WO2001029085A2

WO2001029085A2 - Cdt6 can inhibit angiogenesis and tumor growth, and can induce or enhance the formation of structures containing collagen v

Info

Publication number: WO2001029085A2
Application number: PCT/NL2000/000674
Authority: WO
Inventors: Ronald Peek
Original assignee: Royal Netherlands Academy Of Arts And Sciences
Priority date: 1999-10-21
Filing date: 2000-10-19
Publication date: 2001-04-26
Also published as: AU1310201A; WO2001029085A3

Abstract

In spite of the classification of CDT6 as a nonangiopoietin member of the fibrinogen domain containing superfamily, we have demonstrated that CDT6 has a potent effect on angiogenesis and tumor growth. CDT6 is capable of inhibiting at least in part angiogenesis. The invention provides the use of this novel angiogenesis antagonist in the treatment of diseases, preferably cancers that form solid tumors.

Description

Title- CDT6 can inhibit angiogenesis and tumor growth, and can induce or enhance the formation of structures containing collagen V.

The invention relates to the field of medicine.

The human cornea consists of three clearly separated cellular components: the epithelium, the stroma, and the endothelium. The epithelium is a self -renewing tissue consisting of five to seven cell layers that are constantly replaced by proliferation and differentiation of the corneal limbal stem cells. The stroma is formed by fibroblast-li e cells, the corneal keratocytes, which are embedded between ordered layers of collagen fibrils surrounded by proteoglycans . Although the keratocytes produce this stromal layer, which constitutes approximately 90% of the corneal thickness, little is known about keratocyte specific gene expression and turn-over. The inner cellular layer, the corneal endothelium, consists of a single layer of hexagonal cells and is essentially amitotic. The human cornea is densely innervated with nerve fibers of sensory origin from the trigeminal nerve. The most striking properties of the cornea - avascularity, transparency, and light refraction - are shared with the eye lens only.

In spite of these striking properties and unique features, only few cornea-specifIC expressed genes have been identified. One such cornea-specific expressed gene is CDT6. CDT6 was isolated from a human cDNA library of total cornea and was shown to be specifically expressed in the stroma of the cornea. The nucleotide sequence of CDT6 snowed distant homology with the recently described family of angiopoietms (Peek et al . , 1998; Valenzuela et al . , 1999) . All angiopoietms known thus far comprise a fibrinogen domain and as such the angiopoietms are classified to belong to the fibrinogen domain- containing superfamily. The fibrinogen domain-containing superfamily comprises a large variety of molecules comprising various functions of which only the angiopoietms posses a function angiogenesis. Analysis of members of the flbr ogen-domain containing superfamily teaches that CDT6 is a nonangiopoietin member of the fibrinogen domam-contammg superfamily (Peek et al . , 1998; Valenzuela et al . , 1999).

In the present invention, however we have observed that expression of CDT6 in tumor cells, at least m part prevents the growth of these tumor cells in mice. Analysis of the mice suggested that vascularisation of the tumor was at least m part impaired. These results suggest that m spite of the classification of CDT6 as a non-angiopoietm member of the flbrmogen-domain superfamily, CTD6 does have a regulating role in angiogenesis. In the present invention we demonstrate that at least part of the angiogenesis inhibiting effect of CDT6 lies in the capacity of CDT6 to induce and/or enhance the formation of a structure that is at least m part impenetrable for blood vessels.

In normal tissue, new blood vessels are formed during tissue growth and repair, during the normal female reproductive cycle, and during the development of the fetus during pregnancy. In cancerous tissue, tumors cannot grow, and m most cases do not spread (metastasize) without the development of new blood vessels. Blood vessels supply tissues with oxygen and nutrients necessary for survival and growth.

For the migration and proliferation of vascular endothelial cells the process of angiogenesis it is essential that the basal membrane surrounding existing blooa vessels is degraded by endothelial cell derived proteases. The next step is the induction by endothelial cell bound tissue factor of fibrin deposition. On this temporary matrix the endothelial cells are capable of forming new vessels to support blood flow in the direction of tumor tissue.

The effects of CDT6 expression in solid tumors appeared to be dramatic. The number of blood vessel sprouts was reduced while the basal membrane of tumor vessels was thickened considerably. Furthermore, tumor growth was greatly reduced while the tumor cells displayed a tortuous, aberrant morphology as the result of CDT6 expression. These effects were apparent in vivo but absent m vitro and therefore strongly suggest that CDTβ produced by the transfected tumor cells does not act on these cells directly but exerts its potent effect on the tumor invading blood vessels.

W th the recognition of a function of the CDT6 molecule at least m part inhibiting angiogenesis, CDT6 or functional equivalents thereof can be administered to individuals at sites of potential angiogenesis whereupon such angiogenesis is at least part prevented. The invention therefore provides CDT6 or a functional equivalent thereof for use as a pharmaceutical . A functional equivalent of CDT6 is a functional part, derivative and/or analogue of CDT6 comprising the same activity in kind but not necessarily in amount . Several other flbrmogenlike domain containing proteins, such as CDT6, use the flbrmogenlike domain to bind to their cognate receptor. The flbrmogenlike domain of CDT6 is therefore likely to be the binding site to the yet to be identified receptor. The leader sequence m the amino- terminal end of the CDT6 protein is probably not essential for CDT6 function as these leader sequences are generally removed during the maturation and secretion of leader sequence containing proteins .

Functional derivatives of CDT6 can be derived from CDT6 through one or more neutral ammo-acid substitution. A neutral ammo-acid substitution is typically a substitution with an ammo-acid comprising a similar charge as the one it replaces. However, non-essential regions usually also non- neutral ammo-acid substitutions are tolerated without affecting the activity m kind though such substitutions can effect the activity m amount. A charge may be a positive charge, a negative charge or a neutral charge. A functional analogue of CDT6 is a molecule comprising CDT6 , or a functional part and/or derivative thereof.

Administration of CDT6 or a functional equivalent thereof, to an individual will lead to a reduction m the potential for novel angiogenesis m said individual at least at the site of administration.

The invention therefor also provides the use of CDT6 or a functional equivalent thereof m the preparation of a medicament for the inhibition of angiogenesis.

In one embodiment of the invention CDT6 or a functional equivalent thereof is used m the preparation of a medicament for the treatment of solid tumors. Solid tumors typically require active angiogenesis for survival and growth. Administration of CDT6 or a functional equivalent thereof preferably at the site of the tumor and/or metastasis thereof can at least m part prevent angiogenesis and thereby at least m part inhibit the growth of said tumor and/or metastasis. Growth of a tumor typically is a balance between cell division and cell death per time unit said tumor. An effect on tumor growth therefor is an effect on the balance of cell division and cell death per time unit said tumor. Preferably, CDT6 has the sequence as depicted figure 1 or a sequence at least 80, preferably 90, more preferably 95, more preferably 99% homologous therewith.

In applications where angiogenesis needs to be at least m part prevented for a prolonged period of time one can deliver a nucleic acid comprising a gene encoding CDT6 or a functional equivalent thereof to cells at or near a site where angiogenesis should at least m part be prevented. Subsequent expression of CDT6 or functional equivalent thereof such cells provides a more or less continuous source of CDT6 thus allowing the effects of CDT6 to extend over a prolonged period of time CDT6 is capable of inducing and/or enhancing the expression of specific genes coding for extracellular matrix proteins. These include collagen I, collagen V and proteoglycans The high level expression of these matrix proteins in addition to the low turn over of such proteins leads to fibrosis of the tissue and aberrant blood vessels These effects turn have a dramatic effect on tumor growth m vi vo . The invention therefore provides a nucleic acid comprising a gene encoding CDT6 or a functional equivalent thereof for use as a pharmaceutical . A gene encoding CTD6 may comprise a sequence coding for CDT6 or a functional equivalent thereof, of which the expression is governed by the CDT6 transcription machinery. However, alternative transcription and/or translation regulating nucleic acid may also be used. Preferably, the gene and/or the nucleic acid encoding CDT6 has the sequence as depicted in figure 1 or a sequence at least 80, preferably 90, more preferably 95, more preferably 99% homologous therewith. In one embodiment the invention provides the use of a nucleic acid comprising a gene encoding CDT6 or a functional equivalent thereof m the preparation of a medicament for the inhibition of angiogenesis, preferably for the treatment of solid tumors and/or metastasis thereof.

Expression of CDT6 tumor cells had a profound effect on the phenotype of the tumor m v vo. Tumor cells were embedded structures that were not present m control tumor not expressing CDT6. The material of the structures was at least m part impenetrable for blood vessels. Through the structures the tumor was at least part physically separated from tne blood supply. Thus the structures were at least part responsible for the angiogenesis inhibiting effect of CDT6. The invention therefor further provides a method for inducing and/or enhancing the formation of a structure, said structure at least m part impenetrable for blood vessels comprising contacting a cell with CDT6 or a functional equivalent thereof At least part of the structures contained collagen V Collagen V is normally found only m cartilage and the cornea. According to the invention collagen V has the property of organizing the structure such that it becomes at least in part impenetrable for blood vessels. Thus preferably, said structure comprises collagen V.

Preferably, said structure further comprises collagen I Collagen I can be expressed by many different cell types.

Structures of the invention comprising collagen I, collagen V and preferably proteoglycans comprise properties of cartilage that they are a deposit of collagenous matrix with fibrils resembling those of cornea and cartilage. Considering that CDT6 is expressed m the cornea and considering that the structures found m the tumor cells comprise proteins that are normally found m the cornea, the function of CDT6 resembles that of a morphogen. A morphogen is capable of inducing a phenotypic change a cell such that it resembles or expresses functionality of a cell of a different lineage. In the present invention CDT6 has the capacity to effectuate a change m the cell such that said cell expressed functionality of a cornea stromal cell. Preferably said cell contacted with CDT6 is a flbroblastoid cell. CDT6 can induce and/or enhance, among others, a high level expression of collagen V. The high level expression of collagen V has a direct influence on blood vessel endothelial cells which is likely to contribute to the relative impenetrability of structures of the invention by blood vessels. Both the adhesion as well as proliferation of blood vessel endothelial cells is inhibited by collagen V (Hashimoto et al , 1991; Fukuda et al , 1988; Ziats et al , 1993) .

In another aspect the invention provides a structure obtainable by a method according to the invention. At least part of the structures were transparent and had a curved shape such that they can be used in at least partial cornea transplants. Thus the invention also provides the use of a structure of the invention for the preparation of a medicament .

Providing cells m the body with CDT6 and/or a nucleic acid encoding CDT6 has many applications. In rheumatoid arthritis cartilage and eventually bone is lost from joints. Administration of CDT6 or a nucleic acid encoding CDT6 (or functional equivalent of CDT6) in for instance the synovium of such affected joints induces the production of cartilage like structure. Cartilage has a similar composition of extra cellular matrix as the Cornea. Production of said cartilagelike structure at least part reduces the rheumatoid symptoms in the treated joint.

Corneal transplantation: during cornea transplantation a "button" which consists of the central part of the donor cornea is placed m the recipients eye of which the cornea has been removed. The donor cornea is than fixed in the recipient's eye by several sutures. The junction between donor and recipient tissue m most cases remains relatively fragile and is easily ruptured by mechanical forces. The administration of CDT6 (or functional equivalent thereof) directly after transplantation induces the formation and deposition of extracellular matrix proteins between the donor and recipient corneal tissue leading to a more stable junction.

Organculture . Cartilage and bone grafts are being used m transplantation settings. CDT6 induced or enhanced formation of cartilage like structures can at least part be used for such purposes. An advantage is that these structures can be made using autologous cells thereby circumventing potential rejection problems now encountered transplantation of natural grafts. In vitro growth of cartilage like structures is currently being investigated. CDT6 administration to such cultures can aid the generation of such grafts.

Other applications and diseases m which the administration of CTD6 or a functional equivalent thereof, and/or the administration of a nucleic acid encoding CTD6 or a functional equivalent thereof to an individual is at least part beneficial to the condition of the patient is given table 1. To efficiently deliver nucleic acid to cells, especially m vivo, said nucleic acid may be combined with a gene delivery vehicle. The invention therefor also provides a gene delivery vehicle comprising a nucleic acid encoding CDT6 or a functional equivalent thereof. Preferably, said gene delivery vehicle is a recombmant virus. Typically, but not necessarily, such a recombmant virus comprises a recombmant nucleic encoding CDT6 or a functional equivalent thereof packaged into a protective shell, the so called envelop and/or capsid, comprising virus encoded proteins. Preferably said gene delivery vehicle is of adenoviral, retroviral or adeno-associated viral origin.

The invention further provides a method for inhibiting angiogenesis comprising providing CDT6 or a functional equivalent thereof at the site of possible angiogenesis m a host. A site of possible angiogenesis is typically a site the body of an individual with suitable conditions for angiogenesis. One such condition may be the production of angiogenesis promoting factors by cells m the vicinity of the site. An example of a site of possible angiogenesis is a solid tumor. The invention therefor also provides a method for inhibiting the growth of a solid tumor m a host comprising providing CDT6 or a functional equivalent thereof at the site of sa d tumor m said host . Preferably said CDT6 or its functional equivalent is provided through a gene delivery vehicle. Preferably, said CDT6 or its functional equivalent is provided through expression of a gene encoding it a cell. In one embodiment said cell is a target cell of

The natural promoter driving expression CDT6 the cornea stroma is not very well suited for obtaining expression of CDT6 m many different cell types. Particularly m cases where relatively high expression of CDT6 is required, it is desired to use a heterologous promoter sequence to drive the expression of CDT6. The invention therefore further provides a nucleic acid encoding CDT6 or a functional equivalent thereof under the control of a heterologous promoter. One example of a strong promoter is a viral promoter or a tissue specific promoter. Such a nucleic acid may be used for the production of CDT6 to prepare a suitable source of CDT6 fur purification purposes. A heterologous promoter may be a bacterial promoter and/or a eukaryotic promoter. Typically, the type of promoter chosen will depend on the application. For instance for purification purposes a promoter will be chosen that enables high expression of CDT6 m cells and/or bacteria m vi tro, whereas for in vivo application the promoter chosen will typically enable a high and preferably also prolonged expression of CDT6 m the cell to which the nucleic acid was delivered. Preferably a nucleic acid encoding CDT6 or a functional equivalent thereof under the control of a heterologous promoter has the sequence as depicted m figure 1 or a CDT6 specific part thereof, or a sequence at least 80, preferably 90, more preferably 95, more preferably 99% homologous therewith. Also provided is a nucleic acid encoding CDT6 or a functional equivalent thereof under the control of a heterologous promoter which has the sequence as depicted m figure 1 or a CDT6 specific part thereof or the complementary strand thereof or a sequence which hybridizes to a sequence as depicted m figure 1 or its complementary strand or a specific part of either strand under stringent conditions. The invention further provides a cell comprising a nucleic acid according to the invention Preferably, said cell expresses functional CDT6 or a functional equivalent thereof . The invention further provides a method for producing recombmant CDT6 or a functional equivalent thereof, comprising culturmg a cell comprising a nucleic acid according to the invention, under suitable conditions and harvesting CDT6 or its functional equivalent from said culture. Said CDT6 protein may of course be modified to allow easy purification and/or detection of the protein. To this end particularly the state of the art technology for fusion protein synthesis is referred to. A fusion protein comprising at least part of CDT6 is therefore also part of the invention . Production of CDT6 can also be accomplished m the milk of transgenic animals. Such a transgenic animal is also part of the invention.

For medicinal purposes the CDT6 protein or functional equivalent or a nucleic acid delivery vehicle or a nucleic acid encoding CDT6 or a functional equivalent thereof or a combination, may be formulated in a pharmaceutically acceptable carrier. Such a composition is therefore also part of the invention. For instance, formulation a solution compatible with the eye, or a slow release formulation such as a slow-release polymer. In case of eye administration such a slow release polymer could be m the form of a contact lens .

Now that the present invention establisned the potent activity of a binding partner of CDT6 can be cloned, such as a receptor.

Examples

Isolation and structural analysis

To isolate tissue-specific gene products from the human cornea a cDNA library (Stratagene, La Jolla, CA) of approximately 200,000 clones was generated from RNA isolated from post-mortem whole corneas Clones representing transcripts showing high tissue-specific expression as analyzed by reversed Northern and conventional Northern blot analysis were subjected to sequencing. The deduced ammo acid sequence of one of these clones, named cornea derived transcript 6 (CDT6) appeared to be homologous to the family of angiogenic factors the angiopoietms (Davis et al . Cell. 1996:87:1161-69, Maisonpierre et al . Science. 1997:277:55- 60) . The expression of CDT6 appeared to be confined to the stromal layer of the human cornea. A detailed report describing the isolation and structural analysis of CDT6 has been published (Peek et al . IOVS 1998:39: 1782-1788).

Functional analysis of CDT6

To clone the open reading frame (ORF) of CDT6 m the appropriate eukaryotic expression vectors, the sequences directly bordering the ATG initiation codon and the TTA stop codon of the CDT6 cDNA sequence were m vitro mutated by PCR using oligo nucleotides with the sequence 5 ' CCACAACATATGCTGAA3 ' and 5 ' GACATATGTTAAGGCTTGAAGTCTTC3 ' . This procedure introduced Ndel restriction enzyme sites bordering the ORF of CDT 6. The resulting PCR product was ligated m pGEMT easy (Pharmacia) . From this clone the ORF of CDT6 was cloned in to the Not I site of the plasmid pORSVCAT (Pharmacia) after the CAT coding sequence was removed by Notl digestion followed by agarose gel purification. Orientation and integrity of the resulting clone (RSV-CDT6) , m which the CDT6 ORF is now under the control of the Rous sarcoma virus promoter, was confirmed by sequence analysis.

The RSV-CDT6 plasmid construct was transfected using the DOSPER method (Boehrmger Mannheim) to a human melanoma cell line MV3 (Muijen et al . 1991:48:85-91) to obtain stable transfectants . In parallel MV3 cells were transfected with the parental construct RSV-CAT, coding for tne prokaryotic protein chloramphenicol acetyl transferase. Stable MV3 transfectants were selected based on resistance to 150 micrograms per milliliter of G418. Expression of CDT6 transcripts by these MV3 transfectants was confirmed by

Northern blot analysis and PCR. Two CDT6 expressing clones were selected; CDT6.9 (low expression) and CDT6.3 (moderate expression) . The m vitro growth rate of these stable transfectants was similar to the parental MV3 cell line. To study the effect of CDT6 on tumor growth and vascularisation , 2 million cells of these cell lines, the MV3-CAT cell line and the parental MV3 cell line were harvested from subconfluent cultures and suspended m 100 micro liters of PBS and inoculated into the lateral thoracic wall of BALB/c nude mice (nu/nu) to obtain xenografts. All experimental groups contained at least 5 mice. Tumor growth was determined three times a week by measuring all three diameters of the subcutaneous tumors. Mice were killed when the subcutaneous tumor reached a volume exceeding 4 cubic centimeters. At autopsy the tumor was removed and parts of these tissues were frozen m liquid nitrogen for RNA extraction or fixed in buffered formalin and embedded m paraffin for microscopic examination.

In the first experiment 3 groups of mice were inoculated with MV3 parental line, MV3-CAT and CDT6.9. After 5 weeks the tumors resulting from the parental MV3 cell line and the control cell line MV3-CAT reached volumes of over 4 cubic centimeters, while the CDT6.9 derived were xenografts 8-fold smaller. Microscopic analyses of the tumor tissue indicated that m addition to a reduced tumor size the expression of CDT6 led to tumor cell showing a tortuous morphology. Furthermore, staining with a monoclonal antibody directed to mouse blood vessel endothelial cells (9F1, unpublished) showed that the blood vessels m CDT6 expressing tumors were abnormal, with lesser sprouts and a thickened basal membrane. Analysis of RNA isolated from the xenografts by PCR indicated that CDT6 was indeed expressed in the MV3 CDT6.9 derived tumors but absent from the control tumors. These experiments were repeated twice, but instead of using the low CDT6 expressing CDT6.9 the MV3 transfectant CDT6.3 cell line was used. In both these experiments CDT6.3 xenografts were tenfold smaller than the control MV3 parental and MV3-CAT xenografts and again the aberrant morphology of tumor cells and blood vessels was apparent.

To further enhance the expression of CDT6 the human melanoma cell line MV3 the coding region of CDT6 was cut from the pORSV vector by Notl digestion and ligated to the expression vector pRc/CMV2 (Invitrogen) . Resulting clones were sequenced for correct orientation and transfected to MV3 cells to obtain stable transfectants . These stable transfectants were analyzed by PCR and Northern blot for the level of CDT6 expression. Three different CDT6 transfectants were obtained; one showing a similar expression level as the CDT6.3 cell line (CDT6.4C), one showing an approximately two fold increase in expression as compared to the CDT6.3 cell line (CDT6.8E) and one showing an approximately ten fold increase in expression as compared to the CDT6.3 cell line (CDT6.1E) . The cell lines were tested m the BALB/c mice assay for xenograft growth and morphology. Tumor growth was shown to be inversely correlated with the expression of CDT6. In addition, CDT6 expressing tumors were shown to have an effect on blood vessel permeability, tumor oxygenation and tumor cell apoptosis.

Molecular mechanisms by which CDT6 influences tumor growth.

In addition to the Rous sarcoma virus promoter (RSV) the CDT6 coding sequence was cloned under the control of the cytomegalovirus early gene promoter (CMV) (see above) . These CMV-CDT6 constructs were stably transfected into the human skin melanoma cell line MV3 m a similar way as performed for pORSV vector constructs. Stable MV3 transfectants which showed detectable CDT6 expression by either Nothern blot analysis or PCR were used to produce xenografts m nude mice (see figure 2) . It appeared that tumor growth was again largely inhibited by CDT6 expression. Tumors derived from MV3 cell lines with high CDT6 expression showed a stronger inhibition of tumor growth than cell lines with moderate or only low CDT6 expression (see figure 3) . Tumors exceeding a volume of 2 cubic centimeters were analyzed by PCR for CDT6 expression and by (immuno) histochemistry with specific antibodies for the expression of several proteins. From these analysis it appeared that expression of CDT6 m the tumor induced a high expression of several extra cellular matrix associated proteins including collagen I, collagen V and proteoglycans but not of collagen IV, lamm or fibronectm. An example of such histochemical analysis is shown m figure 4) . Electron microscopy revealed that these extra cellular matrix associated proteins are derived from both the tumor cells as well as from the invading mouse blood vessel endothelial cells. The co-expression of collagen I and collagen V m combination with high levels of proteoglycans is unique for the human cornea and essential for transparency (Birk et al , 1986; Birk et al 1988; Marchant et al , 1996) . These results suggest that CDT6 acts as a so-called morphogen; a protein which programs a certain cell to differentiate into a particular tissue. In tumor tissue expression of CDT6 leads to fibrosis m which morphology and function of blood vessels is impaired and tumor growth is partly inhibited.

Production and purification of eukaryotic cell produced recombmant CDT6. To purify the CDT6 protein from eukaryotic cells the ORF of CDT6 was cut from the pORSV vector and cloned m the Notl site of the expression vector pcDNA4/HιsMax A (Invitrogen) . This construct in which the CDT6 protein is tagged on the N- terminus with an Xpress epitope and a His tag was transfected to MV3 cells and stable transfectants were obtained by selection with Zeocin. CDT6 protein produced by these cells was purified according to the protocol supplied by the manufacturer of the pcDNA4/HisMaxA plasmid system (Invitrogen) .

To obtain a CDT6 specific antibody the ORF of CDT6 was cloned in several bacterial and eukaryotic expression vectors, such as baculovirus, retrovirus and adenovirus vectors. This protein and the vectors will be used to immunize rabbits and obtain an antibody. This antibody will be used to localize the CDT6 protein in xenografts and in in vitro assays to positively identify CDT6 as the effector molecule .

The purified protein was provided to MV3 cells, that were prior to transfection not able to bind the CDT6 protein, transfected with a cDNA library of endothelial cells. The cells were fixed and incubated with an antibody recognizing the tagged CDT6 protein. Positive cells were picked and the cDNA was amplified with primers recognizing the constant part of the cDNA containing vehicle. Primers were chosen such that they spanned the region containing the cDNA and contained unique restriction sites. The amplified cDNA was cloned into an expression vector and transfected into MV3 cells for a new round of selection.

The same procedure of selection and amplification was performed. The resulting amplificate was cloned into an expression vector and transformed into bacteria. Individual clones were sequenced and transfected into MV3 cells. The transfected MV3 cells were incubated with the tagged recombmant CTD6 and stained with the antibody recognizing the recombmant CDT6 protein. Expression vectors used to stam MV3 cells that showed positive upon transfection of the expression plasmid contain a receptor for CDT6. Receptors were sequenced and characterized. At least one receptor showed some homology to an angiopoietm receptor.

Receptors were used to study the ability of CDT6 to influence processes of blood vessel formation vitro.

Furthermore, the recombmant CDT6 protein was used to administer in the blood or directly in the tumor of BALB/c mice with a MV3 derived xenograft. Tumor growth was at least in part inhibited. Next to tumor volume, additional parameters such as permeability of tumor blood vessels, the level of tumor oxygenation and tumor cell apoptosis were determined.

In addition, the recombmant CDT6 protein was formulated into solution compatible with the eye. Delivery of the solution to a cornea transplanted animal showed improved grafting of said cornea.

The effect of CDT6 protein on blood vessel formation m the cornea was assessed m a rat model.

Table 1. Examples of angiogenesis-dependent diseases

Name of the Disease Brief description of the disorder

Angiofibroma Abnormal blood vessels which are prone to bleeding

Neovacular Glaucoma Abnormal growth of blood vessels m the eye

Arthritis Including Rheumatoid arthritis,

Lupus and other connective tissue disorders .

Osier-Weber syndrome Genetic condition resulting m abnormal blood vessels which are prone to bleeding Psoriasis : Common chronic skin disorder

Corneal graft neovascularisation : Complication of corneal replacement surgery

Retrolental fibroplasia Diabetic retinopathy : Leading cause of blindness in diabetic

Solid tumors : Trachoma : Leading cause of blindness in third world Hemophilic joints : Vascular adhesions : Hypertrophic scars : Neovascular diseases of the eye

Brief description of the drawings

Fig. 1.

Sequences and structure of CDT6. (A) Nucleotide sequence of the CDT6 cDNA and deduced ammo acid sequence. The putative polyadenylation signals are underlined, consensus N- glycosylation sites are indicated by asterisks .

Fig. 2

PCR analysis of CDT6 expression m transfected MV3 cells. As a control the expression of the beta-actm gene is shown.

Fig. 3.

Tumor growth of xenografted nude mice. Tumors expressing CDT6 grow much slower than either the parental line (MV3) or vector control transfectant .

Fig. 4

Histochemical analysis of the expression of lamm, collagen

IV and collagen V m tumors derived from the parental line (MV3) and the CDT6 transfectants . Only expression of collagen

V shows a marked increase m the CDT6 expressing tumor.

References

Birk DE , Fitch JM, Lensenmayer TF . Organization of collagen types I and type V m the embryonic chicken cornea. 1986;27:10 pp 1470-1477.

Birk DE, Fitch JM, Babiarz JP, L senmayer TF . Collagen type I an type V are present m the same fibril m the avian corneal stroma. J. Cell Biol . 1988. Vol 106: pp 999-1008.

Fukuda K, Koshihara Y, Oda H, Ohayama M, Ooyama T. Biochem. Biophys . Res. Commun . 1988 Vol 151; pp 1060-1068.

Hashimoto K, Hatai M, Yaoi Y. Cell. Struct. Funct . 1991 Vol 16; pp 391-7.

Marchant JK, Hahn RA, Lmsenmayer TF, Birk DE . Reduction of type V collagen using a dominant -negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal -specific fibril morphology. J. Cell Biol. 1996 Vol 135:5 pp 1415-1426.

Peek R, van Gelderen BE, Brumenberg M, Kijlstra A. Molecular cloning of a new angiopoietinlike factor from the human cornea. Invest Ophthalmol Vis Sci . 1998:39:1782-1788.

Davids S, Aldπch TH, Jones PF, Acheson A, Compton D, Jam V, Ryan TE, Bruno J, Radzie ewski C, Maisonpierre PC,

Yancopoulos GD. Isolation of Angιopoιetm-1 a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 1996:87:1161-1169.

Maisonpierre PC, Sun C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClam J, Aldrich TH,

Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD . Angιopoιetm-2 , a natural antagonist for Tιe2 that disrupts m vivo angiogenisis . Sci ence 1997:277:55-60.

Muijen GNP, Jansen KFJ, Cornelissen IMH, Smeets DFC, Beck JLM, Ruiter DJ . Establishment and characterization of a human melanoma cell line (MV3) which is highly metastic m nude mice. Int. J. Cancer 1991:48:85-91. Valenzuela DM, Griffiths JA, Ro as J, Aldrich TH, Jones PF, Zhou H, McClam J, Copeland NG , Gilbert DJ, Jenkins NA, Huan T, Papadopoulus N, Maisonpierre PC, Davis S, Yancopoulos GD . Angiopoietms 3 and 4: diverging gene counterparts in mice and humans. Proc Na tl Acad Sci USA 1999:96:1904-1909.

Ziats NP, Anderson JM . J. Vascular Surg . 1993: Vol 17:4; pp 710-718.

Claims

Cl a ims

1. CDT6 or a functional equivalent thereof for use as a pharmaceutical .

2. Use of CDT6 or a functional equivalent thereof in the preparation of a medicament for the inhibition of angiogenesis.

3. Use of CDT6 or a functional equivalent thereof m the preparation of a medicament for the treatment of a solid tumor .

4. A nucleic acid comprising a gene encoding CDT6 or a functional equivalent thereof for use as a pharmaceutical.

5. Use of a nucleic acid comprising a gene encoding CDT6 or a functional equivalent thereof m the preparation of a medicament for the inhibition of angiogenesis.

6. Use of a nucleic acid comprising a gene encoding CDT6 or a functional equivalent thereof in the preparation of a medicament for the treatment of a solid tumor.

7. Use according to claim 2 or claim 5, wherein said angiogenesis is at least m part inhibited through a structure that is at least m part impenetrable for blood vessels.

8. Use according to claim 3 or claim 6, wherein growth of said solid tumor is at least m part inhibited through a structure that is at least in part impenetrable for blood vessels .

9. A method for inducing and/or enhancing the formation of a structure, said structure at least n part impenetrable for blood vessels comprising administering to a cell, CDT6 or a functional equivalent thereof.

10. A method according to claim 9, wherein said structure comprises collagen V.

11. A method according to claim 9 or claim 10, wherein said structure comprises collagen I .

12. A method according to any one of claim 9-11, wherein said structure is transparent.

13. A method according to any one of claims 9-12 wherein said cell is a fibroblastoid cell

14. A structure obtainable by a method according to any one of claims 9- 13.

15. A structure according to claim 14, wherein said structure is transparent.

16. Use of a structure according to claim 14 or claim 15 for the preparation of a medicament .

17. Use according to claim 16, wherein said medicament comprises a transplant.

18. A gene delivery vehicle comprising a nucleic acid encoding CDT6 or a functional equivalent thereof.

19. A gene delivery vehicle according to claim 18, which is a recombmant virus .

20. A gene delivery vehicle according to claim 19 which is of adenoviral, retroviral or adeno-associated viral origin.

21. Use according to any one of claims 1-3, 7 or 8 , wherein CDT6 comprises a sequence as depicted m figure 1 or a sequence at least 80, preferably 90, more preferably 95, more preferably 99% homologous therewith.

22. Use according to any one of claims 4-8, wherein the gene encoding CDT6 comprises a sequence as depicted m figure 1 or a sequence at least 80, preferably 90, more preferably 95, more preferably 99% homologous therewith.

23. A method for inhibiting angiogenesis comprising providing CDT6 or a functional equivalent thereof at the site of possible angiogenesis in a host.

24. A method for inhibiting the growth of a solid tumor in a host comprising providing CDT6 or a functional equivalent thereof at the site of said tumor said host.

25. A method according to claim 23 or 24, wherein CDT6 or its functional equivalent is provided through a gene delivery vehicle .

26. A method according to any one of claims 23-25, whereby CDT6 or its functional equivalent is provided through expression of a gene encoding it m a cell .

27. A method according to claim 26, whereby said cell is a target cell of said host.

28. A nucleic acid encoding CDT6 or a functional equivalent thereof under the control of a heterologous promoter, such as a viral promoter.

29. A nucleic acid according to claim 28, which has the sequence as depicted m figure 1 or a CDT6 specific part thereof, or a sequence at least 80, preferably 90, more preferably 95, more preferably 99% homologous therewith.

30. A nucleic acid according to claim 28, which has the sequence as depicted m figure 1 or a CDT6 specific part thereof or the complementary strand thereof or a sequence which hybridizes to a sequence as depicted m figure 1 or its complementary strand or a specific part of either strand under stringent conditions.

31. A cell comprising a nucleic acid according to any one of claims 28-30.

32. A cell according to claim 31 which expresses functional CDT6 or a functional equivalent thereof.

33. A method for producing recombmant CDT6 or a functional equivalent thereof, comprising culturmg a cell according to claim 32 under suitable conditions and harvesting CDT6 or its functional equivalent from said culture.

34. Use of CDT6 or a functional equivalent thereof for inducing and/or enhancing the formation of a collagen V containing structure .