US20040141978A1 - Angiogenesis related molecules - Google Patents
Angiogenesis related molecules Download PDFInfo
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- US20040141978A1 US20040141978A1 US10/720,273 US72027303A US2004141978A1 US 20040141978 A1 US20040141978 A1 US 20040141978A1 US 72027303 A US72027303 A US 72027303A US 2004141978 A1 US2004141978 A1 US 2004141978A1
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- the present invention relates to the field of angiogenesis, and more specifically to novel molecules, such as proteins and peptides, whereby novel anti-angiogenic substances may be developed.
- the invention also relates to methods for developing such substances.
- Angiogenesis The generation of new blood vessels from existing ones is called angiogenesis.
- Angiogenesis has been associated to cancer and the formation of tumors as well as to other conditions, such as diabetes retinopathy, rheumatoid arthritis and even some inflammatory conditions. Accordingly, a considerable research effort is made world-wide to find ways of preventing and inhibiting the angiogenic process. If this were possible, tumor growth could be controlled and useful therapies could be developed regarding the above mentioned conditions.
- angiogenesis is triggered by factors secreted by the tumor cells. It has recently been discovered that tumors through unleashed proteolytic activity generates peptide fragments, which show anti-angiogenic activities.
- angiostatin which is a fragment of plasrninogen.
- Plasminogen is a substance in blood plasma which, when activated, forms plasrnin or fibrinolysin, an enzyme involved in the coagulation of blood. Plasminogen itself lacks any detectable anti-angiogenic activity. It has been found (Judah Folkman et al, Harvard Medical School, Boston) that a part of this endogenous protein, more specifically the first four kringle domains, is capable of preventing the endothelial cells from dividing. This part of plasminogen has been denoted angiostatin, and a great deal of research within this field is centred around this molecule. The prior art has shows that angiostatin inhibits endothelial cells specifically in vitro and blocks angiogenesis in vivo.
- angiostatin shows two levels of specificity: it induces apoptosis specifically in endothelial cells in vitro (Claesson-Welsh et al., Proc. Natl. Acad. Sci., USA, vol. 95, p. 5579, 1998) and only affects endothelial cells active in angiogenesis in vivo. It has not shown to negatively affect cells in established vessels.
- Agiostatin does indeed exhibit some advantageous properties, inter alia as it is an endogenous substance.
- the disadvantages associated with its possible use for medical purposes cannot be neglected.
- These two disadvantages are in themselves strong motives for directing further research towards the finding of alternative, smaller and/or more efficient molecules to be used as medicaments.
- the present invention solves the problems associated with angiostatin as defined above by providing a human protein, which has been named “ABP-1”, defined by its ability to bind a fragment of plasminogen, preferably the first four Kringle domains (K1-K4) thereof, the said fragment being characterized by anti-angiogenic biological activity.
- ABP-1 comprises an amino acid sequence substantially similar to that shown in SEQ ID NO. 2. Variants and fragments of ABP-1 are encompassed in the present invention.
- Also encompassed in the present invention are the homologs of ABP-1 in other species, especially in other mammals.
- the invention provides isolated nucleic acid molecules comprising a sequence that codes for ABP-1 or for a polypeptide substantially similar to ABP-1, including its variants, fragments and homologs.
- nucleic acid probes whose sequence is derived from SEQ ID NO.1; these probes may be used as research tools as well as in diagnostic methods, for example to detect and measure ABP-1 biosynthesis in tissues and cells.
- the present invention also includes screening methods for identifying a compound capable of interacting with ABP-1 or its variants and fragments.
- the screening method can be in any configuration well known to those skilled in the art.
- It is a further object of the present invention to provide a pharmaceutical composition comprising as active ingredient the compound identified with the above screening method.
- angiogenesis relates to the generation of new blood vessels into a tissue or organ. As mentioned above, under normal physiological conditions, humans and animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is nornally observed in wound healing, fetal and embryonal development and formation of the corpus luteum and placenta.
- endothelium relates to the thin layer of flat epithelial cells, that lines serous cavities, lymph vessels and blood vessels.
- endothelial inhibiting activity relates to the capability of inhibiting the growth of endothelial capillary endothelial cells.
- an angiogenesis associated protein relates to a protein capable of inter-acting in the angiogenesis, such as a receptor binding an anti-angiogenic substance.
- substantially similar when used in reference to the amino acid sequence of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, means an amino acid sequence having an high degree of sequence homology to SEQ ID NO.2, SEQ ID NO.3 and SEQ ED NO.4.
- a high degree of homology means at least approximately 80% amino acid homology, preferably at least approximately 90% amino acid homology, more preferably at least approximately 95% amino acid homology and most preferably at least approximately 98% amino acid homology.
- the term “specifically hybridizing to” refers to the binding, duplexing or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when the sequence is present in a complex mixture (e.g. total cellular) of DNA or RNA.
- stringent conditions relates to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
- the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium).
- stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for longer probes. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
- FIG. 1 shows the binding of 125 I-labelled angiostatin and plasminogen to bovine microcapillary endothelial cells in vitro. Units in abscissa represent the fold molar excess of cold angiostatin and plasminogen, respectively.
- FIG. 2 illustrates activation of His3 and LacZ in the yeast two hybrid system upon binding of angiostatin binding protein.
- FIG. 3 shows the growth of positive yeast clones under selective conditions.
- FIG. 4 compares the binding of recombinant “Big-3” to angiostatin and plasminogen, respectively.
- FIG. 5 is a map over “Big-3”.
- FIG. 6 shows the open reading frame (Frame 2) of the gene encoding the angiostatin receptor according to the invention.
- the other possible frames do not produce any putative protein.
- FIG. 7 shows the expression patterns of fetal and adult mRNA as well as endothelial cells.
- FIG. 8 shows the relative quantitation of ABP-1 gene expression in different tissue. See experimental section for more details.
- FIG. 9 illustrates (A) the cellular localization of GFP-tagged ABP-1 receptor in transiently transfected HeLa cells and (B) the reorganization of GFP-labeled ABP-1 after incubation with angiostatin.
- FIG. 9C shows the binding of angiostatin to ABP-1. See the experimental section for more details.
- FIG. 9D is an immunostaining of ABP-1 in Human umbilical cord endothelial (HUVE) cells together with staining against F-actin with rhodamin-labelled phalloidin.
- ABP-1 is localized in focal adhesions and membrane ruffles (arrows).
- FIG. 10 illustrates the down regulation of ABP-1 associated kinase activity after addition of anoiostatin.
- FIG. 11 is an autoradiography of an SDS-PAGE demonstrating that ABP-1 mediates angiostatin-induced focal adhesion kinase activity. See experimental section for more details.
- the present invention relates to an isolated human angiogenesis-associated protein capable of binding a fragment of plasminogen, preferably an N-terminal fragment, such as kringle domains 1 to 4 and/or kringle 5.
- the protein of the invention acts as a receptor of plasminogen fragments, in particular as a receptor of angiostatin and/or kringle 5 domain of plasminogen.
- the protein of the invention can be synthesized by biological or chemical methods (e.g. recombinant gene expression and peptide synthesis).
- Recombinant techniques include gene cloning or amplification by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS) or the self-sustained sequence replication system (SSR).
- PCR polymerase chain reaction
- LCR ligase chain reaction
- TAS transcription-based amplification system
- SSR self-sustained sequence replication system
- a wide variety of cloning and in vitro amplification methodologies are well-known to those skilled in the art. Examples of these techniques are e.g. found in Berger and Kimmel, Guide to Molecular Cloning Techniques , Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger).
- the present invention includes proteins which comprise amino acid sequences substantially similar to those shown in SEQ D NO.2, SEQ ID NO.3 and SEQ ID NO.4.
- polypeptides capable of binding a fragment of plasminogen and having an amino acid sequence which has at least approximately 80% sequence homology, preferably approximately 90% sequence homology, more preferably approximately 95% sequence homology and most preferably approximately 98% sequence homology to SEQ ID Nos.2, 3 or 4, are contemplated as being included in the present invention.
- These variant forms may result, e.g., from alternative splicing or differential expression in different tissue of the same source organism.
- the variant forms may be characterized by, e.g., amino acid insertion, deletion or substitution.
- a preferred variant form of ABP-1 is illustrated in SEQ ID NO. 3 (see below).
- a particularly preferred embodiment of the present invention is a fragment of ABP-1 named Big-3 which will be described in greater details below and which includes the angiostatin-binding domain of ABP-1 (SEQ ID NO. 4).
- the present invention provides peptides which comprises at least about 5, preferably at least about 10 contiguous amino acid residues of SEQ ID NO 2 or any variant or fragment thereof.
- the most advantageous size of the peptide will be depending on the intended future use thereof and the present invention is not limited to the above stated number of amino acids residues.
- homologs of ABP-1 and Big-3 are also included in the present invention.
- the term “homolog” refers to proteins exerting substantially the same biological function of the proteins of the invention, regardless of the homology existing between the corresponding amino acid sequences.
- the present invention relates to isolated nucleic acid molecules comprising a sequence that codes for the proteins and peptides of the invention, including the variants, fragments and homologs as defined above.
- the nucleic acid molecule has the sequence of SEQ ID NO. 1. This sequence presents a 5′ (from nucleotide 1 to nucleotide 796) and a 3′ (from nucleotide 2825 to nucleotide 6463) untranslated regions.
- the nucleic acid molecule has the sequence from nucleotide 797 to nucleotide 2824 of SEQ ID NO. 1.
- the nucleic acid sequence encodes the ABP-1 fragment named Big-3 (illustrated in SEQ ID NO. 4) and it comprises the nucleotide sequence from position 2180 to position 2608 of SEQ ID NO. 1. All the nucleotide sequences encoding the polypeptides of the invention and differing from the nucleotide sequence of SEQ ID NO. 1 or fragment thereof by way of the degeneracy of the genetic code, are considered part of the invention. Sequencing analysis has revealed the presence of a possible polymorfism in the codon 1199-1201 of SEQ ID NO. 1, wherein a codon for Asn, Ser or Asp may be present; a further region corresponding to nucleotide positions 1238 to 1246 of SEQ ID NO.
- ABP-1 has been found to code for the tripeptide Glu-Leu-Ala or for the tripeptide Thr-Trp-Pro. These variations in the amino acid sequence of ABP-1 are illustrated in SEQ ID NO. 3 which constitutes another preferred protein of the invention. Also included in the present invention are nucleotide molecules encoding a homolog of ABP-1 or fragment thereof.
- the present invention further comprises a nucleic acid capable of specifically hybridizing, under stringent conditions, to any one of the nucleic acid molecules of the invention described above.
- markers may include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- the markers may be incorporated by any of a number of means well known to those of skill in the art. Methods for detecting such markers are also well known in the art and disclosed in the literature.
- the probes find a useful application in diagnostic application, for example to detect and measure ABP-1 biosynthesis in tissues and cells.
- the present invention provide screening methods or assays, wherein molecules that exhibit the same properties as angiostatin and/or kringle 5 are screened for.
- a compound capable of activating the angiostatin signal transduction pathway is identified through a high throughput cell-based screen.
- Such screen rely on a reporter gene driven by an angiostatin responsive element that is stably transfected into an angiostatin-responsive cell line.
- the responsive element is linked to a reporter gene, e.g. the gene for luciferase; upon binding of the compound to the protein of the invention, the intracellular pathway is activated thus causing an increase in the reporter gene activity that can be detected.
- the compounds identified through the screening method hereby described are within the scope of the present invention.
- Such compounds are preferably low molecular weight molecules of peptidic or non-peptidic nature. Thanks to their small size they are more practical for use for medicinal purposes, compared to angiostatin. These molecules and the uses thereof are described in more detail below.
- the proteins and peptides according to the invention may be synthesized using standard chemical peptide synthesis techniques.
- solid phase synthesis see e.g. Barany and Merrifield, Solid - Phase Peptide Synthesis , pp 3-284 in The Peptides: Analysis, Synthesis Biology , Vol. 2 : Special Methods in Peptide Synthesis , Part A.
- the proteins, peptides and polypeptides according to the invention are synthesized using recombinant DNA methodology, which generally involves creating a DNA sequence that encodes the protein or peptide, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein or peptide in a host, isolating the expressed protein or peptide and, if required, renaturing the product.
- the recombinant peptides or proteins can be purified according to standard procedures in the art.
- another aspect of the present invention is a vector, such as a virus or a plasmid, comprising a nucleic acid according to the invention.
- the invention also encompasses a recombinant cell transformed or transfected with the said vector expressing the present protein or peptide as well as a recombinant cell expressing the present antibody, which will be defined in more detail below.
- Vectors encoding the peptides and proteins according to the invention are useful in expressing those molecule to provide immunogens for antibody production.
- the vectors according to the invention are also useful for transforming cells in vitro or in vivo to express the present peptides and proteins.
- Cells expressing any one of the present nucleic acids, such as the gene defined by SEQ ID NO 1, may be used in a wide variety of contexts and are also within the scope of the present invention. Such cells may be eucaryotic or procaryotic.
- the proteins and peptides according to the invention can be used as antigens for raising antibodies against the same. Consequently, the invention also encompasses an antibody which specifically binds a peptide according to the invention. Such antibodies are useful for immunoassays, e.g. for the isolation of peptides or polypeptides.
- the peptides according to the invention may also be used in assays, such as amplification specific assays, immunological assays etc.
- the antibodies according to the invention may be monoclonal or polyclonal and include individual, allelic, strain or species variants, or fragments thereof, both in their naturally occurring (full-length) forms and recombinant forms. Additionally, the antibodies are raised to the present peptides or polypeptides in either their native configuration or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons skilled in the art. For techniques for preparing monoclonal antibodies, see e.g. Stiites et al (eds.), Basic and Clinical Immunology (4 th ed), Lange Medical Publications, Los Altos, Calif., and references cited therein. For techniques that involve selection of libraries of recombinant antibodies in phage or similar vectors, see e.g. Huse et al. (1989) Science 246:1275-1281.
- the molecules according to the invention may be used in pharmaceutical preparations, especially for the treatment and/or prevention of angiogenesis related disorders. Accordingly, the invention relates to a peptide, polypeptide, protein or antibody according to the invention for use as a medicament as well as to the use of said molecules in the manufacture of a medicament directed towards an angiogenesis related disease or disorder. The invention also relates to a pharmacological preparation comprising a molecule according to the invention together with a pharmaceutically acceptable carrier.
- the molecules used as medicaments according to the invention may be anyone of the above described peptides, polypeptides, proteins or antibodies as well as any novel substance identified in a screening method using the same and described above.
- the most preferred use of the present molecules is in assays, wherein novel substances are screened for.
- Compounds may be identified which exhibit similar anti-angiogenic effects as angiostatin and/or kringle 5, but which are smaller, more efficient and preferably exhibits a longer half time in a human or animal body than angiostatin. The shorter half time may be due to a lower tendency to be degraded by proteases.
- a molecule according to the invention is used as a “lead” compound.
- the design of mimetics to known pharmaceutically active compounds is a well known approach in the development of pharmaceuticals based on such “lead” compounds. Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.
- novel molecules are preferably used as medicaments that may be administered in much lower doses than angiostatin, and which may be administered less frequently, such as e.g. once every fourteen days, which is to be compared to the half time of angiostatin which is about ten times shorter.
- the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a pharmaceutical preparation may be prepared thereof for oral intake, such as in tablets.
- the pharmaceutical preparations according to the invention may however be prepared for any route of administration, e.g oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular or intraperitoneal.
- routes of administration e.g oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular or intraperitoneal.
- the nature of the carrier or other ingredients will depend on the specific route of administration. (Examples of techniques and protocols that are useful in this context are inter alia found in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A (ed.), 1980.)
- the dosage of these low molecular weight compounds will depend on the disease state or condition to be treted and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound For treating human or animals, between approximately 0.5 mg/Kg of body weight to 500 mg/Kg of body weight of the compound can be administered.
- the present compounds and methods are advantageously used in relation to all kind of angiogenesis related disorders and/or diseases, such as tumor conditions, diabetes, rheumatoid arthritis and even some inflammatory diseases, such as psoriasis, chronic, inflammations of the intestines, asthma etc. It is also suggested that they may be used in order to treat and cure, or prevent, obesity.
- angiogenesis related disorders and/or diseases such as tumor conditions, diabetes, rheumatoid arthritis and even some inflammatory diseases, such as psoriasis, chronic, inflammations of the intestines, asthma etc. It is also suggested that they may be used in order to treat and cure, or prevent, obesity.
- the present molecules are used in gene therapy.
- gene therapy procedures see e.g. Anderson, Science ; (1992) 256:808-813.
- a further advantageous use of the present invention is to develop methods of regulating the signalling of the present angiostatin and/or kringle 5 receptor in the body and therefore, the invention also relates to methods of treating a human or animal patient suffering from an angiogenesis related disease or disorder as well as to methods preventing such conditions.
- angiostatin acts by blocking angiogenic factors interaction with endothelial cells.
- human angiostatin (cringle domains 1-4) or plasminogen was labeled with iodine 125 by the Iodogen method according to the protocol of the manufacturer (Pierce Inc.). The labelled protein was then purified on a G50 sepharose column (Pharmacia Inc.) The specific activity was estimated at 90 000 cpm/ng protein.
- bovine capillary endothelial cells were grown to confluency in 12 well plates. The cells were washed with PBS containing 1 mg/ml bovine serum albumine (BSA). The cells were then incubated with 10 ng/ml radio-labeled angiostatin or plasminogen.
- Angiostatin retains activity even after reduction, suggesting that three dimensional folding is not vital for binding and activity. This favours screening procedures wherein a large number of clones may be screened although protein refolding may be less accurate.
- yeast two hybrid system was employed to screen for molecules that bind kringle domains 1-4 of plasminogen. This is further disclosed below with reference to FIG. 2, Approximately 2 ⁇ 10 6 clones from a two hybrid cDNA library from human term placenta (Stratagene) were screened. Positives clones were identified in the following way:
- Big3 sequence has been expressed in E. coli as fusion protein with the glutathione S-transferase (GST) domain from Schistosoma japonicum.
- GST glutathione S-transferase
- Big3 fragment (429 bp) was obtained by PCR amplification using pUC18-Big3 plasmid as template.
- Primer sequences were: BamHI-NH 2 5′ TAC GGA TCC GAA TCG AAC AAA ACT GCA GCT G 3′ (SEQ ID NO. 5) XhoI- COOH 5′ ATA CTC GAG TCA TGG AGC TGG ACT TGG AGC CA 3′ (SEQ ID NO. 6)
- the cycling protocol used was: 94° C. 3′, 60° C. 1′, 72° C. 1′ 1 cycle 94° C. 30′′, 60° C. 1′, 72° C. 2′ 30 cycles 94° C. 30′′, 60° C. 1′, 72° C. 5′ 1 cycle
- Taq polymerase native Pfu DNA polymerase (Stratagene)
- PCR fragment was digested with BamHI and XhoI, purified and ligated to PGEX-6P-2 plasmid (Pharmacia Biotech) digested with the same restriction enzymes. Ligation mixture was used to transform XL1-Blue cells (Stratagene). Recombinant plasmids were verified by restriction analysis and automated sequencing.
- DH5 ⁇ cells (Clontech) were transformed with one verified clone, named pGEX-Big3, and induced for 6 hours, room temperature with 0.1 mM IPTG.
- the fermentation broth was centrifuged at 4000 ⁇ rpm 15 min and the cell pellet was resuspended in 10% w/v of lysis buffer (50 mM TRIS.HCl pH 8.0, 100 mM NaCl, 20 mM DTT, 1 mM EDTA, protease inhibitor mix), and lysed by sonication.
- the total lysate was centrifuged at 15000 ⁇ g for 20 min at 4° C.
- the clarified supernatant was applied to a Glutathione-Sepharose column (1 ml for 20 ml of lysate) preequilibrated with lysis buffer.
- the resin was washed with 10 column volumes (CV) of lysis buffer and the fusion protein was eluted with 3 CV of elution buffer (100 mM TRIS.HCl pH 8.0 20 mM reduced glutathione).
- GST-Big3 protein can be cleaved by incubation of the glutathione-Sepharose bound fusion protein with 20 ul/ml resin of PreScission protease in PS buffer containing 50 mM TRIS.HCl pH 7.0. 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF.
- the eluted proteins behave like a single peak in rp-HPLC (10-90% gradient of acetonitrile in water plus 0.1% TFA), show the expected molecular size by mass analysis (electrospray) and the correct NH2 sequence.
- rp-HPLC 10-90% gradient of acetonitrile in water plus 0.1% TFA
- the yields obtained from 1 litre of fermentation are about 10 man of GST-Big3 fusion protein and about 3 mg of the cleaved Big3.
- the overnight culture was diluted 1:10 into 100 ml fresh medium and after 1 hour of incubation at 37° C., IPTG was added to a final concentration of 0.5 mM and incubation continued for 3 to 7 hours.
- the culture was then centrifuged at 5000 g for 10 minutes, the pellet was resuspended in 3-5 ml of ice-cold PBS and cells were lysed by sonication. Triton X100 was added to a final concentration of 0.5-1% and 1.5 ml aliquots of the suspension were centrifuged at 14000 rpm for 10 minutes.
- Binding assay 0.7 ml B.S., 0.1% calf serum, 500 ng angiostatin were mixed with 30-50 ⁇ l 50% slurry of Big3-immobilized glutathione-agarose beads for 1-3 hour at 4° C.
- the resin was washed 5-7 times with ice cold B.S., then used for Western blot analysis using anti-human plasminogen antibodies (Dako Inc.).
- FIG. 4 shows how purified recombinant Big 3 binds angiostatin in vitro. Interestingly, binding of plasminogen could also be detected, but only after reduction of disulphide bonds by treating it with DTT.
- FIG. 5 illustrates the scheme for cloning the full-length sequence of ABP-1.
- the whole gene was cloned by screening a placenta cDNA phage library with a Big3 probe (with repetitive sequences removed) together with 5′ RACE PCR (Gibco) using mRNA from human umbilical cord endothelial cells.
- the full sequence of the cDNA clone is disclosed in SEQ ID NO. 1, whereas the encoded protein is shown in SEQ ID NO. 2. Details of the experimental protocol are as follows.
- RACE procedures have been used for amplification and cloning unknown sequences between the GSP2 and GSP3 and the 5′-end of the mRNA ABP-1. This sequence was then used to design new primers for the next set of RACE RCR: Primers: GSP1-(5′ to 3′) GGTGGCAGCGGACAGGCAGGATAC (SEQ ID NO. 13) GSP2 GAGGCGGAGAGAACTAAGAGAAGA (SEQ ID NO. 14) GSP3 GAGCGGAGATGGAGGAGTAATTCA (SEQ ID NO. 15)
- Clones A2-2, A2-1 and A1-C with overlapping sequences were isolated.
- the A2-1 sequence was used as probe for the second screening of the placenta phage library. From this screening we isolated another 2 clones (7-10 and 6-5). As shown in FIG. 6, of the six possible frame the only one producing a complete open reading frame is frame 2, yielding a putative protein of 675 amino acid residues.
- a commercially obtained multiple human adult and fetal tissue (#7760-1 and #7756-1) mRNA blots (2 mg mRNA/well) was probed for ABP-1 The blots were hybridized with the ExpressHyb hybridization solution (Clontech, Inc.) according to the protocol of the manufacturer. The blots were probed with the 7-2 5′PstI fragment.
- FIG. 7 shows the results of the experiment; sizes are 9.5 and 7.5 kb.
- ABP-1 was also detected in human umbilical cord, bovine aortic and bovine microcapillary endothelial cells. Little or no expression was detected in the immortalized endothelial cell-line EaHy926 and in human fetal fibroblasts. Neither of the cell lines respond to angiostatin or exhibit any binding of FITC-labeled angiostatin.
- the 5′ nuclease assay uses a nonextendable oligonucleotide hybridization probe (TaqMan Probe).
- the TaqMan probe consists of an oligonucleotide with a 5′-reporter dye and a 3′-quencher dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence, primarily by a Foster-type energy transfer.
- forward and reverse primers hybridize to a specific sequence of the target DNA.
- the TaqMan probe hybridizes to a target sequence within the PCR product.
- the Taq Polymerase cleaves the TaqMan probe with its 5′-3′ nuclease activity. The reporter dye and quencher dye are separated upon cleavage, resulting in increased fluorescence of the reporter.
- RNA isolation and purification of total RNA from tissues and cells listed below was performed using the Ultraspec RNA isolation system (Biotex).
- the RT reaction was performed using TaqMan Reverse Transcription Reagent (PE Applied Biosystems) with Random hexamers, as RT primers.
- the reaction volume for Reverse Transcription step was 100 ⁇ l and the total RNA amount was 1 ⁇ g for sample.
- ABP-1 Forward Primer 5′GTTTGACCTGCAATCCAGACAA3′ (SEQ ID NO. 7) Final concentration 300 nM
- ABP-1 Reverse Primer 5′CCCAGGATCTGAATGGGAGTT3′ (SEQ ID NO. 8) Final concentration 900 nm
- ABP-1 TaqMan Probe 5′ (FAM dye)-CAGATGGGCCTGTGTTCCACTCCAA-(TAA dye)3′ (SEQ ID NO. 9) Final probe concentration 200 nM
- the Comparative method use an arithmetic formula to achieve the result for relative quantitation without need for Standard curve.
- resin-immobilised ligand was produced as follows: Big3 polypeptide was diluted in 0.1M sodium bicarbonate, 0.5 M NaCl, pH8.3, at a concentration of 5 mg/ml in a total volume of 2 ml. This was reacted for two hours at room temperature with 2 ml of cyanogen bromide-activated Sepaharose CL4B resin (Sigma Chemical Co. St Louis, Mo.). After reaction, the resin was washed three times in 10 volumes of 100 mM Tris, 500 mM NaCl, pH 7.5.
- Immune serum was incubated with the affinity resin for two hours at 4° C., after which resin was washed in a 5 ml glass chromatography column (Bio-Rad, Richmond Calif.) for with 25 volumes of 100 nm-M Tris, 500 mM NaCl, pH 7.5. Specific antibodies were eluted in 1 ml aliquots with 100 mM Glycine/HCl, pH 2.8. Elution of antibody was followed by monitoring the optical density at 280 nM (OD280), and fractions with an OD280 of greater than 1 were pooled and dialysed (Slide-A-lyzer cassettes, Pierce) for 36 hours at 4° C. against 1 liter PBS, with three changes of buffer.
- OD280 optical density at 280 nM
- FIGS. 9A and 9B show data of binding of FITC-labeled angiostatin to ABP-1 transfected HeLa and EaHy926 cells transfected with the vector control.
- FIG. 9A shows the cellular localization of ABP-1 fused to green fluorescent protein (GFP) in transiently transfected cells. The protein can be detected in the endoplasmic reticulum and the cell membrane.
- GFP green fluorescent protein
- DABP-1 contains a 500 bp deletion in the 5′ end of the gene, which disrupts the previously described localization of ABP-1.
- FIG. 9B shows HeLa cells transfected with ABP-1 GFP. Incubation with 2.5 mg of angiostatin for 60 minutes at 0° C. and subsequent incubation for 15 minutes at 37° C. induces internalization of ABP-1-GFP.
- FIG. 9C shows the binding of angiostatin to ABP-1.
- FITC fluorescein isothiocyanate
- FIG. 9C shows the binding of angiostatin to ABP-1.
- FITC fluorescein isothiocyanate
- FIG. 9D shows immunostaining of ABP-1 in Human umbilical cord endothelial (HUVE) cells together with staining against F-actin with rhodarmin-labelled phalloidin.
- HUVE Human umbilical cord endothelial
- HUVE cells were fixed in 4% formaldehyde, washed in PBS and preblocked in 5% horse serum. The blocking solution was removed and replaced by rabbit polyclonal antibodies against the angiostatin-binding domain Big3 diluted in 5% horse serum. Positive staining was visualized with a fluorescence-labeled secondary antibody (Dako, Inc.). Rhodamin-conjugated phalloidin (Molecular Probes, Inc.) was stained simultaneously with the secondary antibody (after permeabilization with 1% triton X100 for 1 minute). Rabbit polyclonal antibodies against green fluorescent protein was used as a negative control. In addition no positive staining could be detected in human fibroblasts.
- ABP-1 is localized in focal adhesions and membrane ruffles (arrows).
- Huve cells were plated subconfluent in 5 cm Petri dishes. Angiostatin was added at 2.5 mg/ml at different time points the following day. All plates including controls were harvested simultanously. The cells were rinsed in ice cold PBS and incubated in 1 ml lysis buffer. The cells were transferred to an eppendorf tube and centrifuged at 14 000 rpm for 5 minutes at 4° C. The supernatant was transferred to another eppendorf tube and 2 ⁇ g of Big 3 rabbit polyclonal antibodies was added. The samples were incubated for 60 min. at 4° C. and subsequently 50 ⁇ l of a protein A sepharose (Pharmacia, Inc.) slurry was added and incubated for another 60 min.
- a protein A sepharose Pharmacia, Inc.
- FIG. 10 show that addition of angiostatin downregulates ABP-1 associated kinase activity within 30 minutes. This is direct proof that angiostatin affects signalling pathways that are mediated by ABP-1.
- ABP-1 Mediates Angiostatin-Induced Focal Adhesion Kinase Activity.
- EaHy926 cells were plated subconfluent in 5 cm Petri dishes. Angiostatin was added at 2.5 ⁇ g/ml at different time points the following day. All plates including controls were harvested simultaneously. The cells were rinsed in ice cold PBS and incubated in 1 ml lysis buffer. The cells were transferred to an eppendorf tube and centrifuged at 14 000 rpm for 5 minutes at 4° C. The supernatant was transferred to another eppendorf tube and 1 ug of FAK monoclonal antibody (Transduction Lab. Inc.) was added. The samples were incubated for 60 min. at 4° C.
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Abstract
The present invention provides the sequence of a protein capable of acting as an angiostatin receptor as well as the nucleic acid sequence thereof. The invention also relates to the use thereof in screening methods, wherein novel substances are created exhibiting the same advantageous anti-angiogenic properties as angiostatin.
Description
- This application is a Divisional of co-pending application Ser. No. 09/332,063, filed on Jun. 14, 1999, the entire contents of which are hereby incorporated by reference and for which priority is claimed under 35 U.S.C. § 120; and this application claims priority of U.S. Provisional Application Nos. 60/089,266 and 60/114,386 filed in the United States on Jun. 15, 1998 and Dec. 29, 1998, respectively under 35 U.S.C. § 119.
- The present invention relates to the field of angiogenesis, and more specifically to novel molecules, such as proteins and peptides, whereby novel anti-angiogenic substances may be developed. The invention also relates to methods for developing such substances.
- Almost all of the tissue of a mammalian body comprises a fine mesh of very thin blood vessels, each of which is thinner than a human hair. Usually, neither the number nor the size of these vessels increase, since the division of the endothelial cells covering the vessels is slow, actually up to several years. The exceptions are for example during wound healing and menstruation, when the vessels grow rapidly. However, that is during a limited period of time and the cell division ceases thereafter.
- The generation of new blood vessels from existing ones is called angiogenesis. Angiogenesis has been associated to cancer and the formation of tumors as well as to other conditions, such as diabetes retinopathy, rheumatoid arthritis and even some inflammatory conditions. Accordingly, a considerable research effort is made world-wide to find ways of preventing and inhibiting the angiogenic process. If this were possible, tumor growth could be controlled and useful therapies could be developed regarding the above mentioned conditions.
- These exists numerous pieces of evidence showing that tumors are depending on de novo formation of blood vessels for expansion beyond a mass of a few mm3. The angiogenesis is triggered by factors secreted by the tumor cells. It has recently been discovered that tumors through unleashed proteolytic activity generates peptide fragments, which show anti-angiogenic activities. One example is the molecule angiostatin, which is a fragment of plasrninogen.
- Plasminogen is a substance in blood plasma which, when activated, forms plasrnin or fibrinolysin, an enzyme involved in the coagulation of blood. Plasminogen itself lacks any detectable anti-angiogenic activity. It has been found (Judah Folkman et al, Harvard Medical School, Boston) that a part of this endogenous protein, more specifically the first four kringle domains, is capable of preventing the endothelial cells from dividing. This part of plasminogen has been denoted angiostatin, and a great deal of research within this field is centred around this molecule. The prior art has shows that angiostatin inhibits endothelial cells specifically in vitro and blocks angiogenesis in vivo. Systemic treatments with subcutaneous injections of angiostatin induces in vivo dormancy in a wide range of tumors in SCID mice (O'Reilly et al., Nature Med., vol. 2, p. 689, 1996). No detectable toxicity has been detected in these animals even after months of treatment. Angiostatin shows two levels of specificity: it induces apoptosis specifically in endothelial cells in vitro (Claesson-Welsh et al., Proc. Natl. Acad. Sci., USA, vol. 95, p. 5579, 1998) and only affects endothelial cells active in angiogenesis in vivo. It has not shown to negatively affect cells in established vessels.
- Agiostatin does indeed exhibit some advantageous properties, inter alia as it is an endogenous substance. However, the disadvantages associated with its possible use for medical purposes cannot be neglected. One is that the half life thereof is very short, it may be counted in hours, thereby requiring a frequent administration thereof. This far, the efficiency thereof has proven to be rather low, which fact necessitates the use of large doses thereof. These two disadvantages are in themselves strong motives for directing further research towards the finding of alternative, smaller and/or more efficient molecules to be used as medicaments.
- The present invention solves the problems associated with angiostatin as defined above by providing a human protein, which has been named “ABP-1”, defined by its ability to bind a fragment of plasminogen, preferably the first four Kringle domains (K1-K4) thereof, the said fragment being characterized by anti-angiogenic biological activity.
- ABP-1 comprises an amino acid sequence substantially similar to that shown in SEQ ID NO. 2. Variants and fragments of ABP-1 are encompassed in the present invention.
- Also encompassed in the present invention are the homologs of ABP-1 in other species, especially in other mammals.
- In a further aspect, the invention provides isolated nucleic acid molecules comprising a sequence that codes for ABP-1 or for a polypeptide substantially similar to ABP-1, including its variants, fragments and homologs.
- It is another object of the present invention to provide nucleic acid probes whose sequence is derived from SEQ ID NO.1; these probes may be used as research tools as well as in diagnostic methods, for example to detect and measure ABP-1 biosynthesis in tissues and cells.
- Accordingly, it is an object of the present invention to provide a diagnostic method for detecting the presence and the amount of ABP-1 or its variants and fragments in tissues and cells.
- The present invention also includes screening methods for identifying a compound capable of interacting with ABP-1 or its variants and fragments. The screening method can be in any configuration well known to those skilled in the art.
- It is a further object of the present invention to provide compounds identified with the said screening method and capable of modulating the biological activity of ABP-1 or its variants and fragments.
- It is a further object of the present invention to provide a pharmaceutical composition comprising as active ingredient the compound identified with the above screening method.
- It is still a further object of the present invention to provide antibodies directed against epitopes present in ABP-1 or its variants and fragments as well as cell producing the antibody.
- It is a further object of the present invention to provide a vector comprising the nucleotide sequence of ABP-1 or its variants and fragments.
- It is a further object of the present invention to provide a cell containing the above vector.
- Definitions
- In the present application, the following terms are used in the meanings defined below.
- As used herein, the term “angiogenesis” relates to the generation of new blood vessels into a tissue or organ. As mentioned above, under normal physiological conditions, humans and animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is nornally observed in wound healing, fetal and embryonal development and formation of the corpus luteum and placenta.
- The term “endothelium” relates to the thin layer of flat epithelial cells, that lines serous cavities, lymph vessels and blood vessels. The term “endothelial inhibiting activity” relates to the capability of inhibiting the growth of endothelial capillary endothelial cells.
- The term “an angiogenesis associated protein” relates to a protein capable of inter-acting in the angiogenesis, such as a receptor binding an anti-angiogenic substance.
- The term “substantially similar”, when used in reference to the amino acid sequence of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, means an amino acid sequence having an high degree of sequence homology to SEQ ID NO.2, SEQ ID NO.3 and SEQ ED NO.4. A high degree of homology means at least approximately 80% amino acid homology, preferably at least approximately 90% amino acid homology, more preferably at least approximately 95% amino acid homology and most preferably at least approximately 98% amino acid homology.
- The term “specifically hybridizing to” refers to the binding, duplexing or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when the sequence is present in a complex mixture (e.g. total cellular) of DNA or RNA. The term “stringent conditions” relates to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for longer probes. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
- FIG. 1 shows the binding of125I-labelled angiostatin and plasminogen to bovine microcapillary endothelial cells in vitro. Units in abscissa represent the fold molar excess of cold angiostatin and plasminogen, respectively.
- FIG. 2 illustrates activation of His3 and LacZ in the yeast two hybrid system upon binding of angiostatin binding protein.
- FIG. 3 shows the growth of positive yeast clones under selective conditions.
- FIG. 4 compares the binding of recombinant “Big-3” to angiostatin and plasminogen, respectively.
- FIG. 5 is a map over “Big-3”.
- FIG. 6 shows the open reading frame (Frame 2) of the gene encoding the angiostatin receptor according to the invention. The other possible frames do not produce any putative protein.
- FIG. 7 shows the expression patterns of fetal and adult mRNA as well as endothelial cells.
- FIG. 8 shows the relative quantitation of ABP-1 gene expression in different tissue. See experimental section for more details.
- FIG. 9 illustrates (A) the cellular localization of GFP-tagged ABP-1 receptor in transiently transfected HeLa cells and (B) the reorganization of GFP-labeled ABP-1 after incubation with angiostatin. FIG. 9C shows the binding of angiostatin to ABP-1. See the experimental section for more details. FIG. 9D is an immunostaining of ABP-1 in Human umbilical cord endothelial (HUVE) cells together with staining against F-actin with rhodamin-labelled phalloidin. ABP-1 is localized in focal adhesions and membrane ruffles (arrows).
- FIG. 10 illustrates the down regulation of ABP-1 associated kinase activity after addition of anoiostatin.
- FIG. 11 is an autoradiography of an SDS-PAGE demonstrating that ABP-1 mediates angiostatin-induced focal adhesion kinase activity. See experimental section for more details.
- The present invention relates to an isolated human angiogenesis-associated protein capable of binding a fragment of plasminogen, preferably an N-terminal fragment, such as
kringle domains 1 to 4 and/or kringle 5. Thus, the protein of the invention acts as a receptor of plasminogen fragments, in particular as a receptor of angiostatin and/or kringle 5 domain of plasminogen. The protein of the invention can be synthesized by biological or chemical methods (e.g. recombinant gene expression and peptide synthesis). Recombinant techniques include gene cloning or amplification by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS) or the self-sustained sequence replication system (SSR). A wide variety of cloning and in vitro amplification methodologies are well-known to those skilled in the art. Examples of these techniques are e.g. found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger). The present invention includes proteins which comprise amino acid sequences substantially similar to those shown in SEQ D NO.2, SEQ ID NO.3 and SEQ ID NO.4. A comparison of the protein sequence of SEQ ID NO. 2 with the sequences present in all available data bases showed a homology of 23.7% with a hypotetical protein of Caenorhabditis elegans encoded by a putative open reading frame located in the central cluster of chromosome III of this organism (described in R. Wilson et al., Nature, vol. 368, 3 Mar. 1994, p. 32-38). No function has been attributed to this hypotetical protein. - According to the definition given above, it is to be understood that all polypeptides capable of binding a fragment of plasminogen and having an amino acid sequence which has at least approximately 80% sequence homology, preferably approximately 90% sequence homology, more preferably approximately 95% sequence homology and most preferably approximately 98% sequence homology to SEQ ID Nos.2, 3 or 4, are contemplated as being included in the present invention. These variant forms may result, e.g., from alternative splicing or differential expression in different tissue of the same source organism. The variant forms may be characterized by, e.g., amino acid insertion, deletion or substitution. A preferred variant form of ABP-1 is illustrated in SEQ ID NO. 3 (see below).
- A particularly preferred embodiment of the present invention is a fragment of ABP-1 named Big-3 which will be described in greater details below and which includes the angiostatin-binding domain of ABP-1 (SEQ ID NO. 4).
- In a further embodiment, the present invention provides peptides which comprises at least about 5, preferably at least about 10 contiguous amino acid residues of
SEQ ID NO 2 or any variant or fragment thereof. However, the most advantageous size of the peptide will be depending on the intended future use thereof and the present invention is not limited to the above stated number of amino acids residues. - Also included in the present invention are homologs of ABP-1 and Big-3 in other species, in particular in other mammals. The term “homolog” refers to proteins exerting substantially the same biological function of the proteins of the invention, regardless of the homology existing between the corresponding amino acid sequences.
- In another aspect, the present invention relates to isolated nucleic acid molecules comprising a sequence that codes for the proteins and peptides of the invention, including the variants, fragments and homologs as defined above. In a preferred embodiment, the nucleic acid molecule has the sequence of SEQ ID NO. 1. This sequence presents a 5′ (from
nucleotide 1 to nucleotide 796) and a 3′ (from nucleotide 2825 to nucleotide 6463) untranslated regions. In another preferred embodiment the nucleic acid molecule has the sequence from nucleotide 797 to nucleotide 2824 of SEQ ID NO. 1. In another preferred embodiment the nucleic acid sequence encodes the ABP-1 fragment named Big-3 (illustrated in SEQ ID NO. 4) and it comprises the nucleotide sequence from position 2180 to position 2608 of SEQ ID NO. 1. All the nucleotide sequences encoding the polypeptides of the invention and differing from the nucleotide sequence of SEQ ID NO. 1 or fragment thereof by way of the degeneracy of the genetic code, are considered part of the invention. Sequencing analysis has revealed the presence of a possible polymorfism in the codon 1199-1201 of SEQ ID NO. 1, wherein a codon for Asn, Ser or Asp may be present; a further region corresponding to nucleotide positions 1238 to 1246 of SEQ ID NO. 1 has been found to code for the tripeptide Glu-Leu-Ala or for the tripeptide Thr-Trp-Pro. These variations in the amino acid sequence of ABP-1 are illustrated in SEQ ID NO. 3 which constitutes another preferred protein of the invention. Also included in the present invention are nucleotide molecules encoding a homolog of ABP-1 or fragment thereof. - The present invention further comprises a nucleic acid capable of specifically hybridizing, under stringent conditions, to any one of the nucleic acid molecules of the invention described above.
- Where the nucleic acids according to the invention are to be used as probes, it is often desirable to label the sequences with detectable markers. Such markers may include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The markers may be incorporated by any of a number of means well known to those of skill in the art. Methods for detecting such markers are also well known in the art and disclosed in the literature. The probes find a useful application in diagnostic application, for example to detect and measure ABP-1 biosynthesis in tissues and cells.
- In another aspect, the present invention provide screening methods or assays, wherein molecules that exhibit the same properties as angiostatin and/or kringle 5 are screened for. In a typical screening method a compound capable of activating the angiostatin signal transduction pathway is identified through a high throughput cell-based screen. Such screen rely on a reporter gene driven by an angiostatin responsive element that is stably transfected into an angiostatin-responsive cell line. The responsive element is linked to a reporter gene, e.g. the gene for luciferase; upon binding of the compound to the protein of the invention, the intracellular pathway is activated thus causing an increase in the reporter gene activity that can be detected.
- The compounds identified through the screening method hereby described are within the scope of the present invention. Such compounds are preferably low molecular weight molecules of peptidic or non-peptidic nature. Thanks to their small size they are more practical for use for medicinal purposes, compared to angiostatin. These molecules and the uses thereof are described in more detail below.
- The proteins and peptides according to the invention may be synthesized using standard chemical peptide synthesis techniques. For solid phase synthesis, see e.g. Barany and Merrifield,Solid-Phase Peptide Synthesis, pp 3-284 in The Peptides: Analysis, Synthesis Biology, Vol. 2: Special Methods in Peptide Synthesis, Part A.
- Preferably, the proteins, peptides and polypeptides according to the invention are synthesized using recombinant DNA methodology, which generally involves creating a DNA sequence that encodes the protein or peptide, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein or peptide in a host, isolating the expressed protein or peptide and, if required, renaturing the product. Once expressed, the recombinant peptides or proteins can be purified according to standard procedures in the art. Thus, another aspect of the present invention is a vector, such as a virus or a plasmid, comprising a nucleic acid according to the invention. Further, the invention also encompasses a recombinant cell transformed or transfected with the said vector expressing the present protein or peptide as well as a recombinant cell expressing the present antibody, which will be defined in more detail below. Vectors encoding the peptides and proteins according to the invention are useful in expressing those molecule to provide immunogens for antibody production. The vectors according to the invention are also useful for transforming cells in vitro or in vivo to express the present peptides and proteins. Cells expressing any one of the present nucleic acids, such as the gene defined by
SEQ ID NO 1, may be used in a wide variety of contexts and are also within the scope of the present invention. Such cells may be eucaryotic or procaryotic. - The proteins and peptides according to the invention can be used as antigens for raising antibodies against the same. Consequently, the invention also encompasses an antibody which specifically binds a peptide according to the invention. Such antibodies are useful for immunoassays, e.g. for the isolation of peptides or polypeptides. The peptides according to the invention may also be used in assays, such as amplification specific assays, immunological assays etc.
- The antibodies according to the invention may be monoclonal or polyclonal and include individual, allelic, strain or species variants, or fragments thereof, both in their naturally occurring (full-length) forms and recombinant forms. Additionally, the antibodies are raised to the present peptides or polypeptides in either their native configuration or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons skilled in the art. For techniques for preparing monoclonal antibodies, see e.g. Stiites et al (eds.),Basic and Clinical Immunology (4th ed), Lange Medical Publications, Los Altos, Calif., and references cited therein. For techniques that involve selection of libraries of recombinant antibodies in phage or similar vectors, see e.g. Huse et al. (1989) Science 246:1275-1281.
- The molecules according to the invention may be used in pharmaceutical preparations, especially for the treatment and/or prevention of angiogenesis related disorders. Accordingly, the invention relates to a peptide, polypeptide, protein or antibody according to the invention for use as a medicament as well as to the use of said molecules in the manufacture of a medicament directed towards an angiogenesis related disease or disorder. The invention also relates to a pharmacological preparation comprising a molecule according to the invention together with a pharmaceutically acceptable carrier. The molecules used as medicaments according to the invention may be anyone of the above described peptides, polypeptides, proteins or antibodies as well as any novel substance identified in a screening method using the same and described above.
- Accordingly, the most preferred use of the present molecules is in assays, wherein novel substances are screened for. Compounds may be identified which exhibit similar anti-angiogenic effects as angiostatin and/or kringle 5, but which are smaller, more efficient and preferably exhibits a longer half time in a human or animal body than angiostatin. The shorter half time may be due to a lower tendency to be degraded by proteases. When an organic compound is designed, a molecule according to the invention is used as a “lead” compound. The design of mimetics to known pharmaceutically active compounds is a well known approach in the development of pharmaceuticals based on such “lead” compounds. Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.
- Thus, such novel molecules are preferably used as medicaments that may be administered in much lower doses than angiostatin, and which may be administered less frequently, such as e.g. once every fourteen days, which is to be compared to the half time of angiostatin which is about ten times shorter. In a particular embodiment, the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a pharmaceutical preparation may be prepared thereof for oral intake, such as in tablets.
- The pharmaceutical preparations according to the invention may however be prepared for any route of administration, e.g oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular or intraperitoneal. The nature of the carrier or other ingredients will depend on the specific route of administration. (Examples of techniques and protocols that are useful in this context are inter alia found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A (ed.), 1980.)
- The dosage of these low molecular weight compounds will depend on the disease state or condition to be treted and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound For treating human or animals, between approximately 0.5 mg/Kg of body weight to 500 mg/Kg of body weight of the compound can be administered.
- The present compounds and methods are advantageously used in relation to all kind of angiogenesis related disorders and/or diseases, such as tumor conditions, diabetes, rheumatoid arthritis and even some inflammatory diseases, such as psoriasis, chronic, inflammations of the intestines, asthma etc. It is also suggested that they may be used in order to treat and cure, or prevent, obesity.
- In a particular embodiment, the present molecules are used in gene therapy. For a review of gene therapy procedures, see e.g. Anderson,Science; (1992) 256:808-813.
- A further advantageous use of the present invention is to develop methods of regulating the signalling of the present angiostatin and/or kringle 5 receptor in the body and therefore, the invention also relates to methods of treating a human or animal patient suffering from an angiogenesis related disease or disorder as well as to methods preventing such conditions.
- Below, the invention will be disclosed in more detail with reference to the drawings.
- All references in the present disclosure and above are hereby incorporated in the present application.
- Evidence that Angiostatin Binds to Endothelial Cells
- By competitive binding assays between angiostatin and the angiogenic factors, VEGF and bPGF, the present inventors have shown that angiostatin does not affect the ligand-receptor interaction of these molecules. The same level of binding could be detected in the presence or absence of unlabeled angiostatin. Thus, it can be ruled out that angiostatin acts by blocking angiogenic factors interaction with endothelial cells.
- The direct binding of angiostatin and its precursor plasminogen has been studied by binding assays using iodinated proteins and analyzing their interaction with bovine microcapillary endothelial cells, see FIG. 1.
- In particular, human angiostatin (cringle domains 1-4) or plasminogen was labeled with iodine 125 by the Iodogen method according to the protocol of the manufacturer (Pierce Inc.). The labelled protein was then purified on a G50 sepharose column (Pharmacia Inc.) The specific activity was estimated at 90 000 cpm/ng protein. For binding assays, bovine capillary endothelial cells were grown to confluency in 12 well plates. The cells were washed with PBS containing 1 mg/ml bovine serum albumine (BSA). The cells were then incubated with 10 ng/ml radio-labeled angiostatin or plasminogen. The binding was competed with increasing concentrations of unlabeled ligand. Cells were incubated on ice for 2 hours. Cells were then washed five times with PBS+1 mg/ml BSA. The cells were lysed with 1% Triton X100 in PBS and radioactivity was measured in a gamma counter. Dissociation constant and receptor numbers were estimated using the RBINDING program (van Zoelen EJ. Anal. Biochem. 1992 Feb. 1;200 (2):393-9).
- Identification of Angiostatin Bindine Molecules Using the Yeast Two Hybrid System
- Angiostatin retains activity even after reduction, suggesting that three dimensional folding is not vital for binding and activity. This favours screening procedures wherein a large number of clones may be screened although protein refolding may be less accurate.
- The yeast two hybrid system was employed to screen for molecules that bind kringle domains 1-4 of plasminogen. This is further disclosed below with reference to FIG. 2, Approximately 2×106 clones from a two hybrid cDNA library from human term placenta (Stratagene) were screened. Positives clones were identified in the following way:
- (1) Screening under selective conditions (His-, Leu- and Trp-) generated 37 positive clones in yeast strain CG1945.
- (2) Seven out of 37 clones displayed high β-galactosidase activity after incubation with ONPG (SIGMA) at 30° C. for 2h.
- (3) The DNA from the seven colonies that contained high β-gal activity was purified and retransfected into yeast strain Y190. Three out of the seven colonies retained activity in the new yeast strain. Sequencing analysis of these clones revealed that they were derived from the same gene.
- (4) Growth in the presence of 50 mM 3-aminotriazol (FIG. 3) as well as β-gal activity (see table below) was assessed in
Big 3 clones and compared to positive and negative controls.β-Gal Binding Domain Activation Domain Activity Angiostatin + Binding Domain Big3 − Activation Domain 15 u Angiostatin + Binding Domain Activation Domain alone <0.04 u Binding Domain alone Big3 − Activation Domain 0.07 u p53 + Binding Domain Big3 − Activation Domain 0.05 u p53 + Binding Domain SV40LT − Activation Domain 90 u - Sequence of Big 3 (Angiostatin-Binding Domain)
- After isolation of the big 3 clone from a placenta yeast two-hybrid library (Stratagene, Inc.), we directly sequenced the cDNA insert from the pGAD activation domain vector with GAL4 AD sequencing primers. The cDNA insert was recloned into pUC18 vector using EcoR1 sites from adaptors and sequencing was repeated using universal and reverse primers. Sequences were analyzed in an ALF automated sequencer (Pharmacia). The sequence of
Big 3 is shown in SEQ ID NO. 4. - Expression and Purification of Big3 and Big3-GST Fusion Protein
- Big3 sequence has been expressed inE. coli as fusion protein with the glutathione S-transferase (GST) domain from Schistosoma japonicum.
- Vector Construction:
- Big3 fragment (429 bp) was obtained by PCR amplification using pUC18-Big3 plasmid as template. Primer sequences were:
BamHI-NH2 5′ TAC GGA TCC GAA TCG AAC AAA ACT GCA GCT G 3′(SEQ ID NO. 5) XhoI- COOH 5′ ATA CTC GAG TCA TGG AGC TGG ACT TGG AGC CA 3′(SEQ ID NO. 6) - The cycling protocol used was:
94° C. 3′, 60° C. 1′, 72° C. 1′ 1 cycle 94° C. 30″, 60° C. 1′, 72° C. 2′ 30 cycles 94° C. 30″, 60° C. 1′, 72° C. 5′ 1 cycle - Taq polymerase: native Pfu DNA polymerase (Stratagene)
- PCR fragment was digested with BamHI and XhoI, purified and ligated to PGEX-6P-2 plasmid (Pharmacia Biotech) digested with the same restriction enzymes. Ligation mixture was used to transform XL1-Blue cells (Stratagene). Recombinant plasmids were verified by restriction analysis and automated sequencing.
- Expression and Purification:
- DH5α cells (Clontech) were transformed with one verified clone, named pGEX-Big3, and induced for 6 hours, room temperature with 0.1 mM IPTG.
- The fermentation broth was centrifuged at 4000×rpm 15 min and the cell pellet was resuspended in 10% w/v of lysis buffer (50 mM TRIS.HCl pH 8.0, 100 mM NaCl, 20 mM DTT, 1 mM EDTA, protease inhibitor mix), and lysed by sonication. The total lysate was centrifuged at 15000×g for 20 min at 4° C. The clarified supernatant was applied to a Glutathione-Sepharose column (1 ml for 20 ml of lysate) preequilibrated with lysis buffer. The resin was washed with 10 column volumes (CV) of lysis buffer and the fusion protein was eluted with 3 CV of elution buffer (100 mM TRIS.HCl pH 8.0 20 mM reduced glutathione).
- GST-Big3 protein can be cleaved by incubation of the glutathione-Sepharose bound fusion protein with 20 ul/ml resin of PreScission protease in PS buffer containing 50 mM TRIS.HCl pH 7.0. 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF.
- The eluted proteins behave like a single peak in rp-HPLC (10-90% gradient of acetonitrile in water plus 0.1% TFA), show the expected molecular size by mass analysis (electrospray) and the correct NH2 sequence. In SDS-PAGE a contaminant appears at the apparent MW of 80 kD; it was demonstrated to be DnaK, a bacterial chaperone, which can be eliminated running an ion exchange chromatography on a HiTrapQ column.
- The yields obtained from 1 litre of fermentation are about 10 man of GST-Big3 fusion protein and about 3 mg of the cleaved Big3.
- In vitro Binding of Recombinant GST-Tagged
Big 3 to Angiostatin - The protocol used for binding is described here below.
- One colony of pGEX-Big3 transformant was inoculated into 10 ml LB medium plus ampicillin and grown overnight at 37° C.
- The overnight culture was diluted 1:10 into 100 ml fresh medium and after 1 hour of incubation at 37° C., IPTG was added to a final concentration of 0.5 mM and incubation continued for 3 to 7 hours.
- The culture was then centrifuged at 5000 g for 10 minutes, the pellet was resuspended in 3-5 ml of ice-cold PBS and cells were lysed by sonication. Triton X100 was added to a final concentration of 0.5-1% and 1.5 ml aliquots of the suspension were centrifuged at 14000 rpm for 10 minutes.
- 0.1-0.3 ml of 50% slurry of glutathione-agarose beads were added to the supernatant and mixed for 3-5 hour at 4° C. The beads were then collected by centrifugation and washed 5 times with ice-old PBS and 2 times with binding solution (B.S.) consisting of: 50 mM Tris pH7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1 mM PMSF).
- Binding assay: 0.7 ml B.S., 0.1% calf serum, 500 ng angiostatin were mixed with 30-50 μl 50% slurry of Big3-immobilized glutathione-agarose beads for 1-3 hour at 4° C.
- The resin was washed 5-7 times with ice cold B.S., then used for Western blot analysis using anti-human plasminogen antibodies (Dako Inc.).
- FIG. 4 shows how purified
recombinant Big 3 binds angiostatin in vitro. Interestingly, binding of plasminogen could also be detected, but only after reduction of disulphide bonds by treating it with DTT. - Cloning of Full-Length Sequence
- FIG. 5 illustrates the scheme for cloning the full-length sequence of ABP-1. The whole gene was cloned by screening a placenta cDNA phage library with a Big3 probe (with repetitive sequences removed) together with 5′ RACE PCR (Gibco) using mRNA from human umbilical cord endothelial cells. The full sequence of the cDNA clone is disclosed in SEQ ID NO. 1, whereas the encoded protein is shown in SEQ ID NO. 2. Details of the experimental protocol are as follows.
- 10 million clones of a placenta lambda phage library (Stratagene, Inc.) were screened using a HinfI fragment of
Big 3 as probe. DNA isolation and Southern blot procedures were performed according to established protocols (Sambrook et al 1989). We isolated 5 clones (7-1, 7-2, 8-2, 9-3, 9-5) with sequences that overlap with Big3. The 5′-sequence of the 7-2 clone was used for designing Gene-Specific Primers (GSP) for 5′RACE PCR (Gibco Life technologies, Inc.). mRNA isolated from Human umbilical cord endothelial cells was used to identify 5′ sequences. - Primers used for the first RACE PCR:
Primers: GSP1- (5′ to 3′) GCTGACAGTTGCCCTGACGCTGCT (SEQ ID NO. 10) GSP2- (5′ to 3′) CGGAGACGGTGCTCTAGCTGCTCA (SEQ ID NO. 11) GSP3- (5′ to 3′) TCCTTCCAACTCTTGCCTCAAGTTCCG (SEQ ID NO. 12) - RACE procedures have been used for amplification and cloning unknown sequences between the GSP2 and GSP3 and the 5′-end of the mRNA ABP-1. This sequence was then used to design new primers for the next set of RACE RCR:
Primers: GSP1-(5′ to 3′) GGTGGCAGCGGACAGGCAGGATAC (SEQ ID NO. 13) GSP2 GAGGCGGAGAGAACTAAGAGAAGA (SEQ ID NO. 14) GSP3 GAGCGGAGATGGAGGAGTAATTCA (SEQ ID NO. 15) - Clones A2-2, A2-1 and A1-C with overlapping sequences were isolated. The A2-1 sequence was used as probe for the second screening of the placenta phage library. From this screening we isolated another 2 clones (7-10 and 6-5). As shown in FIG. 6, of the six possible frame the only one producing a complete open reading frame is
frame 2, yielding a putative protein of 675 amino acid residues. - mRNA Expression Pattern
- A commercially (Clontech, Inc.) obtained multiple human adult and fetal tissue (#7760-1 and #7756-1) mRNA blots (2 mg mRNA/well) was probed for ABP-1 The blots were hybridized with the ExpressHyb hybridization solution (Clontech, Inc.) according to the protocol of the manufacturer. The blots were probed with the 7-2 5′PstI fragment. FIG. 7 shows the results of the experiment; sizes are 9.5 and 7.5 kb.
- ABP-1 was also detected in human umbilical cord, bovine aortic and bovine microcapillary endothelial cells. Little or no expression was detected in the immortalized endothelial cell-line EaHy926 and in human fetal fibroblasts. Neither of the cell lines respond to angiostatin or exhibit any binding of FITC-labeled angiostatin.
- Real Time RT-PCR
- The 5′ nuclease assay (TaqMan assay) uses a nonextendable oligonucleotide hybridization probe (TaqMan Probe). The TaqMan probe consists of an oligonucleotide with a 5′-reporter dye and a 3′-quencher dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence, primarily by a Foster-type energy transfer. During PCR, forward and reverse primers hybridize to a specific sequence of the target DNA. The TaqMan probe hybridizes to a target sequence within the PCR product. The Taq Polymerase cleaves the TaqMan probe with its 5′-3′ nuclease activity. The reporter dye and quencher dye are separated upon cleavage, resulting in increased fluorescence of the reporter.
- Real Time RT-PCR Analysis
- RNA Preparation
- Isolation and purification of total RNA from tissues and cells listed below was performed using the Ultraspec RNA isolation system (Biotex).
HDMEC/2 h. dermal microvascular EC poly A+ A2780 h. ovarian carcinoma TOTAL A375 h. melanoma TOTAL HDF H. dermal fibroblasts TOTAL HELA h. cervix carcinoma TOTAL DU145 h. prostate carcinoma TOTAL HUVEC h. umbelical vein EC TOTAL ECV304 immortalized EC TOTAL CEM h. acutelymphobl.leuk. TOTAL K562 h. erythroleukemia TOTAL JURKAT h. acuteT-cellleukemia TOTAL THP-1 h. monocyte(acute leu) TOTAL EaHy926 h. dermal cells TOTAL S35/K9 human colon cancer TOTAL Brain human normal tissue TOTAL Colon human normal tissue TOTAL Prostate human normal tissue TOTAL Skel.Muscle human normal tissue TOTAL Uterus human normal tissue TOTAL Placenta human normal tissue TOTAL - cDNA Synthesis
- The RT reaction was performed using TaqMan Reverse Transcription Reagent (PE Applied Biosystems) with Random hexamers, as RT primers. The reaction volume for Reverse Transcription step was 100 μl and the total RNA amount was 1 μg for sample.
- The RT was performed using the following cycling parameter:
- 10′ at 25° C.
- 45′ at 48° C.
- 5′ at 95° C.
- PCR Reaction
- After a primer concentration optimization, the PCR reaction on 10 ng of the cDNA was performed using TaqMan Universal PCR Master LMix (PE Applied Biosystems) and the following oligonucleotides
- ABP-1 Forward Primer: 5′GTTTGACCTGCAATCCAGACAA3′ (SEQ ID NO. 7)
Final concentration 300 nM - ABP-1 Reverse Primer; 5′CCCAGGATCTGAATGGGAGTT3′ (SEQ ID NO. 8) Final concentration 900 nm
- ABP-1 TaqMan Probe: 5′ (FAM dye)-CAGATGGGCCTGTGTTCCACTCCAA-(TAA dye)3′ (SEQ ID NO. 9)
Final probe concentration 200 nM - The cycling protocols was:
- 2′ at 50° C.; 10′ at 95° C. 1 cycle
- 15″ at 95° C.; 1′ at 60° C. 40 cycles
- Relative Quantitation of Gene Expression
- The Comparative method use an arithmetic formula to achieve the result for relative quantitation without need for Standard curve.
- The result, depicted in FIG. 8, indicate how many fold the sample X expresses the target relative to the Calibrator which is the sample that shows the lowest level of expression of the target. In this experiment EaHY926 sample was chosen as calibrator.
- Antibodies Against Big-3
- For immunisation of New Zealand White rabbits, 100 micrograms of Big3-GST fusion protein were dissolved in 1 ml phosphate buffered saline (PBS) homogenised with 1 ml of Freund's Complete Adjuvant (Gibco). The resulting emulsion was injected subcutaneously on
day 0 and this treatment was repeated on days 15 and 28. Blood was removed from rabbits on day 35, allowed to coagulate overnight at 4° C., and the resulting serum stored at −20° C. - For purification of specific antibodies, resin-immobilised ligand was produced as follows: Big3 polypeptide was diluted in 0.1M sodium bicarbonate, 0.5 M NaCl, pH8.3, at a concentration of 5 mg/ml in a total volume of 2 ml. This was reacted for two hours at room temperature with 2 ml of cyanogen bromide-activated Sepaharose CL4B resin (Sigma Chemical Co. St Louis, Mo.). After reaction, the resin was washed three times in 10 volumes of 100 mM Tris, 500 mM NaCl, pH 7.5. Immune serum was incubated with the affinity resin for two hours at 4° C., after which resin was washed in a 5 ml glass chromatography column (Bio-Rad, Richmond Calif.) for with 25 volumes of 100 nm-M Tris, 500 mM NaCl, pH 7.5. Specific antibodies were eluted in 1 ml aliquots with 100 mM Glycine/HCl, pH 2.8. Elution of antibody was followed by monitoring the optical density at 280 nM (OD280), and fractions with an OD280 of greater than 1 were pooled and dialysed (Slide-A-lyzer cassettes, Pierce) for 36 hours at 4° C. against 1 liter PBS, with three changes of buffer.
- Binding and Signalling of Angiostatin via the ABP-1 Protein
- FIGS. 9A and 9B show data of binding of FITC-labeled angiostatin to ABP-1 transfected HeLa and EaHy926 cells transfected with the vector control. In particular FIG. 9A shows the cellular localization of ABP-1 fused to green fluorescent protein (GFP) in transiently transfected cells. The protein can be detected in the endoplasmic reticulum and the cell membrane. DABP-1 contains a 500 bp deletion in the 5′ end of the gene, which disrupts the previously described localization of ABP-1.
- FIG. 9B shows HeLa cells transfected with ABP-1 GFP. Incubation with 2.5 mg of angiostatin for 60 minutes at 0° C. and subsequent incubation for 15 minutes at 37° C. induces internalization of ABP-1-GFP.
- FIG. 9C shows the binding of angiostatin to ABP-1. We have shown that fluorescein isothiocyanate (FITC)-labelled angiostatin binds specifically to endothelial cells (data not shown). We have transfected HeLa cells with either the ABP-1 expression construct or the vector control (p RC/CMV, In Vitrogen, Inc.). We incubated live HeLa cells FITC-labelled angiostatin (10 ug/ml) in DMEM+10% fetal calf serum at 0° C. for 60 minutes. The cells were then incubated at 37° C. at 15 minutes to aggregate bound angiostatin. Binding was analyzed with a fluorescent microscope. Binding of angiostatin was detected in ABP-1 transfected cells but not in the vector control.
- FIG. 9D shows immunostaining of ABP-1 in Human umbilical cord endothelial (HUVE) cells together with staining against F-actin with rhodarmin-labelled phalloidin.
- Immunostaining Protocol:
- HUVE cells were fixed in 4% formaldehyde, washed in PBS and preblocked in 5% horse serum. The blocking solution was removed and replaced by rabbit polyclonal antibodies against the angiostatin-binding domain Big3 diluted in 5% horse serum. Positive staining was visualized with a fluorescence-labeled secondary antibody (Dako, Inc.). Rhodamin-conjugated phalloidin (Molecular Probes, Inc.) was stained simultaneously with the secondary antibody (after permeabilization with 1% triton X100 for 1 minute). Rabbit polyclonal antibodies against green fluorescent protein was used as a negative control. In addition no positive staining could be detected in human fibroblasts.
- As can be seen in FIG. 9D, ABP-1 is localized in focal adhesions and membrane ruffles (arrows).
- In Vitro Kinase Data
- Huve cells were plated subconfluent in 5 cm Petri dishes. Angiostatin was added at 2.5 mg/ml at different time points the following day. All plates including controls were harvested simultanously. The cells were rinsed in ice cold PBS and incubated in 1 ml lysis buffer. The cells were transferred to an eppendorf tube and centrifuged at 14 000 rpm for 5 minutes at 4° C. The supernatant was transferred to another eppendorf tube and 2 μg of
Big 3 rabbit polyclonal antibodies was added. The samples were incubated for 60 min. at 4° C. and subsequently 50 μl of a protein A sepharose (Pharmacia, Inc.) slurry was added and incubated for another 60 min. in a rotating incubator. The immunoprecipitates were collected by brief centrifugation and washed two times in lysis buffer, once in washing buffer and once in kinase buffer. Residual buffer was removed with a syringe. 25 μl of kinase buffer was added to each tube together with 1 μCi gammaATP (Amersham, Inc.). The samples were incubated at R.T. for 20 minutes. The reaction was stopped by adding 2×SDS PAGE sample buffer. The samples were boiled under denaturing conditions and subsequently analyzed by SDS PAGE. - The data illustrated in FIG. 10 show that addition of angiostatin downregulates ABP-1 associated kinase activity within 30 minutes. This is direct proof that angiostatin affects signalling pathways that are mediated by ABP-1.
- ABP-1 Mediates Angiostatin-Induced Focal Adhesion Kinase Activity.
- The data illustrated by FIG. 11 show that addition of 2.5 μg/ml of angiostatin upregulates FAK activity within 1 hour after addition.
- EaHy926 cells were plated subconfluent in 5 cm Petri dishes. Angiostatin was added at 2.5 μg/ml at different time points the following day. All plates including controls were harvested simultaneously. The cells were rinsed in ice cold PBS and incubated in 1 ml lysis buffer. The cells were transferred to an eppendorf tube and centrifuged at 14 000 rpm for 5 minutes at 4° C. The supernatant was transferred to another eppendorf tube and 1 ug of FAK monoclonal antibody (Transduction Lab. Inc.) was added. The samples were incubated for 60 min. at 4° C. and subsequently rabbit anti-mouse IgG+50 μl of a protein A sepharose (Pharmacia, Inc.) slurry was added and incubated for another 60 min. in a rotating incubator. The immunoprecipitates were collected by brief centrifugation and washed two times in lysis buffer, once in washing buffer and once in kinase buffer. Residual buffer was removed with a syringe. 25 μl of kinase buffer was added to each tube together with 1 μCi γATP (Amersham, Inc.). The samples were incubated at R.T. for 20 minutes. The reaction was stopped by adding 2×SDS PAGE sample buffer. The samples were boiled under denaturing conditions and subsequently analyzed by SDS PAGE.
- As can be seen in FIG. 11, addition of angiostatin upregulates FAK activity within 1 hour. It should be noted that EaHy926 cells are not expressing ABP1 as demonstrated by reverse transcriptase PCR analysis.
-
1 15 1 6463 DNA Homo sapiens CDS (797)..(2824) 1 ccaggagctg ccttggcagt cacgcccctt ccttccgagg agctttctgg ctgcctaaac 60 tggtagaccc cctgaattac tcctccatct ccgctctctt tcgcctcctc ttctcttagt 120 tctctccgcc tccccctcaa ctaccaccac ctccagtcag tctcgcctcc ggctatccgc 180 tgctccaccc tctggcccgg tatcctgcct gtccgctgcc accaaggaga gcccggacgg 240 agcagcgagg aggggagcag ccgggagttg gggcttcccc cctgcccatc cctggccgct 300 ggcccgggac cgaagccact tgagcgagca gagagtcgtc accttgtctt ctttgccttc 360 agggagctgc taagaaggac aaataagata gcagagtgaa agagcttttg tctccttaga 420 aggaaggctg agaaactaaa ggccagcgca ggacatctca ttgccattgt cagccaggaa 480 ctcgcagcct cacagcccta cttcttctct gacctctggg gggtccttgc ccttgctaca 540 atctccacca tccactagat tgtctcctgc ccgacacccc ttggtcccaa accagggaga 600 ccattcagct cacctgccta ggccgcagca gcatttcctt cctaatcagg ctcaccaggg 660 ggatcattac cgtctctccc aacctggcct gagtcagcag cagcagcaac agcagcagca 720 gcaccatcat caccatcacc accaacaaca gcagcagcag cagccacagc agcagccagg 780 agaagcctat tcagct atg cct cgg gct cag cca tcc tct gct tct tat cag 832 Met Pro Arg Ala Gln Pro Ser Ser Ala Ser Tyr Gln 1 5 10 cca gtg cca gca gac cct ttt gcc att gtt tcc aga gcc cag cag atg 880 Pro Val Pro Ala Asp Pro Phe Ala Ile Val Ser Arg Ala Gln Gln Met 15 20 25 gtt gag atc ctc tca gac gag aac cgg aac ttg agg caa gag ttg gaa 928 Val Glu Ile Leu Ser Asp Glu Asn Arg Asn Leu Arg Gln Glu Leu Glu 30 35 40 gga tgc tat gag aag gtg gca aga ctg cag aag gtg gag aca gaa atc 976 Gly Cys Tyr Glu Lys Val Ala Arg Leu Gln Lys Val Glu Thr Glu Ile 45 50 55 60 cag cgc gtc tcg gag gca tat gag aac ctc gtg aag tca tcc tcc aaa 1024 Gln Arg Val Ser Glu Ala Tyr Glu Asn Leu Val Lys Ser Ser Ser Lys 65 70 75 aga gag gcc cta gag aaa gcc atg aga aac aag cta gag ggc gag att 1072 Arg Glu Ala Leu Glu Lys Ala Met Arg Asn Lys Leu Glu Gly Glu Ile 80 85 90 cgg agg atg cat gat ttc aac agg gat ctg aga gag cgt cta gag act 1120 Arg Arg Met His Asp Phe Asn Arg Asp Leu Arg Glu Arg Leu Glu Thr 95 100 105 gcc aac aag cag ctt gca gag aag gaa tat gag ggg tca gag gac acc 1168 Ala Asn Lys Gln Leu Ala Glu Lys Glu Tyr Glu Gly Ser Glu Asp Thr 110 115 120 aga aaa acc atc tcg cag ctc ttt gca aaa aat aaa gaa agc cag cgt 1216 Arg Lys Thr Ile Ser Gln Leu Phe Ala Lys Asn Lys Glu Ser Gln Arg 125 130 135 140 gag aag gag aag ctg gaa gcg gag ctg gcc act gcc cgt tct acc aat 1264 Glu Lys Glu Lys Leu Glu Ala Glu Leu Ala Thr Ala Arg Ser Thr Asn 145 150 155 gag gac caa aga cga cac atc gaa atc cga gat cag gcc ctg agt aat 1312 Glu Asp Gln Arg Arg His Ile Glu Ile Arg Asp Gln Ala Leu Ser Asn 160 165 170 gcc cag gcc aag gtg gta aag ctg gaa gaa gag ctg aaa aag aag caa 1360 Ala Gln Ala Lys Val Val Lys Leu Glu Glu Glu Leu Lys Lys Lys Gln 175 180 185 gtg tac gtt gac aag gtg gag aag atg cag cag gcc ctt gta cag ctc 1408 Val Tyr Val Asp Lys Val Glu Lys Met Gln Gln Ala Leu Val Gln Leu 190 195 200 cag gca gca tgt gaa aaa cgt gag cag cta gag cac cgt ctc cgg aca 1456 Gln Ala Ala Cys Glu Lys Arg Glu Gln Leu Glu His Arg Leu Arg Thr 205 210 215 220 cga ctg gag agg gaa ctg gaa tcc ctg aga atc cag cag cgt cag ggc 1504 Arg Leu Glu Arg Glu Leu Glu Ser Leu Arg Ile Gln Gln Arg Gln Gly 225 230 235 aac tgt cag ccc acc aac gtt tca gaa tac aat gct gcc gca ctg atg 1552 Asn Cys Gln Pro Thr Asn Val Ser Glu Tyr Asn Ala Ala Ala Leu Met 240 245 250 gag ctc ctt cgg gag aaa gag gag agg att ctg gct ctg gaa gct gat 1600 Glu Leu Leu Arg Glu Lys Glu Glu Arg Ile Leu Ala Leu Glu Ala Asp 255 260 265 atg aca aag tgg gag cag aaa tat ttg gag gag aat gtg atg aga cat 1648 Met Thr Lys Trp Glu Gln Lys Tyr Leu Glu Glu Asn Val Met Arg His 270 275 280 ttt gct ctg gat gct gct gca act gtg gct gct cag agg gac aca aca 1696 Phe Ala Leu Asp Ala Ala Ala Thr Val Ala Ala Gln Arg Asp Thr Thr 285 290 295 300 gtc atc agt cac tct cct aac acc agc tat gac aca gct cta gaa gct 1744 Val Ile Ser His Ser Pro Asn Thr Ser Tyr Asp Thr Ala Leu Glu Ala 305 310 315 cgc atc cag aaa gag gag gaa gaa atc ttg atg gcc aat aag cgt tgc 1792 Arg Ile Gln Lys Glu Glu Glu Glu Ile Leu Met Ala Asn Lys Arg Cys 320 325 330 ctt gac atg gag ggc agg att aag acc ctc cat gcc cag att att gag 1840 Leu Asp Met Glu Gly Arg Ile Lys Thr Leu His Ala Gln Ile Ile Glu 335 340 345 aag gat gcc atg atc aaa gta ctc cag cag cgt tcc cgg aag gag ccg 1888 Lys Asp Ala Met Ile Lys Val Leu Gln Gln Arg Ser Arg Lys Glu Pro 350 355 360 agc aag aca gag cag ctg tcg tgc atg cgg cca gcg aag tct ctg atg 1936 Ser Lys Thr Glu Gln Leu Ser Cys Met Arg Pro Ala Lys Ser Leu Met 365 370 375 380 tcc att tcc aat gct gga tca ggc ttg ctc tcc cac tca tcc acc ctg 1984 Ser Ile Ser Asn Ala Gly Ser Gly Leu Leu Ser His Ser Ser Thr Leu 385 390 395 act ggc tcc ccc atc atg gaa gaa aag cga gac gac aag agc tgg aag 2032 Thr Gly Ser Pro Ile Met Glu Glu Lys Arg Asp Asp Lys Ser Trp Lys 400 405 410 ggg agc cta ggc att ctc ctg ggt gga gac tac cgt gct gaa tat gtc 2080 Gly Ser Leu Gly Ile Leu Leu Gly Gly Asp Tyr Arg Ala Glu Tyr Val 415 420 425 cct tcc aca ccc tcg cct gtg cca ccc tcg act ccc ctg ctc tcg gct 2128 Pro Ser Thr Pro Ser Pro Val Pro Pro Ser Thr Pro Leu Leu Ser Ala 430 435 440 cac tcc aag aca ggc agc cga gac tgc agt acc caa act gaa cgt ggg 2176 His Ser Lys Thr Gly Ser Arg Asp Cys Ser Thr Gln Thr Glu Arg Gly 445 450 455 460 acg gaa tcg aac aaa act gca gct gtt gct ccc atc tct gtt cct gct 2224 Thr Glu Ser Asn Lys Thr Ala Ala Val Ala Pro Ile Ser Val Pro Ala 465 470 475 cca gtt gct gct gcc gcc act gct gcc gcc atc act gcc act gct gcc 2272 Pro Val Ala Ala Ala Ala Thr Ala Ala Ala Ile Thr Ala Thr Ala Ala 480 485 490 acc atc acc acc acc atg gta gct gct gct cca gtt gct gtt gct gct 2320 Thr Ile Thr Thr Thr Met Val Ala Ala Ala Pro Val Ala Val Ala Ala 495 500 505 gct gct gct cca gct gct gct gct gcc ccg tct cca gcc act gcc gct 2368 Ala Ala Ala Pro Ala Ala Ala Ala Ala Pro Ser Pro Ala Thr Ala Ala 510 515 520 gct act gct gct gct gtt tct cca gct gct gct ggt cag att cca gct 2416 Ala Thr Ala Ala Ala Val Ser Pro Ala Ala Ala Gly Gln Ile Pro Ala 525 530 535 540 gct gcc tct gtt gcc tca gct gct gcc gtt gct cct tct gct gct gct 2464 Ala Ala Ser Val Ala Ser Ala Ala Ala Val Ala Pro Ser Ala Ala Ala 545 550 555 gct gct gct gtt cag gtt gct cca gct gct ccg gct cca gtt cca gct 2512 Ala Ala Ala Val Gln Val Ala Pro Ala Ala Pro Ala Pro Val Pro Ala 560 565 570 ccg gct ctg gtt ccg gtt cca gct cca gca gcg gct cag gct tct gct 2560 Pro Ala Leu Val Pro Val Pro Ala Pro Ala Ala Ala Gln Ala Ser Ala 575 580 585 cct gct cag act cag gca cca act tca gct ccg gct gtg gct cca act 2608 Pro Ala Gln Thr Gln Ala Pro Thr Ser Ala Pro Ala Val Ala Pro Thr 590 595 600 cca gct cca act cca act cca gct gtg gct cag gct gag gtt cct gca 2656 Pro Ala Pro Thr Pro Thr Pro Ala Val Ala Gln Ala Glu Val Pro Ala 605 610 615 620 agt cca gct acc ggt cct gga cca cat cgt ttg tct ata cca agt ttg 2704 Ser Pro Ala Thr Gly Pro Gly Pro His Arg Leu Ser Ile Pro Ser Leu 625 630 635 acc tgc aat cca gac aaa aca gat ggg cct gtg ttc cac tcc aat act 2752 Thr Cys Asn Pro Asp Lys Thr Asp Gly Pro Val Phe His Ser Asn Thr 640 645 650 ctg gaa aga aaa act ccc att cag atc ctg gga caa gag cct gat gca 2800 Leu Glu Arg Lys Thr Pro Ile Gln Ile Leu Gly Gln Glu Pro Asp Ala 655 660 665 gag atg gtg gaa tat ctc atc taa acggccaaat caagagctgc agattatcag 2854 Glu Met Val Glu Tyr Leu Ile 670 675 caaaaatgct tttaatcatt ttcccccttt tattggttct tgttttgagg gtgaggacaa 2914 gggttgtggg gaggggatgt tttttaacag gactttttat tggaacaatg tactacttga 2974 gtaataccat gtgaacacca gtctattttg gtatgcttag ggagtacctc taaagacaga 3034 ttaatcagaa tgtgctctaa agcttattgt ttgaatttat acgaatactg ggactgttaa 3094 caggtggcta tacatcgacg ttttcaatgt gcttaaattt gtttaaattt tccatattct 3154 agatcatttt ttattgaaga gcacagtatg tgtggaagac agtgtataac acgtagtttg 3214 gaagtgggaa gctagagaga attgagtgtg tgctgttttg tatagttact atcctgtgca 3274 gcagctggag aaagcactca cctcaggctt acaaaaggga atagtttcag gagctatgta 3334 agctggaaaa aaggtaggga gttttggggt gcagaagggt actggagcta attttttctt 3394 ccagtttccc agctaccctg ccccagggaa ttgtgtttgt cttcatttca gtggtgcttt 3454 ggaaatggat tcttttggtt ccctcctgga ggttcataca ttcatatata tgctctggag 3514 taatttatgc atttggataa ttaatatatt gctttcagat gctgggagag tacattaact 3574 gagtgatgcg caacttcctc tctcttaggg aattagacca tcagaggcct tgatggagag 3634 ttgcatgggg tgctatatgc agacttccat ggtttgtgtg tagccatgaa cacagcttgc 3694 ttgcatttag taagaccaat cagcttagtg tttatttctt ctacagcaca gattcactgg 3754 ctgggtctcc agtctcaaat tgccaatcat ttgcaaagtg aggaaggatc tttgttgaca 3814 ggttgaatgc tttgaatttc tggtgactac tttgaaataa cttgttttgt ttgtcaaatt 3874 ctaagcatat gtcttaaaag gcatttttga ctatcacctc caagggaata gcttgagaaa 3934 cccaaagtac tatgctgcag tcgggggaga ggtggattgc agcagtatcc tcaactacct 3994 cttctcactg tcagtgacac catcttggaa tacctttggg aagcagcagg aaatgtgcat 4054 gtgggtagag atcaaaggag gcaatggctc caagccttgc catagggctg cctccaagga 4114 cacagaagga tgccagttgc cacaggtccc tgccctgtgt cacctgtctg cccttcatta 4174 aggtgagaaa tctgcagata gcatcattaa gatcagtttt aaggggtata gggagggtga 4234 gggaagtggg gggtgttagg taagggttgg gggtagaggt tttgggatgt cttagttaga 4294 aaccagatta atagaagagt aggcctgata tattacatca tgagccatag tggtgggaaa 4354 gaactttagc aatatagccc tacctcctca ttttagtgat gaggaatctg agaactggag 4414 aggttcagtg actttttgaa agtcatacaa cacagctaac cattatgcca atcaccatgc 4474 ttattttggg aaactcttta tcttttttaa attccatttt atgaaaaggc atcttcatgg 4534 tccagggaat atgtatcttg taaaatgtac ctggttggag tagcttgtcc agtcttgaca 4594 aactactgaa tttctgtctt gcctctcctt cagtgccttt taaaaggttt tcccttttct 4654 gatctgcatt tcaacataga gtcacataaa tgtccccctg agaaaccaat cccacttctt 4714 tctaggagat tgggtatctt agataatctt ttggggttcc tctgtgagta taggaatggt 4774 atccttccta attatcttcc aaaggaatta ttttgtgtgt gtgcctgtgt gtgtgtagag 4834 acataaagga gggtgatgtg attttcagct agtcctttca cattttcaat aatgaggtaa 4894 tcatgttaca tacacattag tcctcagtta taaagtgaat ctcagataga aattaaaagt 4954 gcagttgtgt taagactctt tcatactacc ctttagtcat aaggagaaaa aaacactcaa 5014 atagtagaag cagcaagtag caaacttcag gagagctact ttctatccaa ataatttaaa 5074 aaacactttt cacctactcc tttcatggtt ataacacatt ggcagacttt ttgctggctc 5134 tgggagccat gattttaatc acattctgca aggtgacaaa tgtcatacat tccacattgt 5194 gtggtagcca tctctttaga ctcatgtgtt ttggggaaag gaagaagttc ttggctgagt 5254 actattttga actttccaga accctctcac accagagaca gttcttctct gttcagtttc 5314 caatccccga taatttgcta aaataacatt gtacatccaa gagagggaag aagagtatgt 5374 cagtatatta tgcagaagat agatacagcc ttttcagaag atctccacta gtttttgttc 5434 caaaaattca agtttatggg agaaatctca attagccacc ttttcacagt tgtgtggata 5494 taacatttgg gggatctttc tggactccta cctatctgtg cattttaccg gcacctcagg 5554 aaaggagggt gaccaggttg tcttagcttg tactgcttgg tgatctctga ggaccttcta 5614 attcagttgt accccagtgt tccatgtata gaaaaacttc attagaacaa actttacttg 5674 atatgaaact cctattaaca gtcttttttt gaaataaaaa gtagcttgag ctttctttta 5734 aaatcatgta tcttgattgt tgatttaatg aaggatttcc ttttaatgct gcttttgagc 5794 ttcaaggtaa taggacagca ggaacctaaa atatctgcca tcatctgcca taggaaagat 5854 acccagagac ccatcatgtt ctctttttgt tgttacactg ttgggtgggt ataacaattg 5914 gaaaatgaac aaactgattg attgtgcaaa ctacttttta tgacaagcct aaaccctcat 5974 aatgcggcag cttaaagtgt atacatatgc actaactttg atcaattata ttctcatatc 6034 tgttagctac acagtctcct attatctcaa ttgcttatgt gcatatggaa tatgttactt 6094 aaaacgtgtg cattcttact gaaaatgttt tcaaaggaag gtatcagctg tgggctaatt 6154 gccaccaatt tcagcctgcc acgattcttg gaaatatgtc ttccaagtgc catccatcat 6214 cagtaggaca agtgtcggga gtttgtttat ttttttccag tagcaacgat gggttacatg 6274 gagccatgaa acctccttct ggcctccctt gtgattaatg gcatgtgttt gtaaaatgga 6334 tagctggggt tggcagatgg ctagagaaga atcgcctttg gtttaaaatg tatgtggtcc 6394 cctaatgatt gtgaccccat tctgtaatca actgagctag ttccaataaa gttaagcagg 6454 tttaaatcc 6463 2 675 PRT Homo sapiens 2 Met Pro Arg Ala Gln Pro Ser Ser Ala Ser Tyr Gln Pro Val Pro Ala 1 5 10 15 Asp Pro Phe Ala Ile Val Ser Arg Ala Gln Gln Met Val Glu Ile Leu 20 25 30 Ser Asp Glu Asn Arg Asn Leu Arg Gln Glu Leu Glu Gly Cys Tyr Glu 35 40 45 Lys Val Ala Arg Leu Gln Lys Val Glu Thr Glu Ile Gln Arg Val Ser 50 55 60 Glu Ala Tyr Glu Asn Leu Val Lys Ser Ser Ser Lys Arg Glu Ala Leu 65 70 75 80 Glu Lys Ala Met Arg Asn Lys Leu Glu Gly Glu Ile Arg Arg Met His 85 90 95 Asp Phe Asn Arg Asp Leu Arg Glu Arg Leu Glu Thr Ala Asn Lys Gln 100 105 110 Leu Ala Glu Lys Glu Tyr Glu Gly Ser Glu Asp Thr Arg Lys Thr Ile 115 120 125 Ser Gln Leu Phe Ala Lys Asn Lys Glu Ser Gln Arg Glu Lys Glu Lys 130 135 140 Leu Glu Ala Glu Leu Ala Thr Ala Arg Ser Thr Asn Glu Asp Gln Arg 145 150 155 160 Arg His Ile Glu Ile Arg Asp Gln Ala Leu Ser Asn Ala Gln Ala Lys 165 170 175 Val Val Lys Leu Glu Glu Glu Leu Lys Lys Lys Gln Val Tyr Val Asp 180 185 190 Lys Val Glu Lys Met Gln Gln Ala Leu Val Gln Leu Gln Ala Ala Cys 195 200 205 Glu Lys Arg Glu Gln Leu Glu His Arg Leu Arg Thr Arg Leu Glu Arg 210 215 220 Glu Leu Glu Ser Leu Arg Ile Gln Gln Arg Gln Gly Asn Cys Gln Pro 225 230 235 240 Thr Asn Val Ser Glu Tyr Asn Ala Ala Ala Leu Met Glu Leu Leu Arg 245 250 255 Glu Lys Glu Glu Arg Ile Leu Ala Leu Glu Ala Asp Met Thr Lys Trp 260 265 270 Glu Gln Lys Tyr Leu Glu Glu Asn Val Met Arg His Phe Ala Leu Asp 275 280 285 Ala Ala Ala Thr Val Ala Ala Gln Arg Asp Thr Thr Val Ile Ser His 290 295 300 Ser Pro Asn Thr Ser Tyr Asp Thr Ala Leu Glu Ala Arg Ile Gln Lys 305 310 315 320 Glu Glu Glu Glu Ile Leu Met Ala Asn Lys Arg Cys Leu Asp Met Glu 325 330 335 Gly Arg Ile Lys Thr Leu His Ala Gln Ile Ile Glu Lys Asp Ala Met 340 345 350 Ile Lys Val Leu Gln Gln Arg Ser Arg Lys Glu Pro Ser Lys Thr Glu 355 360 365 Gln Leu Ser Cys Met Arg Pro Ala Lys Ser Leu Met Ser Ile Ser Asn 370 375 380 Ala Gly Ser Gly Leu Leu Ser His Ser Ser Thr Leu Thr Gly Ser Pro 385 390 395 400 Ile Met Glu Glu Lys Arg Asp Asp Lys Ser Trp Lys Gly Ser Leu Gly 405 410 415 Ile Leu Leu Gly Gly Asp Tyr Arg Ala Glu Tyr Val Pro Ser Thr Pro 420 425 430 Ser Pro Val Pro Pro Ser Thr Pro Leu Leu Ser Ala His Ser Lys Thr 435 440 445 Gly Ser Arg Asp Cys Ser Thr Gln Thr Glu Arg Gly Thr Glu Ser Asn 450 455 460 Lys Thr Ala Ala Val Ala Pro Ile Ser Val Pro Ala Pro Val Ala Ala 465 470 475 480 Ala Ala Thr Ala Ala Ala Ile Thr Ala Thr Ala Ala Thr Ile Thr Thr 485 490 495 Thr Met Val Ala Ala Ala Pro Val Ala Val Ala Ala Ala Ala Ala Pro 500 505 510 Ala Ala Ala Ala Ala Pro Ser Pro Ala Thr Ala Ala Ala Thr Ala Ala 515 520 525 Ala Val Ser Pro Ala Ala Ala Gly Gln Ile Pro Ala Ala Ala Ser Val 530 535 540 Ala Ser Ala Ala Ala Val Ala Pro Ser Ala Ala Ala Ala Ala Ala Val 545 550 555 560 Gln Val Ala Pro Ala Ala Pro Ala Pro Val Pro Ala Pro Ala Leu Val 565 570 575 Pro Val Pro Ala Pro Ala Ala Ala Gln Ala Ser Ala Pro Ala Gln Thr 580 585 590 Gln Ala Pro Thr Ser Ala Pro Ala Val Ala Pro Thr Pro Ala Pro Thr 595 600 605 Pro Thr Pro Ala Val Ala Gln Ala Glu Val Pro Ala Ser Pro Ala Thr 610 615 620 Gly Pro Gly Pro His Arg Leu Ser Ile Pro Ser Leu Thr Cys Asn Pro 625 630 635 640 Asp Lys Thr Asp Gly Pro Val Phe His Ser Asn Thr Leu Glu Arg Lys 645 650 655 Thr Pro Ile Gln Ile Leu Gly Gln Glu Pro Asp Ala Glu Met Val Glu 660 665 670 Tyr Leu Ile 675 3 675 PRT Homo sapiens VARIANT (135)..(135) Residue 135 = Asn, Ser or Asp 3 Met Pro Arg Ala Gln Pro Ser Ser Ala Ser Tyr Gln Pro Val Pro Ala 1 5 10 15 Asp Pro Phe Ala Ile Val Ser Arg Ala Gln Gln Met Val Glu Ile Leu 20 25 30 Ser Asp Glu Asn Arg Asn Leu Arg Gln Glu Leu Glu Gly Cys Tyr Glu 35 40 45 Lys Val Ala Arg Leu Gln Lys Val Glu Thr Glu Ile Gln Arg Val Ser 50 55 60 Glu Ala Tyr Glu Asn Leu Val Lys Ser Ser Ser Lys Arg Glu Ala Leu 65 70 75 80 Glu Lys Ala Met Arg Asn Lys Leu Glu Gly Glu Ile Arg Arg Met His 85 90 95 Asp Phe Asn Arg Asp Leu Arg Glu Arg Leu Glu Thr Ala Asn Lys Gln 100 105 110 Leu Ala Glu Lys Glu Tyr Glu Gly Ser Glu Asp Thr Arg Lys Thr Ile 115 120 125 Ser Gln Leu Phe Ala Lys Xaa Lys Glu Ser Gln Arg Glu Lys Glu Lys 130 135 140 Leu Glu Ala Xaa Xaa Xaa Thr Ala Arg Ser Thr Asn Glu Asp Gln Arg 145 150 155 160 Arg His Ile Glu Ile Arg Asp Gln Ala Leu Ser Asn Ala Gln Ala Lys 165 170 175 Val Val Lys Leu Glu Glu Glu Leu Lys Lys Lys Gln Val Tyr Val Asp 180 185 190 Lys Val Glu Lys Met Gln Gln Ala Leu Val Gln Leu Gln Ala Ala Cys 195 200 205 Glu Lys Arg Glu Gln Leu Glu His Arg Leu Arg Thr Arg Leu Glu Arg 210 215 220 Glu Leu Glu Ser Leu Arg Ile Gln Gln Arg Gln Gly Asn Cys Gln Pro 225 230 235 240 Thr Asn Val Ser Glu Tyr Asn Ala Ala Ala Leu Met Glu Leu Leu Arg 245 250 255 Glu Lys Glu Glu Arg Ile Leu Ala Leu Glu Ala Asp Met Thr Lys Trp 260 265 270 Glu Gln Lys Tyr Leu Glu Glu Asn Val Met Arg His Phe Ala Leu Asp 275 280 285 Ala Ala Ala Thr Val Ala Ala Gln Arg Asp Thr Thr Val Ile Ser His 290 295 300 Ser Pro Asn Thr Ser Tyr Asp Thr Ala Leu Glu Ala Arg Ile Gln Lys 305 310 315 320 Glu Glu Glu Glu Ile Leu Met Ala Asn Lys Arg Cys Leu Asp Met Glu 325 330 335 Gly Arg Ile Lys Thr Leu His Ala Gln Ile Ile Glu Lys Asp Ala Met 340 345 350 Ile Lys Val Leu Gln Gln Arg Ser Arg Lys Glu Pro Ser Lys Thr Glu 355 360 365 Gln Leu Ser Cys Met Arg Pro Ala Lys Ser Leu Met Ser Ile Ser Asn 370 375 380 Ala Gly Ser Gly Leu Leu Ser His Ser Ser Thr Leu Thr Gly Ser Pro 385 390 395 400 Ile Met Glu Glu Lys Arg Asp Asp Lys Ser Trp Lys Gly Ser Leu Gly 405 410 415 Ile Leu Leu Gly Gly Asp Tyr Arg Ala Glu Tyr Val Pro Ser Thr Pro 420 425 430 Ser Pro Val Pro Pro Ser Thr Pro Leu Leu Ser Ala His Ser Lys Thr 435 440 445 Gly Ser Arg Asp Cys Ser Thr Gln Thr Glu Arg Gly Thr Glu Ser Asn 450 455 460 Lys Thr Ala Ala Val Ala Pro Ile Ser Val Pro Ala Pro Val Ala Ala 465 470 475 480 Ala Ala Thr Ala Ala Ala Ile Thr Ala Thr Ala Ala Thr Ile Thr Thr 485 490 495 Thr Met Val Ala Ala Ala Pro Val Ala Val Ala Ala Ala Ala Ala Pro 500 505 510 Ala Ala Ala Ala Ala Pro Ser Pro Ala Thr Ala Ala Ala Thr Ala Ala 515 520 525 Ala Val Ser Pro Ala Ala Ala Gly Gln Ile Pro Ala Ala Ala Ser Val 530 535 540 Ala Ser Ala Ala Ala Val Ala Pro Ser Ala Ala Ala Ala Ala Ala Val 545 550 555 560 Gln Val Ala Pro Ala Ala Pro Ala Pro Val Pro Ala Pro Ala Leu Val 565 570 575 Pro Val Pro Ala Pro Ala Ala Ala Gln Ala Ser Ala Pro Ala Gln Thr 580 585 590 Gln Ala Pro Thr Ser Ala Pro Ala Val Ala Pro Thr Pro Ala Pro Thr 595 600 605 Pro Thr Pro Ala Val Ala Gln Ala Glu Val Pro Ala Ser Pro Ala Thr 610 615 620 Gly Pro Gly Pro His Arg Leu Ser Ile Pro Ser Leu Thr Cys Asn Pro 625 630 635 640 Asp Lys Thr Asp Gly Pro Val Phe His Ser Asn Thr Leu Glu Arg Lys 645 650 655 Thr Pro Ile Gln Ile Leu Gly Gln Glu Pro Asp Ala Glu Met Val Glu 660 665 670 Tyr Leu Ile 675 4 143 PRT Homo sapiens 4 Glu Ser Asn Lys Thr Ala Ala Val Ala Pro Ile Ser Val Pro Ala Pro 1 5 10 15 Val Ala Ala Ala Ala Thr Ala Ala Ala Ile Thr Ala Thr Ala Ala Thr 20 25 30 Ile Thr Thr Thr Met Val Ala Ala Ala Pro Val Ala Val Ala Ala Ala 35 40 45 Ala Ala Pro Ala Ala Ala Ala Ala Pro Ser Pro Ala Thr Ala Ala Ala 50 55 60 Thr Ala Ala Ala Val Ser Pro Ala Ala Ala Gly Gln Ile Pro Ala Ala 65 70 75 80 Ala Ser Val Ala Ser Ala Ala Ala Val Ala Pro Ser Ala Ala Ala Ala 85 90 95 Ala Ala Val Gln Val Ala Pro Ala Ala Pro Ala Pro Val Pro Ala Pro 100 105 110 Ala Leu Val Pro Val Pro Ala Pro Ala Ala Ala Gln Ala Ser Ala Pro 115 120 125 Ala Gln Thr Gln Ala Pro Thr Ser Ala Pro Ala Val Ala Pro Thr 130 135 140 5 31 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for PCR reaction 5 tacggatccg aatcgaacaa aactgcagct g 31 6 32 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for PCR reaction 6 atactcgagt catggagctg gagttggagc ca 32 7 22 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for PCR reaction 7 gtttgacctg caatccagac aa 22 8 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for PCR reaction 8 cccaggatct gaatgggagt t 21 9 25 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for PCR reaction 9 cagatgggcc tgtgttccac tccaa 25 10 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for RACE PCR reaction 10 gctgacagtt gccctgacgc tgct 24 11 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for RACE PCR reaction 11 cggagacggt gctctagctg ctca 24 12 27 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for RACE PCR reaction 12 tccttccaac tcttgcctca agttccg 27 13 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for RACE PCR reaction 13 ggtggcagcg gacaggcagg atac 24 14 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for RACE PCR reaction 14 gaggcggaga gaactaagag aaga 24 15 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide primer for RACE PCR reaction 15 gagcggagat ggaggagtaa ttca 24
Claims (18)
1. An antibody or antibody fragment which specifically binds an isolated mammalian protein having anti-angiogenic activity and that is a receptor for an N-terminal fragment of plasminogen comprising kringle domains 1-4 and/or 5 wherein said protein does not cleave plasminogen kringle domains and wherein said protein comprises an amino acid sequence having 80% sequence homology or greater to SEQ ID NO: 4.
2. The antibody or antibody fragment according to claim 1 , wherein said protein comprises an amino acid sequence wherein said sequence has 80% sequence homology or greater to SEQ ID No: 4 and has sequence homology equal to or greater than 80% to SEQ ID Nos: 2 or 3.
3. The antibody or antibody fragment according to claim 2 , wherein said protein comprises the amino acid sequence of SEQ ID NO. 2.
4. The antibody or antibody fragment according to claim 2 , wherein said protein comprises the amino acid sequence of SEQ ID NO. 3, and wherein the amino acid residue in position 135 is Asn, Ser or Asp and the three amino acid residues in positions 148 to 150 are the tripeptide Glu-Leu-Ala or the tripeptide Thr-Trp-Pro.
5. The antibody or antibody fragment according to claim 1 , wherein said protein comprises the amino acid sequence of SEQ ID NO. 4.
6. An antibody or antibody fragment to a peptide capable of binding an N-terminal fragment of plasminogen and which has an amino acid sequence comprising at least 10 contiguous amino acid residues of SEQ ID NO. 2.
7. The antibody according to claim 1 which is a monoclonal antibody or fragment thereof.
8. The antibody according to claim 2 which is a monoclonal antibody or fragment thereof.
9. The antibody according to claim 3 which is a monoclonal antibody or fragment thereof.
10. The antibody according to claim 4 which is a monoclonal antibody or fragment thereof.
11. The antibody according to claim 5 which is a monoclonal antibody or fragment thereof.
12. The antibody according to claim 6 which is a monoclonal antibody or fragment thereof.
13. A recombinant cell expressing the antibody according to claim 1 .
14. A recombinant cell expressing the antibody according to claim 2
15. A recombinant cell expressing the antibody according to claim 3 .
16. A recombinant cell expressing the antibody according to claim 4 .
17. A recombinant cell expressing the antibody according to claim 5 .
18. A recombinant cell expressing the antibody according to claim 6.
Priority Applications (1)
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US10/720,273 US20040141978A1 (en) | 1998-06-15 | 2003-11-25 | Angiogenesis related molecules |
Applications Claiming Priority (4)
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US8926698P | 1998-06-15 | 1998-06-15 | |
US11438698P | 1998-12-29 | 1998-12-29 | |
US09/332,063 US6908898B1 (en) | 1998-06-15 | 1999-06-14 | Angiogenesis related molecules |
US10/720,273 US20040141978A1 (en) | 1998-06-15 | 2003-11-25 | Angiogenesis related molecules |
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US09/332,063 Division US6908898B1 (en) | 1998-06-15 | 1999-06-14 | Angiogenesis related molecules |
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US20040141978A1 true US20040141978A1 (en) | 2004-07-22 |
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US09/332,063 Expired - Fee Related US6908898B1 (en) | 1998-06-15 | 1999-06-14 | Angiogenesis related molecules |
US10/720,273 Abandoned US20040141978A1 (en) | 1998-06-15 | 2003-11-25 | Angiogenesis related molecules |
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US09/332,063 Expired - Fee Related US6908898B1 (en) | 1998-06-15 | 1999-06-14 | Angiogenesis related molecules |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5185438A (en) * | 1991-04-02 | 1993-02-09 | The Trustees Of Princeton University | Nucleic acids encoding hencatoporetic stem cell receptor flk-2 |
US5679350A (en) * | 1992-05-28 | 1997-10-21 | The University Of Toledo | Method of delivery of a medicament to a cancer cell using a pathway of plasminogen activator material |
US5916572A (en) * | 1995-11-14 | 1999-06-29 | Corixa Corporation | Compounds and methods for the detection and prevention of T. cruzi infection |
US6500613B1 (en) * | 1995-09-15 | 2002-12-31 | University Of Alabama At Birmingham | Pneumococcal surface proteins and uses thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL8501682A (en) | 1985-06-11 | 1987-01-02 | Tno | NEW, BLOOD ISOLATED PROTEIN, PROCESS FOR PREPARING THIS PROTEIN, ANTI-BODIES ANTI-BODIES AND PHARMACEUTICAL PREPARATIONS CONTAINING THE PROTEIN OR ANTIBODIES. |
-
1999
- 1999-06-14 US US09/332,063 patent/US6908898B1/en not_active Expired - Fee Related
-
2003
- 2003-11-25 US US10/720,273 patent/US20040141978A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5185438A (en) * | 1991-04-02 | 1993-02-09 | The Trustees Of Princeton University | Nucleic acids encoding hencatoporetic stem cell receptor flk-2 |
US5679350A (en) * | 1992-05-28 | 1997-10-21 | The University Of Toledo | Method of delivery of a medicament to a cancer cell using a pathway of plasminogen activator material |
US6500613B1 (en) * | 1995-09-15 | 2002-12-31 | University Of Alabama At Birmingham | Pneumococcal surface proteins and uses thereof |
US5916572A (en) * | 1995-11-14 | 1999-06-29 | Corixa Corporation | Compounds and methods for the detection and prevention of T. cruzi infection |
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US6908898B1 (en) | 2005-06-21 |
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