EP1037985A1 - Restine et ses methodes d'utilisation - Google Patents
Restine et ses methodes d'utilisationInfo
- Publication number
- EP1037985A1 EP1037985A1 EP98962966A EP98962966A EP1037985A1 EP 1037985 A1 EP1037985 A1 EP 1037985A1 EP 98962966 A EP98962966 A EP 98962966A EP 98962966 A EP98962966 A EP 98962966A EP 1037985 A1 EP1037985 A1 EP 1037985A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- restin
- cells
- ofthe
- disease
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Definitions
- tumor-associated (O'Reilly et al. (1994) Cell 79:315-28; O'Reilly et al. (1997) Cell 88:277-85).
- Angiostatin a potent endogenous inhibitor of angiogenesis generated by tumor-infiltrating macrophages that upregulate matrix metalloelastase (Dong et al. (1997) Cell 88:801-10), inhibits the growth of a wide variety of primary and metastatic tumors (Lannutti et al (1997) Cancer Res. 57:5277-80; O'Reilly et al.
- endostatin an angiogenesis inhibitor from a murine hemangioendothelioma cell line (EOMA). Circulating levels of a fragment of human endostatin have been detected in patients with chronic renal insufficiency with no detectable tumor, but this fragment had deletions, and no anti-angiogenic activity (Standker et al. (1997) FEBS Lett. 420:129-33).
- the amino terminal sequence of endostatin corresponds to the carboxy terminal portion of collagen XVIII. Endostatin is a specific inhibitor of endothelial proliferation and angiogenesis.
- Restin a novel anti-angiogenic protein is described, as well as fragments, mutants, derivatives and fusion proteins, thereof. Restin is a proteolytic fragment of the C-terminal fragment of collagen XV, and is approximately 20 kDa. One fragment, designated "apomigren,” was found to have anti-angiogenic activity equal or superior to that of endostatin. Methods for expression ofthe proteins at high titer are also described.
- Restin comprises about 170 to about 200 amino acid residues, and has at least 70% sequence identity with the C-terminus ofthe NC10 domain ofthe ⁇ l chain of human Type XV collagen.
- Apomigren is a fragment of restin, and comprises the last 80 to 90 contiguous amino acids corresponding to the C-terminus ofthe NC10 domain ofthe ⁇ l chain of human Type XV collagen.
- the invention also comprises isolated polynucleotides encoding restin and apomigren, complementary sequences and sequences that hybridize those sequences described herein, operably linked to expression sequences, and host cells transformed with such a construct. Antibodies to restin and apomigren are also disclosed.
- the invention also relates to processes for producing restin and apomigren, fusion proteins containing restin and apomigren, and compositions comprising restin and apomigren or fusion products thereof.
- the invention also discloses methods of producing polypeptides encoding restin and apomigren.
- the invention comprises methods for inhibiting angiogenic activity in mammalian tissue, comprising contacting the tissue with a composition comprising restin and apomigren, particularly to inhibit angiogenesis, which occurs in many diseases and conditions, including cancer.
- the invention also discloses use of restin and apomigren to induce apoptosis, or antibodies of EM 1 to prevent apoptosis.
- the invention further discloses use of restin and apomigren in methods of gene therapy.
- the cells targeted may be any mammalian cells, particularly lymphocytes, blood cells, TIL cells, bone marrow cells, vascular cells, tumor cells, liver cells, muscle cells, and fibroblast cells.
- Fig. 1 is a diagram ofthe restin nucleotide sequence (SEQ ID NO: ).
- Fig. 2 is a diagram ofthe restin amino acid sequence (SEQ ID NO: ).
- Fig. 3 is a schematic diagram ofthe Pichia pastoris expression vector from InVitrogen (San Diego, California, USA).
- Fig. 4 is a schematic diagram ofthe modified Pichia pastoris expression vector.
- Fig. 5 is a schematic representation ofthe cloning of restin into the modified Pichia pastoris expression system.
- Fig. 6 is a SDS-PAGE electrophoresis gel stained with Coomassie Blue, showing protein expression from the various libraries.
- Fig. 7 consists of four plots ofthe hydrophilicity, surface probability, flexibility, and antigenic index ofthe restin protein.
- Fig. 8 consists of three plots showing the amphiphilic helix, amphiphilic sheet, and secondary structure ofthe restin protein.
- Fig. 9 is a schematic presentation ofthe full length l chain of human collagen type XV in context with the C-terminal 85-amino acid protein apomigren.
- Fig. 10 is an alignment ofthe amino acid sequences of apomigren, and the C- terminus of mouse and human endostatin.
- Fig. 11 is a graph showing the elution profile of recombinant apomigren from the Ni-NTA column.
- Fig. 12 is a photograph of an SDS-PAGE gel. Sizes in kDa are shown on the left. Lane 1 contains size marker, lane 2, apomigren protein lysate prior to loading on the Ni-NTA column; lane 2, flowthrough from the column; lane 4, fraction 4; lanes 5 and 6, fraction 23, non-reduced and reduced, respectively; lane 7, fraction 24; lane 8, fraction 27.
- Figs. 13 A, 13B and 13C are a set of three photographs showing the changes in C-PAE cell morphology following apomigren treatment as monitored by phase contrast microscopy. Fig. 13 A, control; Fig.
- FIG. 13B treatment with 0.05 ⁇ g/ml apomigren
- Fig. 13C treatment with 0.7 ⁇ g/ml apomigren.
- Pictures were taken 24 hours after treatment, and are shown at 400X magnification.
- Fig. 14 is a graph showing thymidine incorporation into C-PAE cells treated with bFGF and purified soluble apomigren protein expressed from bacteria.
- Fig. 15 is a bar graph showing cell cycle analysis with apomigren.
- the x- axis shows concentration of apomigren, ranging from 0 to 1000 ng/ml, and the y- axis shows percentage of cells in S-phase.
- C-PAE cells are represented by dark bars and IMR-90 cells by white bars.
- Fig. 16 is a graph showing inhibition of endothelial cell migration with different concentrations of apomigren, and mouse and human endostatin.
- concentration ofthe various proteins is on the x-axis and percent cells that migrated in response to bFGF on the y-axis.
- the control is apomigren-treated IC-21 cells ( ⁇ ), and treatments consist of apomigren (•) and mouse (D) and human (o) endostatin on ECV304 cells.
- Figure 17 is a bar graph showing inhibition of all migration in ECV304 cell traeted with restin. Amount of cell migration is shown on the y-axis, and the x-axis shows ECV304 cells treated with either nothing (-bFGF, control), bFGF (positive control), and restin at 3.0, 62.5, 12, 25 and 50 ⁇ g/ml.
- Figure 18 is a graph showing the effects of restin on renal cancer cell (RCC) tumor growth, as compared to endostatin.
- Tumor volume is mm 3 is sown on the y- axis, plotted against days after treatment (x-axis).
- Restin (•) compared favorably with endostatin (o) in slowing tumor growth, relative to untreated control tumors
- Figure 19 is a bar graph showing the effects of varying concentrations of apomigren (x-axis) on angiogenic response (y-axis) as measured by the chorioallantoic membrane (CAM) assay.
- Apomigren 0.5, 1, 5, 10, 20 and 50 ⁇ g/mesh was tested against vehicle alone, and VEGF and FGF-2.
- Figure 20A-B is a chart showing the constructs, primers, cloning sites, and vectors, used to clone and express various anti-angiogenic proteins. The amino acid sequences ofthe expressed proteins are also given.
- a wide variety of diseases are the result of undesirable angiogenesis. Put another way, many diseases and undesirable conditions could be prevented or alleviated if it were possible to stop the growth and extension of capillary blood vessels under some conditions, at certain times, or in particular tissues.
- Several anti- angiogenic proteins have been discovered (e.g., angiostatin, endostatin), but problems have been reported regarding (1) the ability to produce the proteins in sufficient quantity to allow for proper testing of their properties, and (2) the reproducibility ofthe anti-angiogenic properties attributed to these proteins.
- restin a novel anti-angiogenic protein
- restin a novel anti-angiogenic protein
- Polynucleotides encoding restin and its fragments are also described, a well as vectors and host cells comprising those polynucleotides.
- Compositions containing restin as a biologically active component are also described, as well as methods for using restin to inhibit angiogenic activity in mammalian tissues, such as in treating diseases and conditions characterized by angiogenesis.
- the present invention includes compositions and methods for the detection and treatment of diseases and conditions that are mediated by or associated with angiogenesis.
- restin is a proteolytic fragment of about 170 to about 200 amino acids, corresponding to the approximately 200 extreme C-terminal amino acids ofthe NC10 domain ofthe ⁇ l chain of human Type XV collagen. Unlike endostatin, restin has no or minimal affinity for heparin.
- the invention describes a protein designated "restin,” which is a protein of about 170 amino acids to about 200 amino acids, corresponding to the approximately 200 amino acids at the C-terminus ofthe ⁇ l chain ofthe NC10 '• domain of human Type XV collagen.
- restin has not been successfully isolated from body fluids, and appears to not exist naturally outside ofthe native collagen XV molecule. Restin can be cloned out of isolated DNA or a cDNA library. Restin has a molecular weight of about 20 kDa as determined by reducing polyacrylamide gel electrophoresis.
- mouse restin, fragments, mutants, derivatives or fusion proteins thereof are also encompassed by the present invention.
- Fig. 1, SEQ ID NO: the polynucleotide sequence encoding restin (Fig. 2, SEQ ID NO: ) can amplified, e.g., with the forward and reverse primers listed in Table 1 , below.
- the template nucleic acid used for the amplification can be from any mammal. Table 1. Constructs and primer sequences used to amplify anti-angiogenic proteins.
- the resulting polynucleotide amplification product can then be cloned into a suitable vector.
- primer denotes a specific oligonucleotide sequence complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence and serve as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.
- Restin is intended to include fragments, mutants, homologs, analogs, and allelic variants ofthe amino acid sequence of (SEQ ID NO: ) as well as mouse restin, fragments, mutants, homologs, analogs and allelic variants of the mouse restin amino acid sequence. It is to be understood that the present invention is contemplated to include any derivatives ofthe restin that have endothelial inhibitory activity (e.g., the capability of a composition to inhibit angiogenesis in general and, for example, to inhibit the growth or migration of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor, angiogenesis-associated factors, or other known growth factors).
- endothelial inhibitory activity e.g., the capability of a composition to inhibit angiogenesis in general and, for example, to inhibit the growth or migration of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor, angiogenesis-associated factors, or other known growth factors.
- the present invention includes the entire restin protein, derivatives ofthe restin protein and biologically-active fragments of the restin protein. These include proteins with restin activity that have amino acid substitutions or have sugars or other molecules attached to amino acid functional groups.
- the present invention also includes genes that code for restin and the restin receptor, and to proteins that are expressed by those genes.
- the invention also encompasses a composition comprising an isolated polynucleotide encoding restin, as well as vectors and host cells containing such a polynucleotide, and processes for producing restin and its fragments, mutants, homologs, analogs and allelic variants.
- vector as used herein means a carrier into which pieces of nucleic acid may be inserted or cloned, which carrier functions to transfer the pieces of nucleic acid into a host cell. Such a vector may also bring about the replication and/or expression ofthe transferred nucleic acid pieces.
- vectors include nucleic acid molecules derived, e.g., from a plasmid, bacteriophage, or mammalian, plant or insect virus, or non-viral vectors such as ligand-nucleic acid conjugates, liposomes, or lipid-nucleic acid complexes. It may be desirable that the transferred nucleic molecule is operatively linked to an expression control sequence to form an expression vector capable of expressing the transferred nucleic acid.
- Such transfer of nucleic acids is generally called "transformation,” and refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included.
- the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
- "Operably linked” refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner, e.g., a control sequence "operably linked" to a coding sequence is ligated in such a manner that expression ofthe coding sequence is achieved under conditions compatible with the control sequence.
- a "coding sequence” is a polynucleotide sequence which is transcribed into mRNA and translated into a polypeptide when placed under the control of (e.g., operably linked to) appropriate regulatory sequences.
- the boundaries ofthe coding sequence are determined by a translation start codon at the 5 '-terminus and a translation stop codon at the 3'-terminus. Such boundaries can be naturally-occurring, or can be introduced into or added the polynucleotide sequence by methods known in the art.
- a coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.
- the vector into which the cloned polynucleotide is cloned may be chosen because it functions in a prokaryotic, or alternatively, it is chosen because it functions in a eukaryotic organism.
- Two examples of vectors which allow for both the cloning of a polynucleotide encoding the restin protein, and the expression of that protein from the polynucleotide are the pET28(a) vector (Novagen, Madison, Wisconsin, USA) and a modified pPICZ ⁇ A vector (InVitrogen, San Diego, California, USA), which allow expression ofthe protein in bacteria and yeast, respectively.
- host cell is meant a cell which has been or can be used as the recipient of transferred nucleic acid by means of a vector.
- Host cells can prokaryotic or eukaryotic, mammalian, plant, or insect, and can exist as single cells, or as a collection, e.g., as a culture, or in a tissue culture, or in a tissue or an organism.
- Host cells can also be derived from normal or diseased tissue from a multicellular organism, e.g., a mammal.
- Host cell, as used herein, is intended to include not only the original cell which was transformed with a nucleic acid, but also descendants of such a cell, which still contain the nucleic acid.
- the isolated polynucleotide encoding the anti-angiogenic protein additionally comprises a polynucleotide linker encoding a peptide.
- linkers are known to those of skill in the art and, for example the linker can comprise at least one additional codon encoding at least one additional amino acid. Typically the linker comprises one to about twenty or thirty amino acids.
- the polynucleotide linker is translated, as is the polynucleotide encoding the anti-angiogenic protein, resulting in the expression of an anti-angiogenic protein with at least one additional amino acid residue at the amino or carboxyl terminus ofthe anti-angiogenic protein.
- Some linkers attached to anti-angiogenic proteins are illustrated in Figure 20. Importantly, the additional amino acid, or amino acids, do not compromise the activity ofthe anti-angiogenic protein.
- the vector After inserting the selected polynucleotide into the vector, the vector is transformed into an appropriate prokaryotic strain and the strain is cultured (e.g., maintained) under suitable culture conditions for the production of the biologically active anti-angiogenic protein, thereby producing a biologically active anti-angiogenic protein, or mutant, derivative, fragment or fusion protein thereof.
- the invention comprises cloning of a polynucleotide encoding an anti-angiogenic protein into the vectors pET17b or pET28a, which are then transformed into bacteria. The bacterial host strain then expresses the anti- angiogenic protein.
- the anti-angiogenic proteins are produced in quantities of about 10-20 milligrams, or more, per liter of culture fluid.
- the eukaryotic vector comprises a modified yeast vector.
- one method uses a pPICz ⁇ plasmid wherein the plasmid contains a multiple cloning site.
- the multiple cloning site has inserted into the multiple cloning site a His. Tag motif.
- the vector can be modified to add a Ndel site, or other suitable restriction sites. Such sites are well known to those of skill in the art.
- Anti-angiogenic proteins produced by this embodiment comprise a histidine tag motif (His.tag) comprising one, or more histidines, typically about 5-20 histidines. Surprisingly, this His.tag does not compromise anti-angiogenic activity.
- a preferred yeast expression system is Pichia pastor es.
- the biologically active protein is typically produced at concentrations of about 10-20 milligrams per liter of culture medium (fluid).
- One method of producing restin is to amplify the polynucleotide of SEQ ID NO: , clone it into an expression vector, e.g., pET28(a), pPICZ ⁇ A, or some other expression vector, transform the vector containing the polynucleotide of SEQ ID NO: into a host cell capable of expressing the polypeptide encoded by the polynucleotide, culturing the transformed host cell under culture conditions suitable for expressing the protein, and then extracting and purifying the protein from the culture.
- an expression vector e.g., pET28(a), pPICZ ⁇ A, or some other expression vector
- the restin protein may also be expressed as a product of transgenic animals, e.g., as a component ofthe milk of transgenic cows, goats, sheep or pigs, or as a product of a transgenic plant, e.g., combined or linked with starch molecules in maize. Restin may also be produced by conventional, known methods of chemical synthesis.
- the synthetically-constructed restin protein sequences by virtue of sharing primary, secondary or tertiary structural and or conformational characteristics with e.g., recombinantly-produced restin, may possess biological properties in common therewith, including biological activity.
- the synthetically-constructed restin protein sequences may be employed as biologically active or immunological substitutes for e.g., recombinantly-produced, purified restin protein in screening of therapeutic compounds and in immunological processes for the development of antibodies.
- the restin protein is useful in inhibiting angiogenesis, as determined in standard assays, and provided in the Examples below.
- Restin does not inhibit the growth of other cells types, e.g., IMR-90 cells, or IC-21 cells.
- Tests have shown that in endothelial cell migration assays with ECV304 cells, treatment with restin resulted in 50% inhibition at levels of 2.0 - 5.0 ⁇ g/ml, e.g., restin has an ED 50 of about 2.0 - 5.0 ⁇ g/ml. This is similar to that of endostatin, but inferior to the activity found for apomigren.
- a renal cancer cell (RCC) model human restin treatment resulted in 20-50% inhibition of tumor growth.
- ED 50 is an abbreviation for the amount of a composition which reduces a biological effect by one-half, relative to the biological effect seen in the absence ofthe composition.
- angiogenesis means the generation of new blood vessels into a tissue or organ, and involves endothelial cell proliferation. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development, and formation ofthe corpus luteum, endometrium and placenta.
- endothelium means a thin layer of flat epithelial cells that lines serous cavities, lymph vessels, and blood vessels. "Anti-angiogenic activity” therefore refers to the capability of a composition to inhibit the growth of blood vessels.
- the growth of blood vessels is a complex series of events, and includes localized breakdown ofthe basement membrane lying under the individual endothelial cells, proliferation of those cells, migration ofthe cells to the location ofthe future blood vessel, reorganization of the cells to form a new vessel membrane, cessation of endothelial cell proliferation, and, incorporation of pericytes and other cells that support the new blood vessel wall.
- Anti-angiogenic activity as used herein therefore includes interruption of any or all of these stages, with the end result that formation of new blood vessels is inhibited.
- Anti-angiogenic activity may include endothelial inhibiting activity, which refers to the capability of a composition to inhibit angiogenesis in general and, for example, to inhibit the growth or migration of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor, angiogenesis-associated factors, or other known growth factors.
- a "growth factor” is a composition that stimulates the growth, reproduction, or synthetic activity of cells.
- An "angiogenesis-associated factor” is a factor which either inhibits or promotes angiogenesis.
- An example of an angiogenesis-associated factor is an angiogenic growth factor, such as basic fibroblastic growth factor (bFGF), which is an angiogenesis promoter.
- bFGF basic fibroblastic growth factor
- angiogenesis-associated factor is an angiogenesis inhibiting factor such as e.g., angiostatin (see, e.g., U.S. Pat. No. 5,801,012, U.S. Pat. No. 5,837,682, U.S. Pat. No. 5,733,876, U.S. Pat. No. 5,776,704, U.S. Pat. No. 5,639,725, U.S. Pat. No. 5,792,845, WO 96/35774, WO 95/29242, WO 96/41194, WO 97/23500) or endostatin (see, e.g., WO 97/15666).
- angiostatin see, e.g., U.S. Pat. No. 5,801,012, U.S. Pat. No. 5,837,682, U.S. Pat. No. 5,733,876, U.S. Pat. No. 5,776,704, U.S. Pat. No. 5,639,725, U.S. Pat.
- compositions have anti-angiogenic activity, and behaves similarly as does restin, as determined in standard assays.
- Standard assays include, but are not limited to, those protocols used in the molecular biological arts to assess anti-angiogenic activity, cell cycle arrest, and apoptosis.
- Such assays include, but are not limited to, assays of endothelial cell proliferation, endothelial cell migration, cell cycle analysis, and endothelial cell tube formation, detection of apoptosis, e.g., by apoptotic cell morphology or Annexin V-FITC assay, chorioallantoic membrane (CAM) assay, and inhibition of renal cancer tumor growth in nude mice.
- assays are provided in the Examples below, and in U.S.S.N. 60/067,888, filed December 8, 1997, U.S.S.N. 60/082,663, filed April 22, 1998, U.S.S.N. 60/108,536, filed November 16, 1998, and in U.S.S.N.
- XX/XXX . XXX "Anti-Angiogenic Peptides and Methods of Use Thereof," by Vikas P. Sukhatme, filed December 8, 1998, and U.S.S.N. XX/XXX,XXX, "Methods of Producing Anti- Angiogenic Proteins," by Vikas P. Sukhatme, filed December 8, 1998, the entire teachings of all of which are herein incorporated by reference.
- the invention also describes fragments, mutants, homologs and analogs of restin.
- a "fragment" of restin is any amino acid sequence shorter that the restin molecule, comprising at least 25 consecutive amino acids ofthe restin polypeptide.
- Such mutants may or may not also comprise additional amino acids derived from the process of cloning, e.g., amino acid residues or amino acid sequences corresponding to full or partial linker sequences.
- additional amino acid residues e.g., amino acid residues or amino acid sequences corresponding to full or partial linker sequences.
- such mutants, with or without such additional amino acid residues must have substantially the same biological activity as the natural or full-length version ofthe reference polypeptide.
- apomigren One such fragment, designated “apomigren”, was found to have anti- angiogenic activity equivalent or superior to that of human or mouse endostatin, as determined by standard assays, apomigren comprises the last approximately 85 amino acid residues of restin itself, from about amino acid 97 to about amino acid 181 of SEQ ID NO: :
- the polynucleotide sequence encoding apomigren therefore corresponds to about nucleotide 289 to about nucleotide 543 of SEQ ID NO: , and can be amplified out of SEQ ID NO: with the forward primer 5'-AAG AAT GCG GCC GCT TAC TTC CTA GCG TCT GTC ATG AAA CTG TTT TCG AT-3' and the same reverse primer as that used for restin. Cloning and expression of apomigren is done as for restin itself, as illustrated in the Examples below. Apomigren provides an advantage in treatment of angiogenic diseases in that increasingly smaller peptides are more potent on a weight basis, and may be able to better penetrate tissues.
- mutant of restin is meant a polypeptide that includes any change in the amino acid sequence relative to the amino acid sequence ofthe equivalent reference restin polypeptide. Such changes can arise either spontaneously or by manipulations by man, by chemical energy (e.g., X-ray), or by other forms of chemical mutagenesis, or by genetic engineering, or as a result of mating or other forms of exchange of genetic information. Mutations include, e.g., base changes, deletions, insertions, inversions, translocations, or duplications.
- Mutant forms of restin may display either increased or decreased anti-angiogenic activity relative to the equivalent reference restin polynucleotide, and such mutants may or may not also comprise additional amino acids derived from the process of cloning, e.g., amino acid residues or amino acid sequences corresponding to full or partial linker sequences.
- analog of restin is meant a non-natural molecule substantially similar to either the entire restin molecule or a fragment or allelic variant thereof, and having substantially the same or superior biological activity.
- Such analogs are intended to include derivatives (e.g., chemical derivatives, as defmed above) ofthe biologically active restin, as well as its fragments, mutants, homologs, and allelic variants, which derivatives exhibit a qualitatively similar agonist or antagonist effect to that ofthe unmodified restin polypeptide, fragment, mutant, homolog, or allelic variant.
- allele of restin is meant a polypeptide sequence containing a naturally- occurring sequence variation relative to the polypeptide sequence ofthe reference restin polypeptide.
- allele of a polynucleotide encoding the restin polypeptide is meant a polynucleotide containing a sequence variation relative to the reference polynucleotide sequence encoding the reference restin polypeptide, where the allele ofthe polynucleotide encoding the restin polypeptide encodes an allelic form ofthe restin polypeptide.
- a given polypeptide may be either a fragment, a mutant, an analog, or allelic variant of restin, or it may be two or more of those things, e.g., a polypeptide may be both an analog and a mutant ofthe restin polypeptide.
- a shortened version ofthe restin molecule e.g., a fragment of restin
- a mutant of restin may be created, which is later discovered to exist as an allelic of restin in some mammalian individuals.
- Such a mutant restin molecule would therefore be both a mutant and an allelic variant of restin.
- Such combinations of fragments, mutants, allelic variants, and analogs are intended to be encompassed in the present invention.
- proteins that have substantially the same amino acid sequence as restin or apomigren or polynucleotides that have substantially the same nucleic acid sequence as the polynucleotides encoding restin or apomigren.
- substantially the same sequence means a nucleic acid or polypeptide that exhibits at least about 70 % sequence identity with a reference sequence, e.g., another nucleic acid or polypeptide, typically at least about 80% sequence identity with the reference sequence, preferably at least about 90% sequence identity, more preferably at least about 95% identity, and most preferably at least about 97% sequence identity with the reference sequence.
- polypeptide indicates a molecular chain of amino acids and does not refer to a specific length ofthe product. Thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term is also intended to include polypeptide that have been subjected to post-expression modifications such as, for example, glycosylations, acetylations, phosphorylations and the like. Restin, in general, has less than 70% amino acid sequence identity with endostatin.
- Sequence identity refers to the subunit sequence similarity between two polymeric molecules, e.g., two polynucleotides or two polypeptides . When a subunit position in both ofthe two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two peptides is occupied by serine, then they are identical at that position.
- the identity between two sequences is a direct function ofthe number of matching or identical positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length), ofthe positions in two peptide or compound sequences are identical, then the two sequences are 50% identical; if 90% ofthe positions, e.g., 9 of 10 are matched, the two sequences share 90% sequence identity.
- the amino acid sequences VRGLQP and HAFLQP have 3 of 6 positions in common, and therefore share 50% sequence identity
- the sequences VRGLQP and AFLQP have 3 of 5 positions in common, and therefore share 60% sequence identity.
- the identity between two sequences is a direct function ofthe number of matching or identical positions.
- sequence identity is often measured using sequence analysis software e.g., BLASTN or
- BLASTP (available at http://www.ncbi.nlm.nih.gov/BLAST/).
- sequence homology it is meant that the two sequences differ from each other only by conservative substitutions.
- conservative substitutions consist of substitution of one amino acid at a given position in the sequence for another amino acid ofthe same class (e.g., amino acids that share characteristics of hydrophobicity, charge, pK or other conformational or chemical properties, e.g., valine for leucine, arginine for lysine), or by one or more non-conservative amino acid substitutions, deletions, or insertions, located at positions ofthe sequence that do not alter the conformation or folding ofthe polypeptide to the extent that the biological activity ofthe polypeptide is destroyed.
- “conservative substitutions” include substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the use of a chemically derivatized residue in place of a non-derivatized residue; provided that the polypeptide displays the requisite biological activity.
- Two sequences which share sequence homology may called “sequence homologs.”
- sequence analysis software e.g., Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705. Protein analysis software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine threonine; lysine, arginine; and phenylalanine, tyrosine. Also encompassed by the present invention are chemical derivatives of restin and apomigren.
- “Chemical derivative” refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized residues include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
- the imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine.
- chemical derivatives those peptides which contain one or more naturally occurring amino acid derivatives ofthe twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substitute for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
- Nucleic acids polypeptides referred to herein as "isolated” are nucleic acids or polypeptides substantially free (i.e., separated away from) the material ofthe biological source from which they were obtained (e.g., as exists in a mixture of nucleic acids or in cells), which may have undergone further processing.
- isolated nucleic acids or polypeptides include nucleic acids or polypeptides obtained by methods described herein, similar methods, or other suitable methods, including essentially pure nucleic acids or polypeptides, nucleic acids or polypeptides produced by chemical synthesis, by combinations of chemical or biological methods, and recombinantly produced nucleic acids or polypeptides which are isolated.
- An isolated polypeptide therefore means one which is relatively free of other proteins, carbohydrates, lipids, and other cellular components with which it is normally associated.
- nucleic acid is not immediately contiguous with (i.e., covalently linked to) both ofthe nucleic acids with which it is immediately contiguous in the naturally-occurring genome ofthe organism from which the nucleic acid is derived.
- the term therefore, includes, for example, a nucleic acid which is incorporated into a vector (e.g., an autonomously replicating virus or plasmid), or a nucleic acid which exists as a separate molecule independent of other nucleic acids such as a nucleic acid fragment produced by chemical means or restriction endonuclease treatment.
- the polynucleotides and proteins ofthe present invention can also be used to design probes to isolate other anti-angiogenic proteins.
- oligonucleotide probe should preferably follow these parameters: (a) It should be designed to an area ofthe sequence which has the fewest ambiguous bases ("N's"), if any, and (b) It should be designed to have a T m of approx. 80° C (assuming 2°C for each A or T and 4 degrees for each G or C).
- the oligonucleotide should preferably be labeled with g- P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity ofthe resulting probe should be approximately 4 x 10 dpm/pmole.
- the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l ofthe stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
- the culture should preferably be grown to saturation at 37 °C, and the saturated culture should preferably be diluted in fresh L-broth.
- Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37 °C. Other known methods of obtaining distinct, well-separated colonies can also be employed. Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them. Highly stringent condition are those that are at least as stringent as, for example, lx SSC at 65 °C, or lx SSC and 50% formamide at 42°C.
- Moderate stringency conditions are those that are at least as stringent as 4x SSC at 65 °C, or 4x SSC and 50% formamide at 42 °C.
- Reduced stringency conditions are those that are at least as stringent as 4x SSC at 50°C, or 6x SSC and 50% formamide at 40°C.
- the filter is then preferably incubated at 65 °C for 1 hour with gentle agitation in ⁇ .times.
- SSC 20x stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter).
- the probe is then added to the hybridization mix at a concentration greater than or equal to 1 x 10 6 dpm/mL.
- the filter is then preferably incubated at 65 °C with gentle agitation overnight.
- the filter is then preferably washed in 500 mL of 2x SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2x SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0. lx SSC/0.5% SDS at 65 °C for 30 minutes to 1 hour is optional.
- the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
- the positive colonies are then picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
- the present invention also includes fusion proteins and chimeric proteins comprising restin, its fragments, mutants, homologs, analogs, and allelic variants.
- a fusion or chimeric protein can consist of a multimer of a single protein, e.g., repeats of restin or repeats of apomigren, or the fusion and chimeric proteins can be made up of several proteins, e.g., restin and apomigren.
- the fusion proteins can comprise a combination of two or more known anti-angiogenic proteins (e.g., angiostatin and endostatin, or biologically active fragments of angiostatin and endostatin), or an anti-angiogenic protein in combination with a targeting agent (e.g.
- endostatin with epidermal growth factor (EGF) or RGD peptides or an anti-angiogenic protein in combination with an immunoglobulin molecule (e.g., endostatin and IgG, specifically with the Fc portion removed).
- the fusion and chimeric proteins can also include restin, apomigren, their fragments, mutants, homologs, analogs, and allelic variants, and other anti-angiogenic proteins, e.g., endostatin, angiostatin, or EM 1 (as described in U.S.S.N. XX/XXX,XXX, "Anti-Angiogenic Peptides and Methods of Use Thereof, by Vikas P.
- fusion protein can also encompass additional components for e.g., delivering a chemotherapeutic agent, wherein a polynucleotide encoding the chemotherapeutic agent is linked to the polynucleotide encoding the anti-angiogenic protein. Fusion proteins can also encompass multimers ofthe anti-angiogenic protein, e.g., a dimer or trimer of endostatin. Such fusion proteins can be linked together via post- translational modification (e.g., chemically linked), or the entire fusion protein may be made recombinantly.
- compositions containing, as a biological ingredient, restin, as well as its fragments, mutants, homologs, analogs, and allelic variants to inhibit or enhance angiogenesis in mammalian tissues and use of such compositions in the diagnosis, prognosis, and treatment of diseases and conditions characterized by, or associated with, angiogenic activity or lack thereof.
- Such methods can involve administration by oral, topical, injection, implantation, sustained release, or other delivery methods.
- the invention includes use of restin, and its fragments, mutants, homologs, analogs, allelic variants, and fusion and chimeric proteins as biologically-active agents in compositions for the p pose of treating diseases or conditions that are associated with angiogenic activity.
- Methods of treating such diseases include contacting the affected tissue with a composition comprising restin, its fragments, mutants, homologs, analogs, or allelic variants.
- the present invention includes the method of treating an angiogenesis- mediated disease with a therapeutically effective amount of restin, or a biologically active fragment thereof, or combinations of restin fragments that possess anti- angiogenic activity, or restin agonists and antagonists.
- Angiogenesis-mediated diseases include, but are not limited to, cancers, solid tumors, blood-born tumors (e.g., leukemias), tumor metastasis, benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas), rheumatoid arthritis, psoriasis, ocular angiogenic diseases (e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis), Osier- Webber Syndrome, myocardial angiogenesis,
- Restin is useful in the treatment of diseases of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars (i.e., keloids). Restin can be used as a birth control agent by preventing vascularization required for embryo implantation. Restin is useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Heliobacte pylori). Restin and apomigren can also be used to prevent dialysis graft vascular access stenosis, and obesity, e.g.
- cancer means neoplastic growth, hype ⁇ lastic or proliferative growth or a pathological state of abnormal cellular development and includes solid tumors, non-solid tumors, and any abnormal cellular proliferation, such as that seen in leukemia.
- cancer also means angiogenesis- dependent cancers and tumors, i.e., tumors that require for their growth (expansion in volume and/or mass) an increase in the number and density ofthe blood vessels supplying them with blood.
- “Regression” refers to the reduction of tumor mass and size.
- therapeutically effective amount means the total amount of each active component ofthe composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration ofthe relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
- the term refers to combined amounts ofthe active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
- an increase in angiogenesis is desired, e.g., in wound healing, or in post-infarct heart tissue
- antibodies or antisera to the restin protein can be used to block localized, native anti-angiogenic proteins and processes, and thereby increase formation of new blood vessels so as to inhibit atrophy of tissue.
- Restin may be used in combination with other compositions and procedures for the treatment of diseases. For example, a tumor may be treated conventionally with surgery, radiation, chemotherapy, or immunotherapy, combined with restin and then restin may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize and inhibit the growth of any residual primary tumor.
- Restin or apomigren, or fragments, antisera, receptor agonists, or receptor antagonists thereof, or combinations thereof, can also be combined with other anti- angiogenic compounds, or proteins, fragments, antisera, receptor agonists, receptor antagonists of other anti-angiogenic proteins (e.g., angiostatin, endostatin, EM 1). Additionally, restin, restin fragments, restin antisera, restin receptor agonists, restin receptor antagonists, or combinations thereof, are combined with pharmaceutically acceptable excipients, and optionally sustained-release matrix, such as biodegradable polymers, to form therapeutic compositions.
- compositions ofthe present invention may also contain other anti-angiogenic proteins or chemical compounds, such as endostatin, angiostatin, EM 1 (as described in U.S.S.N. XX/XXX,XXX, "Anti-Angiogenic Peptides and Methods of Use Thereof, by Vikas P. Sukhatme, filed December 8, 1998, the entire teachings of which are herein inco ⁇ orated by reference), and mutants, fragments, and analogs thereof.
- the compositions may further contain other agents which either enhance the activity ofthe protein or compliment its activity or use in treatment, such as chemotherapeutic or radioactive agents.
- compositions may be included in the composition to produce a synergistic effect with protein ofthe invention, or to minimize side effects.
- administration ofthe composition ofthe present invention may be administered concurrently with other therapies, e.g., administered in conjunction with a chemotherapy or radiation therapy regimen.
- the invention includes methods for inhibiting angiogenesis in mammalian tissues by contacting the tissue with a composition comprising the proteins ofthe invention. By “contacting” is meant not only topical application, but also those modes of delivery that introduce the composition into the tissues, or into the cells of the tissues.
- timed release or sustained release delivery systems are also included in the invention. Such systems are highly desirable in situations where surgery is difficult or impossible, e.g., patients debilitated by age or the disease course itself, or where the risk-benefit analysis dictates control over cure.
- a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
- the sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
- a preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).
- the angiogenesis-modulating composition ofthe present invention may be a solid, liquid or aerosol and may be administered by any known route of administration.
- solid compositions include pills, creams, and implantable dosage units.
- the pills may be administered orally, the therapeutic creams may be administered topically.
- the implantable dosage unit may be administered locally, for example at a tumor site, or which may be implanted for systemic release ofthe angiogenesis-modulating composition, for example subcutaneously.
- liquid composition include formulations adapted for injection subcutaneously, intravenously, intraarterially, and formulations for topical and intraocular administration.
- aerosol formulation include inhaler formulation for administration to the lungs.
- the restin proteins and protein fragments with the anti-angiogenic activity described above can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g. , intravenous, intraspinal, subcutaneous or intramuscular) route.
- the restin may be inco ⁇ orated into biodegradable polymers allowing for sustained release ofthe compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the restin is slowly released systemically.
- Osmotic minipumps may also be used to provide controlled delivery of high concentrations of restin through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor.
- the biodegradable polymers and their use are described, for example, in detail in Brem et al. (1991) (J. Neurosurg. 74:441-446), which is hereby inco ⁇ orated by reference in its entirety.
- compositions containing a polypeptide of this invention can be administered intravenously, as by injection of a unit dose, for example.
- unit dose when used in reference to a therapeutic composition ofthe present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier or vehicle.
- Modes of administration of the compositions of the present inventions include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
- compositions for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
- aqueous and nonaqueous carriers, diluents, solvents or vehicles examples include water, ethanol, polyois (e.g., glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters such as ethyl oleate.
- polyois e.g., glycerol, propylene glycol, polyethylene glycol and the like
- carboxymethylcellulose and suitable mixtures thereof examples include water, ethanol, polyois (e.g., glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters such as ethyl oleate.
- vegetable oils e.g., olive oil
- injectable organic esters such as ethyl oleate.
- Proper fluidity may be maintained,
- microorganisms Prevention ofthe action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged abso ⁇ tion ofthe injectable pharmaceutical form may be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay abso ⁇ tion. Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).
- biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).
- Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
- the injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter or by inco ⁇ orating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use.
- the therapeutic compositions ofthe present invention can include pharmaceutically acceptable salts ofthe components therein, e.g., which may be derived from inorganic or organic acids.
- salts are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.
- Pharmaceutically acceptable salts are well-known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences (1977) 66:1 et seq., which is inco ⁇ orated herein by reference.
- Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups ofthe polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. The salts may be prepared in situ during the final isolation and purification ofthe compounds ofthe invention or separately by reacting a free base function with a suitable organic acid.
- inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like.
- Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example
- Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptonoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxymethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate.
- the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
- lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
- dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates
- long chain halides such as dec
- compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal with a minimum of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
- physiologically tolerable and grammatical variations thereof as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal with a minimum of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
- the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation.
- Such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
- the preparation can also be emulsified.
- the active ingredient can be mixed with excipeints which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
- Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
- the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness ofthe active ingredient.
- the restin polypeptides ofthe present invention can also be included in a composition comprising a prodrug.
- prodrug refers to compounds which are rapidly transformed in vivo to yield the parent compound, for example, by enzymatic hydrolysis in blood.
- the term "pharmaceutically acceptable prodrug” refers to (1) those prodrugs ofthe compounds ofthe present invention which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like, commensurate with a suitable benefit-to-risk ratio and effective for their intended use and (2) zwitterionic forms, where possible, ofthe parent compound.
- the dosage ofthe restin ofthe present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
- about 10 mg/kg of body weight to about 20 mg/kg of body weight of the restin protein or the apomigren protein can be administered.
- combination therapies e.g., the restin protein or apomigren protein ofthe invention in combination with radiotherapy, chemotherapy, or immunotherapy, it may be possible to reduce the dosage, e.g., to about 0.1 mg/kg of body weight to about 0.2 mg/kg of body weight.
- the restin can be administered between several times per day to once a week.
- the present invention has application for both human and veterinary use.
- the methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
- restin and/or apomigren can be administered in conjunction with other forms of therapy, e.g., chemotherapy, radiotherapy, or immunotherapy.
- the restin formulations include those suitable for oral, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, mtradermal, intracranial, intratracheal, and epidural) administration.
- the restin formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
- Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood ofthe intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
- the formulations may be presented in unit-dose dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets ofthe kind previously described.
- the restin protein ofthe present invention When a therapeutically effective amount of protein ofthe present invention is administered orally, the restin protein ofthe present invention will be in the form of a tablet, capsule, powder, solution or elixir.
- the pharmaceutical composition ofthe invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
- the tablet, capsule, and powder contain from about 5 to 95% protein ofthe present invention, and preferably from about 25 to 90% protein ofthe present invention.
- a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
- the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
- the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein ofthe present invention.
- protein ofthe present invention When a therapeutically effective amount of protein ofthe present invention is administered by intravenous, cutaneous or subcutaneous injection, protein ofthe present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
- the preparation of such parenterally acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
- a prefened pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
- the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
- the amount of protein ofthe present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity ofthe condition being treated, and on the nature of prior treatments which the patient has undergone.
- the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein ofthe present invention and observe the patient's response. Larger doses of protein ofthe present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
- the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity ofthe disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein ofthe present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition ofthe present invention.
- Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations ofthe present invention may include other agents conventional in the art having regard to the type of formulation in question. Optionally, cytotoxic agents may be inco ⁇ orated or otherwise combined with restin proteins, or biologically functional protein fragements thereof, to provide dual therapy to the patient.
- the therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins ofthe present invention.
- Cytotoxic agents such as ricin, are linked to restin, and high affinity restin protein fragments, thereby providing a tool for destruction of cells that bind restin. These cells may be found in many locations, including but not limited to, micrometastases and primary tumors. Proteins linked to cytotoxic agents are infused in a manner designed to maximize delivery to the desired location. For example, ricin-linked high affinity restin fragments are delivered through a cannula into vessels supplying the target site or directly into the target.
- Such agents are also delivered in a controlled manner through osmotic pumps coupled to infusion cannulae.
- a combination of restin antagonists may be co-applied with stimulators of angiogenesis to increase vascularization of tissue.
- This therapeutic regimen provides an effective means of destroying metastatic cancer.
- Additional treatment methods include administration of restin, restin fragments, restin analogs, restin antisera, or restin receptor agonists and antagonists linked to cytotoxic agents. It is to be understood that the restin can be human or animal in origin. Restin can also be produced synthetically by chemical reaction or by recombinant techniques in conjunction with expression systems. Restin can also be produced by enzymatically cleaving isolated plasminogen or plasmin to generate proteins having anti-angiogenic activity.
- Restin may also be produced by compounds that mimic the action of endogenous enzymes that cleave plasminogen to restin. Restin production may also be modulated by compounds that affect the activity of plasminogen cleaving enzymes. These protein sequences are compared to known sequences using protein sequence databases such as GenBank, Brookhaven Protein, SWISS-PROT, and PIR to determine potential sequence homologies. This information facilitates elimination of sequences that exhibit a high degree of sequence homology to other molecules, thereby enhancing the potential for high specificity in the development of antisera, agonists and antagonists to restin.
- the present invention also encompasses gene therapy whereby a polynucleotide encoding restin or apomigren, or a mutant, fragment, or fusion protein thereof, is introduced and regulated in a patient.
- gene therapy Various methods of transferring or delivering DNA to cells for expression ofthe gene product protein, otherwise referred to as gene therapy, are disclosed in Gene Transfer into
- Gene therapy encompasses inco ⁇ oration of DNA sequences into somatic cells or germ line cells for use in either ex vivo or in vivo therapy. Gene therapy functions to replace genes, augment normal or abnormal gene function, and to combat infectious diseases and other pathologies.
- Strategies for treating these medical problems with gene therapy include therapeutic strategies such as identifying the defective gene and then adding a functional gene to either replace the function ofthe defective gene or to augment a slightly functional gene; or prophylactic strategies, such as adding a gene for the product protein that will treat the condition or that will make the tissue or organ more susceptible to a treatment regimen.
- a gene such as restin may be placed in a patient and thus prevent occurrence of angiogenesis; or a gene that makes tumor cells more susceptible to radiation could be inserted and then radiation ofthe tumor would cause increased killing ofthe tumor cells.
- Gene transfer methods for gene therapy fall into three broad categories: physical (e.g., electroporation, direct gene transfer and particle bombardment), chemical (e.g., lipid-based carriers, or other non-viral vectors) and biological (e.g., virus-derived vector and receptor uptake).
- non-viral vectors may be used which include liposomes coated with DNA.
- liposome/DNA complexes may be directly injected intravenously into the patient. It is believed that the liposome/DNA complexes are concentrated in the liver where they deliver the DNA to macrophages and Kupffer cells. These cells are long lived and thus provide long term expression ofthe delivered DNA. Additionally, vectors or the "naked" DNA of the gene may be directly injected into the desired organ, tissue or tumor for targeted delivery ofthe therapeutic DNA. Gene therapy methodologies can also be described by delivery site.
- ex vivo gene transfer cells are taken from the patient and grown in cell culture. The DNA is transfected into the cells, the transfected cells are expanded in number and then reimplanted in the patient.
- in vitro gene transfer the transformed cells are cells growing in culture, such as tissue culture cells, and not particular cells from a particular patient. These "laboratory cells" are transfected, the transfected cells are selected and expanded for either implantation into a patient or for other uses.
- In vivo gene transfer involves introducing the DNA into the cells ofthe patient when the cells are within the patient. Methods include using virally mediated gene transfer using a noninfectious virus to deliver the gene in the patient or injecting naked DNA into a site in the patient and the DNA is taken up by a percentage of cells in which the gene product protein is expressed. Additionally, the other methods described herein, such as use of a "gene gun,” may be used for in vitro insertion of restin DNA or restin regulatory sequences.
- Chemical methods of gene therapy may involve a lipid based compound, not necessarily a liposome, to transfer the DNA across the cell membrane.
- Lipofectins or cytofectins lipid-based positive ions that bind to negatively charged DNA, make a complex that can cross the cell membrane and provide the DNA into the interior of the cell.
- Another chemical method uses receptor-based endocytosis, which involves binding a specific ligand to a cell surface receptor and enveloping and transporting it across the cell membrane. The ligand binds to the DNA and the whole complex is transported into the cell.
- the ligand gene complex is injected into the blood stream and then target cells that have the receptor will specifically bind the ligand and transport the ligand-DNA complex into the cell.
- viral vectors to insert genes into cells.
- altered retrovirus vectors have been used in ex vivo methods to introduce genes into peripheral and tumor-infiltrating lymphocytes, hepatocytes, epidermal cells, myocytes, or other somatic cells. These altered cells are then introduced into the patient to provide the gene product from the inserted DNA.
- Viral vectors have also been used to insert genes into cells using in vivo protocols. To direct the tissue-specific expression of foreign genes, cis-acting regulatory elements or promoters that are known to be tissue-specific can be used. Alternatively, this can be achieved using in situ delivery of DNA or viral vectors to specific anatomical sites in vivo.
- gene transfer to blood vessels in vivo was achieved by implanting in vitro transduced endothelial cells in chosen sites on arterial walls.
- the virus infected surrounding cells which also expressed the gene product.
- a viral vector can be delivered directly to the in vivo site, by a catheter for example, thus allowing only certain areas to be infected by the virus, and providing long-term, site specific gene expression.
- retrovirus vectors has also been demonstrated in mammary tissue and hepatic tissue by injection ofthe altered virus into blood vessels leading to the organs.
- Viral vectors that have been used for gene therapy protocols include but are not limited to, retroviruses, other RNA viruses such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, he ⁇ es viruses, SV 40, vaccinia and other DNA viruses.
- Replication-defective murine retroviral vectors are the most widely utilized gene transfer vectors.
- Murine leukemia retroviruses are composed of a single strand RNA complexed with a nuclear core protein and polymerase (pol) enzymes, encased by a protein core (gag) and surrounded by a glycoprotein envelope (env) that determines host range.
- retroviral vector systems exploit the fact that a minimal vector containing the 5' and 3' LTRs and the packaging signal are sufficient to allow vector packaging, infection and integration into target cells providing that the viral structural proteins are supplied in trans in the packaging cell line. Fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, precise single copy vector integration into target cell chromosomal DNA, and ease of manipulation ofthe retroviral genome.
- the adenovirus is composed of linear, double stranded DNA complexed with core proteins and surrounded with capsid proteins. Advances in molecular virology have led to the ability to exploit the biology of these organisms to create vectors capable of transducing novel genetic sequences into target cells in vivo.
- Adenoviral- based vectors will express gene product proteins at high levels.
- Adenoviral vectors have high efficiencies of infectivity, even with low titers of virus. Additionally, the virus is fully infective as a cell free virion so injection of producer cell lines are not necessary. Another potential advantage to adenoviral vectors is the ability to achieve long term expression of heterologous genes in vivo.
- DNA delivery include fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion, lipid particles of DNA inco ⁇ orating cationic lipid such as lipofectin, polylysine-mediated transfer of DNA, direct injection of DNA, such as microinjection of DNA into germ or somatic cells, pneumatically delivered DNA-coated particles, such as the gold particles used in a "gene gun," and inorganic chemical approaches such as calcium phosphate transfection. Particle-mediated gene transfer methods were first used in transforming plant tissue.
- a motive force is generated to accelerate DNA-coated high density particles (such as gold or tungsten) to a high velocity that allows penetration ofthe target organs, tissues or cells.
- Particle bombardment can be used in in vitro systems, or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs.
- Another method, ligand-mediated gene therapy involves complexing the DNA with specific ligands to form ligand-DNA conjugates, to direct the DNA to a specific cell or tissue.
- Non-integration of the transfected DNA would allow the transfection and expression of gene product proteins in terminally differentiated, non-pro liferative tissues for a prolonged period of time without fear of mutational insertions, deletions, or alterations in the cellular or mitochondrial genome. Long-term, but not necessarily permanent, transfer of therapeutic genes into specific cells may provide treatments for genetic diseases or for prophylactic use.
- the DNA could be reinjected periodically to maintain the gene product level without mutations occurring in the genomes ofthe recipient cells.
- Non-integration of exogenous DNAs may allow for the presence of several different exogenous DNA constructs within one cell with all ofthe constructs expressing various gene products.
- Electroporation for gene transfer uses an electrical current to make cells or tissues susceptible to electroporation-mediated mediated gene transfer.
- a brief electric impulse with a given field strength is used to increase the permeability of a membrane in such a way that DNA molecules can penetrate into the cells.
- This technique can be used in in vitro systems, or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs.
- Carrier mediated gene transfer in vivo can be used to transfect foreign DNA into cells.
- the carrier-DNA complex can be conveniently introduced into body fluids or the bloodstream and then site-specifically directed to the target organ or tissue in the body. Both liposomes and polycations, such as polylysine, lipofectins or cytofectins, can be used.
- Liposomes can be developed which are cell specific or organ specific and thus the foreign DNA carried by the Hposome will be taken up by target cells. Injection of immunoliposomes that are targeted to a specific receptor on certain cells can be used as a convenient method of inserting the DNA into the cells bearing the receptor.
- Another carrier system that has been used is the asialoglycoportein/polylysine conjugate system for carrying DNA to hepatocytes for in vivo gene transfer.
- the transfected DNA may also be complexed with other kinds of carriers so that the DNA is carried to the recipient cell and then resides in the cytoplasm or in the nucleoplasm.
- DNA can be coupled to carrier nuclear proteins in specifically engineered vesicle complexes and carried directly into the nucleus.
- Gene regulation of restin may be accomplished by administering compounds that bind to the restin gene, or control regions associated with the restin gene, or its corresponding RNA transcript to modify the rate of transcription or translation.
- cells transfected with a DNA sequence encoding restin may be administered to a patient to provide an in vivo source of restin.
- cells may be transfected with a vector containing a nucleic acid sequence encoding restin.
- the transfected cells may be cells derived from the patient's normal tissue, the patient's diseased tissue, or may be non-patient cells.
- tumor cells removed from a patient can be transfected with a vector capable of expressing the restin protein ofthe present invention, and re- introduced into the patient.
- the transfected tumor cells produce restin levels in the patient that inhibit the growth ofthe tumor.
- Patients may be human or non-human animals.
- Cells may also be transfected by non-vector, or physical or chemical methods known in the art such as electroporation, ionoporation, or via a "gene gun.”
- restin DNA may be directly injected, without the aid of a carrier, into a patient.
- restin DNA may be injected into skin, muscle or blood.
- the gene therapy protocol for transfecting restin into a patient may either be through integration ofthe restin DNA into the genome ofthe cells, into minichromosomes or as a separate replicating or non-replicating DNA construct in the cytoplasm or nucleoplasm ofthe cell. Restin expression may continue for a long-period of time or may be reinjected periodically to maintain a desired level of the restin protein in the cell, the tissue or organ or a determined blood level.
- the invention encompasses antibodies and antisera, which can be used for testing of novel anti-angiogenic proteins, and can also be used in diagnosis, prognosis, or treatment of diseases and conditions characterized by, or associated with, angiogenic activity or lack thereof.
- antibodies and antisera can also be used to up-regulate angiogenesis where desired, e.g., in post-infarct heart tissue, antibodies or antisera to the restin protein can be used to block localized, native anti- angiogenic proteins and processes, and increase formation of new blood vessels and inhibit atrophy of heart tissue.
- restin and apomigren can be used to induce apoptosis, or antibodies thereof can be used to prevent apoptosis in a cell or tissue.
- antibody or “antibody molecule” refers to a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.
- Passive antibody therapy using antibodies that specifically bind restin can be employed to modulate angiogenic-dependent processes such as reproduction, development, and wound healing and tissue repair.
- antisera directed to the Fab regions of restin antibodies can be administered to block the ability of endogenous restin antisera to bind restin.
- the restin ofthe present invention also can be used to generate antibodies that are specific for the inhibitor and its receptor.
- the antibodies can be either polyclonal antibodies or monoclonal antibodies. These antibodies that specifically bind to the restin or restin receptors can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect or quantify the restin or restin receptors in a body fluid or tissue. Results from these tests can be used to diagnose or predict the occurrence or recunence of a cancer and other angiogenic mediated diseases.
- the invention also includes use of restin, antibodies to restin, and compositions comprising restin and/or its antibodies in diagnosis or prognosis of diseases characterized by angiogenic activity.
- prognostic method means a method that enables a prediction regarding the progression of a disease of a human or animal diagnosed with the disease, in particular, an angiogenesis dependent disease.
- diagnostic method means a method that enables a determination ofthe presence or type of angiogenesis-dependent disease in or on a human or animal.
- the restin can be used in a diagnostic method and kit to detect and quantify antibodies capable of binding restin.
- kits would permit detection of circulating restin antibodies which indicates the spread of micrometastases in the presence of restin secreted by primary tumors in situ. Patients that have such circulating anti-restin antibodies may be more likely to develop multiple tumors and cancers, and may be more likely to have recurrences of cancer after treatments or periods of remission.
- the Fab fragments of these anti-restin antibodies may be used as antigens to generate anti-restin Fab-fragment antisera which can be used to neutralize anti-restin antibodies. Such a method would reduce the removal of circulating restin by anti-restin antibodies, thereby effectively elevating circulating restin levels.
- the present invention also includes isolation of receptors specific for restin.
- Protein fragments that possess high affinity binding to tissues can be used to isolate the restin receptor on affinity columns. Isolation and purification ofthe restin receptor is a fundamental step towards elucidating the mechanism of action of restin. Isolation of an restin receptor and identification of restin agonists and antagonists will facilitate development of drugs to modulate the activity of the restin receptor, the final pathway to biological activity. Isolation ofthe receptor enables the construction of nucleotide probes to monitor the location and synthesis ofthe receptor, using in situ and solution hybridization technology. Further, the gene for the restin receptor can be isolated, inco ⁇ orated into an expression vector and transfected into cells, such as patient tumor cells to increase the ability of a cell type, tissue or tumor to bind restin and inhibit local angiogenesis.
- Restin proteins are employed to develop affinity columns for isolation ofthe restin receptor from cultured tumor cells. Isolation and purification ofthe restin receptor is followed by amino acid sequencing. Using this information the gene or genes coding for the restin receptor can be identified and isolated. Next, cloned nucleic acid sequences are developed for insertion into vectors capable of expressing the receptor. These techniques are well known to those skilled in the art. Transfection ofthe nucleic acid sequence(s) coding for restin receptor into tumor cells, and expression ofthe receptor by the transfected tumor cells enhances the responsiveness of these cells to endogenous or exogenous restin and thereby decreasing the rate of metastatic growth.
- Angiogenesis-inhibiting proteins of the present invention can be synthesized in a standard microchemical facility and purity checked with HPLC and mass spectrophotometry. Methods of protein synthesis, HPLC purification and mass spectrophotometry are commonly known to those skilled in these arts. Restin proteins and restin receptors proteins are also produced in recombinant E. coli or yeast expression systems, and purified with column chromatography.
- Different protein fragments ofthe intact restin molecule can be synthesized for use in several applications including, but not limited to the following; as antigens for the development of specific antisera, as agonists and antagonists active at restin binding sites, as proteins to be linked to, or used in combination with, cytotoxic agents for targeted killing of cells that bind restin.
- the amino acid sequences that comprise these proteins are selected on the basis of their position on the exterior regions ofthe molecule and are accessible for binding to antisera.
- the amino and carboxyl termini of restin, as well as the mid-region ofthe molecule are represented separately among the fragments to be synthesized.
- the synthetic protein fragments of restin have a variety of uses.
- the protein that binds to the restin receptor with high specificity and avidity is radiolabeled and employed for visualization and quantitation of binding sites using autoradiographic and membrane binding techniques. This application provides important diagnostic and research tools. Knowledge ofthe binding properties ofthe restin receptor facilitates investigation ofthe transduction mechanisms linked to the receptor.
- Restin and restin-derived proteins can be coupled to other molecules using standard methods.
- the amino and carboxyl termini of restin both contain tyrosine and lysine residues and are isotopically and nonisotopically labeled with many techniques, for example radiolabeling using conventional techniques (tyrosine residues-chloramine T, iodogen, lactoperoxidase; lysine residues-Bolton-Hunter reagent). These coupling techniques are well known to those skilled in the art.
- tyrosine or lysine is added to fragments that do not have these residues to facilitate labeling of reactive amino and hydroxyl groups on the protein.
- the coupling technique is chosen on the basis ofthe functional groups available on the amino acids including, but not limited to amino, sulfhydral, carboxyl, amide, phenol, and imidazole.
- Various reagents used to effect these couplings include among others, glutaraldehyde, diazotized benzidine, carbodiimide, and p-benzoquinone.
- Restin proteins are chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, chemiluminescent, bio luminescent and other compounds for a variety of applications.
- the efficiency ofthe coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabeling of an restin protein with I is accomplished using chloramine T and Na I of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted on disposable columns. The labeled protein is eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity measured in a gamma counter. In this manner, the unreacted Na I is separated from the labeled restin protein.
- the protein fractions with the highest specific radioactivity are stored for subsequent use such as analysis ofthe ability to bind to restin antisera.
- labeling restin proteins with short lived isotopes enables visualization of receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques to locate tumors with restin binding sites.
- Systematic substitution of amino acids within these synthesized proteins yields high affinity protein agonists and antagonists to the restin receptor that enhance or diminish restin binding to its receptor.
- Such agonists are used to suppress the growth of micrometastases, thereby limiting the spread of cancer.
- Antagonists to restin are applied in situations of inadequate vascularization, to block the inhibitory effects of restin and promote angiogenesis.
- the restin protein ofthe present invention can also be used as a nutritional source or supplement. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
- the restin protein ofthe invention can be added to the food of a particular organism, or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
- the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
- Example 1 Cells and cell lines
- C-PAE cow pulmonary artery endothelial, ATCC No. CCL-209 cells, obtained from ATCC (American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, 20110-2209, USA) were maintained in DMEM medium with 10% fetal calf serum, 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, and 2 mM L-glutamine. The cells were incubated in a humidified environment at 37°C in the presence of 5% CO 2 . IMR-90, lung fibroblast and IC-21, macrophage precursor cells were purchased from ATCC and maintained under similar conditions as the C-PAE cells. HMVE-L cells (human microvascular endothelial cells of lung origin, Clonetics, San Diego, California, USA) were maintained in EGM-2MV medium.
- HFK human fetal kidney library 786-0 - human renal cell carcinoma
- MAB human adult brain 293 - control
- Primers conesponding to C-terminal portion ofthe Collagen XV NC10 domain were synthesized from GIBCO-BRL (Life Technologies, Gaithersburg, Maryland, USA).
- the primers used were 5'-TTT TTT GAA TTC ATT TCA AGT GCC AAT TAT GAG AAG CCT GCT CTG CAT TTG-3' (upstream primer) and 5'- AAG AAT GCG GCC GCT TAC TTC CTA GCG TCT GTC ATG AAA CTG TTT TCG AT-3' (downstream primer).
- Amplification was carried out using standard protocols. Briefly, 100 ng of DNA from each cDNA library was used as template. Amplification was carried out for 30 cycles with the following parameters: 94 °C for denaturation, 60 °C for annealing, and 72 °C for extension, each for 1 minute.
- the 540 bp amplified fragment designated "restin” herein, was purified and digested with EcoRI and Notl. Cloning of Collagen XV into the Pichia expression vector (pPICZ ⁇ A, InVitrogen, San Diego, California, USA) is shown schematically Fig. 5. The vector pPICZ ⁇ A was also digested with the above restriction enzymes and ligated with the restin fragment. Initial transformation was carried out with the host strain Top 10F' (InVitrogen, San Diego, California, USA). Positive clones, designated "pPICZ ⁇ A-CXV,” were sequenced to confirm the modification.
- the plasmid was then linearized with S ⁇ cl enzyme and used for homologous recombination into the yeast host strain GS115 (InVitrogen, San Diego, California, USA) via lithium chloride transformation.
- the recombination was carried out as described in the Pichia expression manual.
- the colonies were allowed to grow for two days at 30 °C on YPD/Zeocin plates. Clones which grew on Zeocin were selected for small scale induction.
- the induction procedure was carried out as suggested by the manufacturer (InVitrogen, San Diego, California, USA). Restin was successfully derived from the K592 and HFK libraries.
- the HFK clone was used for all subsequence investigations.
- a single colony was inoculated from YPD/Zeocin into 25 ml of BMGY/Zeo (100 ug/ml) in a 500 ml baffled flask.
- the culture was grown at 30 °C in a shaking incubator (250 ⁇ m) until the culture reached an OD600 2-6 (approximately 16-24 hours).
- the cells in the log phase of growth were used in a 1 : 100 dilution to inoculate a 1 -liter culture.
- 500 ml of BMGY medium was added, and the cells were grown in a 2-liter baffled flask and the culture reached an OD600 15-20 (2-3 days).
- the cells were harvested by centrifugation at 5000 ⁇ m for 10 minutes at room temperature.
- the supernatant was decanted, and the cell pellet resuspended in 300 - 400 ml of BMMY medium to induce expression of the recombinant protein. To maintain induction, 100% methanol was added to a final concentration of 0.5% every 24 hours. Supernatant was collected on the second, third, and fourth days after induction, and analyzed for protein expression by Coomassie stained SDS-PAGE.
- the supernatant was concentrated by precipitation with 60-70% ammonium sulfate.
- the precipitated protein was centrifuged at 10,000 ⁇ m for 10 min and the pellet was resuspended in phosphate-buffered saline and dialyzed overnight at 4°C.
- the final dialysis was done with 10 mM Tris and 50 mM NaCl, pH 7.4.
- a polyprep column was packed with 5 ml of Heparin-Sepharose resin (Pharmacia Biotech, Inc., Piscataway, New Jersey, USA) and equilibrated with 10 mM Tris, 50 MM NaCl, pH 7.4.
- the concentrated crude protein sample was loaded in 20-30 ml batches onto the column at a flow rate of 10-15 ml/hour.
- the column was washed with equilibration buffer until the absorbance at 280 nm was greater than 0.001.
- the elution of bound proteins was carried by step-wise gradients of NaCl (0.2M, 0.6M and 1.0 M).
- the elution profile failed to show any distinct protein peak, and when the samples were analyzed by SDS-PAGE, a majority ofthe protein did not bind to the column. Lowering the sodium chloride concentration and changing the pH did not change the binding profile.
- the recombinant protein did not bind to the heparin column.
- Example 5 Construction of His.Tag Restin recombinant protein
- a His.Tag motif to the restin molecule at the amino terminus to simplify purification.
- Two complementary oligonucleotide primers were synthesized, 5'-AAT TCC ATC ACC ATC ACC ATC ACG-3' (upstream), and 5'-AAT TCG TGA TGG TGA TGG TGA TGG-3' (downstream). These primers code for histidine residues (six histidine residue at a stretch), flanked by the EcoRI restriction site.
- the construct pPICZ ⁇ A-CXV was digested with EcoRI. Fifty ⁇ l of each primer (100 ⁇ M) was mixed and denatured at 95 °C for 5 minutes and cooled immediately on ice.
- the annealed primer was ligated into EcoRI digested vector backbone at 16°C overnight.
- the ligase was heat-inactivated at 70 °C for 10 minutes and the excess primer was removed using Glass Max purification column (Gibco/BRL, Gaithersburg, Maryland, USA).
- the ligated vector was transformed using the host strain Top 10F' (InVitrogen, San Diego, California, USA).
- the recombinant clones were screened by PCR and restriction enzyme analysis. The presence of His.Tag motif was further confirmed by sequencing, and the construct was designated "pPICZ ⁇ A/His.Restin.”
- the recombinant pPICZ ⁇ A/His.Restin was linearized with Sacl and recombination was carried out using the strain GS 115 (InVitrogen, San Diego, California, USA).
- GenBank was searched for sequences that showed homology to endostatin.
- One such sequence was the C-terminal region of human type XV collagen.
- Fig. 9 shows the 85 C-terminal amino acids ofthe full length ⁇ l chain of collagen XV in relation to the entire chain.
- Fig. 10 shows the homology between the 85 extreme C-terminus amino acids of human collagen XV and human and mouse endostatin. This region of human collagen XV has been designated herein as apomigren. About 60% amino acid identity exists between the C-terminal amino acids of these proteins. When conservative substitutions are accounted for, these regions share about 75% amino acid identity.
- Example 7 Cloning, expression and purification of human apomigren
- Amplification was performed for 30 cycles with denaturation at 94°C, annealing at 60°C and extension at 72°C, each for 1 minute, with the following primers: 5'-TTC CAT ATG ATA TAC TCC TTT GAT GGT CGA GAC ATA ATG ACA-3' and 5'-AAG AAT GCG GCC GCT TAC TTC CTA GCG TCT GTC ATG AAA CTG TTT TC GAT-3'
- the amplified DNA (255 bp) was purified using a QIAquick purification kit (Qiagen, Hilden, Germany) digested with NJel and Notl (these sites are underlined in the primer), and ligated into a NJel and Notl predigested pET28(a) expression vector (Novagen, Madison, Wisconsin, USA) so as to give an expected protein sequence of MGSSHHHHHHSSGLVPAGSHM- Apomigren, which is designated herein as "apomigren".
- FIG. 10 A histidine tag was used from the expression vector to assist in the purification procedures, since there was no guarantee that apomigren would bind heparin, as does endostatin.
- the expression, processing and purification ofthe recombinant apomigren was performed using a Ni-NTA column in the presence of 8 M urea as described by Dhanabal et al. (1999) ("Cloning, Expression and in vitro Activity of Human Endostatin," Cancer Research, in press), and also in U.S.S.N. XX/XXX,XXX, "Methods of Producing Anti-Angiogenic Proteins," by Vikas P.
- Fig. 11 is a graph showing the elution pH (left y-axis) and absorbance at 280 nM (right y-axis) for each fraction (x-axis). A small peak was observed around fraction 4, and a large peak at fraction 23. Samples of 15 ml were taken from selected fractions were analyzed by SDS-PAGE, and the results are shown in Fig. 12. Lane 1 contains size marker, lane 2, apomigren protein lysate prior to loading on the Ni-NTA column; lane 2, flowthrough from the column; lane 4, fraction 4; lanes 5 and 6, fraction 23, 99
- Fractions containing purified apomigren fractions 20-26 were pooled and refolded slowly with step-wise dialysis in decreasing urea concentrations. The final dialysis was carried out against sterile IX PBS, pH 7.4 at 4°C. During dialysis, although some protein precipitated out of solution, a significant fraction remained in soluble form. The soluble protein was stored at -70 °C in small aliquots and a fresh aliquot was used for each assay. The concentration ofthe protein was determined by BCA protein assay (Pierce Chemical Co., Rockford, Illinois, USA).
- Endotoxin levels were determined on the purified soluble protein by performing the LAL assay (Associates of Cape Cod, Inc., Falmouth, Massachusetts, USA) and the levels were about 6 units/ml, with a protein concentration of 200 ⁇ g/ml. In previous experiments, these levels had no visible effects on any ofthe assays used. Western blot analysis was performed on the purified protein with a histidine tag antibody and showed positive immunoreactivity.
- Example 8 Apomigren alters the mo ⁇ hology of endothelial cells When apomigren protein (0.5 - 1.0 ⁇ g/ml) was added to endothelial cells, a dramatic change occurred in the mo ⁇ hology of these cells. The cells appeared rounded, detached, and membrane blebbing, cytoplasmic shrinkage, and chromatin condensation was observed, as is typically seen in apoptotic cells. These changes are shown in Fig. 13, which is a set of three photomicrographs.
- Confluent C-PAE cells were trypsinized, plated at approximately 50% confluency in 2% FBS containing 3 ng/ml bFGF with simultaneous addition of apomigren at 0.05 ⁇ g/ml and 0.7 ⁇ g/ml. Pictures were taken 24 hours after treatment (400X magnification). Increasing concentrations of apomigren showed increasing number of cells with apoptotic mo ⁇ hology.
- Figs. 13B and 13C show the apomigren treated cells (0.05 mg/ml and 0.07 mg/ml, respectively) in comparison with untreated control endothelial cells (Fig. 13 A). Such changes were not observed in non-endothelial cells such as IMR-90.
- C-PAE cells were plated in 24- well plates coated with fibronectin (10 mg/ml) coated plates at 12,500 cells per well in 0.5 ml DMEM containing 2% FBS. After a 24-hour incubation at 37°C, the medium was replaced with fresh DMEM and 2% FBS containing 3 ng/ml of bFGF (R & D systems, Minneapolis, Minnesota, USA) with or without the protein being tested. The cells were pulsed with 1 mCi of 3 H-thymidine for 24 hours. Medium was aspirated, cells were washed three times with PBS, and then solubilized by addition of 1.5 N NaOH (100 ml per well) and incubated at 37 °C for 30 minutes. Cell-associated radioactivity was determined with a liquid scintillation counter.
- Example 10 Cell cycle assay C-PAE and IMR-90 cells were growth arrested by contact inhibition for 48 hours in complete medium. The cells were harvested by trypsinization and seeded into a 6 well plate coated with fibronectin (10 ⁇ g/ml). Each well was seeded with 0.2 to 0.3 x 10 6 cells in 1% FCS supplemented with 3 ng/ml of bFGF. For the dose response study, different concentrations of human apomigren were added and the cells harvested at 21-24 hours after treatment. The cells were washed in PBS buffer and fixed in 70% ice cold ethanol. The fixed cells were rehydrated at room temperature for 30 minutes in PBS buffer containing 2% FCS and 0.1% Tween-20.
- the cells were then centrifuged at 1500x g for 10 minutes and resuspended in 0.5 ml ofthe above PBS buffer to which RNase (5 ⁇ g/ml) was added. RNase digestion was carried at 37°C for 1 hour, followed by staining with propidium iodide (5 ⁇ g/ml). The cells were analyzed using a Becton Dickinson FACStar plus flow cytometer (Becton Dickinson, Waltham, Massachusetts, USA). For calculating the percentage of cells in different phases ofthe cell cycle, the ModFit software was used.
- Fig. 15 is a bar graph.
- the x-axis shows concentration of apomigren, ranging from 0 to 1000 ng/ml, and the y-axis shows percentage of cells in S-phase.
- C-PAE cells are represented by dark bars and IMR- 90 cells by white bars.
- Control cells treated with bFGF and 1% FBS alone were considered as 100%.
- the contact-inhibited cells showed 7% cells in S-phase.
- plating out at about 50% confluence, and on stimulation with bFGF (3 ng/ml) and 1% FBS a 4.8-fold increase in S-phase cells was observed after 21 hours (Fig. 15).
- a dose-dependent Gl cell cycle anest was seen with apomigren concentrations ranging from 0.06-1.0 ⁇ g/ml, with an ED 50 of approximately 500 ng/ml. To check the specificity of this effect, apomigren was also tested on IMR-90 fibroblast cells.
- Example 11 Apoptosis of endothelial cells and Annexin V - FITC assay
- PS and therefore apoptosis, can be detected by staining with an FITC conjugate of Annexin V, a calcium-dependent phospholipid binding protein of 38 kDa protein that has a high affinity for PS.
- PS can generally be detected prior to bleb formation and DNA fragmentation.
- Annexin V a calcium dependent phospholipid binding protein with a high affinity for phosphtidylserine (PS) was used to detect early stage apoptosis. Briefly, 200,000 cells were plated onto a fibronectin coated 6-well plate in DMEM containing 2% FBS and 3 ng/ml of bFGF.
- the migration assay was performed using 12-well Boyden chemotaxis chambers (Neuro-Probe, Inc., Cabin John, Maryland, USA) with a polycarbonate membrane (25 x 80 mm PVD free, 8 ⁇ pores, Poretics Co ⁇ ., Livermore, California, USA).
- the non-specific binding of growth factor to the chambers was prevented by coating the chambers with a solution containing 0.5% gelatin, 1 mM CaCl 2 and 150 mM NaCl at 37 °C overnight.
- ECV304 cells were grown in 10% FBS containing 5 ng/ml Dil (l,l'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate DiIC18, Molecular Probes, Eugene, Oregon, USA) overnight and washed with PBS containing 0.5% BSA. Following trypsinization, the cells were counted using Coulter-Counter Zl, (Luton, U.K.) and diluted to 300,000 cells/ml in Medium 199 (Gibco/BRL, Gaithersburg, Maryland, USA) containing 0.5% FBS. The lower chamber was filled with Medium 199 containing 25 ng/ml bFGF.
- the upper chamber was seeded with 15,000 cells/well with different concentrations of recombinant protein. Cells were allowed to migrate for 4 hours at 37°C. At that time, the cells on the upper surface ofthe membrane were removed with a cell scraper and the (migrated) cells on the lower surface were fixed in 3% formaldehyde and washed in PBS. Images ofthe fixed membrane were obtained using fluorescence microscopy at 550 nM with a digital camera and the number of cells on each membrane was determined using the OPTIMAS (version 6.0) software (Media Cybernetics, L.P., Silver Spring, MD, USA).
- Fig. 16 is a graph showing concentration of various proteins on the x-axis and percent cells that migrated in response to bFGF on the y-axis.
- the control is apomigren-treated IC-21 cells ( ⁇ ), and treatments consist of apomigren (•) and mouse (D) and human (o) endostatin on ECV304 cells.
- Apomigren had no effect on the migration of IC-21 macrophage precursor cells at these doses, but dramatic inhibition of migration was observed in endothelial cells at doses of 0.15-1.25 ⁇ g/ml, with 150 ng/ml resulting in about 80% inhibition. (Fig. 16).
- endostatin inhibits migration of ECV304endothelial cells, but at higher doses of (ED 50 ⁇ 5 ng/ml and 80% inhibition at 10-15 ⁇ g/ml) (Dhanabal et al. (1999) "Cloning, Expression and in vitro Activity of Human Endostatin," Cancer Research, in press).
- Example 13 Effect of Angiostatin in a Renal Tumor Model
- Athymic nude mice were transplanted subcutaneously with renal tumor cell line 786-0. Approximately 5 million cells in 200 1 of phosphate buffered saline were inoculated in the flank of each mouse. Each tumor was allowed to reach approximately 200 cubic millimeters.
- apomigren The ability of apomigren to block bFGF induced angiogenesis in vivo was tested using the chorioallantoic membrane (CAM) assay. Fertilized white Leghorn chicken eggs (SPAFAS, Inc., Norwich CT) were opened on 100 mm" petri dishes and allowed to grow until day 11 in a humidified incubator at 38%C. Pellets containing vitrogen (Collagen Biomaterials, Palo Alto, CA) at a concentration of 0.73 mg/ml and supplemented with vehicle alone, VEGF + FGF-2, or his. apomigren (0.5, 1, 5, 10, 20, or 50 ⁇ g/ml) were allowed to polymerize at 37°C for 2 hours.
- CAM chorioallantoic membrane
- the pellets were placed on a nylon mesh and oriented on the periphery ofthe CAM. Embryos were returned to the incubator for 24 hours. Invasion of new capillaries on the collagen mesh was assessed by injection of FITC-dextran into the circulation of the chicken embryo. At the end ofthe experiment, the meshes were dissected and evaluation of vascular density was done using the program NTH Image 1.59. Assays were performed in triplicate and four independent experiments were conducted. The results are shown in Fig. 19.
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US6788897P | 1997-12-08 | 1997-12-08 | |
US67888P | 1997-12-08 | ||
US8266398P | 1998-04-22 | 1998-04-22 | |
US82663P | 1998-04-22 | ||
US10853698P | 1998-11-16 | 1998-11-16 | |
US108536P | 1998-11-16 | ||
PCT/US1998/026058 WO1999029856A1 (fr) | 1997-12-08 | 1998-12-08 | Restine et ses methodes d'utilisation |
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EP98962966A Withdrawn EP1037985A1 (fr) | 1997-12-08 | 1998-12-08 | Restine et ses methodes d'utilisation |
EP98962932A Expired - Lifetime EP1038011B1 (fr) | 1997-12-08 | 1998-12-08 | Procede de production de proteines anti-angiogeniques, dont l'endostatine, l'angiostatine ou la restine, au moyen d'un systeme d'expression de la levure pichia |
EP98962006A Withdrawn EP1037983A1 (fr) | 1997-12-08 | 1998-12-08 | Mutants d'endostatine "em 1" presentant une activite anti-angiogenique et leurs methodes d'utilisation |
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EP98962932A Expired - Lifetime EP1038011B1 (fr) | 1997-12-08 | 1998-12-08 | Procede de production de proteines anti-angiogeniques, dont l'endostatine, l'angiostatine ou la restine, au moyen d'un systeme d'expression de la levure pichia |
EP98962006A Withdrawn EP1037983A1 (fr) | 1997-12-08 | 1998-12-08 | Mutants d'endostatine "em 1" presentant une activite anti-angiogenique et leurs methodes d'utilisation |
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JP (3) | JP2001526040A (fr) |
KR (3) | KR20010015868A (fr) |
CN (3) | CN1322246A (fr) |
AT (1) | ATE319840T1 (fr) |
AU (3) | AU742866B2 (fr) |
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US6852691B1 (en) | 1997-12-08 | 2005-02-08 | Beth Israel Deaconess Medical Center | Anti-angiogenic peptides and methods of use thereof |
US6797488B1 (en) | 1997-12-08 | 2004-09-28 | Beth Israel Deaconess Medical Center | Methods of producing anti-angiogenic proteins |
JP2001526040A (ja) * | 1997-12-08 | 2001-12-18 | ベス イスラエル ディーコネス メディカル センター | ピヒア酵母発現系を用いる抗血管形成タンパク質:エンドスタチン、アンジオスタチンまたはレスチンの生産方法 |
WO2000017240A1 (fr) * | 1998-09-21 | 2000-03-30 | Haemopep Pharma Gmbh | Endostatine ncf pour l'inhibition de la croissance des tumeurs et de la proliferation capillaire et pour le diagnostic des maladies vasculaires et tumorales |
IT1312077B1 (it) | 1999-04-15 | 2002-04-04 | Univ Degli Studi Milano | Polipeptidi ad attivita' antiangiogenica. |
JP2001031586A (ja) * | 1999-07-14 | 2001-02-06 | Sunstar Inc | 動脈硬化症及び動脈硬化症に起因する疾患の予防又は治療組成物 |
ATE303820T1 (de) * | 1999-12-30 | 2005-09-15 | Aventis Pharma Sa | Verwendung von einem für einem anti-angiogenen faktor codierenden vektor zur behandlung von vaskularisation der hornhaut |
FR2803207B1 (fr) * | 1999-12-30 | 2004-04-30 | Aventis Pharma Sa | Utilisation d'un vecteur comprenant un acide nucleique codant pour un facteur anti-angiogenique pour le traitement des neovascularisations corneennes |
AU3681601A (en) * | 2000-02-10 | 2001-08-20 | Zymogenetics Inc. | Anti-angiogenic intestinal peptides, zdint5 |
CN1311228A (zh) * | 2000-03-02 | 2001-09-05 | 上海博德基因开发有限公司 | 一种新的多肽——人精子特异蛋白em1,em6-48和编码这种多肽的多核苷酸 |
EP1666595A1 (fr) | 2000-10-26 | 2006-06-07 | Beth Israel Deaconess Medical Center, Inc. | P97 (GAB2) gène et méthodes d'utilisation |
KR100579660B1 (ko) * | 2003-08-18 | 2006-05-15 | 정인식 | 제미니바이러스 발현시스템을 이용한 재조합 신생혈관생성억제 단백질의 제조방법 |
DE602004015142D1 (de) | 2003-08-29 | 2008-08-28 | Childrens Medical Center | Antiangiogene peptide zur behandlung oder vorbeugung von endometriose |
US7524811B2 (en) | 2003-08-29 | 2009-04-28 | Children's Medical Center Corporation | Anti-angiogenic peptides from the N-terminus of endostatin |
ES2325344B1 (es) * | 2004-11-02 | 2010-06-09 | Univ Madrid Autonoma | Inhibidores de angiogenesis multifuncionales y multivalentes. |
WO2009145489A2 (fr) * | 2008-04-04 | 2009-12-03 | 주식회사 프로셀제약 | Protéine recombinante d'endostatine à cellules perméables |
JP6336389B2 (ja) * | 2011-09-09 | 2018-06-06 | チンファ ユニヴァーシティTsinghua University | Atp結合部位に変異を有するエンドスタチン変異体 |
EP2628748A1 (fr) | 2012-02-14 | 2013-08-21 | Szilak Laboratories Bioinformatics & Molecule-Design Ltd. | Chimères d'angiostatine et utilisations associées |
CN104926933B (zh) * | 2014-03-19 | 2018-09-07 | 北京仁和天通生物科技有限公司 | Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用 |
KR20180033499A (ko) | 2015-06-05 | 2018-04-03 | 아이바이오, 인크. | 섬유증을 치료하는데 사용하기 위한 엔도스타틴 단편 및 변이체 |
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US5945403A (en) * | 1997-05-30 | 1999-08-31 | The Children's Medical Center Corporation | Angiostatin fragments and method of use |
CA2188813C (fr) * | 1994-04-26 | 2010-08-03 | Michael S. O'reilly | Proteine de type angiostatine; acides nucleiques la codant et methodes de detection |
DE69637179T2 (de) * | 1995-04-26 | 2008-04-10 | The Children's Medical Center Corp., Boston | Angiostatinfragmente und verfahren für deren verwendung |
KR19990066981A (ko) * | 1995-10-23 | 1999-08-16 | 윌리엄 뉴우 | 치료용 맥관형성억제 조성물 및 방법 |
AU9673998A (en) * | 1997-10-01 | 1999-04-23 | G.D. Searle & Co. | Fusion proteins comprising an angiostatin moiety and their use in anti-tumor treatment |
WO1999026480A1 (fr) * | 1997-11-20 | 1999-06-03 | Genetix Pharmaceuticals, Inc. | Vecteurs de therapie genique anti-angiogenique et leurs applications dans le cadre du traitement d'affections liees a l'angiogenese |
JP2001526040A (ja) * | 1997-12-08 | 2001-12-18 | ベス イスラエル ディーコネス メディカル センター | ピヒア酵母発現系を用いる抗血管形成タンパク質:エンドスタチン、アンジオスタチンまたはレスチンの生産方法 |
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CA2312287A1 (fr) | 1999-06-17 |
WO1999029878A3 (fr) | 1999-09-16 |
IL136668A0 (en) | 2001-06-14 |
CN1284130A (zh) | 2001-02-14 |
JP2001526031A (ja) | 2001-12-18 |
AU753889B2 (en) | 2002-10-31 |
CN1284134A (zh) | 2001-02-14 |
KR20010015867A (ko) | 2001-02-26 |
WO1999029855A1 (fr) | 1999-06-17 |
CA2313251A1 (fr) | 1999-06-17 |
AU749904B2 (en) | 2002-07-04 |
AU742866B2 (en) | 2002-01-17 |
IL136665A0 (en) | 2001-06-14 |
CN1322246A (zh) | 2001-11-14 |
CA2313705A1 (fr) | 1999-06-17 |
DE69833794D1 (de) | 2006-05-04 |
AU1808899A (en) | 1999-06-28 |
JP2002503449A (ja) | 2002-02-05 |
WO1999029878A2 (fr) | 1999-06-17 |
ATE319840T1 (de) | 2006-03-15 |
IL136667A0 (en) | 2001-06-14 |
KR20010015869A (ko) | 2001-02-26 |
AU1718099A (en) | 1999-06-28 |
KR20010015868A (ko) | 2001-02-26 |
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EP1037983A1 (fr) | 2000-09-27 |
JP2001526040A (ja) | 2001-12-18 |
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