JP2002542762A - peptide - Google Patents

Abstract

  (57) [Summary] Non-natural type I repeat peptide (TRP) and its expression method.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a Type I Repeat Peptides (TRP) that can suppress endothelial cell proliferation and migration. More particularly, the present invention relates to the expression of TRP in vitro and in vivo, and their use in preventing and / or treating diseases, ie, conditions associated with angiogenesis and / or cancer.

[0002] Angiogenesis is an essential component of the metastatic pathway. Most solid tumors, when growing,
It produces new blood vessels, so it is well formed. For many tumors, a prognostic indicator for metastatic potential, such as a hypervascularized tumor having a higher metastatic incidence than a hypovascularized tumor, can be obtained from the vessel density. Tumor angiogenesis is regulated by the production of angiogenic activators and inhibitors (Zetter, BR 1
998 Annu, Rev. Med. , 49: 407-24).

It is believed that both regulated and unregulated angiogenesis occurs in a similar manner.
Endothelial cells and pericytes surrounded by the basement membrane form capillaries. Angiogenesis begins with erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells filling the vessel lumen are then extruded through the basement membrane. In certain pathologies, abnormal cell growth results in the local production of factors that can continue to grow by promoting angiogenesis. As an example, vascular endothelial growth factor (vascular e
ndothelial growth factor; VEGF), PDGF and bFGF (Kuwab)
Angiogenic stimulants such as ara et al. PNAS 1995 92 4606) can induce endothelial cells to migrate through the eroded basement membrane. Cell migration results in the formation of "buds" from the parent vessels where endothelial cells divide and proliferate.
The endothelial sprouts merge with each other to form a capillary loop and thus create new blood vessels.

It is clear that angiogenesis plays a major role in cancer metastasis. The growth of cancer, and especially carcinomas, requires angiogenesis to continue growth. One approach to controlling disease progression in cancer is to limit the neovascularization process. If this angiogenic activity could not be suppressed or eliminated, cancer would not proliferate, even if present. In other disease states, prevention of angiogenesis could prevent damage due to invasion of new microvasculature. Thus, these diseases can be alleviated by treatments aimed at regulating the angiogenic process.

[0005] Tissue growth is limited by nutrient supply. During growth, vascular growth maintains the pace of tissue growth to ensure that cells receive adequate levels of oxygenation and nutrient supply. When cell growth exceeds the vascular supply, cells become
Oxygen will be limited. This oxygen limitation, called hypoxic stress, results in changes in gene expression. Many of these changes are related to glycolysis, which is capable of atelectasis, but is accompanied by increased expression of genes that also include factors that promote angiogenesis / angiogenesis.

As an example, VEGF transcription is associated with increased stability of the HIF-1 transcription factor and the resulting enhancer of this transcription factor, hypoxia by binding to the hypoxia responsive element (HRE) in the 5′UTR of the VEGF gene. (Forsy
the et al. MCB 1996 16 4604, Maxwell et al. PNAS 19
97 94 8104). Other examples also include PDGF and bFGF (Kuwabara et al. PNAS 1995 92 4606). Although there is some information about the signaling mechanisms that effect these changes in gene expression, this process is not yet fully understood.

[0007] Therefore, what is needed is a method of controlling the unwanted growth of blood vessels, especially the formation of tumors. This can occur directly by using inhibitors of pro-angiogenic factors or indirectly by identifying compounds / small molecules that can inhibit the induction of such factors. This includes methods to overcome the activity of endogenous growth factors (eg, αFGF, βFGF, VEGF, IL-8, GM-CSF) in pre-metastatic tumors and prevent capillary formation in tumors. , Thereby inhibiting tumor growth. This would also include methods of regulating capillary formation in other angiogenic processes such as wound healing and regeneration.

[0008] A considerable amount of research has been done on the anti-angiogenic properties of the extracellular matrix molecule thrombospondin. Thrombospondin (TSP) has been identified as an inhibitor of keratinocyte proliferation, motility and morphogenesis (Baga).
bandos and Wilks 1990 Biochem Bioph
ys Res Commun 170: 867-72; Vogel et al., 1993.
J Cell Biochem 53: 74-84; Iruela-Aris.
Pe et al., 1991 Proc Natl Acad Sci 88: 5026.
30). Expression of TSP was dependent on p53 expression and on human bladder cancer (Gross
Feld et al., 1997: J. Am. Natl. Cancer Inst. 89: 219
) And angiogenesis in gliomas.

[0009] Although internal peptides mapped to the type 1 repeat (TRP) of human TSP-1 have been reported to reduce cell proliferation and integument cell migration (Tolsma).
Et al., 1993 J Cell Biol 122: 497-511), cDNA
Expression of these peptides from E. coli and the use of such cDNAs in gene therapy has not been reported.

The present invention seeks to provide TRPs that can be expressed under in vitro and in vivo conditions and have anti-angiogenic activity that can be used for the prevention and / or treatment of angiogenesis and / or cancer. Strive.

[0011] Aspects of the present invention are introduced in the following claims and the following description.

Thus, in one aspect, the invention relates to non-natural type 1 repeat peptides (T
RP).

As used herein, the term “type 1 repeat peptide (TRP)” refers to TRP
Type 1 present in one or more copies (TRP sequence) in the containing protein
Means an internal peptide containing repeats. According to the present invention, "TRP" may itself be a peptide or may be part of a fusion protein.

The term “unnatural” TRP according to the present invention means that when it is in its natural environment,
And when the nucleotide sequence is under the control of its native promoter, also in its native environment, T
Means RP.

Thus, the present invention relates to the invention when it is in its natural environment, and when it is expressed by a native nucleotide coding sequence also in its natural environment, and when the nucleotide sequence is also in its natural environment. Does not include the native TRP according to the invention when under the control of its natural promoter.

[0016] Preferably, the TRP is an isolated TRP and / or a purified TRP.

[0017] TRP, whether natural or not, can be obtained or produced from any suitable source. That is, TRP may be a synthetic TRP, a semi-synthetic TRP, an induced TRP, or a recombinant TRP. Preferably, the non-natural TRP comprises a TRP at least in part prepared by recombinant DNA technology, or a TRP produced by chemical synthesis technology, or a mixture thereof.

[0018] More preferably, the non-natural TRP has been prepared by the use of recombinant technology.

The term “derivative” as used herein encompasses chemical modifications of TRP. Examples of such modifications include replacement of hydrogen by an alkyl, acyl or amino group.

[0020] The sequence of the TRP of the present invention may be the same as the natural one, or may be a mutant, homologue, fragment or derivative thereof.

As used herein, “amino acid sequence” refers to a peptide, polypeptide or protein sequence or a portion thereof.

[0022] In one preferred aspect, the invention provides a polypeptide consisting essentially of TRP.

The present invention also includes the nucleotide sequence (s) encoding the TRPs of the present invention. The nucleotide sequences of the present invention are non-natural. Accordingly, the present invention does not include a native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of a native promoter, which is also in its natural environment.

The sequence of the nucleotide sequence encoding TRP may be the same as the native form, or may be a variant, homolog or derivative thereof. Preferably, the sequence encoding TRP is an isolated TRP coding sequence and / or a purified TRP coding sequence. A TRP coding sequence, whether natural or non-natural, can be obtained or produced from any suitable source, ie, it can be synthetic, semi-synthetic, or recombinant.

As used herein, the term “nucleotide sequence” refers to oligonucleotide or polynucleotide sequences, and variants, homologues, fragments and derivatives thereof (parts thereof). The nucleotide sequence may be double-stranded or represent a sense strand, an antisense strand, or a single strand, and may be DNA or RNA of genomic or synthetic or recombinant origin. Can be. The nucleotide sequence need not be a completely natural DNA sequence. Therefore,
The DNA sequence can be, for example, a synthetic DNA sequence, a recombinant DNA sequence (ie, prepared by use of recombinant DNA technology), a cDNA sequence or a partial genomic DNA.
, And may include combinations thereof. The DNA sequence need not be a coding region. If it is a code area, it need not be all the code area. In addition, the DNA sequence can be in a sense orientation or an antisense orientation. Preferably, the DNA sequence is in the sense orientation. Preferably, the DNA is or comprises cDNA.

Preferably, the term “nucleotide sequence” means DNA. More preferably, the term "nucleotide sequence" refers to DNA prepared by the use of recombinant DNA technology (ie, recombinant DNA). Thus, preferably, the invention relates to a DNA sequence (preferably a cDNA sequence) encoding TRP. In particular, the present invention relates to cDNA sequences encoding TRP.

In one preferred aspect, the invention provides a nucleotide sequence consisting essentially of a sequence encoding TRP.

The term “recombinant type 1 repeat peptide (TRP)” means a TRP prepared by the use of recombinant DNA technology.

A polynucleotide sequence encoding a modified TRP that can be used in accordance with the present invention can include different nucleotide residues so that, even if there is a deletion, insertion or substitution in the same TRP or TRP. A good functionally equivalent polynucleotide encoding TRP results.

As used herein, “deletion” is defined as a change in either the nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues are absent, respectively.

As used herein, “insertion” or “addition” refers to the addition of one or more nucleotides or amino acid residues, each, as compared to a naturally occurring TRP, ie, the nucleotide sequence encoding TRP. A change in the nucleotide or amino acid sequence that results.

As used herein, a “substitution” results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

[0033] Alleles of TRP are included within the scope of the present invention. “Allele” or “allelic sequence” as used herein is another type of TRP. The allele results from a mutation, i.e., a change in the nucleotide sequence,
And generally results in a modified mRNA or polypeptide that may or may not be modified in structure or function. A given gene may be completely absent or have one or many allele types. The usual mutagenic changes that give rise to alleles generally result from amino acid deletions, additions or substitutions. Each of these types of changes may occur alone, may occur in conjunction with others, or may occur one or more times in a predetermined order.

The present invention also relates to a DNA sequence comprising the DNA sequence of SEQ ID NO: 1 or an allelic variant of such a sequence.

A very 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.

The present invention provides a DNA comprising the DNA sequence of SEQ ID NO: 1 or an allelic variant thereof.
Related to

The present invention also relates to a non-natural DNA comprising the DNA sequence of SEQ ID NO: 1 or an allelic variant thereof.

A very preferred aspect of the present invention relates to a recombinant DNA comprising the DNA sequence of SEQ ID NO: 1 or an allelic variant thereof.

As used herein, “immunological activity” refers to natural, recombinant or synthetic TRP.
Or any fragment thereof is defined as the ability to induce a specific immune response in the appropriate animal or cell and / or to bind to a specific antibody.

[0040] In one preferred embodiment, the nucleotide sequences and / or their TRPs are in isolated and / or purified form.

As used herein, the terms “isolated” and “purified” refer to at least one of the species that has been removed from the natural environment and / or isolated, and / or to which they are naturally associated. Refers to any molecule, nucleic acid or amino acid sequence that has been separated from other components.

The TRPs of the present invention can be isolated from body fluids such as serum, ascites or urine of a patient, or the TRPs can be prepared using recombinant DNA methods or synthetic peptide chemistry well known to those of ordinary skill in the art. It can be manufactured by a method. Protein purification methods are also well known in the art.

The present invention also relates to a TRP produced by expressing in a transformed host cell an integrated DNA sequence as described above or a variant thereof.

The term “vector” includes expression vectors and transformation vectors.

The term “expression vector” refers to a cassette capable of expression in vivo or in vitro.

The term “transformation vector” refers to a cassette that can be converted from one species to another.

Preferred vectors for use in accordance with the present invention are recombinant viral vectors,
Specifically, it is a recombinant retrovirus vector (RRV) such as a lentivirus vector or an adenovirus vector, which includes a combination of retrovirus vectors.

The term “recombinant retroviral vector”
(RRV) is a vector that has sufficient retroviral genetic information to allow packaging of the RNA genome into viral particles capable of infecting target cells in the presence of the packaging components. Point. Infection of target cells includes reverse transcription and integration into the target cell genome. RRV carries non-viral coding sequences that will be delivered to target cells by the vector. In RRV, independent replication cannot produce infectious retroviral particles in the final target cell. Usually, RRV is essentially a functional g for replication g
Lack of ag-pol and / or env genes.

In a broader aspect, the invention provides a hybrid viral vector system for in vivo gene delivery. The system includes one or more primary viral vectors encoding a secondary viral vector, wherein the primary vector or multiple primary vectors infect a first target cell and contain a secondary viral vector therein. Can be expressed, and the secondary vector is capable of transducing a second target cell.

Preferably, the primary vector can be obtained from or based on an adenoviral vector, and / or the secondary viral vector can be obtained from a retroviral vector, preferably a lentiviral vector. Or based on it.

The term “targeting vector” refers to the ability to infect / transfect / transduce a cell or to be expressed in a target cell, usually within a host organism.
Refers to a vector that is restricted to certain cell types within cells with a common or similar phenotype.

[0052] Preferably, the nucleotide sequence of the present invention is operably linked to a transcription unit.

As used herein, the term “transcription unit (s)” refers to a nucleic acid region containing coding sequences and signals to achieve expression of those coding sequences that have all other coding sequences uniquely. It is. Thus, each transcription unit generally includes at least a promoter, any enhancers, and a polyarydenylation signal.

The term “promoter” is used in the standard sense in the art,
For example, an RNA polymerase binding site. The promoter may contain an enhancer.

The term “enhancer” includes a DNA sequence that binds to other protein components of the transcription initiation complex and thus facilitates the initiation of transcription as directed by its associated promoter.

The term “cell” includes any suitable organism. In a preferred embodiment, the cells are mammalian cells. In a highly preferred embodiment, the cells are human cells.

The term “transformed cell” refers to a cell that has a modified genetic structure. In the context of the present invention, a cell has a modified genetic structure when the vector according to the invention is 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.

As used herein, the term “transgenic organism” refers to an organism that contains a modified genetic structure. In the context of the present invention, the organism has a modified genetic structure since the vector according to the invention was introduced into the organism.

[0060] The invention also provides a method comprising transforming a host cell with a nucleotide sequence of the invention.

The invention relates to a transformed host cell-the cell has been transformed with a nucleotide sequence according to the invention under conditions suitable for the expression of the TRP encoded by said nucleotide sequence. A method comprising culturing-is also provided.

The present invention relates to a transformed host cell-which comprises a nucleotide sequence according to the invention,
Or a derivative thereof, a homologue, or a mutant thereof,-culturing under conditions suitable for the expression of the TRP encoded by the nucleotide sequence; and Also provided is a method comprising recovering said TRP from a transformed host cell.

The present invention also provides a method for producing TRP, comprising: a) transforming a host cell with a nucleotide sequence according to the invention or a derivative, homologue, variant or fragment thereof. And b) culturing the transformed host cells under conditions suitable for producing the TRP; and c) recovering the TRP from the host cell culture.

The terms “variant”, “homolog”, “derivative” with respect to the nucleotide sequence of the present invention
Or "fragment" includes any substitution, mutation, modification, substitution, deletion, or addition of one (or more) nucleic acids from or to that sequence, provided that the resulting The expression product of the nucleotide sequence has endothelial cell growth inhibitory activity and migration inhibitory activity, and is preferably at least the same as the expression product of the sequence covered by SEQ ID NO: 1 (FIG. 1). It is required to have the activity of inhibiting movement.

In particular, the term “homolog” encompasses structural and / or functional identity, provided that the expression product of the resulting nucleotide sequence has endothelial cell growth inhibitory activity and migration inhibitory activity. I do. With respect to sequence identity (ie, similarity), there is preferably at least 75%, preferably at least 85%, more preferably at least 90% sequence identity. More preferably, at least 95
%, More preferably at least 98% sequence identity. These terms are
Allelic variants of the sequence are also included.

The sequence identity with respect to SEQ ID NO: 1 is a simple “visual” comparison (ie, a strict comparison) of any one or more sequences with another sequence such that the other sequence
It can be determined by examining for at least 75% sequence identity to the sequence (s). Relative sequence identity can be calculated using any suitable algorithm for determining identity, for example, using the default parameters, to calculate the percent identity between two or more sequences. Can also be determined by a computer program. A typical example of such a computer program is CLUSTAL. It is advantageous to use the BLAST algorithm with parameters set to default values. BLA
The ST algorithm is available at http: // www. ncbi. nih. gov / BL
AST / blast_help. html, which description is incorporated herein by reference. The search parameters are defined as follows and can be advantageously set to the defined default parameters.

It is advantageous if the “substantial identity” as assessed by BLAST is equal to a sequence that meets EXPECT values of at least about 7, preferably at least about 9, more preferably 10 or more. The default threshold value for EXPECT in a BLAST search is typically 10.

BLAST (Basic Local Alignment Search Tool)
It is a heuristic search algorithm used by the programs blastp, blastn, blastx, tblastn and tblastx. The significance of these programs was determined by the statistical method of Karlin and Altschul (http://www.ncbi.nih.gov/BLAST/b) with several refinements.
last_help. html). The BLAST program was created to search for sequence similarity, eg, to identify homologs to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Natu.
See re Genetics 6: 119-129.

[0069] http: // www. ncbi. nlm. nih. The following tasks were performed by the five BLAST programs available on gov.

Blastp-Compare amino acid query sequences against protein sequence databases.

Blastn—compares nucleotide query sequences against nucleotide sequence databases.

Blastx—compares six-frame concept translation products of nucleotide query sequences (both strands) against a protein sequence database.

[0073] The tblastn-protein query sequence is compared against a nucleotide sequence database that is dynamically translated in all six reading frames (both strands).

Tblastx—compares a six-frame translation of a nucleotide query sequence with a six-frame translation of a nucleotide sequence database.

BLAST uses the following search parameters: HISTOGRAM—displays a histogram of scores for each search. Default is yes (see parameter H in the manual page).

DESCRIPTIONS—Restricts the number of short descriptions of matching sequences reported to the specified number. The default limit is 100 descriptions (see parameter V on the manual page).

EXPECT—Statistically valid threshold for reporting matches against database sequences. Default value is 10, Karlin and Altschul
According to the Probability Model (1990), a match of 10 is simply expected to be found by chance. If the statistical validity threshold by matching is greater than the EXPECT threshold, no match will be reported. The lower the EXPECT threshold, the more severe and the fewer chances of a match that will be 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 isolated HSPs whose statistical validity threshold has a score equal to the CUTOFF value.
Only report against database sequences if at least as high as the original. The higher the CUTOFF value, the harsher and the fewer opportunities for matching that will be reported. (See parameter S in the BLAST manual). Typically, effective thresholds are handled more intuitively using EXPECT.

ALIGNMENTS—Restricts database sequences to the number specified to report high scoring segment pairs (HSPs). The default limit is 50. Only if more database sequences happen to meet the statistical validity threshold for reporting (see EXPECT and CUTOFF below), a match with the maximum statistical validity threshold is reported. (See parameter B in the BLAST manual).

MATRIX-BLASTP, BLASTX, TBLASTN and TBLA
Defines an alternative scoring matrix for STX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 199).
2). Valid alternative choices include PAM40, PAM120, PAM250 and IDENTITY. In BLASTN, there is no matching alternative scoring available. To identify the MATRIX indication in BLASTN, an error response must be returned.

Restrict the STRAND-TBLASTN search to the very first or last strand of the database sequence, or BLASTN, BLASTX or TBLAST
Limit the X search to just the reading frame in the first or last strand of the query sequence.

The FILTER-Wooton & Federhen SEG program (
1993) Computer and Chemistry 17: 149-1
A segment of the control sequence of low compositional complexity as determined by 63, or C
laverie & States (1993) Computers and
Chemistry 17: 191-201 by the XNU program, or for BLASTN, the DUST of Tatusov and Lipman
Mask out segments consisting of short-period internal repeats determined by the program (see http://www.ncbi.nln.nih.gov). Filtering removes statistically valid but not biologically interesting reports (eg, hits for common acidic, basic or proline-rich regions) from the output of the blast and removes the database sequence Resulting in a more biologically interesting region of the query sequence available for matching the identity to

The low complexity sequences found by the filter program are replaced with the letter “N” in the nucleotide sequence (eg, “NNNNNNNNNNNNNN”).
NN "), replaced in the protein sequence with the letter" X "(eg,"
XXXXXXXXXX ").

The filtering is applied not only to the query sequence (or its translation result) but also to the database sequence. The default filtering is BLASTN
Has DUST, and the other programs have SEG.

When applied to sequences in SWISS-PROT, it is not uncommon for none to be masked by SEG, XNU, or both,
You should not expect filtering to always have an effect. Further, in some cases, the entire sequence is masked, suggesting that the statistical validity of any matches reported for the unfiltered query sequence should be doubted.

The NCBI-gi-NCBI gi identifier is indicated in the output, and the acceptance and / or locus name is indicated.

Most preferably, http: // www. ncbi. nlm. nih. gov
Perform a sequence comparison using the simple BLAST search algorithm provided in / BLAST.

Other computer programming methods for determining identity and similarity between two sequences include the GCG program (Devereux et al. 1984 Nucle
ic Acid Research 12: 387) and FASTA (Ats).
Chul et al., 1990 J Molec Biol 403-410).

In some aspects of the invention, no gap penalties are used when determining sequence identity.

The present invention also encompasses nucleotide sequences complementary to the sequences set forth herein, or any fragments or derivatives thereof. If the sequences are complementary to their fragments, the sequences can be used as probes to identify similar promoter sequences in other organisms.

The present invention also encompasses nucleotide sequences capable of hybridizing to the sequences set forth herein, or any fragments or derivatives thereof.

Hybridization is based on “a method in which a nucleic acid strand binds to a complementary strand through base pairing” (Coombs J (1994) Dictionary of).
Biotechnology, Stockton Press, New Yo
rk NY), and Dieffenbach CW and GS Dvek
sler (1995, PCR Primer, a Laboratory Ma)
Nual, Cold Spring Harbor Press, Plainv
view NY) means a method of amplification as performed in the polymerase chain reaction technique.

[0093] Nucleotide sequences capable of hybridizing to the nucleotide sequences set forth herein under conditions intermediate to maximum stringency are also within the scope of the invention. Hybridization conditions were determined by Berger and Kimmel (1987, Guide to Molecular Cloth).
Ning Technologies, Methods in Enzymolog
y, Vol 152, Academic Press, San Diego C
Based on the melting point (Tm) of the nucleic acid bound to the complex as taught in A),
It provides the defined "stringency" described below.

Typically, maximum stringency occurs at about Tm-5 ° C (5 ° C below the Tm of the probe), high stringency occurs at about 5-10 ° C below the Tm, Strength occurs at about 10 ° C to 20 ° C below Tm, and low stringency occurs at about 20 ° C to 25 ° C below Tm. As will be appreciated by one of skill in the art, maximum stringency hybridization can be used to identify or detect identical nucleotide sequences, while intermediate (or low) stringency hybridization is similar or similar. It can be used to identify or detect related nucleotide sequences.

[0095] In a preferred aspect, the present invention relates to a method wherein stringent conditions (eg, 65 ° C)
Under 0.1 × SSC) inclusive of nucleotide sequences capable of hybridizing to the nucleotide sequences of the present invention.

The present invention also encompasses any nucleotide sequence capable of hybridizing to a sequence complementary to the sequence set forth herein, or any fragment or derivative thereof. Similarly, the invention includes nucleotide sequences complementary to sequences capable of hybridizing to the sequences of the invention. These types of nucleotide sequences are examples of variant nucleotide sequences.

In this regard, the term “variant” refers to a nucleotidic designated herein as
Sequence complementary to a sequence that can hybridize to the
. However, preferably, the term “variant” is used under stringent conditions (eg,
0.1 × SSC ｛1 × SSC = 0.15 M NaCl, 0.015Na at 65 ° C. _Three Citrate pH 7.0 °), the nucleoside designated herein as
Includes a sequence that is complementary to a sequence capable of hybridizing to the peptide sequence
You.

[0098] The present invention demonstrates that TRP can be expressed under in vitro and in vivo conditions. Expression of the TRPs of the present invention under conditions where TRP is in vitro and in vivo indicates that TRP can be administered to cancer-bearing humans or animals as an anti-angiogenic therapy, and (ii) prognostic signs (Iii) Serum and urine of cancer patients can be used as a base for analysis of similar anti-angiogenic molecules, as markers can be monitored in human, animal serum, urine or tissues. (Iv) In the treatment of angiogenesis and / or cancer, it can be used alone or in combination with an agent useful for the treatment, which is particularly advantageous.

The present invention relates to a cerebral angiogenesis inhibitor (Brain Angiogenesis Inhibi) containing TRP capable of suppressing endothelial cell proliferation activity and migration activity.
The surprising discovery of a new isoform of tor (BAI) 3 is also demonstrated.

The term “angiogenesis” refers to the creation of new blood vessels in a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific and limited situations. For example, angiogenesis is commonly observed in wound healing of the corpus luteum, endometrium and placenta, fetal / embryonic development and formation.

The term “angiogenesis inhibitor” is used to describe any agent that can prevent the growth of blood vessels into tissues, such as non-vascularized or vascularized tumors. The term generally describes the ability of the molecule to inhibit angiogenesis and, for example, the growth of bovine and / or human capillary endothelial cells in culture in the presence of an angiogenic stimulant. be able to. The term includes agents such as substrate metalloproteinases (MMPs) that are able to degrade extracellular matrix and thus naturally generate inhibitors of both angiogenesis and tumor metastasis.

The term “endothelium” refers to a thin layer of flat epithelial cells lining serous cavities, lymph vessels and blood vessels.

The term “angiogenesis stimulating agent” includes vascular endothelial growth factor (VEGF), PDGF and bGFG (Kuwabara) that can stimulate endothelial cell proliferation and migration.
PNAS 1995 92 4606), but are not limited thereto. The term "angiogenesis stimulating agent" can be used interchangeably with the term "pro-angiogenic factor".

The nucleotide sequences of the present invention may be used for various reasons including, but not limited to, alterations to improve cloning, processing and / or expression of the gene product.
Can be designed to modify the nucleotide sequence encoding For example, to modify the glycosylation pattern using techniques well known in the art, such as site-directed mutagenesis to insert new restriction sites, leading to the mutation, or Codon preferences can be changed.

In another embodiment of the present invention, a nucleotide sequence encoding a native, modified or recombinant form of TRP can be ligated to a heterologous sequence to encode a fusion protein. For example, to screen a peptide library for inhibitors of TRP activity, it may be useful to encode a chimeric TRP that expresses a heterologous epitope identified by a commercially available antibody.

The present invention relates to TRP, or glutathione-S-transferase (GST)
, Biotin, His6, c-myc tag (Emrich et al. 1993 Bioce
m Biophys Res Commun 197 (1): 214-220), hemagglutinin (HA) (Wilson et al (1984) C
cell 37 767) or the FLAG epitope (Ford
1991 Protein Expr Purif Apr; 2 (2): 95.
Also encompasses the use of fusion proteins comprising their enzymatically active fragments or derivatives attached to an affinity tag such as -107).

In another preferred aspect, the fusion recombinant proteins are GST, β-galactosidase, or a relatively large accessory protein, Haemophilus insolubilized, which facilitates their production and purification.
fluenzae). Alternatively, the fusion protein may include a carrier protein such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). In certain embodiments of the invention, the marker sequence is a pQE vector (Qia
gen Inc) and Gentz et al (1989 PN
AS 86: 821-824). Such fusion proteins can be readily expressed in yeast cultures (Mitchell et al. 1993 Yeast 5: 715-723),
And easily purified by affinity chromatography. The fusion protein can also be designed to include a cleavage site located between the nucleotide sequence encoding TRP and the heterologous protein sequence so that the TRP can be cleaved from the heterologous moiety and purified away. In another embodiment, analysis for the target protein can be performed using total binding protein.
Alternatively, accessory proteins can act as adjuvants in the sense of providing a systemic stimulation of the immune system. The auxiliary protein can be linked to either the amino or carboxy terminus of the first protein.

The presence / absence of marker gene expression suggests that the nucleotide sequence and / or its TPR is also present, but its presence and expression should be confirmed.
For example, if a nucleotide sequence encoding TRP is inserted within a marker gene sequence, recombinant cells containing the coding region for TRP can be identified by the presence of marker gene function. Alternatively, the marker gene can be placed in parallel with the nucleotide sequence encoding TRP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of TRP.

Additional methods for quantifying the expression of an identified molecule include radiolabeling (Mel
by PC et al. 1993 J Immunol Methods 159: 235
-44) or biotinylation (Duplaa C et al. 1993 Anal Bio
chem 229-36) nucleotides, co-amplification of control nucleic acids, and standard curves with experimental results. The TRP of interest is present at various dilutions,
Quantitation of multiple samples can be speeded up by performing the analysis in an ELISA format where a spectroscopic or calorimetric response provides rapid quantification.

The present invention relates to expression vectors and nucleotide sequences encoding TRP or variants thereof for generating TRP in vivo or in vitro, homologues, for the purpose of screening for agents capable of affecting TRP expression or activity. body,
It also relates to a transformed host cell containing the fragment or derivative.

In accordance with the present invention, using a nucleotide sequence encoding TRP, a TRP fragment, a fusion protein or a functional equivalent thereof to produce recombinant DNA molecules that direct their expression in a suitable host cell. Can be. In one embodiment of the invention, the nucleotide sequence encoding TRP of the invention is operably linked to a promoter sequence capable of directing the expression of the nucleotide sequence encoding TRP in a suitable host cell. When inserted into a host cell, the transformed host cell can be cultured under suitable conditions until the TRP reaches a sufficient level after lysing the cell and isolating TRP.

The nucleotide sequence encoding TRP itself includes the use of oligonucleotide probes and PCR amplification techniques to identify known target sequences, or sequences suspected of having substantially homologous sequences to the target sequence. Genetic material can be isolated from cells using well-known techniques for cleavage and sequence analysis.

One example of a method for producing TRP using recombinant DNA technology includes: (1) identifying and purifying a TRP containing a protein such as thrombospondin (TSP); and (2) TRP. Synthesizing 5 ′ and 3 ′ DNA oligonucleotide primers for the sequence, (3) amplifying the TRP nucleotide sequence using a polymerase, and (4) nucleotide sequence in a suitable vector such as an expression vector. Inserting an amplified TRP encoding
Inserting the vector containing the nucleotide sequence encoding TRP into a microorganism or other expression system capable of expressing the nucleotide sequence encoding TRP; and (7) simply recombining the recombinantly produced TRP. Release step. The above technology is described in "Molecular Cloning:
A Laboratory Manual "Second Edition
by Sambrook et al., Cold Spring Harbor Pres.
s, 1989.

The TRP nucleotide sequence can be used to (1) isolate messenger RNA from tissue,
(2) using reverse transcriptase to generate the corresponding DNA sequence, and then (3
A) PCR using appropriate primers can also be used to isolate from cells or tissues that express high levels of TRP (such as tumor cells) by amplifying the nucleotide sequence encoding TRP.

[0115] Yet another method for producing TRP or a biologically active fragment thereof is by way of peptide synthesis. Using an analysis system described more fully below, T
Once a biologically active fragment of RP has been found, it can be sequenced, for example, by automated peptide sequencing. Alternatively, isolating the nucleotide sequence encoding the TRP by the method described above allows the DNA sequence to be determined, thereby providing information about the amino acid sequence. Thus, if the biologically active fragment is produced by a method of identification, such as trypsin digestion, or if the fragment is N-terminally sequenced, the remaining amino acid sequence can be determined from the corresponding DNA sequence. .

[0116] Modified TRP nucleotide sequences that can be used in accordance with the present invention include deletions, insertions or substitutions of different nucleotide residues, resulting in a nucleotide sequence encoding the same or a functionally equivalent TRP. .

[0117] Proteins may have deletions, insertions or substitutions of amino acid residues that produce minor changes and result in a functionally equivalent TRP. Amino acid substitutions can be made deliberately based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic similarity of residues, as long as the biological activity of TRP is maintained. . For example, negatively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, Threonine, phenylalanine, and tyrosine.

[0118] TRP can also be expressed as a recombinant protein with one or more additional polypeptide regions added to facilitate protein purification. In such a purification promoting region, a histidine-tryptophan basic unit (porath J (1992) Protein Ex) which can be purified using an immobilized metal is provided.
metal chelating peptides such as pr Purif 3-26328 1), protein A regions that can be purified using immobilized immunoglobulins, and FLA
GS extension / affinity purification system (Immunex Corp, Seattle,
Areas used in WA) include, but are not limited to. Between the purified region and TRP, factor XA or enterokinase (Invitrogen,
Inclusion of a splittable linker sequence (San Diego, CA) is useful to facilitate purification.

[0119] Host cells transformed with a nucleotide sequence encoding TRP can be cultured under conditions suitable for expression and recovery of TRP from cell culture. The protein produced by the recombinant cell may be secreted or contained intracellularly, depending on the sequence and / or the vector used. As will be appreciated by those skilled in the art, expression vectors containing a nucleotide sequence encoding TRP are designed with a signal sequence that directs secretion of the nucleotide sequence encoding TRP through the identified prokaryotic or eukaryotic cell membrane. be able to. Other recombination structures allow the nucleotide sequence encoding TRP to be joined to a nucleotide sequence encoding a polypeptide region that will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell B).
iol 12: 441-53, see also discussion above on vectors containing fusion proteins).

Once the amino acid sequence of TRP is known, techniques well known in the art, for example, “Solid Phase Peptide Synthesis”
: A Practical Approach "E. Atherton an
dR. C. Shoppard, IRL Press, Oxford Engl
and can synthesize the fragment. Similarly, multiple fragments can be synthesized and subsequently ligated together to produce larger fragments. These synthetic peptide fragments can also be prepared using amino acid substitutions at identified positions for testing for agonist and antagonist activity in vitro and in vivo. TRP receptors can be isolated by affinity columns using peptide fragments with high affinity to bind tissue.

The isolation and purification of the TRP receptor is a useful step to elucidate the mechanism of action of TRP. By isolating the TRP receptor, the structure of the nucleotide probe allows the location and synthesis of the receptor to be monitored using in situ and solution hybridization techniques.

The present invention includes any TRP derivative having endothelial cell proliferation and migration inhibitory activity. The present invention includes all TRPs, TRP derivatives and biologically active TRP fragments. These include TRPs with endothelial cell growth inhibition and migration activity having amino acid substitutions or sugars or other molecules attached to amino acid functional groups. The invention also encompasses nucleotide sequences encoding TRP and TRP receptor, and TRP expressed by these nucleotide sequences.

Different TRP fragments can be synthesized for use in some applications. These uses include use as antigens to develop identified antisera, use as agonists and antagonists active at the TRP binding site, and binding to cytotoxic drugs to target and kill cells that bind to TRP. Uses include, but are not limited to, peptides. The amino acid sequences containing these peptides are selected based on their position in the outer regions of the molecule and are accessible for binding to antisera. Tyrosine or lysine, in addition to fragments without these residues, facilitates the labeling of reactive amino and hydroxyl groups on the peptide. GenBank, Brookhaven Protein, SWISS-
These peptide sequences are compared to known sequences using the PROT and PIR databases to determine potential sequence homology. This information facilitates the deletion of sequences that exhibit high degrees of sequence homology to other molecules, thereby increasing the potential for high specificity in developing antisera, agonists and antagonists to TRP.

[0124] TRP or TRP fragments can be synthesized in standard microchemical facilities, and purity can be confirmed using HPLC and mass spectrometry. Methods of peptide synthesis, HPLC purification and mass spectrometry are generally known to those skilled in the art.

TRP and TRP fragments are produced in recombinant E. coli or yeast expression systems and can be purified using column chromatography.

TRP and TRP fragments can be coupled to other molecules using standard methods. The amino and carboxyl termini of TRP can be isotopically and non-isotopically labeled by a number of techniques, for example radiolabeling using conventional methods (tyrosine residues-chloramine T, iodogen, lactoperoxidase; lysine residues). -Boulton-Hunter reagent). These coupling techniques are well known to those skilled in the art. Coupling techniques are selected based on available functional groups on the amino group, which include, but are not limited to, amino, sulfhydral, carboxyl, amide, phenol, and imidazole. The various reagents used to achieve these couplings include glutaraldehyde, diazotized benzidine, carbodiimide, and p-benzoquinone, among others.

TRP and TRP fragments are chemically conjugated to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, radionucleotides and other compounds for various uses. Coupling reaction efficiencies are determined using different techniques appropriate to the reaction of identification. For example, radiolabeled of TRP peptides using ¹²⁵ I is accomplished using chloramine T and Na ¹²⁵ I of high specific activity. The reaction is stopped with sodium metabisulfite and the mixture is desalted using a disposable column. The labeled peptide is eluted from the column and the fraction is collected. A fixed amount is taken from each fraction, and the radioactivity is measured in a γ counter. In this way,
Unreacted Na ¹²⁵ I is separated from the labeled TRP peptide. The peptide fraction with the higher specific activity is stored for later use, such as analysis of the ability to bind to TRP antiserum.

[0128] TRP and TRP fragments are utilized to develop tee columns for the isolation of TRP receptors from cultured tumor cells. Following isolation and purification of the TRP receptor, the amino acid sequence is determined. Next, a nucleotide probe is created for insertion into a vector for expression of the receptor. These techniques are well-known to those skilled in the art. Transfection of TRP receptors into tumor cells increases the responsiveness of these cells to endogenous or exogenous TRP, thereby reducing the rate of metastatic growth.

[0129] Synthetic TRP fragments have a variety of uses. T with high specificity and avidity
The TRP or TRP fragment that binds to the RP receptor is radiolabeled and used for visualization and quantification of the binding site using autoradiography and membrane binding techniques. This application provides important diagnostic and research tools. Knowledge of TRP receptor binding facilitates investigation of the transduction mechanism associated with the receptor.

In addition, labeling these peptides with short-lived isotopes makes it possible to visualize the receptor binding site in vivo using positron emission tomography or other state-of-the-art radiography techniques, and use the TRP binding site to generate tumors. Can be located.

[0131] Systematic substitution of amino acids in these synthetic peptides will result in T
This results in high affinity peptide agonists and antagonists to the TRP receptor that increase or decrease RP binding. Such agonists are used to suppress the spread of micrometastases and, consequently, limit cancer development. Antagonists to TRP are applied in the context of inappropriate vascularization, inhibiting the suppressive effect of TRP and possibly promoting angiogenesis. This treatment has the therapeutic effect of promoting wound healing in diabetic patients.

Another use for peptide conjugation is for the production of polyclonal antisera. For example, TRP or a TRP fragment containing a lysine residue binds to purified bovine serum albumin using glutaraldehyde. Reaction efficiency is determined by measuring the uptake of the radiolabeled peptide. Unreacted glutaraldehyde and peptides are separated by dialysis. The conjugate is stored for later use.

The TRP or TRP fragment of the present invention can also be used to generate antibodies specific for TRP. Antibodies can be polyclonal antibodies,
It can also be a monoclonal antibody. These antibodies, which specifically bind to TRP or TRP receptor, can be used in diagnostic methods and kits well known to those of ordinary skill in the art to detect their presence or to detect TRP or TRP in body fluids or tissues. Receptors can be quantified. The results from these tests can be used to diagnose or predict the occurrence or recurrence of cancer or other angiogenesis-mediated diseases.

[0134] Antisera to TRP can be generated. After peptide synthesis and production, both monoclonal and polyclonal antisera are generated using off-the-shelf techniques known to those skilled in the art. For example, polyclonal antisera can be raised in rabbits, sheep, goats, or other animals. TRP conjugated to a carrier molecule such as bovine serum albumin, or TRP itself, is mixed with an adjuvant mixture, emulsified, and injected subcutaneously at multiple sites on the back, neck, flank, and sometimes feet. Booster injections are given at regular intervals, such as every 2-4 weeks. Blood samples are obtained by venipuncture, for example, using a periaural vein after dilation about 7-10 days after each injection. Blood samples were allowed to clot overnight at 4 ° C.
Centrifuge for about 2400 times at 4 ° C. for 30 minutes. Serum is removed, aliquoted and stored at 4 ° C for immediate use or stored at -20 ° C to -90 ° C for later analysis.

[0135] Serum samples from polyclonal antiserum production or media samples from monoclonal antiserum production were all analyzed to determine titer. The titer can be determined by several methods, such as using dot blot and density analysis, and using Protein A, secondary antiserum, cold ethanol or charcoal dextran to precipitate the radiolabeled peptide-antibody complex, Then, it is set by measuring the radioactivity with a γ counter. The highest titer antiserum is also purified using a commercial Affi-Tee column. TRP binds to the gel in the affinity column. The antiserum sample passes through the column, and the anti-TRP antibody remains bound to the column. These antibodies are then eluted, collected and evaluated to determine titer and specificity.

The highest titer TRP antiserum was tested to: a) optimal antiserum dilution for highest specific and lowest non-specific binding of the antigen; b) binding to increase the amount of TRP in the standard displacement curve. C) potential cross-reactivity with related peptides and proteins, including also plasminogen and related species of TRP, d) the ability to detect TRP in plasma, urine, tissue extracts, and cell cultures .

Using passive antibody therapy with antibodies that specifically bind TRP, regeneration, development,
And modulate angiogenesis-dependent processes such as wound healing and tissue repair. In addition, an antiserum for the Fab region of the TRP antibody is administered to allow the endogenous T
RP antisera can inhibit the ability to bind to TRP.

The present invention also includes diagnostic methods and kits for detecting and measuring TRP in biological fluids and tissues, and for locating TRP in tissues.

[0139] TRP can be used in diagnostics and kits to detect and quantify antibodies capable of binding TRP. With these kits, circulating TRs showing the spread of micrometastases in the presence of TRP secreted by the primary tumor in situ
Detection of P antibody is possible. Patients with such circulating anti-TRP antibodies will be more likely to develop tumors and cancer, and will develop after treatment or remission. The anti-TRP Feb fragment antiserum can be generated using the Fab fragments of these anti-TRP antibodies as antigens, and the antiserum can be used to neutralize the removal of circulating TRP by the anti-TRP antibodies.

A kit for measuring TRP is also included as a part of the present invention. An antiserum with the highest titer and specificity and capable of detecting TRP in plasma, urine, tissue extracts and cell cultures is a fast, reliable and specific measurement and location of TRP Further testing will be done to establish the ease of use of the kit for the determination. These assay kits include the following technologies: competitive and non-competitive assays, radiolabeled immunoassays, bioluminescence and chemiluminescence assays, fluorescence assays, sandwich assays,
Include, but are not limited to, 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 Not done. For each kit, set the range, sensitivity, precision, reliability, specificity and reproducibility of the analysis. Intraassay and interassay variation values are set at 20%, 50% and 80% points on the standard curve of displacement or activity.

One example of an analysis kit commonly used in research and in clinics is a radiolabeled immunoassay (RIA) kit. After successful radioiodination and purification of TRP or a TRP fragment, the antiserum with the highest titer, at several dilutions, emits a relatively constant amount of radiation (eg, 10,000 cpm) in a suitable buffer system. Add to a functional test tube. Other tubes contain buffer or pre-immune serum to determine non-specific binding. After incubation at 4 ° C. for 24 hours, protein A is added, the tubes are vortexed and incubated at room temperature for 90 minutes, about 2000-25
Centrifugation at g for 4 times at 4 ° C. precipitates the antibody complex bound to the labeled antigen. The supernatant is removed by suction, and the radioactivity in the pellet is counted with a gamma counter. About 10-40% of labeled TRP or TRP after subtraction of non-specific binding
The properties of the diluted antiserum solution that bind to the fragments are further determined.

Next, the T used for the generation of a dilution range (about 0.1 pg to 10 ng) of antiserum was
The RP or TRP fragment is evaluated by adding a known amount of peptide to a tube containing radiolabeled peptide and antiserum. After a further incubation period, for example 24-48 hours, Protein A is added, the tubes are centrifuged, the supernatant is removed and the radioactivity in the pellet is counted. Radioactive sign TRP
Alternatively, displacing the binding of the TRP fragment with unlabeled TRP or a TRP fragment (standard) generates a standard curve. Several concentrations of other TRP fragments, TRP from different species, and homologous peptides are added to analytical tubes to characterize the specificity of the TRP antiserum.

Another aspect of the invention is a method of inhibiting the action of excess endogenous TRP. This can be done by passively immunizing a human or animal with an antibody specific for the unwanted TRP in the body. This treatment may be important in treating abnormal ovulation, menstruation and placentation, and angiogenesis. This provides a useful tool to test the effect of TRP removal on the metastatic process. TR
The Fab fragment of the P antibody has a TRP binding site. This fragment is isolated from the TRP antibody using techniques known to those skilled in the art. The Fab fragment of the TRP antiserum is used as an antigen to generate anti-Fab fragment serum production. Therapeutic benefit is that by inhibiting the binding of TRP to the Fab fragment of anti-TRP, endogenous anti-TR
Obtained by neutralizing the P antibody. The net effect of this treatment is to promote the ability of endogenous circulating TRP to reach the target cells, and thus reduce the spread of metastases.

Another kit is used for locating TRP in tissues and cells. The TRP immunohistochemistry kit includes serum and secondary antibodies that bind to instructions, TRP antisera, and fluorescent compounds such as fluorescein isothiocyanate, or some other reagents used to visualize primary antisera. Provide for potentially inhibiting serum. This TRP immunohistochemistry kit can confirm the location 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, a biopsy is performed on the tumor, harvested, a tissue section is cut using a microtome, and the site of the TRP product is examined. Such information is useful for clinical and potential therapeutic purposes in detecting and treating cancer.

The invention also encompasses gene therapy in which the nucleotide sequence encoding TRP is modulated in vivo. For example, modulation of expression applies a compound that binds to the nucleotide sequence encoding TRP, or a regulatory region associated with the nucleotide sequence encoding TRP, or its corresponding RNA transcript to improve transcription or translation rate. Can be achieved by:

As an example, the nucleotide sequence encoding the TRP of the present invention may be under the control of expression by an expression control element, usually a promoter or promoter and enhancer. The enhancer and / or promoter may be hypoxic, such that the nucleotide sequence encoding TRP is preferentially expressed in the identified tissue of interest, such as those in the environment such as a tumor, ischemic joint or other site. Or it can act preferentially in an ischemic or low glucose environment. Therefore, TR
Any biological or detrimental effects of the nucleotide sequence encoding P on the individual to be treated can be reduced or eliminated. Similarly, or in addition, the enhancer and / or promoter may preferentially act on one or more specific cell types, such as any one or more macrophages, endothelial cells or a combination thereof. Can be. Additionally, examples include terminally terminally differentiated non-replicating cells such as respiratory airway epithelial cells, hepatocytes, myocytes, cardiomyocytes, synovial cells, primary breast epithelial cells, and macrophage neurons.

The promoter and / or enhancer may be structurally effective,
It may be a tissue, ie a tissue whose activity is temporarily restricted. Examples of suitable tissue-restricted promoters / enhancers are those that are highly active in tumor cells, such as promoters / enhancers from the MUC1 gene, the CEA gene or the 5T4 antigen gene. Examples of temporally restricted promoters / enhancers are those that respond to ischemia and / or hypoxia, such as hypoxia response elements or promoters / enhancers from the grp78 or grp94 genes. The α-fetoprotein (AFP) promoter is also a tumor-specific promoter. One preferred promoter-enhancer combination is the human cytomegalovirus (hCMV) primary immediate early (MIE) promoter / enhancer combination.

Preferably, the promoter of the invention is tissue-specific. In other words, the promoter maintains T silence in one tissue while maintaining “silence” mainly in other tissue types.
The nucleotide sequence encoding RP can be transcribed.

The term “tissue specificity” does not limit activity to a single tissue type, but nevertheless, they are active in one group of tissues and less active or silent in another group It means a promoter showing selectivity. A desirable property of the promoter of the present invention is that it has relatively low activity in the presence of an activated hypoxia-regulated enhancer element, even within the target composition. One means of accomplishing this is to use a "silencer" element that suppresses the activity of the selected promoter under non-hypoxic conditions.

The term “hypoxia” refers to a condition in which an identified organ or tissue receives an insufficient supply of oxygen.

[0151] The expression level of a nucleotide sequence encoding TRP under the control of the identified promoter can be regulated by manipulating the promoter region. For example, different domains within a promoter region can have different gene regulatory activities. Typically, the role of these different regions is analyzed using vector constructs with different variants of the promoter with the identified region deleted.
Ie, deletion analysis). Using this approach, for example, the smallest region that can confer tissue specificity or the smallest region that confer hypoxia sensitivity can be identified.

A number of the above-described tissue-specific promoters will be particularly advantageous in the practice of the present invention. Most often, these promoters can be isolated as convenient restriction digest fragments suitable for cloning on the selected vector. Alternatively, the promoter fragment can be isolated using the polymerase chain reaction. Cloning of the amplified fragment can be facilitated by incorporating a restriction site at the 5 'end of the primer.

[0153] TRP or TRP fragments can be used in combination with other compositions and procedures for treating disease. As an example, TRP or a TRP fragment can be used in combination with either an inhibitor of a pro-angiogenic factor (angiogenesis stimulating agent) or a compound / small molecule capable of inhibiting the induction of such factor. As already mentioned, the expression of some of these pro-angiogenic factors / angiogenic stimulants is induced by hypoxia. As an example, VEGF transcription is due to the increased stability of the HIF-1 transcription factor and the resulting binding of this transcription factor to enhancer elements, a hypoxia responsive element (HRE) in the 5′UTR of the VEGF gene. , Induced by hypoxia (Forsythe et al. MCB 1996 16
4604, Maxwell et al., PNAS 1997 94 8104). HIF /
Potential antagonists of the HRE signaling pathway are expected to suppress hypoxia-mediated induction of angiogenic factors and will therefore be inhibitors of tumor angiogenesis.
A series of reporter structures have been found to bind to the HRE and function in different vector backbones and different cell types (see PCT / GB98 / 02885). These reporter structures can be adapted for small molecule screens to find antagonists that can be conveniently mixed with the TRPs and TRP fragments of the present invention.

TRP and TRP fragments can also be used in combination with the usual treatment of disease. As an example, tumors may be treated conventionally with TRP or TRP fragments in conjunction with surgery, radiation or chemotherapy, or TRP or TRP
The fragments may be administered to the patient at a later time to silence micrometastases and stabilize any remaining primary tumor.

TRP can be delivered at the same moment, at the same site, with a therapeutically effective agent. Alternatively, the TRP and therapeutically effective agent can be delivered at different times and to different sites. The TRP and the therapeutically effective agent can even be delivered in the same delivery vehicle for the prevention and / or treatment of angiogenesis and / or cancer.

The TRP or TRP fragment can be used in combination with a cytotoxic agent for the prevention and / or treatment of angiogenesis and / or cancer. TRP, high affinity TRP
Fragments, TRP antisera, cytotoxic agents such as ricin conjugated to TRP receptor agonists and antagonists provide tools for destroying cells that bind TRP. These cells can be found at many sites, including but not limited to micrometastases and primary tumors.

[0157] TRP or a TRP fragment can be used in gene therapy in combination with a prodrug enzyme. Instead of, or in conjunction with, selective expression in a target cell, TRP or a TRP fragment may comprise another nucleotide sequence of interest (NOI
) Or encodes a prodrug activating enzyme or enzymes that have no significant or detrimental effect until the individual is treated with one or more prodrugs once the enzyme or enzymes are acted upon It can be used in combination with the NOI. Treatment of an individual with an appropriate prodrug in the presence of active NOI
Intensifies the reduction of tumor growth or survival.

[0158] Prodrug activating enzymes can be delivered to a tumor site for the treatment of cancer. The appropriate prodrug in each case is used for treating the patient in combination with the appropriate prodrug activating enzyme. The appropriate prodrug is administered with the vector. Examples of prodrugs include etoposide phosphate (along with alkaline phosphatase, Center et al. 1988 Proc Natl Acad Sci 85
: 4842-4846); 5-fluorocytosine (along with cytosine deaminase, Mullen et al. 1994 Cancer Res 54: 1503-1506).
); Doxorubicin-Np-hydroxyphenoxyacetamide (along with Penicillin-V-amidase, Kerr et al. 1990 Cancer Immunol)
Immunother 31: 202-206); para-N-bis (2-chloroethyl) aminobenzoylglutamate (with carboxypeptidase G2); cephalosporin nitrogen mustard carbamate (with β-lactamase); SR4233 (with P450 reductase); (
Borrelli et al. 1988 Proc Na with HSV thymidine kinase.
tl Acad Sci 85: 7572-7576); mustard prodrug with nitroreductase (Friedlos et al. 1997 J Med Ch).
em 40: 1270-1275) and cyclophosphamide (along with P450, Chen et al. 1996 Cancer Res 56: 1331-1340).

Examples of suitable prodrug activating enzymes for use in the present invention include thymidine phosphorylase, which activates the 5-fluorouracil prodrug capsetabin and furturon; thymidine kinase from herpes simplex virus, which activates ganciclobia; Cytochrome P450, which activates prodrugs such as cyclophosphamide to be DNA damaging agents; and cytosine deaminase, which activates 5-fluorocytosine. Preferably, a human-derived enzyme is used.

Other suitable NOI sequences for use in the present invention include cytokines, chemokines, hormones, antibodies, specially designed immunoglobulin-like molecules, single-chain antibodies, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulators. Molecules, antisense RNAs, transdominant negative mutants of target proteins, toxins, conditional toxins, antigens, tumor suppressor proteins and growth factors, membrane proteins, vasoactive proteins and peptides, antiviral proteins and ribozymes, and their derivatives ( (Including but not limited to, related reporters, etc.) for therapeutic and / or diagnostic uses. When listing such a code sequence,
These can typically be operably linked to a suitable promoter.
Suitable promoters may be those that express the ribozyme (s), may be different promoters, such as one or more specific cell types, or multiple promoters.

[0161] The expression product encoded by the NOI may be a protein secreted from the cell. Alternatively, the NOI expression product is not secreted and is active in the cell.
In both cases, the NOI expression product exhibits a third-party effector or third-party distant effect, ie, the production of the expression product in one cell is contiguous or remote (eg, Preferably, it results in the killing of another related cell (of the metastasis).

Suitable NOIs for use in the present invention for treating and preventing cancer include:
Destroys target cells (eg, ribosomal toxin); tumor suppressor (wild-type p53)
Activators of anti-tumor immune mechanisms (such as cytokines, costimulatory molecules and immunoglobulins), act as inhibitors of angiogenesis; or result in enhanced drug sensitivity (prodrug activating enzymes); by natural effector cells NOIs that encode proteins that indirectly stimulate the destruction of target cells (eg, prodrug activating enzymes). The encoded protein also destroys third-party tumor cells (
For example, indirectly stimulates the destruction of third-party tumor cells (eg, cytokines that stimulate the immune system or procoagulant proteins that cause local vascular occlusion), along with secreted anti-tumor antibody-ribosome toxin fusion proteins. Alternatively, the precursor is changed to a toxin substance that destroys third-party tumor cells (eg, an enzyme that activates a prodrug into a diffusible drug).

It also interferes with the expression of cellular genes for tumor survival (eg, for ectopic myc transcription in Burkitt's lymphoma or for bc in chronic myeloid leukemia).
N encoding antisense transcript or ribozyme (for r-abl transcription)
Delivery of OI. Use of such a combination of NOIs is also envisioned.

[0164] Suitable NOIs for use in treating or preventing ischemic heart disease include NOIs that encode plasminogen activators. Suitable NOIs for the treatment and prevention of rheumatoid arthritis or cerebral malaria include anti-inflammatory proteins, antibodies directed to tumor necrosis factor (TNF) α, anti-adhesion molecules (antibody molecules specific for adhesion molecules). Or a gene encoding a receptor or the like).

An example of a hypoxia-regulated therapeutic NOI is PCT / GB95 / 00322 (WO-A-
95/21927).

As is well known in the art, a vector is a tool that allows or facilitates 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 technology can transfer an entity (eg, a heterologous DNA segment, such as a heterologous cDNA) to a target cell, such as a DNA fragment. Optionally, once inside the target cell, the vector helps maintain heterologous DNA within the cell and can serve as a unit of DNA replication. Examples of vectors used for recombinant DNA technology include plasmids, chromosomes, artificial chromosomes or viruses.

[0167] Vectors can be delivered by viral or non-viral techniques.

[0168] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes a method of delivering a gene to a target mammalian cell using a non-viral vector.

Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic drug-mediated, cationic facial amphipathic Substance (CFA), (Nature
Biotechnology 1996 14; 556), polyvalent cations such as spermine, cationic lipids or polylysine, 1,2-bis (oleoyloxy).
-3- (trimethylamino) propane (DOTAP) -cholesterol complex (
Wolff and Truvetskay 1998 Nature Bio
technology 16: 421) and combinations thereof.

[0170] Viral delivery systems include adenovirus vectors, adeno-associated viruses (
AAV), herpesvirus, retrovirus, lentivirus or baculovirus vectors,
Not limited to them.

Examples of retroviruses include mouse leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary adenocarcinoma virus (MMTV), Rous sarcoma virus (RSV), Fujinami Sarcoma virus (Fu
SV), Moloney murine leukemia virus (Mo-MLV), FBR mouse osteosarcoma virus (FBR MSV), Moloney mouse sarcoma virus (Mo-MSV),
Abelson mouse leukemia virus (A-MLV), avian myeloid cell virus
29 (MC29), and avian erythroblastosis virus (AEV).

A detailed list of retroviruses can be found in Coffin et al (“Retor
ovirense "1997 Cold Spring Harbor Lab
laboratory Press Eds: JM Coffin, SM Hughe
s, HE Varmus pp 758-763).

[0173] Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include human immunodeficiency virus (HIV), the causative agent of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV
), But are not limited thereto. The non-primate lentivirus group includes the prototype "slow virus" visna / maedivirus (VMV), and related goat arthritis-encephalitis virus (CAEV), goat infectious anemia virus (EIAV)
), And the feline immunodeficiency virus (FIV) and bovine menequisen virus (BIV) that have been described more recently.

The difference between the lentivirus lineage and other types of retroviruses is that the lentivirus has the ability to infect both dividing and non-dividing cells (L
ewis et al. 1992 EMBO. J 11: 3053-3058; Lewis
and Emerman 1994 J.M. Virol. 68: 510-516).
. In contrast, other retroviruses-such as MLV-are, for example, muscle, brain,
Unable to infect non-dividing cells, such as those that make lung and liver tissue.

Other examples of vectors include semi-vivo delivery systems, including, but not limited to, electroporation, DNA biolistics, lipid-mediated transfection, and DNA transfection methods such as compacted DNA-mediated transfection. Can be

[0176] The vector may be a plasmid DNA vector. Alternatively, the vector may be a recombinant viral vector. Suitable recombinant viral vectors include adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, or preferred retroviral vectors. In the case of viral vectors, gene delivery is mediated by viral infection of target cells.

[0177] The vectors of the present invention are formed as split intron vectors. Split intron vectors are described in PCT Patent Application GB98 / 02885 (WO99 / 1568).
4) and GB / 98/02867 (WO 99/15638).

When the characteristics of adenovirus are used in combination with the genetic stability of retrovirus / lentivirus, essentially a transient retrovirus that can stably infect adjacent cells using adenovirus Transduce target cells to be production cells. Such retroviral producer cells engineered to express the TRP and / or NOI (s) of the invention are implanted into an organism such as an animal or human for use in treating angiogenesis and / or cancer. be able to.

The dosage of the TRP of the present invention will depend on the condition or condition of the disease to be treated and the body weight,
It will depend on other clinical factors such as the condition of the human and animal and the route of administration of the compound. Depending on the half-life of TRP in the animals and humans identified, TRP can be administered between several times a day and once a week. It will be appreciated that the present invention has both human and veterinary use applications. The method of the present invention contemplates single as well as multiple administrations, which may be administered simultaneously or over time.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions containing antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended host; Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations may be presented in single or multi-dose containers, for example, sealed ampules and vials, or may be used in sterile liquid carriers, for example, if only addition of water for injection is required. It may be stored in a freeze-dried (freeze-dried) state until immediately before. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

TRP is mediated by angiogenesis or is effective in treating diseases or processes involving angiogenesis. The present invention relates to an effective amount of TRP or TRP agonist and / or
Alternatively, a method of treating an angiogenesis-mediated disease using an antagonist is also included. T
The RP and TRP fragments can be provided as isolated, substantially purified proteins and protein fragments in pharmaceutically acceptable compositions using conventional formulation methods known to those skilled in the art. These include oral, rectal, ocular (including in vivo or intraocular), nasal, topical (including buccal and sublingual), intrauterine, vaginal, or parenteral (subcutaneous, intraperitoneal, intramuscular, intravenous) (Including intra, intradermal, intracranial, intratracheal, and epidural), transdermal, intraperitoneal, intracranial, intraventricular, intracerebral, intravaginal, intrauterine, or parenteral (
(Intravenous, intrathecal, subcutaneous or intramuscular) routes include, but are not limited to.

The TRP formulations can be conveniently presented in unit dosage form and can be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient with the pharmaceutical carrier (s) or 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 formulated with a biodegradable polymer that allows for sustained release of the compound, wherein the polymer is used where drug delivery is desired, such as a tumor. Implanted near the site or implanted so that TRP is slowly released throughout the body. Biodegradable polymers and their uses are described, for example, in Brem et al. Neurosurg 74: 441-446 (1991)
In more detail. Using an osmotic minipump, high concentrations of TRP can be controlledly delivered through a cannula to a site of interest, for example, to the site of metastatic growth or directly to the blood vessels supplying the tumor.

The peptides that bind to the cytotoxic agent are infused in a manner designed to maximize delivery to the desired site. For example, a ricin-binding high-affinity TRP fragment is delivered through a cannula to a vessel filling the target site or directly to the target. These agents are also delivered in a controlled manner through an osmotic pump coupled to an infusion cannula.

Preferred unit dosage formulations contain a daily dose or unit of the component to be administered, a daily sub-dose as recited herein above, or an appropriate proportion thereof. Is what you do. It will be appreciated that, with respect to additions to the components specifically mentioned above, the formulations of the present invention may include other agents common in the art that have ligands for the type of formulation in question.

A pharmaceutical composition comprising an effective amount of a TRP expression product or a nucleotide sequence encoding a TRP of the present invention can be used for the treatment of angiogenesis and / or cancer.
Persistent and unregulated angiogenesis occurs in a large number of disease sites, tumor metastases and abnormal growth by endothelial cells, supporting the pathological disorders seen in these conditions. A variety of pathological diseases in which unregulated angiogenesis exists are grouped together as angiogenesis-mediated diseases. Diseases mediated by angiogenesis include solid tumors; blood-borne tumors such as leukemia; tumor metastases; benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachoma and purulent granulomas; rheumatoid arthritis; Ocular angiogenic diseases, such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, post-corneal transplant rejection, neovascular glaucoma, retro lens fibrosis, rubeosis; Osler-Weber syndrome;
Myocardial angiogenesis; plaque angiogenesis; telangiectasia; haemophilia arthropathy; angiofibroma; wound granulation; coronal side branch; cerebral side branch; arteriovenous malformation; Diabetic angiogenesis; including, but not limited to, helioobacter-related diseases, fractures, angiogenesis, hematopoiesis, ovulation, menstruation and placenta formation.

TRP is useful for treating diseases involving excessive or abnormal stimulation of endothelial cells.
These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, ie, keloids. TRP is
It can be used as a birth control drug because it prevents angiogenesis necessary for implantation. TRP is cat scratch disease (Rochele minalia quintos)
It is useful in the treatment of diseases having angiogenesis as a pathological result, such as a) and ulcers (Helobacter pylori).

The present invention will now be further described, by way of example only, by reference to the following.

EXAMPLES Example 1-Construction of Expression Vector Encoding Type 1 Repeat Fragment Human thrombospondin 1 with open reading frame (FIG. 2)
A cDNA corresponding to TRP of TSP-1) is amplified from human poly A + mRNA. First, mRNA is reverse transcribed into cDNA using reverse transcriptase (RT), and then
RT-PCR to amplify this cDNA by polymerase chain reaction (PCR)
Performs amplification. The primer used for this reaction (FIG. 3) was a commercially available vector pSecTag2. Hi of A (Invitrogen, FIG. 4)
In frame with an ndIII site and at the 3 'end the N of the same vector
Designed in-frame to have an otI site. The resulting plasmid is called pSec.
Called Tag2-TRP1.

5 'HindIII XXA AGC TTX 3'NotI XGC GGC CGC

A vascular instability regulatory peptide (or non-TRP containing peptide) corresponding to the C-terminal of human TSP-1 (FIG. 2) is also cloned in the same frame into the above vector. A set of four synthetic oligonucleotides encoding the control peptide was designed (FIG. 5) and digested with the enzymes HindIII and NotI in pSecTag. Cloning into 2A (FIG. 4), pSecTag2-TR
This produces P2.

The vector pSecTag2 contains a mouse Igκ chain leader sequence that allows for secretion of TRP. This vector also contains a c-myc epitope that is integrated at the C-terminus of TRP. Thereby, the fusion protein can be detected using the anti-c-myc antibody. Finally, pSecTag2 contains a 6x histidine tag downstream of the c-myc epitope, which facilitates high affinity binding to Ni2 + chelating resins for easy purification of the fusion protein. These structures allow efficient and high level expression of recombinant polypeptides from human CMV adjacent to the early gene promoter-enhancer.

[0193] Plasmids are isolated by standard methods (ie, calcium phosphate) at 293.
T cells are transfected and the expression of the fusion polypeptide in the transfected cells is confirmed by Western blotting. Prostate specific antigen (PSA) expression plasmid, pSecTag2. A based on pSecTag2 / PSA (Invi
trogen) is used as a control. The medium in which the 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 20 ul of control medium is poured on 10-12% SDS / PAGE. Gel is immobilon membrane (
Millipore Inc. ) Electroblot up. C-myc mouse monoclonal antibody (Boehringer) at 1: 1000 dilution and 1/2
Western blot analysis is performed using rabbit HRP-conjugated anti-mouse IgG (Dako) at a dilution of 000. Detection by chemiluminescence is performed using an ECL kit (Amersh).
am International, UK). The amount of recombinant fusion protein produced in the total protein extract of transfected cells is also investigated. Transfected cells were washed in PBS, dissolved in RIPA buffer containing 1 mM freshly prepared PMSF, and Bradford's BioRad reagent (Bio
Determine total protein concentration using Rad) according to the manufacturer's specifications. Run cell lysate (20-50 ug of total protein) on a 10-12% SDS / polyacrylamide gel. The protein is transferred to Immobilon membrane and the above Western blot is performed.

Example 2 In Vitro Endothelial Cell Proliferation Assay The anti-angiogenic effect of different recombinant TRPs is determined in vitro by an endothelial cell proliferation assay. Similar methods for anti-angiogenic factors such as endostatin, endogenous inhibitors of angiogenesis (O'Reilly et al. 1997 Cell 88: 1) or interleukin 4 (Volpert et al. 1998 J Exp Med 188: 1039-1046) As previously described.

A human kidney cell line, 293T, is used to produce high levels of secreted recombinant TRP using the vector expressing pSecTRP. 293T cells are transiently transfected with 20 ug of each vector per 10 cm ² tissue culture by the calcium phosphate method. After transfection for 48 hours, the medium from the transfected cells was filtered with a 0.45 micron pore size filter (Millipore).
e) Filter through and add to endothelial cells. Endothelial cells for inclusion in the in vitro endothelial cell proliferation assay of the present invention include human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HDMVEC), and bovine adrenal capillary endothelial cells (BA
CE), but are not limited thereto. For proliferation analysis, the endothelial cells are washed in PBS and dispersed in a 0.05% trypsin solution. Cell suspension (25,000 cells / ml) using appropriate endothelial cell medium containing 10% bovine calf serum.
) And plated in 24-well tissue culture dishes covered with gelatin (0
. 5 mL / well) and grow for 24 hours. In the presence of appropriate growth factors, 2
Serial dilution of control or base medium from 93T transfected cells 0.5
Replace the medium with mL. The endothelial cells were grown for 72 hours and then diffused into trypsin to obtain Hematall (Fisher Scientific, Pitts).
(Burgh, PA). Count the cells using a Coulter counter.

Example 3 In Vitro Endothelial Cell Migration Assay This example determines whether different recombinant TRPs have anti-angiogenic effects by inhibiting endothelial cell migration in vitro. Migration of different vascular endothelial cell types is determined by adding recombinant TRP to cultured endothelial cells, ie, bovine adrenal capillary endothelial cells (BACE), HUVE cells and HDMVE cells.

Using a protocol similar to that outlined in Example 2, recombinant TRP is produced in a human hepatocyte cell line, 293T. The medium from the transfected cells was filtered through a 0.45 micron pore size filter (Millipore)
Add to endothelial cells. Endothelial cells are plated on gelatin-coated nucleopore membranes (5 micron pore size for BACE, 8 micron pore size for others) in an inverted modified Boyden chamber. After 2 hours, the chamber is inverted again and the test substance is added above each well. Specifically, the cell population was either culture medium alone (control), culture medium containing 10 ng / mL bFGF (basal fibroblast growth factor), or culture medium containing increasing concentrations of recombinant TRP and 10 ng / bFGF. Expose one. The cells are allowed to move for 3-4 hours. Thereafter, the membrane is fixed, stained, and the number of migrated cells is counted.

Example 4 Identification of Type 1 Repeat Peptides Sequence analysis was performed using the third amino acid sequence of the type 1 repeat of hTSP-1. Search the National Biotechnology Information Center (NC)
BI) Advanced BLAST 2.0 Network Service (A
ltschul et al., 1997 Nucleic Acids Res. 25,3
389-3402). Alignment was performed using the nr database. In this database, sequences are derived by conceptually translating sequences from the nucleotide sequence database.

A BLAST search performed using TSP-1 TRP (FIG. 6) shows agreement with human TSP-1, a TSP-1 precursor from different species and TSP-related proteins. This search is based on the human semaphorin F homolog, Sco-spondin (Bo
staurus) and F-spondin, all of which have been shown to be proteins expressed in the central nervous system and responsible for axonal guidance. TSP-1 TRP also shows homology to cerebral angiogenesis inhibitor (BAI) 1, 2 and 3 proteins and two novel human proteins, KIAA0550 and KIAA0688 proteins (FIG. 7), all of which are Expressed in tissue.

Example 5 Further Identification of Type 1 Repeat Peptides The molecular cloning and characterization of the BAI 1 gene has been described previously (Nishimori et al., 1997 Oncogene, 15, 2145-2).
150). BAI 1 has been shown to be specifically expressed in the brain and suppresses experimental angiogenesis in a rat corneal model. The expression of the BAI1 gene is absent or significantly reduced in the neuroglioblastoma cell line. Just recently,
The cloning and characterization of two other genes in this line have been reported, and BAI
2 and BAI3 (Shiratsuchi et al, 1997 Cyto)
(Genet Cell Genet, 79, 103-108), the latter not being present in some neuroglioblast cell lines.

Analysis and alignment of the second sequence was performed using the same BLAST network system at NCBI, using the KIAA0550 protein (accession number AB0111).
22) or KIAA0688 (accession number AB014588). Analysis performed with KIAA0688 shows agreement with secreted proteins with TSP-1 type 1 repeats and regions of homology with cell surface antigens MS2 and metalloproteinases (FIG. 8). Substrate metalloproteinases (MMPs) are capable of degrading extracellular matrix and thus naturally occur inhibitors of both angiogenesis and tumor metastasis (Powell WC and Matrisian)
LM 1996 CUrr Top Microbiol Immunol
213: 1-21).

Unexpectedly, analysis performed with KIAA0550 showed agreement with the BAI3, BAI2 and BAI1 proteins (FIG. 9). KIAA0550 (985a
a) Comparison between the protein and the BAI3 (1523aa) protein shows that these two proteins show high sequence identity (FIG. 10). KIA
A0550 has about two-thirds spanning the first 967 amino acids of both proteins.
BAI3 and have the same type 1 repeat. However, BAI3 has a 22 amino acid insertion downstream of the type 1 repeat. BAI3 also differs in the C-terminal domain, where BAI3 extends an additional 536 amino acids.
These observations suggest that BAI3 and KIAA0550 may be two isoforms of the same protein resulting from alternative splicing of the same RNA transcript.

Example 6 Construction of Viral Vectors for Delivery of Type 1 Repeat Fragments MLV-Based Vectors The MLV-based retroviral vectors are pSec as NcoI-PmeI fragments.
Constructed by cloning recombinant TRP from TRP into OBM80 (FIG. 11). The nucleotide sequence encoding TRP was replaced with human cytochrome P45.
Insert into OBM 80 instead of 0. This vector allows expression of recombinant TRP from the MLV LVR promoter in transduced cells. The vector genome is constructed as a single transcription unit containing the IRES and the bacterial LacZ gene. β-galactosidase expression is used as a control for transduction.

In all of the following examples, the three plasmid transfection methods described previously (Soneoka et al., 1995 Nucl. Acids. Res.
. , 23: 628) to generate a VSV-G pseudotyped vector. The plasmids used in this experiment are as follows. pHIT60 and pRV6
7 encodes the MLV gag-pol nucleotide coding sequence and the VSV-G envelope (env) glycoprotein nucleotide coding sequence, respectively (Soneoka et al., 1995 ibid). In a first experiment, 8 ug of the OBM80-TRP vector genome is co-transfected with 8 ug of each of pHIT60 and pRV67 per 6-well tissue culture dish.

Transfection is performed in the human kidney cell line 293T as described previously (Soneoka et al., 1995 ibid) to generate vector particles. Culture supernatant was harvested 36 hours after transfection and
. Filter through a 45 micron filter (Millipore Inc.). Two cell lines (using HT1080 and D17 cells) and a canine melanoma cell line are used to determine the titer of a pseudotyped retroviral vector of VSV-G.

Cells are seeded in 6-well tissue culture plates at 3 × 10 ⁵ cells per well one day prior to infection. The supernatant of the virus, prepared by transfecting 293T cells with the appropriate plasmid into the pseudotype MLV-TRP vector, is added to these cells. Polybrene (8 ug / mL) is added to each well simultaneously with transfection to make 1 ml of culture supernatant used for infection. Twelve hours after infection, the culture supernatant is replaced with fresh medium. Cells are washed, fixed and stained for 48 hours post-infection to determine virus titer. Virus titers are estimated from the number of β-galactosidase positive colonies.

Adenovirus Vectors The primary recombinant adenovirus vector is usually a left inverted terminal repeat (IT
It consists of a deletion of the E1 and E3 regions of the virus, which allows the insertion of heterologous DNA into the left arm of the virus adjacent to R). Viral packaging signal (18
4-358 nt) overlaps with the Ela enhancer and is therefore present in most El deleted vectors. This sequence can be translocated to the right end of the viral genome (Heering and Shenk, 1983, Cell 33, 69).
5). Therefore, in the E1 deletion vector, 3.2 kb can be deleted (
358-3525 nt).

[0208] Adenovirus can be packaged into a 105% long genome, thus adding an additional 2.1 kb. Therefore, the cloning capacity of the E1 / E3-deleted virus vector is 7 to 8 kb. Recombinant adenoviruses cannot replicate in non-E1 complementing cell lines because they lack the essential E1 early gene. The 293 cell line was obtained from Graham et al (1977, J Ge
nVirol 36, 59) and contains about 4 kb from the left end of the Ad5 genome, including the ITRs, packaging signals, E1a, E1b and pIX. The cells stably express the E1a and E1b gene products, even if the pI
Even if the X sequence is within Elb, late proteins are not expressed. In non-complementing cells, the E1-deleted virus transduces the cell and is transported to the cell nucleus.
There is no expression from the deleted genome.

[0209] The scheme of FIG. 12 is the Microbix Biosystem-NBL Gene.
1 shows a general strategy used to generate recombinant adenovirus using the Science system.

This general strategy involves cloning the heterologous DNA into an E1 shuttle vector.
In this case, replacement of the E1 region (402 to 3328 bp) with a heterologous DNA cassette is included. Thereafter, the recombinant plasmid was added together with the pJM17 plasmid for 2 hours.
Transfect into 93 cells. pJM17 has the E3 region deleted,
A prokaryotic pBRX vector (with ampicillin resistance and bacterial ori sequences) has been inserted into the E1 region of 3.7 map units. Thus, this 40 kb plasmid is too large to be packaged in an adenonucleocapsid, but can be packaged in bacteria. Intracellular recombination in 293 cells replaces the ampr and ori sequences with inserts of heterologous DNA.

In the examples cited herein, two transfer vectors were used. Mic
The first one obtained from Robix is called pE1sp1A and Qquantum B
The second one from iotechnologies is called pADBN. pA
DBN plasmids have the advantage that heterologous DNA can be inserted in either direction. In both cases, the second DNA is a plasmid (eg, pJM17
) As well as a portion of the viral DNA (eg, the right arm of Ad5) is a defective version of the adenovirus. Homologous recombination results in the final gene transfer vector.

A single transcription unit containing a therapeutic gene and a reporter gene has the following structure: TRP-IRES-La driven by a constitutive promoter such as CMV
cZ. These structures were converted into a pADBN transfer vector (Quantum B
iotechnologies), and AdeOBRP-1, -2,-
Yields 3 and -4. Homologous recombination is generated in vivo by linearizing the recombinant AdeOBRP transfer vector (Asel) and transfecting 293 cells with the purified right arm of the Ad5 virus (ClaI site), resulting in the desired set Form a recombinant adenovirus. The virus titer is Lac
Determined by the number of target cells expressing Z. It does not exclude that these structures have a different reporter gene or another therapeutic gene in place of LacZ.

The lentiviral vector TRP is an EIAV lentiviral vector, pONY4 in which the two genes are expressed as a single transcription unit (FIG. 13) and the related vector pONY2.1 (FIG. 1).
4). Both are infectious provirus EIAV clones p
SPEIAV19 (Payne et al. 1998, J. Virol 72, 483). The signal transcription unit has the following structure, TRP-IRES-LacZ. It is not essential that the lentivirus contains a reporter gene instead of including the second therapeutic gene in place of LacZ. Viral particles containing pONY-TPR were transfected with the transient three plasmid system described above (Soneoka et al., 19
95 ibid). The VSV-G pseudotype EIAV vector is titered using D17 cells. Virus titers are estimated from the number of colonies positive for β-galactosidase.

Regulatory Viral Vectors The present invention encompasses the use of regulatory viral vectors to deliver anti-angiogenic TRP. For example, NOI and TRP encoding nucleotide sequences are expressed under the control of a regulatable promoter OBHREI. The Xia-Gen-P450 vector genome is used as a starting molecule. TRP is cloned into Xia-Gen-P450 instead of P450 (FIG. 15). This structure allows for the expression of TRP in transduced cells under the control of HRE. RRV also contains a reporter gene (LacZ or GFP), and a selectable marker (neomycin resistance gene). Viral particles were generated as outlined above and transduced D1
Use for 7 cells. The transduced cell population was split and left overnight in normoxia (21% oxygen).
) And hypoxia (0.1% oxygen). Cells are stained by X-gal histochemistry and endpoint titers are calculated. Titers are measured for β-galactosidase gene expression and reflect changes in gene expression between cell populations under hypoxic and normoxic conditions.

Example 7-Construction of a viral vector encoding TRP in conjunction with a prodrug activating enzyme The invention is not limited to the generation of a viral vector encoding TRP. TR
Combinations of P with prodrug activating enzymes can be formed in these vectors. Xia-Gen-P450 (FIG. 15) contains a therapeutic gene for human cytochrome P450 that activates the anticancer compound cyclophosphamide. CY
The P2B6 gene is driven by the CMV promoter. The therapeutic gene is T
RP is cloned downstream of the IRES and expressed as a single transcription unit replacing the LacZ gene. Thus, the structure is P450-IRES-TRP. Any other prodrug activating enzyme can be used for this structure.
The use of a constitutive promoter is optional within the present invention. Another structure includes the hypoxia-regulated promoter HRE capable of expressing the single transcription unit described above.

Example 8 In Vitro Transduction of Glioma Cell Line a) Transduction of Glioma Cell Line with Recombinant Viral Vector The human glioma cell line U87MG expresses either TRP or human cytochrome P450 of the invention, or both. Transduce with a recombinant viral vector. 10 ⁵ U87MG cells are seeded into 6-well dishes and virus particles are generated as described above and added to the cells over 24-48 hours. Stable transformed cells are selected for 2 weeks in the presence of Genestin 400 mg / mL. Expression of TRP and / or P450 in stable G418 resistant clones is determined by Northern and Western blots, and the highest expressing cell line is selected for use in subsequent experiments.

B) In Vitro Endothelial Cell Proliferation Assay HUVEC cells (10 ⁵ ) were plated in 6-well plates and stably transduced into glioma cells U87MG, U87MG-neo, U87MG
1 mL of TRP, U87MG-P450 and U87MG-TRP / P450
To the specified HUVE cell culture. Three days later, HUVE cells were transformed with 2 mCi of [ ³
[H] thymidine, and the incorporation of this compound into DNA was quantified by scintillation counting.

In the co-culture analysis, the same glioma cells (2 × 10 ⁵ ) were plated in the upper chamber of the cell culture insert and HUVE (10 ⁵ ) cells were plated in the lower chamber. Plate. After 3 days, HUVE cells are labeled as described above and the rate of proliferation determined.

C) In vitro sensitivity of U87MG to cyclophosphamide (CPA) U87MG, U87M stably transduced into glioma cells (5 × 10 ³ )
G-neo, U87MG-TRP, U87MG-P450 and U87MG-T
RP / P450 is inoculated into microwell dishes and 24 hours later, various amounts of CPA (
(0-1000 mM) for 24 hours. Thereafter, the cells are washed and fresh medium is added. After 48 hours, cell death is assessed by trypan blue exclusion analysis.

Example 9 In Vitro Inhibition of Angiogenesis in Tumors a) Transduction of tumors in the brain with vectors encoding TRP a. 1) Transplantation of subcutaneous tumor U87MG, U87MG-TRP, U8 resuspended in 100 mL of HBBS
7MG-P450 and U87MG-TRP / P450 glioma cells (10 ⁶
) Is injected intradermally into the right flank of Balb C nude mice (Nu / Nu). Twenty days after implantation, mice are sacrificed and tumor volume is determined using the following formula. Tumor volume (mm ³ ) = length [mm] × (width [mm]) ² × 1/2.

A. 2) Transplantation of Adrenal Tumor Balb C nude mice (Nu / Nu) were anesthetized by intraperitoneal injection of hyponome and resuspended in 100 mL HBBS U87MG, U87MG-TRP, or U87MG-P450 or U87MG-TRP / P450 glioma. Inject 10 ^{6 of the} cells into the adrenal cortex of the left kidney. Twenty days later, mice are sacrificed and both livers are removed to determine tumor volume, weight and vascularity. Tumor vascularity is determined in paraformaldehyde fixed sections reacted with antibodies to factor VIII-related antigen (von Willebrand factor) and visualized by biotinylated secondary and ABC. The following hematoxylin counterstained microvessels are counted using a light microscope with a visual field of 200x in the tumor region with the highest density vascular staining.

A. 3) Transplantation of brain tumors Balb C nude mice (Nu / Nu) were anesthetized using hyponom and hyponovel in a low-position fixed injection device, and U87MG glioma cells (5 × 10 ³⁾ resuspended in 10 mL of HBBS ) Coordinates: Fregma, 2.25 from dura
Inject into the right caudate nucleus through the foramen of the skull using 2.5 mm lateral, 2.5 mm vertical.
Injections are made using a Hamilton syringe over a 5 minute period, with an additional 5 minutes in place on the left before showing slow regression.

B) Transfection of brain tumors with a vector encoding P450B6 together with a vaso-stable polypeptide and treatment with CPA. Mice receive 10 mL of (1) neo or (2) TRP or (3) P450
Or (4) a recombinant virus (10 ⁸ ) encoding any of TRP / P450
Particles or LFU / mL) are divided into five different groups four days after receiving the same skull tunnel and stereotactic coordinates. Groups (3) and (4)
Half of the animals also receive an intraperitoneal injection of CPA (100 mg / kg) 2-4 days later. The survival of these animals is confirmed, and after acute or late death, all groups are examined histologically for tumors. The statistical advantage between the different groups is K
Evaluate using aplan-Meier survival curve long rank analysis.

Example 10 Agents that Can Inhibit Pro-Angiogenic Factors, or TRPs of the Invention
Identification of agents that can suppress the introduction of such factors that can be combined with the fragment TRP Selection of indicator cell lines for screening for agents that increase or interfere with transcription depending on HRE as outlined in FIG. The indicator cell line is selected to satisfy the following requirements.

(I) Optimal indicator cell lines are capable of mediating strong responses to hypoxic stimuli and require stable integration of a transcriptional cassette upstream of a suitable reporter (analytical readout). This transcription unit, in conjunction with the intrinsic properties of the cell line, can exhibit low basal activity at normoxia while exhibiting high inducible reporter activity under hypoxic stimulation. This provides an appropriate range of uses to assess changes in transcriptional response in the presence of the compound / small molecule to be screened. As an example, the transcription control element OB HRE1 (PCT / GB98 / 0
The properties of 2885 (see WO 99/15684) would meet this requirement.

(Ii) Preferred target cells preferably show high responsiveness to hypoxic stimulation. This is due to transient transfection analysis, or endogenous levels of hypoxia responsiveness as defined by band shift analysis or western analysis of the bHLH-PAS line, eg, HIF1α, HIF2α, HIF3α. It can be defined first by the transposable element.

(Iii) The cell line is preferably biologically stable so as to ensure analytical reproducibility with easy production parameters with respect to culture parameters. In addition, the cell line is preferably selected such that its clinical target tissue is appropriate to be recognized as a benchmark cell line for a wide range of applications and applications. It is necessary to introduce the reporter cassette in such a way that the hypoxia-responsive control element is kept intact. As a result, standard transfection techniques that allow multiple copies and random recombination may not be appropriate. A preferred method of introducing such a transcription cassette is by targeted integration by single copy retroviral transduction. Single copy integration can be selected if transduction of the parent cell line is performed at a low multiplicity of infection (MOI).

Isolation of clones with characteristics as outlined above: Incorporation of a selectable marker facilitates isolation of clones with characteristics as outlined above. The selectable marker can be an antibiotic resistance marker, for example, neomycin, in which case clones can be selected by culture in G418. More preferably, the marker is a FACS technology, eg, GFP
Alternatively, it can be selected using LacZ (using an FDG substrate). Ideally, the selectable marker is controlled in the same way as the reporter for analysis. This regulation can help generate a co-regulated bicistronic message using the IRES sequence.

By using FACS selectable markers, clones that exhibit high levels of expression under hypoxia induction and low basal activity under normoxia can be rapidly selected. A round of positive and negative selection facilitates the production of a pool of cells that can exhibit low basal and high inducing activity. A single clone is isolated from these cell pools and the final selection is developed. Cloning is a condition required for cell-based analytical screens.

Construction of hypoxia-regulated retrovirus The preparation and transduction methodology of the virus is general (PCT / GB98 /
02885 (WO 99/15684)). FIG. 17 shows the genome structure used for transduction. Suitable cell lines for use as host analysis cells include the T47D cell line (PCT
/ GB98 / 02885 (WO 99/15684)) including but not limited to the mouse skeletal muscle cell line C2C12 and the rat muscle cell line H9C2.

Selection of an Appropriate Integrated HRE-regulated Retroviral Genome Cells showing good hypoxia regulation are selected by FACS sorting using LacZ expression. Briefly, transduced cells are induced by culturing overnight in hypoxia (0.1%). The response to this stimulus expresses LacZ (see FIG. 17). LacZ expression was determined by Fluo according to the manufacturer's protocol.
reporter lac Z flow cytometry staining kit (Molec
(Ultra Probes). The cells showing the highest level of expression are isolated, after which a further round of positive FACS selection is developed. X-gel staining of sorted cells in normoxic and hypoxic conditions,
Help monitor your choices. This results in a heterologous line showing tight regulation. Allogeneic cell populations can be derived by single cell cloning. This is necessary to ensure analysis accuracy and system stability.

Screening Analysis Depending on the choice of LacZ regulation, cells that also regulate luciferase as a result of genomic structure are isolated. Analytical readout detection of luciferase expression using the luciferase microtiterminometry assay (PCT / GB98 / 02885) is performed as follows:

[0233] Briefly, "indicator cells" were plated at about 10 ⁴ cells per well in a 96-well plate such that the cells were 70% confluent after plating for 24-48 hours. (Depending on cell type). The target compound is added to the homogenous well (x4 or more) along with the relevant control (compound buffer). Two plates are prepared, one incubated overnight under normoxic conditions (eg, 18 hours), and the other incubated overnight under hypoxic conditions. Cells are lysed in situ by adding luciferase assay lysis buffer (reviewed in the manufacturer's protocol) and then analyzed for luciferase activity.

Data obtained from normoxic and hypoxic plates show a modulation of hypoxia induction when compared to controls (no compound added or control buffer added).

Results Suppression of Hypoxia-Responsive Gene Expression A high-throughput assay was designed that allows rapid screening for molecules capable of blocking the HIF / HRE signaling pathway as shown in FIG. This analysis can identify candidate compounds for use in treating neovascularization diseases, such as cancer, by interfering with HRE transcription by the gene. These molecules can be used in combination with the TRPs or TRP fragments of the invention for the treatment of angiogenesis and / or cancer.

This analysis can be applied to identify compounds that can act as agonists or antagonists (ie, modulators) in other therapeutic pathways, some of which are outlined in FIG. These therapeutic pathways include ubiquitination, proteolysis, kinase / phosphase, dimerization,
DNA binding, formation of transcription complexes, interaction with p300, oxygen sensor analysis using protein-containing heme, redox reactions and / or kinase signaling pathways.

Sequence Listing SEQ ID NO: 1 is the nucleotide sequence encoding human TSP-1 shown in FIG.

SEQ ID NO: 2 shows the consensus TRP amino acid sequence shown in FIG. The consensus amino acid sequence (SEQ ID NO: 2) was derived from 20 different TRP sequences of the same length that were easily and clearly aligned.

[0239] SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 are also shown in FIG. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system herein without departing from the scope and spirit of the invention will be apparent to those skilled in the art.
Although the invention has been described in connection with a preferred embodiment of identification, it will be understood that the invention as claimed should not be unduly limited to such an embodiment of identification. Rather, various modifications of the described methods for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

[Brief description of the drawings]

FIG. 1 is the nucleotide sequence of a type 1 repeat from the hTSP-1 gene shown as SEQ ID NO: 1. HindII and NotI restriction are added by PCR to facilitate cloning of these fragments into a mammalian expression vector.

FIG. 2. Human-thrombospondin 1 (hTSP-1) type 1 repeat peptide (TRP)
).

FIG. 3 shows primers used for amplification of hTSP-1 TRP.

FIG. 4. Plasmid pSecTag. 2A shows a representative diagram. This plasmid contains a leader sequence and a c-myc epitope tag.

FIG. 5 shows primers used for amplification of a hTSP-1 regulatory peptide.

FIG. 6 shows a BLAST search result using the third type 1 repeat of TSP.

FIG. 7 shows the consensus TRP amino acid sequence shown as SEQ ID NO: 2. Human TS
P-1 (SEQ ID NO: 3), KIAA0550 (SEQ ID NO: 4), and KIAA06
88 (SEQ ID NO: 5) is also shown.

FIG. 8 shows a BLAST search result using KIAA0688 protein.

FIG. 9 shows a BLAST search result using KIAA0550 protein.

FIG. 10 shows a comparison diagram between KIAA0550 protein and BAI3 protein.

FIG. 11 is a representation of the structure of plasmid OB80 in the retroviral vector genome.

FIG. 12 is a schematic diagram for creating a recombinant adenovirus vector. As an example, adeno PGKLacZ is an OBHRELacZ cassette from the OB37 plasmid inserted into the Microbix transfer vector pE1sp1A.

FIG. 13 shows a representative diagram of the plasmid pONY4 lentiviral vector genome.

FIG. 14 is a representative drawing of a plasmid pONY2.1 lentiviral vector gag-pol expression cassette.

FIG. 15. Retrovirus Xia containing a therapeutic gene under the control of the OBHRE promoter
1 shows a map of the Gen-P450 vector.

FIG. 16 shows the HIF / HRE signaling pathway.

FIG. 17 shows the structure of the integrated transcription cassette containing the reporter and marker for analysis for clone selection.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/47 C12N 1/19 4C084 19/00 1/21 4H045 C12N 1/15 C12P 21/02 C 1 / 19 C12Q 1/02 1/21 G01N 33/15 Z 5/10 33/50 Z C12P 21/02 33/68 C12Q 1/02 C12N 15/00 ZNAA G01N 33/15 5/00 A 33/50 A61K 37 / 02 33/68 37/48 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA , CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE , SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Martin-Lendon, Enca UK, OEX 44 ES Oxon, Sandford-on-Thames , Buckler Place 4 (72) Inventors Kingsman, Susan Mary United Kingdom, OEX 135 Peep Oxon, Appleton, Oaksmere, Keepers House (No address) F-term (reference) 2G045 AA40 CA25 CA26 CB01 CB03 DA36 FB03 4B024 AA01 CA02 CA04 EA04 GA11 HA03 4B063 QA18 QR72 QS12 QX07 4B064 AG01 CA19 DA01 4B065 AB01 BA02 CA44 4C084 AA01 MA02 NA14 NA15 ZB26 4H045 AA10 AA20 AA30 BA41 EA28 FA74

Claims (38)

[Claims] 1. Non-natural type 1 repeat peptide (TRP). 2. An amino acid sequence represented by SEQ ID NO: 2 or a variant thereof,
2. The TRP of claim 1, comprising at least a homolog, fragment or derivative. 3. The TRP of claim 2, wherein the TRP comprises the sequence shown as SEQ ID NO: 2. 4. An amino acid sequence represented by SEQ ID NO: 3 or a variant thereof,
2. The TRP of claim 1 comprising a homolog, fragment or derivative. 5. The TRP according to any one of claims 1 to 3, wherein the TRP comprises the amino acid sequence shown as SEQ ID NO: 4 or a mutant, homolog, fragment or derivative thereof. 6. The TRP according to claim 1, wherein the TRP comprises the amino acid sequence shown as SEQ ID NO: 5 or a mutant, homolog, fragment or derivative thereof. 7. A nucleotide sequence encoding the TRP according to any one of claims 1 to 6. 8. The sequence shown as SEQ ID NO: 1 or a mutant or homolog thereof,
Nucleotide sequence encoding TRP, including fragments or derivatives. 9. A nucleotide sequence encoding TRP, comprising the sequence shown as SEQ ID NO: 1. 10. A nucleotide sequence capable of hybridizing to the nucleotide sequence according to any one of claims 7 to 9, or a sequence complementary to a nucleotide sequence capable of hybridizing. 11. The 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 a nucleotide sequence according to any one of claims 7 to 11. 13. A vector comprising the nucleotide sequence according to any one of claims 7 to 11. 14. A plasmid comprising the nucleotide sequence according to any one of claims 7 to 11. 15. A host cell comprising a nucleotide sequence according to any one of claims 7 to 11. A host organism comprising a nucleotide sequence according to any one of claims 7 to 11. 17. TRP comprising expressing a nucleotide sequence according to any one of claims 7 to 11, or optionally in the presence of an invention according to any one of claims 12 to 13. A method for preparing TRP according to any one of claims 1 to 6, comprising isolating and / or purifying TRP. 18. A TRP produced by the method of claim 17. 19. A TRP that immunologically reacts with an antibody produced against the purified TRP according to any one of claims 1 to 6 or 18. 20. An analytical method for identifying whether a substance is TRP, comprising: (a) under conditions that endothelial cells can enter a long logarithmic growth phase in culture;
Incubating endothelial cells for a sufficient time; (b) introducing an angiogenic stimulant into the culture to induce endothelial cell proliferation and migration; (c) introducing a substance; 2.) Determine if the substance can inhibit endothelial cell proliferation and inhibit migratory activity in vitro, as indicated by inhibition of endothelial cell proliferation and migration activity in vitro, indicating that the substance is a TRP. A method comprising steps and. 21. A TRP useful for treating angiogenesis and / or cancer
21. The analysis method according to claim 20, wherein the screening is performed. 22. (a) performing the assay of claim 20 or 21; and (b) providing one or more TRPs capable of inhibiting endothelial cell proliferation and migratory activity in vitro. Identifying; and (c) preparing an amount of the one or more TRPs thus identified. 23. (a) carrying out the analysis according to claim 20 or 21; and (b) providing one or more TRPs capable of suppressing endothelial cell proliferation and migration activity in vitro. Identifying; and (c) preparing a pharmaceutical composition comprising one or more of these identified TRPs. 24. (a) performing the assay of claim 20 or 21; and (b) providing one or more TRPs capable of inhibiting endothelial cell growth inhibition and migration activity in vitro. (C) modifying one or more identified TRPs capable of inhibiting endothelial cell proliferation and migration activity in vitro; and (d) one or more of these modified TRPs. Preparing a pharmaceutical composition comprising TRP. 25. A TRP identified by the analysis method according to claim 20 or 21. 26. The TRP is an angiogenesis inhibitory protein (such as human thrombospondin 1, cerebral angiogenesis inhibitor 1, cerebral angiogenesis inhibitor 2, and cerebral angiogenesis inhibitor 3). Or a TRP according to any one of claims 18 or 19 or 25. 27. The TRP according to claim 26, wherein the cerebral angiogenesis inhibitor is an isoform of the cerebral angiogenesis inhibitor 3. 28. A method of causing TRP to act on angiogenesis and / or cancer in vivo, wherein TRP can suppress endothelial cell proliferation and suppress migration activity in an in vitro assay method. A method characterized in that the in vitro analysis method is the analysis method defined in claim 20 or 21. 29. Use of a TRP according to any one of claims 1 to 6 or 18 or 19 or 25 to 27 for preparing a pharmaceutical composition. 30. A pharmaceutical composition comprising TRP and another therapeutically useful agent. 31. The pharmaceutical composition according to claim 30, wherein the other therapeutically useful agent is a prodrug activating enzyme. 32. The pharmaceutical composition according to claim 31, wherein the other therapeutically useful agent is an agent capable of inhibiting pro-angiogenic factors or inhibiting the induction of such factors. 33. A TRP according to claim 1 to claim 6, claim 18, or claim 1.
29. The pharmaceutical composition according to claim 29 or claim 30, which is the TRP according to any one of claims 25 to 27. 34. A prodrug-activating enzyme, or a nucleotide sequence of interest (NOI) encoding a prodrug-activating enzyme, or a pro-angiogenic factor for the treatment of a condition associated with angiogenesis and / or cancer. Use of TRP in combination with an agent capable of inhibiting the induction of such factors. 35. Use of TRP in the preparation of a pharmaceutical composition according to claim 29 or 33 for the treatment of a condition associated with angiogenesis and / or cancer. 36. The TRP is not thrombospondin (TSP) TRP,
TRP obtainable from a TRP-containing protein. 37. A fusion protein comprising TRP. 38. A TRP substantially as herein described and with reference to the accompanying drawings.