US 20060O88532A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0088532 A1 Alitalo et al. (43) Pub. Date: Apr. 27, 2006 (54) LYMPHATIC AND BLOOD ENDOTHELIAL Related U.S. Application Data CELL GENES (60) Provisional application No. 60/363,019, filed on Mar. (76) Inventors: Kari Alitalo, Helsinki (FI); Taija 7, 2002. Makinen, Helsinki (FI); Tatiana Petrova, Helsinki (FI); Pipsa Publication Classification Saharinen, Helsinki (FI); Juha Saharinen, Helsinki (FI) (51) Int. Cl. A6IR 48/00 (2006.01) Correspondence Address: A 6LX 39/395 (2006.01) MARSHALL, GERSTEIN & BORUN LLP A6II 38/18 (2006.01) 233 S. WACKER DRIVE, SUITE 6300 (52) U.S. Cl. .............................. 424/145.1: 514/2: 514/44 SEARS TOWER (57) ABSTRACT CHICAGO, IL 60606 (US) The invention provides polynucleotides and genes that are (21) Appl. No.: 10/505,928 differentially expressed in lymphatic versus blood vascular endothelial cells. These genes are useful for treating diseases (22) PCT Filed: Mar. 7, 2003 involving lymphatic vessels, such as lymphedema, various inflammatory diseases, and cancer metastasis via the lym (86). PCT No.: PCT/USO3FO6900 phatic system. Patent Application Publication Apr. 27, 2006 Sheet 1 of 2 US 2006/0088532 A1 integrin O9 integrin O1 KIAAO711 KAAO644 ApoD Fig. 1 Patent Application Publication Apr. 27, 2006 Sheet 2 of 2 US 2006/0088532 A1 CN g uueleo-gº US 2006/0O88532 A1 Apr. 27, 2006 LYMPHATIC AND BLOOD ENDOTHELLAL CELL lymphatic vessels, such as lymphangiomas or lymphang GENES iectasis. Witte, et al., Regulation of Angiogenesis (eds. Goldber, I. D. & Rosen, E. M.) 65-112 (Birkauser, Basel, BACKGROUND OF THE INVENTION Switzerland, 1997). The VEGFR-3 tyrosine kinase receptor 0001) 1. Field of the Invention is expressed in the normal lymphatic endothelium and is upregulated in many types of vascular tumors, including 0002 The invention relates to polynucleotides and pro Kaposi's sarcomas. Jussila, et al., Cancer Res 58, 1955-1604 teins specifically expressed in lymphatic endothelial cells. (1998); Partanen, et al., Cancer 86:2406-2412 (1999). 0003 2. Description of the Related Art Absence or dysfunction of lymphatic vessels which can result from an infection, Surgery, radiotherapy or from a 0004 Recent evidence on the association of lymphangio genetic defect, causes lymphedema, which is characterized genic growth factors with intralymphatic growth and by a chronic accumulation of protein-rich fluid in the tissues metastasis of cancers (Mandriota, et al., EMBO J. 20:672 that leads to swelling. The importance of VEGFR-3 signal 682 (2001); Skobe, et al., Nat. Med. 7:192-198 (2001): ing for lymphangiogenesis was revealed in the genetics of Stacker, et al., Nat. Med. 7: 186-191 (2001). Karpanen, et familial lymphedema, a disease characterized by a hypopla al., Cancer Res.61:1786-1790 (2001)) has raised hopes that sia of cutaneous lymphatic vessels, which leads to a disfig lymphatic vessels could be used as an additional target for uring and disabling Swelling of the extremities. Witte, et al., tumor therapy. Cancer cells spread within the body by direct Regulation of Angiogenesis (eds. Goldber, I. D. & Rosen, E. invasion to Surrounding tissues, spreading to body cavities, M.) 65-112 (Birkauser, Basel, Switzerland, 1997); Rockson, invasion into the blood vascular system (hematogenous S. G, Am. J. Med. 110, 288-295 (2001). Some members of metastasis), as well as spread via the lymphatic system families with lymphoedema are heterozygous for missense (lymphatic metastasis). Regional lymph node dissemination mutations of the VEGFR3 exons encoding the tyrosine is the first step in the metastasis of several common cancers and correlates highly with the prognosis of the disease. The kinase domain, which results in an inactive receptor protein. lymph nodes that are involved in draining tissue fluid from Karkkainen, et al., Nature Genet. 25:153-159 (2000); the tumor area are called sentinel nodes, and diagnostic Irrthum, et al., Am. J. Hum. Genet. 67:295-301 (2000). measures are in place to find these nodes and to remove them 0007. There is a need in the art for information on the in cases of Suspected metastasis. However, in spite of its transcriptional program which controls the diversity of clinical relevance, little is known about the mechanisms endothelial cells, and into the mechanisms of angiogenesis leading to metastasis via the bloodstream or via the lym and lymphangiogenesis. There is also a need in the art for phatics. new vascular markers, which may be used as valuable targets in the study of a number of diseases involving the 0005. Until recently, the lymphatic vessels have received lymphatic vessels, including tumor metastasis. much less attention than blood vessels, despite their impor tance in medicine. Lymphatic vessels collect protein-rich fluid and white blood cells from the interstitial space of most SUMMARY OF THE INVENTIONTO BE tissues and transport them as a whitish opaque fluid, the REVISED UPON FINALIZATION OF CLAIMS) lymph, into the blood circulation. Small lymphatic vessels 0008. The compositions of the present invention include coalesce into larger vessels, which drain the lymph through isolated polynucleotides, in particular, lymphatic endothelial the thoracic duct into large veins in the neck region. Lymph genes, polypeptides, isolated polypeptides encoded by these nodes serve as filtering stations along the lymphatic vessels polynucleotides, recombinant DNA molecules, cloned genes and lymph movement is propelled by the contraction of or degenerate variants thereof, especially naturally occurring Smooth muscles Surrounding collecting lymphatic vessels variants such as allelic variants, and antibodies that specifi and by bodily movements, the direction of flow being cally recognize one or more epitopes present on Such secured by valves as it is in veins. The lymphatic capillaries polypeptides. are lined by endothelial cells, which have distinct junctions with frequent large interendothelial gaps. The lymphatic 0009. The compositions of the present invention addi capillaries also lack a continuous basement membrane, and tionally include vectors, including expression vectors, con are devoid of pericytes. Anchoring filaments connect the taining the polynucleotides of the invention, cells geneti abluminal surfaces of lymphatic endothelial cells to the cally engineered to contain Such polynucleotides and cells perivascular extracellular matrix and pull to maintain vessel genetically engineered to express such polynucleotides. patency in the presence of tissue edema. The absence or 0010. In selected embodiments, such isolated polynucle obstruction of lymphatic vessels, which is usually the result otides of the invention represent a polynucleotide compris of an infection, Surgery, or radiotherapy and in rare cases, a ing a nucleotide sequence set forth in the sequence listing, genetic defect, causes accumulation of a protein-rich fluid in e.g., any of SEQ ID NOS:1-30. tissues, lymphedema. The lymphatic system is also critical 0011. The polynucleotides of the present invention also in fat absorption from the gut and in immune responses. include, but are not limited to, a polynucleotide that hybrid Bacteria, viruses, and other foreign materials are taken up by izes to the complement of the nucleotide sequence of SEQ the lymphatic vessels and transported to the lymph nodes, ID NOS:1-30 under highly stringent hybridization condi where the foreign material is presented to immune cells and tions; a polynucleotide that hybridizes to the complement of where dendritic cells traverse via the lymphatics. There has the nucleotide sequence of SEQ ID NOS:1-30 under mod been slow progress in the understanding of and ability to erately stringent hybridization conditions; a polynucleotide manipulate the lymphatic vessels. which is an allelic variant of any polynucleotide recited 0006 Abnormal development or function of the lym above; a polynucleotide which encodes a species homologue phatic ECs can result in tumors or malformations of the of any of the proteins recited above; of a polynucleotide that US 2006/0O88532 A1 Apr. 27, 2006 encodes a polypeptide comprising a specific domain or tiguous nucleotides of a sequence selected from the group truncation of the polypeptide encoded by any one of SEQID consisting of SEQID NOS:1-30, 45, 47,49, 51, 82,93, 111, NOS:1-30. Exemplary high stringency hybridization condi 188, 208, 212, 236, 242, 294, and 392, or a complement tions are hybridization at 42°C. for 20 hours in a solution thereof, under the following stringent hybridization condi containing 50% formamide, 5xSSPE, 5x Denhardt’s solu tions: (i) hybridization at 42°C. for 20 hours in a solution tion, 0.1% SDS and 0.1 mg/ml denatured salmon sperm containing 50% formamide, 5xSSPE, 5x Denhardt’s solu DNA, with a wash in 1xSSC, 0.1% SDS for 30 minutes at tion, 0.1% SDS and 0.1 mg/ml denatured salmon sperm 650 C. DNA, and (ii) washing for 30 minutes at 65° C. in 1xSSC, 0.1% SDS; and (b) detecting hybridization of the candidate 0012 Another aspect of the invention is drawn to LEC LEC nucleic acid and the polynucleotide, thereby identify and BEC polypeptides, including polypeptides encoded by ing a LEC nucleic acid. the polynucleotides described above. In some embodiments, the polypeptides are the mature forms of the polypeptides of 0016. The invention also provides a method of identify the invention. Expressly contemplated is a purified and ing a LEC protein comprising: (a) contacting a biological isolated polypeptide