KR20010092800A - Peptides - Google Patents

Abstract

The present invention relates to type I repeat peptides (TRPs) capable of inhibiting proliferation and migration of endothelial cells. More particularly, the present invention relates to the expression of TRPs in vitro and in vivo and the use and / or angiogenesis and / or disease associated with cancer in the present invention. Or the treatment of a condition.

Description

Peptides {Peptides}

Angiogenesis is an essential component of the metastasis pathway. Most solid tumors are well-formed and produce new ones as they grow. For many tumors, the higher blood vessel density can provide an indication of metastatic potential that higher angiogenesis has a higher incidence of metastasis than weaker angiogenesis. Tumor angiogenesis is regulated by the production of angiogenic activators and inhibitors (see Zetter BR 1998 Annu. Rev. Med., 49: 407-24).

It is believed that both regulated and unregulated angiogenesis proceeds in a similar manner. Endothelial cells and pericytes surrounded by a basement membrane form capillaries. Angiogenesis begins with erosion of the basal membrane by enzymes secreted by endothelial cells and leukocytes. Then endothelial cells along the lumen of the blood vessel protrude through the basal membrane. In certain pathologies, cell proliferation of abnormal development results in local production of urea, which makes it possible to sustain growth through enhanced angiogenesis. For example, angiogenic stimulants such as vascular endothelial growth factor (VEGF), PDGF and bFGF (Kuwabara et al PNAS 1995 92 4606) can induce endothelial cells to migrate through the eroded basement membrane. The migrated cells form an ancestral vascular outlet through which endothelial cells divide and proliferate. Endothelial vents fuse with each other to form capillary rings, thus creating new blood vessels.

It is clear that angiogenesis plays an important role in metastasis of cancer. Cancer, and in particular cancer development, requires angiogenesis that allows for sustained growth. One approach to inhibiting disease progression in cancer is to limit new angiogenesis processes. If such angiogenic activity can be inhibited or eliminated, the tumor will not be able to grow even if present. Prevention of angiogenesis in other disease states can prevent damage caused by invasion of new microvascular systems. Treatment directed to the regulation of angiogenic processes in this way leads to alleviation of these diseases.

Tissue growth is limited by nutrition. Vascular growth during development maintains the rate of tissue growth that ensures that the cells receive the correct level of oxygenation and nutrition. If cell proliferation precedes blood supply, the cells are constrained by oxygen. This oxygen restriction, called hypoxic stress, causes changes in gene expression. Many of these changes include factors that enhance angiogenesis as well as increased expression of genes, including those involved in glycolysis to enable inorganic respiration.

For example, VEGF transcription is induced by hypoxia through the increased stability of HIF-1 transcription factors by binding these transcription factors to enhancer elements and hypoxia response elements present in the 5 'UTR of the VEGF gene (Forsythe et al. MCB 1996 16 4604, Maxwell et al PNAS 1997 94 8104). Other examples also include PDGF and bFGF (Kuwabara et al PNAS 1995 92 4606). There is some information on the signaling mechanisms that cause these changes in gene expression, but this process is not yet fully understood.

Therefore, what is needed is a method of inhibiting the growth of blood vessels, which is undesirable, especially in tumors. This can be done directly by the use of inhibitors of pro-angiogenic factors or indirectly by demonstrating ingredients / small molecules that can inhibit the induction of such factors. This should include a method of overcoming the activity of endogenous growth factors (ie, αFGF, βFGF, VEGF, IL-8, GM-CSF) in the premetastatic tumor and prevent the formation of capillaries in the tumor to prevent tumor growth. It must be restrained. It should also include methods for regulating capillary formation within other angiogenic processes such as wound healing and regeneration.

A significant amount of research has been done on the anti-angiogenic properties of the outer matrix molecule thrombospondin. Thrombospondin (TSP) has been identified as an inhibitor of endothelial cell proliferation, motility and morphogenesis (Bagavandoss and Wilks 1990 Biochem Biophy Res Commun 170: 867-72; Vogel et al , 1993 J Cell Biochem 53: 74-84; Iruela -Arispe et al , 1991 Proc Natl Acad Sci 88: 5026-30). Expression of TSP appears to be inverse to p53 expression and angiogenesis in human bladder cancer and glioma.

It has been reported that internal peptides formulated within Type I repeats (TRP) of human TSP-1 reduce cell proliferation and endothelial cell migration, but there are no reports of expression of such peptides from cDNAs and the use of such cDNAs in gene therapy.

The present invention relates to type I repeat peptides (TRPs) capable of inhibiting proliferation and migration of endothelial cells. More particularly, the present invention relates to the expression of TRPs in vitro and in vivo and the use and / or angiogenesis and / or disease associated with cancer in the present invention. Or the treatment of a condition.

The invention will be further described only by the examples referred to by the following figures.

1 is a nucleotide sequence of type 1 repeats from the hTSP-1 gene shown in SEQ ID NO: 1. HindⅡ and NotⅠ limit is by PCR to facilitate cloning of this segment was added to the mammalian expression vector;

2 is the amino acid sequence (SEQ ID NO: 3) of human thrombospondin-1 (hTSP-1) type I repeat peptide (TRP);

3 shows the primers used for amplification of hTSP-1 TRP;

4 shows a schematic of plasmid pSecTag.2A. Such plasmids contain a leader sequence and a c-myc epitope tag;

5 shows the primers used for amplification of the hTSP-1 control peptide;

6 shows BLAST search results using a third TSP Type 1 iteration;

7 shows the consensus TRP amino acid sequence shown in SEQ ID NO: 2. It also represents TRPs from human TSP-1 (SEQ ID NO: 3), KIAA0550 (SEQ ID NO: 4) and KIAA0688 (SEQ ID NO: 5);

8 shows BLAST search results using KIAA0688 protein;

9 shows BLAST search results using KIAA0550 protein;

10 shows a schematic comparison between KIAA0550 protein and BAI3 protein;

11 shows a schematic of the shape of plasmid OB80 in the retroviral vector genome;

12 is a schematic diagram for constructing a recombinant adenovirus vector. As an illustration adeno PGK Lac Z is an OBHRE Lac Z cassette inserted into the Microbix transfer vector pE1sp1A from the OB37 plasmid;

13 shows a schematic of the plasmid pONY4 lentiviral vector genome;

14 shows a schematic of the plasmid pONY2.1 lentiviral vector gag-pol expression cassette;

15 shows a map of a retroviral XiaGen-P450 vector composed of therapeutic genes under the control of the OBHRE promoter;

16 shows the HIF / HRE signal path; And

17 shows the shape of an integrated transcription cassette composed of analytical reporters and markers for clonal selection.

It is an object of the present invention to provide TRPs having anti-angiogenic activity which can be expressed under laboratory and in vivo conditions and can be used for the prevention and / or treatment of angiogenesis and / or cancer.

Aspects of the invention are indicated in the accompanying claims and the following detailed description.

Thus, in one aspect, the present invention provides a non-naturally occurring Type I repeat peptide (TRP).

As used herein, the term "type I repeat peptide (TRP)" refers to an internal peptide consisting of type I repeats present in one or more repeats (TRP configuration) in the TRP containing the protein.

In addition, according to the invention “TRP” may be not only a part of the fusion protein but also the peptide itself.

The term "non-naturally occurring" TRP according to the present invention is not expressed by a natural nucleotide coding sequence when the TRP is in a natural environment and the nucleotide sequence is under the control of a natural promoter present in the natural environment. Does not mean TRP.

Thus, the present invention does not include the natural TRP according to the present invention when expressed by a natural nucleotide coding sequence when present in a natural environment and whose nucleotide sequence is under the control of a natural promoter present in the natural environment. .

Preferably the TRP is an isolated TRP and / or purified TRP.

The TRP may be natural or non-synthetic or synthetic TRP, semi-synthetic TRP, derived TRP, or recombinant TRP or may be obtained or produced from any suitable raw material. Preferably, the non-native TRP induces at least some TRP prepared by recombinant DNA technology or TRP produced by chemical synthesis techniques or a combination thereof.

More preferably non-native TRP was prepared by the use of recombinant technology.

The term "derivative" as used herein includes chemical modifications of TRP. Examples of such modifications are hydrogen return by alkyl, acyl or amino groups.

The sequence of the TRP of the present invention may be identical to that of a naturally occurring form or may be a variant, homologue, segment or derivative thereof.

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

In one preferred aspect, the present invention provides peptides consisting essentially of TRP.

The invention also encompasses the nucleotide sequence (s) encoding the TRP of the invention. Nucleotide sequences of the present invention are in non-naturally occurring form. The present invention therefore does not include the native nucleotide coding sequence according to the invention in its natural environment when under the control of the native promoter present in its natural environment.

The sequence of the TRP encoding nucleotide sequence may be identical to the naturally occurring form or is a variant, homologue, segment or derivative thereof. Preferably the TRP encoding sequence is an isolated TRP encoding sequence and / or a purified TRP coding sequence. The TRP coding sequence may be natural or non-synthetic, synthetic, semi-synthetic or recombinant or obtained from any suitable source or produced.

As used herein, “nucleotide sequence” refers to an oligonucleotide sequence or polynucleotide sequence and variants, homologues, segments or derivatives thereof (such as portions thereof). The nucleotide sequence may be genomic or synthetic or recombinant origin DNA or RNA, meaning sense or antisense or may be double-helix or single-helix. Nucleotide sequences do not always require completely natural DNA sequences. Thus, for example, the DNA sequence can be a synthetic DNA sequence, a recombinant DNA sequence (ie, prepared by the use of recombinant DNA technology), a cDNA sequence or a partial genomic DNA sequence or a combination thereof. DNA sequences do not require coding regions. If it is a coding region, it does not need to be the entire coding region. Furthermore, the DNA sequence can be in sense orientation or antisense direction. Preferably in the sense direction. Preferably it is DNA or consists of cDNA.

Preferably the term "nucleotide sequence" means DNA. More preferably, the term "nucleotide sequence" refers to DNA (ie, recombinant DNA) prepared by the use of recombinant DNA technology. Accordingly, the present invention preferably 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 present invention provides a nucleotide sequence consisting essentially of the TRP coding sequence.

The term "recombinant type I repeat peptide (TRP)" means TRP prepared by the use of recombinant DNA technology.

Modified TRP encoding polynucleotide sequences that may be used in accordance with the present invention may include other nucleotide residues to result in polynucleotides in which the TRP encodes the same or functionally identical TRP with deletions, insertions or substitutions.

As used herein, “deletion” is defined as a change in the disappearance of one or more nucleotide or amino acid residues, respectively, in a nucleotide or amino acid sequence.

As used herein, “insertion” or “addition” is defined as the change in which one or more nucleotide or amino acid residues are added, respectively, as compared to a naturally occurring TRP or TRP encoding nucleotide sequence within the nucleotide or amino acid sequence.

As used herein, “substitution” is caused by the replacement of one or more nucleotides or amino acids with each other nucleotide or amino acid.

Alleles of TRP are included within the scope of the present invention. As used herein, an “allele” or “allele sequence” is another form of TRP. Alleles result from mutations, ie changes in nucleotide sequences, and generally produce modified mRNAs or polynucleotides with or without altering structure or function. It may not have one gene allele or may have one or many. Conventional mutagenic changes that cause alleles are generally due to deletion, insertion or substitution of amino acids. Each type of such change occurs independently, or in combination with another, one or more times the given sequence.

The invention also relates to a DNA segment consisting of the DNA sequence of SEQ ID NO: 1 or allelic modifications of such sequence.

In a very preferred aspect the present invention relates to a TRP consisting of the amino acid sequence of SEQ ID NO: 2. For example, the present invention relates to an isolated TRP consisting of the amino acid sequence of SEQ ID NO: 2.

The present invention also relates to non-native DNA consisting of the DNA sequence of SEQ ID NO: 1 or an allelic modification thereof.

In a very preferred aspect the present invention relates to recombinant DNA consisting of the DNA sequence of SEQ ID NO: 1 or allelic modifications thereof.

As used herein, "immune activity" is defined as the ability of an intrinsic, recombinant or synthetic TRP or segment thereof to induce a specific immune response against a suitable animal or cell or to bind to a particular antibody.

In one embodiment the nucleotide sequence and / or its TRP is in isolated and / or purified form.

As used herein, the terms “isolated” and “purified” refer to molecules such as nucleic acid or amino acid sequences that have been separated and separated from at least one or more components that have been removed from their natural environment or naturally bound.

The TRP of the present invention can be isolated from body fluids such as serum, ascites or urine of a patient or TRP can be produced by recombinant DNA methods or synthetic peptide chemical methods well known to those skilled in the art. Protein purification methods are also well known in the art.

The present invention also relates to TRP produced by expression in transformed host cells integrated with the above DNA sequences or allelic modifications thereof.

The term "vector" includes expression vectors and transformation vectors.

The term "expression vector" refers to a construct that can be expressed in a laboratory or in vivo.

The term "transformation vector" means a structure capable of being transformed from one species to another.

Preferred vectors for use according to the invention are recombinant retroviral vectors (RRVs) such as lentiviral vectors, especially adenovirus vectors, including combinations of recombinant vectors, in particular retroviral vectors.

The term "recombinant retroviral vector" (RRV) refers to a vector with sufficient retroviral genetic information to package the RNA genome into viral particles capable of infecting target cells in the presence of a packaging component.

Infection of target cells includes reverse transcription and integration into the target cell genome. RRVs carry non-viral coding sequences that are delivered to target cells by vectors. RRV is incapable of independent replication producing infectious retroviral particles in the final target cells. In general, RRVs do not have the functional gag-pol and / or env genes and / or other genes essential for replication.

In a broader sense, the invention is the first vector or vectors capable of expressing a second viral vector capable of infecting a first target cell and transducing a second target cell therein, the second vector encoding a second viral vector. A hybrid viral vector system for gene delivery in vivo, a system consisting of one or more first viral vectors, is provided.

Preferably the first vector is obtainable or based on adenos virus or the second vector is obtainable or based on a retroviral vector, preferably a lentiviral vector.

The term "targeted vector" refers to a vector that can infect / transfect / transduce a cell or be expressed in a target cell that is generally restricted to a particular cell type with a common or similar phenotype in the host organism. .

Preferably the nucleotide sequence of the invention is operably linked to a transcription unit.

As described herein, the term "transcriptional unit (s)" is a region of nucleic acid that contains a signal to achieve expression of such coding sequence, independent of coding sequences and other coding sequences. Thus, each transcription unit generally consists of at least one promoter, selective enhancer and polyadenylation signal.

The term "promoter" is used in the general sense in this field, namely as RNA polymerase binding site. The promoter may contain an enhancer element.

The term "enhancer" includes DNA sequences that bind other protein components of a transcription initiation complex to promote transcription initiation directed by its associated promoter.

The term "cell" includes any suitable organism. In a preferred embodiment the cell is a mammalian cell. In a more preferred embodiment the cell is a human cell.

The term "transformed cell" means a cell with a modified genetic structure. In the present invention, the cell has a modified genetic structure when the vector according to the present invention is introduced into the cell.

The term "living organism" means any suitable organism. In a preferred embodiment the organism is a mammal. In a more preferred embodiment the organism is a human.

As used herein, the term "organism" refers to an organism composed of a modified genetic structure. In the present invention, the organism has a modified genetic structure because the vector according to the present invention is introduced into the organism.

The present invention also provides a method consisting of transforming a host cell with the nucleotide sequence of the present invention.

The invention also provides a method consisting of culturing a transformed host cell, wherein the cells have been transformed with the nucleotide sequence according to the invention under conditions suitable for the expression of the TRP encoded by said nucleotide sequence.

The present invention also provides a method of culturing a host cell transformed under conditions suitable for expression of TRP encoded by the nucleotide sequence, wherein the cell is transformed with a nucleotide sequence according to the present invention or a derivative, homologue, variant, or segment thereof. and; There is then provided a method consisting in recovering the TRP from the transformed host cell culture.

The present invention further relates to a method of transforming a host cell with a nucleotide sequence or a derivative, homologue, variant or segment thereof according to the present invention; b) culturing the transformed host cell under suitable conditions for the production of said TRP; c) recovering the TRP from the host cell culture.

The term "variant", "homolog", "derivative" or "segment" for the nucleotide sequence of the present invention refers to the activity of the sequence covered by endothelial cell proliferation and migration inhibitory activity, preferably SEQ ID NO: 1. Substituting, mutating, modifying, replacing, deleting or inserting one or more nucleic acids from or into the sequence that provides the expression product of the resulting nucleotide sequence having at least the same endothelial cell proliferation and migration inhibitory activity (FIG. 1) as the expression product. Include.

In particular, the term “homolog” includes identity in structure and / or function to provide an expression product of the resulting nucleotide sequence with endothelial cell proliferation and migration inhibitory activity. Regarding sequence identity (ie, similarity), there is preferably at least 75% or more, more preferably 85% or more, particularly even more preferably 90% sequence identity. Also more preferably there is at least 95%, more preferably at least 98% sequence identity. Such terms also include allelic variations of sequences.

Sequence identity with respect to SEQ ID NO: 1 may be determined by simple “eye” comparison (ie, a precise comparison) of one or more sequences with other sequences, for example, to see whether other sequences have at least 75% or more sequence identity. Can be.

Relative sequence identity can also be determined by a commercially available computer program capable of calculating the percent identity between two or more sequences using any suitable algorithm for determining identity using, for example, default parameters. . A common example of such a computer program is CLUSTAL. Advantageously, the BLAST algorithm can be used with parameters set to default values. The BLAST algorithm is described in http: //www.ncbi.nih.gov.BLAST/blast_help.

It is described in detail in html. The search parameters defined below can be advantageously set to the defined default parameters.

Advantageously "substantial identity" as assessed by BLAST is equivalent to a sequence matched with an EXPECT value of at least about 7 or more, preferably at least 9 or most preferably 10 or more. The default threshold for an EXPECT in a BLAST search is generally ten.

Basic Local Alignment Search Tool (BLAST) is a heuristic search algorithm used by programs blastp, blastx, tblastn and tblastx; This program is a bit of enhancement with Karlin and Altschul (http: //www.ncbi.

Note their findings using the statistical method in nih.gov.BLAST / blast_help.html). The BLAST program was created for sequence similarity searches, for example, to demonstrate homology to the sequences in question. See Altschul et al (1994) Nature Genetics 6: 119-129 for a discussion of the underlying issues in similarity search of sequence databases.

The five BLAST programs available at http://www.ncbi.nih.gov. perform the following tasks:

Compare amino acid question sequences against blastp -protein sequence database.

Compare nucleotide question sequences to the blastn -nucleotide sequence database.

Compare the 6-frame conceptual translation of the nucleotide question sequence (both strands) against the blastx -protein sequence database.

tblastn —Compare protein question sequences against a dynamically translated nucleotide database within all 6 reading frames (both strands).

Compare the 6-frame translation of the nucleotide question sequence to the 6-frame translation of the tblastx -nucleotide sequence database.

BLAST uses the following search parameters:

HISTOGRAM-shows a histogram of scores for each search; The default is yes (see parameter H in the BLAST booklet).

DESCRIPTIONS-limits the number of simple descriptions of matched sequences reported in characterized numbers; The default limit is 100 descriptions (see parameter V in the book pages).

EXPECT-statistical significance threshold for the report is matched against the database sequence; The default value is 10, and according to the stochastic model of Karlin and Altschul (1990), 10 matches are expected to be found only by chance. If the statistical significance of the match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent and result in fewer reported match opportunities. Fractional values can be accepted (see parameter E in the BLAST booklet).

CUTOFF-Cuts off the score for reporting high-scoring segment pairs. The default value is calculated from the EXPECT value (see above). HSPs are reported for database sequences when the statistical significance is at least equal to the size of the isolated HSP with a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, resulting in fewer chances of a reported match (see parameter S in the BLAST booklet). In general, significance thresholds can be more strongly managed using EXPEXT.

ALIGNMENTS—limit database sequence to the specified number of high-segment segments (HSPs) reported; The default limit is 50. If more database sequences meet the statistical significance threshold for reporting, only the match of the largest statistical significance is reported (see parameter B in the BLAST booklet).

MATRIX-characterizes different scoring matrices for BLASTP, BLASTX, TBLASTN and TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). Other choices available include PAM40, PAM120, PAM250 and IDENTITY. There is no other scoring matrix available for BLASTN; Characterizing MATRIX instructions within BLASTN requests results in an error response.

STRAND-limit TBLASTN search to only the top or bottom of the database sequence; Or limiting LASTP, BLASTX, or TBLASTX searches to only reading frames on top or bottom of the query sequence.

FILTER—segment or segment of question sequences with low compositional complexity, as measured by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17: 149-163. Claverie & States (1993) Computers and Chemistry 17: 191. Mask off segments consisting of short-period interval repetitions as measured by the DUST program of the Tatusov and Lipman (see http://www.ncbi.nih.gov.) mask off). Filtering removes statistically significant but not biologically interesting reports from the blast output (i.e. common acid-, basic- or proline-rich regions), and of query sequences useful for specific matching to database sequences. Leaves more area of biological interest.

The low complexity sequences found by the filter program are replaced using the letter "N" in the nucleotide sequence (ie "NNNNNNNNNNNNN") and using the letter "X" in the protein sequence (ie "XXXXXXXXX").

Filtering applies only to the query sequence (or its translation product), not to the database sequence. The default filtering is DUST for BLASTN and SEG for other programs.

When applied to sequences in SWISS-PROT, filtering is not always effective because it is not at all specific to anything masked by SEG or XNU or both. Moreover, in some cases, the sequence is fully masked, indicating that the statistical significance of any match reported against the unfiltered query sequence may be suspected.

NCBI-gi-Causes the NCBI-gi identifier to appear in the output in addition to the access and / or locus name.

Most preferably the sequence comparison is performed using a simple BLAST search algorithm provided at http: //www.ncbi.nih.gov.BLAST.

Other computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387) and FASTA (Atschul et al 1990 J Molec Biol 403-410). It is not an enemy.

In some aspects of the invention no gap penalty is used when measuring sequence identity.

The invention also encompasses nucleotide sequences or segments or derivatives thereof that are complementary to the sequences set forth herein. When a sequence is complementary to its segment, that sequence can be used as a probe to demonstrate similar promoter sequences in other organisms.

The present invention also encompasses nucleotide sequences or segments or derivatives thereof that can hybridize to the sequences set forth herein.

Hybridization is the same as that performed in a polymerase chain reaction as described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratoory Manual, Cold Spring Harbor Press, Plainview NY). As well as the amplification process, the strand of nucleic acid binds together with the complementary strand through base pairs (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY).

Also included in the scope of the present invention are nucleotide sequences capable of hybridizing to the nucleotide sequences set forth herein under maximum stringency conditions. Hybridization conditions are based on the melting point (Tm) of the nucleic acid binding complex as known by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA). , "Stringency" as defined below.

Maximum stringency generally occurs at about Tm-5 ° C. (below 5 ° C. of the Tm of the probe); High stringency is about 5-10 ° C. Tm; Intermediate stringency is about 10-20 ° C. or less, Tm; Low stringency is about 20-25 ° C. or less, Tm. As will be appreciated by those skilled in the art, maximum stringency hybridization may be used to verify or detect the same nucleotide sequence, while medium (or low) stringency hybridization may be used to identify similar or related nucleotide sequences. Can be used to prove or detect.

In a preferred aspect, the present invention includes nucleotide sequences capable of hybridizing to the nucleotide sequences of the present invention under stringent conditions (ie, 65 ° C. and 0.1 × SSC).

The invention also encompasses nucleotide sequences or segments or derivatives thereof capable of hybridizing to the complementary sequences of the sequences presented herein. Likewise, the present invention includes nucleotides that are complementary to sequences capable of hybridizing to the sequences of the present invention. This type of nucleotide sequence is an example of a nucleotide variant.

In this respect the term "variant" includes sequences complementary to sequences capable of hybridizing to the nucleotide sequences set forth herein. Preferably, however, the term “variant” can hybridize to the sequences set forth herein under stringent conditions (ie 65 ° C. and 0.1 × SSC {1 × SSC = 0.15M NaCl, 0.015 citric acid Na ₃ pH 7.0}). Include sequences complementary to the sequence in which they reside.

The present invention demonstrates that TRPs can be expressed in the laboratory and in vivo. Expression of the TRPs of the invention in the laboratory and in vivo, the TRP is:

(Iii) can be administered to a human or animal with a tumor, such as anti-angiogenic therapy

(Ii) can be monitored in serum, urine or tissue of a human or animal as an indication marker;

(Iii) can be used as a basis for analyzing serum and urine of patient cancers for similar anti-angiogenic molecules

(Iii) It is advantageous because it can be used alone or in combination as a therapeutically useful agent in the treatment of angiogenesis and / or cancer.

The present invention also describes the surprising discovery of a new isoform of brain angiogenesis inhibitor (BAI) 3 composed of TRPs capable of inhibiting endothelial cell proliferation and migration activity.

The term "angiogenesis" refers to the creation of new blood vessels within a tissue or organ. Under normal physiological conditions, humans or animals develop angiogenesis only within very specific limited circumstances. For example, angiogenesis is generally found within wound treatment, fetal / embryonic development and formation of the corpus luteum, endometrium and placenta.

The term “angiogenesis inhibitor” refers to an agent that is capable of preventing the growth of new blood vessels into tissues, such as nonangiogenic and angiogenic tumors. The term may generally refer to molecules capable of inhibiting angiogenesis and inhibiting bovine and / or human capillary endothelial cell growth in culture, for example in the presence of an angiogenic stimulant. This term includes agents such as matrix metalloproteins (MMPs) that naturally generate inhibitors of both angiogenesis and tumor metastasis because of their ability to degrade external matrices.

The term "endothelium" refers to a thin layer of flat epithelial cells aligned in the serous cavity, lymphatic vessels and blood vessels of the intima.

The term “angiogenic stimulant” includes but is not limited to factors such as vascular endothelial growth factor (VEGF), PDGF and bFGF (Kuwabara et al PNSA 1995 92 4606) that can stimulate endothelial cell proliferation and migration. The term "angiogenic stimulant" is interchangeable with the term "pro-angiogenic factor".

Nucleotide sequences of the invention can be designed to alter TRP encoding nucleotide sequences of various causes, including, but not limited to, changes that modify the cloning, processing, and / or expression of gene products. Mutations, for example, to change glycosylation patterns or to change codon priorities, are well known in the art, namely site-directed, inserting new restriction sites. Mutagenesis can be introduced.

In another embodiment of the present invention, TRP native, modified or recombinant encoding nucleotide sequences may be ligated to heterologous sequences encoding fusion proteins. For example, for screening peptide libraries for TRP activity inhibitors, it may be useful to encode chimeric TRP expressing heterologous epitopes recognized by commercially available antibodies. have.

In addition, the present invention is glutathione-S-transferase (glutathione-S-transferase)

(GST)), biotin, His6, c-myc tag (see Emrich et al 1993 Biocem Biophys Res Commun 197 (1): 214-220), hemagglutinin (HA) (Wilson et al (1984 Cell 37 767) the same) as those described in or FLAG epitope (Ford et al 1991 Protein Expr Purif Apr; 2 (2): 95-107) and the affinity (affinity) TRP, or a tag such as an enzyme linked to And use of a fusion protein consisting of an actively active segment or derivative.

In another preferred aspect, the fused recombinant protein is a relatively large co-protein, GST, beta-galactosidase from Haemophilus influesnzae , which dissolves and promotes its products and purified products. Or antigenic co-proteins such as lipoprotein D. The fused protein may also consist of a carrier protein, such as bovine serum albumin (BSA) or keyhole limpet haemocyaini (KLH). In a particular embodiment of the invention the labeling sequence is a hexa-histidine peptide provided in a pQE vector (Qiagen Inc) and described in Gentz et al (1989 PNSA 86: 821-824). Such fusion proteins can be readily expressed in yeast cultures (as described in Mitchell et al 1993 Yeast 5: 715-723) and are easily purified by affinity chromatography. The fusion protein may also be designed to contain a cleavage site located between the TRP encoding nucleotide sequence and the heterologous protein sequence so that the TRP is cleaved and purified from the heterologous moiety. Co-proteins can also act as adjuvants in providing generalized stimulation of the immune system. Co-proteins may be attached to the amino or carboxyl terminus of the first protein.

Although the presence / absence of label gene expression suggests that the nucleotide sequence and / or its TRP is also present, its presence and expression is assured. For example, when a TRP encoding nucleotide sequence is inserted into the label gene sequence, recombinant cells containing the TRP coding region can be demonstrated by the absence of label gene fusion. The marker gene can also be located in association with a TRP encoding nucleotide sequence under the control of a single promoter. In general, expression of marker genes for induction or selection indicates TRP expression.

Additional methods of quantifying the expression of specific molecules are radiolabeled (Melby PC et al 1993 J Immunol Methods 159: 235-44) or biotin attached (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides. , Standard curves with coamplification of control nucleic acids and experimental results. Quantification of complex samples can be faster as the TRP of interest is analyzed in an ELISA format with varying dilution concentrations, and spectrophotometirc or calorimetric reactions allow for rapid quantification.

The invention also relates to expression vectors and traits consisting of TRP encoding nucleotide sequences or variants, homologues, segments or derivatives thereof for the in vivo or laboratory production of TRP in terms of screening agents that may affect TRP expression or activity. It relates to a converted host cell.

According to the present invention, TRP encoding nucleotide sequences, TRP segments, fusion proteins or functional equivalents thereof can be used to generate recombinant DNA molecules capable of directing expression in a suitable host cell. In one embodiment of the invention the nucleotide sequence encoding the TRP of the invention is operably linked to a promoter sequence capable of directing the expression of the TRP encoding nucleotide sequence in a suitable host cell. When inserted into a host cell, the transformed host cell may be cultured under suitable conditions until the cell is degraded and TRP is isolated until a sufficient level of TRP is achieved.

The TRP encoding nucleotide sequence itself is well known for segmentation and sequencing of genetic material, including the use of oligonucleotide probes and PCR amplification techniques to prove a known target sequence or suspicious sequence having sequences substantially homologous to the target sequence. Known techniques can be used to separate from cells.

One example of a method for generating TRP using recombinant DNA technology includes the steps of (1) verifying and purifying TRP including a protein such as thrombospondin (TSP), (2) 5 'and 3' DNA oligonucleotide primers of the TRP sequence. synthesizing (primer), (3) amplifying the TRP nucleotide sequence using a polymerase, (4) inserting the amplified TRP encoding nucleotide sequence into a suitable vector, such as an expression vector, (5 ) Inserting the vector containing the TRP encoding nucleotide sequence into an organism or other expression system capable of expressing the TRP encoding nucleotide sequence and (6) separating the recombinantly generated TRP. This technique is described in more detail in an experiment such as "Molecular Cloning: a Laboratory Manula" Second Edition by Sambrook et al , Cold Spring Harbor Press, 1989.

The TRP nucleotide sequence also uses PCR as a suitable primer to (1) isolate messenger RNA from tissue, (2) use reverse transcription to generate the corresponding DNA sequence, and (3) amplify the TRP encoding nucleotide sequence. By separating from cells or tissues (such as tumors) that express high levels of TRP.

Another method of generating TRP or its biologically active fragments is by peptide synthesis. If a biologically active segment of TRP is found using an assay system described in more detail below, it can be sequenced by, for example, automated peptide sequencing methods. In addition, the separation of the nucleotide sequence that encodes TRP, for example by the method described above, determines the DNA sequence and provides information about the amino acid sequence. Thus, if a biologically active segment such as trypsin digestion is produced by a particular method or if the segment is an N-terminal sequence, the remaining amino acid sequence can be determined from its corresponding DNA sequence.

Modified TRP nucleotide sequences according to the present invention include the deletion, insertion or substitution of other nucleotide residues resulting in a sequence encoding the same or functionally identical TRP.

The invention may also include the deletion, insertion or substitution of amino acid residues that produce a silent change and result in a functionally identical TRP. Careful amino acid substitutions should be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues so that the biological activity of the TRP is maintained for the same period of time. For example, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine, arginine; Amino acids with uncharged polar hair groups with similar hydrophilicity values include leucine, erucine, valine, glycine, alanine, arparagine, glutamine, serine, threonine, phenylalanine and trocin.

TRP can also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Domains facilitating such purification include histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that enable purification on immobilized immunoglobulins, and the FLAGS expansion / affinity purification system (Immunex Corp.). , Metal chelating peptides such as, but not limited to, domains used in Seatle, WA). The inclusion of cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego, Calif.) Between the purification domain and TRP is useful for facilitating purification.

Host cells transformed with TRP encoding nucleotide sequences can be cultured under conditions suitable for expression and recovery of TRP from cell culture. Proteins produced by recombinant cells can be secreted or contained internally depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors containing TRP encoding nucleotide sequences can be designed as signal sequences that direct the secretion of TRP encoding nucleotide sequences through certain prokaryotic or eukaryotic cell membranes. Other recombinant constructs can combine TRP encoding nucleotide sequences with nucleotide sequence encoding polypeptide domains that will facilitate purification of water soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: 441-53 and vectors containing fusion proteins). See discussion above).

Once the amino acid sequence of the TRP is known, the segment can be synthesized by techniques well known in the art as illustrated by "Solid Phase Peptide Synthesis: A Practical Approach" by E. Atherton and RC Sheppard, IRL Press, Oxford England. . In brief, multiple segments can be synthesized so that they are connected to each other to form larger segments. Such synthetic peptide fragments can also cause amino acid substitutions at specific locations to test effective and opposing activities in the laboratory and in vivo. Peptide segments with high affinity binding to tissue can be used to separate TRP receptors on affinity columns.

Isolation and purification of TRP is a useful step in explaining the mechanism of action of TRP. This promotes the development of drugs that modulate the activity of potential TRP receptors. Isolation of TRP receptors can be used to dry nucleotide probes that monitor the location and synthesis of receptors using in situ and solution hybridization techniques.

The present invention includes TRP derivatives having endothelial cell proliferation and migration inhibitory activity. The present invention includes whole TRP, TRP derivatives and biologically-active TRP segments. These include TRP with endothelial cell proliferation inhibition and migration activity with sugars or other molecules having amino acid substitutions or attached to amino acid functional groups. The present invention also includes nucleotide sequences encoding TRP and TRP receptors and TRPs expressed by such nucleotide sequences.

Other TRP segments are synthesized for use in some applications as antigens for the development of specific anti-serum, as agonists and antagonists at the TRP binding site, and as peptides linked to cytotoxic agents for targeted killing cells that bind to TRP. It may, but is not limited to. Amino acid sequences composed of such peptides are selected based on their position on the outer region of the molecule and are likely to bind antiserum. Tyrosine or lysine is added to segments that do not have residues that promote the levels of reactive amino and hydroxyl groups on the peptide. Such peptide sequences are compared to known sequences using Genbank, Brookhaven Protein, SWISS-PROT, and PIR databases to determine potential sequence homology. This information promotes the removal of sequences that exhibit a high degree of sequence homology to other molecules, enhancing the potential for high specificity in the development of antisera, agonists and antagonists.

TRPs or TRP segments can be synthesized in standard microchemical equipment and purity irradiated with HPLC and mass spectrophotometry. Peptide synthesis methods, HPLC purification and mass spectrophotometry are commonly well known in the art.

TRPs or TRP segments can be coupled with other molecules using standard methods. The amino and carboxyl termini of the TRP are for example radiolabeled using conventional techniques (tyrosine residues-chlororamine T, iodogen, lactoperoxidase; lysine residues-Bolton-Hunter reagent) And isotopically and non-isotopically labeled as such. This coupling technique is selected based on useful functional groups on amino acids, including, but not limited to, amino groups, sulfhydryl groups, carboxyl groups, amide groups, phenol groups, pheno groups, and imidazole groups. It is not an enemy. Various reagents used to influence this coupling include glutaraldehyde, diazotized benzidine, carboiimide, and p-benzoquinone. .

TRPs and TRP segments are chemically coupled with isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, radionucleotides and other compounds for a variety of applications. The efficiency of the coupling reaction is measured using other techniques suitable for the particular reaction. For example, antiseptic labeling of TRP peptides with ¹²⁵ I is achieved with high specific activity of chloramine T and Na ¹²⁵ I. The reaction is terminated by sodium bisulfate and the mixture is desalted on a disposable column. Labeled peptides are eluted from the column and fractions are collected. Subsamples are withdrawn from each fraction and radioactivity is measured in a gamma counter. Na ¹²⁵ I that did not react in this manner is isolated from the labeled TRP peptide. Peptide fractions with the highest specific radioactivity are stored for other uses, such as assay for binding to TRP antiserum.

TRPs and TRP segments are used to develop affinity columns for the separation of TRP receptors from cultured tumor cells. Isolation and purification of TRP receptors is followed by amino acid sequencing. Nucleotide probes are then developed for insertion into a vector for expression of the receptor. This technique is well known to those skilled in the art. Transfection of TRP receptors into tumor cells enhances the responsiveness of these cells to endogenous or external TRPs, thereby reducing the rate of metastatic growth.

Synthetic TRP segments have a variety of uses. TRP or TRP segments that bind to the TRP receptor with high specificity and binding can be radiolabeled and used for visualization and quantification using radiographic and cell membrane binding techniques. This application provides an important diagnostic and research tool. Knowledge of the binding properties of TRP receptors facilitates the study of transduction mechanisms linked to receptors.

Moreover, labeling these peptides with short-lived isotopes to locate tumors with TRP binding sites can visualize receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques. Can be.

Systemic substitution of amino acids in such synthesized peptides yields high affinity peptide agonists and antagonists for TRP receptors that enhance or reduce TRP binding to its receptor. These agents inhibit the growth of micrometastases and limit the spread of cancer. Antagonists against TRP are applied in situations of insufficient angiogenesis to block the inhibitory effects of TRP and promote angiogenesis. This treatment has a therapeutic effect that promotes wound healing in diabetes.

Another application of peptide conjugation is the production of polyclonal antisera. For example, TRPs or TRP segment glutaraldehyde containing lysine residues are linked to purified bovine serum albumin. The efficiency of the reaction is determined by measuring the incorporation of radiolabeled peptides. Unreacted glutaraldehyde and peptides are separated by dialysis. The conjugate is stored for other use.

The TRPs or TRP segments of the invention can also be used to generate antibodies specific for TRP. The antibody may be a polyclonal or monoclonal antibody. Such antibodies that specifically bind to TRPs or TRP segments can be used in diagnostic methods and kits well known to those skilled in this distribution to confirm or quantify the presence of TRP or TRP receptors in body fluids or tissues. The results of these tests can be used to diagnose or predict the onset or recurrence of cancer and other angiogenesis related diseases.

Antisera against TRP can be produced. After peptide synthesis and purification, both monoclonal and polyclonal antiserum are increased using techniques well known in the art. For example, polyclonal antiserum may be increased in rabbits, sheep, goats or other animals. TRPs conjugated to a carrier molecule, such as bovine serum albumin or TRP itself, are combined with the adjuvant mixture, emulsified, and injected subcutaneously at multiple sites in the back, neck, flanks and sometimes footpads. Second injections are made at regular intervals, such as once every 2-4 weeks. Blood samples are obtained by paternity using, for example, ear margin veins after dilation, almost 7-10 days after each injection. Blood samples are coagulated overnight at 4 ° C. and centrifuged at nearly 2400 × g at 4 ° C. for about 30 minutes. Such serum is recovered and partitioned and stored at 4 ° C. for immediate use or at −90 to −20 ° C. for other analysis.

All serum samples from polyclonal antiserum production or media samples from production of monoclonal antiserum were analyzed for determination of titer. Titration is achieved via some means using, for example, dot blot and density analysis and also radiolabeled peptide-antibody complexes using protein A, second antiserum, cold ethanol or charcoal-dextran By precipitation and activity measurement by a gamma counter. The highest titer of antiserum is also purified on commercially available affinity columns. TRPs are coupled to the gel in the affinity column. Antisera samples flow through the column and the anti-TRP antibody remains bound to the column. These antibodies are then eluted and collected and evaluated for determination of titer and specificity.

The highest TRP antiserum titer is a) optimal antiserum dilution for highest specific binding and lowest non-specific binding of the antigen, b) binding capacity to increase the amount of TRP within the standard substitution curve, c) plasmin Related peptides and proteins, including plasminogen, and also potential cross-reactivity with TRP of related species, d) tested to establish the ability to detect TRPs in plasma, urine, extracts of tissues and in cell culture media.

Passive antibody treatments with antibodies specifically binding to TRPs can be used to regulate angiogenesis-development processes and tissue repair such as regeneration, development and wound healing. Moreover, antisera directed to the Fab region of TRP antibodies can be used to block the ability of endogenous TRP antisera to bind TRPs.

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

TRPs can also be used in diagnostic methods and kits for detecting and quantifying antibodies capable of binding to TRP. Such kits enable detection of the circulation of TRP antibodies that exhibit the spread of micrometastasis in the presence of anti-TRP secreted by the first tumor in situ. Patients with such circulating anti-TRP are more likely to develop tumors and cancer, and are more likely to recur cancer after a treatment or remission period. Fab fragments of such anti-TRP antibodies can be used as antigens to generate anti-TRP Fab-segment antisera that can be used to negate the removal of circulating TRP by anti-TRP antibodies.

Kits for the measurement of TRPs are also contemplated as part of the present invention. Antisera with the highest titer and specificity and capable of detecting TRP in plasma, urine, tissue extracts and cell culture media make TRP's fast, reliable, sensitive and specific measurement and location kit easy to use. Is further measured. Such assay kits include, but are not limited to, the following techniques; Competitive and non-competitive assays, radioimmunoassays, bioluminescence and chemiluminescence assays, fluorescence assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, Antibody-coated strips or dipsticks and immunocytochemistry for rapid monitoring of urine or blood. For each kit, the scope, sensitivity, precision, reliability, specificity and reproducibility of the assay are established. Intraassay and interassay variations were defined as 20%, 50% and 80% points on a standard curve of substitution or activity.

One example of an assay kit typically used in laboratories and hospitals is a radioimmunoassay (RIA) kit. After successful radioiodination and purification of the TRP or TRP segment, the antiserum with the highest titer is added to the test tubes containing relatively constant amounts of radioactivity, such as 10,000 cpm, in appropriate buffer systems. Other tubes contain buffer or pre-immune serum to measure non-specific binding. After 24 hours of incubation at 4 ° C., protein A is added, the test tubes are mixed, incubated at room temperature for 90 minutes, and precipitated at about 2000-2500 × g at 4 ° C. to precipitate a complex of antibody bound to the labeled antigen. Centrifuge at Supernatant is removed by aspiration and the radioactivity in the pellet is calculated at the gamma counter. Antiserum dilution bound to about 10-40% of labeled TRP or TRP segments after non-specific binding is further characterized.

Next, the dilution range (about 0.1 pg to 10 ng) of the TRP or TRP segment used in the development of antiserum is determined by adding radiolabeled peptides and known amounts of peptides to the test tubes containing antiserum. For example, after an additional incubation period of 24 to 48 hours, protein A is added, the test tube is centrifuged and the supernatant is removed and the radioactivity in the pellet is calculated. Substitution of the binding of radiolabeled TRP or TRP segments by unlabeled TRP or TPR segments (standard) provides a standard curve. Different concentrations of different TRP segments, TRP from different species and homologous peptides, etc., are added to the assay tube to characterize the specificity of TRP antiserum.

Another aspect of the invention is a method of blocking the activity of excessive endogenous TRP. This is done by passively immunizing humans and animals with antibodies specific for unwanted TRP in the system. Such treatment may be important in the treatment of abnormal ovulation, menstruation and placental formation. This provides a useful means of measuring the effect of TRP removal on the transition process. Fab fragments of TRP antibodies contain a binding site for TRP. This segment is isolated from TRP antibodies using techniques known to those skilled in the art. Fab segments of TRP antiserum are used as antigens to generate anti-Fab segment serum. Injection of this antiserum into the Fab segment of TRP prevents TRP from binding to the TRP antibody. Therapeutic benefits are obtained by nullifying endogenous anti-TRP antibodies by blocking the binding of TRP to the Fab segment of anti-TRP. The net effect of this treatment is to promote the ability of endogenous circulating TRP to reach target cells, thereby reducing the spread of metastases.

Another kit is that used for localization of TRP in tissues and cells. This TRP immunohistochemistry kit provides instructions, TRP antiserum and second antisera linked to fluorescent molecules such as blockable serum and fluorescent isothiocyanate or other reagents used to visualize the first antiserum. Immunohistochemical techniques are well known to those skilled in the art. This TRP immunohistochemistry kit allows for TRP localization in tissue sections and cultured cells using both optical and electron microscopy. It is used for both research and clinical purposes. For example, tumors are biopsied or collected and cut into microtome to measure the site of TRP production. This information is useful for diagnostic and possible therapeutic purposes in the detection and treatment of cancer.

The present invention also encompasses gene therapy in which the TRP encoding nucleotide sequence is regulated in vivo. For example, expression control is achieved by a compound that allows binding to a TRP encoding nucleotide sequence or a corresponding regulatory region that modulates the rate of transcription or translation or regulatory regions associated with a TRP encoding nucleotide sequence.

By way of example, the TRP encoding nucleotide sequence of the present invention is under the control of expression of an expression regulatory element, generally a promoter or enhancer. Enhancers and / or promoters are particularly active in hypoxic or ischemic or hypoglycemic environments such that TRP encoding nucleotide sequences are preferentially expressed in certain tissues of interest, such as tumors, joints or other ischemic sites. Thus, the significant biological or detrimental effects of the TRP encoding nucleotide sequence on the individual to be treated can be reduced or eliminated. Enhancer elements or other elements endowed with regulated expression are present in many copies. Likewise or in addition, enhancers and / or promoters are particularly active within one or more specific cell types, such as one or more macrophages, endothelial cells or combinations thereof. Further examples include post-mitotic terminally differentiated non-abdominal cells such as respiratory pathway endothelial cells, hepatocytes, muscle cells, cardiomyocytes, synoviocytes, first mammary endothelial cells, and macrophage neurons. Include.

Promoters and / or enhancers are structurally effective or tissue or time limited in their activity. Examples of suitable tissue limited promoters / enhancers are those that are very active in tumor cells, such as promoters / enhancers from the MUC1 gene, CE4 gene or 5T4 antigen gene. Examples of time-limited promoters / enhancers are those that respond to ischemia and / or hypoxia such as hypoxic response elements or promoters / enhancers of the grap78 or grap94 genes. Alpha fetal protein (AFP) promoters are also tumor-specific promoters. One preferred promoter-enhancer combination is human cytomegalovirus (hCMV) major immediate early (MIE) promoter / enhancer combination.

Preferably the promoter of the invention is tissue specific. That is, it is possible to modulate the transcription of TRP encoding nucleotide sequences within one tissue while being very "quiet" within another tissue type.

The term "tissue specific" refers to a promoter whose activity is not limited to a single tissue type but which is active within one group of tissues or less active or serene selectivity in other groups. A preferred property of the promoters of the invention is that they have relatively low activity even in the presence of target tissues in the absence of activated hypoxic-regulatory enhancer elements. One means of achieving this is to use a "noise" element that inhibits the activity of the selected promoter in the absence of hypoxia.

The term "hypoxic" means that a particular organ or tissue is under conditions of inadequate oxygen supply.

The expression level of the TRP encoding nucleotide sequence under the control of a specific promoter can be controlled by manipulating the promoter region. For example, other domains within the promoter region may have different gene regulatory activities. The role of these other regions can generally be assessed using vector constructs with other variants of the promoter with specific regions deleted (ie, deletion analysis). This approach is used to demonstrate, for example, the smallest zone that can confer tissue specificity or the smallest zone that confer hypoxic sensitivity.

Many tissue specific promoters described above are particularly advantageous for practicing the present invention. In most cases these promoters can be separated into convenient restriction digest segments suitable for cloning in the selected vector. Promoter segments can also be isolated using polymerase chain reaction. Cloning of the amplified segment is facilitated by integrating the restriction site at the 5 'end of the primer.

TRP or TRP segments can be used in combination with other compositions and procedures for the treatment of a disease. By way of example, TRP or TRP segments may be used in combination with inhibitors of pro-angiogenic factors (angiogenic stimulants) or with compounds / small molecules capable of inhibiting the induction of such factors. As already described, the expression of some of these angiogenesis / angiogenic stimulants is induced by hypoxia. For example, VEGF transcription is induced by hypoxia through the increased stability of HIF-1 transcription factors and the binding of these transcription factors to hypoxic response elements (HREs), enhancer elements present in the 5 'UTR of the VEGF gene (Forsythe et. al MCB 1996 16 4604, Maxwell et al PNAS 1997 94 8104). Potential angiogenesis of the HIF / HRE signaling pathway is expected to inhibit hypoxia-mediated angiogenesis factor induction and thus will be an inhibitor of tumor angiogenesis. The series of receptor shapes are linked to HREs and function in various vector backbones and various cell types (see PCT / GB98 / 02885). This receptor shape can be adapted to small molecule screens that find antagonists that can advantageously bind to the TRPs and TRP segments of the invention.

TRPs or TRP segments can also be used in combination with conventional disease treatment. For example, a tumor can be conventionally treated with surgical, radiation, or chemotherapy combined with a TRP or TRP segment, or the TRP or TRP segment may be The patient may later be administered to postpone the rest and stabilize the first remaining tumor.

TRP can be delivered with a therapeutically effective agent at the same time and at the same site. TRP and therapeutically effective agents can also be delivered at different times and at different sites. TRP and therapeutically effective agents may be delivered in the same delivery vehicle for the prevention and / or treatment of angiogenesis and / or cancer.

TRP or TRP segments can be used in combination with cytotoxic agents for the prevention and / or treatment of angiogenesis and / or cancer. Cytotoxic agents linked to TRP, such as lysine, high affinity TRP segments, TRP antiserum, TRP receptor agonists and antagonists, provide a means for the destruction of cells that bind to TRP. Such cells are found in many locations including but not limited to micrometastasis and the first tumor.

TRP or TRP segments can be used in combination with pre-dose activating enzymes in gene therapy. Instead of or in addition to being selectively expressed in target tissues, a TRP or TRP segment may have a significant or detrimental effect until the individual is treated with one or more pre-dose with pre-dose activating enzymes or enzymes or efficacies. It can be used in combination with another dose of interest nucleotide (NOI) or NOIs encoding a pre-dose activating enzyme or enzymes that do not exhibit a. Treatment with an appropriate pre-dose of an individual in the presence of active NOI leads to an enhanced decrease in tumor growth and survival.

Pre-dose activating enzymes are delivered to the tumor site for the treatment of cancer. In each case a suitable pre-dose is used in the treatment of the patient in combination with a suitable pre-dose activating enzyme. Suitable pre-dose is administered in combination with the vector. Examples of pre-dose include epitope phosphates (along with alkaline phosphates, Senter 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 Immunoother 31: 202-206); Para-N-bis (2-chloroethyl aminobenzonyl glutamate (with carboxypeptide G2); cephalosporin nitrogen mustard carbamate (β) With lactamase; SR4233 (with P450 reductase); with ganciclovir (HSV thymidine kinase, Borrelli et al 1988 Proc Natl Acad Sci 85: 7572-7576) Mustard pre-dose with nitroreductase (Friedlos et al 1997 J Med Chem 40: 1270-1275) and cyclophosphamide (with P450, Chen et al 1996 Cancer Res 56: 1331 -1340).

Examples of suitable pre-dose activating enzymes for use in the present invention include thymidine phosphatase, which activates 5-fluoro-uracil pre-dose capcetabine and furtulon; Thymidine kinase from Herpes Simplex virus that activates livercyclovir; Cytochrome P450, which activates pre-dose, such as cyclophosphamide, with a DNA damaging agent; And cytosine deaminase that activates 5-fluorocytosine and the like. Preferably enzymes of human origin are used.

Other suitable NOI sequences for use in the present invention include cytosine, chemokines, hormones, antibodies, designed immunoglobulin-like molecules, single chain antibodies, fusion proteins, enzymes, immune co-stimulatory molecules, immunoregulatory molecules, anti Sense RNA, transdominant negative mutations of target proteins, toxins, conditional toxins, antigens, tumor suppressor proteins and growth factors, membrane proteins, vascular proteins and peptides, anti-viral proteins and ribozymes ), And derivatives thereof (such as bound receptor groups), and the like, such as, but not limited to, therapeutic and / or diagnostic applications. Such coding sequences, when included, may be operably linked to other promoters or promoters, such as within a suitable promoter or one or more specific cell types, which generally modulate the expression of ribozyme (s).

The expression product encoded by NOIs may be a protein secreted from the cell. NOI expression products can also be active intracellularly without being secreted. In both cases the NOI expression product describes a bystander effector or a separate bystander effector; That is, expression products in one cell that lead to the death of neighboring or distinct (ie metastatic) additional and related cells possessing a common phenotype.

Suitable NOIs for use in the present invention in the treatment or prevention of cancer destroy target cells (eg ribosomal toxin); Tumor suppressor (such as wild type p53); Activators of anti-tumor immune mechanisms (such as cytokines, co-stimulatory molecules and immunoglobulins); Act as an angiogenesis inhibitor; Or provide enhanced drug sensitivity (such as pre-dose activating enzymes); Natural effector cells indirectly stimulate destruction of target cells (eg, strong antigens that stimulate the immune system) or convert precursor materials into toxic substances that destroy target cells (eg, pre-dose activating enzymes) NOI encoding proteins. The enclosed protein may also destroy bystander tumor cells (eg, secretion of anti-tumor antibody-ribosome toxin fusion proteins), indirectly stimulate the destruction of bystander tumor cells (eg, cytokines that stimulate the immune system, or Or precursors that cause local vascular occlusion) or convert to precursors (i.e., enzymes that activate the pre-dose into diffuseable drugs).

In addition, delivery of NOI (s) encoding antisense transcripts or ribozymes that interfere with the expression of cellular genes for tumor persistence (e.g., against aberrant myc transcripts of Burkitts lymphoma or of chronic myelogenous leukemia The use of such a combination of NOIs (as opposed to bcr-abl electron bodies) is also observed.

Suitable NOIs for use in the treatment and prevention of ischemia heart disease include NOIs encoding plasminogen activators. Suitable NOIs for use in the treatment and prophylaxis of rheumatoid arthritis or brain malaria include anti-inflammatory proteins, antibodies to tumor necrosis factor (TCF) alpha, and anti-adhesion molecules (such as antibody molecules or receptors of specific adhesion molecules). Contains the gene that encodes.

Examples of hypoxic adjustable therapeutic NOIs are shown in PCT / GB95 / 00322 (WO-A-95 / 21927).

As is well known in the art, a vector is a means of enabling or facilitating the transfer of an entity from one environment to another. Some vectors used according to the invention and by way of example in recombinant DNA techniques transfer entities such as DNA segments (such as heterologous cDNA segments, such as heterologous DNA segments) to target cells. Optionally, within the target cell, the vector may maintain heterologous DNA in the cell or act as a unit of DNA replication. Examples of vectors used in recombinant DNA techniques include plasmids, chromosomes, artificial chromosomes and viruses.

Vectors can be delivered by viral or non-viral techniques.

Non-viral delivery systems include, but are not limited to, DNA translation methods. Transfection here involves the use of a non-viral vector to deliver the gene to the target mammalian cell.

Common methods of infection include electrophoration, DNA biolistics, lipid-mediated cell infection, dense DNA-mediated cell infection, liposomes, immunoliposomes, lipofectin, cationic drug-mediated cations Amphiphiles (CFAs) (Nature Biotechnology 1996 14: 556), polyvalent cations such as spermine, cationic lipids or polylysine, 1,2-bis (oleoyloxy) -3- (Trimethylammonio propane (DOTAP) -cholesterol complex (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.

Viral delivery systems include, but are not limited to, adenovirus vectors, adeno-binding virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentiviral vectors, or baculoviral vectors.

Examples of retroviruses include murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), murine mammalian tumor virus (MMTV), Rous sacroma virus (RSV) Fujinami Sacroma Virus (FuSV), Moloney Rat Leukemia Virus (Mo-MLV), FBR Rat Osteosacroma Virus (FBR MSV), Moroni Rat Sacroma Virus (Mo- MSV), Abelson Rat Leukemia Virus (A-MLV), Avian Myelomyosis Virus-29 (MC29), and Avian Red Leukemia Virus (AEV).

A detailed list of retroviruses is presented in Coffin et al ("Retroviruses" 1997 Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).

Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), causative agents of human autoimmune deficiency syndrome (AIDS), and apes immunodeficiency virus (SIV). The non-primate lentiviral group includes the Goat Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV) and the recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV), as well as the original "slow virus". Visna / maedi virus (VMV).

The difference between lentiviral and other types of retroviruses is that lentiviral has the ability to infect both dividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, other retroviruses, such as MLVs, cannot infect non-dividing cells that form, for example, muscle, brain, lung, and hepatic qualities.

Other examples of vectors include ex vivo delivery systems, including but not limited to methods of DNA cytofection, such as electroporation, DNA bioistics, lipid-mediated transfection, dense DNA-mediated cell infection.

The vector may be a plasmid DNA vector. The vector can also be a recombinant viral vector. Suitable recombinant viral vectors include adenovirus vectors, adeno-binding virus (AAV) vectors, Hulps-virus vectors or preferably retroviral vectors. In the case of viral vectors, gene transfer is mediated by viral infection of target cells.

The vector of the present invention is shaped as a split-intron vector. Split intron vectors are described in PCT patent applications GB98 / 02885 (WO99 / 15684) and GB98 / 02867 (WO99 / 15683).

When the shape of the adenovirus is combined with the genetic stability of the retrovirus / lentiviral, the adenovirus can be used for transduced target cells that essentially become transient retrovirus producing cells capable of stably infecting neighboring cells. . Retroviral producing cells designed to express such TRP and / or NOIs of the invention may be transplanted into an organism such as an animal or human for use in the treatment of angiogenesis and / or cancer.

The dosage of TRP of the present invention will vary depending on the disease state or condition being treated and other clinical factors such as the weight and condition of the human or animal and the route of administration of the compound. Depending on the half-life of TRP in certain animals or humans, TRP may be administered from several times a day to once a week. It is understood that the present invention is applicable to both human and veterinary use. The methods of the invention contemplate multiple administrations as well as multiple administrations given concurrently or in an extended period of time.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions containing anti-oxidants, buffers, bacteriostatic agents and solutes to make the formulation of the recipient's blood and isotonic; Aqueous and non-aqueous sterile suspensions containing suspending and thickening agents. The formulation may be present in unit- or multi-dose containers such as sealed ampules and vials, for example requiring only the addition of a sterile solution carrier such as water for injection just prior to use. Stored in freeze-dried conditions. Instant injection solutions and suspensions can be prepared from sterile powders, particulates and tablets of the kind described above.

TRPs are effective in the disease or process being treated that is mediated by or including angiogenesis. The present invention encompasses methods of treating angiogenic mediated diseases with effective amounts of TRP or TRP agonists and / or antagonists. TRPs and TRP segments can be provided as proteins and protein segments separated and subsequently purified into pharmaceutically acceptable compositions using formulation methods generally known to those skilled in the art. Such compositions may be administered by standard routes. They may be oral, rectal, eye (including intravitreal or intracameral), nose, topical (including oral and sublingual), intrauterine, vaginal or parenteral (subcutaneous, intraperitoneal, Intramuscular, intravascular, intradermal, intracranial, intratracheal and epidural), transdermal, intraperitoneal, intracranial, cerebral, cerebral, intravaginal, intrauterine or parenteral (ie, intravascular, spinal, subcutaneous or muscular) My) including, but not limited to, routes.

TRP formulations may conveniently be presented in unit dose and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient with the drug carrier (s) or excipient (s). In general, the formulation is prepared constantly, the active ingredient is combined with the liquid carrier or the finely divided solid carrier or both, and the product is then shaped if necessary.

Moreover, the TRPs of the present invention can be incorporated into biodegradable polymers, such as, for example, the site of a tumor, to be implanted near the intended site or implanted to release TRP synthetically slowly, thereby maintaining release of the compound. Biodegradable polymers and their use are described in detail in, for example, Brem et al , J. Neurosurg 74: 441-446 (1991). Osmotic minipumps are also used to provide controlled delivery of high concentrations of TRPs directly to the site of interest, such as through cannulae, directly to metastatic growth or to vascular supply of tumors.

Peptides linked to cytotoxic agents are injected in a designed manner to maximize delivery to the intended location. For example, ricin-linked high affinity TRP segments are delivered directly to the target or to blood vessels supplied to the target site via the cannula. Such agents are also delivered in a controlled manner via an osmotic pump paired with an infusion cannula.

Preferred unit dosage formulations contain the daily administration or unit, daily sub-administration, or a suitable fraction thereof of the components to be administered as listed above. It is to be understood that in addition to the components, in particular, as mentioned above, the formulations of the present invention may include other agents conventional in the art, depending on the formulation type in question.

Pharmaceutical compositions consisting of effective amounts of TRP expression products or TRPs encoding the nucleotide sequences of the invention can be used for the treatment of angiogenesis and / or cancer. Long and unregulated angiogenesis occurs variously in disease states, tumor metastases and abnormal growth by endothelial cells, and supports the pathological damage seen within these conditions. Various pathological disease states in which unregulated angiogenesis are present have been grouped with angiogenic mediated diseases. Diseases mediated by angiogenesis include solid tumors; Blood producing tumors such as leukemia; Benign tumors such as, for example, hemangiomas, auditory neuromas, neurofibromas, trachoma and purulent granulomas; Rheumatoid arthritis; psoriasis; Visual angiogenic diseases such as, for example, diabetic retinopathy, prematurity retinopathy, spot degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibrosis, and rubeosis; Osler-Webber syndrome; Myocardial angiogenesis; Plaque angiogenesis; Capillary dilator; Hemophilic joints; Fibrotic hemangioma; Wound angiogenesis; Coronal side; Cerebral flanks; Arteriovenous malformation; Ischeniic limb angiogenesis; Neovascular glaucoma; Retrorental fibrosis; Diabetic neoangiogenesis; Heliobacter-related diseases include, but are not limited to, fractures, angiogenesis, blood formation, ovulation, menstruation and placental formation.

TRPs are useful for treating diseases of excessive or abnormal stimulation of endothelial cells. Such diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma and hypertrophic scars, ie keloids. TRP can be used as birth control by preventing angiogenesis necessary for culture transplantation. TRP is useful for the treatment of diseases with angiogenesis as a pathological consequence, such as cat scratch disease ( Rochele minalia quintosa ) and ulcers ( Helobacter pylori ).

Example 1 Expression Vector Structure Encoding Type 1 Repeat Segments

The cDNA segment corresponding to the TRPs of the human thrombospondin-1 (TSP-1) open reading frame is amplified from human poly A + mRNA. Amplification is performed by RT-PCR using reverse transcriptase (RT) firstly mRNA is reverse transcribed into cDNA and secondly cDNA is amplified by polymerase chain reaction (PCR). The specific primers used in this reaction (FIG. 13) were in-frame at the Hind III site of the conventional vector pSecTag.2A (Invitrogen, FIG. 4) at the 5 ′ end, and also the same vector at the 3 ′ end. Designed in-frame on the Not I site.

5 ' Hind III XXA AGC TTX

3 ' Not Ⅰ XGC GGC CGC

Also non-angiogenic control peptides (or non-TRP containing peptides) corresponding to the C-terminus of human TSP-1 (FIG. 2) are cloned in the same frame to the vector described above. A set of four synthetic oligonucleotides encoding control peptides was designed (FIG. 5) and cloned with pSecTag.2A digested with the enzyme Hind III to produce pSecTag.2A-TRP2.

The vector pSecTag2 contains a murine Ig kappa chain leader sequence that enables the secretion of TRPs. It also contains a c-myc epitope that will pass through the C-terminus of TRPs. This enables the detection of fusion proteins using anti- c-myc antibodies. Finally pSecTag2 contains a 6 × histidine tag located below from the c-myc epitope that promotes binding with high affinity to the Ni2 + chelating resin for easy purification of the fusion protein. Such constructs allow for efficient and high-level expression of recombinant polypeptides from human CMV immediate early gene promoter enhancers.

Plasmids are transfected with 293T cells by standard methods (ie calcium phosphate), and expression of the fusion polypeptide in the transfected cells is confirmed by Western blotting. pSecTag2 / PSA (Invitrogen), a prostate-specific antigen (PSA) expression plasmid based on pSecTag2.A, is used as a control. The medium in which the infected cells are grown is filtered through a 0.45 micron pore-size filter (Millipore Inc.) and analyzed for secreted fusion proteins. 20 μl aliquots of conditioned media are run on 10-12% SDS / PAGE. Gels are electroblotted on Immobilon membrane (Millipore Inc.). Western blot analysis is performed with anti- c-myc murine monoclonal antibody (Boehirnger) at 1: 1000 dilution and anti-rat IgG (Dako) conjugated with rabbit HRP at 1/2000 dilution. Detection by chemiluminescence is performed using an ECL kit (Amersham International, UK). The production of recombinant fusion proteins is also examined in total protein extracts of the infected cells. Transfected cells are washed in PBS, digested in RIPA buffer containing 1 mM PMPM freshly prepared, and total protein concentration is measured using Bradford's BioRad Reagent (BioRad) according to the manufacturer's instructions. . Cytolytics (20-50 μg of total protein) run on 10-12% SDS / polyacrylamide gels. Proteins are transferred to the immobilon membrane and western blotted as described above.

Example 2 Endothelial Cell Proliferation Assay in a Laboratory

The anti-angiogenic effect of other recombinant TRPs is measured in the laboratory by endothelial cell proliferation assay. Analogous methods are angiogenesis factors such as endostatin, an endogenous suppressor of angiogenesis (O'Reilly et al 1997 Cell 88: 1) or Interleukin 4 (Volpert et al 1998 J Exp Med 188: 1039- 1046).

Human kidney cell line, 293T, is used to generate high levels of secreted recombinant TRPs using pSecTRP expression vectors. 293T cells are instantaneously transfected with 20 μg of each vector per 10 cm 2 tissue culture dish by the calcium phosphate method. After 48 hours—after cell infection, the medium from the infected cells is filtered through a 0.45 micron pore-size filter (Millipore) and added to endothelial cells. Endothelial cells for inclusion in endothelial cell proliferation assays in the laboratory of the present invention include human umbilical vein endothelial cells (HUVEC); Human skin microvascular endothelial cells (HDMVEC); And bovine adrenal capillary endothelial cells (BACE).

Endothelial cells are washed in PBS for proliferation analysis and dispersed into 0.05% trypsin solution. Cell suspensions (25,000 cells / ml) are made with appropriate endothelial cell medium containing 10% calf serum, plated onto gelatinized 24-well tissue culture dishes (0.5 ml / well) It was grown for 24 hours. The medium is replaced with a continuous dilution of 0.5 ml of conditioned medium from 293T transfected cells or basal medium with the appropriate growth factor present. Endothelial cells are grown for 72 hours and redispersed in Hematall (Fisher Scientific, Pittsburgh, PA) before they are dispersed into trypsin. Cells are counted on the culture counter.

Example 3 Endothelial Cell Migration Assay in the Laboratory

This example measures whether other recombinant TRPs have anti-angiogenic effects by inhibiting endothelial cell migration in the laboratory. Migration of other vascular endothelial cell types is measured by adding recombinant TRPs to cultured endothelial cells, namely bovine adrenal capillary endothelial cells (BACE), HUVE cells and HDMVE cells.

Recombinant TRPs are generated in human kidney cell line, 293T, using a protocol similar to that outlined in Example 2. Medium from the transfected cells is filtered through a 0.45 micron pore-size filter (Millipore) and added to endothelial cells. Endothelial cells are plated on gelatinized Nucleopore membranes (5 micron holes for BACE, 8 micron holes for other) in an inverted modified Boyden chamber. After 2 hours, the chamber is inverted and test material is added to the top of each well. In particular, the cell population is exposed to a single culture medium (control), a culture medium containing 10 ng / ml bEGF (basic fibroblast growth factor) or a culture medium containing increasing concentrations of recombinant TRPs and 10 ng / ml bEGF. . The cells are allowed to migrate for 3 to 4 hours. The membrane is then fixed, stained and the number of migrated cells is counted.

Example 4 Demonstration of Type 1 Repeated Peptides

The amino acid sequence of the third type 1 repeat of hTSP-1 was used to perform sequencing. The search was performed at the National Center for Biotechnology Information (NCBI) through the use of the Advanced BLAST 2.0 network service (Altschul et al , 1997 Nucleic Acids Res. 25: 3389-3402). Alignment was performed using an nr database derived from the conceptual translation of sequences from the nucleotide database.

BLAST searches performed with TSP-1 TRP (FIG. 6) show the identity of human TSP-1, TSP-1 precursors of other species and TSP-related proteins. It is also expressed in the central nervous system and exhibits homology to human semaphorin F homologs, Sco-spondin (Bos taurus) and F-spondin, which are important for the indication of axons. TSP-1 TRP also shows homology with brain angiogenesis inhibitors (BAI) 1, 2 and 3 proteins and two new human proteins, KIAA0550 and KIAA0688 proteins (FIG. 7), all of which are expressed in brain tissue.

Example 5 Type 1 Repeated Peptide Plus Proof

Molecular cloning and characterization of the BAI gene has been described previously (Nishimori et al , 1997 Oncogene, 15: 2145-2150). BAI 1 has been shown to be specifically expressed in the brain and inhibits experimental angiogenesis in rat corneal models. Expression of BAI 1 is absent or significantly reduced in glial cell line. More recently, cloning and characterization of two other genes of this line, BAI 2 and BAI 3 have been reported, the latter being absent in some glial cell lines.

A second sequence analysis and alignment was performed using KIAA0550 protein (Accession No. AB011122) or KIAA0688 (Accession No. AB014588) using the same BLAST network system in NCBI. Assays performed with KIAA0688 show concordance with TRP-1 type 1 repeats and secretory proteins containing regions homologous to the cell surface of antigen MS2 and metalloproteinase (FIG. 8). Matrix metalloproteinases (MMPs) constitute another example of naturally occurring inhibitors of both angiogenesis and tumor metastasis because of their ability to degrade their outer matrix.

The analysis performed surprisingly by the KIAA0550 protein shows agreement with the BAI 3, BAI 2 and BAI 1 proteins (FIG. 9). Comparison between KIAA0550 protein (985 aa) and BAI 3 protein (1523 aa) shows that these two proteins have a high degree of sequence identity (FIG. 10). KIAA0550 represents nearly two thirds of BAI 3 that interferes with the first amino acid of both proteins and contains the same type 1 repeats. However, BAI 3 contains an insertion of 22 amino acids at the bottom of the type 1 repeat. They also show differences in the C-terminal domain where the BAI 3 extra 536 amino acids are expanded. This observation suggests that BAI 3 and KIAA0550 may be two isoforms of the same protein produced by another splicing of the same RNA transcript.

Example 6 Structure of Viral Vector for Delivery of Type 1 Repeat Segments

MLV Foundation Vector

The MLV based retroviral vector is rescued by cloning recombinant TRPs from pSecTRPs when the NcoI- Pme I segment is inserted into OBM80 (FIG. 11). The nucleotide sequence encoding the TRP is inserted into OBM80 at the position of human cytochrome P450. Such vectors enable expression of recombinant TRP from the MNV LTR promoter in transduced cells. The vector genome is dried into a single transcription unit containing IRES and the LacZ gene of bacteria. Expression of beta-galactosidase is used as a control of transduction.

In all of the following examples as previously described, three plasmid transfection methods (Soneoka et al , 1995 Nucl. Acids. Res., 23: 628) are used to generate VSV-G pseudotype vectors. . The plasmids used in this experiment were as follows: pHIT60 encoding the MLV gag-pol nucleotide encoding sequence and pRV67 and VSV-G envelope ( env ) glycoprotein nucleotide encoding sequences (Soneoka et al , 1995 ibid ) . In initial experiments 8 μg of OBM80-TRP vector genomes were co-transfected with 8 μg pHIT60 and pRV67 per well of 6-well tissue culture dishes, respectively.

Transfection is performed in human kidney cell line 293T as previously described to generate vector particles (Soneoka et al , 1995 ibid ). Culture supernatants are harvested after 36 hours post-transfection and filtered through a 0.45 micron pore-size filter (Millipore). Two cell lines and one dog melanoma cell line using HT1080 cells and D17 cells are used to titrate pseudotyped retroviral vectors with VSV-G.

Cells are seeded into 6-well tissue culture plates at 3 × 10 ⁵ cells per well before the day of infection. Viral supernatants prepared by transfecting 293T cells with the appropriate plasmid to pseudotype the MLV-TRP vector are added to these cells. Polybrene (8 μg / ml) is added to each well at the time of transduction into 1 ml of tissue supernatant used for infection. After 12 hours of post-infection, the tissue supernatant is replaced with fresh medium. To determine virus titer, cells are washed, fixed, stained and infected 48 hours later. Viral titers are calculated from the number of beta-galactosidase positive colonies.

Adenovirus vector

The first recombinant adenovirus vector generally consists of the deletion of the E1 and E3 regions of the virus, allowing insertion of foreign DNA into the left arm of the virus adjacent to the left inverted terminal repeat (ITR). . Virus package signals 184 to 358 nt overlap with the E1a enhancer and are mostly present in the vector from which E1 has been deleted. Such sequences can be translocated at the right end of the viral genome (Hearing and Shenk, 1983, Cell 33: 695). Therefore, 3.2 kb may be deleted in the vector from which E1 is deleted (358 to 352 nt).

Adenovirus can package 105% of the genome length, thus allowing the insertion of extra 2.1 kb. Therefore, the cloning capacity in the viral vector from which E1 / E3 has been deleted is 7-8kb. Recombinant adenoviruses cannot replicate within non-E1 complementary cell lines because they lack the essential E1 early genes. The 293 cell line was developed by Graham et al (1977, J Gen Virol 36:59) and contains nearly 4 kb from the left end of the Ad5 genome, including ITRs, package signals, E1a, E1b and pIX. The cells stably express the El and Elb gene products but do not express late protein IX even if the pIX sequence is present in Elb. Viruses in which E1 is deleted in non-complementary cells transduce cells and are transported to the cell nucleus but have no expression from the genome from which E1 is deleted.

The schematic in FIG. 12 shows the general strategy used to generate recombinant adenovirus using the Microbix Biosystems-NBL Gene Science System.

The general strategy involves cloning foreign DNA into the El shuttle vector where the El region (402-3328 bp) is replaced by an outer DNA cassette. The recombinant plasmid is then co-transfected with the pJM17 plasmid into 293 cells. pJM17 contains deletion of the E3 region and insertion of 3.7 map units into the E1 region of the prokaryotic pBRX vector (including ampicillin resistance and bacterial duck sequence). Thus this 440 kb plasmid is so large that it cannot be packaged into adeno nucleotides but can be propagated into bacteria. Intracellular recombination in 293 cells results in replacement of the ampr and ori sequences with insertion of foreign DNA.

In the examples cited here, two transfer vectors were used. The first obtained from Microbix is called pE1sp1A and the second obtained from Quantum Biotechnology is called pADBN. The pAFBN plasmid has the advantage that external DNA can be inserted in both directions. In both cases the second DNA is a defective version of the adenovirus genome either as a plasmid (eg pJM17) or as part of a viral DNA (eg right cancer of Ad5). Homologous recombination produces the final gene transfer vector.

A single transcription unit containing a therapeutic gene and a reporter gene has a TRP-IRES- LacZ , indicated by a constitutive promoter, such as CMV, in the following form. This shape is inserted into the pADBN transfer vector (Quantum Biotechnology) to produce AdeOBTRP-1, -2, -3 and -4. Recombinant AdeOBTRPs transfer vectors were linearized ( Ase I) to allow topological recombination in vivo to result in the formation of the desired recombinant adenovirus and co-simulated to 293 cells along the right arm of the purified Ad5 virus (from the Cla I site). Cell infections Viral titer is determined by the number of targeted cells expressing LacZ . This shape does not exclude having another reporter gene or another therapeutic gene at the position of LacZ .

Lentiviral vectors

TRPs are shaped as pONY4 (FIG. 13), an EIAV lentiviral vector in which two genes are expressed in a single transcription unit and associated vector pONY2.1 (FIG. 14). Both are derived from the infectious pre-viral EIAV clone pSPEIAV19 (Payne et al , 1998, J. Virol 72: 483). The single transcription unit has the following shape TRPs-IRES- LacZ . It is not essential for the lentiviral vector to contain a reporter gene and instead a second therapeutic gene may be included at the position of LacZ . As described above, viral particles containing the pONY-TRP genome are produced in an instantaneous three plasmid system (Soneoka et al , 1995 ibid ). VSV-G pseudotype EIAV vectors are titrated on D17 cells. Viral titers are calculated from the number of beta-galactosidase positive colonies.

Regulated virus vector

The present invention includes the use of regulated viral vectors for the delivery of anti-angiogenic TRPs. For example, NOI and TRP encoding nucleotide sequences are expressed under the control of the regulated promoter OBHRE1. The Xia-Gen-P450 vector genome is used as the starting molecule. TRPs are cloned into Xia-Gen-P450 at the position of P450 (FIG. 15). This shape allows for the expression of TRPs under the control of the HRE promoter in the transduced cells. The RRV also contains selectable markers such as reporter genes such as LacX or GFP and neomycin resistance genes. Viral particles are generated as outlined above and used to transduce into D17 cells. The transduced cell population is divided and incubated overnight in normal oxygen (21% oxygen) and low oxygen (0.1% oxygen). Cells are stained by X-gal histology and endpoint titration is calculated. Titration is measured by beta-galactosidase gene expression and represents a change in gene expression between cell groups under conditions of hypoxic and normal oxygen.

Example 7 Structure of Viral Vector Encoding TRPs in Combination with Pre-Dose Activating Enzyme

The present invention is not limited to the generation of viral vectors encoding TRPs. Combinations of pre-dose activating enzymes and TRPs can be shaped within these vectors. Xia-Gen-P450 (FIG. 15) contains a therapeutic gene of human cytochrome P450 that activates the anti-cancer compound cyclophosphamide. The CYP2B6 gene is directed by the CMV promoter. The therapeutic gene is expressed as a single transcription unit in which TRPs are cloned downstream from the IRES and within the location of the LacZ gene. Thus the shape is P450-IRES-TRPs. Another pre-dose activating enzyme can be used for this configuration. The use of constitutive promoters is optional for the present invention. Another shape includes the hypoxic regulatory promoter HRE, which can direct the expression of a single transcription unit described above.

Example 8 Transduction of Glioma Cell Lines in a Laboratory

a) Transduction of Glioma Cell Lines into Recombinant Viral Vectors

Human glioma cell line U87MG is transduced with a recombinant viral vector expressing TRPs, human cytochrome P450, or both of the present invention. 10 ⁵ U87MG cells are sown in 6 well dishes and the virus particles produced as described above are added to the cells for 24 to 48 hours. Stable transformed cells are selected in the presence of 400 mg / ml of geneticin for 2 weeks. Expression of TRPs and / or P450 in stable G418 resistant clones is measured by Northern and Western blots, the highest expression cell lines are selected and used for the next experiment.

b) Endothelial Cell Proliferation Assay in the Laboratory

Culture supernatants of U87MG, U87MG- neo , U87MG-TRPs, U87MG-P450 and U87MG-TRP / P450 with HUVEC cells (10 ⁵ ) plated in 6-well plates and stably transduced glioma cells HUVE cell culture medium is added. After 3 days, HUVE cells are labeled with 2mCi of [ ³ H] thymidine and the integration of these compounds into DNA is quantified by scintillation counting.

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

c) the sensitivity of U87MG to cyclophosphamide (CPA) in the laboratory

U87MG, U87MG- neo and U87MG- stably transduced glioma cells (5 × 10 ⁵ )

TRPs, U87MG-P450 and U87MG-TRP / P450 are sprayed into microwell dishes and treated for 24 hours with various doses of CPA (0-1000 mM) after 24 hours. The cells are subsequently washed and fresh medium is added. After 48 hours cell death is examined by trypan blue exclusion assay.

Example 9 Inhibition of Angiogenesis of Tumors in Vivo

a) transduction of cerebral tumors into a vector encoding TRPs

a.1) Transplantation of Subcutaneous Tumors

U87MG, U87MG- neo , U87MG-TRPs, U87MG-P450 and U87MG-TRP / P450 glioma cells (10 ⁶ ) resuspended in 100 ml of HBSS were found in the right flank of Balb C nude mice ( Nu / Nu ). Injected into the skin. Mice were sacrificed 20 days post-transplantation and tumor volume was measured using the formula: Tumor volume (length) = length [mm] x (width [mm]) ² x 1/2.

a.2) Transplantation of Lower Kidney Tumors

Balb C nude rats ( Nu / Nu ) were anesthetized by ip injection of hypnorma and hypnovel and resuspended in 100 ml of HBSS U87MG, U87MG- neo , U87MG-TRPs, U87MG-P450 or 10 ⁶ of U87MG-TRP / P450 glioma cells are injected into the lower kidney capsule of the left kidney. Mice are sacrificed after 20 days and both kidneys are removed to measure tumor volume, weight and vasculature. Tumor vasculature is measured in sections fixed with paraformaldehyde that reacts with antibodies to factor VIII-associated antigen (Von Willebrand factor) and visualized by biotinylated second and ABC. Microtubules counterstained with hematoxylin below were counted using an optical microscope at x200x within the tumor range with the highest density of vascular staining.

a.3) Transplantation of Cerebral Tumors

Balb C nude rats ( Nu / Nu ) were anesthetized by ip injection of hypnoma and hyponobel located in a stereotaxic injection apparatus and resuspended in U87MG glioma cells (5 × 10 ³ ) in 10 ml of HBSS. The injection is delivered from the epidural to the right tail trachea through a small quartz hole using a vestibule of 2.25 mm lateral and 2.5 mm vertical. Injection is performed via a Hamilton syringe for at least 5 minutes and left for another 5 minutes prior to slow contraction.

b) Transduction of tumors in the cerebrum with vectors encoding both CPA-treated angiogenic peptides

After 4 days, 10 ml of recombinant virus (10 ⁸ particles or LFU / ml) encoding (1) neo , (2) TRPs, (3) P450 or (4) TRP / P450 using the same silica hole and stereotactic coordinates. Intratumoral injections into the) are divided into five different groups awarded. In addition, half of the animals in groups (3) and (4) receive ip injections of CPA after 2-4 days. The survival of these animals is measured, and the measurement of tumors in all groups is made histologically after acute or late killing. Statistical significance between different groups is examined using log rank analysis of Kaplan-Meier survival curves.

(Example 10) An agent capable of inhibiting a proliferative agent or an agent capable of inhibiting the induction of such factor which may be combined with TRPs of the TRP segment of the present invention.

1. Indicator cell line selection to screen for agents that augment or interfere with HRE dependent transcription as described in FIG. 16. The selection of the indicator cell lines is selected to satisfy the following requirements:

(Iii) Optimal indicator cell lines can mediate strong responses to hypoxic stimuli and require stable integration of transcription cassettes on top of appropriate reporters (analytical readout). Such transcription units can co-operate with the inherent features of cell lines that exhibit low basal activity in normal oxygen and exhibit a tension of highly induced reporter activity under hypoxic stimulation. This provides a suitable range of action for examining changes in the transcriptional reaction in the presence of the expected compound / screened small molecule. By way of example, the properties of the transcriptional regulatory element OB HRE1 (see PCT / GB98 / 02885 (WO 99/15684)) are suitable for this requirement.

(Ii) Preferred target cell lines preferably respond highly to hypoxic stimulation. This may be initially defined by the endogenous levels of hypoxic reactive transcription factors exemplified by the bHLH-PAS system, which is HIF1α, HIF2α, HIF3α defined by instantaneous cytometry analysis or bandshift analysis or Western analysis.

(Iii) Cell lines are preferably biologically stable to ensure reproducibility assays with easy production parameters relative to culture parameters. Moreover, the cell lines are preferably selected to be suitable for clinical target tissue in order to be recognized as reference cell lines of widespread use and adaptability. Introduction of the reporter cassette needs to be made to maintain complete hypoxic reactivity regulatory elements. As a result standard transfection techniques that allow multiple replications and random recombination can be considered inadequate. A preferred method of introducing such transcription cassettes is by targeted integration via single copy retroviral cell infection. Single replication components can be selected if the parent cell line is performed with a low majority of infection (MOI).

Isolation of clones with the characteristics as described above:

Incorporation of a selection marker facilitates the isolation of clones with the properties described above. The selection may be an antibiotic resistance marker, ie neomycin, in which case the clone may be selected by culturing in G418. More preferred markers can be stored by using FACS markers, ie GFP or Lac Z (using FDG substrate). Ideally the selection marker is adjusted in the same way as the reporter for analysis. Such regulation can be facilitated using IRES sequences that produce co-regulated bicistronic messages.

The use of a marker that can be a sorted FACS allows for the rapid selection of clones that exhibit low expression levels under hypoxic induction and low basal activity in normal oxygen. The traversal of positive selection and negative selection promotes the creation of cell pools that can exhibit low basal and high inducible activity. Monoclones from these cell pools can be isolated and expanded for final selection. Clonality is a requirement on cell based assay screens.

Hypoxia Regulated Retrovirus Structure

Virus preparation and transduction are standardized (see PCT / GB98 / 02885 (WO99 / 15684)). Genomic shape on transduction is shown in FIG. 17.

Suitable cell lines for use as host assay cells include, but are not limited to, T47D cell line (see PCT / GB98 / 02885 (WO99 / 15684)), rat skeletal muscle cell line C2C12 and rat muscle cell line H9C2.

Selection of the Appropriate Integrated HRE Modulated Retroviral Getom

Cells exhibiting good hypoxic regulation are selected by FACS-storage with expression of LacZ . Simply transduced cells are induced by overnight culture in hypoxic conditions (0.1%). Cells that respond to this stimulus express Lac Z (see FIG. 17). Lac Z expression can be detected on FACS using a Fluoreporter lac Z flow hemocytometer stain kit (Molecular Probes) according to the manufacturer's protocol. Cells showing the highest expression level are isolated and expanded for further traversal of positive FACS selection. X-galvan staining of cells stored in normal and hypoxic allows monitoring of selection. Cells that are not induced and exhibit Lac Z expression under normal oxygen conditions are eliminated by negative selection of FACS. This results in a heterogeneous line indicating tight control. Single cell cloning allows the derivation of homologous cell populations. This is necessary to ensure analysis consistency and line stability.

Screening Analysis

The choice of LacZ regulation also isolates cells that similarly regulate luciferase as a result of genomic shape. Analysis of Luciferase Expression Using Luciferase microtitre luminometry analysis (see PCT / GB98 / 02885) Readout detection is performed as follows:

Briefly, 'indicator cells' are plated at 96-well plates at about 10 ⁴ cells per well (depending on cell type), and after plating for 24 to 48 hours, the cells are 70% fused. Target compounds are added to replicate wells (× 4 or greater) with appropriate controls (compound buffer). The plates are placed in duplicate, one is incubated at normal oxygen overnight (eg 18 hours), and the other is incubated at low oxygen over night. Cells are lysed in situ by addition of luciferase assay lysis buffer (according to the manufacturer's protocol) and then assayed for luciferase activity.

Data obtained from normal oxygen and hypoxic plates indicate the control of hypoxic induction by comparison with the control (without addition of compound or addition of control buffer).

result

Inhibition of hypoxic reactive gene expression

As shown in FIG. 16, a high throughput analysis was devised that enables a fast screen of molecules capable of inhibiting the HIF / HRE signal pathway. This analysis can demonstrate candidate compounds used in the treatment of new-angiogenic dependent diseases such as cancer by interfering with the transcription of HRE dependent genes. Such molecules can be used in combination with the TRP or TRP segments of the invention for the treatment of angiogenesis and / or cancer.

Such assays can be applied to demonstrate compounds that can act as agonists or antagonists within other therapeutic target pathways as outlined in FIG. 16. These therapeutic target pathways include ubiquitination, proteolytic degradation, kinase / phosphatase, duplexing, DNA binding, transcription complex formation, interaction with p300, and oxygen sensor analysis using heme-containing proteins. , Redox reactions and / or kinase signaling pathways.

Sequence list

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

SEQ ID NO: 2 is the Consequences TRP amino acid sequence shown in FIG. 7. The consensus amino acid sequence (SEQ ID NO: 2) was derived from twenty different TRP sequences of the same length that were easily and clearly aligned. Identical sequences were divided by at least 10 sequences.

SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 are shown in FIG.

All publications described in the above specification are incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed is not overly limited to this particular embodiment. Indeed, various modifications of the described methods for carrying out the invention which are apparent to those skilled in molecular biology or related art should be within the scope of the following claims.

Claims (38)

Non-naturally occurring Type 1 Repeat Peptides (TRP) The TRP according to claim 1, which is composed of at least the amino acid sequence represented by SEQ ID NO: 2 or a variant, homologue, segment or derivative thereof. The TRP according to claim 2, consisting of the sequence shown in SEQ ID NO: 2. The TRP according to claim 1, comprising an amino acid sequence as shown in SEQ ID NO: 3 or a variant, homologue, segment or derivative thereof. The TRP according to any one of claims 1 to 3, which is composed of the amino acid sequence shown in SEQ ID NO: 4 or a variant, homologue, segment or derivative thereof. The TRP according to any one of claims 1 to 3, which is composed of the amino acid sequence shown in SEQ ID NO: 5 or a variant, homologue, segment or derivative thereof. The nucleotide sequence of any one of claims 1 to 6, wherein the nucleotide sequence encodes a TRP. Nucleotide sequence encoding a TRP characterized by consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a variant, homologue, segment or derivative thereof Nucleotide sequence encoding a TRP characterized by consisting of the sequence shown in SEQ ID NO: 1 10. The sequence according to any one of claims 7 to 9, wherein the nucleotide sequence capable of hybridizing to the nucleotide sequence or the sequence complementary to the hybridizable nucleotide sequence The nucleotide sequence of claim 7, wherein the nucleotide sequence is operably linked to a promoter. 12. Structure according to any one of claims 7 to 11, characterized in that it consists of said nucleotide sequence The vector of any one of claims 7 to 11, wherein the vector consists of the nucleotide sequence. 12. A plasmid according to any one of claims 7 to 11, consisting of said nucleotide sequence. 12. A host cell according to any one of claims 7 to 11 consisting of said nucleotide sequence. 12. A host organism according to any one of claims 7 to 11, consisting of said nucleotide sequence. The method according to any one of claims 1 to 6, wherein the nucleotide sequence according to any one of claims 7 to 11 is expressed or the TRP is selectively isolated when the invention of any one of claims 12 to 13 is present. Or TRP preparation process consisting of refining 18. The TRP of claim 17, wherein the TRP is generated by the process. The TRP according to any one of claims 1 to 6 or 18, characterized in that it reacts immunologically with the antibody produced against the purified TRP. (a) incubating the endothelial cells under conditions and for a sufficient time to allow the endothelial cells to enter the log phase of growth in the culture medium; (b) introducing an angiogenic stimulant into the culture medium to induce endothelial cell proliferation and migration; (c) introducing an entity; (d) Inhibition of endothelial cell proliferation and migration activity in the laboratory indicates that the entity is a TRP, thus determining whether an entity can inhibit endothelial cell proliferation and migration activity in the laboratory. Analytical method to prove whether an entity consisting of one is TRP The method of claim 20, wherein the assay is for screening TRPs useful for the treatment of angiogenesis and / or cancer. (a) carrying out the analysis according to claim 20 or 21; (b) demonstrating one or more TRPs capable of inhibiting endothelial cell proliferation and migration activity in the laboratory; (c) preparing an amount of one or more of these TRPs A process of steps (a) carrying out the analysis according to claim 20 or 21; (b) demonstrating one or more TRPs capable of inhibiting endothelial cell proliferation and migration activity in the laboratory; (c) preparing a pharmaceutical composition consisting of one or more such certified TRPs A process of steps (a) carrying out the analysis according to claim 20 or 21; (b) demonstrating one or more TRPs capable of inhibiting endothelial cell proliferation and migration activity in the laboratory; (c) modifying one or more of these proven TRPs capable of inhibiting endothelial cell proliferation and migration activity in the laboratory; (d) preparing a pharmaceutical composition consisting of one or more such modified TRPs A process of steps TRP certified by the method of analysis of claim 20 or 21 26. An angiogenesis-inhibiting protein according to any one of claims 1 to 6 or 18 or 19 or 25 (human thrombospondin-1, cerebral angiogenesis inhibitor 1, cerebral angiogenesis inhibitor 2). And cerebral angiogenesis inhibitor 3). 27. The TRP of claim 26 wherein the cerebral angiogenesis inhibitor is isoform of cerebral angiogenesis inhibitor 3. TRP can inhibit endothelial cell proliferation and migration activity in laboratory assays; Method in vivo to affect angiogenesis and / or cancer with TRP in vivo, characterized in that the analytical method as defined in claim 20 or 21 28. Use of a TRP to prepare a pharmaceutical composition according to any one of claims 1 to 6 or 18 or 19 or 25 to 27. Pharmaceutical composition consisting of TRP and another therapeutically useful agent The pharmaceutical composition of claim 30, wherein the other therapeutically useful agent is a pre-dose activating enzyme. 32. A pharmaceutical composition according to claim 31, wherein the other therapeutically useful agent is a pro-angiogenic agent or an agent capable of inhibiting the induction of such an agent. The pharmaceutical composition according to claim 29 or 30, wherein the TRP is a TRP according to any one of claims 1 to 6 or 18 or 19 or 25 or 27. TRP in combination with a pre-dose activating enzyme or a nucleotide sequence of interest (NOI) or a pro-angiogenic factor of interest encoding an agent or an agent capable of inhibiting the induction of such a factor for the treatment of conditions associated with angiogenesis and / or cancer Use of 34. Use of TRP in the preparation of a pharmaceutical composition according to claim 29 or 33 for the treatment of conditions associated with angiogenesis and / or cancer. TRP obtained from a protein containing TRP rather than thrombospondine (TSP) TRP Fusion protein consisting of TRP TRP as described above and referenced by the accompanying drawings