDTA, 500 mM sucrose) containing a cocktail of protease inhibitor (Roche, Basel, Switzerland). The sdab concentrations were estimated by spot spot analysis using the P5D4 mouse monoclonal anti-VSV-G, rabbit anti-mouse immunoglobulin conjugated with alkaline phosphatase (Dako, Denmark) and spotted NBT / BCIP. Sdabs were tested in ELISA with respect to peptide-PLXNDI specificity. The single domain antibodies A12 and F8 did not bind to irrelevant peptides, not to bovine serum albumin, and not to human immunoglobulin G (Figure 5B). The dissociation constants (kd's) of the link between the single domain antibodies A12 and F8 were determined using the Biacore 2000 biosensor (Uppsala, Switzerland). The sensor chip and protein coupling chemistries were purchased from Biacore AB. The conjugate PLXND1-peptide-KLH (27 μg / ml in Na-Acetate, pH 4.0) or BSA (1 μg / ml in Na-Acetate, pH 5.0) was coupled to activated CM5 surfaces using carbodiimide of / V-ethyl-AT - (dimethylaminopropyl), N-hydroxysuccinimide, under conditions recommended by the manufacturer. The non-reactivated groups were deactivated through 1M ethanolamine, pH 8.5. Kinetic measurements were carried out at a temperature of 25 ° C with a flow range of 10 ml / min in HBS-EP buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20). Six concentrations of purified sdabs with Ni affinity (within the range of 1 mM to 50 μm) were used to determine the dissociation constants (Kds) of the interaction with the peptide-PLXNDI. After each experiment, the regeneration of the sensor surface was carried out with 10 mM NaOH. The specific binding, defined by the link to a surface-PLXNDI minus the link to a control-BSA surface, was analyzed using Bl Aevaluation 4.1 software and a 1: 1 Langmuir link model. The affinities of single domain antibodies A12 and F8 were 2.1 x 10"8 and 3.5 x 1 O" 8 M, respectively (Figure 6). EXAMPLE 4 Immunohistochemical staining with single-domain antibodies A12 and F8 Single-domain antibodies are labeled at the carboxyterminal end with a VSV-His tag, allowing immunohistochemical staining using an anti-VSV antibody. The protocol found below was followed by immunohistochemical staining with single-domain antibodies A12 and F8. After deparaffinization, the endogenous peroxidase activity was blocked by 0.03% H202 incubation. Antigen retrieval was carried out by pronase treatment according to standard protocols. Subsequently, the sliders were incubated previously with normal horse and goat serum (to block non-specific binding sites in human and mouse tissue sections, respectively), followed by incubation with sdabs for 1 hour. Sdabs were detected by incubation in 1 hour sequences with a mouse or rabbit anti-VSV-G antiserum (Sigma-Aldrich Chemie BV, Zwijndrecht, Holland), biotinylated anti-mouse or anti-rabbit antibody as appropriate (Vector , Burlingame, CA), and avidin-biotin peroxidase complex (Vector, Burlingame, CA). Finally, peroxidase was visualized by peroxidase reaction 3-amino-9-ethylcarbazole (ScyTek, Utah, USA) with hematoxylin as a counter-stain. All steps were carried out at room temperature. The specificity of the A12 and F8 antibody for plexin D1 in immunohistochemical staining was first checked by staining of mouse embryos, in which the plexin D1 expression patterns at the RNA level were well characterized (Van der Zwaag et al. (2002)). , supra). and comparing profiles with immunoblots with antibody Anti-endothelial anti-CD31 (DAKO, Glostrup, Denmark). In the trabecular bone growth plate of mouse embryos in E16.5, immunostaining was observed in positive blood vessels-CD31. It was correlated with in situ hybridization for plexin D1 transcription (Figure 7A). The origin of the blood vessel of the PLXND1 expression was further confirmed by carrying out spotting in serial sections with sdabs and anti-human anti-CD31 antibody (anti-human CD-31). EXAMPLE 5 Staining of tumor cells with F8 Sections of four μ? T were stained? of cerebral mouse xenografts of the human melanoma cell line Me157-VEGF-A (Kusters et al., (2003), supra). with F8 single domain antibody, according to the protocol exemplified in Example 4. The antibody clearly recognized plexin D1 in tumor blood vessels (Figure 7B). To additionally investigate the expression of the plexin D1 protein in tumors, tumor tissue of different origins was immunostained or embedded in paraffin (multiform glioblastoma (FIG. 8A), melanoma brain metastasis (FIG. 8B) colon carcinoma (FIG. 8C) and renal cell carcinoma (Figure 8D) with anti-PLXND1 sdabs Immunohistochemistry using the A12 antibody and comparison with the anti-human CD31 stains Sections in series, showed expression in all the tumors reviewed and plexin D1 expression was confirmed at the protein level in tumor cells and in tumor blood vessels. EXAMPLE 6 Plexin Expression Timing in Malignant Cells To investigate whether the expression of plexin D1 occurring in pre-malignant cells, a series of melanoma in progress was stained, consisting of benign nevi, dysplastic nevi, melanoma of radial growth phase, invasive melanoma and disseminated melanoma. The melanocytes in benign nevi and nevi dysplastic did not express the protein, while the transformed cells in malignant form, both in tumors of the radial growth phase and the vertical growth phase, are positive in terms of protein (Figure 9 and Table 1). II). EXAMPLE 7 Activation status of cells expressing plexin D1 Expression of plexin D1 is related to the activation state of endothelial cells in tumor blood vessels. Treatment with ZD6474, an inhibitor of VEGFR2 and EGFR, previously showed blockage to angiogenesis in mouse brain tumor model, resulting in a change of the co-option phenotype from angiogenic to non-angiogenic vessel (43). Treatment with ZD6474 resulted in a decrease in plexin D1 expression in blood vessels associated with tumor in a dose-dependent manner (figure 10). Therefore, the expression plexin D1 is a characteristic of activated endothelial cells. EXAMPLE 8 Immunohistochemistry with A12 in normal tissues Expression of plexin D1 in brain, heart, skin, kidney, vessel, intestine, normal endometrium was checked by immunohistochemistry using antibody A12. The D1 plexin vessels expressed in proliferative myometrium, show that plexin D1 is associated not only with pathological angiogenesis, but also with physiological angiogenesis (not shown). In some cases, co-immunoblots were performed with CD68 macrophage marker. These stains revealed that a subpopulation of macrophages expressed the protein (figure 11). It was also discovered that fibroblasts in the skin and certain intestinal proliferating epithelial cells express plexin D1 (not shown). EXAMPLE 9 Staining of Macrophages in Inflammatory Diseases To further review the involvement of plexin D1 in diseases with prominent macrophage involvement, immunohistochemical stains were performed on atherosclerotic plaques, multiple sclerosis and rheumatoid arthritis. Macrophages express plexin D1.

EXAMPLE 10 Access to plexin D1 in tumor vessels by intravenous injection The expression of plexin D1 protein in tumor blood vessels suggests that plexin D1 is accessible through intravenous injection. To test this, microsurgical injections were injected into the right internal carotid artery of BALB / C deprotected mice, Me157 cells stably transfected 2x105 expressing the VEGF-A16s isoform. After 18 days, when the animals showed neurological symptoms (Kusters and associates, (2003) supra). 1012 PLXND1-binding phages from clones A12, F8 or non-relevant phages were injected into the tail vein of unprotected mice, carrying established Me157-VEGF-A165 brain metastases (n = 2 for A12, n = 4 for F8 , n = 3 for control phage). In two other groups of mice, we injected intravenously 30 μg of F8 sdab or a control sdab (n = 2 for each group). After 5 minutes, the mice were anesthetized using isoflurane, the breasts were opened, and the unbound phages were flushed from the system by cardiac propulsion with 15 ml of phosphate buffered saline (PBS). Subsequently, the mice were sacrificed by cervical dislocation, and the parts of the brains, hearts, lungs, livers, vessels and kidneys were frozen under pressure in liquid nitrogen.

Other formalin parts were fixed to be embedded in paraffin. After a stain of short hematoxylin, the tumors were dissected from brain sections of 10 μ? using laser capture dissection microscopy (Leica laser dissection microscope). Equivalent areas were dissected from unaffected brain, contralateral to the tumor. Subsequently, the phages were eluted from tissue samples dissected using the trypsin treatment and used to infect TG1 cells. The numbers of phages that form colonies were counted and used with a measure of tumor accommodation. To quantitatively assess tumor accommodation by phages or sdabs, sections of 4 μ ?? were stained, in series with sections used for laser dissection, with anti-M13 p8 antibody (Abcam Limited, Cambridge, UK) to detect bound phages, or anti-VSV-G antibodies, (Sigma-Aldrich) to detect single domain antibodies. Intravenous injection of phage M13 showing single domain antibody an \\ - PLXND1 F8, but not phages carrying irrelevant single domain antibodies, in mice bearing angiogenic melanoma lesions resulted in phage accumulation in tumor vessels but not the specific detectable presence of phages in normal brain vessels, nor blood vessels in liver, vessel, kidney (Figure 12A, D and not shown). This indicates that plexin D1 expresses on the luminal side of the endothelial cell specifically in tumor blood vessels and can therefore be used as an airborne marker. The injection of the partially purified simple domain antibody led correspondingly to a preferential tumor location (Figure 12C). In the latter situation, it should be considered that the small molecular weight of 20 kDa of the single domain antibodies allows the extravasation of the highly permeable tumor vessels and the accumulation in the tumor interstitium. The latter effect is non-specific and is also observed with non-relevant single-domain antibodies. Antibodies of small molecular weight and relatively low affinities are considered to have a higher penetration capacity through tumors and are more suitable for targeting the tumor cell compartment. EXAMPLE 11 Accumulation of F8 in tumor blood vessels Mice were injected transcranially with E98, a glioma xenograft line. E98 tumors were maintained as subcutaneous tumors. An attentive Balbc / c nu / nu mouse bearing a subcutaneous E98 tumor was killed and the tumor was removed. The tumor chopped pieces with a sterile scalpel and the homogenate was passed through a nylon mesh filter sterile 70 μ ?? They were injected transcranially into the brain of the unprotected mouse, twenty μ? of the resulting cell suspension, which contains 150,000 cells. After 3 weeks, M13 phage displaying the F8 single-domain antibody was injected intravenously, and after five minutes the mice underwent cardiac perfusion with 15 ml of phosphate-buffered saline. The mice were killed, the brains were removed and fixed in formalin. Sections of four μ? to immunohistochemistry with anti-M13 antibody, and serial sections stained immunohistochemically with antibodies against CD34 (endothelial marker) and glut-1 (a marker for preexisting brain endothelial cells (Kusters, B and associated, Cancer Res 62 : 341-345 (2002).) Phages carrying single-domain anti-plexin D1 antibodies accumulated specifically in blood vessels associated with tumor, but not in normal vessels (Figure 13). they accumulated in tumor blood vessels that were positive in glut-1, and which therefore can be considered as pre-existing blood vessels, instead of newly formed blood vessels.This indicates that not only angiogenic blood vessels are subject to direction with anti-plexin D1 antibodies, but also not Angiogenic, blood vessels still activated in tumors. EXAMPLE 12 Recombinant D1 plexin ectodomains inhibit angiogenesis Me157 human melanoma cells were transfected with the VEGF-A16s coding sequence in pIREShyg vector. Stably transfected cells were selected by culturing in 200 μg / ml hygromycin in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and penicillin / streptomycin. Because expression of the hydromycin resistance gene is linked to that of cDNA VEGF-A through the internal ribosomal entry site (IRES), all cells resistant to hydromycin will also produce the VEGF-A protein. Me157-VEGF cells stably transfected were subsequently infected with pIRESneo-PlexinDI ED. The vector containing the cDNA encoding the extracellular domain of nucleotides 1-2745 was ligated through IRES for expression of the neomycin resistance gene. Double transfectants were injected into the right carotid artery of unprotected mice, and the tumors were fixed. In the emergence of neurological symptoms (approximately 18 days) the mice underwent denial of magnetic resonance imaging enhanced with Gadolinium-DTPA. Subsequently, the mice were sacrificed, the brains were fixed in formalin and subjected to immunohistochemical staining. to examine the vasculature of the tumor. When compared with the controls, which consist of tumors that express only VEGF-A, the Gd-DTPA improvement in MRI-weighted magnetic resonance imaging (MRI) or smaller (compare figure 14A, which represents two examples of tumors Me157- VEGF-A165, with 14B representing two examples of VEGF-A165 / PLEXIND1 -ED tumors.In tumors expressing VEGF-A165 and Plexin D1 ectodomain, the vasculature showed activation of the endothelial marker CD34, (an unmistakable hallmark of endothelial activation by VEGF-A165.) The vasculature in tumors expressing only VEGF-A165, is negative for the glut-1 brain endothelial cell marker, which is consistent with the fact that these vessels are newly elaborated and Consequently, they lack specific endothelial cell-brain markers, as can be seen in Figure 12B, the vessels that are associated with tumors that also express plexin D1 ectodomain, express glutathione 1. This is a strong indication that these vessels are actually pre-existing. Therefore, the plexin D1 ectodomain does not prevent the activation of endothelial cells by VEGF-A165, but prevents the formation of neovasculature. EXAMPLE 13 High Affinity Antibodies against Plexin D1 A protein sequence, corresponding to amino acids 47-506 (the 459 amino-terminal amino acids of the majority of the mature protein) was expressed in E. coli M15 pREP4 cells, using the expression vector pQE16 (Qiagen). The recombinant protein, which was produced in the bacterial cell as inclusion bodies, was dissolved in the denaturing buffer, which contains 4M urea and 1 mM dithiothreitol (DTT) and subsequently dialyzed gradually against PBS. The protein was used to immunize BALB c / c 25 mice, according to standard procedures. Figure 15 shows the characteristics of mouse serum. As shown in Figure 15B, the mouse immune serum specifically recognized the recombinant protein E. coli 47-506 (52 kDa, column 1), and a second recombinant plexin D1 sequence of 18 kDa, comprising amino acids 225 -388 (therefore resting completely within the sequence that was used for immunization, column 2). The pre-immune serum did not show reactivity (panel A). When tested in immunohistochemical staining of brain metastasis of a soft alveolar tissue sarcoma, the mouse immune serum (panel D), but not the pre-immune serum (panel C), showed positivity towards blood vessels and tumor cells, a machado pattern that was similar to that of the A12 simple domain antibody. Therefore, the B-lymphocytes of these mice were considered adequate to generate Hybridomas of B-vessel lymphocytes with myeloma cell line SP2 / 0. From these hybridomas, a number of cell lines producing antibodies based on reactivity against protein 47-506 in ELISA were selected, and analyzed for their potential to detect plexin D1 in frozen sections of human tumors. Of these, 11F5H6 and 17E9C12, both antibodies of the IgM subtype, showed strong positivity in sarcoma and melanoma brain metastases, as shown in figure 16. The inserts in the CF panel represent control stains in which the patient was omitted. primary antibody. Panels A and B show that these antibodies do not noticeably recognize vessel structures in normal brain tissue. EXAMPLE 14 Monoclonal Antibody 11F5H6 Having the Capacity to Recognize Tumor Blood Vessels To additionally assess whether monoclonal antibody 11F5H6 has the ability to recognize tumor blood vessels, angiogenic Me157-VEGF-A tumors were grown in brains of unprotected mice, essentially as described in Example 10. Antibody 11F5H6 (1 mg) was injected into the lateral tail vein and allowed to circulate for 15 minutes. After this period, the mice were anesthetized with 1.3% isoflurane and The chest was opened, at which time a cardiac perfusion was carried out with 20 ml of phosphate-buffered saline. After this procedure the mice were decapitated, and the brains were removed and snap frozen and fixed in formalin. The frozen sections of 4 μG? they were stained with anti-IgM antibody. In Figure 17A it is shown that antibody 11F5H6 is housed and accumulated in tumor vessels but not in normal vessels (compare anti-lgM staining in Figure 17A with anti-endothelial staining CD31 in Figure 17B). Such staining is not observed when anti-IgM staining is carried out in non-injected mice. Therefore, 11F5H6 is a promising antibody that allows tumor targeting. EXAMPLE 15 Expression of plexin D1 in rheumatoid arthritis plexin D1 was expressed in macrophages in mouse models of rheumatoid arthritis (figure 18). A subset of macrophages in human atherosclerotic plaques also expresses plexin D1 (Figure 19). The smears were carried out with single domain antibody A12. In Figure 19, a double spotting was carried out, displaying plexin D1 in red and the macrophage marker CD68 in blue. The purple color indicates coexpression.

S EC U E N C IAS A 12 (SEQ ID NO: 1): ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCC ATGGCCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTG AGACTCTCCTGTGCAGCCTCTGGAAGCAGTATCAGTATCAATAACTGGGGCTGGTACCGC CAGGCTCCAGGAAAACAGCGCGAGCGGGTCGCAGCTATATCTGGTGGTGGTAAAACAGTC TATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTG TATCTGCAAATGAACAGCCTGAAACCTGAGGATACGGCCGTCTATTACTGTAGAGCAGTC CGGAAAAGTACGGGTTGGCTTAGGGGGCTTGACGTCTGGGGCCAGGGGACCCAGGTCACC GTCTCCGCAGAACCCAAGACACCAAAACCACAACCAGCGGCCGCACATCATCACCATCAT CACCATCATTATACAGACATAGAGATGAACCGACTTGGAAAGGGGGCCGCATAG MKYLLPTAAAGLLLLAAQPA MAQVQLQESGGGLVQPGGSL RLSCAASGSSISINNWGWYR QAPGKQRERVAAISGGGKTV YADSVKGRFTISRDNAKNTV YLQMNSLKPEDTAVYYCRAV RKSTGWLRGLDVWGQGTQVT VSAEPKTPKPQPAAAHHHHH HHHYTDIEMNRLGKGAA @ F8 (SEQ ID NO: 2): ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCC ATGGCCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGAGACTCTCTG AGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTACTTTGATTATGGCCTGGTTCCGC CAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCGGCGATTAGCCGGGGTGGCGGTAGCACA AGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACGCG GTGTATCTACAAATGAACAGCCTGAAACCTGATGACACGGCCGTCTATTACTGTAATGCC CGGTACGGTAGCCGAATTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCC AAGACACCAAAACCACAACCAGCGGCCGCACATCATCACCATCATCACCATCATTATACA GACATAGAGATGAACCGACTTGGAAAGGGGGCCGCATAG MKYLLPTAAAGLLLLAAQPA MAQVQLQESGGGLVQAGDSL RLSCAASGRTFSTLIMAWFR QAPGKEREFVAAISRGGGST SYADSVKGRFTISRDNSKNA VYLQMNSLKPDDTAVYYCNA RYGSRIY GQGTQVTVSSEP KTPKPQPAAAHHHHHHHHYT DIEMNRLGKGAA6 Single-chain antibody sequence, derived from antibody 1 1 F5H6 (S E Q I D N O: 3) MKYLLPTAAAGLLLLAAQPA ADYKDIV TQTPLSLPVSLGDQASISCRSSQSIVHSNGNT YLE YLQKPGQSPKLLIYKVFNRLSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPLTFGAGTKLELKRGGGGSGGGGSGGGGRAPGGGGSEVQLQQSGPELVKPGASMKISCK ASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNGGTSYNQKFKGKATLTVDKSSSTAYMELL SLTSEDSAVYYCARAITTDGWFAYWGQGTLVTVSAAAAHHHHHHHHYTDIEMNRLGKGAA Single chain antibody sequence, antibody derivative 1 7E9 C 12 (S EQ I D N O: 4) MKYLLPTAAAGLLLLAAQPAMADYKDIQMTQTPSSLAVSAGEKVTMSCKSSQSVLYSSNQK NYLA YQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQY LSSWTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLQQSGAELVKPGASVKLSCTAS GFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQG ATITADTSS TAYLQLSSL TSEDTAVYYCAMDY GQGTSVTVSSAAAHHHHHHHHYTDIEMNRLGKGAA Table 1 PLXND1 expression in human tissues Table 2 PLXND1 expression in melanoma progress series

Claims (27)

1. CLAIMS 1. Plexin D1 to be used as a targeting protein in the treatment or diagnosis of disorders involving the expression of plexin D1.
2. 2. Plexin D1 as described in claim 1, characterized in that the diagnosis is carried out by detecting the presence of plexin D1 in the body or a body tissue or fluid.
3. 3. Plexin D1 as described in claim 1, characterized in that the treatment is carried out by directing plexin D1 for the delivery of therapeutics to the site where the treatment is needed.
4. 4. Plexin D1 as described in claim 1, characterized in that the treatment is carried out by interfering with the interaction of plexin D1 and its ligands.
5. 5. Plexin D1 as described in claim 1, characterized in that the treatment is carried out by interfering with the plexin function D1.
6. 6. Plexin D1 as described in claim 1, characterized in that the treatment is carried out by interfering in the expression of the gene encoding plexin D1.
7. 7. The use of plexin D1 binding molecules, a plexin D1 encoding nucleic acid or a plexin D1 ligand for the preparation of a therapeutic composition for the treatment or diagnosis of disorders involving the plexin expression D1.
8. 8. The use as described in claim 7, characterized in that the disorders comprise disorders in which plexin D1 is expressed in tumor cells, tumor blood vessels or activated macrophages.
9. 9. The use as described in claim 7 or 8, characterized in that the disorders are selected from brain tumors, in particular astrocytomas, oligodendrogliomas and hemagioblastomas, colon carcinomas, in particular colon ductal carcinomas, prostate carcinomas, carcinomas of renal cell, in particular renal clear cell carcinomas, breast carcinomas, in particular ductal carcinomas of the breast, ovarian carcinomas, squamous cell carcinomas, melanomas, lung carcinomas, in particular small cell lung carcinomas and lung carcinomas of non-small cell, soft tissue sarcomas. The use as described in claim 7 or 8, characterized in that the disorders are inflammatory disorders. 11. The use as described in claim 10, characterized in that the inflammatory disorder is an autoimmune disease. 12. The use as described in claim 11, characterized in that the autoimmune disease is arthritis rheumatoid 13. The use as described in claim 10, characterized in that the inflammatory disease is atherosclerosis or multiple sclerosis. The use as described in claim 7, characterized in that the plexin D1 binding molecules are selected from antibodies, antibody fragments, protein domains, peptides, small molecules, DNA or RNA aptamers. 15. The use as described in claim 7, characterized in that the molecules that bind to the nucleic acid encoding plexin D1, are selected from siRNA, antisense RNA, antisense phosphothio oligonucleotides. 16. The use as described in claim 7, characterized in that the molecules that bind a plexin D1 ligand are selected from antibodies against the ligand, the soluble ectodomain of plexin D1, peptides with affinity for the plexin D1 binding site. 17. The use as described in any of claims 7 to 16, characterized in that the binding molecule is labeled with a detectable label. 18. The use as described in claim 12, characterized in that the detectable label is selected from a radioactive label, a paramagnetic label, a fluorescent label, a chemiluminescent label. 19. The use as described in any of claims 7 to 18, characterized in that the binding molecule is supplied with an effector compound or a nanoaparate comprising an effector compound. 20. Use as described in claim 19, characterized in that the effector compound is a toxin, a compound that induces thrombosis, a chemotherapeutic agent, a radioactive portion, a peptide that induces apoptosis, in particular (KLAKLAK) 2. 21. The use as described in claim 20, characterized in that the toxin damages or exterminates endothelial cells to induce thrombosis, and in particular is ricin. 22. The use as described in claim 20, characterized in that the compound that induces thrombosis is truncated tissue factor. 23. The use as described in claim 20, characterized in that the chemotherapeutic agent is selected from doxorubicin, cisplatin, bleomycin sulfate, carmustine, chlorambucil and hydroxyurea of cyclophosphamide. 24. The use as described in claim 20, characterized in that the radioactive entity is selected from: tecnetium 99m, iodine-123, iodine-131, rhenium-186 or -188, gallium-67, yttrium-90, lutetium- 177 25. The use as described in claim 19, characterized in that the nanoapats are liposomes, polymersomes, in particular polymersomes composed of block copolymers. 26. Molecules that link plexin D1. 27. Molecules that bind plexin D1 as described in claim 26, characterized in that they comprise the plexin D1 binding part of A12 (SEQ ID NO: 1), F8 (SEQ ID NO: 2), 11F5H6 (SEQ ID NO: 3) or 17E9C12 (SEQ ID NO: 4). 29. The plexin binding molecules as described in claim 26 or 27, coupled to a detectable label as described in claim 18. 30. The plexin binding molecules as described in claim 26 or 27, coupled to an effector molecule or a nanoareate comprising an effector compound, wherein the effector compound is as defined in any of claims 20 to 24. 31. The plexin binding molecules as described in claim 26 or 27, characterized in that the nanoareate is as defined in claim 25. 32. A diagnostic composition comprising a plexin binding molecule as described in claim 29. 33. A therapeutic composition comprising a plexin binding molecule as described in any of claims 30 to 32. 34. The single chain antibody A12 (SEQ ID NO: 1). 35. The single chain antibody F8 (SEQ ID NO: 2). 36. The single chain antibody 11F5H6 (SEQ ID NO: 3). 37. The single chain antibody 17E9C12 (SEQ ID NO: 4) 38. A method for identifying molecules that have the ability to bind to plexin D1, wherein the method comprises contacting a collection of molecules with plexin D1 and selecting the collection molecules that show plexin D1 binding, in the form of molecules of plexin link D1.