Abrogen polypeptides, nucleic acids encoding them and methods for using them to inhibit angiogenesis

Abstract

The present invention relates to novel nucleic acids encoding novel amino acid fragments of polypeptides, called abrogens. The present invention also relates to novel, potent in vitro and in vivo inhibitors of endothelial cell proliferation, and compositions of them and their use. The present invention further provides methods for modulating angiogenesis and/or inhibiting unwanted angiogenesis. Polypeptides according to the present invention are useful for developing cell growth-modulating compositions and methods and for treating and/or preventing cancer, tumor growth, or other angiogenic dependent or angiogenesis associated diseases.

Description

FIELD OF THE INVENTION AND INTRODUCTION

[0002] The present invention relates to novel nucleic acids encoding novel amino acid fragments of polypeptides, called abrogens. The present invention also relates to novel, potent in vitro and in vivo inhibitors of endothelial cell proliferation, and compositions of them and uses of them. The present invention further provides methods effective for modulating angiogenesis and/or inhibiting unwanted angiogenesis. Polypeptides according to the present invention are useful for treating and/or preventing cancer, tumor growth, or other angiogenic dependent or angiogenesis associated diseases.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis is the generation of new blood vessels from preexisting vessels into a tissue or organ. Angiogenesis is required and observed under normal physiological conditions, such as in wound healing, fetal and embryonic development, female reproduction, i.e., formation of the corpus luteum, endometrium and placenta, organ formation, and tissue regeneration and remodeling (Risau W et al., Nature, 1997, 386, 671-674).

[0004] Angiogenesis begins with local degradation of the basement membrane of capillaries followed by invasion of stroma by underlying endothelial cells in the direction of an angiogenic stimulus. Subsequent to migration, endothelial cells proliferate at the leading edge of a migrating column and then organize to form new capillary tubes.

[0005] Persistent, unregulated angiogenesis occurs in a multiplicity of pathological conditions, tumor metastasis and abnormal growth by endothelial cells, and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases. Outgrowth of new blood vessels under pathological conditions can lead to the development and progression of diseases such as tumor growth, diabetic retinopathy, tissue and organ malformation, obesity, macular degeneration, rheumatoid arthritis, psoriasis, and cardiovascular disorders.

[0006] Several studies have produced direct and indirect evidence that tumor growth and metastasis are angiogenesis-dependent (Brooks et al., Cell, 1994, 79, 1154-1164; Kim KJ et al., Nature, 1993, 362, 841-844). Expansion of the tumor volume requires the induction of new capillary blood vessels. Tumor cells promote angiogenesis by the secretion of angiogenic factors, in particular basic fibroblast growth factor (bFGF) (Kandel J. et al., Cell, 1991, 66, 1095-1104) and vascular endothelial growth factor (VEGF) (Ferrara et al., Endocr. Rev., 1997, 18: 4-25). Tumors may produce one or more of these angiogenic peptides that can synergistically stimulate tumor angiogenesis (Mustonen et al., J Cell Biol., 1995, 129, 865-898). Therefore, expression or administration of anti-angiogenic factors, by gene therapy, for instance, should counteract the tumor-induced angiogenesis.

[0007] Various anti-angiogenic polypeptides have been discussed and used to treat human angiogenic dependent or angiogenic associated diseases. For example, angiostatin and endostatin are proteolytic fragments of plasminogen (Pgn) and collagen XVIII, respectively (O'Reilly et al., Cell, 1994, 79:315-328; O'Reilly et al., Cell, 1997, 88:1-20). Angiostatin contains the first four disulfide-linked structures of plasminogen, which are known as kringle structures, and which display differential effects on the suppression of the endothelial cell growth. For example, kringle 1 was shown to exhibit some inhibitory activity, while kringle 4 is an ineffective fragment. Hua L et al., (BBRC, 1999, 258 :668-673) has characterized another kringle structure within plasminogen but outside of angiostatin, e.g., kringle 5. The kringle 5 was shown to inhibit endothelial cell proliferation and migration. Also, Renhai et al. (PNAS, 1999, Vol. 96, No. 10, pp. 5728-5733) has demonstrated a synergistic effect on endothelial inhibition when angiostatin and kringle 5 were coincubated with capillary endothelial cells. It was, however, stated that such association did not completely arrest tumor growth or tumors at a dormant stage.

[0008] The prothrombin kringle-2 domain, which is a fragment released from prothrombin by factor Xa cleavage, was identified as having anti-endothelial cell proliferative activity by Lee TH et al. (JBC, 1998, vol 273, No. 44, pp. 25505-25512) using in vitro angiogenesis assay system with bovine capillary endothelial (BCE) cell proliferation. The prothrombin kringle-2 domain was, however, described as having endothelial cell suppression activities comparable with those of angiostatin.

[0009] An amino terminal portion of the urokinase plasminogen activator uPA, termed ATF, has also been disclosed (Li et al., Hum Gen Ther 10: 3045-53, 1999; Griscelli et al., Hum. Gen. Ther., 1999, Vol 10, No. 18, pp. 3045-53) as inhibiting angiogenesis. uPA is composed of three domains, a serine proteinase domain, a kringle domain, and a growth-factor-like domain. The urokinase plasminogen binds to its receptor (uPAR) by its growth-factor-like domain, and initiates a proteolytic cascade at the surface of migrating cells to stimulate intracellular signaling responsible for cell migration and proliferation. The uPA lacking the growth-factor-like domain was, however, unable to associate with uPAR and was rapidly cleared from the cell surface (Poliakov et al., Biochem J., 2001, 355:639-45).

[0010] Binding of uPA to its receptor greatly potentiates plasminogen/plasmin conversion at the cell surface. Plasmin is a broadly specific serine protease, which can directly degrade components of the extracellular matrix. uPA and plasmin are somehow involved in cell morphogenesis by activating or inducing the release of morphogenic factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), or fibroblast growth factor (FGF). Clinical observations correlate the presence of enhanced uPA activity at the invasive edge of the tumors (Schmitt M et al, Fibrinolysis, 1992, 6, 3-26). ATF is capable of mediating disruption of the uPA/uPAR complex and inhibiting tumor cell migration and invasion in vitro (H. Lu et al., FEBS Letter, 1994, 356, 56-59). However, the ATF molecule retains the EGF growth factor binding domain, which interacts with the uPAR receptor. Such interactions may facilitate tumor growth, as suggested in the scientific literature (Rabbani et al., J Biol. Chem 275:16450-58 (1992)).

SUMMARY OF THE INVENTION

[0011] The present invention provides kringle-containing polypeptides, called abrogens, that are potent inhibitors of endothelial proliferation and/or angiogenesis. The abrogen polypeptides are capable of inhibiting or reducing cell proliferation induced by both bFGF and VEGF in a specific endothelial cell proliferation assay, whereas angiostatin only inhibits bFGF induced proliferation in this assay. Furthermore, vectors that express abrogen polypeptides in vivo reduce tumor metastasis in two lung cancer models. Thus, aspects of the invention include novel polypeptides, nucleic acids that encode them, vectors containing them, and methods of using any of these aspects to express polypeptides, alter growth or other characteristics of cells, or treat or prevent disease.

[0012] Embodiments of the abrogen activity include a region of urokinase plasminogen activator encompassing the kringle domain. The mammalian urokinase plasminogen activator (uPA) kringle domain (ATF-kringle) has not been previously identified as a separate molecule with anti-angiogenic activity. Rather, it was previously shown to be a potent source of attraction of smooth muscle cells [2]. Surprisingly, the Applicant has identified and showed that the ATF-kringle retains a very potent anti-angiogenic activity, while not containing the growth-factor-like domain acting as binding site to the uPAR, thereby allowing uPA/uPAR complex disruption. As demonstrated in Example 3, for example, ATF-kringle containing polypeptides can inhibit endothelial cell activation and/or proliferation mediated by several different proangiogenic proteins, such as bFGF and VEGF, in a species independent manner.

[0013] The use of the kringle domain allows greater specificity in the anti-angiogenic mode of action. Our data from in vitro studies shows that the ATF-kringle molecule possesses a new activity that inhibits both bFGF and VEGF induced tube formation and/or cell proliferation in a specific endothelial cell assay. This assay also distinguishes the species-specific activity of other anti-angiogenic polypeptides. The abrogen polypeptides, and in particular those of SEQ ID No.: 1, 3, 5, 7, 9, and 10, do not show a species-specific response and both mouse and human derived polypeptides, for example, function in a mouse model system. This can be advantageous in developing human therapeutic compositions based upon a mouse model system. In another contrast over previous polypeptides, anti-angiogenic factors such as endostatin or angiostatin only inhibit bFGF-induced activity in this assay (Chen et al., Hum Gen Ther 11: 1983-96 (2000)). In general terms, the invention encompasses the production of, identification of, and use of polypeptides, as well as the nucleic acids that encode them, that possess this new activity, referred to as abrogens.

[0014] Thus, in one aspect, the invention comprises an isolated abrogen polypeptide, such as one with an amino acid sequence of SEQ ID NO.: 1, 3, 5, 7, 9, or 10 comprises repeated sequence thereof, between 2 to 5, and preferably between 3 to 4 repeated sequences of one or more of SEQ ID NO.: 1, 3, 5, 7, 9, or 10, the polypeptide being in a form that does not exist in nature and has not been previously disclosed. The abrogen polypeptide can be in purified form, so that, for example, it is no longer inside a cell that produces it, it is in an extract derived from a cell that produces it, it is at least partially separated from a final reaction mixture that produces it, or one or more components of a mixture containing it have been substantially or to a measurable extent removed. A purified form can also be a form suitable for pharmaceutical research use, such as a form substantially free of antigenic or inflammatory components. A purified form can also be the result of an affinity purification process.

[0015] The invention also includes a nucleic acid comprising or consisting of a sequence that encodes an abrogen polypeptide, such as the sequences of SEQ ID NO.: 2, 4, 6, or 8, or comprising repeated sequences thereof, between 2 to 5, and preferably between 3 to 4 repeated sequences of one or more of SEQ ID NO.: 2, 4, 6, or 8. The nucleic acid can be DNA, RNA, or DNA or RNA comprising modified nucleotide bases. A nucleic acid encoding an abrogen polypeptide can also be operably linked to a variety of one or more sequences used in expression vectors, and/or cloning vectors, and/or other vectors. For example, the abrogen encoding nucleic acid can be linked to a promoter, enhancer, a sequence encoding a signal sequence, and/or a sequence encoding an affinity purification sequence. One of ordinary skill in the art is familiar with selecting appropriate sequence(s) or vector(s) and using them. The invention also encompasses cells that contain or comprise an abrogen polypeptide or abrogen encoding nucleic acid.

[0016] The cell can be transduced with, transfected with, or have an introduced into it a vector that comprises the abrogen encoding nucleic acid. Progeny of the cell, for example cells that result from cultured cell splitting or maintenance procedures, are also included in the invention. The cell can be a cultured primary cell, an established cell line cell, a transformed cell, a tumor cell, an endothelial cell, or a variety of other mammalian cells.

[0017] The invention also comprises a novel purified polypeptide that comprises one or more fragments of a mammalian or human kringle-containing protein, and for example comprises a 2 to 5 repeated sequences and preferably 3 to 4 repeated sequences of one or more kringle fragments, the fragments having a kringle domain that is capable of inhibiting tube formation in endothelial cell cultures induced by bFGF and VEGF, and/or capable of reducing cell proliferation induced by bFGF and VEGF, and/or capable of inhibiting metastasis of mammalian tumors. This fragment does not contain an EGF-binding domain, such as the EGF-binding domain of uPA or the amino terminal fragment (ATF) of uPA. The novel purified polypeptide does not contain the exact amino acid sequence of the kringle 5 domain of human plasminogen, the exact sequence of kringle 2 from human prothrombin, the exact 80 amino acids beginning at residue 462 of human plasminogen, or the exact sequence of any of the previously disclosed kringle-containing polypeptides, peptides, or proteins. The polypeptide can comprise or consist of any one of SEQ ID NO: 1, 3, 5 or 7, or one or more of the sequences listed in FIG. 2, or repeated sequences thereof. The novel polypeptides can advantageously be used in a number of instances where inhibiting or reducing cell proliferation associated with bFGF and VEGF treatment is desired, and/or where inhibiting angiogenesis or tumor metastasis is desired.

[0018] In another aspect, the invention comprises a recombinant kringle-containing polypeptides consisting of 2 to 5, preferably 3 to 4 repeated kringle fragments.

[0019] In still another aspect, the invention comprises nucleic acids that encode these novel polypeptides, vectors containing them, and cells containing them. Preferably, inhibiting tube formation in endothelial cell cultures induced by bFGF and VEGF, reducing cell proliferation induced by bFGF and VEGF, and/or inhibiting metastasis of mammalian tumors is measured in culture with established endothelial cell lines or tumor cell lines. However, other types of measurements, including measurements in vivo, can also be used. In this and other aspects of the invention involving cells, a preferred embodiment employs or involves human umbilical vein endothelial cells or mammary or lung tumor cells.

[0020] Preferably, the kringle-containing protein is a human protein or fragment thereof, such as a human plasminogen activator, like urokinase plasminogen activator or tissue plasminogen activator. Other human proteins from which the novel polypeptides and nucleic acids of the invention can be derived are ApoArgC, Factor XII, hepatocyte growth factor activator, hyaluronan binding protein, macrophage stimulating protein, thrombin, retinoic acid receptors 1 and 2, and kringle-containing domains from extended sequence tag database or other databases. In preferred examples, these polypeptides comprise a kringle domain having a region of SEQ ID NO.: 1 from Asn 53 to Asp 59 [NYCRNPD], and further comprise one or more regions within a particular amino acid sequence identity range to a region of SEQ ID NO.: 1, 3, 5, or 7. In particular, the regions of SEQ ID NO.: 1 that may be modified include from Cys 3 to Trp 27, Asn 53 to Cys 84, Lys 1 to Thr 2, and Ala 85 to Asp 86. However, these derivatives contain the conserved 6 Cys residues that are thought to help properly fold the kringle domain into a characteristic structure. Various regions are quite amenable to modification by substitution, deletion, and/or addition, including the region from about Asn 28 to about His 52 or Lys 51, and the terminal 2 residues from each of the N terminus and C-terminus of SEQ ID NO.:1. Particularly preferred derivatives include those with a region of approximately 50% amino acid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region of approximately 40% amino acid identity to the region of SEQ ID NO.: 1 from Asn 53 to Cys 84; a region of approximately 55% amino acid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region of approximately 45% amino acid identity to the region of SEQ ID NO.: 1 from Asn 53 to Cys 84; a region of approximately 35% amino acid identity to the region of SEQ ID NO.: 1 from Cys 3 to Trp 27 and a region of approximately 35% amino acid identity to the region of SEQ ID NO.: 1 from Asn 53 to Cys 84. For each of the abrogen regions identified here or elsewhere in this disclosure, one of skill in the art can clearly select an optimum or desirable range or specific sequence identity difference from that listed in the previous sentence. Thus, the 50% percent amino acid identity noted here and elsewhere can also be 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or from about 50-55%, or 55-60%, or 60-65%, or 65-70%, or 70-75%, or 75-80%, or 80-85%, or 85-90%, or 90-95%, or 95-98%, or 98-99%. Similarly, the 40% noted here or elsewhere can be 45%, 50%, and above and in various ranges as just listed, and the 35% noted here and elsewhere can be 40%, or 45% and above and in various ranges as just listed. Additional examples include an abrogen polypeptide with amino acid sequence of SEQ ID NO.: 1 modified to contain 1 to about 15 amino acid changes of substitutions, deletions, or additions, wherein the amino acid changes occur in the amino acids from Asn 28 to His 52, Lys 1 to Thr 2, Ala 85 to Asp 86. Furthermore, derivatives may merely contain or may additionally contain 1 to about 5, 1 to about 10, 1 to about 15, or 1 to about 20 amino acid changes outside of the consensus region from Asn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD] that are conservative amino acid substitutions.

[0021] The polypeptides and the nucleic acids that encode them may additionally have or encode a selected signal sequence region and/or an affinity purification sequence region. As used herein, the term “signal sequence or signal peptide” is understood to mean a peptide segment which directs the secretion of the abrogen polypeptides or abrogen fusion polypeptides and thereafter is cleaved following translation in the host cells. The signal sequence or signal peptide thus initiates transport of a protein across the membrane of the endoplasmic reticulum. Signal sequences have been well characterized in the art and are known typically to contain 16 to 30 amino acid residues, and may contain greater or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal peptide across the membrane lipid bilayer during transport of the nascent polypeptide. Following initiation, the signal peptide is usually cleaved within the lumen of the endoplasmic reticulum by cellular enzymes known as signal peptidases (von Heijne (1986) Nucleic Acids Res., 14: 4683). Numerous examples exist including the well known poly-His tag sequence, the immunoglobulin signal sequence, and the human interleukin 2 (IL2) signal sequence.

[0022] The polypeptide and the sequence encoding the polypeptide used in a specific vector encoding the given kringle domain may also be linked to stabilizing elements or polypeptides or the sequences that encode them, such as those from human serum albumin or the immunoglobulin Fc portion of an IgG molecule.

[0023] The abrogen polypeptides according to the present invention may be advantageously linked to one or more human serum albumin (HSA) protein sequences one or more other fusion partners. Such fusion polypeptides comprise the abrogen polypeptide fused at its C- or N-terminal, or both, with HSA. The amino acid sequence of HSA is well known in the art and is inter alia disclosed by Meloun et al. (Complete Amino Acid Sequence of HSA, FEBS Letter: 58:1. 136-137, 1975) and Behrens et al. (Structure of HSA, Fed. Proc. 34,591, 1975), and more recently by genetic analysis (Lawn et al., Nucleic Acids Research, 1981, 9, 6102-6114). Shorter forms or variants of HSA, as described in EP 322 094, may also be used to produce the abrogen fusion protein of the invention. Any abrogen polypeptide noted here can be used to prepare an abrogen fusion protein or polypeptide of the invention. Construction of such fusion proteins is well known in the art and is disclosed inter alia, in U.S. Pat. No. 5,876,969. Fusion proteins so obtained possess a particularly advantageous distribution in the body, while modifying the pharmacokinetic properties of the abrogen poplypeptide and compositions containing them, and favors the development of their biological activity.

[0024] An abrogen fusion protein or polypeptide according to the present invention may also comprise an N-terminal signal peptide, such as the IL2 signal peptide providing for secretion into the surrounding medium, followed or preceded by a HSA or a portion thereof, or a variant thereof and the sequence of the abrogen polypeptides. The abrogen polypeptides may be coupled either directly or via an artificial peptide or linker to albumin, at the N-terminal end or the C-terminal end or both.

[0025] The chimeric molecule may be produced by eucaryotic, prokaryotic, or cellular hosts that contain a nucleotide sequence encoding the abrogen fusion protein, and then harvesting the polypeptide produced. Animal cells, yeast, fungi may be used as eucaryotic hosts. In particular, yeasts of the genus of Saccharomyces, Kluveromyces, Pichia, Schwanniomyces, or Hansenula may be cited. Animal cells, such as for example, COS, CHO, 293 cell lines, and C127 cells, and the like may be used. Fungi such as Aspergillus sp., or Trichoderma ssp may be used. Bacteria, such as Esherichia coli, or bacteria belonging to the genera of Corynebacterium, Bacillus, or Streptomyces may be used as prokaryotic cells.

[0026] In another fusion protein or polypeptide example, the abrogen polypeptide is fused to one or more immunoglobulin Fc regions as described in WO 00/01133. Immunoglobulin Fc region is understood to mean the carboxylterminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region may comprise: 1) an immunoglobulin constant heavy 1 (CH1) domain, an immunoglobulin constant heavy 2 (CH2) domain, and an immunoglobulin constant heavy (CH3) domain; 2) a CH1 domain and a CH2 domain; 3) a CHI domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; and/or 5) a combination of two or more domains and an immunoglobulin hinge region. In a preferred embodiment the Fc region used in the DNA construct encoding the abrogen polypeptide also enclodes an immunoglobulin hinge region, CH2 and CH3 domains, and depending upon the type of immunoglobulin used to generate the Fc region, optionally a CH4 domain. More preferably, the immunoglobulin Fc region comprises a hinge region, and CH2 and CH3 domains. Immunoglobulin from which the heavy chain constant region is preferably derived is IgG of subclasses 1, 2, 3, or 4, and most preferably of subclass 2, most preferably the murin or human immunoglobulin Fc region from IgG2a. Other classes of immunoglobulin, IgA, IgD, IgE and IgM, may be used. The choice of appropriate or advantageous immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art. The Fc region used in the fusion protein is preferably from a mammalian species, for example from murine origin, and preferably from human origin, or from a humanized Fc region.

[0027] The fusion proteins of the invention preferably are generated by conventional recombinant DNA methodologies. The fusion proteins preferably are produced by expression in a host cell of a DNA molecule encoding a signal sequence, an immunoglobulin Fc region or HSA for example, and an abrogen polypeptide. The constructs may encode in a 5′ to 3′ direction, the signal sequence, the immunoglobulin Fc region or HSA for example, and the abrogen polypeptide. Alternatively, the constructs may encode in a 5′ to 3′ direction, the signal sequence, the abrogen polypeptide and the immunoglobulin Fc region or HSA for example. As noted above, other fusion partners or stabilizing elements or polypeptides can be selected for use. The abrogen polypeptide may be coupled either directly or via a linker to the immunoglobulin Fc region or HSA, for example. The fusion of the abrogen with the immunoglobulin Fc region are produced by introducing into mammalian cell such constructs, and culturing the mammalian cells to produce the fusion proteins. The resulting fusion protein can be harvested, refolded if necessary, and purified using conventional purification techniques well known and used in the art. The resulting abrogen polypeptides exhibit longer serum half-lives, presumably due to their larger molecular sizes, and other advantageous properties.

[0028] The abrogen polypeptides and either the HSA or the immunoglobulin Fc region, for example, may be linked by a polypeptide linker. As used herein the term “polypeptide linker” is understood to mean a peptide sequence that can be used to link two proteins together or a protein and an Fc region. The polypeptide linker preferably comprises a plurality of amino acids such as glycine and/or serine. Preferably, the polypeptide linker comprises a series of glycine and serine peptides about 10-15 residues in length. See, for example, U.S. Pat. No. 5,258,698, the disclosure of which is incorporated herein by reference. More preferably, the linker sequence is as set forth in SEQ ID NO: 12 or 16, or comprises an Asp-Ala or an Arg-Leu sequence. It is contemplated however, that the optimal linker length and amino acid composition may be determined by routine experimentation.

[0029] The present invention also provides methods for producing abrogen from non-human species and as fusion proteins, such as with HSA and Fc regions. Non-human angiogenesis inhibitor fusion proteins are useful for preclinical studies of angiogenesis inhibitors because efficacy and toxicity studies of a protein drug must be performed in animal model systems before testing in humans. A human protein may elicit an immune response in mouse, and/or exhibit different pharmacokinetics, skewing the test results. Therefore, the equivalent mouse protein is the best surrogate for the human protein for testing in a mouse model.

[0030] Additionally, various promoter/enhancer and RNA transcript stabilizing elements may be included in the vector.

[0031] In another aspect, the invention comprises methods for analyzing or identifying a polypeptide that reduces or inhibits endothelial cell proliferation induced by bFGF and VEGF, and/or reduces or inhibits tube formation induced by bFGF and VEGF, and/or reduces or inhibits tumor metastasis. In general, the method may comprise selecting a polypeptide having a kringle domain from a mammalian protein, the kringle domain comprising amino acid residues Asn 53 to Asp 59 of SEQ ID NO.: 1 [NYCRNPD], the kringle domain also containing 6 Cys residues and 2 Trp residues, and introducing the polypeptide to an endothelial cell, for example by employing an expression vector such as a recombinant adenoviral vector, a recombinant adeno-associated viral vector, or a plasmid vector. Any method for measuring the relative inhibition of tubule formation, the relative inhibition of cell proliferation, or the relative inhibition of tumor metastasis can be employed to detect a polypeptide having the appropriate characteristic or even a combination of characteristics. The invention specifically includes polypeptides and nucleic acids encoding these polypeptides that are identified or are capable of being identified by these methods.

[0032] Moreover, an abrogen polypeptide and compositions comprising it may be used as a therapeutic. The polypeptide and the method for expressing it in a cell can be, therefore, used in methods to treat or prevent a variety of angiogenesis related diseases or conditions, including, but not limited to hemangioma, solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, rheumatoid arthritis, diabetic neovascularization, diabetic retinopathy, macular degeneration, wound healing, obesity, peptic ulcer, Helicobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, psiorasis, ovulation-related disorders, menstruation-related disorders, placentation, psoriasis, and cat scratch fever.

[0033] In general, the use can also be for abrogating tumor vasculature growth or angiogenesis associated with a tumor. One skilled in the art is familiar with polypeptide expression and purification systems as well as methods for administering polypeptides and vectors in appropriate pharmaceutical compositions.

[0034] In addition, combination treatments where one or more angiogenesis inhibitor polypeptides of the invention, or abrogen polypeptides, are administered with one or more therapeutic compounds, polypeptides, or proteins. Also, the abrogen polypeptides or fusion proteins thereof can also be used in combination with the use of other therapeutic agents and in a combination with multiple, different abrogen or fusion polypeptides. Any existing or available therapeutic treatments can be combined with the polypeptides, combinations, or methods described here. Numerous examples exist and the compounds and the treatment methods can be selected from those available, such as those in the Physician's Desk Reference, Remington's Pharmaceutical Sciences, or Remington's Science and Practice of Pharmacy. A combination with an erythropoietin is specifically noted. Combinations with treatments or compounds that implicate angiogenesis or anti-angiogenesis mechanisms are preferred, but other tumor suppressing treatments and anti-cancer treatments or treatments used in cancer patients can also be selected.

[0035] In another aspect, the nucleic acids encoding an abrogen polypeptide can be used in a gene transfer method. The examples show how recombinant plasmid and adenoviral vectors, for example, can be used to affect metastasis in a lung tumor model. Various gene transfer and gene therapy vectors can be used in conjunction with the nucleic acids of the invention to either analyze the activity of an abrogen polypeptide in vivo or treat, prevent, or ameliorate an angiogenesis-related disease or condition in an animal. Preferably, the animal is human or mouse. More particularly, a nucleic acid encoding an abrogen of SEQ ID NO.: 1, 3, 5, 7, 9, or 10 or a repeat thereof, can be cloned into a vector, preferably an adenoviral vector, an adeno-associated virus (AAV), a plasmid, or other suitable viral or non-viral vector. In one embodiment, the vector is administered to a tumor bearing or non-tumor bearing animal by direct intratumoral injection, intravenous injection, intramuscular injection, electrotransfer-mediated administration, or other suitable method. The efficacy of the abrogen expressed from the vector can be assessed in the context of, for example, reduction of the primary tumor and/or abrogation of metastatic dissemination.

[0036] Accordingly, the invention comprises gene transfer methods and methods for expressing abrogen polypeptides in a cell of an animal. These methods may comprise inserting a selected abrogen encoding sequence, such as one encoding SEQ ID NO.: 1, 3, 5, 7, 9, 10 or one selected from FIG. 2, into a mammalian expression vector or the expression cassette of an appropriate vector. The vector is administered to a cell of the animal by any number of methods available, including intratumoral injection, electrotransfer, infusion, subcutaneous injection, intramuscular injection, or intravenous administration. The effect of the expressed abrogen polypeptide can then be measured and compared to control. These methods can be used to treat any one of a number of angiogenesis related diseases or disorders, such as those listed above.

[0037] While the production of kringle-containing polypeptides has been previously discussed, the successful and efficient production of soluble forms of biologically active abrogen polypeptides from E. coli has not. An aspect of the invention, therefore, is the use of expression vectors and fusion protein constructs to efficiently produce soluble abrogen polypeptides from E. coli. A related aspect of the invention is the novel constructs and vectors that encode abrogen polypeptides and fusion proteins of abrogen polypeptides that can be used to express soluble abrogen polypeptides and fusions from E. coli. Advantageously, the methods, vectors, and constructs described and exemplified produce comparatively high levels of soluble fusion protein per gram of wet cell pellet. Furthermore, the ability to directly express measurable or high levels of soluble fusion protein from E. coli simplifies the purification and production of protein.

[0038] It is known that peptides and proteins may be produced via recombinant means in a variety of expression systems, such as various strains of bacterial, fungal, mammalian or insect cells. The production of small heterologous peptides recombinantly for effective research and therapeutic use encounters however several difficulties. They may be for example subject to intracellular degradation by proteases and peptidases present in the host cell. In particular, it has been previously reported that the various kringles of human plasminogen, i.e, kringles 2 (Eur. J. Biochem. 1994 219 p455), or kringle 3 (Eur. J. Biochem. 1994 219 p 455) or kringles 2 and 3 (Biochemistry 1996 35 p2357), or again kringle 4 (Biochemistry 2000 39 p7414-7419), are unable to adopt a stable soluble conformation when produced in E. coli. Therefore, these kringle polypeptides are generally accumulated and are found in the insoluble or “inclusion bodies” fraction, which render them almost useless for screening purposes in biological or biochemical assays. Furthermore, these inclusion bodies usually require further manipulations in order to solublize and properly refold the heterologous proteins. These additional steps are technically difficult and expensive and essential render useless any high throughput applications or project, that is for the practical production of recombinant proteins for therapeutic, diagnostic or other research use.

[0039] Several different fusion protein partners with a desired heterologous peptide to protein are proposed in the art to enable the recombinant expression and or secretion of an heterologous protein. These fusions protein include inter alia LacZ, trpE fusion proteins, maltose binding protein fusions (MBP, Bedouelle et al., Eur.J.Biochem, 1988, 171(3): 541-9), the glutathione-S-transferase fusion protein (GST, Smith et al., Gene, 1988, 67(1): 31-40), the Z domain from the protein A (Z, Nilson et al., Protein Eng., 1987, 1:107-113), thioredoxin (TrxA, La Vallie et al., Biotechnology, 1993, 11: 187-193; Hoog et al., Biosci. Rep. 4:917, 1984), NusA (Davis et al., Biotechnol. Bioeng., 1999, 65: 382-388), and the Gb-i domain from the protein G (Gb1, Huth et al., Protein Sci., 1997, 6:2359-64), at the amino- or the carboxy- termini.

[0040] In this regard, Hammarstrom et al. (Protein Science 11:313 (2002) provides some discussion as to the effect of different fusions, namely GST, NusA, ZZ (double Z domain of protein A), Gb1, MBP, and TrxA, upon expression and solubilization of 32 potentially interesting human proteins having various characteristics in terms of size, cysteine content, and their solubility probability. While none appear outstanding, MBP seems to be somewhat better than the other fusion partners.

[0041] Kapust et al. (Protein Science 8:1668, 1999) also compared three soluble fusion partners MBP, TrxA, and GST to inhibit aggregation of six diverse proteins that normally accumulate in an insoluble form, and reports that MBP is far more effective for solubilizing than the two other partners, in that the MBP fusion partners invariably proved to be more soluble than GST and TrxA, and thus rendered the protein capable of adopting a stably folded conformation.

[0042] However, neither Hammarstrom et al. nor Kapust et al. have specifically addressed the problem of solubility of peptides having a cysteine content of around at least 7% and post-translational modifications such as the formation of disulfide bonds, although this refolding can be critical to produce or retain the activity of the protein.

[0043] The Applicant has now discovered and shown that among the existing fusion partners the thioredoxin (TrxA) is in fact capable of providing a very advantageous effect in terms of solubility of proteins having a cysteine content of around 7% and comprising 3 disulfide bonds, such as the abrogen polypeptides. This superior result was unexpected, as the previously existing guidelines have been found to be only partially predictable for producing stable, soluble and biologically active protein forms.

[0044] The invention thus comprises a method for producing a soluble abrogen polypeptide that comprises preparing a nucleic acid fusion construct comprising at least a TrxA encoding sequence fused in frame to an abrogen polypeptide sequence, as for example any one of SEQ ID NO: 1, 3, 5, 7, 9, 10, or those listed in FIG. 2. As in other aspects of the invention, the fusion partner encoding sequence can be located at the N-teminus, the C-terminus, or both ends of the abrogen encoding sequence, and different combinations of fusion partners can be selected for use. Preferably, the TrxA fusion partner is fused to the N-terminal of the abrogen. The amino acid sequence of the TrxA fusion partner is provided in SEQ ID NO: 22. The TrxA-abrogen fusion according to the invention may further comprise a linker peptide between the TrxA sequence and the abrogen sequence, which advantageously provides a selected cleavage site. Preferred cleavage site used is a thrombin cleavage site comprising the following amino acid sequence LVPRGS (SEQ ID NO: 23).

[0045] The present invention thus provides for an efficient method of increasing solubility of recombinant abrogen peptides. The abrogen produced by the method according to the present invention is obtained in an unexpected highly soluble form. The fusion protein is cytoplasmic and can be easily recovered by lysing the bacteria or host cell, purified and cleaved using for example the thrombin cleavage site. The nucleic acid construct can be incorporated into a vector or otherwise manipulated into a cell in order to express the fusion abrogen polypeptide. To produce the TrxA-abrogen fusion protein of this invention, a host cell is either transformed with, or has integrated in its genome, a DNA molecule comprising the TrxA-abrogen fusion protein, preferably under the control of an expression control sequence capable of directing the expression of the fusion protein production. Any one of a number of available expression control sequences can be selected for use. In preferred embodiments, the expression control sequences can operate in bacterial cells, such as E. coli, in order to express soluble fusion protein in E. coli cultures or cells.

[0046] Host cells suitable for the present invention are preferably bacterial cells, such as the various strains of E.coli, which are well known host cells in the field of biotechnology. The E.coli strain BL21 lambda DE3, used in the Examples, is preferably used, and most preferably the E.coli BL21 lambda DE3 trxB− (Novagen), which has a mutation in the thioredoxine reductase (trxB gene) is used, thereby allowing for the fonnation of disulfide bond in E.coli cytoplasm.

[0047] The trxA-abrogen fusion protein may be purified by conventional procedures including selective precipitation solubilization and column chromatography methods. Preferably, a purification tag is included between the trxA and the abrogen sequence, eventually in upstream or downstream position of the cleavage proteolytic site for the thrombin (SEQ ID NO: 23). Purification tag sequences are well known in the art and include inter alia Arg-tag, calmodulin-binding peptide, cellulose binding domain, DsbA, c-myc-tag, FLAG-tag, HAT-tag, HIS-tag, and Strep-tag (Terpe K., Appl. Microbiol. Biotechnol, 2003, 60(5): 523-33). Preferably, the purification tags, such as a His tag sequence, which comprises 6 histidine residues, and the streptokinase tag comprising a nine-amino acid peptide having intrinsic streptavidin binding activity, such as for examples the sequences AWRHPQFGG or WSHPQFEK (Lamla et al., Mol.Cell.Proteomics, 2002, 1(6): 466-71) are used or incorporated into the fusion protein construct or encoded by the vector. One or more cleavage sites to liberate abrogen polypeptide from the fusion protein can also be used in the fusion protein construct or encoded by the vector.

[0048] The invention also comprises administration of one or more recombinant abrogen polypeptides in a cell of an animal. These methods may comprise administering the abrogen peptide as in SEQ ID NO: 1, 3, 5, 7, 9, or 10, (plus the peptide produced in E. coli with 4 extra amino acids at the N terminal sequence as listed in Example 12) or an abrogen fusion construct thereof as in SEQ ID NO: 13, 14, 15, 17, 18, 20, or 21, by any well-known method in the art, including for example, direct injections of the peptide at a specific site, i.e., by ophthalmic (including intravitreal or intraorbital), intraperitoneal, intramuscular, or intratumoral injections.

[0049] The invention also includes compositions comprising the abrogen polypeptides or nucleic acids, and the derivatives and nucleic acids encoding derivatives, such as those having the sequences of sequences listed herein or an abrogen fusion construct thereof as in SEQ ID NO: 13, 14, 15, 17, 18, 20, or 21, or nucleic acids encoding them. The abrogen polypeptides or derivatives can be recombinant polypeptides or purified polypeptides. The compositions of the present invention may be provided to an animal by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., parenterally or orally). Where the composition is to be provided parenterally, such as by intravenous, subcutaneous, ophthalmic (including intravitreal or intracameral), intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracistemal, intracapsular, intranasal or by aerosol administration, the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution. Thus, the carrier or vehicle is physiologically acceptable so that in addition to delivery of the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance. The fluid medium for the agent thus can comprise normal physiologic saline (e.g., 9.85% aqueous NaCl, 0.15 M, pH 7-7.4). In one embodiment, the composition is a pharmaceutically acceptable composition. One skilled in the art is familiar with selecting and testing pharmaceutically acceptable compositions for use with recombinant polypeptides and nucleic acids.

[0050] The abrogen formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s).

[0051] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0052] It is another aspect or object of the present invention to provide a method of treating diseases and processes that are mediated by angiogenesis.

[0053] It is yet another aspect of the present invention to provide a method and composition for treating diseases and processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, rheumatoid arthritis, diabetic neovascularization, diabetic retinopathy, macular degeneration, wound healing, peptic ulcer, Helicobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, placentation, psoriasis, obesity and cat scratch fever.

[0054] It is another aspect of the present invention to provide a composition for treating cancer or repressing the growth of a cancer.

[0055] It is still another aspect of the present invention to provide a method for treating ocular angiogenesis related diseases such as macular degeneration or diabetic retinopathy by direct ophthalmic injections of the recombinant abrogen peptides.

[0056] Another aspect of the present invention is to provide a method for targeted delivery of abrogen compositions to specific locations.

[0057] Yet another aspect of the invention is to provide compositions and methods useful for gene therapy for the modulation of angiogenic processes.

[0058] Throughout this disclosure, applicants refer to journal articles, patent documents, published references, web pages, sequence information available in databases, and other sources of information. One skilled in the art can use the entire contents of any of the cited sources of information to make and use aspects of this invention. Each and every cited source of information is specifically incorporated herein by reference in its entirety. Portions of these sources may be included in this document as allowed or required. However, the meaning of any term or phrase specifically defined or explained in this disclosure shall not be modified by the content of any of the sources. The description and examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.

BRIEF DESCRIPTION OF THE FIGURES

[0059] FIG. 1: The proliferative response of transduced HUVEC human endothelial cells to human abrogen (hATF-K; SEQ ID NO. 1) and mouse abrogen (mATK-K; SEQ ID NO.: 3). Cultured cells were transduced with adenoviral vectors containing an expression cassette for producing the abrogen polypeptide (hATF-K and mATF-K), a control, CMV promoter only vector (CMV), and the full amino terminal fragment of plasminogen (hATF or mATF). In FIG. 1A, the left axis indicates the degree of cell proliferation and each of the boxes represents the level of cell proliferation under a treatment regimen as indicated by the addition of bFGF, VEGF, or both. The reduction in cell proliferation in all samples where the human abrogen polypeptide is expressed (hATF-K) is markedly reduced compared to controls (CMV, hATF, and mATF). The proliferation in the mouse abrogen expressing cells (mATF-K) is also markedly reduced. FIG. 1B shows representative cell cultures from mouse and human full ATF polypeptides and mouse and human ATF-Kringle containing abrogen polypeptides (see Examples). The first page shows Control (full human ATF treated with FGF) compared to hATF-Kringle containing polypeptide treated with FGF. The remaining pages list the adenoviral vector used to transduce the cells (see Examples).

[0060] FIG. 2: Various human protein sequences having a kringle domain possessing the consensus region from Asn 53 to Asp 59 of SEQ ID NO.: 1 and the with the 6 conserved Cys, 2 conserved Trp, and conserved Gly and Arg residues aligned. These proteins and homologs, isoforms, and derivatives of them, can be used in methods of the invention and used to produce polypeptides and polynucleotides of the invention.

[0061] FIG. 3: Effect of anti-angiogenic polypeptides on tubule growth in endothelial cells.

[0062] Because culture conditions rapidly deplete anti-angiogenic factors if they are added as a recombinant or purified polypeptide, HUVECs are directly transduced with adenoviral vectors to provide consistent protein expression and secretion for the duration of the assay (7-10 days). HUVECs are transduced with Adenovirus expressing: human abrogen, hATF-K (as in SEQ ID NO.: 1), mouse abrogen, mATF-K (as in SEQ ID NO.: 3), and human endostatin (FIG. 3A) or human Angiostatin (FIG. 3B). Control adenovirus containing the LacZ or no gene of interest (empty control) is also included. The transduced cells are then cultured in a 3-dimensional matrix of fibrin with recombinant VEGF or bFGF added, as indicated. Tubule formation as a marker for activation and proliferation of endothelial cells is then visualized and recorded. Tubule formation in both the bFGF and VEGF treated cells is markedly inhibited in only the abrogen expressing cultures.

[0063] FIG. 4: Prevention of tumor metastasis in mouse 4T1 lung cancer model. Control empty plasmid and abrogen (hATF-K or mATF-K) expression cassette containing plasmid introduced via electrotransfer 6 days prior to injection of 4T1 tumor cells. Approximately 250,000 tumor cells are injected subcutaneously. Fifteen days after injection, primary tumors are removed in a surgical procedure. Lungs are harvested 35 days post tumor injection and the size and number of metastatic tumor colonies measured.

[0064] FIG. 5: Prevention of tumor metastasis in mouse 4T1 lung cancer model. Control empty plasmid compared to mATF-K expression plasmid. The assay protocol is the same as in FIG. 4.

[0065] FIG. 6: Prevention of tumor metastasis in mouse 3LL Boston lung cancer model. Control empty plasmid compared to mATF-K expression plasmid. The assay protocol is the same as FIG. 4, with the exception that 3LL Boston cells are used.

[0066] FIG. 7: Prevention of tumor metastasis in mouse 3LL Boston lung cancer model. Control empty plasmid compared to experimental control mEndostatin expression plasmid. The assay protocol is the same as FIG. 6.

[0067] FIG. 8: Measurement of size and number of metastasis in the 4T1 lung tumor model described for FIG. 4. Each spot represents the weight of the lung from each animal surveyed (C57BL/6 mice), indicating the relative size of the tumor nodules present. The left axis indicates the number of visible tumor nodules for each of the animals. With the exception of one animal in the hATF-K sample, the abrogen expressing vector treatment animals show a reduction in both the size and number of metastatic tumor nodules as compared to control. The hATF-K animals with abnormally high number of nodules were not further examined for experimental or procedural error or expression of hATF-K. Here the controls are empty plasmid (Control) and an alkaline phosphatase expressing control plasmid (mSEAP).

[0068] FIG. 9: Measurement of size and number of metastasis in the 3LL Boston lung tumor model described for FIG. 4 using the graphical representation method described for FIG. 7. Controls are the same as in FIG. 7. Again, the use of both the mouse and human abrogen expressing vectors (mATF-K and hATF-K) results in significant reduction in tumor metastasis.

[0069] FIG. 10: Measurement of size and number of metastasis in the 3LL Boston lung tumor model as described for FIG. 9. These data indicate that treatment with mouse endostatin or angiostatin, or either mouse or human ATF-K, reduce the number and size of the lung metastatic nodules compared to control treatment. The fact that both mouse and human abrogen encoding vectors are efficacious indicates that the species-specific characteristics that limit the use of the endostatin and angiostatin polypeptides are not present in the abrogen polypeptides. Furthermore, the abrogen polypeptides appear at least as efficacious as the either endostatin or angiostatin and much more efficacious than a combined endostatin/angiostatin treatment (mEndo/mAngio).

[0070] FIG. 11: Systemic expression of mouse or human derived abrogen polypeptides (here listed as MuPAK or HuPAK) from vector introduced into muscle significantly reduces the formation of spontaneous lung metastases in the 3LL-B tumor model. Systemic expression of therapeutic transgenes from the muscle is established 6 days before C57BL/6 mice are injected with a tumorigenic dose of 3LL-B tumor cells. The primary tumor is carefully excised 15 days post cell injection. The study is terminated on day 35 and lung metastases were counted. Panel A: lungs from mice treated with empty expression vector; Panel B: mice treated with human derived ATF-Kringle abrogen expressing vector (HuPAK); and Panel C: with treated with mouse derived ATF-Kringle abrogen expressing vector (MuPAK); Panel D: graphically shows the number and size of metastatic nodules present as the diameter of each “bubble” represents the lung weight.

[0071] FIG. 12: Systemic expression of mouse or human abrogen (here listed as MuPAK or HUPAK) from muscle significantly reduces the formation of spontaneous lung metastases in the MDA-MB-435 tumor model. Systemic expression of therapeutic transgenes from the muscle is established 10 days after SCID/bg mice are injected with a tumorigenic dose of MDA-MB-435 (human breast adenocarcinoma tumor cells). The primary tumor is carefully excised when a volume of 250 to 350 mm3 is reached. The study is terminated on day 89 and lung metastases measured. Panel A: lungs from mice treated with control mSEAP; Panel B: with treated with mouse derived ATF-Kringle abrogen expressing vector (here MuPAK); Panel C: mice treated with human derived ATF-Kringle abrogen expressing vector (HuPAK); and Panel D: graphically shows lung metastases counts as noted above.

[0072] FIG. 13A: is a schematic representation of the plasmid pXL2996.

[0073] FIG. 13B: is a schematic representation of the plasmid pMB063.

[0074] FIG. 13C is a schematic representation of the plasmid pBA140.

[0075] FIG. 14: is a schematic representation of the plasmid pMB060 and fusion construct.

[0076] FIG. 15: is a schematic representation of the plasmid pMB059 and fusion construct.

[0077] FIG. 16 is a schematic representation of the plasmid pMB056 and fusion construct.

[0078] FIG. 17: is a schematic representation of the plasmid pMB055 and fusion construct.

[0079] FIG. 18: is a schematic representation of the plasmid pMB060m prepro and fusion construct.

[0080] FIG. 19: is a schematic representation of the plasmid pMB053 and fusion construct.

[0081] FIG. 20: is a schematic representation of the plasmid pMB057 and fusion construct.

[0082] FIG. 21: is a schematic representation of the plasmid pXL4128.

[0083] FIG. 22: is a schematic representation of the plasmid pET28-Trx, which can be used in the methods to produce abrogen fusion protein.

[0084] FIG. 23: is a schematic representation of plasmids pXL4189 (top) and pXL4215 (bottom).

[0085] FIG. 24: is a schematic representation of plasmids pXL4190 (top) and pXL4219 (bottom).

[0086] FIG. 25: Production of Fusion Proteins. This Figure shows the expression products from various plasmids separated by gel electrophoresis. The far left lane of the gel image (lane #M) shows the molecular weight markers, indicated by the numbers on the left side (Kda). Lane #2 is the total cell extract from cell expression using pXL4189 (TrxA-abrogen N43 fusion), for expressing abrogen N43. Lane #8 is the soluble fraction from the cell expression of Lane #2. The results show that a substantial percentage of fusion protein is soluble and can be cleaved to produce soluble abrogen N43. Lane #9 is the remaining cell pellet from Lane #2. Lane #5 is the total cell extract from cell expression using pXL4190 (TrxA-K4 angiostatin fusion), for expressing K4 kringle domain from angiostatin. Lane #10 is the soluble fraction from the cell expression of Lane #5. The results show that a substantial percentage of fusion protein is soluble and can be cleaved to produce soluble K4 polypeptide. Lane #11 is the remaining cell pellet from Lane #5.

[0087] FIG. 26: Analysis of Purified Abrogen by SDS-PAGE. This Figure shows the production and purification results from an abrogen expression and cleavage method as described in Example 12. The various levels of protein loaded on the gel indicate the purity of the abrogen D43 polypeptide, that the fusion protein is no longer present, and that no other protein components are visible.

[0088] FIG. 27: Biological effect of recombinant kringle domains produced in E. coli on tubule growth in HUVE cell spheroids. Recombinant kringle domains are added to HUVEC spheroids in a 3-dimensional matrix of fibrin containing VEGF and bFGF. Tubule formation is then visualized and recorded at day 11 post treatment with the test product. Tubule formation in the bFGF and VEGF treated cells is markedly inhibited only in the presence abrogen D43 and plasminogen K5 kringle domain.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0089] A number of Kringle domain containing proteins and polypeptides have been described and used in a variety of methods, including therapeutic methods. As shown here, a kringle-containing abrogen polypeptide can be identified and used to inhibit or reduce tumor metastasis, inhibit or reduce endothelial cell proliferation, and/or inhibit or reduce endothelial cell tubule formation. As an abrogen polypeptide or nucleic acid encoding an abrogen polypeptide, specific examples include the mouse or human derived kringle domains of uPA (SEQ ID NO.: 1-8). Additional examples have been mentioned and/or are described below in their structure and/or method of making and identifying. Functionally, an abrogen polypeptide can be distinguished by the ability to inhibit tumor metastasis. A more specific set of abrogen polypeptides include those that inhibit the endothelial cell proliferation induced by both of bFGF and VEGF, either in separate assays or together in one assay. An abrogen polypeptide can be either secreted or expressed inside a cell.

[0090] In making and using aspects and embodiments of this invention, one skilled in the art may employ conventional molecular biology, cell biology, virology, microbiology, and recombinant DNA techniques. Exemplary techniques are explained fully in the literature. For example, one may rely on the following general texts to make and use the invention: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Sambrook et al., Third Edition (2001); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation, Hames & Higgins, eds. (1984); Animal Cell Culture (RI. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); Gennaro et al. (eds.) Remington's Pharmaceutical Sciences, 18th edition; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (2001), Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, Inc. (2001); W. Paul et al. (eds.) Fundamental Immunology, Raven Press; E. J. Murray et al. (ed.) Methods in Molecular Biology: Gene Transfer and Expression Protocols, The Humana Press Inc. (1991); J. E. Celis et al., Cell Biology: A Laboratory Handbook, Academic Press (1994); J. E. Coligan et al. (Eds.) Current Protocols in Protein Science, John Wiley & Sons (2001); and J. S. Bonifacino et al. (Eds.) Current Protocols in Cell Biology, John Wiley & Sons, Inc. (2001). Additional information sources are listed below or are referred to by citation number corresponding to the references at the end of the specification.

[0091] As used herein, a “vector” means any nucleic acid or nucleic acid-bearing particle, cell, or organism capable of being used to transfer a nucleic acid into a host cell and/or used to cause the expression of a polypeptide in a host cell. The term “vector” includes both viral and nonviral products and means for introducing the nucleic acid into a cell. A “vector” can be used in vitro, ex vivo, or in viva. Non-viral vectors include plasmids, cosmids, and can comprise liposomes, electrically charged lipids (cytofectins), DNA protein complexes, and biopolymers, for example. Viral vectors include retroviruses, lentiviruses, adeno-associated virus, pox viruses, baculovirus, reoviruses, vaccinia viruses, herpes simplex viruses, Epstein-Barr viruses, and adenovirus vectors, for example. Vectors can also comprise the entire genome sequence or recombinant genome sequence of a virus. A vector can also comprise a portion of the genome that comprises the functional sequences for production of a virus capable of infecting, entering, or being introduced to a cell to deliver nucleic acid therein

[0092] The abrogen derivatives of this invention include those having one or more conservative amino acid substitutions. For example, one or more amino acid residues within a sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent when the substitution results in no significant change in activity in at least one selected biological activity or function.

[0093] Substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0094] “Isolated,” when referring to a nucleic acid or polypeptide, means that the indicated molecule is present in the substantial absence of at least one other molecule with which it naturally occurs or necessarily occurs because of its method of preparation. Thus, for example, an “isolated abrogen polypeptide” refers to a molecule substantially free of a macromolecule existing in a cell used to produce the abrogen polypeptide. However, the preparation or sample containing the molecule may include other components of different types. In addition, “isolated from” a particular molecule may also mean that a particular molecule is substantially absent from a preparation or sample. Varying degrees of isolation can be prepared from methods known in the art. Similarly, a “purified” form of a molecule is at least partially separated from a final reaction mixture that produces it, or one or more components of a mixture containing it have been substantially or to a measurable extent removed. A purified form can also be a form suitable for pharmaceutical research use, such as a form substantially free of antigenic or inflammatory components. A purified form can also be the result of an affinity purification process or any other purification step or process.

[0095] The “derivatives” noted here can be produced using homologue sequences, modifications of an existing sequence, or a combination of the two. The term “homologue” is used herein to refer to similar or homologous sequences, whether or not any particular position or residue is identical to or different from the molecule similarity or homology is measured against. A nucleic acid or amino acid sequence alignment may include spaces. Preferably, alignment is made using the consensus residues listed in FIG. 2, or the 6 Cys residues of the kringle domain. One way of defining a homologue is through “percent identity” between two nucleic acids or two polypeptide molecules. This refers to the percent defined by a comparison using a basic blastn or blastp or blastx algorithm at the default setting, unless otherwise indicated (see, for example, NCBI BLAST home page: http://www.ncbi.nlm.nih.gov/BLAST/). Aligning a Cys residue in abrogen, for example, can be performed by comparing sequences where the first amino acid residue or codon is for a particular Cys, or where the particular Cys residue is set at the same position as that of the abrogen Cys residue. For example, the blastp algorithm was used to generate homologue sequences, as in those of FIG. 2, by selecting the Blosum62 matrix, gap costs set at Existence: 11 and Extension: 1 (the default settings when performed). Typically, the default setting is used unless otherwise indicated. “Homology” can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions allowing for the formation of stable duplexes between homologous regions and determining of identifying double-stranded nucleic acid.

[0096] A “functional homologue” or a “functional equivalent” of a given polypeptide or sequence includes molecules derived from the native polypeptide sequence, as well as recombinantly produced or chemically synthesized polypeptides, which function in a manner similar to the reference molecule or achieve a similar desired result. Thus, a “functional homologue” or a “functional equivalent” of a given kringle nucleotide region includes similar regions derived from a different species, nucleotide regions derived from an isoform, or from a different cellular source, or resulting from an alternative splicing event, as well as recombinantly produced or chemically synthesized nucleic acids that function in a manner similar to the reference nucleic acid region in achieving a desired result, such as a result in a particular assay or cell characteristic.

[0097] A “recombinant” molecule is one that has undergone at least one molecular biological manipulation, as known in the art. Typically, this manipulation occurs in vitro but it can also occur within a cell, as with homologous recombination. A recombinant polypeptide is one that is produced from a recombinant DNA or nucleic acid. A “coding sequence” or “sequence that encodes” is a sequence capable of being transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′(carboxyl) terminus.

[0098] A “nucleic acid” is a polymeric compound comprised of covalently linked nucleotides, from whatever source. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or doublestranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The term “nucleic acid” also captures sequences that include any of the known base analogues of DNA and RNA.

[0099] A cell has been “transfected” by a vector or exogenous or heterologous nucleic acid when the vector or nucleic acid has been introduced inside the cell. A cell has been “transformed” or “transduced” by a vector or exogenous or heterologous nucleic acid when the vector or nucleic acid effects a phenotypic change or detectable modification in the cell, such as expression of a polypeptide.

[0100] Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques 7:980-990 (1992)). Preferably, the viral vectors are replication defective or conditionally replication defective, that is, they are unable to replicate autonomously in the target cell or unable to replicate autonomously under certain conditions. In general, the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome necessary for encapsulating the viral particles.

[0101] DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-330 (1991)), defective herpes virus vector lacking a glyco-protein L gene, or other defective herpes virus vectors (PCT Publication WO 94/21807 and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626-630 (1992); see also La Salle et al., Science 259:988-990 (1993)); a defective adeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101 (1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski et al., Mol. Cell. Biol. 8:3988-3996 (1988)); and a conditional replicative recombinant vectors (see, for example, U.S. Pat. Nos. 6,111,243, 5,972,706, and published PCT documents WO 00136650, WO 0024408).

[0102] Recombinant adenoviruses display many advantages for use as transgene expression systems, including a tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts (see e.g., Berkner, K. L., Curr. Top. Micro. Immunol., 158:39-66 (1992); Jolly D., Cancer Gene Therapy, 1:51-64 (1994)).

[0103] It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter or eletrotransfer device (see, e.g., Wu et al., J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)). Naked plasmids or cosmids can be used in a number of gene transfer protocols and these plasmids and cosmids can be used in embodiments of this invention (see, in general, Miyake et al., PNAS 93:1320-1324 (1996); U.S. Pat. No. 6,143,530; U.S. Pat. No. 6,153,597; Ding et al., Cancer Res., 61:526-31 (2001); and Crouzet et al., PNAS 94:1414-1419 (1997). Among the preferred plamid vectors are those described in WO9710343 and WO9626270. Plasmids can also be combined with lipid compositions, pharmaceutically acceptable vehicles, and used with electrotransfer technology, as known in the art (see, for example, U.S. Pat. Nos. 6,156,338 and 6,143,729, and WO9901157 and the related devices in WO9901175).

[0104] As noted above, a number of compositions comprising one or more of the abrogen polypeptides of the invention can be prepared. Combinations of two or more isolated or purified abrogen polypeptides can be prepared. In addition, combinations of one or more abrogen polypeptides with another biologically active compound, such as a therapeutic compound, can be prepared.

[0105] The combination according to the present invention can be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above.

[0106] Therapeutic agents for possible combination are especially one or more cytostatic or cytotoxic compounds, for example a chemotherapeutic agent or several selected from the group comprising an inhibitor of polyamine biosynthesis, an inhibitor of protein kinase, especially of serine/threonine protein kinase, such as protein kinase C, or of tyrosine protein kinase, such as epidermal growth factor receptor tyrosine kinase, a cytokine, a negative growth regulator, such as TGF-β or IFN-β, an aromatase inhibitor, a classical cytostatic, and an inhibitor of the interaction of an SH2 domain with a phosphorylated protein.

[0107] The pharmaceutical compositions according to the present invention for use in a method for the prophylactic or especially therapeutic treatment of angiogenesis related disease; especially those mentioned hereinabove, as well as tumor diseases.

[0108] Preference is given to the use of solutions of the active ingredient, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilised compositions comprising the active ingredient alone or together with a carrier, for example mannitol, can be made up before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers.

[0109] Suspensions in oil comprise as the oil component the vegetable, synthetic, or semi-synthetic oils customary for injection purposes. In respect of such, special mention may be made of liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brassidic acid or linoleic acid, if desired with the addition of antioxidants, for example vitamin E, p-carotene or 3, 5-di-tert-butyl-4-hydroxytoluene. The alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di-or trivalent, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, but especially glycol and glycerol. As fatty acid esters, therefore, the following are mentioned: ethyl oleate, isopropyl myristate, isopropyl palmitate, “Labrafil M 2375” (polyoxyethylene glycerol trioleate from Gattefoss, Paris), “Labrafil M 1944 CS” (unsaturated polyglycolized glycerides prepared by alcoholysis of apricot kernel oil and consisting of glycerides and polyethylene glycol ester; Gattefossé, France),“Labrasol” (saturated polyglycolized glycerides prepared by alcoholysis of TCM and consisting of glycerides and polyethylene glycol ester; Gattefossé, France), and/or “Miglyol 812” (triglyceride of saturated fatty acids of chain length C9 to C12 from Huis AG, Germany), but especially vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and more especially groundnut oil.

[0110] The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.

[0111] Pharmaceutical compositions for oral administration can be obtained, for example, by combining the active ingredient with one or more solid carriers, if desired granulating a resulting mixture, and processing the mixture or granules, if desired or necessary, by the inclusion of additional excipients, to form tablets or tablet cores.

[0112] Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

[0113] Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient.

[0114] Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as cornstarch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxyethylene sorbitan fatty acid ester type, may also be added.

[0115] For parenteral administration, aqueous solutions of an active ingredient in water-soluble form, for example of a water-soluble salt, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers, are especially suitable. The active ingredient, optionally together with excipients, can also be in the form of a lyophilizate and can be made into a solution before parenteral administration by the addition of suitable solvents.

[0116] Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.

[0117] Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic acid or benzoic acid.

[0118] The invention relates likewise to a process or a method for the treatment of one of the pathological conditions mentioned hereinabove, especially angiogenesis related diseases or neoplastic disease.

[0119] A polypeptide or combination can be administered as such or especially in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a patient requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.05 g to approximately 5 g, preferably from approximately 0.25 g to approximately 1.5 g, of a compound of the present invention.

EXAMPLES

[0120] Previous studies have shown that the ATF molecule can be effective as an anti-tumoral and anti-angiogenic molecule especially when delivered by gene therapy vectors [6]. However the presence of the EGF like domain may lead to the activation of intracellular pathways in both tumor cells [3] and endothelial cells [7]. These activities are counterproductive to an anti-angiogenic treatment. We have assessed the potency of the kringle domain from human and mouse uPA (ATF-kringle) by in vitro and in vivo assays for its potential as an anti-angiogenic therapeutic. The kringle domain of human uPA was previously shown to be a potent source of attraction for smooth muscle cells [2]. This activity again is counterproductive to use as an anti-angiogenic agent. Surprisingly, our data now shows that ATF-kringle containing polypeptides can inhibit endothelial cell activation and/or proliferation mediated by several different proangiogenic proteins, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), and in a species independent manner. We have designated the name Abrogen to this activity.

Example 1

Cloning and Manipulating Abrogen Nucleic Acids

[0121] Exemplary primary nucleotide and polypeptide structures for both the mouse and human abrogens sequences are shown below.

Amino acid sequence of human abrogen N43
ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yha hrs nal qlg SEQ ID NO.: 1
lgk hny crn pdn rrr pwc yvq vgl kpl vqe cmv hdc ad
Nucleotide sequence of human abrogen N43
aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQ ID NO.: 2
ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac
agatctaatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac
cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg
gtgcatgact gcgcagat
Amino acid sequence of mouse abrogen
ktc yhg nyd syr gka ntd tkg rpc law nap avl qkp yna hrp dai slg SEQ ID NO.: 3
lgk hny crn pdn qkr pwc yvq igl rqf vqe cmv hdc sl
Nucleotide sequence of mouse abrogen
aaaacctgct atcatggaaa tggtgactct taccgaggaa aggccaacac tgataccaaa SEQ ID NO.: 4
ggtcggccct gcctggcctg gaatgcgcct gctgtccttc agaaacccta caatgcccac
agacctgatg ctattagcct aggcctgggg aaacacaatt actgcaggaa ccctgacaac
cagaagcgac cctggtgcta tgtgcagatt ggcctaaggc agtttgtcca agaatgcatg
gtgcatgact gctctctt
Amino acid sequence of human abrogen
ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yha hrs dal qlg SEQ ID NO.: 5
lgk hny crn pdn rrr pwc yvq vgl kpl vqe cmv hdc ad
Nucleotide sequence of human abrogen D43
aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQ ID NO: 6
ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac
agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac
cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc cgcttgtcca agagtgcatg
gtgcatgact gcgcagat
Amino acid sequence of human abrogen D43 and L74
ktc yeg ngh fyr gka std tmg rpc lpw nsa tvl qqt yha hrs dal qlg lgk SEQ ID NO: 7
hny crn pdn rrr pwc yvq vgl kll vqe cmv hdc ad
Nucleotide sequence of abrogen D43 and L74
aaaacctgct atgaggggaa tggtcacttt taccgaggaa aggccagcac tgacaccatg SEQ ID NO: 8
ggccggccct gcctgccctg gaactctgcc actgtccttc agcaaacgta ccatgcccac
agatctgatg ctcttcagct gggcctgggg aaacataatt actgcaggaa cccagacaac
cggaggcgac cctggtgcta tgtgcaggtg ggcctaaagc tgcttgtcca agagtgcatg
gtgcatgact gcgcagat

[0122] Exemplary polypeptide sequences of the fusion proteins comprising the human abrogen having sequence of SEQ ID NO: 1 fused to the IL-2 signal peptide and to human serum albumin or immunoglobulin IgG2 Fc region, as well as linker peptide sequences, are listed below.

human abrogen as secreted from pMB063
AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSDALQLGL SEQ ID NO: 9
GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCAD
human abrogen as secreted from pBA140
AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ ID NO: 10
GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCAD
human HSA amino acid sequence
DAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLVNEVTEF SEQ ID NO: 11
AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN
ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF
YAPELLFFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK
CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD
LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP
ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL
AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG
EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA
EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP
KEFNAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD
DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGL
Linker DA(G4S)3
DAGGGGSGGGGSGGGGS                SEQ ID NO: 12
Fusion HSA—linker DA(G4S)3—abrogen (pMB060)
ADAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLNEVTEF SEQ ID NO: 13
AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN
ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF
YAPELLEFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK
CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD
LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP
ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL
AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG
EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA
EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP
KEFNAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD
DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGLDAGG GGSGGGGSGG
GGSKTCYEGN GHFYRGKAST DTMGRPCLPW NSATVLQQTY HAHRSNALQL
GLGKHNYCRN PDNRRRPWCY VQVGLKPLVQ ECMVHDCAD
Secreted fusion HSA—linker DA—abrogen from pMB059
(IL2sp is not on the sequence below but has been introduced into all the mammalian expression vector)
ADAHKSEVAH RFKDLGEENF KALVLIAFAQ YLQQCPFEDH VKLVNEVTEF SEQ ID NO: 14
AKTCVADESA ENCDKSLHTL FGDKLCTVAT LRETYGEMAD CCAKQEPERN
ECFLQHKDDN PNLPRLVRPE VDVMCTAFHD NEETFLKKYL YEIARRHPYF
YAPELLFFAK RYKAAFTECC QAADKAACLL PKLDELRDEG KASSAKQRLK
CASLQKFGER AFKAWAVARL SQRFPKAEFA EVSKLVTDLT KVHTECCHGD
LLECADDRAD LAKYICENQD SISSKLKECC EKPLLEKSHC IAEVENDEMP
ADLPSLAADF VESKDVCKNY AEAKDVFLGM FLYEYARRHP DYSVVLLLRL
AKTYETTLEK CCAAADPHEC YAKVFDEFKP LVEEPQNLIK QNCELFEQLG
EYKFQNALLV RYTKKVPQVS TPTLVEVSRN LGKVGSKCCK HPEAKRMPCA
EDYLSVVLNQ LCVLHEKTPV SDRVTKCCTE SLVNRRPCFS ALEVDETYVP
KEFNAETFTF HADICTLSEK ERQIKKQTAL VELVKHKPKA TKEQLKAVMD
DFAAFVEKCC KADDKETCFA EEGKKLVAAS QAALGLDAKT CYEGNGHFYR
GKASTDTMGR PCLPWNSATV LQQTYHAHRS NALQLGLGKH NYCRNPDNRR
RPWCYVQVGL KPLVQECMVH DCAD
Fusion abrogen—HSA (pMB056)
AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEO ID NO: 15
GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCADDAH KSEVAHRFKD
LGEENFKALV LIAFAQYLQQ CPFEDHVKLV NEVTEFAKTC VADESAENCD
KSLHTLFGDK LCTVATLRET YGEMADCCAK QEPERNECFL QHKDDNPNLP
RLVRPEVDVM CTAFHDNEET FLKKYLYEIA RRHPYFYAPE LLFFAKRYKA
AFTECCQAAD KAACLLPKLD ELRDEGKASS AKQRLKCASL QKFGERAFKA
WAVARLSQRF PKAEFAEVSK LVTDLTKVHT ECCHGDLLEC ADDRADLAKY
ICENQDSISS KLKECCEKPL LEKSHCIAEV ENDEMPADLP SLAADFVESK
DVCKNYAEAK DVFLGMFLYE YARRHPDYSV VLLLRLAKTY ETTLEKCCAA
ADPHECYAKV FDEFKPLVEE PQNLIKQNCE LFEQLGEYKF QNALLVRYTK
KVPQVSTPTL VEVSRNLGKV GSKCCKHPEA KRMPCAEDYL SVVLNQLCVL
HEKTPVSDRV TKCCTESLVN RRPCFSALEV DETYVPKEFN AETFTFHADI
CTLSEKERQI KKQTALVELV KHKPKATKEQ LKAVMDDFAA FVEKCCKADD
KETCFAEEGK KLVAASQAAL GL
Linker (G4S)3
GGGGSGGGGSGGGGS                  SEQ ID NO: 16
Secreted fusion abrogen—(G4S)3—HSA
AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ ID NO: 17
GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCADGGG GSGGGGSGGG
GSDAHKSEVA HRFKDLGEEN FKALVLIAFA QYLQQCPFED HVKLVNEVTE
FAKTCVADES AENCDKSLHT LFGDKLCTVA TLRETYGEMA DCCAKQEPER
NECFLQHKDD NPNLPRLVRP EVDVMCTAFH DNEETFLKKY LYEIARRHPY
FYAPELLFFA KRYKAAFTEC CQAADKAACL LPKLDELRDE GKASSAKQRL
KCASLQKFGE RAFKAWAVAR LSQRFPKAEF AEVSKLVTDL TKVHTECCHG
DLLECADDRA DLAKYICENQ DSISSKLKEC CEKPLLEKSH CIAEVENDEM
PADLPSLAAD FVESKDVCKN YAEAKDVFLG MFLYEYARRH PDYSVVLLLR
LAKTYETTLE KCCAAADPHE CYAKVFDEFK PLVEEPQNLI KQNCELFEQL
GEYKFQNALL VRYTKKVPQV STPTLVEVSR NLGKVGSKCC KHPEAKRMPC
AEDYLSVVLN QLCVLHEKTP VSDRVTKCCT ESLVNRRPCF SALEVDETYV
PKEFNAETFT FHADICTLSE KERQIKKQTA LVELVKHKPK ATKEQLKAVM
DDFAAFVEKC CKADDKETCF AEEGKKLVAA SQAALGL
HSA—DA(G4S)3—abrogen
DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA SEQ ID NO: 18
KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE
CFLQHKDDNP NLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY
APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC
ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDL
LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA
DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA
KTYETTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE
YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE
DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSA LEVDETYVPK
EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDD
FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGLDAGGG GSGGGGSGGG
GSKTCYEGNG HFYRGKASTD TMGRPCLPWN SATVLQQTYH AHRSNALQLG
LGKHNYCRNP DNRRRPWCYV QVGLKPLVQE CMVHDCAD
murine IgG2a Fe region
EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSED SEQ ID NO: 19
DPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVN
NKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYV
EWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN
HHTTKSFSRTPGK
Fusion IgG2a-abrogen
ARLEPRGPTI KPCPPCKCPA PNLLGGPSVF IFPPKIKDVL MISLSPIVTC SEQ ID NO: 20
VVVDVSEDDP DVQISWFVNN VEVHTAQTQT HREDYNSTLR VVSALPIQHQ
DWMSGKEFKC KVNNKDLPAP IERTISKPKG SVRAPQVYVL PPPEEEMTKK
QVTLTCMVTD FMPEDIYVEW TNNGKTELNY KNTEPVLDSD GSYFMYSKLR
VEKKNWVERN SYSCSVVHEG LHNHHTTKSF SRTPGKKTCY EGNGHFYRGK
ASTDTMGRPC LPWNSATVLQ QTYHAHRSNA LQLGLGKHNY CRNPDNRRRP
WCYVQVGLKP LVQECMVHDC AD
abrogen—RL—IgG2a
AKTCYEGNGH FYRGKASTDT MGRPCLPWNS ATVLQQTYHA HRSNALQLGL SEQ ID NO: 21
GKHNYCRNPD NRRRPWCYVQ VGLKPLVQEC MVHDCADRLE PRGPTIKPCP
PCKCPAPNLL GGPSVFIFPP KIKDVLMISL SPIVTCVVVD VSEDDPDVQI
SWFVNNVEVH TAQTQTHRED YNSTLRVVSA LPIQHQDWMS GKEFKCKVNN
KDLPAPIERT ISKPKGSVRA PQVYVLPPPE EEMTKKQVTL TCMVTDFMPE
DIYVEWTNNG KTELNYKNTE PVLDSDGSYF MYSKLRVEKK NWVERNSYSC
SVVHEGLHNH HTTKSFSRTP GK
amino acid sequence of thioredoxin
SDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY SEQ ID NO: 22
QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL
KEFLDANLAG
thrombin cleavage site
LVPRGS                           SEQ ID NO: 23

[0123] The cDNA sequence can be obtained from GenBank or a number of available sources.

[0124] PCR based methods can be used to retrieve the cDNA from an appropriate library. The cDNA can then be conveniently stored in a vector such as the pGEM or pGEX vectors by standard ligation or plasmid manipulation methods. The polypeptide encoding regions are then transferred into an appropriate, selected expression cassette or vector. Specific examples of vectors for various applications exist, including gene therapy (Chen et al., Hum Gen Ther 11: 1983-96 (2000); MacDonald et al., Biochecm Biophys Res Comm 264:469-477 (1999); Cao et al., J Biol Chem 271:29461-67 (1996); Li et al., Hum Gene Ther 10:3045-53 (1999)). For the examples that follow, the method of Crouzet et al., Proc. Natl. Acad. Sci. USA 94:1414-1419 (1997), is used to prepare recombinant adenovirus with E1/E3 deletion, CMV expression promotor and SV40 polyA. The plasmid vector used below contains the Amp resistance gene, the CMV promotor, the SV40 poly A sequence, and the IL-2 signal sequence for efficient secretion. The fairly robust adenoviral system can be selected for its ability to be used in a variety of cell types, whereas the plasmid system is selected for its relative efficiency of vector introduction. One skilled in the art is familiar with selecting or modifying vectors with these or other elements for use.

[0125] Once cloned and inserted into an appropriate vector, any of the abrogen encoding sequences or abrogen derivatives encoding sequences can be assayed for specific activity related to anti-angiogenesis using the Examples below or an assay mentioned here or in the references.

[0126] In a preferred embodiment for expressing a recombinant abrogen polypeptide, a vector comprising the coding region for human serum albumin linked to the C-terminus of the abrogen encoding region is used (see, for example, Lu et al., FEBS Lett. 356: 56-9 (1994)). Other fusion proteins or chimeric proteins can also be used. In another embodiment of a fusion protein, the abrogen encoding region is linked to an immunogenic peptide or polypeptide encoding region. These fusions can be used in created antibodies or monoclonal antibodies against an abrogen. Methods for preparing antibodies are well known in the art and both the purified abrogen polypeptides and fusion of them can be used to prepare antibodies. Monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et ai., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide expressing cell. The mice splenocytes are extracted and fused with a suitable myeloma cell line, such myeloma cell line SP20, available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned by limiting dilution as described (Wands et al., Gastroenterology 80:225-232 (1981)).

[0127] The hybridoma cells obtained through such a selection are then assayed to identify clones, which secrete antibodies capable of binding the polypeptide. Additional fusions can be used to ease purification of abrogen polypeptides, including poly-His tracks, constant domain of immunoglobulins (IgG), the carboxy terminus of either Myc or Flag epitope (Kodak), and glutathione-S-transferase (GST) fusions. Plasmids for this purpose are readily available.

[0128] A relatively simple method for preparing recombinant or purified abrogen polypeptide involves the baculovirus expression system or the pGEX system (Nesbit et al., Oncogene 18:6469-6476 (1999), Nesbit et al., J of Immunol 166:6483-90 (2001)). In the baculovirus system, plasmid DNA encoding the abrogen polypeptide is cotransfected with a commercially available, linearized baculovirus DNA (BaculoGold baculovirus DNA, Pharmingen, San Diego, Calif.), using the lipofection method (Felgner et al., PNAS 84:7413-7417 (1987)). BaculoGold virus DNA and the plasmid DNA are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). 10 μl Lipofectin and 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature.

[0129] The transfection mixture is added drop-wise to Sf 9 insect cells (ATCC CRL 1711), and seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27° C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The cells are cultured at 27° C. for four days. The cells can then be selected for appropriately transduction and assayed for the expression of abrogen polypeptide. If a fusion polypeptide was desired, the fusion polypeptide can be purified by known techniques and used to prepare monoclonal antibodies.

Example 2

Proliferation Analysis of Transduced HUVEC Using Alamar Blue

[0130] A number of different assays for analyzing cell proliferation, tubule formation, cell migration, endothelial cell growth, and tumor metastasis exist. Some of them are described in the references cited.

[0131] Human umbilical vein endothelial cells (HUVEC: Clonetics, San Diego) are seeded at 5×105 cells/ well of 6-well-plate in EGM-2 medium. The cells are incubated overnight at 37° C., 5% CO2. Endothelial Cell Basal Medium (EBM) and Endothelial Cell Growth Medium (EGM) are available (Clonetics, San Diego). The medium is aspirated off and 500 ul of ECM medium containing 100 IT/cell viruses put over cells. The cells are incubated at 37° C. for 2 hours, then aspirated and 1.5 ml EGM-2 medium is added. The cells are again incubate overnight at 37° C.

[0132] The cells are trypsinized, counted, and seeded at 2000cell/well of 96-well-plate in EGM-2 medium. The cells are incubated at 37° C. for 3 hours. The medium is changed into 200 μl of the following medium: Control=ECM+0.5% FBS; Test 1=control medium with bFGF 10 ng/ml; Test 2=control medium with VEGF 10 ng/ml; Test 3=control medium with bFGF 10 ng/ml+VEGF 10 ng/ml. After changing the medium, the cells are incubated at 37° C. for 5 days. 20 μl Alamar Blue (BioSource International) for each well is added. Plates are incubated at 37° C. for 6 hours and then the OD read at 570 nm and 595 nm.

[0133] Typical results are depicted in FIG. 1. From the results of this proliferation assay, both human and mouse ATF-K polypeptides (SEQ ID NO.: 1, 3, 5, and 7) are very effective in abrogating the proliferation of endothelial cells induced by bFGF and VEGF.

Example 3

Assay of Transduced HUVEC Embedded in Fibrin Gel

[0134] In an assay that distinguishes the abrogen activity from angiostatin, human umbilical vein endothelial cells (HUVEC: Clonetics, San Diego) are seeded (passage 3, growing in EGM-2 medium) at 5×105 cells/ well of 6-well-plate in EGM-2 medium. The embedded cell assay also or alternatively provides data concerning the invasiveness of the endothelial cells in response to certain treatments. Endothelial cell tubule formation induced by pro-angiogenic factors such as FGF and VEGF, a characteristic measured by this assay, can be directly correlated to angiogenesis. The abrogen polypeptides used here can inhibit or reduce angiogenesis by inhibiting tubule formation. The use of virally transduced HUVEC can provide very detailed information as to the effects that a selected abrogen polypeptide or derivative has on primary cell types. The potential anti-angiogenic agents are introduced by transduction of the cells (m-ATF, h-ATF, m- ATF-K and h-ATF-K, CMV empty was included as a control) using a recombinant human adenovirus.

adenovirus	VP/ml	vp/IT	IT/ml	cell/flask	ul/flask
Ad CMV	5.85E+12	100	5.85E+10	5.00E+06	8.55
Ad hATF	2.76E+12	100	2.76E+10	5.00E+06	18.12
Ad mATF	5.00E+12	100	5.00E+10	5.00E+06	10
Ad hATF-K	2.02E+12	161	1.25E+10	5.00E+06	5
Ad mATF-K	5.19E+12	51	1.02E+11	5.00E+06	40

[0135] The fibrin gel includes PBS (control), VEGF or bFGF. HUVEC cells are split ½ to ⅓ the day before transduction. On the day of the transduction, the cells are washed with PBS. 10 ml of serum free medium containing 100:1 (IT: cell ratio) of virus is incubated with the HUVEC for 2 hours to transduce the cells. The medium is then removed and the cells washed with PBS and 20 ml of full HUVEC medium placed in each T150 flask.

[0136] 48 hours following transduction the cells are trypsinized and the concentration of each cell solution adjusted to 5×10 5 cell/ml. The assay is performed in a 24 well plate. Each well is coated with 200 μl of fibrinogen solution (12 mg/ml) and 8 ul of thrombin (50 U/ml). Then in each well is added (according to the conditions):

[0137] VEGF165 (2 μl), b-FGF(2 μl) or nothing (final [growth factor]=1 ug/ml)

[0138] Thrombin (20 ul) of a 1000 U/ml solution.

[0139] 250 μl cell solution for a final concentration of 5×5 cells/ml

[0140] 250 μl of fibrinogen

[0141] Gels set in about 30 seconds. Then, 1.5 ml of medium is added on top. Each type of infected cells was assayed with VEGF165 alone, b-FGF alone or without any growth factor other than those already present in the medium.

[0142] After 6 days medium is removed and cells subjected to staining with Dif-Quick for enhanced visualization under microscopy. Fibrin plugs are fixed in 10% formalin, and then subjected to the 3 Dif Quick stains for 15 mins each before being rinsed in PBS and then fixed with 10% formalin again.

[0143] Representative photographs of cells are depicted in FIG. 1B. Tubules can be seen in control cells, whereas no tubules are detected in the hATF-K and mATF-K transduced cells. Tubule formation can be correlated with endothelial cell invasiveness, a characteristic of angiogenic activity. Thus, the lack of tubule formation in the abrogen polypeptide samples (human ATF Kringle and mouse ATF Kringle) demonstrates an inhibition of endothelial cell invasiveness, correlating to an inhibition of angiogenesis and metastasis. In the FIG. 1B pictures, transduced HUVEC are treated with control PBS, bFGF, or VEGF, which give the following results. For CMV control: limited structure is visible when PBS is in the fibrin gel; with VEGF there is robust proliferation showing the phenotype generated; tubules are clearly visible and are ubiquitous throughout the gel, some extensions are quite long; in the presence of bFGF the response is not as robust, the structures, which form, are long and spindle like in appearance. For full human ATF polypeptide: in PBS there are a considerable number of structures formed; the response is far more than that seen with control CMV transduced endothelial cells, also in relation to the CMV control there has been a robust response with the addition of bFGF, which is definitely synergistic with the human ATF transduced cells in comparison to those transduced with CMV; in the presence of VEGF there has been a considerable drop in the number of visible structure when compared to the CMV transduced cells. For full mouse ATF polypeptide: regardless of condition there are no structures forming in any of the gels. For human ATK Kringle (abrogen of SEQ ID NO.: 1): regardless of condition there are no structures forming in any of the gels. For mouse ATF Kringle (abrogen of SEQ ID NO.: 3): regardless of condition there are no tubule structures forming in any of the fibrin gels.

[0144] Without limiting the scope of the invention to any particular mode of action or mechanism, applicants offer the following possible explanation of these results. Human ATF still has the EGF like growth factor domain and may stimulate the growth of endothelial cells, which are human in origin. This growth is potentiated in the presence of ubiquitous bFGF in this assay, as one of the downstream effects of bFGF is the upregulation of uPAR. This synergy is observed when cells are transduced with human ATF in the presence of bFGF. In the absence of bFGF, human ATF can stimulate low level uPAR and presumably inhibits growth through the action of the kringle. Hence the observed decrease in number of structures when compared to CMV control. Mouse ATF does not cross react with human uPAR. Therefore, the mode of action is mediated through the kringle domain. With human and mouse ATF-K, there is no growth factor domain so no proliferative events can be initiated. This is specific to both bFGF and VEGF induced proliferative responses.

Example 4

In vivo Expression of Abrogen Polypeptides Using Adenoviral Vectors

[0145] For in vivo documentation of the activity of abrogen, a first experiment involves the systemic injection iv of 1×1011 VP of hATF-K expressing adenovirus. Circulating levels of HATF-K as shown by Western can be measured. Exemplary expression levels at d4 can be between 500-1000 ng/ml in either SCID or SCID/Beige mice. The 4T1 spontaneously metastatic breast cell line in SCID mice is used in which animals are injected with 2×105 cells sub-cutaneously in the right flank. At d7, when tumors were 20-40 mm3, adenovirus is injected at 1×1011 vp: Tris, CMV1.0 control Ad; mATF-K; and hATF-K. A second and third iv administration of adenovirus can be performed. Lung metastasis is then measured at about day 35, as described below.

Example 5

In vivo Expression of Abrogen Polypeptides Using Plasmid Vectors

[0146] Two tumor models are used, employing 4T1 tumor cells and 3LL Boston tumor cells. In the assay, the anti-tumor activity of abrogen polypeptide in the prophylactic murine Lewis lung carcinoma model, 3LL-B, in C57BL/6 mice is tested. The assay is designed to assess whether circulating levels of abrogen prevent and/or reduce the formation and growth of spontaneously formed metastases from subcutaneously implanted primary tumors. The tumor cells are cultured in DMEM containing 10% FCS, sodium pyruvate, nonessential amino acids, Pen-Strep, and L-Glutamine until prepared for injection using a buffered saline solution. The tumor cells are injected into the right flank of 8-10 week old C57BL/6 or BALB/c female mice via subcutaneous injection of a suspension of 2.5×105 tumor cells. Six days prior to tumor cell injection, the 25 ul of the plasmid solutions (25 ug DNA in Tris EDTA with 10% glycerol) are injected into the tibialis cranialus muscle. The injection site is then exposed to 4 pulses (1 pulse per second) at 100 mV using a square wave pulse generator (the electrotransfer method, ET). Alternatively, the electrotransfer enhancement can utilize four electric pulses of 100 V (250 V/cm) at 1 Hz with a pulse length of 20 msec. On about day 15 post cell injection, the primary subcutaneous tumor was surgically removed. At day 35, the lungs are collected and tumor nodules measured. Expression levels are measured on day −1, 7, and 14 relative to electrotransfer. A control alkaline phosphatase expressing plasmid (mSEAP) is used to assay expression.

[0147] The results of one set of experiments are depicted in FIGS. 4-10. The empty expression plasmid and the mSEAP control plasmid treatments resulted in many lung tumor nodules. In both the 4T1 and 3LL tumor models, the mATF-K and HATF-K abrogen polypeptides reduced the size and number of metastasis. The reduction in size and number is at least equivalent to those of the known anti-angiogenic polypeptides endostatin and angiostatin (FIG. 10).

[0148] Another set of assays with 3-LL Boston cells employing electrotransfer enhancement with four electric pulses of 100 V (250 V/cm) at 1 Hz with a pulse length of 20 m sec are shown in FIG. 11. Metastases were counted using a dissecting microscope. The FIG. 11 pictures of the lungs show that the formation of spontaneous lung metastases from the primary subcutaneous tumor was significantly reduced in the two therapeutic groups receiving plasmid DNA encoding either mouse of human ATF Kringle (listed as MuPAK or HuPAK here). Lung metastases counts as well as lung weights, reflected by the diameter of the “bubble” in panel C, were reduced in both treatment groups. Delivery of plasmid DNA encoding either the murine secreted alkaline phosphatase (mSEAP) or no protein as control to the T. cranialis muscle did not result in a significant reduction of lung metastases. Similar results can be obtained in the prophylactic 4T1 mammary tumor model (data not shown).

[0149] To assess the anti-tumor activity of systemically expressed abrogen polypeptides in a human breast adenocarcinoma xenograft model of SCID/bg mice, MDA-MB-435 tumor cells are used. These cells are significantly less aggressive as compared to the 4T1 and 3LL-B syngeneic mouse tumor models. However, spontaneous lung metastases formation is established in the time frame of 35 days post subcutaneous cell injection. Subcutaneous palpable MDA-MB-435 tumors are established by injecting SCID/bg mice with 106 tumor cells. On day 10 post injection, plasmid DNA was transferred to the Tibialis cranialis muscle using electrotransfer as described previously. Briefly, 25 μg of plasmid DNA (a total of 50 μg) in a 25 μl volume are injected directly into each T. cranialis muscle followed by four electric pulses of 100 V (250 V/cm) at 1 Hz with a pulse length of 20 msec. The primary tumor is carefully removed when the volume reached between 250 and 350 mm3, i.e. on day 39 or 44 post cell injections depending on the growth of the primary tumor. The study is terminated on day 89 and lungs harvested carefully and fixed in Bouin's solution. Metastases are counted using a dissecting microscope. FIG. 12 shows pictures of the lungs.

[0150] Lungs from mice treated with either mouse or human AFT-Kringle containing polypeptide, FIG. 12 panels B and C, bear significantly fewer metastases compared to the control group (panel A) treated with the plasmid encoding mSEAP. Overall lung metastases counts are significantly reduced as shown in panel D. By the time of treatment at day 10, no lung metastases have been formed in the lung of SCID/bg mice, so it is most likely that the systemic expression of abrogen from the muscle prevents the formation and/or growth of distant lung metastases from the primary subcutaneous tumor. This demonstrates an inhibition of angiogenesis, a hallmark for the growth of metastatic tumors.

Example 6

Production of Derivative Abrogen Polypeptides by PCR Based Site-directed Mutagenesis

[0151] In one method for generating an abrogen derivative, four oligonucleotide primers are used. Two of these are primers that flank the ends of the cDNA (SEQ ID NO.: 2, 4, 6, or 8 ) and contain convenient restriction sites for cloning into a desired vector. The other two mutagenic primers are complementary and contain the mutation(s) of interest. Typically, the mutagenic primers overlap by about 24 base pairs. Two separate PCR reactions are performed, each using a different outside primer and a different mutagenic primer that anneal to opposite strands of the DNA template. The amplified product from both PCR reactions are purified and added to a new primerless PCR mix.

[0152] After a few PCR cycles, the two products are annealed and extended at the region of overlap yielding the derivative product. The two outside primers are then added to this mixture to amplify the cDNA product by PCR. This method can be used to introduce amino acid substitutions at any point in an abrogen sequence.

[0153] In addition to the conservative amino acid substitutions noted throughout the disclosure, one skilled in the art is familiar with numerous methods for analyzing and selecting homologs and derivative sequences to use as abrogen sequences. For example, the sequence identified as “Putative-K1 (Est)” in FIG. 2 can be identified by searching for homologs using GenBank, an EST database, or any cDNA or genomic DNA database available. The EST can be pulled from a library, PCR amplified using primers specific for the EST, or synthesized using automated methods. Once isolated, the polypeptide encoding region can be cloned into an appropriate vector and tested as described above.

Example 7

Construction of IL2sp-abrogen Polypeptide

[0154] The combined techniques of site-directed mutagenesis and PCR amplification allowed to construct a chimeric gene encoding a chimeric peptide resulting from the translational coupling between the first 20 amino acids of the interleukin 2 signal peptide, which represent a signal sequence or signal peptide that is cleaved to produce the mature factor (Tadatsugu, T. et al. (1983) Nature 302:305) and the abrogen sequences as set forth in SEQ ID NO: 4 and 6 (IL2sp-abrogen). These hybrid genes were preferably bordered in 5′ of the translational initiator ATG and in 3′ of the translational stop codon and encode chimeric proteins of the IL2sp-abrogen. The hybrid gene is cloned in the pXL2996 (FIG. 13A), under the control of the human CMV Enhancer/promoter (−522/+72) and upstream of a SV40 late poly A signal. The resulting plasmid pMB063 as described in FIG. 13B was obtained. The abrogen peptide secreted from the plasmid pMB063 retained an alanine from the IL-2 signal peptide (IL2sp) at the N-terminus, and thus contains a 87 amino acid sequence as set forth in SEQ ID NO: 9.

[0155] The hybrid nucleotide sequence comprising the interleukine 2 signal peptide sequence and the abrogen sequence as set forth in SEQ ID NO: 2 was cloned in plasmid pXL 2996 downstream of the human CMV enhancer/promoter (−522/+72) and upstream of a SV40 late poly A signal. The resulting plasmid pBA140 as described in FIG. 13C was obtained. The abrogen peptide secreted from the plasmid pBA140 also retained an alanine from the IL-2 signal peptide at the N-terminus, and thus contains a 87 amino acid sequence as set forth in SEQ ID NO: 10.

Example 8

Construction of Fusion Proteins of Abrogen and HSA

[0156] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the nucleotide sequence encoding the human HSA as set forth in SEQ ID NO: 11, a linker, and the abrogen sequence as set forth in SEQ ID NO: 2 was cloned in plasmid pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The linker DA(G4S)3 was used (SEQ ID NO: 12). The construct of the fusion protein IL2sp-HSA-linker-abrogen and the resulting plasmid designated pMB060 are shown in FIG. 14. The fusion protein HSA/abrogen secreted from the plasmid pMB060 has the sequence as set forth in SEQ ID NO: 13.

[0157] Another linker DA (Asp-Ala) was used. The chimeric construct of the fusion protein IL2sp-HSA-DA linker-abrogen and the resulting plasmid is designated pMB059 are displayed in FIG. 15. The fusion protein HSA/abrogen secreted from the plasmid pMB059 has the sequence as set forth in SEQ ID NO: 14.

[0158] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the abrogen nucleotide sequence as set forth in SEQ ID NO: 2, and the sequence of the human HSA (SEQ ID NO: 11), was cloned in pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB056 and construct are displayed in FIG. 16. The fusion protein abrogen- HSA secreted from the plasmid pMB056 has the sequence as set forth in SEQ ID NO: 15.

[0159] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the abrogen nucleotide sequence having the sequence as set forth in SEQ ID NO: 2, a (G4S)3 linker (as set forth in SEQ ID NO: 16) and the sequence of the human HSA, was cloned downstream to the human CMV promoter and upstream of a SV40 polyA. The chimeric construct of the fusion protein IL2sp-abrogen-linker-HSA and the resulting plasmid designated pMB055 are displayed in FIG. 17. The fusion protein abrogen/HSA secreted from the plasmid pMB055 has the sequence as set forth in SEQ ID NO: 17.

[0160] Alternatively, a nucleotide sequence containing from 5′ to 3′ the prepro signal of HSA, the human HSA, a sequence encoding a DA(G4S)3 linker and the abrogen nucleotide sequence as set forth in SEQ ID NO: 2 was cloned in the plasmid pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB060m and the fusion protein prepro HSA—human HSA- DA(G4S)3 linker-abrogen are displayed in FIG. 18. The fusion protein HSA/abrogen secreted from the plasmid pMB060m has the sequence as set forth in SEQ ID NO: 18.

[0161] A fusion protein encoding plasmid may also comprise the bacteriophage T7 promoter suitable for the production of the abrogen polypeptide in E coli. Such plasmids are also described in U.S. Pat. No. 6,143,518. The plasmid pYG404 as described in the Patent application EP 361 991, which comprises the sequence encoding the prepro-HSA gene, may be used. For example, the C-terminal of HSA is coupled in phase with a linker sequence and the kringle polypeptide nucleotide sequence. The resulting plasmid can also be used for production of the polypeptide in yeasts, for example.

Example 10

Construction of Fusion Proteins of Abrogen and IG2a

[0162] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the murin IgG2a Fc region (SEQ ID NO: 19) and the human abrogen nucleotide sequence having the sequence as set forth in SEQ ID NO: 2 was cloned in pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB053 and the fusion construct are displayed in FIG. 19. The fusion protein IgG2alabrogen secreted from the plasmid pMB053 has the sequence as set forth in SEQ ID NO: 20.

[0163] A nucleotide fragment containing from 5′ to 3′ the IL-2 signal peptide, the human abrogen nucleotide sequence having the sequence as set forth in SEQ ID NO: 2, the nucleotide sequence coding for a RL (Arginine-Leucine) linker, the murin (mu) IgG2a Fc region was cloned in pXL2996 downstream to the human CMV promoter and upstream of a SV40 polyA. The resulting plasmid is designated pMB057 and the fusion construct are shown in FIG. 20. The fusion protein abrogen/IgG2a secreted from the plasmid pMB057 has the sequence as set forth in SEQ ID NO: 21.

Example 11

Construction of Plasmids Suitable for the Production of Recombinant Abrogen or Fusion Polypeptide

[0164] The plasmid pXL4128, which is represented in FIG. 21 and comprises the bacteriophage T7 promoter was also constructed, and is suitable for the production of the abrogen peptide in E coli. Such plasmids for the production in E.coli are also described in U.S. Pat. No. 6,143,518. The plasmid pYG404 as described in the Patent application EP 361 991, which comprise the sequence encoding the prepro-HSA gene may be used. For example, the C-terminal of HSA is coupled in transitional phase with a linker sequence and the abrogen nucleotide sequence. The resulting plasmid is used for production of the peptide in yeasts, for example.

Example 12

Construction of Fusion Protein Construct of trxA and Abrogen Polypeptide

[0165] The polypeptide sequence of abrogen N43 and abrogen is used for incorporation into a fusion protein. The abrogen polypeptide can then be expressed in soluble form, or substantially soluble form, in E. coli cells with the use of a bacterial expression vector, such as pET28-Trx (see FIG. 22; Novagen). K4 from angiostatin and K5 of plasminogen are also incorporated into a fusion protein and used as a control.

[0166] The sequences are amplified by PCR and the amplified fragments digested by NdeI-BamHI and cloned into pET28-Trx digested with NdeI-BamHI. Alternatively, sequences can be prepared using synthetic methods or a combination of synthetic and other methods, such as PCR or recombinant manipulation. The following Table presents the sequences selected and the primers used for cloning in an exemplary expression method. The plasmids obtained for the expression of each kringle polypeptide are also listed in the Table. Templates for the kringle sequences are available from a number of sources.

		Plasmid for
Sequence	Primers	expression
Abrogen N43	Sense:	AAACATATGGCCAAAACCTGCTATGAGGG	pXL4189
	Antisense:	AAAGGATCCTTAATCTGCGCAGTCATGCA
Abrogen D43	Sense:	AAACATATGGCCAAAACCTGCTATGAGGG	pXL4215
	Antisense:	AAAGGATCCTTAATCTGCGCAGTCATGCA
K4 from	Sense:	AAAAGCTTCATATGGCCCAGGACTGCTA	pXL4190
angiostatin	Antisense:	AAATCTAGAGGATCCTTATCCTGAGCA
K5 from	Sense:	AACATATGGAAGAAGACTGTATGTTTGGGAA	pXL4219
plasminogen	Antisense:	CCGGATCCTTAGGCCGCA

[0167] The plasmids for expression are also described in FIGS. 23-24. These plasmids can be sequenced to verify that they encode the expected protein. Exemplary fusion proteins are represented below and comprise a TrxA sequence (from amino acid 2 to 110; see Hoog et al., Biosci. Rep. 4:917 (1984)), a poly-histidine sequence (amino acids 118 to 123), a thrombin cleavage site (amino acids 127-132), followed by the abrogen sequence or by the K4 or K5 domain.

TrxA-Abrogen N43: Translation of pXL4189
1   GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDETADEY
51  QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL
101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMAKTCYE GNGHFYRGKA
151 STDTMGRPCL PWNSATVLQQ TYHAHRSNAL QLGLGKHNYC RNPDNRRRPW
201 CYVQVGLKPL VQECMVHDCA D
Abrogen N43
    GSHMAKTCYE GNCHFYRGKA STDTMGRPCL PWNSATVLQQ TYHAHRSNAL
    QLGLGKHNYC RNPDNRRRPW CYVQVGLKPL VQECMVHDCA D
TrxA-Abrogen D43: Translation ofpXL4215
1   GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY
51  QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL
101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMAKTCYE GNGHFYRGKA
151 STDTMGRPCL PWNSATVLQQ TYHAHRSDAL QLGLGKHNYC RNPDNRRRPW
201 CYVQVGLKPL VQECMVHDCA D
Abrogen D43
    GSHM AKTCYE GNGHFYRGKA STDTMGRPCL PWNSATVLQQ TYHAHRSDAL
    QLGLGKHNYC RNPDNRRRPW CYVQVGLKPL VQECMVHDCA D
TrxA-K4 kringle from angiostatin: Translation of pXL4190
1   GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY
51  QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL
101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMAQDCYH GDGQSYRGTS
151 STTTTGKKCQ SWSSMTPHRH QKTPENYPNA GLTMNYCRNP DADKGPWCFT
201 TDPSVRWEYC NLKKCSG
K4 kringle from angiostatin
    GSHMAQDCYH GDGQSYRGTS STTTTGKKCQ SWSSMTPHRH QKTPENYPNA
    GLTMNYCRNP DADKGPWCFT TDPSVRWEYC NLKKCSG
TrxA-K5 kringle from plasminogen: Translation of pXL4219
1   GSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY
51  QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL
101 KEFLDANLAG SGSMGSSHHH HHHSSGLVPR GSHMEEDCMF GNGKGYRGKR
151 ATTVTGTPCQ DWAAQEPHRH SIFTPETNPR AGLEKNYCRN PDGDVGGPWC
201 YTTNPRKLYD YCDVPQCAA
K5 kringle from plasminogen
    GSHMEEDCMF GNGKGYRGKR ATTVTGTPCQ DWAAQEPHRH SIFTPETNPR
    AGLEKNYCRN PDGDVGGPWC YTTNPRKLYD YCDVPQCAA

[0168] The plasmids are introduced into bacteria cells, such as E. coli BL21 λDE3trxB− (Novagen). Isolated clones are inoculated in LB media containing kanamycin for selection at 37° C. After dilution, cultures are grown until an OD600 nm reaches 0.6-1.5. Expression of the fusion protein is initiated at 30° C. by adding IPTG to a final concentration of 1 mM, and continues for 3 hours. Cells are pelleted and an aliquot used to extract total protein, or to separate soluble from insoluble fractions. These samples are analyzed after separation on a polyacrylamide gel (Novex 4-12%) and staining with Coomassic Brilliant Blue.

[0169] FIG. 25 represents the results obtained with TrxA-abrogenN43 and TrxA-K4 from angiostatin. The results show that the proteins are expressed at the appropriate molecular weight (around 24 kD) and that they are soluble (around 50% for Trx-AbrogenN43 and 90% for Trx-K4). Similar results were obtained with Trx-abrogenD43 and TrxA-K5 from plasminogen.

Example 13

Purification of Abrogen From a Fusion Protein

[0170] The abrogen polypeptide can be liberated from the fusion protein using a cleavage site present in the fusion protein sequence and an appropriate cleavage enzyme. A variety of cleavage sites and related methods for cleaving a protein are available, including chemical cleavage and terminal peptidases. This example employs the thrombin cleavage site. A cell pellet of 25 grams (centrifugation pellet) from the E. coli BL21 λDE3trxB− (pXL4215) cells are taken up with 100 ml of 20 mM potassium phosphate (pH 7.4)-0.5 M NaCl (buffer A), containing 12,500 units of Benzonase™, 35 mg of lysozyme, 0.1% Triton X-100 and 0.5 mM EDTA. The suspension thereby obtained is incubated for 30 min at 37° C., and then centrifuged at 12,000×g for 60 min at +4° C. The supernatant is collected and injected onto a column of Sephadex G-25 (Amersham Biosciences) equilibrated with buffer A and the protein fraction is collected and loaded onto a Hi Trap Chelating HP column (Amersham Biosciences) previously loaded with Ni2+ and equilibrated with buffer A containing 10 mM imidazole. The Hi Trap Chelating column is washed with buffer A containing 100 mM imidazole, and the fraction containing fusion protein is eluted with 300 mM imidazole in buffer A. This fraction is chromatographed on a Sephadex G25 column (Amersham Biosciences) equilibrated with buffer A, collected, mixed with 2 μg of thrombin per mg of protein, and incubated for 16 h at 25° C. The resulting solution is injected onto a Hi Trap Benzamidine Sepharose Fast Flow column (Amersham Biosciences), equilibrated, and eluted with buffer A. The fraction that is not retained on the column is collected and loaded onto a second Hi Trap Chelating HP column previously loaded with Ni2+ and equilibrated with buffer A. The liberated kringle polypeptide is eluted from the column with a linear gradient of 0 to 150 mM imidazole in buffer A over 10 column volumes. Purified kringle polypeptide is buffer exchanged by gel filtration on a column of Sephadex G25 equilibrated with PBS (pH 7.4), filtered through a 0.2 μm filter and stored at +4° C. until use.

[0171] After this step, the polypeptide is substantially purified. Gel electrophoresis analysis shows a single band by SDS-PAGE after Coomassie staining, centered at a molecular weight estimated at around 10,000. It is unambiguously identified by N-terminal sequencing (10 amino-acids). Protein concentration is quantitated by Coomassie Blue staining with the Bradford reagent.

TABLE 1
Typical purification of uPA kringle from E. coli
BL21 λDE3trxB− (pXL4215)
		Volume
	Step	(mL)	Total protein (mg)
	Crude lysate	102	1020
	First Hi Trap Chelating HP	20	113
	column eluate
	Hi Trap benzamidine column	110	95
	eluate
	Second Hi Trap Chelating HP	8.0	34
	column eluate

[0172] The yield of purification of the peptides from the E.coli BL21 lambda DE3 trxB−, is set forth in the following Table 2.

TABLE 2
Purification of various kringle domains from E. coli
BL21 λDE3trxB−
		Wet cell pellet	Purified protein
peptides	Plasmid	(g)	obtained (mg)
Abrogen (N43)	pXL4189	7.5	8.7
Abrogen (D43)	pXL4215	25.0	34
Angiostatin	pXL4190	13.4	15.8
kringle 4
Plasminogen	pXL4219	24.5	45.6
kringle 5

[0173] These data demonstrate the successful production of soluble fusion protein, in an advantageously high percentage compared to prior methods, and the successful generation of biologically active abrogen polypeptide from this fusion protein.

Example 14

Biological Activity of Kringle Domains Produced in E. coli

[0174] The biological activity of abrogen D43 kringle domain produced in E. coli was assayed on a modified three-dimensional spheroid model (see Korff et al., FASEB J. 15:447 (2001)) using 800 HUVE (Human Umbilical Vein Endothelial) cells per spheroid in a fibrin matrix in the presence of pro-angiogenic factors bFGF and VEGF. This assay measures cell tubule formation induced by pro-angiogenic factors such as FGF and VEGF, which can be directly correlated to angiogenesis. Luminized tubule formation or inhibition of tubule formation is measured 11 days after addition, to the spheroid, of pro-angiogenic factors or of pro-angiogenic factors plus the test product. The plasminogen K5 kringle domain (Cao, Y., et al., J. Biol. Chem. 272(36):22924 (1997)) and the angiostatin K4 domain (Cao Y., et al., J. Biol. Chem. 271(56): 29461(1996)), also produced in E. coli were included as positive and negative inhibitors of angiogenesis, respectively. Tubule formation in the bFGF and VEGF treated HUVE cell spheroids is markedly inhibited only in the presence abrogen D43 and Plasminogen K5 kringle domains (see FIG. 27).

REFERENCES

[0175] The references cited below may be referred to above by the reference number. Each of the references is specifically incorporate herein by reference.

[0176] 1. Andreasen, P. A., et al., The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer, 1997. 72(1): p. 1-22.

[0177] 2. Mukhina, S., et al., The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. Characterization of interactions and contribution to chemotaxis. J Biol Chem, 2000. 275(22): p. 16450-8.

[0178] 3. Rabbani, S. A., et al., Structural requirements for the growth factor activity of the amino-terminal domain of urokinase. J Biol Chem, 1992. 267(20): p. 14151-6.

[0179] 4. Quax, P. H., et al., Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding. Arterioscler Thromb Vasc Biol, 1998. 18(5): p. 693-701.

[0180] 5. Min, H. Y., et al., Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. Cancer Res, 1996. 56(10): p. 2428-33.

[0181] 6. Li, H., et al., Systemic delivery of antiangiogenic adenovirus AdmATF induces liver resistance to metastasis and prolongs survival of mice. Hum Gene Ther, 1999. 10(18): p. 3045-53.

[0182] 7. Tang, H., et al., The urokinase-type plasminogen activator receptor mediates tyrosine phosphorylation of focal adhesion proteins and activation of mitogenactivated protein kinase in cultured endothelial cells. J Biol Chem, 1998. 273(29): p. 18268-72.

[0183] 8. Soff, G. A., Angiostatin and angiostatin-related proteins. Cancer Metastasis Rev, 2000.19(1-2): p.97-107.

[0184] 9. Kleiner, D. E., Jr. and W. G. Stetler-Stevenson, Structural biochemistry and activation of matrix metalloproteases. Curr Opin Cell Biol, 1993.5(5): p.891-7.

[0185] 10. Aguirre Ghiso, J. A., et al., Deregulation of the signaling pathways controlling urokinase production. Its relationship with the invasive phenotype. Eur J Biochem, 1999.263(2): p.295-304.

[0186] 11. Dong, Z., et al., Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell, 1997.88(6): p.801-10.

[0187] 12. Cao, Y., et al., Kringle domains of human angiostatin. Characterization of theanti-proliferative activity on endothelial cells. J Biol Chem, 1996. 271(46): p. 29461-7.

[0188] 13. Cao, Y., et al., Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem, 1997.272(36): p.22924-8.

[0189] 14. Nesbit, M., Abrogation of tumor vasculature using gene therapy. Cancer Metastasis Rev, 2000.19(1-2): p.45-9.

[0190] 15. Lee, T. H., T. Rhim, and S. S. Kim, Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells. J Biol Chem, 1998.273(44): p.28805-12.

[0191] 16. Rhim, T. Y., et al., Human prothrombin fragment 1 and 2 inhibit bFGF-induced BCE cell growth. Biochem Biophys Res Commun, 1998.252(2): p.513-6.

[0192] 17. Xin, L., et al., Kringle 1 of human hepatocyte growth factor inhibits bovine aortic endothelial cell proliferation stimulated by basic fibroblast growth factor and causes cell apoptosis. Biochem Biophys Res Commun, 2000.277(1): p.186-90.

[0193] 18. Chen, C. T., et al., Antiangiogenic gene therapy for cancer via systemic administration of adenoviral vectors expressing secretable endostatin. Hum Gene Ther, 2000. 11(14): p.1983-96.

[0194] The additional references below are also specifically incorporated herein by reference and cab be used, or any parts ussed, to make and use aspects of this invention.

[0195] Lee T-H, Rhim T, Kim SS. Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells. J Biol Chem 1998; 273(44): 28805-28812.

[0196] Lu H, Dhanabal M, Volk R, Waterman MJF, Ramchandran R, Knebelmann B, Segal M, Sukhatme VP. Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells. Biochem. Biophys. Res. Com. 1999; 258: 668-673.

[0197] Cao Y, Chen A, Seong Soo AA, Richard-Weidong J, Davidson D, Cao Y, Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem 1997; 272(36): 22924-22928.

[0198] Sauter BV, Martinet O, Zhang W-J, Mandeli J, Woo SLC. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases. Proc. Natl. Acad. Sci. 2000; 97(9): 4802-4807.

[0199] Li H, Lu H, Griscelli F, Opolon P, Sun L-Q, Ragot T, Legrand Y, Belin D, Soria J, Soria C, Perricaudet M, Yeh P. Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice. Gene Therapy 1998; 5: 1105-1113.

[0200] Dong Z, Yoneda J, Kumar R, Fidler IJ. Angiostatin-mediated suppression of cancer metastases by primary neoplasms engineered to produce granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 1998; 188(4): 755-763.

[0201] Cao Y, Ji RW, Davidson D, Schaller J, Marti D, Sohndel S, McCance SG, O'Reilly MS, Llinas M, Folkmann J. Kringle domains of human angiostatin. J. Biol. Chem. 1996; 271(56): 29461-29467.

[0202] Mukhina S, Stepanova V, Traktouev D, Poliakov A, Beabealashvilly R, Gursky Y, Minashkin M, Shevelev A, Tkachuk V. The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. J. Biol. Chem. 2000; 275(22): 16450- 16458.

[0203] Fischer K, Lutz V, Wilhelm O, Schmitt M, Graeff H, Heiss P, Nishiguchi T, Harbeck N, Luther T, Magdolen V, Reuning U. Urokinase induces proliferation of human ovarian cancer cells: characterization ofstructual elements required for growth factor function. FEBS Lett. 1998; 438(1-2): 101-105.

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[0206] Korff T, Kimmina S, Martiny-Baron G and Augustin H. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smopth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 2001; 15: 447-457.

Claims

1. A method of inhibiting angiogenesis in a subject comprising administering an abrogen polypeptide or a fusion construct thereof to a subject.

2. The method of claim 1, wherein the amino acid sequence of the abrogen polypeptide consists of one of SEQ ID NO.: 1, 3, 5, 7, 9, or 10 or the fusion construct consists of one of SEQ ID NO: 13, 14, 15, 17, 18, 20, or 21.

3. A method of expressing a soluble abrogen polypeptide-containing fusion protein comprising providing a vector or nucleic acid encoding a fusion protein, which comprises a thioredoxin sequence and an abrogen polypeptide sequence, whereby the fusion protein can be expressed in a bacterial cell, inserting the vector or nucleic acid into a bacterial cell to express the fusion polypeptide, and detecting the presence of soluble fusion protein.

4. The method of claim 3, wherein a substantial fraction of the total fusion protein is expressed in a soluble, stable form.

5. The method of claim 4, wherein the bacterial cell is E. coli, the thioredoxin has the sequence of SEQ ID NO: 22, and the abrogen polypeptide has the sequence of one of SEQ ID NO: 1, 3, 5, 7, 9, or 10.

6. A method of preparing an abrogen polypeptide composition, comprising the method of claim 5, wherein the vector or nucleic acid further comprises a proteolytic cleavage site for liberating the abrogen polypeptide sequence from the fusion polypeptide, and further comprising incubating the fusion protein with an appropriate cleavage enzyme to generate abrogen polypeptide molecules.

7. The method of claim 6, wherein the cleavage site is a thrombin cleavage site.

8. The method of claim 6, wherein the nucleic acid further comprises a purification tag placed in between the thioredoxine sequence and the cleavage site, and further comprising purifying the abrogen polypeptide from other components by chromatography.

9. The method of claim 8, further comprising adding a pharmaceutically acceptable excipient or carrier to the purified abrogen polypeptide.

10. The abrogen polypeptide obtained by the method of claim 8, wherein the abrogen peptide is substantially soluble and stable.

11. A composition comprising the abrogen polypeptide of claim 10, and a suitable carrier or excipient.

12. A method of inhibiting angiogenesis comprising administering an effective an animal or cell.