CA2359750A1

CA2359750A1 - Peptides with anti-angiogenic activity

Info

Publication number: CA2359750A1
Application number: CA002359750A
Authority: CA
Inventors: Nicholas Mazarakis; Enca Martin-Rendon; Susan Mary Kingsman
Original assignee: Individual
Current assignee: Oxford Biomedica UK Ltd
Priority date: 1999-02-15
Filing date: 2000-02-15
Publication date: 2000-08-17
Also published as: GB9903408D0; KR20010092800A; CN1340059A; GB0120713D0; WO2000047622A2; AU2559600A; NO20013933L; IL144376A0; JP2002542762A; GB2362887A; EP1161454A2; WO2000047622A3; NO20013933D0

Abstract

A non-naturally occuring Type I Repeat Peptide (TRP) and expression methods thereof.

Description

2 PCT1GBOOI00520 PEPTIDES
The present invention relates to Type I Repeat Peptides (TRPs) capable of inhibiting the proliferation and migration of endothelial cells. More particularly, the present invention relates to the in vitro and in vivo expression of TRPs and their use in the prevention and/or treatment of a disease or condition associated with angiogenesis and/or cancer.
Angiogenesis is an essential component of the metastatic pathway. The majority of solid tumours are well vascularised, generating new blood vessels as they grow. For many tumours the vascular density can provide a prognostic indicator of metastatic potential, with highly vascularised tumours having a higher incidence of metastasis than poorly vascular ones. Tumor angiogenesis is regulated by the production of angiogenic activators and inhibitors (for a review, see Zetter, BR 1998 Annu.
Rev.
Med., 49: 407-24).
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. In certain pathologies aberrant cellular proliferation results in the local production of factors that permit continued growth through enhancement of angiogenesis. By way of example, angiogenic stimulants such as vascular endothelial growth factor (VEGF), PDGF and bFGF (Kuwabara et al PNAS 1995 92 4606) can induce the endothelial cells to migrate through the eroded basement membrane.
The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary Loops, thus creating new blood vessels.

It is clear that angiogenesis plays a major role in the metastasis of a cancer. Cancer and in particular carcinoma development requires angiogenesis to permit continued growth. One such approach to inhibit disease progression in cancer is to limit the neovascularisation process. If this angiogenic activity could be repressed or eliminated, then a tumor, although present, would not grow. In other disease states, the prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. In this way, therapies directed at control of angiogenic processes could lead to the mitigation of these diseases.
Tissue growth is limited by nutrient supply. During development vascular growth maintains pace with tissue growth to ensure that cells receive the correct level of oxygenation and nutrient supply. If cellular proliferation outpaces that of vascular supply then cells become oxygen limited. This oxygen limitation, called hypoxic stress, causes changes in gene expression. Many of these changes involve an increase 1 ~ in the expression of genes including those involved in glycolysis to enable anaerobic respiration but also factors that promote angiogenesis/vasculogenesis.
By way of example, VEGF transcription is induced by hypoxia through the increased stability of the HIF-1 transcription factor and the consequent binding of this transcription factor to an enhancer element, the hypoxia response element (HRE), that resides in the 5' UTR of the VEGF gene (Forsythe et al MCB 1996 16 4604, Maxwell et al PNAS 1997 94 8104). Other examples also include PDGF and bFGF (Kuwabara et al PNAS 1995 92 4606). Although there is some information on the signalling mechanism that leads to these changes in gene expression this process is yet to be fully understood.
What is needed therefore is a way of inhibiting the unwanted growth of blood vessels, especially into tumours. This can occur either directly by the use of inhibitors of proangiogenic factors or indirectly by identifying compounds/small molecules that can inhibit the induction of such factors. This should include ways of overcoming the activity of endogenous growth factors (e.g., aFGF, ~iFGF, VEGF, IL-8, GM-CSF), in J
premetastatic tumours and prevent the formation of capillaries in the tumours thereby inhibiting the growth of the tumours. It should also include ways of modulating the formation of capillaries in other angiogenic processes, such as wound healing and reproduction.
A considerable amount of research has been carried out on the anti-angiogenic properties of the extracellular matrix molecule thrombospondin. Thrombospondin (TSP) has been confirmed to be an inhibitor of endothelial cell proliferation, motility and morphogenesis (Bagavandoss and Wilks 1990 Biochem Biophys Res Commun 170: 867-72; Vogel et al, 1993 J Cell Biochem 53: 74-84; Iruela-Arispe et al, Proc Natl Acad Sci 88: 5026-30). T'he expression of TSP has been shown to be inversely related to p53 expression and to angiogenesis in human bladder cancer (Grossfeld et al, 1997: J. Natl. Cancer Inst., 89:219), and gliomas.
Although internal peptides mapping in the Type 1 Repeats (TRPs) of the human has been reported to reduce cell proliferation and endothelial cell migration (Tolsma et al, 1993 J Cell Biol 122:497-S11), there has been no report of the expression of these peptides from cDNA and the use of such cDNA in gene therapy.
The present invention seeks to provide TRPs with anti-angiogenic activity which can be expressed under in vitro and in vivo conditions and which can be used in the prevention andlor treatment of angiogenesis and/or cancer.
Aspects of the present invention are presented in the accompanying claims and in the following description.
Thus, in one aspect, the present invention provides a non-naturally occuring Type I
Repeat Peptide (TRP).
As used herein, the term "Type I Repeat Peptide (TRP)" means an internal peptide comprising Type I repeats which are present in one or more copies (TRP arrays) in TRP containing proteins. In accordance with the present invention, the "TRP"
can be the peptide per se also as well as being part of a fusion protein.
The term "non-naturally occuring" 'fRP according to the present invention means a TRP which is not expressed by a natural nucleotide coding sequence when it is in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.
Thus, the present invention does not cover the native TRP according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.
Preferably, the TRP is an isolated TRP andlor purified TRP.
The TRP can be obtainable from or produced by any suitable source, whether natural or not, or it may be a synthetic TR.P, a semi-synthetic TRP, a derivatised TRP, or a recombinant TRP. Preferably, the non-native TRP includes a TRP at least a portion of which has been prepared by recombinant DNA techniques or a '1~RP produced by chemical synthesis techniques or combinations thereof.
More preferably, the non-native TRP has been prepared by use of recombinant techniques.
The term "derivative" as used herein includes chemical modification of a TRP.
Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or ammo group.

The sequence of the TRP of the present invention may be the same as that of the naturally occuring form or it may be a variant, homologue, fragment or derivative thereof.
5 As used herein "amino acid sequence" refers to peptide, polypeptide or protein sequences or portions thereof.
In one prefered aspect. the present invention provides a polypeptide consisting essentially of a TRP.
The present invention also covers nucleotide sequences) coding for the 'fRP of the present invention. The nucleotide sequence of the present invention is a non-naturally occuring form. Thus, the present invention does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under 1 ~ the control of its native promoter which is also in its natural environment.
The sequence of the TRP encoding nucleotide sequence may be the same as the naturally occuring forni, or is a variant, homologue, fragment or derivative thereof.
Preferably, the TRP encoding sequence is an isolated TRP encoding sequence and/or a purified TRP coding sequence. The TRP coding sequence can be obtainable from or produced by any suitable source, whether natural or not, or it may be synthetic; semi-synthetic or recombinant.
The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or 2~ polynucleotide sequence, and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand. The nucleotide sequence need not necessarily be a complete naturally occuring DNA sequence. Thus, the DNA
sequence can be, for example, a synthetic DNA sequence, a recombinant DNA sequence (i.e.
prepared by use of recombinant DNA techniques), a cDNA sequence or a partial wo oora~62z Pc~rrcaooroos2o genomic DNA sequence, including combinations thereof. The DNA sequence need not be a coding region. If it is a coding region, it need not be an entire coding region.
In addition, the DNA sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation. Preferably, the DNA is or comprises eDNA.
Preferably, the term "nucleotide sequence" means DNA. More preferably, the term "nucleotide sequence" means DNA prepared by use of recombinant DNA techniques (i.e. recombinant DNA). Thus, preferably, the present invention relates to a DNA
sequence (preferably a cDNA sequence) encoding a TRP. In particular, the present invention relates to cDNA sequences encoding a TRP.
In one prefered aspect. the present invention provides a nucleotide sequence consisting essentially of a TRP coding sequence.
1~
The term ''recombinant Type I Repeat Peptide (TRP)" means a TRP prepared by use of recombinant DNA techniques.
Altered TRP encoding polynucleotide sequences which may be used in accordance with the invention may include different nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent TRP which TRP
may have deletions, insertions or substitutions therein.
As used herein a "deletion" is defined as a change in either nucleotide or amino acid 2~ sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
As used herein an "insertion" or "addition" is a change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring TRP or TRP
encoding nucleotide sequence.

As used herein "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
Included within the scope of the present invention are alleles of the TRP. As used herein, an "allele" or "allelic sequence" is an alternative form of the TRf.
Alleles result from a mutation, i.e., a change in the nucleotide sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered.
Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
The present invention also relates to DNA fragments comprising the DNA
sequence of SEQ ID No. 1 or allelic variations of such sequences.
A highly preferred aspect of the present invention relates to a TRP comprising the amino acid sequence of SEQ ID No. 2. For example, the present invention relates to an isolated TRP comprising the amino acid sequence of SEQ ID No. 2.
Tlie present invention also relates to DNA comprising the DNA sequence of SEQ
ID No.
1 or an allelic variation thereof.
The present invention also relates to non-native DNA comprising the DNA
sequence of SEQ ID No. 1 or an allelic variation thereof.
A highly preferred aspect of the present invention relates to recombinant DNA
comprising the DNA sequence of SEQ ID No. 1 or an allelic variation thereof.
As used herein, "immunological activity" is defined as the capability of the natural, recombinant or synthetic TRP or any fragment thereof, to induce a specific immune response in appropriate animals or cells and/or to bind with specific antibodies.

_ _ .

In one preferred embodiment, the nucleotide sequence andlor the TRP thereof are in an isolated and/or purified form.
As used herein, the terms "isolated" and "purified" refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment and/or isolated and/or separated from at least one other component with which they are naturally associated.
The TRPs of the present invention can be isolated from body fluids such as blood serum, ascites or urine of patients or the TRPs can be produced by recombinant DNA
methods or synthetic peptide chemical methods that are well known to those of ordinary skill in the art. Protein purification methods are also well known in the art.
The present invention also relates to TRPs produced by expression in a transformed host cell into which has been incorporated the foregoing DNA sequences or allelic variations thereof.
The term "vector" includes expression vectors and transformation vectors.
The term "expression vector" means a construct capable of in vivo or in vitro e~cpression.
The term "transformation vector" means a construct capable of being transferred from one species to another.
2~ Preferred vectors for use in accordance with the present invention are recombinant viral vectors, in particular recombinant retroviral vectors (RRV) such as lentiviral vectors, adenoviral vectors including a combination of retroviral vectors.
The term "recombinant retroviral vector" (RRV) refers to a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell.

. . i WO 00!47622 PCT/GB00/00520 Infection of the target cell includes reverse transcription and integration into the target cell genome. The RRV carries non-viral coding sequences which are to be delivered by the vector to the target cell. An RRV is incapable of independent replication to produce infectious retroviral particles within the final target cell. Usually the RRV
lacks a functional gag pol andlor env gene and/or other genes essential for replication.
In a further broad aspect, the present invention provides a hybrid viral vector system for in vivo gene delivery, which system comprises one or more primary viral vectors which encode a secondary viral vector, the primary vector or vectors capable of infecting a first target cell and of expressing therein the secondary viral vector, which secondary vector is capable of transducing a secondary target cell.
Preferably the primary vector is obtainable from or is based on an adenoviral vector and/or the secondary viral vector is obtainable from or is based on a retroviral vector preferably a lentiviral vector.
The term "targeted vector'' refers to a vector whose ability to infect/transfect/transduce a cell or to be expressed in a target cell is restricted to certain cell types within the host organism, usually cells having a common or similar phenotype.
Preferably the nucleotide sequence of the present invention is operably linked to a transcription unit.
The term "transcription unit(s)" as described herein are regions of nucleic acid containing coding sequences and the signals for achieving expression of those coding sequences independently of any other coding sequences. Thus, each transcription unit generally comprises at least a promoter, an optional enhancer and a polyadenylation signal.
The term "promoter" is used in the normal sense of the art, e.g. an RNA
polymerase binding site. The promoter may contain an enhancer element.

s WO 00/47622 PCTlGB00/00520 The term "enhancer" includes a DNA sequence which binds to other protein components of the transcription initiation complex and thus facilitates the initiation of transcription directed by its associated promoter.
5 The term ''cell" includes any suitable organism. In a preferred embodiment, the cell is a mammalian cell. In a highly preferred embodiment, the cell is a human cell.
The term "transformed cell" means a cell having a modified genetic structure.
With the present invention, a cell has a modif ed genetic structure when a vector according 10 to the present invention has been introduced into the cell.
The term ''organism" includes any suitable organism. In a preferred embodiment, the organism is a mammal. In a highly preferred embodiment, the organism is a human.
Here the term "transgenic organism" means an organism comprising a modified genetic structure. With the present invention, the organism has a modified genetic structure since a vector according to the present invention has been introduced into the organism.
The present invention also provides a method comprising transforming a host cell with the nucleotide sequence of the present invention.
The present invention also provides a method comprising culturing a transformed host cell - which cell has been transformed with a nucleotide sequence according to the present invention under conditions suitable for the expression of the TRP
encoded by said nucleotide sequence.
The present invention also provides a method comprising culturing a transformed host cell - which cell has been transformed with a nucleotide sequence according to the present invention or a derivative, homologue, variant or fragment thereof -under WO 00/47622 PCTlGB00100520 conditions suitable for the expression of the TRP encoded by said nucleotide sequence;
and then recovering said TRP from the transformed host cell culture.
The present invention also provides a method for producing a '1 RP, the method comprising the steps ~of a) transforming a host cell with a nucleotide sequence according to the present invention or a derivative, homologue, variant or fragment thereof; b) culturing the transformed host cell under conditions suitable for the production of said TRP and c) recovering said TRP from the host cell culture.
The terms "variant", "homologue", "derivative" or "fragment" in relation to the nucleotide sequence of the present invention include any substitution of variation of, modification of, replacement of, deletion of or addition of one (or morej nucleic acid from or to the sequence providing the expression product of the resultant nucleotide sequence has endothelial cell proliferation and migration inhibitory activity, preferably having at least the same endothelial cell proliferation and migration inhibitory activity as the expression product of a sequence covered by SEQ LD. No. 1 (Figure 1).
In particular, the term "homologue" covers identity with respect to structure andlor function providing the expression product of the resultant nucleotide sequence has endothelial cell proliferation and migration inhibitory activity. With respect to sequence identity (i.e. similarity), preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass allelic variations of the sequences.
Sequence identity with respect to SEQ ID No. 1 can be determined by a simple "eyeball" comparison (i.e. a strict comparison) of any one or more of the sequences with another sequence to see if that other sequence has, for example, at least 75%
sequence identity to the sequence(s).

WO 00!47622 PCTIGB00/00520 Relative sequence identity can also be deternzined by commercially available computer programs that can calculate % identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters. A
typical example of such a computer program is CLUSTAL. Advantageously, the S BLAST algorithm is employed, with parameters set to default values. The BLAST
algorithm is described in detail at http:1/www.ncbi.nih.govBLAST/blast help.html, which is incorporated herein by reference. The search parameters are defined as follows, can be advantageously set to the defined default parameters.
Advantageously, "substantial identity" when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST
searching is usually 10.
1S BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.govBLAST/blast help.html) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119-129.
The five BLAST programs available at http://www.ncbi.nlm.nih.gov perform the following tasks:
blastp - compares an amino acid query sequence against a protein sequence database.
blastn - compares a nucleotide query sequence against a nucleotide sequence database.

blastx - compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
tblastn - compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands).
tblastx - compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM - Display a histogram of scores for each search; default is yes.
(See parameter H in the BLAST Manual).
1 ~ DESCRIPTIONS - Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).
EXPECT - The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT
threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
CUTOFF - Cutoff score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as high as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported.

(See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.
ALIGNMENTS - Restricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CI1TOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).
MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN
and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, t992). The valid alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate scoring matrices are available for BLASTIv'; specifying the MATRIX
directive in BLASTN requests returns an error response.
STRAND - Restrict a TBLASTN search to just the top or bottom strand of the database sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading frames on the top or bottom strand of the query sequence.
FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short-periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.

WO 00/47622 PCTlGB00100520 Low complexity sequence found by a filter program is substituted using the letter "N"
in nucleotide sequence (e.g., " ") and the letter "X" in protein sequences (e.g., "XX~XXX~~X").
5 Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default f ltering is DUST for BLASTN, SEG for other programs.
It is not unusual for nothing at all to be masked by SEG, XNU, or both, when applied to sequences in SWISS-PROT, so filtering should not be expected to always yield an 10 effect. Furthermore, in some cases, sequences are masked in their entirety, indicating that the statistical significance of any matches reported against the unfiltered query sequence should be suspect.
NCBI-gi - Causes NCBI gi identifiers to be shown in the output, in addition to the 1 ~ accession and/or locus name.
Most preferably, sequence comparisons are conducted using the simple BLAST
search algorithm provided at http:/Iwww.ncbi.nlm.nih.gov/BLAST.
Other computer program methods to determine identify and similarity between the two sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387) and FASTA (Atschul et a! 1990 J Molec Biol 403-410).
In some aspects of the present invention, no gap penalties are used when determining sequence identity.
The present invention also encompasses nucleotide sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar promoter sequences in other organisms.

The present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences presented herein. or any fragment or derivative thereof.
Hybridization means a "process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J ( 1994) Dictionary of Biotechnology, Stockton Press, New York NY) as well as the process of amplification as carried out in polymerase chain reaction technologies as described in Dieffenbach CW and US Dveksler (199, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY).
Also included within the scope of the present invention are nucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in 1~ Berger and Kimmel (19$7, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.
Maximum stringency typically occurs at about Tm-S°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm;
intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related nucleotide sequences.
In a preferred aspect. the present invention covers nucleotide sequences that can hybridise to the nucleotide sequences of the present invention under stringent conditions (e.g. 65°C and O.IxSSC).

a WO 00!47622 PCT/GB00100520 The present invention also encompasses nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof. Likewise, the present invention encompasses nucleotide sequences that are complementary to sequences that are capable of hybridising to the sequence of the present invention. These types of nucleotide sequences are examples of variant nucleotide sequences.
In this respect, the term ''variant" encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.
Preferably, however, the term ''variant" encompasses sequences that are complementary to sequences that are capable of hydridising under stringent conditions (eg.
65°C and O.IxSSC { IxSSC = 0.15 M NaCI, 0.015 Na3 citrate pH 7.0}) to the nucleotide sequences presented herein.
The present invention demonstrates that TRPs can be expressed under in vitro and in vivo conditions. The expression of the TRPs of the present invention under in vitro and in vivo conditions is advantageous because TRPs can be:
(i) administered to tumor-bearing humans or animals as anti-angiogenic therapy.
(ii) monitored in human or animal serum, urine, or tissues as prognostic markers.
(iii) used as the basis to analyze serum and urine of cancer patients for similar anti-angiogenic molecules.
(iv) used either alone or in combination with therapeutically useful agents in the treatment of angiogenesis and/or cancer.
The present invention also demonstrates the surprising finding of a novel isoform of Brain Angiogenesis Inhibitor (BAI) 3 comprising TRPs capable of inhibiting endothelial cell proliferation and migration activity.

The term "angiogenesis" means the generation of new blood vessels in a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal/embryonal development and formation of the corpus luteum, endometrium and placenta.
The term ''angiogenesis inhibitor" is used to describe any agent which can prevent the growth of blood vessels into tissues such as unvascularised or vascularised tumours.
The term can described the capability of a molecule to inhibit angiogenesis in general and, for example, to inhibit the growth of bovine and/or human capillary endothelial cells in culture in the presence of an angiogenic stimulant. This term includes agents such as matrix metalloproteinases (MMPs) which are naturally occuring inhibitors of both angiogenesis and tumour metastasis because of their ability to degrade the extracellular matrix.
IS
The term "endothelium" means a thin layer of flat epithelial cells that Lines serous cavities, lymph vessels, and blood vessels.
The term "angiogenic stimulant" includes but is not limited to factors, such as vascular endothelial growth factor (VEGF), PDGF and bFGF (Kuwabara et al PNAS 1995 92 4606) which are capable of stimulating endothelial cell proliferation and migration.
The term "angiogenic stimulant" is used interchangeably with the term "proangiogenic factor".
2~ The nucleotide sequences of the present invention may be engineered in order to alter a the TRP encoding nucleotide sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product. For example, mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change codon preference.

In another embodiment of the invention, a TRf natural, modified or recombinant encoding nucleotide sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for inhibitors of the TRP activity, it may be useful to encode a chimeric TRP expressing a heterologous S epitope that is recognized by a commercially available antibody.
The present invention also encompasses the use of fusion proteins comprising the TRP
or an enzymatically active fragment or derivative thereof linked to an affinity tag such as glutathione-S-transferase (GST), biotin, His6, a c-myc tag (see Emrich et al 1993 Biocem Biophys Res Commun 197(1): 214-220), hemagglutinin (HA) (as described in Wilson et al (1984 Cell 37 767) or a FLAG epitope (Ford et al 1991 Protein Expr Purif Apr; 2(2):95-107).
In another preferred aspect, the fused recombinant protein comprises an antigeme co-t ~ protein such as GST, beta-galactosidase or the lipoprotein D from Haemophilus influenzae which are relatively large co-proteins, which solubilise and facilitate production and purification thereof. Alternatively, the fused protein may comprise a carrier protein such as bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). In certain embodiments of the present invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen Inc) and described in Gentz et al {1989 PNAS 86: 821-824). Such fusion proteins are readily expressable in yeast culture (as described in Mitchell et al 1993 Yeast 5: 715-723) and are easily purified by affinity chromatography. A fusion protein may also be engineered to contain a cleavage site located between the nucleotide sequence encoding the TRP and the heterologous protein sequence, so that the TRP may be cleaved and purified away from the heterologous moiety, In another embodiment, an assay for the target protein may be conducted using the entire, bound fusion protein. Alternatively, the co-protein may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.

Although the presence/absence of marker gene expression suggests that the nucleotide sequence and/or its TRP is also present, its presence and expression should be confirmed. For example, if the TRP encoding nucleotide sequence is inserted within a marker gene sequence, recombinant cells containing the TRP coding regions may be identified by the absence of marker gene function. Alternatively, a marker gene may be placed in tandem with a the TRP encoding nucleotide sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the TRP as well.
10 Additional methods to quantitate the expression of a particular molecule include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated.
Quantitation of multiple samples may be speeded up by running the assay in an ELISA
15 format where the TRP of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantitation.
The present invention also relates to expression vectors and transformed host cells comprising TRP encoding nucleotide sequences or variant, homologue, fragment or 20 derivative thereof for the in vivo or in vitro production of a TKP with a view to screening for agents that can affect TRP expression or activity.
In accordance with the present invention, TRP encoding nucleotide sequences, TRP
fragments, fusion proteins or functional equivalents thereof, may be used to generate 2~ recombinant DNA molecules that direct the expression thereof in appropriate host cells. In one embodiment of the present invention, a nucleotide sequence encoding the TRP of the present invention is operably linked to a promoter sequence capable of directing expression of the TRP encoding nucleotide sequence in a suitable host cell.
When inserted into the host cell, the transformed host cell may be cultured under suitable conditions until sufficient levels of the TRP are achieved after which the cells may be lysed and the TRP isolated.

WO 00/47622 PCTlGB00/00520 The T RP encoding nucleotide sequence itself may be isolated from cells using familiar techniques for the fragmentation and sequence analysis of genetic material including the use of oligonucleotide probes and PCR amplification techniques to identify known target sequences or suspected sequences having substantial sequence homology with the target sequence.
One example of a method of producing TRP using recombinant DNA techniques entails the steps of (1) identifying and purifying a TRP containing protein such as Thrombospondin (TSP), (2) synthetically generating ~' and 3' DNA
oligonucleotide primers for the TRP sequence, (3) amplifying the TRP nucleotide sequence using polymerase, (4) inserting the amplified TRP encoding nucleotide sequence into an appropriate vector such as an expression vector, (5) inserting the vector containing the TRP encoding nucleotide sequence into a microorganism or other expression system capable of expressing the TRP encoding nucleotide sequence, and (7) isolating the recombinantly produced TRP. The above techniques are more fully described in laboratory manuals such as "Molecular Cloning: A Laboratory Manual" Second Edition by Sambrook et al, Cold Spring Harbor Press, 1989.
The TRP nucleotide sequence may also be isolated from cells or tissue (such as tumor cells) that express high levels of TRP by ( 1 ) isolating messenger RNA from the tissue, (2) using reverse transcriptase to generate the corresponding DNA sequence and then

(3) using PCR with the appropriate primers to amplify the TRP encoding nucleotide sequence.
2~ Yet another method of producing TRP, or biologically active fragments thereof, is by peptide synthesis. Once a biologically active fragment of an TRP is found using the assay system described more fully below. it can be sequenced, for example by automated peptide sequencing methods. Alternatively, once the nucleotide sequence which encodes the TRP is isolated, for example by the methods described above, the DNA sequence can be determined, which in turn provides information regarding the amino acid sequence. Thus, if the biologically active fragment is generated by specific methods, such as tryptic digests, or if the fragment is N-terminal sequenced, the remaining amino acid sequence can be determined from the corresponding DNA
sequence.
Altered TRP nucleotide sequences which may be used in accordance with the present invention include deletions, insertions or substitutions of different nucleotide residues resulting in a nucleotide sequence that encodes the same or a functionally equivalent TRP.
The protein may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent TRP.
Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobiciry, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of the TRP is retained. For example, 1 ~ negatively charged amino acids include aspariic acid and glutamic acid;
positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophiliciry values include leucine, isoteucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
The TRP may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J
(1992) Protein Expr Purif 3 - 26328 1), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affiniry purification system (Immunex Corp, Seattle, WA). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the TRP is useful to facilitate purification.

WO 00/47622 PCTlGB00/00520 Host cells transformed with the TRP encoding nucleotide sequence may be cultured under conditions suitable for the expression and recovery of the TRP from cell culture.
The protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing the TRP
encoding nucleotide sequences can be designed with signal sequences which direct secretion of the TRP encoding nucleotide sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the TRP encoding nucleotide sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Knoll DJ et al (1993) DNA Cell Biol 12:441-53, see also above discussion of vectors containing fusion proteins).
Once the amino acid sequence of the TRP is known, the fragment can be synthesized by techniques well known in the art, as exemplified by "Solid Phase Peptide 1 ~ Synthesis: A Practical Approach" E. Atherton and R. C. Sheppard, IRL
Press, Oxford England. Similarly, multiple fragments can be synthesized which are subsequently linked together to form larger fragments. These synthetic peptide fragments can also be made with amino acid substitutions at specific locations in order to test for agonistic and antagonistic activity in vitro and in vivo. Peptide fragments that possess high affinity binding to tissues can be used to isolate the TRP receptor on affinity columns.
Isolation and purification of a TRP receptor is a useful step towards elucidating the mechanism of action of TRP. This facilitates development of drugs to modulate the activity of potential TRP receptors. Isolation of TRP receptors enables the 2~ construction of nucleotide probes to monitor the location and synthesis of the receptor, using in situ and solution hybridization technology.
The present invention includes any TRP derivatives that have endothelial cell proliferation and migration inhibitory activity. The present invention includes the entire TRP, TRP derivatives and biologically-active TRP fragments. These include TRP with endothelial cell proliferating inhibitory and migratory activit~~
that have WO 00!47622 PCT/GB00100520 amino acid substitutions or have sugars or other molecules attached to amino acid functional groups. The present invention also inctucies nuc~eonae sequences mai encode TRP and TRP receptors, and TRPs that are expressed by those nucleotide sequences.
Different TRP fragments can be synthesized for use in several applications including, but not limited to the following; as antigens for the development of specific antisera, as agonists and antagonists active at TRP binding sites, as peptides to be linked to cytotoxic agents for targeted killing of cells that bind TRP. The amino acid sequences that comprise these peptides are selected on the basis of their position on the exterior regions of the molecule and are accessible for binding to antisera. A tyrosine or lysine is added to fragments that do not have these residues to facilitate labeling of reactive amino and hydroxyl groups on the peptide. These peptide sequences are compared to known sequences using the GenBank, Brookhaven Protein, SWISS-PROT, and PIR
1 ~ databases to determine potential sequence homologies. This information facilitates elimination of sequences that exhibit a high degree of sequence homology to other molecules, thereby enhancing the potential for high specificity in the development of antisera, agonists and antagonists to TRP.
TRPs or TRP fragments can be; synthesized in a standard microchemical facility and purity checked with HPLC and mass spectrophotometry. Methods of peptide synthesis, HPLC purification and mass spectrophotometry are commonly known to those skilled in these arts.
TRPs and TRP fragments may be produced in recombinant E. coli, or yeast expression systems, and purified with column chromatography.
TRPs and TRP fragments can be coupled to other molecules using standard methods.
The amino and carboxyl termini of TRP may be isotopically and nonisotopically labeled with many techniques, for example radiolabeling using conventional techniques (tyrosine residues- chlorarnine T, iodogen, lactoperoxidase; lysine residues-Bolton-Hunter reagent). These coupling techniques are well known to those skilled in the art. The coupling technique is chosen on the basis of the functional groups available on the amino acids including, but not limited to amino, sulfhydral, carboxyl, amide, phenol, and imidazole. Various reagents used to effect these couplings include among others, glutaraldehyde, diazotized benzidine, carbodiimide, and p-benzoquinone.
TRPs and TRP fragments are chemically coupled to isotopes, enzymes, canter proteins, cytotoxic agents, fluorescent molecules, radioactive nucleotides and other 10 compounds for a variety of applications. The efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction.
For example, radiolabeling of an TRP peptide with lz'I is accomplished using chloramine T and Nalzsl of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted on disposable columns. The labeled peptide 15 is eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity measured in a gamma counter. In this manner. the unreacted Na 'z' I is separated from the labeled TRP peptide. The peptide fractions with the highest specific radioactivity are stored for subsequent use such as analysis of the ability to bind to TRP antisera.
TRPs and TRP fragments are employed to develop affinity columns for isolation of the TRP receptor from cultured tumor cells. Isolation and purification of the TRP
receptor is followed by amino acid sequencing. Next, nucleotide probes are developed for insertion into vectors for expression of the receptor. These techniques are well known to those skilled in the art. Transfection of the TRP receptor into tumor cells enhances the responsiveness of these cells to endogenous or exogenous TRP and thereby decreasing the rate of metastatic growth.
'the synthetic TRP fragments have a variety of uses. The TRP or TRP fragment that binds to a TRP receptor with high specificity and avidity is radiolabeled and employed for visualization and quantitation of binding sites using autoradiographic and membrane binding techniques. This application provides important diagnostic and research tools. Knowledge of the binding properties of the TRP receptor facilitates investigation of the transduction mechanisms linked to the receptor.
In addition, labeling these peptides with short lived isotopes enables visualization of receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques in order to locate tumours with TRP binding sites.
Systematic substitution of amino acids within these synthesized peptides yields high affmiry peptide agonists and antagonists to the TRP receptor that enhance or diminish TRP binding to its receptor. Such agonists are used to suppress the growth of micrometastases, thereby limiting the spread of cancer. Antagonists to TRP are applied in situations of inadequate vascularization, to block the inhibitory effects of TRP and possibly promote angiogenesis. This treatment may have therapeutic effects I S to promote wound healing in diabetics.
Another application of peptide conjugation is for production of polyclonal antisera.
For example, TRPs or TRP fragments containing lysine residues are linked to purified bovine serum albumin using glutaraldehyde. The efficiency of the reaction is determined by measuring the incorporation of radiolabeled peptide. Unreacted glutaraldehyde and peptide are separated by dialysis. The conjugate is stored for subsequent use.
The TRPs or TRP fragments of the present invention also can be used to generate antibodies that are specific for the TRP. The antibodies can be either polyclonal antibodies or monoclonal antibodies. These antibodies that specifically bind to the TRPs or TRP receptors can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect the presence or quantify the TRP or TRP
receptors in a body fluid or tissue. Results from these tests can be used to diagnose or predict the occurrence or recurrence of a cancer and other angiogenic mediated diseases.

WO 00!47622 PCT/GB00100520 Antiserum against TRPs can be generated. After peptide synthesis and purification, both monoclonal and polyclonal antisera are raised using established techniques known to those skilled in the art. For example, polyclonal antisera may be raised in rabbits, sheep, goats or other animals. TRPs conjugated to a carrier molecule such as bovine serum albumin, or TRP itself, is combined with an adjuvant mixture, emulsified and injected subcutaneously at multiple sites on the back, neck, flanks, and sometimes in the footpads. Booster injections are made at regular intervals, such as every 2 to 4 weeks. Blood samples are obtained by venipuncture, for example using the marginal ear veins after dilation, approximately 7 to 10 days after each injection. The blood samples are allowed to clot overnight at 4°C and are centrifuged at approximately 2400 times g at 4°C for about 30 minutes. The serum is removed, aliquoted, and stored at

4°C for immediate use or at -20° to -90°C for subsequent analysis.
All serum samples from generation of polyclonal antisera or media samples from production of monoclonal antisera are analyzed for determination of titer.
Titer is established through several means, for example, using dot blots and density analysis, and also with precipitation of radiolabeled peptide-antibody complexes using protein A, secondary antisera, cold ethanol or charcoal-dextran followed by activity measurement with a gamma counter. The highest titer antisera are also purified on affinity columns which are commercially available. TKPs are coupled to the gel in the affinity column. Antiserum samples are passed through the column and anti-TRP
antibodies remain bound to the column. These antibodies are subsequently eluted, collected and evaluated for determination of titer and specificity.
The highest titer TRP antisera is Tested to establish the following: a) optimal antiserum dilution for highest specific binding of the antigen and lowest non-specific binding, b) the ability to bind increasing amounts of TRPs in a standard displacement cun~e, c) potential cross-reactivity with related peptides and proteins, including plasminogen and also TRPs of related species, d) ability to detect TRPs in extracts of plasma, urine, tissues, and in cell culture media.

Passive antibody therapy using antibodies that specifically bind TR.Ps can be employed to modulate angiogenic-dependent processes such as reproduction, development, and wound healing and tissue repair. In addition, antisera directed to the Fab regions of TRP antibodies can be administered to block the ability of endogenous 'rRP
antisera to bind TRPs.
The present invention also includes diagnostic methods and kits for detection and measurement of TRPs .in biological fluids and tissues, and for localization of TRPs in tissues.
TRPs can also be used in a diagnostic method and kit to detect and quantify antibodies capable of binding TRP. These kits permit detection of circulating TRP
antibodies which indicates the spread of micrometastases in the presence of TRP secreted by primary tumours in situ. Patients that have such circulating anti-TRP
antibodies may 1 S be more likely to develop tumours and cancers, and may be more likely to have recurrences of cancer after treatments or periods of remission. The Fab fragments of these anti-TRP antibodies may be used as antigens to generate anti-TRP Fab-fragment antisera which can be used to neutralize the removal of circulating TRP by anti-TRP
antibodies.
Kits for measurement of TRPs are also contemplated as part of the present invention.
Antisera that possess the highest titer and specificity and can detect TRPs in extracts of plasma, urine, tissues, and in cell culture media are further examined to establish easy to use kits for rapid, reliable, sensitive, and specific measurement and localization of TRPs. These assay kits include but are not limited to the following techniques; competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots. enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry. For each kit the range, sensitivity, precision, reliability, specificity and reproducibility of the assay are established.
Intraassay and WO OO14~622 PCT/GB00/00520 interassav variation is established at 20%, 50% and 80% points on the standard curves of displacement or activity.
One example of an assay kit commonly used in research and in the clinic is a radioimmunoassay (RIA) kit. After successful radioiodination and purification of TRP
or a TRP fragment, the antiserum possessing the highest titer is added at several dilutions to tubes containing a relatively constant amount of radioactivity, such as 10,000 cpm, in a suitable buffer system. Other tubes contain buffer or preimmune serum to determine the non-specific binding. After incubation at 4°C
for 24 hours, protein A is added and the tubes are vortexed, incubated at room temperature for 90 minutes, and centrifuged at approximately 2000-2500 times g at 4°C to precipitate the complexes of antibody bound to labeled antigen. The supernatant is removed by aspiration and the radioactivity in the pellets counted in a gamma counter.
The antiserum dilution that binds approximately 10 to 40 % of the labeled TRP or TRP
1 ~ fragment after subtraction of the non-specific binding is further characterized.
Next, a dilution range (approximately 0.1 pg to 10 ng) of the TRP or TRP
fragment used for development of the antiserum is evaluated by adding known amounts of the peptide to tubes containing radiolabeled peptide and antiserum. After an additional incubation period, for example, 24 to 48 hours, protein A is added and the tubes centrifuged, supernatant removed and the radioactivity in the pellet counted.
The displacement of the binding of radiolabeled TRP or TRP fragment by the unlabeled TRP or TRP fragment (standard) provides a standard curve. Several concentrations of other TRP fragments, TRP from different species, and homologous peptides are added to the assay tubes to characterize the specificity of the TRP antiserum.
Another aspect of the present invention is a method of blocking the action of excess endogenous TRP. This can be done by passively immunizing a human or animal with antibodies specific for the undesired TRP in the system. This treatment can be important in treating abnormal ovulation, menstruation and placentation, and vasculogenesis. This provides a useful tool to examine the effects of TRP
removal on WO 00/47622 PCTlGB00/00520 metastatic processes. The Fab fragment of TRP antibodies contains the binding site for TRP. This fragment is isolated from TRP antibodies using techniques known to those skilled in the art. The Fab fragments of TRP antisera are used as antigens to generate production of anti-Fab fragment serum. Infusion of this antiserum against the

5 Fab fragments of TRP prevents TRP from binding to TRP antibodies.
Therapeutic benefit is obtained by neutralizing endogenous anti-TRP antibodies by blocking the binding of TRP to the Fab fragments of anti-TRP. The net effect of this treatment is to facilitate the ability of endogenous circulating T'RP to reach target cells, thereby decreasing the spread of metastases Another kit is used for localization of TRP in tissues and cells. This TRP
immunohistochemistry kit provides instructions, TRP antiserum, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum.
Immunohistochemistry techniques are well known to those skilled in the art.
This TRP
immunohistochemistry kit permits localization of TRP in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical purposes. For example, tumours are biopsied or collected and tissue sections cut with a microtome to examine sites of TRP production. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer.
The present invention also encompasses gene therapy whereby the TRP encoding nucleotide sequence is regulated in vivo. For example, expression regulation may be accomplished by administering compounds that bind to the TRP encoding nucleotide sequence, or control regions associated with the TRP encoding nucleotide sequence, or its corresponding RNA transcript to modify the rate of transcription or translation.
By way of example, the TRP encoding nucleotide sequence of the present invention may be under the expression control of an expression regulatory element, usually a promoter or a promoter and enhancer. The enhancer andlor promoter may be preferentially active in a hypoxic or ischaemic or low glucose environment, such that WO 00/4'7622 PCTlGB00/00520 the TRP encoding nucleotide sequence is preferentially expressed in the particular tissues of interest, such as in the environment of a tumour, arthritic joint or other sites of ischaemia. Thus, any significant biological effect or deleterious effect of the TRP
encoding nucleotide sequence on the individual being treated may be reduced or eliminated. The enhaneer element or other elements conferring regulated expression may be present in multiple copies. Likewise, or in addition, the enhancer and/or promoter may be preferentially active in one or more specific cell types -such as any one or more of macrophages, endothelial cells or combinations thereof. Further examples include include respiratory airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cess and post-mitotically terminally differentiated non-replicating cells such as macrophages neurons.
The promoter and/or enhancer may be constitutively efficient, or may be tissue or temporally restricted in their activity. Examples of suitable tissue restricted promoters/enhancers are those which are highly active in tumour cells such as a promoter/enhancer from a MUC1 gene, a CEA gene or a .iT=l antigen gene.
Examples of temporally restricted promoters/enhancers are those which are responsive to ischaemia and/or hypoxia, such as hypoxia response elements or the promoter/enhancer of a grp78 or a grp94 gene. The alpha fetoprotein (AFP) promoter is also a tumour-specific promoter. One preferred promoter-enhancer combination is a human cytomegalovirus (hCMV) major immediate early (MIE) promoter/enhancer combination.
Preferably the promoters of the present invention are tissue specific. That is, they are capable of driving transcription of a TRP encoding nucleotide sequence in one tissue while remaining largely "silent" in other tissue types.
The term "tissue specific" means a promoter which is not restricted in activity to a single tissue type but which nevertheless shows selectivity in that they may be active in one group of tissues and less active or silent in another group. A desirable characteristic of the promoters of the present invention is that they posess a relatively WO 00!47622 PCT/GB00/00520 low activity in the absence of activated hypoxia-regulated enhancer elements, even in the target tissue. One means of achieving this is to use "silencer" elements which suppress the activity of a selected promoter in the absence of hypoxia.
The term "hypoxia" means a condition under which a particular organ or tissue receives an inadequate supply of oxygen.
The level of expression of a TRP encoding nucleotide sequence under the control of a particular promoter may be modulated by manipulating the promoter region. For example, different domains within a promoter region may possess different gene regulatory activities. The roles of these different regions are typically assessed using vector constructs having different variants of the promoter with specific regions deleted (that is, deletion analysis). This approach may be used to identify, for example, the smallest region capable of conferring tissue specificity or the smallest region I 5 conferring hypoxia sensitivity.
A number of tissue specific promoters, described above, may be particularly advantageous in practising the present invention. In most instances, these promoters may be isolated as convenient restriction digestion fragments suitable for cloning in a selected vector. Alternatively, promoter fragments may be isolated using the polymerase chain reaction. Cloning of the amplified fragments may be facilitated by incorporating restriction sites at the S' end of the primers.
TRPs or TRP fragments may be used in combination with other compositions and 2~ procedures for the treatment of diseases. By way of example, TRPs or TRP
fragments may be used in combination with either inhibitors of proangiogenic factors (angiogenic stimulants) or compounds/small molecules that can inhibit the induction of such factors. As already mentioned, the expression of some of these proangiogenic factorslangiogenic stimulants are induced by hypoxia. By way of example, VEGF
transcription is induced by hypoxia through the increased stability of the HIF-transcription factor and the consequent binding of this transcription factor to an JJ
enhancer element, the hypoxia response element (HRE) that resides in the 5' UTR of the VEGF gene (Forsythe et al MCB 1996 16 4604, Maxwell et al PNAS 1997 94 8104). Potential antagonists of the HIF/HRE signalling pathway would be expected to inhibit the hypoxia mediated induction of angiogenic factors and would therefore, be inhibitors of tumour vascularisation. A series of reporter configurations have been linked to HREs and have been shown to function in a variety of vector backbones and in a variety of cell types (see PCT/GB98/028$5). These reporter configurations can be adapted to a small molecule screens to fmd antagonists which can be advantageously combined with the TRPs and TRP fragments of the present invention.
TRPs or TRP fragments may also be used in combination with conventional treatments of diseases. By ways of example, a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with a TRP or TRP fragment or a TRP or TRP
fragment may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor.
The TRP can be delivered with a therapeutically effective agent at the same moment in time and at the same site. Alternatively, the TRP and the therapeutically effective agent may be delivered at a different time and to a different site. The the TRP and the therapeutically effective agent may even be delivered in the same delivery vehicle for the prevention and/or treatment of angiogenesis and/or cancer.
TRPs or TRP fragments may be used in combination with cytotoxic agents for the prevention and/or treatment of angiogenesis and/or cancer. Cytotoxic agents such as 2~ ricin, linked to TRP, high affinity TRP fragments, TRP antisera, TRP
receptor agonists and antagonists provide a tool for the destruction of cells that bind TRP.
'these cells may be found in many locations, including but not limited to, micrometastases and primary tumours.
TRPs or TRP fragments may be used in combination with a pro-drug activating enzyme in gene therapy. Instead of or as well as being selectively expressed in target tissues. the TRP or TRP fragments may be used in combination with another nucleotide sequences of interest (NOI) or NOIs which encode a pro-drug activation enzyme or enzymes which have no significant effect or no deleterious effect until the individual is treated with one or more pro-drugs upon which the enzyme or enzymes act. In the presence of the active NOI, treatment of an individual with the appropriate pro-drug leads to enhanced reduction in tumour growth or survival.
A pro-drug activating enzyme may be delivered to a tumour site for the treatment of a cancer. In each case, a suitable pro-drug is used in the treatment of the patient in combination with the appropriate pro-drug activating enzyme. An appropriate pro-drug is administered in conjunction with the vector. Examples of pro-drugs include:
etoposide phosphate (with alkaline phosphatase, Senter et al 1988 Proc Natl Acad Sci 85: 4842-4846); 5-fluorocytosine (with cytosine deaminase, Mullen et al 1994 Cancer Res 54: 1503-1506); Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerr et al 1990 Cancer Immunol Immunother 31: 202-206); Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (with (3-lactamase); SR4233 (with P450 Reductase);
Ganciclovir (with HSV thymidine kinase, Borrelli et al 1988 Proc Natl Acad Sci 85:
7572-7576); mustard pro-drugs with nitroreductase (Friedlos et al 1997 J Med Chem 40: 1270-1275) and Cyclophosphamide (with P450 Chen et al 1996 Cancer Res 56:
1331-1340).
Examples of suitable pro-drug activation enzymes for use in the invention include a thymidine phosphorylase which activates the 5-fluoro-uracil pro-drugs capcetabine and furtulon; thymidine kinase from Herpes Simplex Virus which activates ganciclovir; a cytochrome P450 which activates a pro-drug such as eyclophosphamide to a DNA
damaging agent; and cytosine deaminase which activates 5-fluorocytosine.
Preferably, an enzyme of human origin is used.
Other suitable NOI sequences for use in the present invention include those that are of therapeutic and/or diagnostic application such as, but are not limited to:
sequences WO 00!47622 PC'r/GB00/00520 encoding cytokines, chemokines, hormones, antibodies, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an 5 antigen, a tumour suppressor protein and growth factors, membrane proteins, vasoactive proteins and peptides, anti-viral proteins and ribozymes, and derivatives therof (such as with an associated reporter group). When included, such coding sequences may be typically operatively linked to a suitable promoter, which may be a promoter driving expression of a ribozyme(s), or a different promoter or promoters, 10 such as in one or more specific cell types.
The expression products encoded by the NOIs may be proteins which are secreted from the cell. Alternatively the NOI expression products are not secreted and are active within the cell. In either event, it is preferred for the NOI expression product to 15 demonstrate a bystander effector or a distant bystander effect; that is the production of the expression product in one cell leading to the killing of additional, related cells, either neighbouring or distant (e.g. metastatic), which possess a common phenotype.
Suitable NOIs for use in the invention in the treatment or prophylaxis of cancer include 20 NOIs encoding proteins which: destroy the target cell (for example a ribosomal toxin), act as: tumour suppressors (such as wild-type p53); activators of anti-tumour immune mechanisms (such as cytokines, co-stimulatory molecules and immunoglobulins);
inhibitors of angiogenesis; or which provide enhanced drug sensitivity (such as pro-drug activation enzymes); indirectly stimulate destruction of target cell by natural 25 effector cells (for example, strong antigen to stimulate the immune system or convert a precursor substance to a toxic substance which destroys the target cell (for example a prodrug activating enzyme). Encoded proteins could also destroy bystander tumour cells (for example with secreted antitumour antibody-ribosomal toxin fusion protein), indirectly stimulated destruction of bystander tumour cells (for example cyrtokines to 30 stimulate the immune system or procoagulant proteins causing local vascular occlusion) or convert a precursor substance to a toxic substance which destroys bystander tumour cells (eg an enzyme which activates a prodrug to a diffusible drug).
Also, the delivery of NOI(s) encoding antisense transcripts or ribozymes which interfere with expression of cellular genes for tumour persistence (for example against aberrant myc transcripts in Burkitts lymphoma or against bcr-abl transcripts in chronic myeloid leukemia. The use of combinations of such NOIs is also envisaged.
Suitable NOIs for use in the treatment or prevention of ischaemic heart disease include NOIs encoding plasminogen activators. Suitable NOIs for the treatment or prevention of rheumatoid arthritis or cerebral malaria include genes encoding anti-inflammatory proteins, antibodies directed against tumour necrosis factor (TNF) alpha, and anti-adhesion molecules (such as antibody molecules or receptors specific for adhesion molecules).
Examples of hypoxia regulatable therapeutic NOIs can be found in PCTlGB95100322 (WO-A-95/21927).
As it is well known in the art, a vector is a tool that allows or faciliates the transfer of an entity from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. Optionally, once within the target cell, the vector may then serve to maintain the heterologous DNA
within the cell or may act as a unit of DNA replication. Examples of vectors used in recombinant DNA techniques include plasmids, chromosomes, artificial chromosomes or viruses.
The vector can be delivered by viral or non-viral techniques.

Non-viral delivery systems include but are not limted to DNA transfection methods.
Here, transfection includes a process using a non-viral vector to deliver a gene to a target mammalian cell.
Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421 ) and combinations thereof.
Viral delivery systems include but are not limited to adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector or a baculoviral vector.
Examples of retroviruses include but are not limited to: marine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney marine leukemia virus (Mo-MLV), FBR marine osteosarcoma virus (FBR MSV), Moloney marine sarcoma virus (Mo-MSV), Abelson marine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
A detailed list of retroviruses may be found in Coffin et al (''Retroviruses"
1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).
Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV}
and the more recently described feline immunodeficiency virus (FIV) and bovine S immunodeficiency virus (BIV).
A distinction between the lentivirus family and other types of retroviruses is that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516).
In contrast, other retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
Other examples of vectors include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection.
The vector may be a plasmid DNA vector. Alternatively, the vector may be a recombinant viral vectors. Suitable recombinant viral vectors include adenovirus vectors, adeno-associated viral (AAV) vectors, Herpes-virus vectors, or retroviral vectors which are preferred. In the case of viral vectors, gene delivery is mediated by viral infection of a target cell.
The vector of the present invention may be configured as a split-intron vector. A split intron vector is described in PCT patent application GB98,~02885 (W099/15684) and 2S GB/98/02867 (W099115683).
If the features of adenoviruses are combined with the genetic stability of retroviruses/lentiviruses then essentially the adenovirus can be used to transduce target cells to become transient retroviral producer cells that could stably infect neighbouring cells. Such retroviral producer cells engineered to express a TRl' andlor an NOI(s) of the present invention can be implanted in organisms such as animals or humans for use in the treatment of angiogenesis and/or cancer.
The dosage of the TRP of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. Depending upon the half life of the 'fRP in the particular animal or human, the TRP can be administered between several times per day to once a week. It is to be understood that the present invention has application for both human and veterinary use. The methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and 1 S solutes which render the formulation isotonic with the blood of the intended recipient;
and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or mufti-dose containers, far example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
TRPs are effective in treating diseases or processes that are mediated by, or involve, angiogenesis. The present invention includes the method of treating an angiogenesis mediated disease with an effective amount of a TR.P or TRP agonists and/or antagonists. TRPs and TRI' fragments can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable compositions using formulation methods known to those of ordinary skill in the art. These compositions can be administered by standard routes. These include but are not limited to: oral, rectal, ophthalmic (including intravitreal or intraeameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradetmal, intracranial, intratracheal, and epidural) transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, or parenteral (e.g., 5 intravenous, intraspinal, subcutaneous or intramuscular) routes.
The TRP formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carriers) or 10 excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
In addition, the TRPs of the present invention may be incorporated into biodegradable 15 polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the TRP is slowly released systemically. The biodegradable polymers and their use are described, for example, in detail in Brem et al, J.
Neurosurg 74:441-446 (1991). Osmotic minipumps may also be used to provide controlled 20 delivery of high concentrations of TRI's through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor.
Peptides linked to cytotoxic agents are infused in a manner designed to maximize delivery to the desired location. For example, ricin-linked high affinity TRP
fragments 25 are delivered through a cannula into vessels supplying the target site or directly into the target. Such agents are also delivered in a controlled manner through osmotic pumps coupled to infusion cannulae.
Preferred unit dosage formulations are those containing a daily dose or unit, daily 30 sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
Pharmaceutical compositions comprising an effective amount of a TRP expression product or a TRP encoding nucleotide sequence of the present invention can be used in the treatment of angiogenesis andlor cancer. Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic mediated diseases. Diseases that are mediated by angiogenesis include. but not limited to: solid tumours; blood born tumours such as leukemias; tumor metastasis; benign tumours, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;
psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis;
plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma;
wound granulation; corornay collaterals; cerebral collaterals; arteriovenous malformations;
ischeniic limb angiogenesis; neovascular glaucoma; retrolental fibroplasia;
diabetic neovascularization; heliobacter related diseases, fractures, vasculogenesis, hematopoiesis, ovulation, menstruation and placentation.
TRPs are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. TRP can be used as a birth control agent by preventing vascularization required for embryo implantation.
TRP is useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helobacter pylori).

Figures The invention will now be further described only by way of example in which reference is made to the following Figures:
Figure 1 which is the nucleotide sequence of the Type-1 repeats from the hTSP-1 gene which is presented as SEQ ID No. 1. The HindII and Notl restriction are added by PCR to facilitate the cloning of these fragments into a mammalian expression vector;
Figure 2 which is the amino acid sequence (SEQ ID No. 3} of human thrombospondin-1 (hTSP-1) Type I repeat peptide (TRP);
Figure 3 which shows the primers used in the amplification of hTSP-1 TRP;
1 ~ Figure 4 which shows a diagrammatic representation of plasmid pSecTag.2A.
This plasmid contains a leader sequence and the c-myc epitope tag;
Figure 5 which shows the primers used in the amplification of hTSP-1 control peptide;
Figure 6 which shows the BLAST search results using the 3rd TSP Type-I repeat;
Figure 7 which shows a consensus TRP amino acid sequence presented as SEQ ID
No.
2. It also shows the TRPs from human TSP-I (SED ID No. 3), KIAAO>j0 (SED ID
No. 4) and KIAA0688 (SED ID No. S).
Figure 8 which shows the BLAST search results using KIAA0688 protein;
Figure 9 which shows the BLAST search results using KIAA05~0 protein;
Figure 10 which shows a schematic comparison between ICIAA0550 and BAI3 proteins;

Figure 11 which is a diagrammatic representation of the configuration of the plasmid 0880 in a retroviral vector genome;
Figure 12 is a schematic diagram for constructing a recombinant adenoviral vector. By way of explanation, Adeno PGKLacZ is the OBHRELacZ cassette from OB37 plasmid inserted into the Microbix transfer vector pE 1 sp 1 A;
Figure 13 shows a diagrammatic representation the plasmid pONY4 lentiviral vector genome;
Figure 14 shows a diagrammatic representation the plasmid pONY2.1 lentiviral vector gag pol expression cassette;
Figure 1 ~ shows a map of a retroviral XiaGen-P450 vector comprising a therapeutic 1 ~ gene under the control of OBHRE promoter;
Figure 16 shows the HIF/I-IRE signalling pathway; and Figure 17 shows the configuration of integrated transcriptional cassette comprising an assay reporter and a marker for clonal selection.
EXAMPLES
Example 1 - Construction of an expression vector encoding Type-1 repeats 2~ fragments The eDNA fragments corresponding to TRPs of human thrombospondin-1 (TSP-1) open reading frame (Figure 2) are amplified from human poly A+ mRNA. The amplification is performed by RT-PCR in which the mRNA is firstly reverse transcribed to cDNA using the enzyme reverse transcriptase (RT) and secondly, this cDNA is amplified by the Polymerase Chain Reaction (PCR). The specific primers WO 00!47622 PCT/GB00/00520 used in this reaction (Figure 3) have been designed in-frame with the HindIII
site of the commercial vector pSecTag2.A (Invitrogen, Figure 4) at the Send and in-frame with the NotI site of the same vector at the 3'end. The plasmid generated is called pSecTag2-TRP 1.
5' HindIII XXA AGC 'ITX
3' NotI XGC GGC CGC
A non-angiostatic control peptide (or non TRP containing peptide) corresponding to the C-terminal part of human TSP-1 (Figure 2) is also cloned in the same frame into the vector described above. A set of four synthetic oligonucleotides encoding the control peptide is designed (Figure 5) and cloned into pSecTag.2A digested with the enzymes HindIII and NotI (Figure 4) to generate pSecTag2-TRP2.
The vector pSecTag2 contains the murine ig kappa chain leader sequence which allows the secretion of the TRPs. It also contains the c-myc epitope which will be incorporated at the C-terminus of the TRPs. This allows the detection of the fusion proteins using anti c-myc antibody. Finally, pSecTag2 contains a 6xHistidine tag downstream from the c-myc epitope which facilitates high affinity binding to Ni2+
chelating resin for easy purification of the fusion proteins. These constructs permit efficient, high-level expression of the recombinant polypeptides from the human CMV
immediate early gene promoter-enhancer.
The plasmids are transfected into 293T cells by standard methods (i.e.;
calcium phosphate) and the expression of the fusion polypeptides in transfected cells is confirmed by Western blotting. A prostate-specific antigen (PSA) expression plasmid, pSecTag2/PSA (Invitrogen), based on pSecTag2.A is used as a control. The medium in which transfected cells are grown is filtered through a 0.45 micron pore-size filter (Millipore Inc.) and analyzed for secreted fusion proteins. An aliquot of 20u1 of the conditioned medium is run on 10-12% SDSIPAGE. The gels are electroblotted onto Immobilon membrane (Millipore Inc.). Western blot analysis is carried out with anti c-myc mouse monoclonal antibody at a dilution of 1:1000 (Boehringer} and rabbit HRP
conjugated anti-mouse IgG at a dilution of 112000 (Dako). Detection by chemiluminescence is performed using ECL kit (Amersham International, UK). The 5 production of the recombinant fusion proteins is also checked in total protein extracts of the transfected cells. Transfected cells are washed in PBS, lysed in RIPA
buffer containing 1mM of freshly prepared PMSF and total protein concentration determined using Bradford's BioRad reagents (BioRad) as per the manufacturer's specifications.
The cell lysates (20-50 ug of total protein) are run on 10-12%
SDS/polyacrylamide gel.
10 The proteins are transfered to Immobilon membrane and western blotted as described above.
Example 2 - In vitro endothelial cell proliferation assay 1 ~ The anti-angiogenic effect of the different recombinant TRPs is determined in vitro by an endothelial cell proliferation assay. Similar methods have been previously described for antiangiogenic factors such as endostatin, an endogenous inhibitor of angiogenesis (O'Reilly et al 1997 Cell 88:1) or Interleukin 4 (Volpert et al 1998 J Exp Med 188:1039-1046).
Human kidney cell line, 293T, is used to produce high level of secreted recombinant TRPs using the pSecTRP expressing vectors. 293T cells are transiently transfected with 20ug of each vector per lOcmZ tissue culture dish by the calcium phosphate method. 48 hours post tranfection, the media from transfected cells is filtered through a 0.45 micron pore-size filter (Millipore) and added to the endothelial cells.
Endothelial cells for inclusion in the in vitro endothelial cell proliferation assay of the present invention include but are not limited to Human Umbilical Vein Endothelial Cells (HUVEC); Human Dermal Microvascular Endothelial cells (HDMVEC); and Bovine Adrenal Capillary Endothelial Cells (BACE}.

For the proliferation assay, the endothelial cells are washed in PBS and dispersed in a 0.05% trypsin solution. A cell suspension (25,000 cells/ml} is made with the appropriate endothelial cell media containing 10% bovine calf serum, plated onto gelatinized 24-well tissue culture dish (0.5 mllwell) and grown for 24 hours.
The media is replaced with 0.5 ml of a serial dilution of conditioned media from transfected cells or base media in the presence of the appropriate growth factors. The endothelial cells are grown for 72 hours prior to dispersing them in trypsin and resuspending them in Hematall (Fisher Scientific, Pittsburgh, PA). Cells are counted on a Coulter counter.
Example 3 - In vitro endothelial cell migration assay This example determines if the different recombinant TRPs have an anti-angiogemc effect by inhibiting endothelial cell migration in vitro. The migration of different vascular endothelial cell types is determined by adding the recombinant TltPs to cultured endothelial cells, i.e., bovine adrenal capillary endothelial cells (BACE), HUVE cells and HDMVE cells.
Using a protocol similar to that outlined in Example 2, recombinant TltPs are produced in Human kidney cell line, 293T. The media from the transfected cells is filtered through a 0.45 micron pore-size filter (Millipore) and added to the endothelial cells. The endothelial cells are plated on gelatinised Nucleopore membranes (5 micron pores for BACE and 8 micron pores for the others) in an inverted modified Boyden chamber. After two hours, the chamber is reinvented and test substances added to the top of the wells of each. Specifically, cell populations are exposed to either culture medium alone (control), culture medium containing lOng/ml bFGF (basic Fibroblast Growth Factor), or culture medium containing an increasing concentration of recombinant TRPs and l Onglml bFGF. The cells are allowed to migrate for 3-4 hours.
Following this, the membranes are fixed and stained, and the number of cells that have migrated counted.

Example 4 - Identification of Type-1 Repeat Peptides We have used the amino acid sequence of the third Type-1 repeat of hTSP-1 to perform sequence analyses. Searches were carried out at the National Center for Biotechnology Information (NCBI) through the use of the Advanced BLAST 2.0 network service (Altschul et al, 1997 Nucleic Acids Res. 2~, 3389-3402). The alignments were carried out using the nr database, where the sequence are derived from conceptual translation of sequences from nucleotide databases.
The BLAST search carried out with the TSP-1 TRP (Figure 6) reveals identity to human TSP-1, TSP-I precursors from different species and TSP-related proteins.
It also shows homology with the human semaphorin F homolog, Sco-spondin (Bos taurus) and F-spondin, all of which are proteins expressed in the central nervous system and which have been shown to be responsible for axonal guidance. TSP-1 TRP
also displays homology with Brain Angiogenesis Inhibitor (BAI) 1, 2 and 3 proteins and two novel human proteins, KIAA0550 and KIAA0688 proteins (Figure 7) all of which are expressed in brain tissue.
Example ~ - Further Identification of Type-1 Repeat Peptides The molecular cloning and characterization of the BAI 1 gene has been previously described (Nishimori et al, 1997 Oncogene,l5, 2145-2150). BAI 1 has been shown to be expressed specifically in the brain and it inhibits experimental angiogenesis in a rat cornea model. The expression of BAI 1 gene is absent or significantly reduced in glioblastoma cell lines. More recently the cloning and characterization of two other genes of this family has been reported, BAI 2 and BAl 3 (Shiratsuchi et al, Cytogenet Cell Genet, 79, 103-108), the latter being absent in several glioblastoma cells lines.
A second sequence analysis and alignment has been carried out using KIAA0550 protein (Accession No AB011122) or KIAA0688 (Accession No AB014588) using the WO 00/47622 PCTlGB00/00520 same BLAST network system at the NCBI. The analysis carried out with KIAA0688 reveals identity with secretory proteins that contain TSP-1 Type 1 repeats and regions of homology with cell surface antigen MS2 and metalloproteinases (Figure 8).
Matrix metalloproteinases (MMPs) constitute another example of naturally occuring inhibitors of both angiogenesis and tumor metastasis (Powell WC and Matrisian LM 1996 Curr Top Microbiol Immunol 213: 1-21), because of their ability to degrade extracellular matrix.
Surprisingly, the analysis carried out with KIAA0550 protein revealed identity to BAI
3, BAI 2 and BAI 1 proteins (Figure 9). The comparison between K1AA0550 (985 aa) and BAI 3 (1523 aa) proteins reveals that these two proteins show high degree of sequence identity (Figure 10). KIAA0550 represents approximately two thirds of BAI
3 spanning the first 967 amino acids of both proteins and contains identical Type-I
repeats. BAI 3, however, contains an insertion of 22 amino acids downstream the 1 ~ type-1 repeats. They also differ in the C-terminal domain, where BAI 3 expands for an extra 536 amino acids. These observations suggest that BAI 3 and KIAA0550 might be two isoforms of the same protein produced by alternative splicing of the same RNA
transcript.
Example 6 - Construction of viral vectors for the delivery of the Type-1 repeats fragments MLV based vectors An MLV based retroviral vector is contracted by cloning the recombinants TRPs from pSecTRPs as NcoI-PmeI fragments into OBM80 (Figure 11 ). The nucleotide sequence encoding TRP is inserted into OBM80 in place of human cytochrome P450. This vector allows the expression of recombinant TRP from the MLV LTR promoter in transduced cells. The vector genome is built as a single transcription unit, containing an IRES and the bacterial LacZ gene. The expression of beta-galactosidase is used as a control for transduction.

WO 00/47622 PC'TJGBOOJ00520 In all the following examples the three plasmids transfection method as described previously (Soneoka et al, 1995 Nucl. Acids. Res., 23: 628) is used to generate VSV-G
pseudotyped vectors. The plasmids used in this experiment are as follows: pI-and pRV67 encode the MLV gag pol nucleotide encoding sequences and VSV-G
envelope (env) glycoprotein nucleotide encoding sequence respectively (Soneoka et al, 1995 ibic~. In initial experiments, Bug of the OBM80-TRP vector genomes are co-transfected with Bug each of pHIT60 and pRV67 per well of a 6-well tissue culture dish.
Transfections are carried out in the human kidney cell line 293T as described previously (Soneoka et al, 199 ibic~ to produce the vector particles. Culture supernatants are harvested 36 hours post-transfection and filtered through 0.45 micron pore-size filters (Millipore). Two cell lines, using HT1080 cells and D17 cells, and a dog melanoma cell line are used to titre the retroviral vectors pseudotyped with VSV-G.
Cells are seeded into 6-well tissue culture plates the day before infection at 3 x 10' cells per well. Viral supernatant prepared by transfecting 293T cells with appropriate plasmids to pseudotype MLV-TRP vectors are added to these cells. Polybrene (8ug/ml) is added to each well at the time of transduction into lmi of the culture supernatant used for infection. 12 hours post infection, the culture supernatant is replaced by fresh medium. To measure the viral titre, cells are washed, fixed and stained 48 hours post infection. Viral titres are estimated from the number of beta-galactosidase positive colonies.
2~
Adenoviral Vectors A first generation recombinant adenovirus vectors consist of a deletion of the E1 and E3 regions of the virus alowing insertion of foreign DNA, usually into the left arm of the virus adjacent to the left Inverted Terminal Repeat (ITR). The viral packaging signal (184-358 nt) overlaps with the Ela enhancer and hence is present in most El deleted vectors. This sequence can be translocated to the right end of the viral genome (Hearing and Shenk, 1983, Cell 33, 695). Therefore, in a E1 deleted vector 3.2kb can be deleted (358-3525nt).
5 Adenovirus is able to package 105% length of the genome, thus allowing the addition of an extra 2.lkb. Therefore, in a EI/E3 deleted viral vector the cloning capacity becomes 7-8kb. Since the recombinant adenovirus lacks the essential E1 early gene it is unable to replicate in non-EI complementing cell lines. The 293 cell line was developed by Graham et al (1977, J Gen Virol 36, 59) and contains approximately 4 kb 10 from the left end of the Ad5 genome including the ITR, packaging signal, E
1 a, E1 b and pIX. The cells stably express E 1 a and E 1 b gene products, but not the late protein IX, even though pIX sequences are within Elb. In non-complementing cells E1 deleted virus transduces the cell and is transported to the nucleus but there is no expression from the E 1 deleted genome.
The diagram in Figure 12 shows the general strategy used - to create recombinant adenoviruses using the Microbix Biosystems-NBL Gene Sciences system.
The general strategy involves cloning the foreign DNA into an E1 shuttle vector, where the E1 region (402-3328 bp) is replaced by the foreign DNA cassette. The recombinant plasmid is then co-transfected into 293 cells with pJMl7 plasmid. pJMl7 contains a deletion of the E3 region and an insertion of the prokaryotic pBRX vector (including the ampicillin resistance and bacterial on sequences) into the E1 region at 3.7 map units. This 40kb plasmid is therefore too large to be packaged into adeno nucleocapsids but can be propagated in bacteria. Intracellular recombination in 293 cells results in replacement of the arnpr and on sequences with the insert of foreign DNA.
In the examples quoted herein two transfer vectors have been used. The first obtained from Microbix is called pElsplA and the second, pADBN from Quantum Biotechnologies. The pADBN plasmid has the advantage that the foreign DNA can be a WO 00!47622 PCTJGB00/00520 inserted in either orientation. In both cases the second DNA is a defective version of the adcnoviral genome, either as a plasmid (for example pJMl7) or as part of the viral DNA (for example the right arm of Ad5). Homologous recombination generates the final gene transfer vector.
A single transcription unit containing the therapeutic gene and a reporter gene has the following configuration, TRP-IRES-LacZ driven by a constitutive promoter such as CMV. These configuration are inserted into pADBN transfer vector (Quantum Biotechnologies) to create AdeOBTRP-1, -2, -3 and -4. The recombinant AdeOBTRPs transfer vector are linearised (Asel) and co-transfected into 293 cells along with the purified right arm of the Ad5 virus (from CIaI site) to allow in vivo homologous recombination to occur resulting in the formation of the desired recombinant adenovirus. The virus titre is determined by the number of targeted cell expressing LacZ. These configurations do not exclude having a different reporter gene or another therapeutic gene in place of LacZ.
Lentiviral vectors The TRPs are configured into an EIAV lentiviral vector, pONY4 (Figure 13) where two genes are expressed as a single transcription unit and the related vector pONY2.l (Figure 14). Both are derived from infectious proviral EIAV clone pSPEIAV 19 (Payne et al, 1998, J. Virol 72, 483). The single transcription unit has the following configuration TRPs-IRES-LacZ. It is not essential that the lentiviral vectors contain a reporter gene, instead a second therapeutic gene could be included in place of LacZ.
Viral particles containing pONY-TRP genomes are produced in a transient three plasmid system (Soneoka et al, 1995 ibicl) as described above. VSV-G
pseudotyped EIAV vectors are titred on D17 cells. Viral titres are estimated from the number of beta-galactosidase positive colonies.

Regulated viral vectors The present invention includes the use of a regulated viral vector for the delivery of anti-angiogenic TRPs. For example, an NOI and a TRP encoding nucleotide sequence are expressed under the control of the regulated promoter OBHRE1. Xia-Gen-P450 vector genome is used as a starting molecule. The TRPs are cloned into Xia-Gen-in place of P450 (Figure 15). This configuration allows the expression of the TRPs under the control of the HRE promoter in transduced cells. The RRV also contains a reporter gene, that is either LacZ or GFP and a selectable marker, that is a neomycin resistance gene. Viral particles are produced as outlined above and used to transduced D17 cells. The transduced cell population is split and incubated overnight in normoxia (21% oxygen) and hypoxia (0.1% oxygen). Cells are stained by X-gal histochemistry and end-point titres calculated. Titre is measured of beta-galactosidase gene expression and reflects changes in gene expression between cell populations under conditions of hypoxia and normoxia.
Example 7 - Construction of viral vectors encoding TRPs in combination with a pro-drug activating enzyme The invention is not restricted to the generation of viral vectors encoding TRPs. The combination of TRPs with a pro-drug activating enzyme can be configured in these vectors. Xia-Gen-P450 (Figure 15) contains a therapeutic gene for human cytochrome P450 which activates the anti-cancer compound cyclophosphamide. The CYP2B6 gene is driven by a CMV promoter. The therapeutic genes are expressed as a single transcription unit wherein the TRPs are cloned downstream from an IRES and in place of the LacZ gene. The configuration is therefore P450-IRIJS-TRPs. Any other pro drug activating enzyme can be used in this configuration. The use of a constitutive promoter is optional in the present invention. Another configuration includes the hypoxia regulated promoter HRIthat is capable of driving the expression of the single transcription unit stated above.

Example 8 - In vitro transduction of glioma cell lines a) Transduction of gliomas cell lines with recombinant viral vectors The human gliorna cell line U87MG is transduced with recombinant viral vectors expressing either the TRPs of the present invention, the human Cytochrome P450 or both. 10' U87MG cells are seeded in 6 well dishes and viral particles produced as stated above are added to the cells for 24-48h. Stable tranformed cells are selected in the presence of 400 mg/ml geneticin for 2 weeks. The expression of TRPs and/or P450 in the stable 6418 resistant clones is determined by Northern and Western Blots and the highest expressing cell lines are selected and used in subsequent experiments.
b) In vitro endothelial cell proliferation assay IS HUVEC cells (10') are plated in six-well plates, and culture supernatants of U87MG, U87MG-neo, U87MG-TRPs, U87MG-P450 and U87MG-TRP/P450 stably transduced glioma cells are added to lml of the normal HUVE cell culture medium. Three days later, the HUVE cells are labelled with 2mCi of [3H] thymidine and the incorporation of this compound into the DNA was quantitated by scintillation counting.
In a co-culture assay, the same glioma cells (2x105) are plated in the upper chamber of cell culture inserts and HLTVE (105) cells in the lower chamber. Three days later HUVE cells are labelled as stated above and proliferation rate determined.
c} In vitro sensitivity of U87MG to cyclophosphamide (CPA) U87MG, U87MG-neo, U87MG-TRP, U87MG-P450 and U87MG-TRP/P450 stably transduced glioma cells (~x 103) are seeded in microwell dishes and after 24 hours treated with varying doses of CPA (0-1000 mM) for 24 h. The cells are subsequently washed and fresh media added to them. 48 hours later cell death is assessed by Trypan blue exclusion assay.

G.

Example 9 - In vivo inhibition of angiogenesis in tumours a) Transduction of intracerebral tumours with vectors encoding TRPs.
a. l ) Implantation of subcutaneous tumours U87MG, U87MG-TRP, U87MG-P450 and U87MG-TRP/P450 glioma cells (106) resuspended in 100 ml of HBSS are injected intradermally in the right flank of Balb C
nude mice (NulNu). The mice are sacrificed 20 days post-transplantation and tumour volume is determined using the formula: tumour volume (mm3)= length [mm] x (width [mm])2 x 112.
a.2) Implantation of subrenal tumours Balb C nude mice (NulNu) are anaesthetised with an i.p. injection of hypnorm and hypnovel and 106 of either U87MG, U87MG-TRPs, or U87 MG-P450 or U87MG-TR.P/P450 glioma cells resuspended in 100 ml of HBSS, are injected in the subrenal capsule of the left kidney. The mice are sacrificed 20 days later and both kidneys removed to determine tumour volume, weight and vascularity. Tumour vascularity is determined in paraformaldehyde-fixed sections reacted with an antibody against factor VIII-related antigen (Von Willebrand factor) and visualised by a biotinylated secondary and ABC. Following hematoxylin counterstaining microvessels are counted using light microscopy on x200 fields in areas of the tumour with the highest density of vascular staining.
a.3) Implantation of intracerebral tumours Balb C nude mice (NulNu) are anaesthetised with hypnorm and hypnove! placed in a stereotaxic injection apparatus and an injection of (5x103) U87MG glioma cells resuspended in 10 ml of HBSS are delivered to the right caudate through a small burr hole using the coordinates: bregma, 2.25 mm lateral, 2.5 mm vertical tiom dura. The WO 00/47622 PCTlGB00/00520 injection is performed through a Hamilton syringe over 5 minutes and left in place for a further 5 minutes prior to slow retraction.
b) Transduction of intracerebral tumours with vectors encoding both angiostatic 5 peptides together with P450 B6 and treatment with CPA.
Mice are subsequently divided in 5 different groups which 4 days later received an intratumoural injection of lOml of recombinant virus (10$ particles or LFUlmI) encoding either (1) neo or (2) TRPs or (3) P450 or (4) TRP/P450, using the same burr 10 hole and stereotaxic coordinates. Half of the animals in groups (3) and (4) also receive an i.p. injection of CPA (100mg/kg) two to four days later. The survival of these animals are determined and examination of the tumours in all groups is made histologically after acute or late death. Statistical significance between different groups is assessed using the log rank analysis of Kaplan-Meier survival curves.
Example 10 - Identification of agents capable of inhibiting proangiogenic factors or agents capable of inhibiting the induction of such factors that can be combined with the TRPs of the TRP fragments of the present invention.
1. Selection of an indicator cell line for screening for agents that augment or interfere with HRF dependent transcription as outlined in Figure 16. The selection of an indicator cell line is chosen to satisfy the following requirements:
(i) The optimal indicator cell line is able to mediate a strong response to hypoxia stimulus and requires the stable integration of a transcriptional cassette upstream of suitable reporter (assay readout). This transcriptional unit is able co-operate with the inherent features of the cell line to display low basal activity in normal oxygen tensions with high induced reporter activity under hypoxic stimulus. This provides suitable working range to assess changes in the transcriptional response in the presence of the prospective compound/small molecule being screened. By way of example the characteristics of the transcriptional control element OB HREI (see (WO 99J15684)) would fit this requirement.
(ii) The preferable target cell line is preferably highly responsive to a hypoxic stimulus. This can be initially defined by transient transfection assay or the endogenous level of hypoxia responsive transcription factors as typified by those of the bHLH-PAS family eg HIFIa, HIF2a, HIF3oc as defined by bandshift assay or Western analysis.
(iii) The cell line is preferably biologically stable to ensure assay reproducibility with easy production parameters with regard to culture parameters. Furthermore the cell line is preferably chosen so that it is appropriate for the clinical target tissue in order for it to be recognised as a benchmark cell line of wide use and applicability.
Introduction of the reporter cassette needs to be made in such a way as to keep the hypoxia responsive control elements intact. As a result, standard transfection techniques that allow multiple copy and random recombination may be deemed inappropriate. T'he preferable method to introduce such a transcriptional cassette is by targeted integration via single copy retroviral transduetion. Single copy integrants can be selected if transduction of the parental cell line is performed at low multiplicity of infection (MOI).
Isolation of clones with the characteristics as outlined above:
The incorporation of a selection marker facilitates the isolation of clones that have the characteristics outlined above. The selection can be an antibiotic resistance marker, eg neomycin, and in this case clones can be selected by culture in 6418. A more preferable marker is one that can be sorted using FACS technology, eg GFP or Lac Z
(using FDG substrates). Ideally, the selection marker is controlled in the same way as the reporter for the assay. This regulation can be facilitated using an 1RES
sequence to ;0 produce a co-regulated bicistronic message.

Using a marker that can be FACS sorted allows rapid selection of clones that show high level of expression under hypoxic induction and low basal activity in normoxia.
Rounds of positive selection and negative selection facilitate the production of a cell pool capable of showing low basal and high inducible activity. From such a cell pool S single clones can be isolated and expanded for final selection. Clonality is a requirement for a cell based assay screen.
Construction of a hypoxia regulated retrovirus Viral preparation and transduction methodologies are standard (see (W099/15684)). The genome configuration on transduction is shown in Figure 17.
Suitable cell lines for use as host assay cells include but are not limited to the T47D
cell line (see PC1'/GB98/02885 (W099/15684)) mouse skeletal muscle cell line l~ C2C12 and the rat myocyte line, H9C2.
Selection of stable integrated HRE regulated retroviral genome Cells that show good hypoxic regulation are selected by FACS-sorting using the expression of LacZ. Briefly the transduced cells are induced by overnight culture in hypoxic conditions (0.1%). The cells responsive to this stimulus express Lac Z
(see Figure 17). Lac Z expression can be detected on the FRCS using the Fluoreporter lac Z flow cytometry staining kit (Molecular Probes) according to the manufacturers protocol. Cells showing the highest levels of expression are isolated and then expanded for further rounds of positive FACS selection. X gal staining of sorted cells in normoxia and hypoxia helps to monitor the selection. Cells that show LacZ
expression under uninduced, normoxic, conditions are removed by negative selection using the FACS. This results in a heterogeneous line that shows tight regulation.
Single cell cloning allows the derivation of a homogeneous cell population.
This is required to ensure assay consistency and line stability.

Screening assay Selection of LacZ regulation also isolates cells that similarly regulate luciferase as a result of the genome configuration. The assay readout detection of luciferase expression using the luciferase microtiter luminometry assay (see PCT/GB98/02885) is carried out as follows:
Briefly, 'indicator cells' are plated in a 96-well plate at approximately 104 cells per well (this will depend on cell rype), such that after plating for 24-48h cells are 70%
confluent. Target compounds are added to replicate wells (x4 or greater) with relevant controls (compound buffers). Plates are set up in duplicate, one incubated overnight (18h for example) in normoxia the other incubated overnight in hypoxia. Cells are lysed in situ by the addition of the luciferase assay lysis buffer (outlined in manufacturers protocol) then assayed for luciferase activity.
Uata obtained from normoxie and hypoxic plates reveal modulations of hypoxic induction by comparison with controls (no addition of compound or addition of control buffer).
Results Inhibition of hypoxia responsive gene expression We have designed a high throughput assay that enables the rapid screen of molecules that can inhibit the HIF/HRE signalling pathway as shown in Figure 16. This assay can identify candidate compounds for use in the treatment of disease that depend on neo-angiogenesis such as cancer by interfering with transcription of HRE
dependent genes. These molecules can be used in combination with TRP or TRP fragments of the present invention for the treatment of angiogenesis andlor cancer .
This assay can be applied to identify compounds capable of acting as agonists or antagonists (i.e. modulators) in other therapeutic target pathways some of which are outlined in Figure 16. These therapeutic target pathways include but are not limited to ubiquitination, proteolytic degradation, kinase/phosphatase, dimerisation, DNA
binding, formation of transcriptional complex, interaction with p300, oxygen sensor assays using heme containing protein, redox reaction and/or the kinase signalling pathway.
SEQUENCE LISTING
SEQ ID No. l is the nucleotide sequence encoding human TSP-1 TRP shown in Figure 1.
SEQ ID No. 2 is a consensus TRP amino acid sequence shown in Figure 7. The consensus amino acid sequence (SEQ ID No 2) was derived from 20 different TRP
sequences of the same length which were easily and unambiguously aligned. The 1 S identical residues are shared by at least I 0 sequences.
SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 are also shown in Figure 7.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

60

1. A non-naturally occuring Type I Repeat Peptide (TRP)

2. A TRP according to claim 1 comprising at least the amino acid sequence presented as SEQ ID No. 2 or a variant, homologue, fragment or derivative thereof.

3. A TRP according to claim 2 wherein the TRP comprises the sequence presented as SEQ ID No. 2.

4. A TRP according to claim 1 comprising an amino acid sequence presented as SEQ ID No. 3 or a variant, homologue, fragment or derivative thereof.

5. A TRP according to any one of claims 1 to 3 wherein the TRP comprises an amino acid sequence presented as SEQ ID No. 4 or a variant, homologue, fragment or derivative thereof.

6. A TRP according to any one of claims 1 to 3 wherein the TRP comprises an amino acid sequence presented as SEQ ID No. 5 or a variant, homologue, fragment or derivative thereof.

7. A nucleotide sequence encoding a TRP according to any one of claims 1 to 6.

8. A nucleotide sequence encoding a TRP wherein the nucleotide sequence comprises the sequence presented as SEQ ID No. 1 or a variant, homologue, fragment or derivative thereof.

9. A nucleotide sequence encoding a TRP wherein the nucleotide sequence comprises the sequence presented as SEQ ID No. 1.

10. A nucleotide sequence capable of hybridising to the nucleotide sequence according to any one of claims 7 to 9, or a sequence that is complementary to the hybridisable nucleotide sequence.

11. A nucleotide sequence according to any one of claims 7 to 10 wherein the nucleotide sequence is operably linked to a promoter.

12. A construct comprising the nucleotide sequence according to any one of claims 7 to 11.

13. A vector comprising the nucleotide sequence of any one of claims 7 to 11.

14. A plasmid comprising the nucleotide sequence of any one of claims 7 to 11.

15. A host cell comprising the nucleotide sequence according to any one of claims 7 to 11.

16. A host organism comprising the nucleotide sequence according to any one of claims 7 to 11.

17. A process of preparing a TRP according to any one of claims 1 to 6 comprising expressing a nucleotide sequence according to any one of claims 7 to 11 or when present in the invention of any one of claims 12 to 13 and optionally isolating and/or purifying the TRP.

18. A TRP produced by a process according to claim 17.

19. A TRP which is immunologically reactive with an antibody raised against a purified TRP according to any one of claims 1 to 6 or claim 18.

20. An assay method for identifying whether an entity is a TRP wherein the assay method comprises:
(a) incubating endothelial cells under conditions and for a sufficient time to allow such cells to enter a log phase of growth in a culture medium;
(b) introducing an angiogenic stimulant into the culture medium to induce endothelial cell proliferation and migration;
(c) introducing the entity;
(d) determining whether the entity is capable of inhibiting in vitro endothelial cell proliferation and migration activity such that inhibition of in vitro endothelial cell proliferation and migration activity is indicative that the entity is a TRP.

21. An assay method according to claim 20 wherein the assay is to screen for a TRP
useful in the treatment of angiogenesis and/or cancer.

22. A process comprising the steps of:
(a) performing the assay according to claim 20 or claim 21;
(b) identifying one or more TRPs capable of inhibiting in vitro endothelial cell proliferation and migration activity; and (c) preparing a quantity of those one or more TRPs.

23. A process comprising the steps of:
(a) performing the assay according to claim 20 or claim 21;

(b) identifying one or more TRPs capable of inhibiting in vitro endothelial cell proliferation and migration inhibitory activity; and (c) preparing a pharmaceutical composition comprising those one or more identified TRPs.

24. A process comprising the steps of:
(a) performing the assay according to claim 20 or claim 21;
(b) identifying one or more TRPs capable of inhibiting in vitro endothelia!
cell proliferation and migration activity;
(c) modifying those one or more identified TRPs capable of inhibiting in vitro endothelial cell proliferation and migration activity; and (d) preparing a pharmaceutical composition comprising those one or more modified TRPs.

25. A TRP identified by the assay method of claim 20 or claim 21.

26. A TRP according to any one of claims 1 to 6 or claim 18 or claim 19 or claim 25 wherein the TRP is an angiogenesis inhibitory protein (such as human Thrombospondin-1, Brain Angiogenesis Inhibitor 1, Brain Angiogenesis Inhibitor 2 and Brain Angiogenesis Inhibitor 3).

27. A TRP according to claim 26 wherein the Brain Angiogenesis Inhibitor is an isoform of Brain Angiogenesis Inhibitor 3.

28. A method of affecting in vivo angiogenesis and/or cancer with a TRP;
wherein the TRP is capable of inhibiting endothelial cell proliferation and migration activity in an in vitro assay method; and wherein the in vitro assay method is the assay method defined in claim 20 or claim 21.

29. Use of a TRP according to any one of claims 1 to 6 or claim 18 or claim 19 or claims 25 to claim 27 to prepare a pharmaceutical composition.

30. A pharmaceutical composition comprising a TRP and another therapeutically useful agent.

31. A pharmaceutical composition according to claim 30 wherein the other therapeutically useful agent is a pro-drug activating enzyme.

32. A pharmaceutical composition according to claim 31 wherein the other therapeutically useful agent is an agent capable of inhibiting proangiogenic factors or the induction of such factors.

33. A pharmaceutical composition according to claim 29 or claim 30 wherein the TRP is a TRP according to any one of claims 1 to 6 or claim 18 or claim 19 or claims 25 to claim 27.

34. Use of a TRP in combination with a pro-drug activating enzyme or a nucleotide sequence of interest (NOI] encoding same or an agent capable of inhibiting proangiogenic factors or the induction of such factors for the treatment of condition associated with angiogenesis and/or cancer.

35. Use of a TRP in the preparation of a pharmaceutical composition according to claim 29 or claim 33 for the treatment of a condition associated with angiogenesis and/or cancer.

36. A TRP obtainable from a TRP containing protein wherein the TRP is not a Thrombospondin (TSP) TRP.

37. A fusion protein comprising a TRP.

38. A TRP substantially as described herein and with reference to the accompanying drawings.