# Patent

## Title
Polypeptide ESDN, polynucleotides encoding the polypeptide, and utility of the polypeptide

## Abstract

The invention discloses a useful and novel factor (polypeptide) which plays an important role for morbid vascular smooth muscle in restenosis after percutaneous transluminal coronany angioplasty (PTCA) and arterial sclerosis in the field of cardiovascular system.

## Background
This is a divisional of application Ser. No. 10/191,436 filed Jul. 10, 2002.


FIELD OF THE INVENTION
The invention is related to a new polypeptide called Endothelial and Smooth muscle cell-Derived Neuropilin-like molecule (hereinafter, simply referred to as “ESDN”), the preparation process, cDNA encoding ESDN, a vector containing the cDNA, host cells transformed by the vector, an antibody of ESDN, pharmaceutical compositions containing the polypeptide or the antibody, methods and reagents of measuring the quantity of ESDN, screening methods using ESDN.
BACKGROUND OF THE INVENTION
Since isolation of vascular endothelial growth factor (VEGF), the complicated network of extracellular signal transduction in angiopoiesis has been drastically elucidated. Research into mice which has a targeted mutation for the tyrosine kinase, for example, this ligand, Flk-1/VEGFR2, and Flt-1/VEGFR1, etc. was a first key point related the first step of angiopoiesis and vascularization.
VEGF receptor-2(VEGFR-2) is necessary in endothelial formation and a targeted mutation for this gene resulted in a shortage of blood cells or organized blood vessels and death in the embryo at 9.5 days post-coitum. However, according to analysis of early marker by RT-PCR revealing early hemopoiesis and the report that endothelial precursory cell was actually formed in absence of VEGFR-2, it is suggested that VEGFR-2 is required for the growth later, but not absolutely essential for hematoblast formation.
VEGF receptor-1 (VEGFR-1) knock-out mice were also dead in the embryo at 9.5 days post-coitum, but differentiation of their endothelial cells was not influenced directly. Instead, it is speculated that movement of the mesenchyme hematoblasts may be stagnant generally and that their overcrowded endothelial precursory cells may result in destroying vascular system severely.
VEGF knock-out mice were also dead in the embryo at 9.5 days post-coitum conspicuously, and differentiation of their endothelial cells were not defective. That is, substantially, it was the same to VEGF-1 knock-out mice, but the phenotype was slighter than that of VEGF-1 knock-out mice. The conspicuous characteristic related to a targeted mutation within this gene was that gene deletion of the heterozygote was lethal to the mouse embryo at 11.5 days post-coitum. This revealed that development in the embryo may depend strictly on the quantity of VEGF expression.
Other tyrosine kinase receptors, such as Tie-2 and its ligand, are specifically expressed in vascular endothelial cells, contributing to the step of vascularization in the late stage of angiopoiesis. Though destruction of these genes, which does not have any influence on angiopoiesis, affects vessel remodeling to cause death in the embryo at 10.5 days post-coitum. They play some roles in interaction between endothelial cells and their surrounding vascular smooth muscle cells (VSMC) or mesenchyme cells. In addition to the system specific to vascular, PDGF-BB, TGF-β and their receptors also play similar roles in interaction between endothelial cells and their surrounding cells. Investigations into the relationship between vascular system and neuropilin-1 (NP-1) which was cloned as an antigen recognized by the monoclonal antibody recognizing the first developed neuron led this field to a new phase.
Then, identification as a receptor of semaphorin 3A (sema3A) has come to attract attention in relation to axon formation, and search for other VEGF receptors led to discovery of NP-1 which is also a co-receptor for VEGFR-2. NP-1 enhances not only the binding to one isoform of VEGF-2, but also chemotaxis and probable mitosis of endothelial cells. Before this identification, it was revealed that chimerical mice over-expressing NP-1 presented the phenotype of hypertrophic vascular formation. NP-1 knock-out mice presented the phenotype of low angiogenesis in the central nervous system and large vascular variations. On the other hand, it has not been reported whether sema3A knock-out mice presented the abnormal vessels, while it is interesting that these mice presented the phenotype of thin myocardium, which was also observed in mice over-expressing NP-1.
Another example is the Eph/Ephrin system, which was first studied extensively in the neurological field. Later, this system was re-identified as an only surface marker discriminating arteries and veins. They are expressed in mesenchyme cells which surround the vessels and contact with endothelial cells. Speculating that there are many extracellular signal transduction molecules in vessels besides the above molecules, the inventors have suggested that proving this speculation would contribute to more understanding of this complex system.
DISCLOSURE OF THE INVENTION
The present inventors have directed their attention and have carried out energetic researches in order to find useful and novel factors (polypeptides) which play an important role for morbid vascular smooth muscle in restenosis after percutaneous transluminal coronany angioplasty (PTCA) and arterial sclerosis in the field of cardiovascular system, especially secretory and membrane protein containing signal sequence.
Conventionally, when a man skilled in the art intends to obtain a particular polypeptide or a cDNA encoding it, he generally utilizes methods by confirming an intended biological activity in a tissue or in a cell medium, isolating and purifying the polypeptide and then cloning a gene or methods by “expression-cloning” with the guidance of the biological activity.
However, physiologically active polypeptides in living body have often many kinds of activities. Therefore, a gene which was cloned with the guidance of a certain activity often turns to be identical to one encoding a polypeptide already known. Moreover, in bone marrow stromal cells, many of factors are expressed in a very small amount or only under specific physiological conditions, which makes it difficult to isolate and purify of the factor and to confirm its biological activity.
The present inventors have studied methods for cloning genes coding proliferation and/or differentiation factors functioning in hematopoietic systems and immune systems. Focusing their attention on the fact that most of the secretory proteins such as proliferation and/or differentiation factors (for example various cytokines) and membrane proteins such as receptors thereof (hereafter these proteins will be referred to generally as secretory proteins and the like) have sequences called signal peptides in the N-termini, the inventors conducted extensive studies on a process for efficiently and selectively cloning a gene coding for a signal peptide. Finally, the present inventors have successfully invented a screening method for cDNAs having sequence encoding signal peptides and named the method signal sequence trap method (hereinafter, simply referred to as “SST”) (See Japanese Patent Application No. 6-13951). The present inventors have also developed yeast SST method on the same concept. By the method using yeast, genes including sequence encoding signal peptide can be identified more easily and effectively (See U.S. Pat. No. 5,536,637).
By using newly amended SST method, the present inventors have achieved isolation of a novel membrane protein produced by vascular cell. The protein of the invention, called ESDN, is a novel type-I transmembrane protein and contains characteristic domains like neuropilin.
As it may be explained in detail subsequently, since ESDN of the present invention is expressed in coronary cells and smooth muscle cells, and in artery smooth muscle cells and tunica media of carotid artery after ballon injury, it is suggested that ESDN of the present invention is useful for treatments in restenosis after PTCA and arterial sclerosis in the field of cardiovascular system.
The cDNA sequence of the invention was identified as the human ESDN shown in SEQ ID NO. 2. Basedon the information obtained from the yeast SST method, it was isolated from cDNA libraries constructed from primary culture of human coronary arterial cells and smooth muscle cells. The human ESDN clone shown in SEQ ID NO.3 is the full-length cDNA containing complete sequence of cDNA coding the secretory protein(shown as the human ESDN protein herein).
The cDNA sequence of the invention was identified as the mouse ESDN shown in SEQ ID NO. 6. Based on the information obtained from the yeast SST method, it was isolated from mouse cDNA libraries. The mouse ESDN clone shown in SEQ ID NO.5 is the full-length cDNA containing complete sequence of cDNA coding the secretory protein (shown as the mouse ESDN protein herein).
The cDNA sequence of the invention was identified as the rat ESDN shown in SEQ ID NO. 9. Based on the information obtained from the yeast SST method, it was isolated from rat cDNA libraries. The rat ESDN clone shown in SEQ ID NO.8 is the full-length cDNA containing complete sequence of cDNA coding the secretory protein (shown as the rat ESDN protein herein).
At GeneBank and NCBI, the rat polypeptide of the invention and the nuclear acid sequence coding the polypeptide were compared by BLASTN, FASTA, and UNIGENE searches to known nuclear acid sequences registered in nucleotide sequence data bases, or by BLASTP, FLy Database, SwissProt searches to amino acid sequences of known polypeptide registered in amino acid sequence data bases. As a result, there was no sequence which corresponded to rat ESDN as the polypeptide of the present invention and nuclear acid sequence coding the polypeptide, so that it was found out that the polypeptide of the invention is a novel secretory protein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an alignment of human, mouse and rat ESDNs.
FIG. 2 shows a schematic view of the respective domain structures in ESDN, neuropilin and Coch.
FIG. 3 shows an alignment of respective LCCL modules from human, mouse, or rat ESDN, Limulus factor C, two LCCL domains of predicted rat Lgl-1, and human, mouse, or chicken Coch.
FIG. 4 shows hydrophobicity profiles of human and mouse ESDNs.
FIG. 5 shows a schematic view of the expression vectors.
FIG. 6 shows Western blot analysis of the recombinant full-length ESDN.
FIG. 7 shows images of surface expression of ESDN.
FIG. 8 shows Westernblot analysis revealing that the longest secretory signal sequence of ESDN is cleavable at the predicted site.
FIG. 9A shows Southern zooblot analysis of ESDN performed with human ESDN cDNA as a probe and 9B shows that human ESDN gene is on chromosome 3.
FIGS. 10A and B show Northern blot analysis with human cDNA and rat cDNA using RNA from human coronary arterial cells and rat organs or cultured aortic smooth muscle cells.
FIG. 11 shows an induction of ESDN mRNA in human coronary artery smooth muscle cells in response to PDGF-BB or FCS stimulation.
FIG. 12 shows up-regulation of ESDN mRNA in balloon-injured rat carotid arteries.
FIG. 13 shows immunohistochemical staining which reveals up-regulation of ESDN protein in the rat carotid artery after balloon injury.
FIG. 14 shows suppression of BrdU uptake in cells over-expressing ESDN.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides:

    (1) A substantially purified form of a polypeptide comprising one of amino-acid sequences shown in SEQ ID NO. 2, 5 or 8, or homologue thereof, fragment thereof, or a polypeptide comprising homologue of the fragment.
    (2) A polypeptide according to (1), comprising the amino-acid sequence shown in SEQ ID NO. 2, 5 or 8.
    (3) A cDNA encoding the polypeptide according to (1) or (2).
    (4) A cDNA according to (3) comprising the nucleotide sequence shown in SEQ ID NO. 3, 6 or 9, or a fragment cDNA selectively hybridized to the sequence.
    (5) A replication or expression vector carrying the cDNA according to (3) to (4).
    (6) A host cell transformed with the replication or expression vector according to (5).
    (7) A method for producing the polypeptide according to (1) or (2) which comprises culturing a host cell according to (6) under a condition effective to express the polypeptide according to (1) or (2).
    (8) A monoclonal or polyclonal antibody against the polypeptide according to (1) or (2).
    (9) A pharmaceutical composition containing the polypeptide according to (1) or (2) or the antibody according to (8), in association with pharmaceutically acceptable diluent and/or carrier.
    (10) A pharmaceutical composition which is therapeutically effective in treatment for restenosis after PTCA, and containing the polypeptide according to (1) or (2) or the antibody according to (8), in association with pharmaceutically acceptable diluent and/or carrier.
    (11) The pharmaceutical composition described in above (10), which is therapeutically effective in treatment for arterial sclerosis, and containing the polypeptide according to (1) or (2) or the antibody according to (8), in association with pharmaceutically acceptable diluent and/or carrier.
    (12) A method for measuring the quantity of the polypeptide according to (1) or (2).
    (13) A immunochemical method for measuring the quantity of the polypeptide according to (1) or (2), comprising using the antibody according to (8).
    (14) A reagent for detecting the polypeptide according to (1) or (2), which is used in the method according to (12) or (13).
    (15) A reagent for examining restenosis after PTCA by the method according to (12) or (13).
    (16) A reagent for examining arterial sclerosis, which is used in the method according to (12) or (13).
    (17) A screening method for selecting reagents having antagonistic or agonistic activity against the polypeptide, comprising using the polypeptide according to (1) or (2).


The selectively hybridizing cDNAs contain complementary sequences against above sequence. The hybridization on stringent condition is preferred.
Generally, a polypeptide comprising amino acid sequence shown in SEQ ID NO. 2, 5 or 8 in substantially purified form means a polypeptide comprising the amino acid sequence No. 2, 5 or 8 in 90% or more, e.g. 95%, 98% or 99%, in the preparation.
A homologue of polypeptide comprising amino acid sequence shown in SEQ ID NO. 2, 5 or 8 is generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the polypeptide over a region of at least 20, preferably at least 30, for instance 40, 60, 80 or 100 more contiguous amino acids. Such a polypeptide homologue is referred to as a polypeptide of the present invention.
Generally, a fragment of polypeptide comprising amino acid sequence shown in SEQ ID NO. 2, 5 or 8, or a fragment of its homologues is at least 10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 amino acids in length, and is also referred to as polypeptide of the present invention.
A cDNA capable of selectively hybridizing to the DNA comprising nucleotide sequence shown in SEQ ID NO. 3, 6 or 9 is generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the cDNA comprising nucleotide sequence shown in SEQ ID NO. 3, 6 or 9 over a region of at least 20, preferably at least 30 or more, for instance 40, 60, 80 or 100, contiguous nucleotides. Such a cDNA is referred to as cDNA of the present invention.
Fragments of the DNA comprising nucleotide sequence shown in SEQ ID NO. 3, 6 or 9 are at least 10, preferably at least 15, for example 20, 25, 30 or 40 nucleotides in length, and are also referred to as cDNA of the present invention as used herein.
A further embodiment of the present invention provides replication and expression vectors carrying cDNA of the present invention. The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said cDNA and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes, for example a ampicillin resistance gene. The vector may be used in vitro, for example, in production of RNA corresponding to the cDNA and in transfection of a host cell.
A further embodiment of the invention provides host cells transformed with the vectors for the replication and expression of the DNA of the invention, including the DNA SEQ ID NO. 3, 6 or 9 or the open reading frame thereof. The cells will be chosen to be compatible with the vector and may for example be bacterial, yeast, insect or mammalian.
A further embodiment of the present invention provides a method for producing a polypeptide which comprises culturing host cells of the present invention under conditions effective to express a polypeptide of the present invention. Preferably, in addition, such a method is carried out under conditions in which the polypeptide of the invention is expressed and then produced from the host cells.
The cDNA of the present invention may also be inserted into the vectors described above in an antisense orientation in order to prove for production of antisense RNA. Such antisense RNA may be used in controlling the level of the polypeptide of the present invention in a cell.
The present invention also provides monoclonal or polyclonal antibodies against a polypeptide of the present invention. The present invention further provides a process for production of monoclonal or polyclonal antibodies to the polypeptides of the present invention. Monoclonal antibodies may be prepared by common hybridoma technology using polypeptides of the present invention or fragments thereof as an immunogen. Polyclonal antibodies may also be prepared by common means which comprises inoculating host animals, for example a rat or a rabbit, with polypeptides of the invention and recovering immune serum.
The present invention also provides pharmaceutical compositions containing a polypeptide of the present invention, or an antibody thereof, in association with a pharmaceutically acceptable diluent and/or carrier.
As the polypeptide of the present invention, those which have deficiency in a part of their amino acid sequence (e.g., a polypeptide comprised of the only essential sequence for revealing a biological activity in an amino acid sequence shown in SEQ ID NO. 2), those which have a part of their amino acid sequence replaced by other amino acids (e.g., those replaced by an amino acid having a similar property) and those which have other amino acids added or inserted into a part of their amino acid sequence, as well as those comprising the amino acid sequence shown in SEQ ID NO. 2, 5 or 8.
As known well, there are one to six kinds of codon encoding one amino acid (for example, one kind of codon for Methionine (Met), and six kinds of codon for leucine (Leu) are known). Accordingly, the nucleotide sequence of cDNA can be changed without changing the amino acid sequence of the polypeptide.
The cDNA of the present invention includes every group of nucleotide sequences encoding polypeptides shown in SEQ ID NO. 2, 5 or 8. There is a probability that yield of a polypeptide is improved by changing a nucleotide sequence.
The cDNA specified in SEQ ID NO. 3, 6 or 9 is an embodiment of the DNA shown in SEQ ID NO. 2, 5 or 8, and indicates the sequence of natural form.
A cDNA carrying nucleotide sequence shown in SEQ ID NO. 3, 6 or 9 is prepared by the following method:
First, Yeast SST method (see U.S. Pat. No. 5,536,637) is briefly described below.
Yeast such as Saccharomyces cerevisiae should secrete invertase into the medium in order to take sucrose or raffinose as a source of energy or carbon (Invertase is an enzyme to cleave raffinose into sucrose and melibiose, sucrose into fructose and glucose.). It is known that many of known mammalian signal sequences make yeast secrete its invertase.
From this knowledge, SST method was developed as a screening method to find novel signal sequence which enables invertase secretion of yeast from mammalian cDNA library with growth of the yeast as index.
Non-secretory type invertase gene SUC2 (GENBANK Accession No. V 01311) lacking initiation codon ATG was inserted to yeast expression vector to prepare yeast SST vector pSUC2. In this expression vector, ADH promoter, ADH terminator (both were derived from AAH5 plasmid (Gammerer, Methods in Enzymol. 101, 192–201, 1983)), 2 μ ori (as a yeast replication origin), TRP1 (as a yeast selective marker), ColE1 ori (as a E. Coli replication origin) and ampicillin resistance gene (as a drug resistance marker) were inserted. Mammalian cDNA was inserted into the upstream of SUC2 gene to prepare yeast SST cDNA library. Yeast lacking secretory type invertase, was transformed with this library.
If inserted mammalian cDNA encodes a signal peptide, the yeast could survive in raffinose medium as a result of restoring secretion of invertase. By culturing yeast in colonies to prepare plasmids and determine the nucleotide sequence of the insert cDNAs, it is possible to identify novel signal peptide rapidly and easily.
Preparation of yeast SST cDNA library is as follows:

    (1) mRNA is isolated from the targeted cells, a double-strand cDNA is synthesized by using random primer with certain restriction enzyme (enzyme I) recognition site,
    (2) the double-strand cDNA is ligated to adapter containing certain restriction endonuclease (enzyme II) recognition site different from enzyme I, digested with enzyme I and fractionated in a appropriate size,
    (3) the obtained cDNA fragment is inserted into yeast expression vector on the upstream region of invertase gene of which signal peptide is deleted and the library is transformed.


Detailed description of each step is as follows:

    In step (1), mRNA is isolated from mammalian organs and cell lines after stimulating them with appropriate stimulator if necessary by known methods (as described in Molecular Cloning (Sambrook, J., Fritsch, E. F. and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) or Current Protocol in Molecular Biology (F. M. Ausubel et al, John Wiley & Sons, Inc.)) unless otherwise specified.


A suitable tissue may be heart of fetal mouse. Double-strand cDNA synthesis using random primer is performed by known methods.
Any sites may be used as restriction endonuclease recognition site I which is linked to adapter and restriction endonuclease recognition site II which is used in step (2), insofar as both sites are different each other. Preferably, XhoI is used as enzyme I and EcoRI as enzyme II.
In step (2), ends of cDNA are blunted with T4 DNA polymerase, and ligated to enzyme II adapter and digested with enzyme I. Fragment cDNA is analyzed with agarose-gel electrophoresis (AGE) and cDNA fraction ranging in size from 300 to 800 bp is selected. As mentioned above, any enzyme may be used as enzyme II insofar as it is not same with the enzyme I.
In step (3), cDNA fragment obtained in step (2) is inserted into yeast expression vector on the upstream region of invertase gene of which signal peptide is deleted. E. coli transformed with the expression vector. Many vectors are known as yeast expression plasmid vector. For example, YEp24 is also functioned in E. Coli. Preferably pSUC2 as described above is used.
Many host E. Coli strains are known as usable for transformation, preferably DH10B competent cell is used. Any known transformation method is available, preferably it is performed by electropolation method. Transformant is cultured by conventional methods to obtain cDNA library for yeast SST method.
However, not all of the cloned cDNA fragment are introduced into this cDNA library. Further, not all of the gene fragments encode unknown (novel) signal peptides. It is therefore necessary to screen a gene fragment encoding for an unknown signal peptide from the library.
Therefore, screening of fragments containing a sequence encoding an appropriate signal peptide is performed by transformation of the cDNA library into Saccharomyces cerevisiae (e.g. YT455 strain) lacking the invertase gene or strain which artificially lack the gene(it may be prepared by known methods.). Transformation of yeast is performed by known methods, e.g. lithium acetate method. Transformant is cultured in a selective medium, then transferred to a medium containing raffinose as a carbon source. Survival colonies are selected and then plasmid is collected. Survival colonies on a raffinose-medium indicates that some signal peptide of secretory protein was inserted to this clone.
With respect to isolated positive clones, the nucleotide is determined. As to a cDNA encoding unknown protein, full-length clone may be isolated by using cDNA fragment as a probe, and then the full-length nucleotide sequence is determined. The manipulation is performed by known methods.
Once the nucleotide sequences shown in SEQ ID NO. 3, 6 or 9are determined partially or preferably fully, it is possible to obtain cDNA encoding mammalian protein itself, homologue or subset. By screening cDNA library or mRNA derived from mammals by PCR method with any synthesized oligonucleotide primers or by hybridization with any fragment as a probe, it is possible to obtain cDNA encoding other mammalian homologue protein from other mammalian cDNA or genome library.
If the cDNA obtained above contains a nucleotide sequence of cDNA fragment obtained by SST (or consensus sequence thereof), it implies that the cDNA encodes signal peptide. Accordingly, it is clear that the length of the cDNA is full or almost full. (All signal sequences exist at N-termini of a protein and are encoded at 5′-termini of open reading frame of cDNA.)
By known methods, the confirmation of full-length may be carried out by Northern analysis with the said cDNA as a probe. The cDNA is assumed to have almost complete length if the length of the cDNA is almost the same with the length of the mRNA obtained in the hybridizing band.
The present invention provides both types of protein, i.e., full-length and mature. The full-length proteins are specified with the amino acid sequences translated from the nucleotides shown in SEQ ID NO.2, 5 or 8. The mature proteins are obtained by expression in suitable mammal cells or other host cells transformed by the full-length DNA shown in SEQ ID NO.3, 6 or 9. Sequences of mature proteins could be predicted from full-length amino acid sequences. (Shown in FIG. 1)
Once the nucleotide sequences shown in SEQ ID Nos. 3, 6 or 9 are determined, cDNAs of the present invention are obtained by chemical synthesis, or by hybridization making use of nucleotide fragments which are chemically synthesized as a probe. Furthermore, cDNAs of the invention are obtained in desired amount by transforming a vector that contains the DNA into a proper host, and culturing the transformant.
The polypeptides of the present invention may be prepared by:

    (1) isolating and purifying from an organism or a cultured cell,
    (2) chemically synthesizing, or
    (3) using recombinant DNA technology, preferably, by the method described in (3) in an industrial production.


Examples of expression system (host-vector system) for producing a polypeptide by using recombinant DNA technology are the expression systems of bacteria, yeast, insect cells and mammalian cells.
In the expression of the polypeptide, for example, in E. Coli, the expression vector is prepared by adding the initiation codon (ATG) to 5′ end of a cDNA encoding mature peptide, connecting the cDNA thus obtained to the downstream of a proper promoter (e.g., trp promoter, lac promoter, λ PL promoter, and T7 promoter), and then inserting it into avector (e.g., pBR322, pUC18 and pUC19 ) which functions in an E. coli strain.
Then, an E. coli strain (e.g., E. coli DH1 strain, E. coli JM109 strain and E. coli HB101 strain) which is transformed with the expression vector described above may be cultured in a appropriate medium to obtain the desired polypeptide. When a signal peptide of bacteria (e.g., signal peptide of pel B) is utilized, the desired polypeptide may be also released in periplasm. Furthermore, a fusion protein with other polypeptide may be also produced easily.
In the expression of the polypeptide, for example, in mammalian cells, for example, the expression vector is prepared by inserting the DNA encoding nucleotide shown in SEQ ID NO. 3, 6 or 9 into the downstream of a proper promoter (e.g., SV40 promoter, LTR promoter and metallothionein promoter) in a proper vector (e.g., retrovirus vector, papilloma virus vector, vaccinia virus vector and SV40 vector). A proper mammalian cell (e.g., monkey COS-1 cell, COS-7 cell, Chinese hamster CHO cell, mouse L cell etc.) is transformed with the expression vector thus obtained, and then the transformant is cultured in a proper medium, the secretory protein of the present invention can be secreted into the culture medium as the aimed polypeptide. Then, by linking to cDNA fragment coding other polypeptides, for example, common region (Fc portion) of antibody, fusion proteins can be produced. Polypeptides obtained by the method above can be isolated and purified by conventional biochemical methods.
INDUSTRIAL APPLICABILITY
The present inventors have confirmed that the polypeptide is more highly expressed in neointima than in tunica media of vascular smooth muscle. So it is suggested that the polypeptide of the present invention is useful for therapeutic treatment in restenosis after PTCA and arterial sclerosis in the field of cardiovascular system.
Further, since the polypeptide of the present invention showed the suppressive activity of cell proliferation in experiments and the structural similarity to that of VEGF, it is suggested that the polypeptide may possess the following activities.
(1) Cytokine Activity and Cell Proliferation/Differentiation Activity
Since the polypeptide of the present invention suppresses cell proliferation in the over-expression system, it may exhibit cytokine activity, cell proliferation (either inducing or inhibiting) activity or cell differentiation (either inducing or inhibiting) activity or may induce or suppress production of other cytokines in certain cell populations.
(2) Immune Stimulating/Suppressing Activity
The polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity. The polypeptide of the invention may be useful in treatment of various immune deficiencies and disorders, for example, in regulating (stimulating or suppressing) growth and proliferation of T and/or B lymphocytes, as well as acting on the cytotoxicity of NK cells and other cell populations. Especially, it is suggested that the polypeptide of the present invention may exhibit suppressing activities against lymphogenous tumor metastasis induced by tumor vascularization.
(3) Suppressing Activity against Ischemic Vascularization
The polypeptide of the present invention may suppress diabetic retinopathy. It is known that VEGF exhibits activity of vascularization induced by ischemia. The vascularization in diabetic retinopathy is also ischemic vascularization after formation of avasocular regions of retina. Structural similarity to neurophilin suggest that the protein of the present invention may suppress growth of blood vessels as a novel VEGF receptor, therefore, may exhibit a therapeutic effect on diabetic retinopathy.
Administration or use of the protein or of cDNA coding the protein (for example, gene therapy (including regenerative therapy) or vectors suitable for cDNA transfection) may provide the effect or biological activities described about the protein of the invention.
Quantitative analysis of the polypeptide of the present invention in vivo can be performed using polyclonal or monoclonal antibodies against the polypeptide of the present invention. It can be used in studies on relationship between this polypeptide and disease, or diagnosis of disease, etc. The polyclonal and the monoclonal antibodies can be prepared using this polypeptide or its fragment as an antigen by conventional methods.
Identification, purification or molecular cloning of known or unknown proteins (ligands) which are connected with the polypeptide of the present invention can be performed using the polypeptide of the present invention by, for example, preparation of the affinity-column.
Identification of molecules which interact with the polypeptide, molecular cloning of the gene may be conducted, for example, by western blot, using the polypeptide, or by yeast two-hybrid method, using the cDNA (desirably cDNA coding the polypeptide).
Screening method, which can identify agonists or antagonists against the polypeptide receptor and inhibitors against interaction between receptors and signal transduction molecules may be performed by using the polypeptide.
For example, the screening method could be performed by the following steps:

    
    
        a) The polypeptide of the invention, compound to be screened and reaction mixture including cells are mixed (the reaction mixture includes peptides except markers which are transferred into cells as the cell grows and except the polypeptide for efficient observation of the function of the polypeptide.) under condition which the cells are normally stimulated by the polypeptide, then,
        b) it is determined whether the compound is a useful agonist or antagonist by measuring the cell growth.
    
    


The cDNA of the invention may be useful not only as an important and essential template in production of the polypeptide of the present invention which is expected to have a considerable utility, but also for diagnoses and treatments of hereditary diseases(treatments of gene deficiency or treatments which anti-sense DNA(RNA) s intercept expression of polypeptides, etc). In addition, genomic DNAs may be isolated by using cDNA of the invention as a probe.
ADMINISTRATION AND DOSING
To practice with diseases mentioned above, administration of the polypeptide of the invention or its antibodies can be carried out in general or local, generally peroral or parenteral ways. Oral, intravenous and intracerebroventricular administration are preferred.
The dosage to be administered depends upon age, body weight, symptom, desired therapeutic effect, route of administration, and duration of the treatment etc. In human adults, one dose per person is generally between 100 μg and 100 mg by oral administration up to several times per day, and between 10 μg and 100 mg by parenteral administration up to several times per day.
As mentioned above, the doses to be used depend upon various conditions. Therefore, there are cases in which doses lower than or greater than the ranges specified above may be used.
The polypeptide or the compounds of the present invention, may be administered as solid compositions, liquid compositions or other compositions for oral administration, as injections, liniments or suppositories etc. for parenteral administration.
Examples of solid compositions for oral administration include compressed tablets, pills, capsules, dispersible powders and granules. Examples of capsules include soft capsules and hard ones.
In such compositions, one or more of the active compound(s) is or are admixed with at least one inert diluent (such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium metasilicate aluminate, etc.). The compositions may also comprise, as is normal practice, additional substances other than inert diluents: e.g. lubricating agents (such as magnesium stearate etc.), disintegrating agents (such as cellulose calcium glycolate, etc.), stabilizing agents (such as human serum albumin, lactose etc.), and assisting agents for dissolving (such as arginine, asparaginic acid etc.).
The tablets or pills may, if desired, be coated with a film of gastric or enteric materials (such as sugar, gelatin, hydroxypropyl cellulose or hydroxypropylmethyl cellulose phthalate, etc.), or be coated with more than two films. And then, coating may include containment within capsules of absorbable materials such as gelatin.
Liquid compositions for oral administration may contain pharmaceutically-acceptable emulsions, solutions, suspensions, syrups and elixirs, and also may contain inert diluent(s) commonly used (purified water, ethanol etc.). Besides inert diluents, such compositions may also comprise adjuvants (such as wetting agents, suspending agents, etc.), sweetening agents, flavoring agents, perfuming agents, and preserving agents.
Other compositions for oral administration include spray compositions which may be prepared by known methods and which comprise one or more of the active compound(s). Spray compositions may comprise additional substances other than inert diluents: e.g. stabilizing agents (sodium sulfite etc.), isotonic buffer (sodium chloride, sodium citrate, citric acid, etc.). For preparation of such spray compositions, for example, the method described in the U.S. Pat. Nos. 2,868,691 or 3,095,355 (herein incorporated in their entireties by reference) may be used.
Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. In such compositions, one or more active compound(s) is or are admixed with at least one inert aqueous diluent(s) (distilled water for injection, physiological salt solution, etc.) or inert non-aqueous diluents(s) (propylene glycol, polyethylene glycol, olive oil, ethanol, POLYSOLBATE 80 TM, etc.).
Injections may comprise additional compound other than inert diluents: e.g. preserving agents, wetting agents, emulsifying agents, dispersing agents, stabilizing agent (such as human serum albumin, lactose, etc.), and assisting agents such as assisting agents for dissolving (arginine, asparaginic acid, etc.).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The invention are illustrated by the following examples, but not limit the invention.
EXAMPLE 1 
Primary culture of normal human coronary artery endothelial cells (hCAEC) and smooth muscle cells (hCASMC) purchased from Clonetech and co-culture (an equal number of endothelial and smooth muscle cells were mixed and maintained in EGM-2-MV (trade name, a product of Bio Whittaker) for two days) were used as the sources for the construction of the cDNA libraries, and the yeast signal sequence trap (See U.S. Pat. No. 5,536,637) screening was carried out. ESDN containing CUB domain was isolated from cDNA library derived from the co-culture.
EXAMPLE 2 
The cloning of 5′- and 3′-end regions of cDNA were performed by 5′- and 3′-RACE (Rapid Amplication cDNA End) methods using the Marathon cDNA Amplification Kit (trade name, a product of CLONTECH) to isolate the full-length cDNA. Mouse and rat counterparts were obtained with RT-PCR using two kinds of primer pairs based on human sequence data.








Mouse primers:









(SEQ ID NO.10)









5′-CTG-CTC-CAA-CTC-CTC-CTC-CTT-C-3′



 








(SEQ ID NO.11)









5′-CTG-CTT-CAT-TCC-TTT-CCA-CCA-ACC-TG-3′



 


Rat primers:









(SEQ ID NO.12)









5′-TGT-GCT-GGT-CAT-GGT-CCT-CAC-TAC-TCT-C-3′



 








(SEQ ID NO.13)









5′-TGT-GCT-TTA-AAA-CGA-TGC-TTT-G-3′.







As a result, it was revealed that alignment of human, mouse, and rat amino-acid sequences showed high homology between them. However, 5′-RACE method could not reach 5′-terminal sequence containing the definitive start codon, ATG, in any species, resulting from high GC-content in the 5′-region of ESDN. Therefore, the inventors conducted mouse genomic library screening with Lambda FIXII library (tradename, a product of Stratagene Inc.), to obtain two positive clones containing translation initiation site (Met). Human and rat counterparts were obtained with RT-PCR using a sense primer of 5′-GCA-CTA-TGC-GGG-CGG-ATT-GC-3′ (SEQ ID NO.14) containing the first methionine of mouse ESDN and an anti-sense primer of 5′-GGA-TGT-AAG-GGT-TCC-ACT-CTC-AGG-3′ (SEQ ID NO.15) situated in the downstream exon (which is not included in genomic clone). Alignment of three products are shown in FIG. 1 with amino-acid homology between human and rodents and between mouse and rat was 84–5% and 92%, respectively.
A motif search revealed that ESDN is a type-I transmembrane protein composed of a CUB domain and a factor FV/VIII-like domain and resembles neuropilin (with two CUB domains, two factor FV/VIII domain and a MAM domain) in the structure. A sequence homology search of the other area revealed that a region has significant homology with Limulus factor C and Coch, which is one of causal genes for deafness disorder, and has the conservative domain with four cysteines. In Coch domain, all four kinds of mutations identified to date in DFNA9 deafness disorder converge. Therefore, this domain has been detected in the protein of Limulus which was disassociated from genealogical tree in the evolution, so that it is suggested that the structure of this domain could be a novel domain structure. (Shown in FIGS. 2 and 3)
EXAMPLE 3 
Human ESDN constructs were cloned into pEF6V5-His (tradename, a product of Invitrogen Corp.), swapping the original V5 epitope with the FLAG tag. These expression vectors were transfected into 293T and COS7 cells with CellPhect (trade name, a product of Amersham BioSciences) and Lipofectamine (tradename, a product of Life Technologies, Inc.), respectively. Further, after preparing cell lysate, the target protein was detected by western analysis using various antibodies.
Rabbit anti-CUB and anti-FV/VIII polyclonal antibodies were raised against KLH-conjugated polypeptides of GERIRIKFGDGDIEDSD (SEQ ID NO.16) and QDKIFQGNKDYHKDVRNN (SEQ ID NO.17), respectively, and affinity-purified against each polypeptide by Sawady Technology. Western analysis was carried out following the ECL (trade name, a product of Amersham BioSciences) or Renaissance (trade name, a product of NEN Life Science) western blot protocols. The other antibody used are anti-FLAG M2 monoclonal antibody (trade name, a product of Sigma-Aldrich Co.). As a result, 127, 106, 93 Kda protein bands, which have not been detected in the lysate from cells transfected with expression vector only were detected. (Shown in FIG. 6)
EXAMPLE 4 
Hydrophobicity profile of ESDN amino acid sequence revealed that the predicted signal sequence of ESDN is very long and a typical. COS7 cells transfected with human full-length cDNA were fixed in 4% paraformaldehyde solution and reacted with the primary antibody for 30 minutes at room temperature, followed by reaction with the second antibody for 30 minutes. Then, they were observed and analyzed under a Bio-Rad confocal laser scanning microscope after double-staining with texas red anti-mouse IgG (tradename, a product of Vector Libraries, Ltd.) and FITC-anti-rabbit IgG (Jackson Laboratories). As a result, it was confirmed that this protein is expressed on the cell-surface. (Shown in FIG. 7)
Then, the inventors tried to confirm the location of a signal sequence cleavage site.
Mouse ESDN constructs was re-cloned into an expression vector pCAGGS with mESDN-Ex (Edns), which was constructed with C-termini of the extracellular portion of mouse ESDN linked with 6×His tag. Further, another construct, mESDN-Ex (CD5), whose signal sequence was replaced by that of human CD5; MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO.18) was prepared. 293T cells were transiently transfected with these constructs, and culture media were analyzed by Western blot using the His-probe H-15 polyclonal antibody (trade name, a product of Santa Cruz Biotechnology Inc.) to detect the target proteins. As a result, it was confirmed that both constructs, mESDN-Ex (Edns) and mESDN-Ex (CD5) yielded the protein product of exactly the same size. (Shown in FIG. 5) As the size of human CD5 is shorter than that of mouse ESDN 39 amino-acid residues, this result supports that the two proteins belong to the same location of signal sequence cleavage sites. (Shown in FIG. 8)
EXAMPLE 5 
A Southern zooblot analysis was performed with a human ESDN probe labeled with [32P]dCTP in all mammals (mouse, rat, rabbit, cow, and human), Xenopus, fly and yeast (at 37° C., washing with 1×SSC). In addition to strong bands observed in all mammals, weak bands were detected in Xenopus. No bands were observed in fly and yeast. (Shown in FIG. 9A)
EXAMPLE 6 
Search for STS (sequence-tagged site) in human ESDN sequence revealed that it contains two independent STS clones, stSG29921 and sts-D29024, which are mapped at the neibourhood of D3S1603-D3S1271 and D3S1552-D3S1603 in radiation hybrid map (sites expected to correspond to chromosome 3q11.2 in cytegenetics map), respectively. To confirm this database result, mouse cell lines A9 (Neo3) and A9 (Neo12) (JCRB Cell Bank) were used for genomic southern hybridization. As human ESDN probe identified a cross-hybridized mouse band in both lanes, mouse ESDN was confirmed in both A9 (Neo3) and A9 (Neo12), whereas human bands were present in the lane of A9(Neo3) only. Thus, these result revealed that human ESDN gene is located within the range which corresponds to cytogenetic region of chromosome 3p11.2(Shown in FIG. 9B)
EXAMPLE 7 
The total RNA prepared from hCAEC, hCASMC and the mixed co-culture using TRIzol (trade name, a product of Life Technologies) were used in Northern analysis of cultured human cells. In Northern analysis of rat organizations or cells, the total RNA prepared from the whole blood and other organizations or cultured cells using TRIzol LS and TRIzol respectively were used. Then, Poly(A)+RNA was purified with OligotexTM-dT30 Super (tradename, a product of Roche Molecular Biochemicals) and Northern analysis (at 65° C., washing with 0.2×SSC) was performed by using human and rat ESDN, their GAPDH probes labeled with [32p]dCTP. ESDN was highly expressed in hCASMC (6.4, 3 kb), whereas the expression in hCAEC was weaker than in hCASMC. Further, no change of ESDN mRNA in the mixed co-culture was observed. (Shown in FIG. 10A)
In northern blot analysis for various rat cells or tissues, no ESDN was detected in whole blood cells, and the expression in liver was very faint. (Shown in FIG. 10B)
EXAMPLE 8 
hCASMC was cultured in DMEM/2mM glutanine depleted of serum for 48 hours, then was stimulated with the medium containing either of PDGF-BB, AT-II or FCS (tradenames, products of Sigma-Alsrich Co. and Life Technologies, Inc.) at the specified concentrations. Total RNA was extracted with TRIzol (trade name, a product of Life Technologies, Inc.), cDNA was synthesized using SuperScript Preamplification System for First Strand cDNA Synthesis Kit (Life Technologies, Inc.). Further, the mRNAs were measured by real-time quantitative RT-PCR using PE Applied Biosystems Prism Model 7700 Sequence Detection System. The nucleotide sequences of forward and reverse primers are as follows.








(SEQ ID NO.19)











ESDN forward: 5′-CCC-AGC-AAG-GTG-ATG-GAT-G-3′




 








(SEQ ID NO.20)











ESDN reverse: 5′-CAA-GAA-TCA-GAA-TCT-TCA-




 



ATG-TCA-AAG-3′



 



ESDN probe:









(SEQ ID NO.21)











5′- (6-FAM) -CCT-GAG-AGT-GGA-ACC-CTT-ACA-




 



TCC-ATA-AAC- (TAMRA)-3′






These were based on the human sequence, but were confirmed to be applicable in quantitative measurements of rodent transcripts as well. The nucleotide sequences of human GAPDH are as follows.








Human GAPDH forward:









(SEQ ID NO.22)









5′-GAA-GGT-GAA-GGT-CGG-AGT-C-3′



 


Human GAPDH reverse:








(SEQ ID NO.23)









5′-GAA-GAT-GGT-GAT-GGG-ATT-TC-3′



 


Human GAPDH probe:








(SEQ ID NO.24)









5′- (VIC) -CAA-GCT-TCC-CGT-TCT-CAG-CC- (TAMRA) -3′







TaqMan Rodent GAPDH Control Reagents (PE biosystems) were used for measurements of rat GAPDH. The mRNA level of ESDN and GAPDH means the number of copies, so standard curves could be prepared from known amount of plasmids with ESDN or GAPDH amplicon subcloned into pBlueScript SK(−) (Stratagene), and the ESDN mRNA level normalized to that of GAPDH was used for further analyses.
The results of quantitative RT-PCR showed that ESDN was up-regulated with PDGF-BB dose-dependently, but not with AT-II stimulation. FCS also up-regulated ESDN expression dose-dependently, but much less than PDGF-BB. (Shown in FIG. 11)
EXAMPLE 9 
The carotid arteries were harvested at 0, 5 or 14 days after balloon injury (n=5 on day 0, 5, n=4, on day 14). Total RNA was extracted with TRIzol (trade name, a product of Life Technologies, Inc.), cDNA was synthesized using SuperScript Preamplification System for First Strand cDNA Synthesis Kit (Life Technologies, Inc.). Further, the mRNAs were measured by real-time quantitative RT-PCR using PE Applied Biosystems Prism Model 7700 Sequence Detection System. Quantitative RT-PCR analysis revealed that rat ESDN mRNA expression showed a tendency of up-regulation at day 5, and an significant increase at day 14 by 30%. Then, to elucidate the expression of ESDN, an immunohistochemical study was performed. (Shown in FIG. 12)
Under anesthetization, treated or untreated rats were perfused with physiological saline cooled at 4° C. in advance. Then, the local perfused tissues were fixed with cold 4% paraformaldehyde, the harvested carotid arteries were embeded in Tissue-Tek O.C.T. Compound (trade name, a product of Sakura Finetechnical Co., Ltd.) on dry-ice/ethanol bath. The embeded carotid arteries were sliced in the thickness of 4 μm and were immunohistochemistrically analyzed by the method using avidin-biotin-alkaline phosphatase complex (Vector Laboratories). The substrate, VectorRed (tradename, a product of Vector Laboratories, Inc.), which reacted with alkaline phosphatase stained the slices, to contrast with methyl-green staining. As primary antibodies, rabbit anti-peptide polyclonal antibodies were used at the concentration of 5–10 μg/ml, and normal rabbit IgG (DAKO) were used at the same concentration for negative controls. The result revealed that tunica media of aorta and common carotid arterie in vascular smooth muscle were stained, and that parts (arrow) of central nervous system, brain and spinal cord, etc and peripheral part, vagus nerves (arrowheads), etc were rather conspicuously stained. (Shown in FIG. 13) A, B, E and F were stained by anti-CUB antibody, and C, D, G and H were stained by an equal concentration of rabbit IgG as a primary antibody (control). M and N shown in E and F indicate tunica media and neointima, respectively.
EXAMPLE 10 
ESDN constructs of human full-length ESDN (hESDN-FL) and the deletion mutants (hESDN-ΔEC, hESDN-ΔCy) cloned into one expression vector were prepared with QIAfilter Plasmid Midi Kit (QIAGEN). Then, they were purified twice by phenol/CIAA extraction and once by CIAA extraction. 293T cells were transfected by these expression vectors using CellPhect (Amersham Life Science). After 12 hours of incubation with transfection solution, the medium was replaced by fresh DMEM+10% FCS and incubated for 2 hours. Cells were collected using trypsin and replated in 96-well plates in duplicate. For one plate, 2-hour-BrdU pulse was applied after 24 hours of culturing, and the incorporated BrdU was measured by Cell Proliferation ELISA, BrdU (colorimetric) (trade name, a product of Roche Diagnostics). The other plate was used to estimate the number of cells using Premix WST-1 assay kit (trade name, a product of TaKaRa Shuzo Co.) 2 hours after replating. (Shown in FIG. 14) The results revealed that uptaking of BrdU was significantly suppressed in cells with ESDN expressed therein, while such suppressing effects is weaker in cells without extracellular domains, and no such effects were observed in cells without intracellular domains.

## Claims

1. A purified polypeptide comprising the amino-acid sequence of SEQ ID NO: 5 or 8, or a homologue that is at least 90% homologous to one of said sequences, said homologue having ESDN (Endothelial and Smooth muscle cell-Derived Neuropilin-like molecule) function.

2. The purified polypeptide according to claim 1, comprising the amino-acid sequence of SEQ ID NO: 5 or 8.

3. A purified polypeptide consisting of the amino-acid sequences of residues 64–769 of SEQ ID NO: 5 or 8, or a homologue that is at least 90% homologous to one of said sequences, said homologue having ESDN function.

4. The purified polypeptide according to claim 3, consisting of one of the amino-acid sequences of residues 64 to 769 shown in SEQ ID NO:5 or 8.