Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth fa

专利摘要:
A method of treating and alleviating melanoma and various cancers characterized by the expression of VEGF-D by tumors, the method comprising the steps of screening for finding an organism having tumor cells expressing VEGF-D and an effective amount of a VEGF-D antagonist Administering to prevent binding of VEGF-D; A method for screening a tumorous disease, wherein the detection of VEGF-D on or in cells, such as tumor cells, vascular endothelial cells, lymphatic endothelial cells, and / or cells with potential tumor growth, indicates tumorous disease. ; To promote and maintain vascularization of normal tissues in an organism by administering VEGF -D or fragments or analogs thereof; A method of screening a tumor for the risk of metastasis, wherein the expression of VEGF-D by the tumor indicates the risk of metastasis; And a method of detecting micro-metastasis of a tumorous disease, wherein the detection of VEG F-D on or in a tissue sample is an indication for metastasis of the tumorous disease.
公开号:KR20020080461A
申请号:KR1020027011490
申请日:2001-03-02
公开日:2002-10-23
发明作者:아첸마크；스태커스티븐
申请人:루드빅 인스티튜트 포 캔서 리서치；
IPC主号:

专利说明:

METHODS FOR TREATING, SCREENING FOR, AND DETECTING CANCERS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR D}
[2] Two major components of the mammalian vascular system are endothelial and smooth muscle cells. Endothelial cells form the lining of the inner surface of all blood and lymphatic vessels in mammals. Formation of new blood vessels can occur by two different processes, vasculogenesis or ang-iogenesis (see Risau W. Nature 386: 671-674, 1997). Angiogenesis is characterized by the differentiation of endothelial precursors into mature endothelial cells and the rounding of these cells to form vasculature, as occurs during the formation of the primary plexus in early embryos. In contrast, angiogenesis, which is the formation of blood vessels by the growth and branching of existing vessels, is important in later embryoogenesis and is a cause of vascular growth in adults. Angiogenesis is a physiologically complex process that includes proliferation of endothelial cells, degeneration of the extracellular matrix, branching of the vessels, and subsequent cell adhesion events. Angiogenesis in adults is tightly controlled and limited to the female reproductive system under normal circumstances. However, angiogenesis can be switched on in response to tissue damage. Importantly, solid tumors can induce angiogenesis in surrounding tissue, thereby sustaining tumor growth and facilitating the formation of metastatases (Folman J. Nature Med. 1: 27-31, 1995). The molecular mechanisms underlying the complex angiogenesis process are not understood at all.
[3] In addition, angiogenesis is implicated in a number of pathological conditions, in which case it plays some role or is directly involved in the different sequelae of the disease. Some examples include neovascularization associated with various liver diseases, neovascular sequelae of diabetes mellitus, neovascular sequelae of hypertension, posttraumatic neovascularization, neovascularization due to head trauma, and chronic hepatitis (e.g. chronic hepatitis). Neovascularization, neovascularization due to burns or frostbite, dysfunction associated with hormonal excess, hemangioma production, and restenosis after angiogenesis.
[4] Because of the critical role of angiogenesis in so many physiological and pathological processes, the factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been known to be involved in the regulation of angiogenesis, these include fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGFα), and hepatocyte growth factor (HGF). It includes. See, eg, Folkman et al., J. Biol. See Chem., 267: 10931-10934, 1992.
[5] Vascular endothelial growth factor (VEGF), a specific family of endothelial cell-specific growth factors, and their corresponding receptors, have been suggested to be the primary cause for stimulation of endothelial cell growth and differentiation and for certain functions of differentiated cells. These factors are members of the PDGF / VEGF family and seem to act primarily by endothelial receptor tyrosine kinase (RTK). The PDGF / VEGF family of growth factors belongs to the cystine-knot superfamily of growth factors, which also includes neurotrophin and transforming growth factor-β.
[6] Eight different proteins have been identified in the PDGF / VEGF family, ie five members closely related to two PDGF (A and B), VEGF, and VEGF. The five members closely related to VEGF include VEGF-B described in international patent application PCT / US96 / 02957 (WO 96/26736) and US Pat. Nos. 5,840,693 and 5,607,918 by the Ludwig Cancer Institute and the University of Helsinki; Joukov et al. EMBO J., 15: 290-298, 1996, Lee et al., Proc. Natl. Acad. Sci. VEGF-C or VEGF2 described in USA, 93: 1988-1992, 1996, and US Patents 5,932,540 and 5,935,540 by Human Genome Sciences; International Patent Application Nos. PCT / US97 / 14696 (WO 98/07832) and Achen et al., Proc. Natl. Acad. Sci. VEGF-D described in USA, 95: 548-553, 1998; Maglione et al., Proc. Natl. Acad. Sci. Placental growth factor (PlGF) described in USA, 88: 9267-9271, 1991; And VEGF3 described in International Patent Application No. PCT / US95 / 07283 (WO 96/39421) by Human Genome Sciences. Each VEGF family member has 30% to 45% amino acid sequence identity with VEGF. VEGF family members share a VEGF homology domain containing six cysteine residues that form a cystine-knot motif. Functional features of the VEGF family include the ability to promote dividing endothelial cells, vascular permeability, and angiogenesis and lymphangiogenic properties.
[7] Vascular endothelial growth factor (VEGF) is a homodimer glycoprotein isolated from several sources. Alternate mRNA splicing of a single VEGF gene results in five VEGF isoforms. VEGF shows very specific promoting activity against endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during angiogenesis and in angiogenesis throughout the adult's lifetime (Carmeliet et al., Nature, 380: 435-439, 1996; Ferrara et al., Nature, 380: 439-442). , 1996; Ferrara and Davis-Smyth, Endocrine Rev., 18: 4-25, 1997). The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele leads to batch death due to failure of vasculature development (Carmeliet et al. Nature 380: 435-439, 1996; Ferrara et al. Nature , 380: 439-442, 1996). The isolation and characterization of VEGF was examined. See Ferrara et al., J. Cellular Bioch em., 47: 211-218, 1991 and Connolly, J. Cellular Biochem., 47: 219-223, 1991.
[8] In addition, VEGF has strong chemoattractant activity towards monocytes, can induce plasminogen activators and plasminogen activator inhibitors in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeation factor (VPF). VEGF is also chemotactic for some hematopoietic cells. Recent literature indicates that VEGF reduces the effectiveness of immune responses against tumors by blocking maturation of dendritic cells (Gabrilovich et al., Blood 92: 4150-4166, 1998; Ga-brilovich et al., Clinical Cancer Research 5: 2963-). 2970, 1999).
[9] VEGF-B has similar angiogenic and other properties as VEGF, but is distributed and expressed in tissues different from VEGF. In particular, VEGF-B is very strongly expressed in the heart and only weakly expressed in the lungs, but vice versa. This suggests that despite the fact that VEGF and VEGF-B are expressed together in many tissues, they may have functional differences.
[10] VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins that interact with cellular retinoic acid-binding protein type I (CRABP-I). Its separation and characterization is described in PCT / US96 / 02957 (WO 96/26736), US Patents 5,840,693 and 5,607,918 by Ludwig Cancer Institute and the University of Helsinki, and Olofsson et al., Proc. Natl. Acad. Sci. USA, 93: 2576-2581, 1996.
[11] VEGF-C proliferates the PC-3 prostate by screening the ability of the medium to cause tyrosine phosphorylation of endothelial cell-specific receptor tyrosine kinase VEGFR-3 (Flt4) using cells transfected to express VEGFR-3. Adenocarcinoma cell line (CRL1435) was isolated from the conditioning medium. VEGF-C was purified by affinity chromatography using recombinant VEGFR-3 and cloned from the PC-3 cDNA library. Its separation and features are described in detail in Joukov et al., EMBO J., 15: 290-298, 1996.
[12] VEGF-D was isolated from human breast cDNA libraries commercially available from Clontech by screening with expressed sequence tags obtained from a human cDNA library designated as hybridization probe "Soares Breast 3NbHBst" (Achen et al., Proc. Natl). Acad.Sci. USA, 95: 548-553, 1998). Its separation and features are described in detail in International Patent Application No. PCT / US97 / 14696 (WO 98/07832).
[13] PCT / US97 / 14696 also describes the separation of biologically active fragments of VEGF-D, designated VEGF-DΔNΔC. This fragment is affinity tag peptide FLAG VEGF-D amino acid residues linked to 93 at 201. The entire specification of international patent application PCT / US97 / 14696 (WO 98/07832) is incorporated herein by reference.
[14] The VEGF-D gene is widely expressed in adult humans, but of course it is not expressed anywhere. VEGF-D is strongly expressed in the heart, lungs and skeletal muscle. Medium levels of VEGF-D are expressed in the spleen, ovary, small intestine and colon, and lower levels in the kidneys, pancreas, thymus, prostate and testes. VEGF-D mRNA was not detected in RNA from brain, placenta, liver or peripheral blood leukocytes.
[15] PlGF was isolated from the latent placental cDNA library. Its separation and features are described in Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267-9271, 1991. Its biological function is currently not well understood.
[16] VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3 is said to have about 36% identity and 66% similarity with VEGF. The method of isolation of genes encoding VEGF3 is not clear and no characterization of biological activity is disclosed.
[17] Similarity of two proteins is determined by comparing amino acid sequences, where one amino acid substitution in the protein is converted to the sequence of the second protein, while identity does not include the converted amino acid substitution.
[18] The main function of the lymphatic system is to provide fluid return from tissues and to transport many extravascular substances back into the blood. In addition, during the maturation process, lymphocytes leave the blood, travel through lymphoid organs and other tissues, enter the lymphatic vessels, and return to the blood through the chest tube. The specialized endothelial vein, the high endothelial vein (HEV), recombines with lymphocytes, causing them to extravasate into the tissue. Thus, lymphatic vessels, especially lymph nodes, play an important role in immunology and in the development of metastases of different tumors. Unlike blood vessels, the exhaust source of the lymphatic system is not clear, and at least three different theories exist about its origin. Lymphatic vessels are difficult to identify because there are no known specific markers available for them.
[19] Lymphatic vessels are most commonly studied with the help of lymph node angiography. In lymph node angiography, X-ray contrast media is injected directly into the lymphatic vessels. The control medium is distributed along the export drainage tube of the lymphatic system and collected in the lymph nodes. The control medium can stay in lymph nodes for up to half a year, during which X-ray analysis allows tracking of lymph node size and structure. This diagnosis is particularly important in cancer patients who have metastasized to lymph nodes and in lymphoid malignancies such as lymphomas. However, improvements in materials and methods for imaging lymphoid tissue are needed in the art.
[20] As noted above, PCGF / VEGF family members act primarily by binding to receptor tyrosine kinases. Generally, receptor tyrosine kinases are glycoproteins and receptors can be regulated by extracellular domains that can bind to specific growth factor (s), typically transmembrane domains that are alpha-helix portions of proteins, such as protein phosphorylation. The proximity membrane, if present, the tyrosine kinase domain that is the enzyme component of the receptor, and the carboxy-terminal tail implicated in the recognition and binding of substrates to tyrosine kinases in many receptors.
[21] Five endothelial cell-specific receptor tyrosine kinases have been identified, namely VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flt-1), VEGFR-3 (Flt4), Tie and Tek / Tie-2. . These receptors differ in their specificity and affinity. All of these have unique tyrosine kinase activity required for signal transduction.
[22] The only receptor tyrosine kinases known to bind VEGF are VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with high affinity and VEGFR-1 also binds VEGF-B and PlGF. VEGF-C is known to be a ligand for VEGFR-3 and also activates VEGFR-2 (Joukov et al., The EMBO Journal, 15: 290-298, 1996). VEGF-D binds to both VEGFR-2 and VEGFR-3 (Achen et al. Proc. Natl. Acad Sci. USA, 95: 548-553, 1998). Ligands for Tek / Tie-2 are described in International Patent Application No. PCT / US95 / 12935 (WO 96/11269) by Regeneron Pharmaceuticals. Ligands for Tie have not yet been identified.
[23] Recently a novel 130-135kDa VEGF isotype specific receptor has been purified and cloned (Soker et al., Cell, 92: 735-745, 1998). The VEGF receptor has been shown to specifically bind VEGF ₁₆₅ isoforms by exon 7 encoded sequences, which shows a weak affinity for heparin (Soker et al., Cell, 92: 735-745, 1998). Surprisingly, this receptor has been shown to be identical to human neurophylline-1 (NP-1), a receptor involved in early stage neuromorphogenesis. PlGF-2 also appears to interact with NP-1 (Migdal et al. J Biol Chem, 273: 22272-22278, 1998).
[24] VEGFR-1, VEGFR-2 and VEGRF-3 are expressed differently by endothelial cells. In general, both VEGFR-1 and VEGFR-2 are expressed in the vascular endothelium (Oelrichs et al. Oncogene 8: 11-18, 1992; Kaipainen et al., J. Exp. Med., 178: 2077-2088, 1993; Dumont et al., Dev. Dyn., 203: 80-92, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996), VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl Acad.Sci. USA, 9: 3566-3570, 1995). VEGFR-3 is also expressed in the vasculature surrounding the tumor.
[25] VEGFR-1 is mainly expressed in endothelial cells during development, but it can also be found in hematopoietic progenitor cells during the early stages of embryogenesis (Fong et al., Nature, 376: 66-70, 1995). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 87: 3336-3343, 1995). In embryos, VEGFR-1 is mostly azi but mostly expressed by vasculature (Breier et al., Dev. Dyn., 204: 228-239, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996) .
[26] Receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development, but embryonic progression is limited to predetermined vein endothelial and then lymphatic endothelial (Kaipainen et al., Cancer Res., 54: 6571-6577, 1994; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570, 1995). VEGFR-3 is expressed on lymphoid endothelial cells of adult tissues. This receptor is essential for angiogenesis during embryogenesis.
[27] An essential and specific role in VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek / Tie-2 in angiogenesis, angiogenesis and / or lymphangiogenesis is the goal of inactivating these receptors in mouse embryos. Proven by one mutation. Destruction of the VEGFR gene results in anomalous vasculature leading to batch death near mid-pregnancy. Analysis of embryos with a fully inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al. Nature 376: 66-70, 1995). However, deletion of the intracellular tyrosine kinase domain of VEGFR-1 results in viable mice with normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95: 9349-9354, 1998). The underlying reason for these differences should still be explained, but receptors signaling by tyrosine kinases are not necessary for proper function of VEGFR-1. Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoiesis and angiogenesis (Shalaby et al., Nature, 376: 62-66, 1995; Shalaby et al. , Cell, 89: 981-990, 1997). Targeted inactivation of both copies of the VEGFR-3 gene in mice results in the formation of defective blood vessels characterized by abnormally organized vasculature with defective lumens, accumulating fluid in the pericardial cavity and 9.5 days after mating To cardiovascular failure (Dumont et al., Science, 282: 946-949, 1998). Based on these findings, it was suggested that VEGFR-3 is necessary for maturation of primary vascular network into larger vessels. However, the role of VEGFR-3 in the development of lymphatic vasculature could not be studied in these mice because the embryo died before the lymphatic system appeared. Nevertheless, it is speculated that VEGFR-3 plays a role in the development and development of lymphatic vessels and provides its specific expression in lymphatic endothelial cells during embryonic development and in the lifetime of an adult. This is supported by the finding that displacement expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice leads to lymphatic endothelial cell proliferation and vasodilation in the dermis. Moreover, this suggests that VEGF-C may have a primary function in lymphatic endothelium and a secondary function in angiogenesis and permeability regulation shared with VEGF (Joukov et al., EMBO J., 15: 290-298, 1996). .
[28] In addition, VEGF-like proteins were identified as being encoded by four different strains of the orf virus. This is the first virus reported to encode a VEGF-like protein. The first two strains are NZ2 and NZ7 and are described in Lyttle et al., J. Virol., 68: 84-92, 1994. The third is D1701 and is described in Meyer et al., EMBO J., 18: 363-374, 1999. The fourth strain is NZ10 and is described in international patent application PCT / US99 / 25869. These viral VEGF-like proteins bind VEGFR-2 on the endothelium of the host (sheep / goat / human), and this binding has been shown to be important for the development of infections (Meyer et al., EMBO J., 18: 363-374, 1999 Ogawa et al., J. Biol. Chem., 273: 31273-31282, 1988; and International Patent Application PCT / US99 / 25869. These proteins exhibit amino acid sequence similarities with VEGF and with each other.
[29] The orf virus is a species of the genus Parapoxvirus that causes highly contagious purulent dermatitis in sheep and goats and can be easily transmitted to humans. Pulmonary dermatitis caused by orf virus infection is characterized by vasodilation, swelling of the local area, and marked proliferation of endothelial cells of the vascular lining. These features are seen in all species infected by orf and can lead to tumor-like growth or formation of sintered nodes due to viral replication in epithelial cells. In general, orf virus infections resolve within a few weeks, but in immunocompromised individuals severe infections appear that cannot be resolved without surgical intervention.
[30] There is great interest in the development of pharmaceutical agents that can antagonize the receptor-mediated action of VEGF (Martiny-Baron and Marme, Curr. Opin. Biotechnol. 6: 675-680, 1995). Monoclonal antibodies against VEGF have been shown to inhibit tumor growth in vivo by inhibiting tumor-associated angiogenesis (Kim et al., Nature 362: 841-844, 1993). Similar inhibitory effects on tumor growth are expressed by antisense RNA of VEGF (Saleh et al., Cancer Res. 56: 393-401, 1996) or dominant-negative VEGFR-2 mutations (Millauer et al., Nature 367: 576-579, 1994). In the model system resulting from
[31] However, tumor inhibition studies with neutralizing antibodies suggested that other angiogenic factors other than VEGF could be involved (Kim, K. et al., Nature 362: 841-844, 1993). Moreover, the activity of angiogenic factors other than VEGF in mammalian melanoma is suggested by the discovery that not all melanoma expresses VEGF (Issa, R. et al., Lab Invest 79: 417-425, 1999).
[32] The biological function of different members of the VEGF family is currently being elucidated. Of particular interest are the properties of VEGF-D and VEGF-C. These proteins bind to both VEGFR-2 and VEGFR-3 (located on vascular and lymphatic endothelial cells, respectively) and are closely related to primary structure (48% amino acid identity). These two factors are accelerating to endothelial cells in vitro. Recently, VEGF-C has been shown to be angiogenic in mouse corneal models and avian ureter (Cao et al. Proc. Natl. Acad. Sci. USA 95: 14389-14394, 1998). (Witzenbichler et al., Am. J. Pathol. 153: 381-394, 1998). Moreover, VEGF-C stimulated lymphangiogenesis in avian urinary membranes (Oh et al. Dev. Biol. 188: 96-109, 1997) and transgenic mouse models (Jeltsch et al. Science 276: 1423-1425, 1997). VEGF-D has been shown to be angiogenic in rabbit corneas (Marconcini et al., Proc. Natl. Acad. Sci. USA 96: 9671-9676, 1999). Lymphangiogenic capacity of VEGF-D has not yet been reported, but VEGF-D binds to and activates VEGFR-3, a receptor thought to be a signal for lymphangiogenesis, like VEGF-C (Taipale et al., Cur. Topics Micro. 237: 85-96, 1999), it is very likely that VEGF-D is lymphangiogenic. VEGF-D and VEGF-C may be particularly important for malignant tumors because metastatase may be propagated by blood vessels or lymphatic vessels, and thus molecules that stimulate angiogenesis or lymphangiogenesis may contribute to malignancy. This has already been seen in the case of VEGF. It is noteworthy that VEGF-D gene expression is induced by c-Fos, a transcription factor known to be important in malignancy (Orlandini et al., Proc. Natl. Acad. Sci. USA 93: 11675-11680, 1996). The mechanism by which c-Fos contributes to malignancy is presumed to be up-regulation of genes encoding pulmonary factor. Tumor cells defective in c-Fos do not induce progression to malignancy because they probably cannot induce angiogenesis (Saez, E. et al., Cell 82: 721-732, 1995). This indicates the presence of angiogenic factors that are upregulated by c-Fos during tumor progression.
[33] As shown in FIG. 1, the dominant intracellular morphology of VEGF-D is a homodimer propeptide consisting of the VEGF / PDGF homology domain (VHD) and the N- and C-terminal propeptide and the sequence of SEQ ID NO: 2 Has After secretion this polypeptide is proteolytically cleaved (Stacker, S. A. et al., J. Biol. Chem. 274: 32127-32136, 1999). Proteolytic processing (positioned by black arrows) results in partially processed and fully processed mature forms, which are composed of the dimers of the VHD. The VHD having the sequence of SEQ ID NO: 3 (ie, residues 93 to 201 of full length VEGF-D) contains binding sites for both VEGFR-2 and VEGFR-3. The mature form binds both VEGFR-2 and VEGFR-3 with much higher affinity than the unprocessed form (Stacker, S. A. et al., J. Biol. Chem. 274: 32127-32136, 1999).
[34] Localization of VEGF-D protein in human cancers has not been studied due to the lack of antibodies specific for VHD of VEGF-D. Antibodies to N- or C-terminal propeptide are inadequate because these regions are cleaved from bioactive VHDs and localize differently than VHDs.
[1] The present invention generally relates to methods of treating and alleviating melanoma and various cancers, methods of screening for neoplastic disease, and methods for promoting and maintaining vascularization of normal tissues.
[77] 1 is a schematic representation of VEGF-D processing.
[78] 2 shows the specificity of MAb 4A5 for the VEGF / PDGF homology domain (VHD) of human VEGF-D as assessed by Western blot analysis.
[79] 3 shows radiographs taken 2 days after exposure to 15.5 day old mouse tissue sections hybridized with VEGF-D antisense and sense RNA.
[80] 4A-4D show the results of analysis of the distribution of VEGF-D mRNA in mouse embryos 15.5 days after mating by in situ hybridization.
[81] 5A-5H show immunohistochemistry results from two malignant melanomas illustrating different response patterns.
[82] 6A-6F show localization of VEGF-D in squamous cell carcinoma of the lung.
[83] 7A-7F show localization of VEGF-D in situ breast duct carcinoma.
[84] 8 shows localization of VEGF-D in situ endometrial adenocarcinoma.
[85] 9A-9F show localization of VEGF-D in normal colon tissue.
[86] 10 shows untransfected parental 293 cells (designated “293”) and 293 cells transfected with expression vectors encoding VEGF-D-FULL-N-FLAG (designated “VEGF-D-293”). The tumor analysis result of SCID mouse resulting from the medical injection is shown.
[87] 11 shows tumors produced by VEGF-DΔN cells.
[88] 12 shows normal tumors.
[35] The present invention generally relates to methods of treating and alleviating melanoma and various cancers, methods of screening for neoplastic disease, and methods of maintaining vascularization of normal tissues.
[36] According to a first aspect, the invention suffers from a tumorous disease characterized by the expression of VEGF-D by tumors including but not limited to melanoma, breast duct carcinoma, squamous cell carcinoma, prostate tumor and endometrial cancer. Provided are methods for treating an organism. The method comprises screening an organism to determine the presence or absence of VEGF-D-expressing tumor cells; Selecting an organism determined to have a tumor expressing VEGF-D at screening; And administering an effective amount of the VEGF-D antagonist near the tumor to prevent binding of VEGF-D to its corresponding receptor.
[37] VEGF-D antagonists can inhibit VEGF-D expression as well as the use of compositions comprising antisense nucleic acids or triple stranded DNA encoding VEGF-D.
[38] In addition, VEGF-D antagonists may inhibit VEGF-D activity as well as the use of compounds comprising competitive or noncompetitive inhibitors of binding of VEGF-D in antibody and / or dimer formation and receptor binding. These VEGF-D antagonists include the VEGF-D modified polypeptides described below, which have the ability to bind VEGF-D and can prevent binding to VEGF-D receptors or bind to VEGF-D receptors. Have the capacity to stimulate endothelial cell proliferation, differentiation, migration or survival. Small molecule inhibitors for VEGF-D, VEGFR-2 or VEGFR-3 and antibodies directed to VEGF-D, VEGFR-2 or VEGFR-3 can also be used.
[39] Certain modified VEGF-D polypeptides may have the ability to bind VEGF-D receptors on cells, including but not limited to endothelial cells, connective tissue cells, myofibroblasts and / or mesenchymal cells, but cell proliferation, differentiation. It is not believed to stimulate migration, motility or survival, or induce vascular proliferation, connective tissue development, or wound healing. These modified polypeptides can act as competitive or noncompetitive inhibitors of VEGF-D polypeptides and PDGF / VEGF family growth factors, and would be useful in situations where prevention or reduction of VEGF-D polypeptides or PDGF / VEGF family growth factor actions is desired. It is expected. Thus, also such receptor-binding but non-mitogenogenic, non-differentiation inducible, non-migration inducible, non-motor inducible, non-viability promoting, non-connective tissue generating, non-wound Variants of VEGF-D polypeptides that are curable or non-vascular proliferative are also within the scope of the present invention and are referred to herein as "receptor-binding but otherwise inactive variants". Since VEGF-D forms a dimer to activate its receptor, one monomer comprises the "receptor-binding but otherwise inactive variant" VEGF-D polypeptide mentioned above and the second monomer is a wild type VEGF. It is thought to include wild-type growth factors of the -D or PDGF / VEGF family. Thus, these dimers can bind to their corresponding receptors but cannot induce downstream signaling.
[40] In addition, wild type growth factors of the wild type VEGF-D or PDGF / VEGF family and their corresponding receptors on cells, including but not limited to endothelial cells, connective tissue cells (such as fibroblasts), myofibroblasts and / or mesenchymal cells. It is believed that there are other modified VEGF-D polypeptides that can prevent the binding of. Thus, these dimers cannot stimulate endothelial cell proliferation, differentiation, migration, survival, or induce vascular permeability, and / or proliferation and / or differentiation and / or motility of connective tissue cells, myofibroblasts or mesenchymal cells. Can not stimulate. These modified polypeptides can act as competitive or noncompetitive inhibitors of VEGF-D growth factor or PDGF / VEGF family growth factor and are useful in situations where prevention or reduction of VEGF-D growth factor or PDGF / VEGF family growth factor action is desired. It is expected to. Such situations include tissue remodeling that occurs while tumor cells penetrate into normal cell populations by primary or metastatic tumor formation. Thus, also such VEGF-D or PDGF / VEGF family growth factor-binding, but non-mitogenic, non-differentiation inducible, non-migration inducible, non-motor inducible, non-promoting survival, non Variants of VEGF-D growth factors that are connective tissue promoting, non-wound healing, or non-vascular proliferation inducing are also within the scope of the present invention and are described herein as "VEGF-D growth factor-dimer forming but otherwise. Variants that are inactive or disruptive.
[41] Possible modified forms of the VEGF-D polypeptide can be prepared by targeting the regions of the VEGF-D polypeptide essential for modification. These essential regions are expected to be in strongly conserved PDGF / VEGF homology domains (VDH). In particular, growth factors of the PDGF / VEGF family, including VEGF, are dimers, and VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A and PDGF-B are complete of the eight cysteine residues in the VHD. Conservation (Olofsson et al., Proc. Natl. Acad. Sci. USA 1996 93 2576-2581; Joukov et al., EMBO J., 1996 15 290-298). These cysteines are thought to be involved in intramolecular and intermolecular disulfide bonds. In addition, there is a less but more strongly conserved cysteine residue in the C-terminal domain. Loops 1, 2, and 3 of each VHD subunit formed by intramolecular disulfide bonds are involved in binding to receptors of growth factors of the PDGF / VEGF family (Anderss-on et al., Growth Factors, 1995 12 159-164). Modified polypeptides can be readily tested for their ability to inhibit the biological activity of VEGF-D by conventional activity assay procedures such as endothelial cell proliferation assays.
[42] In addition, VEGF-D antagonists useful in the present invention may include a molecule comprising a polypeptide corresponding to the VEGF-D binding domain of VEGFR-2 (Flk1) or VEGFR-3 (Flt4). For example, Achen et al., Proc. Natl. Acad. Sci. Soluble Ig fusion proteins containing extracellular domains of human VEGFR-2 and human VEGFR-3, described in USA 95: 548-553, 1998, and binding to VEGF-DΔNΔC can be suitably used as VEGF-D antagonists. .
[43] In addition, methods of treating and alleviating melanoma and various cancers can occur by targeting tumors expressing VEGF-D, VEGFR-2 and / or VEGFR-3 leading to death. This includes coupling the antibody of the invention, VEGF-D, VEGFR-2 or VEGFR-3, or a small molecule with respect to VEGF-D, VEGFR-2 or VEGFR-3 and a cytotoxic agent, thereby VEGF Kill tumors expressing D, VEGFR-2 and / or VEGFR-3. Such cytotoxic agents include, but are not limited to, plant toxins (eg lysine A chains, saporins), bacterial or fungal toxins (eg diphtheria toxins) or radionucleotides (eg 211-astatin, 212-bismus, 90-yttrium, 131-iodine, 99-technium), alkylating agents (eg chlorambucil), anti-mitotic agents (eg vinca alkaloids), and DNA inclusion agents (eg adriamycin) ).
[44] In addition, polypeptides, VEGF-D antagonists or antibodies that inhibit the biological activity of VEGF-D can be used in combination with their pharmaceutically acceptable non-toxic salts and pharmaceutically acceptable solid or liquid carriers or adjuvants. Preferred pharmaceutical compositions will inhibit or interfere with at least the biological activity induced by VEGF-D.
[45] Examples of such carriers or auxiliaries include, but are not limited to, saline, buffered saline, Ringer's solution, mineral oil, talc, cornstarch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, dextrose, Water, glycerol, ethanol, thickening agents, stabilizers, suspending agents, and combinations thereof. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, plasters, elixirs, syrups, wafers, ointments, or other conventional forms. The formulations may of course be adapted according to known principles to suit the mode of administration. Compositions comprising the peptides of the present invention contain from about 0.1% to 90%, most typically from about 10% to 30%, of the active compound (s).
[46] The dose (s) and route of administration depend on the nature of the patient and the condition to be treated and is at the discretion of the attending physician or veterinarian. Suitable routes include oral, subcutaneous, intramuscular, intraperitoneal or intravenous injection, parenteral, topical application, transplantation, and the like. For example, an effective amount of a peptide or antibody of the invention is administered to an organism in need thereof at a dose of 0.1 to 100 mg / kg body weight, more preferably 1 to 10 mg / kg body weight. For humanized antibodies, which typically have long computational half-lives, it is specifically contemplated to administer at intervals ranging from daily to monthly, more preferably weekly, or biweekly, or every three weeks. Monitoring the progress of therapy, patient side effects, and computational antibody levels provide additional guidance for optimal dosing regimens. In addition, data from open and ongoing clinical trials of other antibody-based cancer therapies provide useful dosage regimen guidance. Topical application of VEGF-D can be used in a similar manner to VEGF.
[47] According to a second aspect, the present invention provides a method for screening and / or diagnosing a neoplastic disease characterized by an increase in vascular endothelial cells of the blood vessels in or around the neoplastic growth. The method comprises obtaining a sample from an organism suspected of a tumorous disease state characterized by an increase in vascular endothelial cells of the blood vessels in or around the tumorous growth; Exposing the sample to a composition comprising a compound that specifically binds VEGF-D; Washing the sample; And screening the disease by detecting the presence, amount, or distribution of the compound in the sample, wherein the VEGF-D is in or on vascular endothelial cells of the vascular endothelial growths and blood vessels surrounding it. Detection is an indication for tumorous disease. The method may further comprise exposing the sample to a second compound that specifically binds to VEGFR-2 and / or VEGFR-3, wherein the screening step comprises blood vessels of the blood vessel and the compound that binds VEGF-D. Detecting a second compound bound to endothelial cells to determine the presence, amount, or distribution of vascular endothelial cells of the vessel having both VEGF-D and VEGFR-2 and / or VEGFR-3 in and around potential tumor growths It involves doing.
[48] For purposes of this specification, it will be apparent that the term "sample" includes, but is not limited to, obtaining a tissue sample, blood, serum, plasma, urine, ascites fluid or pleural effusion. The tissue is preferably human tissue and the compound is preferably a monoclonal antibody. It is highly appreciated that the use of a second compound helps the doctor confirm that VEGF-D found in the tumor or in the vessel near it is due to receptor-mediated uptake, which is secreted by tumor cells. It supports the hypothesis that -D binds to and accumulates in target endothelial cells to establish a secretory mechanism that regulates tumor angiogenesis.
[49] According to a third aspect, the present invention provides a method for screening and / or diagnosing a tumorous disease characterized by an increased expression of VEGF-D. The method comprises obtaining a sample from an organism suspected of a disease state characterized by an increased expression of VEGF-D; Exposing the sample to a composition comprising a compound that specifically binds VEGF-D; Washing the sample; And screening the disease by detecting the presence, amount, or distribution of the compound in the sample, wherein detection of VEGF-D in the potential tumor growth and in the cells surrounding it is performed on the tumor disease. The detection of VEGF-D in or on the vascular endothelial cells in or around potential tumor growths is an indication for tumorous disease.
[50] According to a fourth aspect, the present invention provides a method for screening and / or diagnosing a tumorous disease characterized by changes in lymphatic endothelial cells. The method comprises obtaining a sample from an organism suspected of a disease state characterized by an increase in lymphatic endothelial cells; Exposing the sample to a composition comprising a compound that specifically binds VEGF-D; Washing the sample; And screening the disease by detecting the presence, amount, or distribution of the compound in the tissue sample, wherein detection of VEGF-D in or on the potential endothelial growth and lymphatic endothelial cells around it. This is an indication for tumorous disease. The method may further comprise exposing the tissue sample to a second compound that specifically binds VEGFR-3, wherein the screening step is a compound that binds VEGF-D and a second that binds to lymphatic endothelial cells Detecting the compound includes measuring the presence, amount, or distribution of lymphoid endothelial cells having both VEGF-D and VEGFR-3 in and around the potential tumor growth.
[51] It is highly appreciated that the use of a second compound helps doctors confirm that VEGF-D found in the tumor or in nearby lymphatic vessels is due to receptor-mediated uptake, which is VEGF secreted by tumor cells. It supports the hypothesis that -D binds to and accumulates in target lymphoid endothelial cells to establish a secretory mechanism that regulates tumor lymphangiogenesis.
[52] According to a fifth aspect, the present invention provides a method for maintaining the vascularization of tissue in an organism, which method requires such treatment for an effective amount of VEGF-D, or fragments or analogs thereof having biological activity of VEGF-D. It comprises the step of administering to the organism.
[53] Especially in elderly patients who may have atrophy of peripheral vasculature, the fifth aspect is considered to be important when VEGF-D / VEGF is restricted in the tissues of the patient. Treatment with an effective amount of VEGF-D may help to maintain the integrity of the vasculature by stimulating endothelial cell proliferation in the aging / damaged vasculature.
[54] Preferably, VEGF-D is full-length unprocessed VEGF-D or fully processed mature form of VEGF-D, as well as full-length VEGF-D and mature having the biological activity of VEGF-D as defined herein. It is expressed as a fragment or analog of both forms of VEGF-D.
[55] For purposes of this disclosure, the paragraph “Fully processed VEGF-D” refers to a mature form of VEGF-D polypeptide, ie VEGF homology domain, having a sequence of SEQ ID NO: 3 that does not have N- and C-terminal propeptide. It will be clearly understood that it means (VHD). The paragraph “protein hydrolysed form of VEGF-D” refers to a VEGF-D polypeptide having no N- and / or C-terminal propeptide, and the paragraph “unprocessed VEGF-D” refers to N- and By full-length VEGF-D polypeptide having the sequence of SEQ ID NO: 2 having both C-terminal propeptide.
[56] The full length VEGF-D polypeptide having the sequence of SEQ ID NO: 2 is optionally FLAG May be linked to a peptide. Full length VEGF-D polypeptide is FLAG When linked to, this fragment is referred to herein as VEGF-D-FULL-N-FLAG. Preferred fragments of VEGF-D are optionally FLAG A portion of VEGF-D (ie, VHD (SEQ ID NO: 3)) from amino acid residue 93 to amino acid residue 201 linked to the peptide. This fragment is FLAG When linked to, the fragment is referred to herein as VEGF-DΔNΔC.
[57] The expression “biological activity of VEGF-D” should be understood to mean the ability to stimulate one or more of endothelial cell proliferation, differentiation, migration, survival or vascular permeability.
[58] It should be clearly understood that polypeptides, including conservative substitutions, insertions or deletions, which still retain the biological activity of VEGF-D, are within the scope of the present invention. Those skilled in the art will be familiar with methods that can be readily used to produce such polypeptides, such as site-directed mutagenesis, or the use of specific enzyme cleavage and ligation. In addition, those skilled in the art will appreciate that the attainable compound of the peptide or a compound in which one or more amino acid residues have been replaced by non-naturally occurring amino acids or amino acid analogs may retain the necessary aspects of the biological activity of VEGF-D. Such compounds can be readily made and tested by methods known in the art and are also within the scope of the present invention.
[59] Preferably, when amino acid substitutions are used, these substitutions are conservative, ie the amino acids are replaced by amino acids having similar size and similar charge properties.
[60] As used herein, the term “conservative substitution” denotes the replacement of amino acid residues by other biologically similar residues. Examples of conservative substitutions include substitution of one hydrophobic moiety with another hydrophobic moiety, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine, or other polarity of one polar moiety. Substitution with residues, for example, arginine to lysine, glutamic acid to aspartic acid, glutamine to asparagine, and the like. Neutral hydrophilic amino acids that may be substituted for one another include asparagine, glutamine, serine and threonine. The term “conservative substitutions” also includes the use of substituted amino acids in place of unsubstituted parent amino acids.
[61] As such, in the context of the present invention, it should be understood that conservative substitutions are recognized in the art as substitutions with one amino acid for another having similar characteristics. Typical conservative substitutions are shown in the following Table A of WO 97/09433.
[62]
[63] Alternatively, conservative amino acids can be classified as described in Lehninger (Biochemistry, 2nd edition; Worth Publishers, Inc. NY: NY (1975), pp. 71-77), set forth in Table B below.
[64]
[65] Typical conservative substitutions are shown in Table C below.
[66]
[67] As is known to occur using VEGF and VEGF-B, possible variant forms of VEGF-D polypeptides that can result from alternating splicing, and naturally occurring allelic variants of nucleic acid sequences encoding VEGF-D are within the scope of the invention. Mine Allelic variants are well known in the art and refer to other forms of nucleic acid sequences that include substitution, deletion or addition of one or more nucleotides, but do not result in any substantial functional alteration of the encoded polypeptide.
[68] Such variant forms of VEGF-D can be prepared by targeting regions of the VEGF-D polypeptide that are not essential for modification. These non-essential areas are expected to be outside of the strongly conserved areas. In particular, growth factors of the PDGF / VEGF family, including VEGF, are dimers, and VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A and PDGF-B are N-terminal domains, namely PDGF / VEGF -Complete conservation of 8 cysteine residues in the analogous domain (Olofsson et al., Proc. Natl. Acad. Sci. USA 1996 93 2576-2581; Jou-kov et al., EMBO J. 1996 15 290-298). These cysteines are thought to be involved in intramolecular and intermolecular disulfide bonds. In addition, there is a less but more strongly conserved cysteine residue in the C-terminal domain. Loops 1, 2, and 3 of each VHD subunit formed by intramolecular disulfide bonds are involved in binding to growth factor receptors of the PDGF / VEGF family (Andersson et al., Growth Factors, 1995 12 159-164).
[69] Thus, those skilled in the art are well aware that these cysteine residues must be conserved in any proposed variant form, and that the active sites present in loops 1, 2 and 3 must also be conserved. However, other regions of the molecule are expected to be less important for biological function, thus providing a suitable target for modification. Modified polypeptides can be readily tested for their ability to exhibit the biological activity of VEGF-D by conventional activity assay procedures such as endothelial cell proliferation assays.
[70] It is known that strong signals of VEGF-D are present in a subset of hematopoietic cells. These cells rush into the peripheral regions of a tumor in some kind of inflammatory response. Thus, inhibition of this process is useful when it is desirable to prevent this inflammatory response.
[71] Accordingly, a sixth aspect of the present invention provides a method of inhibiting the inflammatory response resulting from this subset of hematopoietic cells of these tumors, which method expresses the expression or activity of VEGF-D by this subset of hematopoietic cells. Inhibiting. Inhibition of this type of inflammatory response may be used to treat autoimmune diseases such as arthritis.
[72] In addition, the antibodies according to the invention can be labeled with a detectable label and used for diagnostic purposes. Antibodies can be covalently or non-covalently coupled with a supermagnetic, paramagnetic, high magnetic density, ecogenic or radioactive agent suitable for imaging. For use in diagnostic assays, radioactive or non-radioactive labels can be used. Examples of radiolabels include radioactive atoms or groups such as ¹²⁵ I or ³² P. Examples of non-radioactive labels include enzyme labels, such as horseradish peroxidase, or fluorescent labels, such as fluorescein-5-isothiocyanate (FITC). Labeling can be direct or indirect, shared or non-covalent.
[73] According to a further aspect of the invention, the invention suffers from a tumorous disease characterized by the expression of VEGF-D by a tumor such as malignant melanoma, breast duct carcinoma, squamous cell carcinoma, prostate cancer or endometrial cancer. A method of treating an organism, such as a mammal, includes administering an effective amount of a VEGF-D antagonist near the tumor to prevent binding of VEGF-D to its corresponding receptor. If desired, cytotoxic agents may be co-administered with the VEGF-D antagonist. Preferred VEGF-D antagonists are monoclonal antibodies that specifically bind VEGF-D and block binding of VEGF-D and VEGF receptor-2 or VEGF receptor-3, in particular antibodies that bind to the VEGF homology domain of VEGF-D to be.
[74] In another aspect, the present invention relates to a method for screening a tumor for risk of metastasis, the method comprising exposing a tumor sample to a composition comprising a compound that specifically binds VEGF-D; Washing the sample; And screening for risk of metastasis by detecting the presence, amount, or distribution of the compound in the sample, wherein expression of VEGF-D by the tumor is an indication of the risk of metastasis. Preferred compounds for use in this aspect of the invention are monoclonal antibodies that specifically bind VEGF-D, in particular antibodies that bind to the VEGF homologous domain of VEGF-D and are labeled with a detectable label.
[75] A further aspect of the invention relates to a method for detecting micro-metastasis of a tumorous disease state characterized by an increased expression of VEGF-D, which method surrounds tumorous growths in an organism of a tumorous disease state. Obtaining a tissue sample from a site remote from the tumorous growth, such as obtaining a lymph node from the tissue; Exposing the sample to a composition comprising a compound that specifically binds VEGF-D; Washing the sample; And screening the metastasis of the tumorous disease by detecting the presence, amount or distribution of the compound in a tissue sample, wherein detection of VEGF-D in the tissue sample is an indication for metastasis of the tumorous disease. . Again, preferred compounds include monoclonal antibodies that specifically bind VEGF-D, especially antibodies that bind the VEGF homologous domain of VEGF-D and are labeled with a detectable label.
[76] For the purposes of this specification, it will be apparent that the word "comprising" means "including but not limited to." The corresponding meaning also applies to the word "comprise".
[89] Example 1
[90] Production of Monoclonal Antibodies That Bind to Human VEGF-D
[91] In order to detect VEGF / PDGF homology domains (VHD) rather than N- and C-terminal properties, residues 93 of the mature form of human VEGF-D (full length VEGF-D (SEQ ID NO: 2), ie 201 N- and C-terminal regions are removed). DNA fragments encoding 201 at residue 93 were amplified by polymerase chain reaction (PCR) using Pfu DNA polymerase. Full length human VEGF-D cDNA (SEQ ID NO: 1) was used as a template. Next, the amplified DNA fragments confirmed correct by nucleotide sequencing were inserted into the expression vector pEFBOS SFLAG (from Dr. Clare McFarlane, Walter and Eliza Hall Institute for Medical Research (WEHI), Melbourne, Australia) to designate the plasmid designated pEFBOSVEGF-DΔNΔC. Generated. The pEFBOSSFLAG vector encodes a signal sequence for protein secretion from the interleukin-3 (IL-3) gene and FLAG Octapeptide (Sigma-Aldrich). FLAG Octapeptides can be recognized as commercially available antibodies, such as the M2 monoclonal antibody (Sigma-Aldrich). VEGF-D PCR fragment was inserted into the vector, IL-3 signal sequence was FLAG It was located just upstream from the octapeptide, which in turn was just upstream from the VEGF-D sequence, truncated. All three sequences were in the same reading frame, so translation of mRNA resulting from transfection of pEFBOSVEGF-DΔNΔC into mammalian cells resulted in a protein with an IL-3 signal sequence at the N-terminus, followed by FLAG Octapeptide and truncated VEGF-D sequences were generated. Cleavage of the signal sequence and subsequent secretion of the protein from the cell resulted in FLAG adjacent the N-terminus VEGF-D polypeptides tagged with octapeptide were generated. Anti-FLAG from medium of COS cells transiently transfected VEGF-DΔNΔC with plasmid pEFBOSVE GF-DΔNΔC Purification by affinity chromatography (see Example 9 of International Patent Application No. PCT / US97 / 14696)
[92] Using purified VEGF-DΔNΔC, female Balb / C mice were immunized 85 days (intraperitoneal), 71 days (intraperitoneal) and 4 days (intravenously) before collecting splenocytes from the immunized mice, and then These splenocytes and mouse myeloma P3X63Ag8.653 (NS-1) cells were fused. Approximately 10 μg of VEGF-DΔNΔC was injected in a 1: 1 mixture of PBS and TiterMax adjuvant (# R-1 study adjuvant; CytRx Corp., Norcross, Georgia) for the first two immunizations, and in PBS for the third immunization. 35 μg of VEGF-DΔNΔC was used.
[93] Monoclonal antibodies against VEGF-DΔNΔC were selected by screening hybrid cells on purified VEGF-DΔNΔC using enzymatic immunoassay. Briefly, 96-well microplates were coated with VEGF-DΔNΔC, incubated at 4 ° C. for 2 hours with the addition of hybrid cell supernatant and washed 6 times in PBS with 0.02% Tween 20. It was then incubated with horseradish peroxidase conjugated anti-mouse Ig (Bio-Rad, Hercules, CA) for 1 hour at 4 ° C. After washing this assay was developed using a 2,2'-azino-di- (3-ethylbenz-thiazoline sulfonic acid) (ABTS) substrate system and was a multi-well plate reader (Flow Laboratories MCC / 340, Virginia McLean). The absorbance at 405 nm was read and quantified. Six antibodies were selected for further analysis and subcloned twice by limiting dilution. These antibodies were designated 2F8, 3C10, 4A5, 4E10, 4H4 and 5F12. Subsamples of these antibodies were measured using the Isotrip ^™ subsample kit (Möllinger Mannheim, Indianapolis). All six antibodies contained kappa light chains.
[94] Hybrid cell lines were grown in DMEM containing 5% v / v IgG-depleted serum (Gibco BRL, Gettysburg, MD), 5 mM L-glutamine, 50 μg / ml gentamycin and 10 μg / ml recombinant IL-6. Antibodies 2F8, 4A5, 4E10 and 5F12 are described by Darby et al., J. Immunol. Purification was carried out by affinity chromatography using protein G-Sepharose according to the methods of Methods 159: 125-129, 1993, and the absorbance at 280 nm was measured to evaluate the yield.
[95] Example 2
[96] Specificity of 4A5
[97] The specificity of MAb 4A5 (renamed VD1) to VHD of human VEGF-D was assessed by Western blot analysis. The derivative of VEGF-D used was FLAG VEGF-DΔNΔC consisting of amino acid residues 593 to 201 of human VEGF-D tagged N-terminus with octapeptide (Example 1), FLAG VEGF-D-FULL-N-FLAG (Stacker, SA et al., J. Biol. Chem. 274: 32127-32136, 1999) consisting of full-length VEGF-D tagged N-terminus, and N-terminus FLAG VEGF-D-CPRO, consisting of C-terminal propeptide, tagged amino acid residues 206 to 354. These proteins were expressed in 293-EBNA-1 cells and described in Achen M. et al., Proc. Natl. Acad. Sci. USA, 95: 548-553, 1998 using the procedure presented in M2 (anti-FLAG ) Purification by affinity chromatography using MAb (IBI / Kodak, Connecticut New Haven). SDS-PAGE using VD1 MAb and biotinylated M2 MAb as controls as 50 ng purified VEGF-D-FULL-N-FLAG (FN), VEGF-DΔNΔC (ΔΔ), and VEGF-D-CPRO (CP) (Reduction) and Western blot analysis (antibodies used for blotting are shown below the panel in FIG. 2). SDS-PAGE and Western blot analysis are described in Stacker, SA et al., J. Biol. Chem. 274: 32127-32136, 1999, as described.
[98] As shown in FIG. 2, the dominant species in the sample of VEGF-D-FULL-N-FLAG contained all of the unprocessed VEGF-D (Mr-53K), N-terminal propeptide and VHD (-31K). Consisting of partially processed VEGF-D, and N-terminal propeptide (~ 10K) (Stack-er, SA et al., J. Biol. Chem. 274: 32127-32136, 1999), all of which are FLAG Detected with M2 MAb because it was tagged with octapeptide (left arrow, numbers indicate Mr of K and subscripts indicate samples where bands were detected). Similarly, VEGF-DΔNΔC (˜21K) and VEGF-D-CPRO (two bands of ˜31 and ˜29K resulting from different glycosylation) are also FLAG It is detected with M2 (left arrow) because it is tagged as. VD1 detects unprocessed VEGF-D, partially processed VEGF-D and VEGF-DΔNΔC, but the N-terminal propeptide (~ 10K) and VEGF-D-CPRO of the VEGF-D-FULL-N-FLAG preparation It does not detect the C-terminal propeptide of the samples (˜31 and ˜29K). Results of VEGF-D-FULL-N-FLAG were analyzed using intestinal (L) and short (S) exposures. The position of the molecular weight marker is shown on the right in FIG.
[99] Thus, MAb VD1 binds to unprocessed VEGF-D, partially processed form containing VHD, and fully processed VEGF-D, but not to N- or C-terminal propeptide. Moreover, MAb VD1 was able to immunoprecipitate native human VEGF-DΔNΔC in enzymatic immunoassays, but did not do the VHD of human VEGF-C (VEGF-DΔNΔC) (Joukov, V. et al., EMBO J 16: 3898-). 3911, 1997), which indicates that VD1 is specific for VEGF-D.
[100] Example 3
[101] In Situ Hybridization Studies on VEGF-D Gene Expression in Mouse Embryos
[102] Patterns of VEGF-D gene expression were studied by in situ hybridization using radiolabeled antisense RNA probes corresponding to 340 to nucleotides of mouse VEGF-D1 cDNA (SEQ ID NO: 4). Antisense RNA was synthesized by in vitro transcription using T3 RNA polymerase and [ ³⁵ S] UTPαs. Mouse VEGF-D is fully described in international patent application PCT / US97 / 14 696 (WO 98/07832). This antisense RNA probe was hybridized with paraffin-embedded tissue sections of 15.5 days old mouse embryos after mating. Radiographic photography was performed on the labeled sections for 2 days. Radiographs of the results for fragments hybridized with antisense RNA and complementary sense RNA (negative control) are shown in FIG. 3. In Figure 3, "L" indicates lung and "Sk" indicates skin, and these two tissue sections shown are consecutive sections. A strong signal of VEGF-D mRNA was detected in the developing lung and related to the skin. Using control sense RNA, no signal was detected.
[103] In FIGS. 4A-4D, sagittal sections were hybridized with VEGF-D antisense RNA probes, then incubated with photographic emulsion, developed and stained. 4A and 4D are enlarged x40, FIG. 4B is x200, and FIG. 4C is x500.
[104] In FIG. 4A, the dark portion of the micrograph shows the strong signal of VEGF-D mRNA in the lung (Lu). Liver (Li) and ribs (R) are also shown. 4B shows higher magnified lungs. The brighter part of the picture shows the bronchus (BR) and bronchial artery (BA). The rectangular black outline magnifies higher to indicate the area of the intercept shown in FIG. 4C. 4C shows bronchial endothelial cells (Ep), developing smooth muscle cells (SM) and mesenchymal cells (Mes) surrounding the endothelial cell layer. Abundant silver granules related to mesenchymal cells are apparent. Thus, microscopic analysis reveals that VEGF-D mRNA is abundant in developing lung mesenchymal cells (FIGS. 4A-4C). In contrast, endothelial cells of the bronchus and bronchioles are negative, as did the developing smooth muscle cells surrounding the bronchus. In addition, endothelial cells of the bronchial artery are also negative.
[105] In FIG. 4D, the dark parts of the picture represent the jia. A strong signal was located just below the skin of the tissue area hatched with fibroblasts and developing melanocytes.
[106] These results indicate that VEGF-D can elicit the growth of blood vessels and lymphatic vessels in the area just below the developing lung and skin. Due to the expression of the VEGF-D gene adjacent to the dermal region, VEGF-D may play a role in inducing angiogenesis associated with malignant melanoma. Malignant melanoma is a very highly vascularized tumor. This suggests that local inhibition of VEGF-D expression, for example with VEGF-D or VEGF receptor-2 or VEGF receptor-3 antibodies, is useful for the treatment of malignant melanoma. In addition, other suitable inhibitors of VEGF-D activity, such as antisense nucleic acids or triple stranded DNA, may also be used.
[107] Example 4
[108] Use of Monoclonal Antibodies Against Human VEGF-D for Immunohistochemical Analysis of Human Tumors
[109] To assess the role of VEGF-D in tumor angiogenesis, immunohistochemistry of VEGF-D MAbs, 4A5, 5F12 and 2F8 (renamed VD1, VD2 and VD3, respectively) of 15 randomly selected invasive malignant melanoma Used for analysis. Also used in the analysis were MAb (human sigma, St. Louis, MO) against human VEGFR-2 and polyclonal antibodies (affinity purified anti-human Flt-4 antibody; R & D system, Minneapolis) against VEGFR-3 . MAb derived from the receptor of granulocyte colony-stimulating factor designated LMM774 (Layton et al., Growth Factors 14: 117-130, 1997) was used as a negative control. Like VEGF-D MAb, LMM774 was a mouse IgG1 subsample and was therefore used as a subsample-matched control antibody. Sections 5 μm thick from formalin fixed and paraffin embedded tissues of cutaneous malignant melanoma were used as test tissues. This section was dewaxed and rehydrated and washed with PBS. Primary antibodies were incubated with tissue sections at concentrations of 5-40 μg / ml depending on incubation time. The anti-VEGF-D MAb was performed in parallel with the uptake control, where the primary antibody was omitted, as with the uptake control, which was incubated with a 40-fold molar excess of VEGF-DΔNΔC for 1 hour at room temperature prior to incubation with the tissue sections. . Subsample-matched controls with LMM744 antibodies were also performed. Detection of alkaline phosphatase-conjugated secondary antibodies was achieved using Fast Red Substrate (Sigma, St. Louis, MO). In some cases, melanin was bleached in tissue sections by incubation for 3 hours in 0.25% potassium permanganate prior to immunohistochemistry followed by 6 minutes in 1% oxalic acid. In these cases, the detection of peroxidase-conjugated secondary antibodies used 3,3'-diaminobenzadine (DAB) (Dako Corp., Calif., California).
[110] A positive response was seen in all three VEGF-D MAbs with essentially the same staining pattern. VEGF-D immunoreactivity was detected in 13 of 15 melanoma tested. Melanoma showed a response pattern ranging from homogeneous staining of the entire lesion to localization in the periphery of the lesion.
[111] 5A-5H show the results of immunohistochemical analysis from two tumors illustrating different response patterns. Antibody detection in FIGS. 5A and 5B used a fast red substrate (red indicates a positive signal) and DAB (brown indicates a positive signal) in FIGS. 5C-5H. Tissue sections shown in FIGS. 5C-5H bleached melanin prior to incubation with antibodies. The VEGF-D antibody used in all panels except for FIGS. 5E and 5G was VD1 (4A5). The scale bar of FIG. 5A displays 150 [mu] m, 20 [mu] m in FIG. 5B-5D and 10 [mu] m in FIG. 5E-5H.
[112] As shown in Figures 5A and 5B, non-uniform staining was apparent through the bulk of the first melanoma. In this tumor, the detected VEGF-D staining is more pronounced in the intradermal nest (white arrowheads) of tumor cells at the periphery of the invasive portion of the primary bulk of the tumor, less intense or undetectable in the central portion. In addition, VEGF-D is a small capillary-sized vessel (white arrow) in the nipple and reticulated dermis adjacent to positive tumor cells (FIG. 5B) and a vessel with thickened walls of pre-capillary and post-capillary lavage veins. Is detected.
[113] As shown in FIG. 5C, in the second melanoma, VEGF-D is more evenly distributed throughout the tumor mass and detected in tumor cells as well as in blood vessels in the tumor. The areas of negatively stained stroms are marked with black asterisks.
[114] In the two tumors mentioned above, the upper dermal capillary vessels and other vessels deep in the middle and deep of the reticulated dermis slightly away from the tumor and far from the tumor and the gland showed very weak vascular wall staining or vascular wall staining. No granular cytoplasmic endothelial staining was seen in the vessels adjacent to the immunoreactive tumor cells. In addition, non-tumorigenic melanocytes were negative, indicating that VEGF-D is not expressed by these cells in adult skin. Figure 5D, a continuous control section for the tissue of Figure 5C, shows that the absorption control negative. In addition, the step omission and subsample-matched control were negative.
[115] Sections of malignant melanoma were analyzed for localization of VEGFR-3, a receptor for VEGF-D expressed on endothelial cells of lymphatic vessels in adult tissues (Lymboussaki, A. et al., Am. J. Pathol. 153: 395-). 403, 1998). As shown in FIG. 5E, VEGFR-3 was detected in endothelial cells (white arrows) of thin-walled vessels in melanoma. In addition, VEGFR-3 positive vasculature adjacent to tumor cells was positive for VEGF-D (FIG. 5F) as assessed by immunohistochemical analysis of successive sections, indicating that the VEGF-D immunoreactivity of these vasculature was endothelial. It may be due to receptor-mediated uptake into cells. In addition, sections were analyzed for localization of VEGFR-2 by immunohistochemistry. VEGFR-2 is known to be upregulated in the endothelium of blood vessels in tumors (Plate K. et al., Cancer Res, 53: 5822-5827, 1993). As shown in FIG. 5G, VEGFR-2 was detected in the endothelium of the blood vessels (white arrow) and in nearby tumors. In addition, some of the vasculature that was immunopositive for VEGFR-2 were also positive for VEGF-D (white arrow in FIG. 5H), indicating that VEGF-D uptake into the tumor vasculature can also be mediated by this receptor.
[116] Example 5
[117] VEGF-D in Lung Cancer
[118] Angiogenesis is thought to be a useful prognostic marker for non-small cell lung carcinoma (NSCLC) (Fontanini, G. et al., Clin Cancer Res. 3: 861-865, 1997). Therefore, localization of VEGF-D by immunohistochemistry was analyzed in cases of squamous cell carcinoma (FIGS. 6A-6F). Immunohistochemistry was performed as in Example 4 except that antibodies against alpha-smooth muscle actin (DAKO Corp., CA Pinteria, CA) were also used for immunostaining. The anti-VEGF-D MAb used for immunostaining in FIGS. 6A and 6D was VD1 (4A5). 6A shows that VEGF-D is detected in tumor cells forming islets in the center of the micrograph, endothelial cells of adjacent aorta, and cells in connective tissue stroma. The connective tissue forming stroms are shown in black brackets, and dashed boxes indicate areas shown with higher power in FIG. 6D. Immunopositive cells in the stroms can be myofibroblasts.
[119] 6B shows that VEGFR-2 is detected in endothelial cells of the vestibule. However, these vessels were negative for VEGFR-3 in this tumor. The dashed box indicates the area shown at higher power in FIG. 6E. Control staining of tissue sections from the same case, where the VEGF-D MAb was preincubated with VHD of 40-fold molar excess of human VEGF-D, did not provide a signal (FIG. 6C).
[120] As mentioned above, the immunopositive cells in the connective tissue forming strom can be myofibroblasts. Thus, connective tissue forming stroms were immunostained using MAbs specific for alpha-smooth muscle actin to detect myofibroblasts. As shown in FIG. 6F, the stroms stained positive, indicating the presence of myofibroblasts. The secretion of angiogenic factors by stromal components can contribute to amplifying angiogenic stimuli produced by the tumor.
[121] Example 6
[122] VEGF-D in Breast Cancer
[123] In addition, localization of VEGF-D was analyzed in breast duct carcinoma by in situ immunohistochemistry and the results are shown in FIGS. 7A-7F. Immunohistochemistry was performed as in Example 4 except that MAb specific for alpha-smooth muscle actin (DAKO Corp., Calif., California) and platelet / endothelial adhesion molecule (PECAM) (DAKO Corp., Calif., California) Also used for immunostaining. The anti-VEGF-D MAb used for immunostaining in FIG. 7A was VD1 (4A5).
[124] As shown in FIG. 7A, VEGF-D was detected in the so-called "neckles" (the black arrowheads) directly adjacent to the tumor cells in the catheter and the base plate of the catheter filled with the tumor. The necklace vasculature was also positive for VEGFR-2 (FIG. 7C), VEGFR-3 (FIG. 7D) and PECAM (FIG. 7E) represented by black arrowheads. PECAM is a classic marker for endothelial and is also found on platelets and white blood cells. PECAM plays a role in the migration of white blood cells to the site of inflammation (Muller et al., J. Exp. Med. 178: 449-460). PECAM antibody staining for "neckless" vessels helps to confirm that these structures are vasculature. The edge of the conduit is confirmed by staining for alpha-smooth muscle actin (FIG. 7B) to detect myofibroblasts. Control staining of tissue sections in series with that shown in FIG. 7A preincubating VEGF-D MAb with VHD of 40-fold molar excess of human VEGF-D did not provide a signal (FIG. 7F). These findings indicate that VEGF-D secreted by tumor cells plays a role in activating its receptors on nearby vessels, leading to the growth of necklaceless vessels very close to the catheter. This may be important for both solid tumor growth and metastatic spread.
[125] Example 7
[126] VEGF-D in Endometrial Cancer
[127] VEGF-D was also detected in endometrial adenocarcinoma (FIG. 8). Immunohistochemistry was performed as in Example 4 using anti-VEGF-D MAb VD1 (4A5). Moderate staining of VEGF-D was seen in gland tumor cells (GL), and very strong reactivity was seen in myofibroblast cells of connective tissue-forming stromal (DM) at the advanced invasive edge of the tumor. It was seen on the endothelium and walls of adjacent blood vessels (black arrows) in the myometrium (Myo). Interestingly, VEGF-D reactivity was particularly strong in myofibroblasts of connective tissue stroma, indicating that adenocarcinoma cells can induce the expression of VEGF-D in these fibroblasts and amplify the tumor's angiogenic potential. . Since expression of VEGF-D in cells of connective tissue-forming stroma was also detected in lung carcinoma (FIG. 6A), there is a possibility that a range of tumors induce VEGF-D in stromal components. This is similar to the developing lung, in which case the mesenchymal cells, which are probably precursors of fibroblasts, are likely to express strongly the VEGF-D gene. Thus, signals from embryonic and tumor tissues can induce the expression of VEGF-D in fibroblasts.
[128] Example 8
[129] VEGF-D in non-tumorigenic tissue
[130] Tissues with high cell turnover and / or metabolic burden, such as the colon, require extensive vascular network structures. Thus, human colon was analyzed for localization of VEGF-D by immunohistochemistry and the results are shown in FIGS. 9A-9F. Immunohistochemistry was performed as in Example 4 except that an antibody specific for alpha-smooth muscle actin (DAKO Corp., Calif., California) was also used for immunostaining. For all tissue sections shown, detection used DAB (brown indicates positive signal) and the VEGF-D antibody used in FIGS. 9A, 9B, 9C and 9F was VD1 (4A5). For clarity, contrast staining was omitted in FIGS. 9A, 9B, 9D and 9F. In FIG. 9A the scale bar shows 120 μm, 40 μm in FIGS. 9B, 9D and 9F, and 6 μm in FIGS. 9C and 9F.
[131] VEGF-D was localized to submucosal vessels (FIG. 9A). Higher power analysis revealed staining of vascular smooth muscle (white arrowheads), but did not stain the endothelial cells of the arteriole (black arrowheads) (FIGS. 9B and 9C). Staining of sections in series with those shown in FIGS. 9A-9C with antibodies specific for alpha-smooth muscle actin detect vascular smooth muscle (white arrowheads) but not endothelial (black arrowheads) (FIGS. 9D and 9E). This stain demonstrates that VEGF-D reactivity was in the vascular smooth muscle cells of the arterioles. Moreover, these endothelial cells did not show immunoreactivity to VEGFR-2 or VEGFR-3, indicating that these cells do not accumulate VEGF-D in a receptor-mediated manner. Preincubation of VEGF-D MAb with 40-fold molar excess of human VEGF-D VHD completely blocks staining of vascular smooth muscle (FIG. 9F).
[132] Various injuries to the colon cause certain injuries, and submucosal VEGF-D can be produced by vascular smooth muscle cells in preparation for vascular regeneration. When endothelial is activated in response to vascular injury, upregulation of VEGFR-2 on these endothelial cells of these vessels causes VEGF-D produced by vascular smooth muscle to induce endothelial cell proliferation and vascular repair. Upregulation of VEGFR-2 by the endothelial of the small arterioles and microvessels in response to arterial injury has been previously reported in connection with ischemic stroke (Issa, R. et al., Lab Invest 79: 417-425, 1999).
[133] Example 9
[134] Role of VEGF-D in Tumor Development
[135] In order to generate a cell line constitutively overexpressing a derivative of VEGF-D, a region of human VEGF-D cDNA was inserted into the mammalian expression vector Apex-3 (Evans et al., Mol Immunol 1995 32 1183-1195). This vector remains episomal when transfected into 293-EBNA human embryonic kidney cells. For expression of mature VEGF-D, the region of pEFBOSVEGF-DΔNΔC containing the sequence encoding the IL-3 signal sequence, FLAG Octapeptide and mature VEGF-D were inserted into the XbaI site of Apex-3 (see Example 9 of international patent application PCT / US97 / 14696 (WO 98/0783 2)). The resulting plasmid is designated pVDApexDΔNΔC (see Stacker, SA et al., J Biol Chem 274: 32127-32136, 1999 and Example 1 of International Patent Application PCT / US98 / 27373). The entire specification of international patent application PCT / US98 / 27373 is incorporated herein by reference. FLAG similar compositions An unprocessed full length tagged with N-terminus was made for expression of VEGF-D. In this construct, the DNA encoding the VEGF-D signal sequence for protein secretion was deleted, and after substitution with the DNA encoding the IL-3 signal sequence, immediately upstream and at residue 24 of VEGF-D, 354 was removed. FLAG within the same reading frame as the DNA you are coding It was substituted with octapeptide and two amino acids (Thr-Arg). This construct was designated pVDApexFULL-N-Flag (see Stacker, SA et al., J Biol Chem 274: 32127-32136, 1999 and Example 1 of International Patent Publication PCT / US98 / 27373). These vectors may be purified by the calcium phosphate method or according to the manufacturer's instructions. (Roche Molycula Biochemicals, Mannheim, Germany) was used to transfect cells of human embryonic kidney cell line 293EBNA-1, and a stable transfectant was selected in the presence of 100 μg / ml hygromycin supplemented DMEM. Cell lines expressing high levels of VEGF-D-FULL-N-Flag and VEGF-DΔNΔC were subsequently identified by metabolic labeling, immunoprecipitation and Western blot analysis (Stacker, SA et al., J Biol Chem 274: 32127- 32136, 1999 and Example 1 of International Patent Application PCT / US98 / 27373).
[136] The mammary fat layer of 6-8 week old SCID mice (ARC, Perth, Australia) was injected subcutaneously with transfected 293 cells or untransfected parental 293 cells 2 × 10 ⁷ in PBS. Tumors were allowed to grow and measured with digital calipers over three weeks. The experiment was terminated after three weeks, when the first animal reached the maximum size allowed by the International Ethics Committee. Tumor size was calculated as width x length x 0.6x (width x length) / 2.
[137] 10 shows injection of 293 cells transfected with constructs encoding untransfected parental 293 cells (designated “293”) or VEGF-D-FULL-N-FLAG (designated “VEGF-D-293”). Results of analysis for tumors of SCID mice resulting from There is a significant difference between tumors derived from 293-VEGF-D-FULL-N-FLAG cells and tumors derived from untransfected 293 cells. After 3 weeks, the mean tumor size of the 293-VEGF-D-FULL-N-FLAG group was 937 ± 555mm ³ (mean ± SD, n = 8), and 136 ± 230mm ³ for 293 cells that were not transfected. (n = 8). Interestingly, tumors generated from 293 cells transfected with the construct encoding VEGF-DΔNΔC did not differ significantly in size 50 ± 76 mm ³ (n = 7) from tumors from untransfected 293 cells.
[138] In addition, the microscopic pattern of tumors derived from untransfected 293 cells was of a faint white surface, compared with tumors derived from 293-VEGF-D-FULL-N-FLAG cells compared to the presence of clear blood vessels throughout the tumor. Together with the appearance of blood.
[139] Sections were also analyzed by immunohistochemistry using an anti-PECAM monoclonal antibody (Pharmingen, San Diego, Calif.), A marker for endothelial cells. Sections of tumors generated using 293-VEGF-D-FULL-N-FLAG cells showed a significant increase in PECAM expression compared to tumors generated using parent 293 cells that were not transfected. This analysis confirms that blood vessels are much richer in tumors expressing unprocessed full-length VEGF-D.
[140] This experiment shows that the unprocessed form of VEGF-D can induce tumor angiogenesis and solid tumor growth in vivo. Interestingly, tumors derived from cells expressing mature fully processed forms of VEGF-D showed no increase in growth compared to 293 untransfected parental cells. This indicates the importance of propeptides (N-pro and C-pro) of VEGF-D for accurate localization or function of VHD of VEGF-D. Interpretation of these results suggests that the propeptide is involved in matrix association and that heparin sulphate proteoglycan is a growth factor capable of inducing angiogenesis and / or lymphangiogenesis only when VEGF-D is correctly located on the extracellular matrix or cell surface. will be. Another interpretation is that propeptide increases the half-life of VEGF-D VHD in vivo.
[141] Example 10
[142] VEGF-D Induction of Tumor Angiogenesis
[143] To determine whether VEGF-D plays a role in tumor angiogenesis, 293EBNA cell lines expressing VEGF or VEGF-D were generated. 293EBNA cell lines normally do not express detectable levels of VEGF, VEGF-C or VEGF-D, ligands that activate VEGFR-2 and / or VEGFR-3 (Stacker, SA et al., Growth Factors 17: 1-11 (1999)). 293EBNA cells produce slow epithelial and epithelial-like epithelial-like tumors in immunodeficient mice. Western blot analysis of conditioning medium from 293EBNA cell lines generated in vitro showed that mature growth factor was secreted.
[144] Female SCID or SCID / nod mice (Animal Resource Center, Corning Vale, Australia; Austin Research Institute, Australia; and Walter and Eliza Hall Institute for Medical Research, Australia) 6 to 21 weeks old are placed in a group of 6 to 10 mice, Cell lines expressing VEGF-293, VEGF-D-293, or control 293 cell lines were used to subcutaneously injected into the mammary fat layer at a concentration of 2.0 to 2.5 × 10 ⁷ in culture medium. Tumor growth and morphology were analyzed over 35 days. Tumors were measured with a digital caliper and tumor volume was calculated by the formula: volume = length × width ² × 0.52. Mice were euthanized three to five weeks after cell line injection and tumors were removed for testing. VEGF-D-293 tumors and 293 tumors were excised and weighed 25 days post mortem.
[145] VEGF-293 cells produced tumors with increased growth rates compared to control 293 cells. VEGF-293 tumors are highly vascularized with extensive edema, consistent with VEGF, a potent tumor angiogenesis and vascular permeability inducer. In addition, VEGF-D-293 cells showed enhanced growth in vivo and tumors were highly vascularized compared to control 293 tumors, but there was no apparent or microscopic evidence for edema.
[146] Tumor growth resulting from the injection of VEGF-D-293 cells was observed twice a week for monoclonal antibody VD1, an antibody specific for the bioactive region of VEGF-D that blocks binding of VEGF-D to VEGFR-2 and VEG FR-3. Blocked by intraperitoneal injection. However, tumor growth was not affected by treatment with a control that was a sub-matched antibody.
[147] As assessed by immunohistochemistry of the endothelial cell marker PECAM-1, treatment with VD1 antibodies reduced the abundance of vasculature in tumors. Western blotting demonstrated the expression of VEGF-D and VEGF respectively in VEGF-D-293 and VEGF-293 tumors, and also demonstrated that VEGF was not upregulated in VEGF-D-293 tumors. Analysis of post-tumor weight was significant between VEGF-D-293 tumor ((0.49 ± 0.22g, n = 7; mean ± SD) and control 293 tumor (0.123 ± 0.118g, n = 9, p = 0.01). Showed a difference.
[148] Example 11
[149] VEGF-D induction of tumor lymphangiogenesis
[150] Since metastasis through lymphatic vessels to local lymph nodes is a common step in the propagation of solid tumors, whether VEGF-D induced tumor lymphangiogenesis or whether expression of VEGF-D in tumor cells led to tumor spread to lymph nodes. An experiment was conducted to determine whether it was.
[151] To analyze the role of VEGF-D in tumor propagation, VEGF-D-293 tumors were screened in SCID / NOD mice (Animal Resource Center, Kerningvale, Australia; Austin Research Institute Australia; and Walter and Eliza Hall Institute for Medical Research, Australia). Induced from. Post hoc analysis revealed metastatic lesions in 14 lateral fluids and lymph nodes and / or chemotaxis in 23 animals in VEGF-D-293 tumors, compared to 16 for VEGF-293 tumors. 0 of 14 animals and 0 of 293 tumors. In some cases, the propagation of metastatic tumor cells from primary tumors in SCID / NOD mice was evident as traces of tumor cells in the lymphatic vessels of the skin between the primary tumor and the lateral fluid and nodes.
[152] Treatment of mice bearing VEGF-D-293 tumors with the VD1 monoclonal antibody (Table 1) blocked metastatic spread to lymph nodes. All 7 mice treated over 25 days with VD1 did not show lymphatic vessel propagation, whereas 6 of 10 mice treated with control subsample-matched monoclonal antibodies showed lymphatic vessel propagation. These results indicate that VEGF-D can promote metastatic spread of these tumors by lymphatic vessels.
[153]
[154] ^a Purified monoclonal antibody was injected twice weekly during the course of the experiment, beginning one day after injection of tumor cells. VD1 is a neutralizing monoclonal antibody against VEGF-D.
[155] ^b LMM774 is a subsample-matched control monoclonal antibody that does not bind VEGF-D.
[156] These data indicate that expression of VEGF-D can promote metastatic spread of tumor cells through lymphatic network. As measured by immunohistochemistry for the lymph-specific marker LYVE-1, activation of VEGFR-3-VEGFR-2-heterodimer cannot be ruled out, but VEGF-D is thought to inhibit the lymphatic receptor VEGFR-3. Through the formation of lymphatic vessels in tumors. Expression of only lymphangiogenic factors is sufficient to induce the formation of lymphatic vessels in the center of the tumor and to promote metastatic spread to lymph nodes.
[157] VEGF-D was localized to the endothelial of tumor cells and vasculature in malignant melanoma, lung and breast cancers (see Examples 4-6).
[158] Example 12
[159] Changes in tumor characteristics induced by different forms of VEGF-D
[160] In addition to the determination of the role of VEGF-D in tumor angiogenesis and lymphangiogenesis, the methods of Examples 10 and 11 were used to show different forms of VEGF-D indicating cleavage of N, C, and N and C both terminal propeptides. Expressing tumors were generated and evaluated. Cell lines injected into mice were 293EBNA, VEGF-D-293, VEGF-DΔNΔC-293, VEGF-DΔC-293 (cells expressing VEGF-D without C-terminal propeptide), and VEGF-DΔN-293 ( Cells expressing VEGF-D without the N-terminal propeptide).
[161] Tumors produced by VEGF-DΔN cells grew faster than tumors produced by control cells. In the morphological examination, the tumor had a red appearance, contained sufficient vascular response, and contained substantial humoral components that were not seen in the control tumor. Tumors produced by VEGF-DΔN cells had significant differences in control and morphological features from control tumors.
[162] The graph of FIG. 11 shows increased growth rates in tumors from VEGF-DΔN cells. The tendency of fluid accumulation in tumors produced by VEGF-DΔN cells can be seen in FIG. 12, which is a picture of such tumors. This can be contrasted with the photograph of FIG. 13 showing normal tumors as produced by control cells.
[163] Tumors produced by VEGF-DΔC cells grew in a manner similar to control cells and did not show excess humor formation.
[164] Tumors produced by VEGF-DΔNΔC cells grew very slowly compared to control tumors. VEGF-DΔNΔC tumors formed at about 70 days, compared with an average of 30-35 days for control tumors and 20-25 days for VEGF-DΔN tumors. Testing of these tumors showed that they had a reduced vascular response and had fewer blood vessels than control tumors by PECAM-1 staining. This tumor gave rise to the lymph network structure indicated by LYVE-1 staining and induced the formation of lymph metastases. The graph of FIG. 14 shows reduced growth rate in tumors from VEGF-DΔNΔC cells.
[165] Since strong signals of VEGF-D were detected in endothelial cells of blood vessels near immunopositive tumor cells, but not in vasculature away from tumor cells, localization of VEGF-D in malignant melanoma was associated with its role in tumor angiogenesis. Matches. This indicates that VEGF-D found in the tumor or nearby vessels may be due to receptor-mediated uptake, which is caused by the binding of VEGF-D secreted by tumor cells to and accumulation in the target endothelial cells. It supports the hypothesis of establishing a secretory mechanism that regulates angiogenesis. Similar patterns of VEGF localization in tumor cells and tumor vessels have been previously reported (Plate, K. et al., Brain Pathology 4: 207-218, 1994). Consistent with the hypothesis that VEGF-D plays a role in tumor angiogenesis, the finding that VEGFR-2, a receptor for VEGF-D, is upregulated in endothelial cells of blood vessels in tumors (Plate, K. et al., Cancer Res) 53: 5822-5827, 1993). Indeed, some of the VEGF-D immunopositive vessels detected in melanoma studied here were also positive for VEGFR-2. Signaling by VEGFR-2 was important for sustaining tumor angiogenesis (Millauer B. et al. Cancer Res 56: 1615-1620, 1996) and angiogenic activity of VEGF-D in vivo (Marconcini. L. et al., Proc. Natl.Acad. Sci. USA 96: 9671-9676, 1999) are most likely mediated by this receptor. As VEGF-D was detected in tumor cells and nearby vasculature, staining patterns similar to those seen in melanoma were observed in squamous cell carcinoma of the lung and in situ breast duct carcinoma (BDCIS). In addition, the vessels near the catheter filled with tumors in the BDCIS and the vessels near the islets of tumor cells in lung carcinomas were positive for VEGFR-2, which in turn controls the control of tumor angiogenesis in a lateral secretory manner. Imply that they can contribute.
[166] In addition, because the vasculature positive for VEGFR-3, a receptor for VEGF-D expressed on the lymphatic endothelium of normal adult tissue, was also positive for VEGF-D, these results suggest that VEGF-D stimulates the growth of lymphatic vessels in the vicinity of malignant melanoma. To play a role. Similar staining patterns were seen in BDCIS as some of the VEGF-D positive vasculature surrounding the tumor filled catheter was also positive for VEGFR-3. Signaling by VEGFR-3 is thought to be important for lymphangiogenesis (Taipale, J. et al., Curr. Top. Microbiol. Immunol. 237: 85-96, 1999), but this receptor is upregulated in the vascular capillaries of cancer. (Valtola, R. et al. Am. J. Path. 154: 1381-1390, 1999). Thus, the secretory regulation mechanism composed of VEGF-D and VEGFR-3 can stimulate both lymphangiogenesis and angiogenesis in cancer. Thus, the pathway by which tumors metastasize can be determined in part by the tumor's ability to induce angiogenesis and / or lymphangiogenesis. If so, expression by tumor cells of purely angiogenic, soluble growth factor (eg, VEGF), as opposed to growth factor (eg, VEGF-D), which may also induce lymphangiogenesis is a pathway of metastatic spread. It may be an important determinant for.
[167] VEGF-D may also play a role in vascular maintenance in non-tumorigenic tissue. In the submucosa of the colon, VEGF-D was localized to vascular smooth muscle but not to the endothelium. The absence of VEGF-D in the endothelial is probably the result of the lack of expression of the VEGF-D receptors VEGFR-2 and VEGFR-3 in endothelial cells. Endothelial activation in response to vascular injury seems to be sufficient to induce the expression of VEGFR-2 by endothelial cells (Issa R. et al., Lab. Invest. 79: 417-425, 1999), which in turn is caused by vascular smooth muscle VEGF-D produced affects vascular repair by enabling endothelial cell proliferation.
[168] The foregoing description and examples have been presented merely to illustrate the invention and are not intended to be limiting. As modifications of the disclosed embodiments which combine the spirit and gist of the invention may occur to those skilled in the art, the invention is to be accorded the broadest scope so as to encompass all modifications falling within the scope of the appended claims and their equivalents.

权利要求:
Claims (44)
[1" claim-type="Currently amended] A method of treating an organism suffering from a tumorous disease characterized by the expression of VEGF-D by a tumor,
Screening the organism to determine the presence or absence of VEGF-D-expressing tumor cells;
Selecting the organism determined to have a tumor expressing VEGF-D at screening; And
Administering an effective amount of VEGF-D antagonist in the vicinity of the tumor to prevent binding of VEGF-D to its corresponding receptor
How to include.
[2" claim-type="Currently amended] The method of claim 1 wherein said organism is a mammal.
[3" claim-type="Currently amended] The method of claim 1, wherein the VEGF-D antagonist is co-administered with a cytotoxic agent.
[4" claim-type="Currently amended] The method of claim 1, wherein the antagonist is administered in a composition further comprising at least one pharmaceutical carrier or adjuvant.
[5" claim-type="Currently amended] The method of claim 1, wherein the tumorous disease is selected from the group consisting of malignant melanoma, mammary carcinoma, squamous cell carcinoma, prostate cancer and endometrial cancer.
[6" claim-type="Currently amended] The method of claim 1, wherein the antagonist is a monoclonal antibody that specifically binds VEGF-D to block binding of VEGF-D and VEGF receptor-2 or VEGF receptor-3.
[7" claim-type="Currently amended] 7. The method of claim 6, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[8" claim-type="Currently amended] A method for screening tumorous disease characterized by an increased expression of VEGF-D,
Obtaining a sample from an organism suspected of having a tumorous disease characterized by increased expression of VEGF-D;
Exposing the sample to a composition comprising a compound that specifically binds VEGF-D;
Washing the sample; And
Screening for the disease by detecting the presence, amount or distribution of the compound in the tissue sample
Wherein the detection of VEGF-D in the cells of or surrounding the potential tumorous growth is an indication for a tumorous disease.
[9" claim-type="Currently amended] The method of claim 8, wherein the compound is a monoclonal antibody that specifically binds VEGF-D.
[10" claim-type="Currently amended] The method of claim 8, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[11" claim-type="Currently amended] The method of claim 8, wherein the compound comprises a detectable label.
[12" claim-type="Currently amended] 9. The method of claim 8, wherein the tumorous disease is selected from the group consisting of malignant melanoma, mammary carcinoma, squamous cell carcinoma, prostate cancer and endometrial cancer.
[13" claim-type="Currently amended] 9. The method of claim 8, wherein said sample is a human tissue sample.
[14" claim-type="Currently amended] A method for screening tumorous disease characterized by an increased expression of VEGF-D,
Obtaining a sample from an organism suspected of having a tumorous disease characterized by increased expression of VEGF-D;
Exposing the sample to a composition comprising a compound that specifically binds VEGF-D;
Washing the sample; And
Screening for said disease by detecting the presence, amount or distribution of said compound in said sample
Wherein the detection of VEGF-D in or on the vascular endothelial cells in or around the potential neoplastic growth is an indication for a tumorous disease.
[15" claim-type="Currently amended] 15. The method of claim 14, wherein said compound is a monoclonal antibody that specifically binds VEGF-D.
[16" claim-type="Currently amended] The method of claim 15, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[17" claim-type="Currently amended] The method of claim 14, wherein said compound comprises a detectable label.
[18" claim-type="Currently amended] A method for screening tumorous diseases characterized by an increase in vascular endothelial cells of blood vessels,
Obtaining a sample from an organism suspected of having a tumorous condition characterized by an increase in vascular endothelial cells of the blood vessel;
Exposing the sample to a composition comprising a compound that specifically binds VEGF-D;
Washing the sample; And
Screening for a disease by detecting the presence, amount, or distribution of said compound in said sample
Wherein the detection of VEGF-D in or on the vascular endothelial cells in or around the potential neoplastic growth is an indication for a tumorous disease.
[19" claim-type="Currently amended] 19. The method of claim 18, wherein said compound is a monoclonal antibody that specifically binds VEGF-D.
[20" claim-type="Currently amended] 20. The method of claim 19, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[21" claim-type="Currently amended] 19. The method of claim 18, wherein said compound comprises a detectable label.
[22" claim-type="Currently amended] 19. The method of claim 18, further comprising exposing the sample to a second compound that specifically binds to at least one of VEGFR-2 and VEGFR-3, wherein the screening step comprises a compound and a blood vessel that binds VEGF-D. The presence, amount, or distribution of vascular endothelial cells having both VEGF-D and at least one of VEGFR-2 and VEGFR-3 in or around the potential tumor growth Method comprising the step of measuring.
[23" claim-type="Currently amended] A method for screening tumorous disease characterized by an increase in lymphatic endothelial cells,
Obtaining a sample from an organism suspected of having a tumorous condition characterized by an increase in lymphatic endothelial cells;
Exposing the sample to a composition comprising a compound that specifically binds VEGF-D;
Washing the sample; And
Screening for said disease by detecting the presence, amount or distribution of said compound in said sample
Wherein the detection of VEGF-D in or on the lymphatic endothelial cells in or around the potential neoplastic growth is an indication for a tumorous disease.
[24" claim-type="Currently amended] The method of claim 23, wherein said compound is a monoclonal antibody that specifically binds VEGF-D.
[25" claim-type="Currently amended] The method of claim 24, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[26" claim-type="Currently amended] The method of claim 23, wherein said compound comprises a detectable label.
[27" claim-type="Currently amended] The method of claim 23, further comprising exposing the sample to a second compound that specifically binds to VEGFR-3, wherein the screening step is a compound that binds VEGF-D and a second that binds to lymphatic endothelial cells. Detecting the compound to determine the presence, amount or distribution of lymphatic endothelial cells having both VEGF-D and VEGFR-3 at or around the potential tumor growth.
[28" claim-type="Currently amended] A method of maintaining vascularization of tissue in an organism, the method comprising administering VEGF-D, or an effective amount of a fragment or analog thereof having a biological activity of VEGF-D, to the organism in need of such treatment.
[29" claim-type="Currently amended] A method of treating an organism suffering from a tumorous disease characterized by the expression of VEGF-D by a tumor, wherein an effective amount of VEGF-D antagonist is administered in the vicinity of the tumor to prevent binding of VEGF-D to its corresponding receptor. Method comprising the steps of:
[30" claim-type="Currently amended] 30. The method of claim 29, wherein said organism is a mammal.
[31" claim-type="Currently amended] 30. The method of claim 29, wherein said VEGF-D antagonist is co-administered with a cytotoxic agent.
[32" claim-type="Currently amended] 30. The method of claim 29, wherein said antagonist is administered in a composition further comprising at least one pharmaceutical carrier or adjuvant.
[33" claim-type="Currently amended] 30. The method of claim 29, wherein the neoplastic disease is selected from the group consisting of malignant melanoma, mammary carcinoma, squamous cell carcinoma, prostate cancer and endometrial cancer.
[34" claim-type="Currently amended] The method of claim 29, wherein the antagonist is a monoclonal antibody that specifically binds VEGF-D to block binding of VEGF-D and VEGF receptor-2 or VEGF receptor-3.
[35" claim-type="Currently amended] The method of claim 34, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[36" claim-type="Currently amended] A method of screening tumors for risk of metastasis,
Exposing the tumor sample to a composition comprising a compound that specifically binds VEGF-D;
Washing the sample; And
Screening for risk of metastasis by detecting the presence, amount or distribution of said compound in said sample
Wherein the expression of VEGF-D by the tumor is an indication of the risk of metastasis.
[37" claim-type="Currently amended] The method of claim 36, wherein said compound is a monoclonal antibody that specifically binds VEGF-D.
[38" claim-type="Currently amended] The method of claim 37, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[39" claim-type="Currently amended] The method of claim 36, wherein said compound comprises a detectable label.
[40" claim-type="Currently amended] A method for detecting micro-metastasis of a tumorous disease state characterized by increased expression of VEGF-D,
Obtaining a tissue sample from a site remote from the neoplastic growth in the organism of the neoplastic disease state;
Exposing the sample to a composition comprising a compound that specifically binds VEGF-D;
Washing the sample; And
Screening for the metastasis of the tumorous disease by detecting the presence, amount or distribution of the compound in the tissue sample
Wherein said detection of VEGF-D in said tissue sample is an indication for metastasis of said tumorous disease.
[41" claim-type="Currently amended] 41. The method of claim 40, wherein said tissue sample is a lymph node from tissue surrounding said neoplastic growth.
[42" claim-type="Currently amended] The method of claim 40, wherein said compound is a monoclonal antibody that specifically binds VEGF-D.
[43" claim-type="Currently amended] The method of claim 42, wherein said antibody binds to the VEGF homology domain of VEGF-D.
[44" claim-type="Currently amended] 41. The method of claim 40, wherein said compound comprises a detectable label.

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公开号 | 公开日

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ES2234818T3|2005-07-01|

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DE60107815D1|2005-01-20|

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AU4194601A|2001-09-12|

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EP1259248A1|2002-11-27|

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AT533056T|2011-11-15|

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EP1259248A4|2003-05-28|

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CA2401665A1|2001-09-07|

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US20010038842A1|2001-11-08|

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EP1259248B1|2004-12-15|

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ES2377119T3|2012-03-22|

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JP2003525248A|2003-08-26|

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NZ520546A|2004-05-28|

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AT284701T|2005-01-15|

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DE60107815T2|2005-12-08|

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WO2001064235A1|2001-09-07|

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PT1259248E|2005-04-29|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-03-02|Priority to US18636100P

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2000-03-02|Priority to US60/186,361

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2001-03-02|Application filed by 루드빅 인스티튜트 포 캔서 리서치

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2001-03-02|Priority to PCT/US2001/006791

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2002-10-23|Publication of KR20020080461A

优先权:

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申请号 | 申请日 | 专利标题

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US18636100P| true| 2000-03-02|2000-03-02|

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US60/186,361|2000-03-02|

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PCT/US2001/006791|WO2001064235A1|2000-03-02|2001-03-02|Methods for treating, screening for, and detecting cancers expressing vascular endothelial growth factor d|