MXPA01001970A - Expression and export of angiostatin and endostatin as immunofusis - Google Patents

Abstract

Disclosed are nucleotide sequences, for example, DNA or RNA sequences, which encode an immunoglobulin Fc-angiogenesis inhibitor fusion protein. The angiogenesis inhibitors can be angiostatin, endostatin, a plasminogen fragment having angiostatin activity, or a collagen XVIII fragment having endostatin activity. The nucleotide sequences can be inserted into a suitable expression vector and expressed in mammaliancells. Also disclosed is a family of immunoglobulin Fc-angiogenesis inhibitor fusion proteins that can be produced by expression of such nucleotide sequences. Also disclosed are methods using such nucleotide sequences and fusion proteins for treating conditions mediated by angiogenesis.

Description

EXPRESSION AND EXPORTATION OF ANGIOGENESIS INHIBITORS AS IMMUNOFUSIN Field of the Invention This invention relates generally to methods and compositions for making and using fusion proteins containing an angiogenesis inhibitor. More particularly, the invention relates to methods and compositions for making and using fusion proteins called immunofusins which contain an immunoglobulin Fe region and an angiogenesis inhibitor.

Background of the Invention Two potent angiogenesis inhibitors, angioestatin (O'Reilly et al. (1994) Cell 79:31 5) and endoestatin (O'Reilly et al. (1997) Cell 88: 277), were discovered and found that are naturally generated by primary tumors. Both proteins are specific inhibitors of endothelial cell proliferation and inhibit tumor growth by blocking angiogenesis, the formation of new blood vessels that nourish tumors. Studies have shown that these angiogenesis inhibitors are non-toxic even at very high doses and that they can eliminate the growth of metastases and primary tumors can return to a latent microscopic state. Both inhibitors were identified as proteolytic fragments of much longer integral molecules. Angioestatin was found to be a fragment of plasminogen, and endoestatin a fragment of collagen XVIII.

These two proteins have generated great interest in the area of cancer because they have been shown to eliminate the growth of several different types of tumors in mice, with no obvious side effects or drug resistance. Generally, traditional chemotherapy is aimed at acquired drug resistance caused mainly by the genetic instability of cancer cells. Instead of targeting cancer cells, therapies that use angiogenesis inhibitors target normal endothelial cells, which support tumor growth. Because endothelial cells are genetically stable, it is possible that angiogenesis inhibitor therapies may result in less drug resistance. Studies indicate that drug resistance did not develop in mice exposed to prolonged anti-angiogenic therapy using endoestatin. In addition, repeated cycles of endo-statin treatment in mice resulted from a tumor lethargy and no recurrence of tumors following discontinuation of therapy (Boehm et al. (1997) Nature 390: 404). In spite of the promising results in mice, it has not been possible to produce active angioestatin and statin, soluble at clinical level, in commercial quantities that use E. coli, baculoviral, yeast, and mammalian expression systems. Expression in E. coli produced aggregates of insoluble protein of undefined composition, which could not be injected into humans. Other production methods, such as mammalian and baculoviral expression systems, produced very low levels of the recombinant proteins (O'Reilly et al. (1997) Cell 88: 277).

The scarce productions of expression systems to date can be explained by both angioestatin and endoestatin, which are internal fragments of much larger proteins. Truncated proteins can not bend properly in the absence of residues that break through precursor molecules. For example, angioestatin has 26 cysteine residues that form numerous disulfide bonds. The expression of angioestatin by itself can not provide the optimum environment for these numerous disulfide bonds to form correctly in the secretory path. Also, the endoestatin recombinant protein produced in E. coli was precipitated during dialysis, possibly due to the hydrophobicity of endostatin (O'Reilly, et al (1997) Cell 88: 277). A major challenge with the use of angiostatin and endoestatin in their present forms is that relatively large amounts of protein have to be injected daily for weeks to months to achieve the desired clinical outcome. For example, in recent mouse models, doses of endoestatin 20 mg / kg / day are necessary to demonstrate optimal efficacy (Boehm et al. (1997) Nature 390: 404). Since there is an urgent need to clinically test endostatin and angioestatin, a production method that can generate large amounts of clinical level material is important. An expression system that has been used to produce high level expression of fusion proteins in mammalian cells is a DNA construction coding, a signal sequence, a Fc immunoglobulin region and a target protein. The fusion product of this construct is generally referred to as an "immunofusin". Various target proteins have been successfully expressed as immunofusins including: IL2, CD26, Tat, Rev, OSF-2, ßlG-H3, IgE Receptor, PSMA, and gp1 20. These expression constructs are described in EU Patent No. 5,541 , 087 and U.S. Patent No. 5,726,044, the descriptions of which are incorporated herein by reference. A primary purpose of recombinant expression proteins in mammalian cells has been attempted to confer new or useful properties to hybrid molecules, eg, suitable folding, increased solubility, target of a cytokine or toxin in vivo, Fc receptor binding , complement fixation, protein A binding, increased augmented circulation light, and increased ability to cross the brain blood barrier. Examples of recombinant fusion proteins produced in mammalian cells included cytokine immunoconjugates (Gillies et al. (1992) Proc. Nati. Acad. Sci. USA 89: 1428; Gillies I went to. (1993) Bioconjugate Chemistry 4: 230), immunoadhesins (Capon et al. (1989) Nature 337: 525), immunotoxins (Chaudhary et al. (1989) Nature 339: 394), and a conjugate of nerve growth factor ( Friden et al. (1993) Science 259: 373). Each of the preceding publications is incorporated herein by reference. It is an object of the invention to provide novel DNAs that facilitate efficient production and secretion of angiogenesis inhibitors in a variety of mammalian host cells. It is another object of the invention to provide methods for treating mammals with nucleic acid coding, or amino acid sequences that define the angiogenesis inhibitor proteins, including non-native, biosynthetic, or otherwise artificial proteins such as proteins that have been created by rational design.

Brief Description of the Invention The present invention shows methods and compositions useful in the manufacture and use of fusion proteins containing an angiogenesis inhibitor protein. Fusion proteins can facilitate high level expression of biologically active angiogenesis inhibitor proteins. The angiogenesis inhibitor proteins can thus break through the fusion protein and combine with a pharmaceutically acceptable carrier prior to administration to a mammal, eg, a human. Alternatively, the coding of nucleic sequences, or amino acid sequences defining the angiogenesis inhibitor that contain the fusion proteins can be combined with a pharmaceutically acceptable carrier and administered to the mammal. In one aspect, the invention provides nucleic acid molecules, e.g., DNA or RNA molecules, that encode a fusion protein of the invention. The nucleic acid molecule encodes a signal sequence, an immunoglobulin Fc region, and at least one target protein, also referred to herein as the angiogenesis inhibiting protein, which is selected from the group consisting of angioestatin, endoestatin, a fragment of plasminogen having angioestatin activity, a fragment of collagen XVIII having endoestatin activity, and combinations thereof. In a preferred embodiment, the nucleic acid molecule encodes, serially in a 5 'to 3' direction, the signal sequence, the immunoglobulin Fc region and the target protein sequence. In another preferred embodiment, the nucleic acid molecule encodes, serially in a 5 'to 3' direction, the signal sequence, the target sequence, and the immunoglobulin Fc region. In another preferred embodiment, the Fc immunoglobulin region comprises an immunoglobulin linkage region and preferably comprises at least one immunoglobulin constant heavy region, eg, an immunoglobulin constant heavy domain 2 (CH2), a heavy domain 3 (CH3) immunoglobulin constant), and depending on the type of immunoglobulin used to generate the Fc region, optionally a heavy region domain 4 (CH4) immunoglobulin constant. In a more preferred embodiment, the immunoglobulin Fc region comprises an articulation region, a CH2 domain and a CH3 domain. Under certain circumstances, the Fc region of immunoglobulin preferably lacks at least the CHi domain. Although the Fc regions of immunoglobulin can be based on any class of immunoglobulin, for example, IgA, IgD, IgE, IgG, and IgM, immunoglobulin Fc regions that are based on IgG are preferred. In another embodiment, the nucleic acid of the invention can be incorporated in operative association into a replicable expression vector that can thus be transfected into a mammalian host cell. In another preferred embodiment, the invention provides host cell hosting such as nucleic acid sequences of the invention. In another aspect, the invention provides a fusion protein comprising an immunoglobulin Fc region linked, either directly through a polypeptide linkage or via a polypeptide linker program, to a target protein that is selected from the group that consists of angioestatin, endoestatin, a fragment of plasminogen having angioestatin activity, a fragment of collagen XVIII having endoestatin activity, and combinations thereof. The target protein can be fused via a C-terminal extremity to an N-terminal extremity of the immunoglobulin Fc region. However, in a more preferred embodiment the target protein is fused via its N-terminal extremity to a C-terminal extremity of the immunoglobulin Fc region. In another embodiment, the fusion protein may comprise a second target protein that is selected from the group consisting of angioestatin., endoestatin, a plasminogen fragment having angioestatin activity, a fragment of collagen XVIII having endoestatin activity. In this type of construction the first and second target fusion proteins can be the same or different proteins. For example, in a preferred embodiment, the fusion protein comprises a first target protein of angioestatin, an Fc region of immunoglobulin and a second target protein of endostatin. The first and second target proteins can be linked together, either directly or by means of a polypeptide linker program. Alternatively, both target proteins can be linked, either directly or via a polypeptide linker program, to the Fc region of immunoglobulin. In the latter case, the first target protein is connected to an N-terminal extremity of the immunoglobulin Fc region and the second target protein is connected to a C-terminal extremity of the immunoglobulin Fc region. In another embodiment, two fusion proteins can be associated, either covalently, for example, with a disulfide or peptide bond, or non-covalently, to produce a multimeric protein. In a preferred embodiment, two fusion proteins are covalently associated by means of one or more disulfide bonds through cysteine residues, preferably located within the immunoglobulin articulation regions disposed within the immunoglobulin Fc regions of both chains. In a preferred embodiment, the target protein comprises a fragment of plasminogen having a molecular weight of about 40 kD and, optionally, comprising an amino acid sequence as set forth in SEQ ID NO: 3. In another preferred embodiment, the target protein comprises a fragment of collagen XVIII having an amino acid sequence set forth in SEQ ID NO: 1. In addition, the target protein may be full length of angioestatin or endoestatin or bioactive fragments thereof. The source of the target protein in the generation of certain fusion proteins will depend on the proposed use of the target protein. For example, if the target protein is to be administered to a human, the target protein is preferably of human origin. In another aspect, the invention provides methods of producing a fusion protein comprising an Fc region of immunoglobulin and a target protein that is selected from the group consisting of angioestatin, endoestatin, a fragment of plasminogen having angioestatin activity, and a fragment of collagen XVIII having endoestatin activity. The method comprises the steps of (a) providing a mammalian cell containing a DNA molecule encoding such as a fusion protein, either with or without a signal sequence, and (b) culturing the mammalian cell to produce the fusion protein. . The resulting fusion protein can thus be cultured, redoubled, if necessary, and purified using conventional purification techniques well known and used in the art. Assuming that the fusion protein comprises a proteolytic cleavage site disposed between the Fc region of immunoglobulin and the target protein, the target can be cleaved from the fusion protein using conventional proteolytic enzymes and if necessary, purified before use. In another aspect, the invention provides methods for treatment of mammals, for example, a human, in need of a therapy based on an angiogenesis inhibitor. For example, it is contemplated that the angiogenesis inhibitors of the invention may be administered to a human suffering from a tumor. Treatment with the angiogenesis inhibitor can slow or stop tumor growth and, under certain circumstances, can cause tumor regression. The treatment may include administering to the mammal an amount of the angiogenesis inhibitor in an amount sufficient to decrease or arrest the growth of the tumor. The angiogenesis inhibitor can be provided in the form of a fusion protein or as a nucleic acid, preferably operatively associated with an expression vector, in combination with a pharmaceutically acceptable carrier. The foregoing and other objects, features and advantages of the present invention will become more apparent from the detailed description, drawings and claims that follow.

Brief Description of the Drawings Figures 1 A-1 F are schematic illustrations of exemplary angiogenesis inhibitor fusion proteins constructed in accordance with the invention (see Examples 10-1 5). The Figures represent, respectively, Figure 1A, Fc-Kringle 1 of Angioestatin; Figure 1 B, Fe-interior Kringle 1 of Angioestatin; Figure 1 C, Fc-Endoestatin-GlySer Kringle 1 internal linker program of Angioestatin; Figure 1 D, Fc-Endoestatin-GlySer linker program-Kringle 1 of Angioestatin; Figure 1 E, Fc-Endoestatin-GlySer linker-Angioestatin program; Figure 1 F, Angioestatin-Fc-Endoestatin. The vertical lines represent the optional disulfide bonds that connect the cysteine residues (C) of disposition within a joint region of the Fc molecule.

Detailed Description of the Invention The invention provides fusion proteins, referred to herein as immunosufins, which are useful in the production of commercial quantities of angiogenesis inhibitors at the clinical level. Angiogenesis inhibitors can break through the immunosufin protein constructs before being used. However, it is contemplated that immunosufins or nucleic acids encoding immunosufins can be administered directly to mammals in need of treatment with an angiogenesis inhibitor. The invention in this manner provides fusion proteins comprising an Fc region of immunoglobulin and at least one target protein, referred to herein as an inhibitor of angiogenesis. The angiogenesis inhibitor is preferably selected from the group consisting of angioestatin, endoestatin, an angioestatin activity of plasminogen fragment, a fragment of collagen XVIII having endoestatin activity. It is contemplated, however, that other polypeptides having angiogenesis inhibitor activity, now known or recently discovered, can be expressed as fusion proteins of the type described herein. Six exemplary embodiments of the protein constructs embodying the invention are illustrated in the drawings as Figures 1A-1F. Because dimeric constructs are preferred, they are all illustrated as dimers degraded by a pair of disulfide bonds between cysteines in adjacent subunits. In the drawings, the disulfide bridges are represented as a bond together with the portions of two immunoglobulin Fc regions via an immunoglobulin articulation region, and thus are characteristic of native forms of these molecules. Although constructs including the Fc joint region are preferred and have shown promise as therapeutic agents, the invention contemplates that crosslinking in other positions may be chosen as desired. In addition, under some circumstances, the dimers or multimers useful in the practice of the invention can be produced by non-covalent association, for example, by hydrophobic interaction. Because homodimeric constructions are important embodiments of the invention, Figure 1 illustrates such constructions. It should be appreciated that heterodimeric structures are also useful but, as is known to those skilled in the art, they can often be difficult to purify. However, viable constructs useful for inhibiting angiogenesis in various mammalian species, including humans, can be constructed comprising a mixture of homodimers and heterodimers. For example, one chain of the heterodimeric structure may comprise endo-statin and the other may comprise angio-statin. Figure 1 A illustrates a dimer construct produced according to the procedure set forth in Example 10. Each monomer of the dimer comprises an immunoglobulin 1 Fc region that includes a hinge region, a CH2 domain and a CH3 domain. Attached directly to the C terminus of the FC 1 region is the first Kringle region of angioestatin 2, both inner and outer rings.

Figure 1 B shows a second embodiment of the invention (see Example 1 1) comprising the same Fc region as in Figure 1 A, this time having only the Kringle inner ring one of angiostatin 3 attached to the terminal extremity C of the Fc 1 region. Figures 1C to 1E represent various embodiments of the protein constructs of the invention, which include plural angiogenesis inhibitors of target protein adjusted in series and connected by a linker program. In Figure 1C, the target protein comprises full length endoestatin 4, a polypeptide 5 linker program, and the Kringle internal ring one of angiostatin 3. Figure 1 D represents a protein comprising an Fc region the same as the Figure 1A and a target protein comprises a full-length endoestatin 4, a polypeptide 5 linker program, and a Kringle complete one angioestatin region (both inner and outer rings) 2. Figure E differs from the construction of Figure 1 D wherein the C-plus protein domain comprises a full length copy of angioestatin 7. Although FIGS. 1 A-1 E represent the Fc-X type constructs, where X is the target protein, It is contemplated that X-Fc constructs may be useful in the practice of the invention. In addition, it is contemplated that the useful proteins of the invention may also be represented by the formula X-Fc-X, wherein the Xs may represent the same or different target proteins. Figure 1 F represent such a construct comprising in a terminal N to C direction, full length human angioestatin 7, an Fc 6 region of human immunoglobulin including an articulation region, and full length human endostatin 4 domain. The term "angiogenesis inhibitor", as used herein, refers to any polypeptide chain that reduces or inhibits the formation of new blood vessels in a mammal. With respect to cancer therapy, the angiogenesis inhibitor reduces or inhibits the formation of new blood vessels in or on a tumor, preferably in or on a solid tumor. It is contemplated that useful angiogenesis inhibitors can be identified by using a variety of well-known and used assays in the art. Such assays include, for example, the bovine capillary endothelial cell proliferation assay, the chick chorioallantoic membrane (CAM) assay or the mouse cornea assay. However, the CAM assay is preferred (see, for example, O'Reilly et al. (1994) Cell 79: 315-328 and O'Reilly et al. (1997) Cell 88: 277-285, descriptions of the which are incorporated herein by reference). In short, embryos with whole yolks are removed from fertilized white eggs three days old and placed in a petri dish. After incubation at 37 ° C, 3% C02 for three days, a methylcellulose disk containing the putative angiogenesis inhibitor is delivered to the chorioallantoic membrane of an individual embryo. After incubation for approximately 48 hours, the chorioallantoic membranes were observed under a microscope for evidence of zones of inhibition. Preferred useful angiogenesis inhibitors in the practice of the invention include, for example, angioestatin (O'Reilly, et al. (1 994) Cell 79: 31 5-328, and US Patent Nos. 5,733,876; 5,837,682 and 5,885,795). , and endoestatin (O'Reilly et al., (1 997) Cell 88: 277-285 and U.S. Patent No. 5,854,205). As previously established, angioestatin and endoestatin are inhibitors of specific endothelial cell proliferation and are capable of inhibiting tumor growth by blocking angiogenesis, the formation of new blood vessels that nourish tumors. Angioestatin has been identified as a proteolytic fragment of plasminogen (O'Reilly et al. (1 994) Cell 79: 31 5-328, and U.S. Patent Nos. 5,733,876; 5,837,682; and 5,885,795, the description of which is incorporated herein by reference). Specifically, angioestatin is an internal 38 kDa fragment of the plasminogen that contains at least three of the Kringle regions of plasminogen. Endoestatin has been identified as a proteolytic fragment of collagen XVIII (O'Reilly et al. (1997) Cell 88: 277-285, the disclosure of which is incorporated herein by reference). Specifically, the endoestatin is a 20 kDa fragment of C-terminal collagen XVIII. The terms "angioestatin" and "endoestatin", as used herein, refer not only to full-length proteins, but also to variants and bioactive fragments thereof, as well as to bioactive fragments of plasminogen and collagen XVIII. , respectively. The term "bioactive fragment", with respect to angioestatin, refers to any plasminogen or angiostatin protein fragment having at least 30%, more preferably at least 70%, and more preferably at least 90% of the full-length angioestatin activity as determined by the CAM assay. The term "bioactive fragment", with respect to endo-statin, refers to any protein fragment of collagen XVIII or endoestatin having at least 30%, more preferably at least 70%, and more preferably at least 90% of the endoestatin activity in length complete as determined by the CAM assay. The term "variants" includes specifications and allelic variants, as well as other naturally occurring or non-naturally occurring variants, for example, generated by conventional genetic engineering protocols, which are at least 70% similar or 60% identical, more preferably at least 75% similar or 65% identical, and more preferably 80% similar or 70% identical to either the naturally occurring endoestatin or angioestatin sequences described herein. To determine whether a candidate polypeptide has the requisite similarity or identity to a reference polypeptide, the candidate amino acid sequence and the reference amino acid sequence are first aligned by using the dynamic programming algorithm described in Smith and Waterman (1981) , J. Mol. Bio. 147: 1 95-197, in combination with the BLOSUM62 substitution binder described in Figure 2 of Henikoff and Henikoff (1992), "Amino acid substitution matrices from protein blocks", Proc. Nati Acad. Sci. USA 89: 10915-10919. For the present invention, a suitable value for the output insertion penalty is -12, and a suitable value for the output extension penalty is -4. Performance alignments of computer programs using the Smith-Waterman algorithm and the BLOSUM62 binder, such as the GCG program series (Oxford Molecular Group, Oxford, England), are commercially available and widely used by those skilled in the art. . Once the alignment between the candidate and reference sequence is done, a similarity percent score can be calculated. The individual amino acids of each sequence are compared sequentially according to their similarity to each other. If the value in the BLOSUM62 binder corresponding to the two aligned amino acids is zero or a negative number, the pair similarity score is zero; or otherwise the pair similarity score is 1 .0. The raw similarity score is the sum of the pairs similarity scores of the aligned amino acids. The raw score is then normalized by dividing it by the number of amino acids in the smaller candidate or reference sequences. The normalized raw score is the percent similarity. Alternatively, to calculate a percent identity, the aligned amino acids of each sequence are again compared sequentially. If the amino acids are not identical, the identity of couples is zero; or otherwise the pair identity score is 1 .0. The raw identity score is the sum of the identical aligned amino acids. The raw score is then normalized by dividing it by the number of amino acids in the smaller candidate or reference sequences. The normalized raw score is the percent identity. Insertions and deletions are ignored for the purposes of calculating percent similarity and identity. According to the above, the exit penalties are not used in this calculation, although they are used in the initial alignment. The proteins described herein are expressed as fusion proteins with an Fc region of an immunoglobulin. As is known, each immunoglobulin heavy chain constant region is comprised of four or five domains. The domains are referred to sequentially as follows: CH1-joint-CH2-CH3 (-CH4). The DNA sequences of the heavy chain domains have cross homology between the immunoglobulin classes, for example, the CH2 domain of IgG is homologous to the CH2 domain of IgA and IgD, and to the CH3 domain of IgM and IgE. As used herein, the term "immunoglobulin Fc region" is understood to mean the carboxyl terminal 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) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3) a C ^ domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a combination of two or more domains and an immunoglobulin articulation region. In a preferred embodiment the Fc region used in DNA construction includes at least one immunoglobulin-binding region, one CH2 domain and one CH3 domain and preferably lacks at least the CH domain. The recently preferred class of immunoglobulin of which the region heavy chain constant is derived is IgG (Ig?) (subclasses and 1, 2, 3, or 4). Other classes of immunoglobulin, IgA (Ig), IgD (Igd), IgE (Ige), and IgM (Igμ), can be used. The choice of suitable immunoglobulin heavy chain constant regions is discussed in detail in the U. Nos. 5,541, 087 and 5,726,044. The choice of immunoglobulin heavy chain constant region sequences particular to certain classes and subclasses of immunoglobulin to achieve a particular result is considered to be within the level of skill in the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of an articulation domain, and preferably at least a portion of a CH3 domain of Fc? or homologous domains in either IgA, IgD, IgE, or IgM. Depending on the application, the constant region genes of other species than the human eg mouse or rat, can be used. The Fc region used as a fusion partner in the construction of immunosufin DNA can generally be of any of the mammalian species. When it is undesirable to deduce an immune response in the host cell or animal against the Fc region, the Fc region can be derived from the same species as the host cell or animal. For example, human Fc can be used when the animal or host cell is human; likewise, murine Fc can be used when the cell or host animal is a mouse. In addition, substitution or deletion of the constructs of these constant regions, in which one or more amino acid residues of the constant region domains are substituted or deleted, would also be useful. One example could be introducing amino acid substitutions in the upper CH2 region to create an Fc variant with reduced affinity for the Fc receptors (Colé et al (1 997) J. Immunol., 59: 361 3). One of ordinary skill in the art can prepare such constructions by using well-known molecular biology techniques. The use of human Fc? 1 as the Fc region sequence has several advantages. For example, if the Fc fusion protein of angiogenesis inhibitor is to be used as a biopharmaceutical, the Fc? 1 domain can confer effector function activities to the fusion protein. Effector function activities include biological activities such as complement fixation, antibody-directed cellular cytotoxicity, placental transfer, and increased serum half-life. The Fc domain is also provided for the detection by ELISA of anti-Fc and purification through binding to protein A Staphylococcus aureus ("Protein A"). In certain applications, however, it may be desirable to suppress specific effector functions of the Fc region, such as Fc receptor binding or complement fixation. In the case of angiogenesis inhibitor immunofusins, a function of the immunoglobulin Fc fusion partner is to facilitate the proper folding of the angiogenesis inhibitor protein to produce active protein of angiogenesis inhibitor and to impact the solubility of the active elements, at least in the extracellular milieu. Since the Fc fusion partner is hydrophilic, the angiogenesis inhibitor immunofusin is easily soluble unlike, for example, the recombinant endoestatin produced in E. coli (O'Reilly (1997) Cell 88: 277). In all the Examples described herein, high levels of production of the immunosufins were obtained. The angiogenesis inhibitor immunofusins were secreted in media at concentrations typically from about 30 to 1000 μg / ml, and could easily be purified to homogeneity by Protein A chromatography. In addition, immunosufins of angiogenesis inhibitor could break through and in addition be purified by using conventional purification protocols using, for example, heparin sepharose, lysine sepharose or affinity purification. In addition to high expression levels, the fusion proteins of the invention also show higher serum half-lives, presumable due to their larger molecular sizes. For example human angioestatin of human Fc has a serum half-life of 33 hours in mice, compared to 4-6 hours for human angioestatin (O'Reilly et al. (1996) Nature Medicine 2: 689). It is believed that the angioestatine with a molecular weight of 40 kD, and the endoestatin with a molecular weight of 20 kD, are sufficiently small to be effectively cleaned by renal filtration. In contrast, the dimeric forms of Fc angioestatin and dimeric Fc endoestatin are 145 kD and 100 kD, respectively, because there are two immunoglobulin Fc regions linked to either two angiostatin molecules or two endoestatin molecules. Such a bivalent structure may show a higher binding affinity to the angioestatin or endo-statin receptor. If the angiogenesis inhibitory activity is mediated by receptor, the Fc fusion proteins are potentially more effective in eliminating the tumors than the monovalent angioestatin or monovalent endoestatin by themselves. In addition, if the angioestatin and / or endostatin belong to a class of dimeric protein binders, the physical bond imposed by the Fc on angioestatin or endoestatin could make the dimerization an intramolecular process, thus shifting the balance in favor of the dimer and improving its union with the receiver. The cysteine residues can also be introduced by the standard recombinant DNA technology to the monomer at suitable sites to stabilize the dimer through the formation of the covalent disulfide bond. As used herein, the term "multivalent" refers to a recombinant molecule that incorporates two or more biologically active segments. The protein fragments that make up the multivalent molecule can be linked through a polypeptide peptide linker program that binds the constituent parts without causing a structure shift and allows each to function independently. As used in this, the term "bivalent" refers to a recombinant multivalent molecule having two target proteins in a fusion construct of the invention, for example, an Fc-X molecule, wherein X is independently selected from angioestatin, endoestatin, or a variant of the same. Since there are two X elements fused to an immunoglobulin Fc region (which typically is itself a dimer of the heavy chain fragments that includes at least a portion of the CH3 domain and articulation region, and optionally the CH2 domain) , the molecule is bivalent (see, for example, Figure 1 A). If the fusion construct of the invention has the Fc-X-X form, the resulting Fc dimer molecule is tetravalent. The two proteins that make up the Fc-X-X molecule can be linked through a peptide linker program. For example, if a portion of endoestatin of an Fc endoestatin can bind to a receptor in a cell with a certain affinity, the second endostatin portion of the same Fc endoestatin can bind to a second receptor in the same cell with much more force. high (apparent affinity). This is due to the physical proximity of the second portion of endoestatin to the recipient after the first portion of endoestatin is already bound. In the case of an antibody binding to antigen binding, the apparent affinity is increased by at least 1 04. As used herein, the terms "multimer" and "multimeric" refer to the stable association of two or more chains of polypeptide either covalently, for example, by means of a covalent interaction, for example, by a disulfide bond or non-covalently, for example, by hydrophobic interaction. The term multimer is proposed to comprise both homomultimers, wherein the polypeptides are the same, as well as heteromultimers, wherein the polypeptides are different. As used herein, the term "dimeric" refers to a specific multimeric molecule where two protein polypeptide chains are stably associated through covalent or non-covalent interactions. It should be understood that the Fc fragment of the immunoglobulin Fc region itself is typically a dimer of the heavy chain fragments that includes at least a portion of the hinge region and the CH3 domain, and optionally the CH2 domain. Several protein binders are known to bind to their receptors as a dimer. If an X protein binder naturally dimerizes, the X element in an Fc-X molecule will dimerize to a much greater extent, since the dimerization process is concentration dependent. The physical proximity of the two X elements connected by the Fc region of associated immunoglobulin could make the dimerization an intramolecular process, mostly shifting the balance in favor of the dimer and improving its binding to the receptor. It is understood that the present invention exploits conventional recombinant DNA methodologies to generate the Fc fusion proteins useful in the practice of the invention. The Fc fusion constructs are preferably generated at a DNA level, and the resulting DNAs were integrated into the expression vectors, and expressed to produce the immunofusins. As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence to be incorporated into a host cell and to recombine with and integrate into the host cell genome or to duplicate autonomously as an episome . Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector include a retrovirus, an adenovirus and an associated adenovirus. As used herein, the term "gene expression" or "expression" of a target protein is understood to mean the transcription of a DNA sequence., translation of mRNA transcription and secretion of an Fc fusion protein product. A useful expression vector is pdCs (Lo e al. (1988) Protein Engineering 1 1: 495, the description of which is incorporated herein by reference) in which the transcription of the Fc-X gene utilizes the enhancer / promoter of the human cytomegalovirus and the SV40 polyadenylation signal. The enhancer and promoter sequence of the human cytomegalovirus used was derived from nucleotides -601 to +7 of the sequence provided in Boshart et al. , 1 985, Cell 41: 521, the description of which is incorporated herein by reference. The vector also contains the mutant dihydrofolate reductase gene as a selection marker (Simonsen and Levinson (1983) Proc. Nati, Acad. Sci USA 80: 2495, the disclosure of which is incorporated herein by reference). An appropriate host cell can be transformed or transfected with the DNA sequence of the invention, and used for the expression and secretion of a target protein. Freshly preferred host cells for use in the invention include immortal hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary cells, Hela cells, and COS cells. The fusion proteins of the invention are preferably generated by conventional recombinant DNA methodologies. The fusion proteins are preferably produced by the expression in a host cell of a DNA molecule encoding a signal sequence, an immunoglobulin Fc region and a target protein (also referred to herein as an angiogenesis inhibitor). Preferred constructs can encode in a 5 'to 3' direction, the signal sequence, the immunoglobulin Fc region and the target protein. Alternatively, the constructs may encode in a 5 'to 3' direction, the signal sequence, the target protein and the immunoglobulin Fc region. As used herein, the term "signal sequence" is understood to mean a segment of a peptide that directs the secretion of the immunofusin protein of angiogenesis inhibitor and opens thereafter following translation in a host cell . The sequence signal of the invention is a polynucleotide, which encodes an amino acid sequence that initiates the transport of a protein through the membrane of the endoplasmic reticulum. Signal sequences that will be useful in the invention include antibody light chain signal sequences, eg, antibody 14.18 (Gillies et al., 1989, Jour.of Immunol.Meth., 125: 191-202), antibody heavy chain signal signal, for example, the MOPC141 antibody heavy chain sequence signal (Sakano et al., 1980, Nature 286: 5774), and any other signal sequences that are known in the art (see for example, Watson, 1984, Nucleic Acids Research 12: 5145). Each of these references are incorporated herein by reference. The signal sequences have been well characterized in the art and are typically known to contain from 16 to 30 amino acid residues, and may contain higher or lower amino acid residues. A typical signal peptide consists of three regions: an N-terminal basic region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains from 4 to 12 hydrophobic residues that anchor the signal peptide through of the membrane lipid bilayer during transport of the growing polypeptide. Following initiation, the signal peptide usually makes its way into the lumen of the endoplasmic reticulum by cellular enzymes known as signal peptidases. The potential dissociation sites of the signal peptide generally follow the "rule (-3, -1)". In this way a typical signal peptide has neutral, small amino acid residues at positions -1 and -3 and lacks proline residues in this region. The signal peptidases will open passage to such a signal peptide between amino acids -1 and +1. In this way, the portion of the DNA encoding the signal sequence can be cleaved from the amino terminus of the immunofusin protein during secretion. This results in the secretion of an immunofusin protein consisting of the Fc region and the target protein. A detailed discussion of the signal peptide sequences is provided by von Heijne (1986) Nucleic Acids Res., 14: 4683 the description of which is incorporated herein by reference. As would be apparent to one skilled in the art, the suitability of a particular signal sequence for use in the invention may require some routine experimentation. Such experimentation will include determining the ability of the signal sequence to direct the secretion of an immunofusin and also a determination of the optimal configuration, genomic or cDNA, of the sequence to be used in order to achieve efficient secretion of immunofusins. Additionally, a person skilled in the art is capable of creating a synthetic signal peptide following the rules presented by von Heijne, referred to above, and testing the efficacy of such a synthetic signal sequence by routine experimentation. A sequence signal can also be referred to as a "signal peptide", "leader sequence", or "leader peptide". The fusion of the signal sequence and the immunoglobulin Fc region is sometimes referred to herein as a secretion cassette. An exemplary secretion cassette useful in the practice of the invention is a polynucleotide that encodes, in a 5 'to 3' direction, a signal sequence of an immunoglobulin light chain gene and an Fcγ1 region of the immunoglobulin gene. 1 human The Fc? 1 region of the Fc? 1 immunoglobulin gene preferably includes at least a portion of the joint domain and at least a portion of the CH3 domain, or alternatively at least portions of the joint domain, CH2 domain and CH3 domain. The DNA encoding the secretion cassette can be found in its genomic configuration or its cDNA configuration. In another embodiment, the DNA sequence encodes a proteolytic cleavage site interposed between the secretion cassette and the angiogenesis inhibitor protein. A dissociation site provides the proteolytic cleavage of the fusion protein encoded in this manner separates the Fc domain from the angiogenesis inhibitor protein. As used herein, the "proteolytic cleavage site" is understood to mean amino acid sequences that are preferably opened by a proteolytic enzyme or other proteolytic cleavage agents. Useful proteolytic cleavage sites include amino acid sequences that are recognized by proteolytic enzymes such as trypsin, plasmin or enterokinase K. Many of the cleavage agent / cleavage site pairs are known. See, for example, U.S. Patent No. 5,726,044, the disclosure of which is incorporated herein by reference. When the target protein sequence is a precursor molecule to angioestatin, endo-statin, or an active variant thereof, the desired protein product can be produced by dissociation with the endogenous proteolytic enzyme, such as elastin or plasmin or urokinase. The present invention also comprises fusion proteins containing different combinations of angioestatin and recombinant endoestatin, fragments thereof, which can be made in large quantities. Despite the demonstrated efficacy in tumor suppressor growth, the mechanism of how angioestatin and endoestatin blocks angiogenesis is not fully understood. Angioestatin has various Kringle structures and endo-statin has different structural motifs, each of which may be solely responsible for or aiding in the binding of proteins to endothelial cells and exert an anti-angiogenic effect. According to the above, this invention includes target proteins which are bioactive fragments of angioestatin, such as Kringle 1, Kringle 2, Kringle 3, and combinations thereof, and endoestatin which shows physiologically similar behavior to angioestatin and long-chain statin complete that occur naturally. Another embodiment of the present invention is provided for bifunctional hybrid constructs of angiogenesis inhibitors. As used in this, a bifunctional hybrid molecule or construction means a protein produced by combining two protein subunits, where the two subunits can be derived from different proteins. Each protein subunit has its own independent function such that in the hybrid molecule, the functions of the two subunits can be additive or synergistic. Such functional hybrid proteins could allow to explore the synergistic effect of angioestatin and endoestatin in animal models. A preferred bifunctional hybrid may comprise at least two different angiogenesis inhibitors linked in series, either directly or by means of a polypeptide linker program. For example, in a preferred embodiment, the target sequence encodes at least a portion of angioestatin bound in the structure with at least a portion of endoestatin and both the angioestatin and endostatin domains show anti-angiogenesis activity or inhibition of angiogenesis. The two units can be linked by a polypeptide linker program. As used herein the term "polypeptide linker program" is understood to mean a peptide sequence that can bind two proteins together or a protein and an Fc region. The polypeptide linker program preferably comprises a plurality of amino acids such as glycine and / or serine. Preferably, the polypeptide linker program comprises a series of glycine and serine peptides of approximately 10-1 5 residues in length. See, for example, U.S. Patent No. 5,258,698, the disclosure of which is incorporated herein by reference. It is contemplated, however, that the length of the optimal linker program and the amino acid composition can be determined by routine experimentation. It was found that when the different parts of the angioestatin are expressed as the Fc fusion molecules, high expression levels are obtained, presumably because the Fc portion acts as a vehicle, helping the polypeptide in the C terms to fold correctly. In addition, the Fc region can be glycosylated and highly charged at physiological pH, in this way the Fc region can help to solubilize the hydrophobic proteins. The present invention also provides methods for the production of angioestatin and endoestatin from non-human species such as Fc fusion proteins. Non-human angiogenesis inhibitor fusion proteins are useful for preclinical studies of angiogenesis inhibitors because protein drug efficacy and toxicity studies should be carried out in animal model systems before being tested in humans. A human protein may not work in a mouse model because the protein can deduce an immune response, and / or show different pharmacokinetics that falsify the test results. Therefore, the equivalent mouse protein is the best substitute for the human protein to be tested in a mouse model. The standard Lewis lung carcinoma model in mice (O'Reilly et al (1 997) Cell 88: 277) was used to compare the huFc-huAngiostatin soluble, huFc-huEstastatin, muFc-muAngiostatin, muFc-muEstastatin with the proteins insolubles produced in an E. co // 7 expression system Soluble Fc fusion proteins were more effective in suppressing tumor growth in the Lewis lung model than the corresponding proteins produced in E. coli. In addition, laboratory mice are consanguineous and their tumors are induced and are not spontaneous. Therefore, efficacy in a mouse model can not be correlated for probable efficacy against human tumors. Preclinical studies in dogs will provide more accurate information about the efficacy of these angiogenesis inhibitors in spontaneous tumors because there are numerous spontaneous canine tumors occurring naturally. Methods to produce murine Fc-mu angiostatin (mu), endo-statin muFc-mu, and canine Fc-ca angioestatin (ca), cafc-ca endoestatin of the present invention will facilitate preclinical studies of angiogenesis inhibitors in both murine and canine systems. The present invention provides methods of treating a condition mediated by angiogenesis by administering the DNA, RNA or proteins of the invention. Conditions mediated by angiogenesis include, for example: solid tumors; tumors of blood origin, tumor metastasis, benign tumors that include hemangiomas, acoustic neuromas, neurofibromas, trachoma, and pyrogenic granulomas: rheumatoid arthritis; psoriasis; ocular angiogenic diseases (diabetic retinopathy, retinopathy of prematurity, macular degeneration, rejection of corneal graft, neovascular glaucoma) retrolental fibroplasia, rubeosis, Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; connections of hemophiliac angiofibroma; and a granulation of injury; and excessive or abnormal stimulation of endothelial cells, intestinal adhesions, atherosclerosis, sclerodermal and hypertrophic scars, that is, keloids. The DNA constructs described herein may be useful in gene therapy procedures in which the endoestatin or angioestatin gene is delivered into a cell by one or several means eg, native DNA associated with a promoter or DNA within a viral vector. Once inside a cell, the gene or fragment of angioestatin and / or endostatin gene is expressed and the protein is produced in vivo to carry out its normal biological function. The DNA construct of the present invention results in high levels of expression of the fusion protein. The fusion proteins of the present invention may also be useful in the treatment of conditions mediated by angiogenesis and may have greater clinical efficacy than the native angiogenesis inhibitors and other recombinant angiogenesis inhibitors because the angiogenesis inhibitor immunofusins of the present invention invention have a longer serum half-life than the other recombinant angiogenesis inhibitors or the unique native angiogenesis inhibitors. The bivalent and dimeric forms of the present invention should have higher binding affinity due to the bivalent and dimeric structure. The bifunctional hybrid molecules of the present invention may have a higher clinical efficacy due to the possible synergistic effects of two different angiogenesis inhibitors connected by the fused Fc region or a flexible polypeptide linker program. The compositions of the present invention can be provided to an animal by any suitable means, directly (eg, locally, such as by injection, implantation or topical administration at a tissue site) or systemically (eg, parenterally or orally). When the composition is provided parenterally, such as by intravenous, subcutaneous, ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intratranase administration; or by aerosol, the composition preferably comprises part of a suspension or solution of physiologically compatible or aqueous fluid. In this way, the vehicle is physiologically acceptable such that in addition to the provision 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 in this manner may comprise normal physiological saline (eg, 9.85% aqueous NaCl, 0.15 M, pH 7-7.4). Preferred doses of the immunofusins per administration are within the range of 50 ng / m2 to 1 g / m2, more preferably 5 μg / m2 to 200 mg / m2, and more preferably 0.1 mg / m2 to 50 mg / m2. Preferred doses of nucleic acids encoding immunofusins by administration are within the range of 1 μg / m2 to 1 00 mg / m2, more preferably 20 μg / m2 to 10 mg / m2, and more preferably 400 μg / m2 to 4 mg / m2. It is contemplated, however, that optimal modes of administration, and doses can be determined by good routine experimentation within the level of experience in the art. The invention is further illustrated by the following non-limiting examples. EXAMPLES Example 1. Expression of huFc-huEstastatin Human endoestatin was expressed as a human fusion protein of human Fc endoestatin (huFc-huEndo) according to the teachings of Lo e al. (1998) Protein Engineering 1 1: 495. Fc refers to the Fc fragment of the human immunoglobulin range (DNA sequence set forth in SEQ ID NO: 1; amino acid sequence set forth in SEQ ID NO: 2). (PCR polymerase chain reactions) was used to adapt the cDNA endoestatin (SEQ ID NO: 3, whose amino acid sequence is described in SEQ ID NO: 4), for expression in an Fc-Endo fusion protein. The front primary was either 5'-CC CCG GGT AAA CAC AGC CAC CGC GAC TTC C (SEQ ID NO: 5); encoded amino acids described in SEQ ID NO: 6) or 5'-C AAG CTT CAC AGC CAC CGC GAC TTC C (SEQ ID NO: 7; encoded amino acids described in SEQ ID NO: 8), where the Xmal site or the Hindlll site were followed by the sequence encoding the N-terminus of endo-statin. The primary one with the Xmal site adopted the cDNA endodestatin for ligation to the Xma l site at the extremity of the CH3 domain of the IgGFc region. The primer with the Hind II site adopted the endoestatin cDNA for ligation to the HindIII site of the pdCs-Fc vector (DK), which contains the enterokinase recognition site Asp4-Lys (LaVallie et al (1993) J. Bio. Chem. 268: 2331 1 -2331 7) in the connection of the fusion protein. The subsequent primer was 5'-C CTC GAG CTA CTT GGA GGC AGT CAT G (SEQ ID NO: 9), which was designed to put a coding DETENTION of translation (anticodon, CTA) immediately after the C-terminus of endoestatin, and This was followed by a site Xohl. The PCR products were cloned and sequenced, and the Xmal-XhoI fragment was ligated to the resulting Xmal and XhoI which extracted the pdCs-Fc vector. Similarly, the endoestatin encoding the HindIII-XhoI fragment was ligated into pdCs-huFc (D4K) vector appropriately extracted. Stable clones expressing Fc-endo or Fc (DK) -estastatin were obtained by electroporation of NS / 0 cells followed by selection in growth medium containing 1 00 nM of metrotrexate. The level of protein expression was assayed by anti-human Fc ELISA (Example 3) and confirmed by SDS-PAGE, which showed a protein product of -52 kD. The best production clones were subcloned by limiting dilutions. Example 2. Cell culture and transfection For transient transfection, the plasmid was introduced into 293 human kidney cells by co-precipitation of plasmid DNA with calcium phosphate (Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual , Cold Spring Harbor, NY) or by lipofection using LipofectAMINE Plus (Life Technologies, Gaithersburg, MD) according to the provider's protocol. In order to obtain stably transfected clones, the plasmid DNA was introduced into the mouse NS / O myeloma cells by electroporation. NS / O cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. Approximately 5 x 1 06 cells were washed once with PBS and resuspended in 0.5 ml PBS. Ten μg of linearized plasmid DNA was thus incubated with the cells in a Gen Pulser Probe (0.4 cm electrode distance, BioRad, Hercules, CA) on ice for 10 min. Electroporation was performed using a Gen Push Button (BioRad, Hercules, CA) with establishments at 0.25 V and 500 μF. The cells were allowed to recover for 10 min. on ice, after which they were resuspended in growth medium and placed on two 96-well plates. Stably transfected clones were selected for growth in the presence of 100 nM metrotrexate (MTX), which was introduced two days post-transfection. The cells were fed every 3 days for three more times, and the clones resistant to MTX appeared for 2 to 3 weeks. The supernatants of clones were tested by anti-Fc ELISA to identify the high producers. The high production clones were isolated and propagated in growth medium containing 100 nM MTX.

Example 3. ELISA procedures Three different ELISAs were used to determine the concentrations of protein products in the supernatants of MTX resistant clones and other test samples. The anti-human Fc ELISA (huFc) was used to measure the amount of proteins containing human Fc. Anti-murine Fc (muFc) and anti-canine Fc (caFc) antibodies were used in ELISAs to measure the amount of proteins containing canine Fc and murine Fc, respectively. The procedure for the anti-huFc ELISA is described below in detail herein. A. Coating plates ELISA plates were coated with AfiniPura Goat anti-human IgG (H + L) (Jackson ImmunoResearch Laboratories, West Grove, PA) at 5 μg / ml in PBS, and 100 μl / perforation in plates. 96 perforations (Nunc-lmmuno píate MaxiSorp ™, Nalge Nunc International, Rochester, NY). The coated plates were covered and incubated at 4 ° C overnight. The plates were thus washed 4 times with 0.05% Tween 20 in PBS, and blocked with 1% BSA / 1% Goat Serum in PBS, 200 μl / well. After incubation with the blocking regulator at 37 ° C for 2 hours, the plates were washed 4 times with 0.05% Tween in PBS and dried by shredding on paper towels. B. Incubation with test samples and secondary antibody The test samples were diluted in appropriate concentrations in a sample buffer, containing 1% BSA / 1% Goat Serum /0.05% Tween in PBS. A standard curve was prepared with a chimeric antibody (with a human Fc), the concentration of which was known. To prepare a standard curve, serial dilutions were made in the sample buffer to give a standard curve reference of 125 ng / ml to 3.9 ng / ml. Diluted and standard samples were added to the plate, 1000 μl / well and the plate was then incubated at 37 ° C for 2 hr. After incubation, the plate was washed 8 times with 0.05% Tween in PBS. For each perforation, 100 μl of secondary antibody was added, the horseradish peroxidase anti-human IgG conjugated with (HRP) (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA), was diluted approximately 1: 120,000 in the regulator of sample. The exact dilution of the secondary antibody had to be determined for each part of the Anti-human IgG conjugated with HRP. After incubation at 37 ° C for 2 hr, the plate was washed 8 times with 0.05% Tween in PBS. C. Development A substrate solution was prepared by dissolving 30 mg (1 tablet) of o-phenylenediamine dihydrochloride (OPD) in 15 ml of 0.025 M citric acid / 0.05 M Na2HPO4 buffer, pH 5, containing 0.03 % of H202 recently added. The substrate solution was added to the plate at 100 μl / well. The color was allowed to develop for 30 min. at room temperature in the dark. The development time can be subject to change, depending on the part to the variability of part of the coated plates, the secondary antibody, etc. The reaction was stopped by adding 4N H2SO4, 100 μl / perforation. The plate was read by a plate reader, which was fixed at both 490 and 650 nm, and was programmed to subtract the OD background at 650 nm from the OD at 490 nm.

The procedure for the anti-muFc ELISA was similar, except that the ELISA plate was coated with anti-murine Goat anti-murine Igini (H + L) (Jackson ImmunoResearch, West Grove, PA) at 5 μg / ml in PBS, and 100 μl / perforation; and the secondary antibody was Fc? goat anti-mulgG conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA), used from 1 in 5000 dilution. Similarly, for the anti-caFc ELISA, the ELISA plate was coated with AfiniPuro rabbit dog anti-IgG, specific Fc fragment (Jackson ImmunoResearch, West Grove, PA) at 5 μg / ml in PBS, and 1000 μl / well; and the secondary antibody was AfiniPuro rabbit dog IgG, specific Fc fragment conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA), used from 1 in 5000 dilution. Example 4. Expression of huCF-huAnqioestatin Human angioestatin (DNA sequence established in SEQ ID NO: 10; amino acid sequence set forth in SEQ ID NO: 1 1) was expressed as a fusion protein of human Fc angioestatin (huFc-huAngium) essentially as described in Example 1. The PCR was used to adapt the angioestatin cDNA (SEQ ID NO: 3), for expression in the pdCs-huFc or pdCs-huFc (D4K) vectors. The respective bootstrap loads were 5'-CC CCG GGT AAG AAA GTG TAT CTC TCA GAG (SEQ ID NO: 12); encoded amino acids described in SEQ ID NO: 13), and 5'-C CCC AAG CTT AAA GTG TAT CTC TCA GAG (SEQ ID NO: 14; encoded amino acids described in SEQ ID NO: 15), where the Xmal site or site HindIII were followed by the sequence encoding the N-terminus of angioestatin. The subsequent primer was 5'-CCC CTC GAG CTA CGC TTC TGT TCC TGA GCA (SEQ ID NO: 16), which was designed to put a coding DETENTION of translation (anticodon, CTA) immediately after the C term of angioestatin, and this was followed by a site Xohl. The PCR products were cloned and sequenced, and the Xmal-XhoI fragment and the resulting HindIII-XhoI fragment encoding angioestatin were ligated for the pdCs-huFc and pdCs-huFc (D4K) vectors, respectively. Stable NS / O clones expressing huFc-huAngio and huFc (D4K) -huAngio were selected and assayed as described in Examples 2 and 3. Example 5. Expression of muFc-mu-Endoestatin The murine endoestatin (established DNA sequence) in SEQ ID NO: 17, amino acid sequence set forth in SEQ ID NO: 18) and murine Fc (DNA sequence set forth in SEQ ID NO: 19, encoded amino acids set forth in SEQ ID NO: 20) were expressed as a protein of Murine Fc murine endoestatin (muFc-muEndo) essentially as described in Example 1. The PCR was used to adapt the endoestatin cDNA (SEQ ID NO: 4), for expression in the vector pdCs-muFc (D K). The primary was 5'-C CCC AAG CTT CAT ACT CAT CAG GAC TTT C (SEQ ID NO: 21); encoded amino acids described in SEQ ID NO: 22), where the Xmal site or the Hindlll site were followed by the sequence encoding the N terminus of endoestatin. The primary primer was 5'-C CCC CTC GAG CTA TTT GGA GAA AGA GGT C (SEQ ID NO: 23), which was designed to put a coding DETENTION of translation (anticodon, CTA) immediately after the endostatin C term , and this was followed by a site Xoh l. The PCR product was cloned and sequenced, and the resulting HindIII-XhoI fragment encoding endoestatin was ligated into the vector pdCs-muFc (D4K). Stable NS / O clones expressing muFc (DK) -huEndo were selected and assayed (anti-muFc ELISA) as described in Examples 2 and 3. Example 6. Expression of muFc-muAngiostatin Murine angioestatin (established DNA sequence) in SEQ ID NO: 24, amino acid sequence set forth in SEQ ID NO: 25) was expressed as a murine Fc murine angioestatin fusion protein (muFc-muAngio) essentially as described in Example 1. PCR was used to adapt the angioestatin cDNA (SEQ ID NO: 6), for expression in the pdCs-Fc vector (D4K). The front primer was 5'-C CCC AAG CTT GTG TAT CTG TCA GAA TGT AAG CCC TCC TGT CTC TGA GCA (SEQ ID NO: 26); encoded amino acids described in SEQ ID NO: 27), where the Hindlll site was followed by the sequence encoding the N-terminus of angioestatin. The subsequent primer was 5'-CCC CTC GAG CTA CCC TCC TGT CTC TGA GCA (SEQ ID NO: 28), which was designed to put a coding DETENTION of translation (anticodon, CTA) immediately after the C term of angioestatin, and This was followed by a site Xohl. The PCR product was cloned and sequenced, and the Hindlll-Xhol fragment encoding angioestatin was ligated to the vector pdCs-muFc (D4K). Stable NS / O clones expressing muFc (DK) -huAngio were selected and tested (anti-muFc ELISA) as described in Examples 2 and 3. Example 7. Expression of canine Fc (caFc) Peripheral blood monocytic cells of canine (PBMCs) isolated from dog blood were used to prepare mRNA. After the synthesis of the first strand of cDNA with oligo reverse transcriptase (dT), PCR was performed to amplify the canine Fc (Kazuhiko et al. (1 992) JP 1 992040894-A1) using the front primary 5'-CC TTA AGC GAA AAT GGA AGA GTT CCT CGC (SEQ ID NO: 29; encoded amino acids described in SEQ ID NO: 30), in which the Aflll site was introduced immediately upstream of the sequence encoding the canine Fc joint region, and the posterior primary 5'-C CTC GAG TCA TTT ACC CGG GGA ATG GGA GAG GGA TTT CTG (SEQ ID NO: 31), in which the Xhol site was introduced after the translational STOP codon (anticodon, TCA) of the canine Fc. The forward primer introduced a silent mutation to create an Xmal restriction site, which facilitates the construction of the pdCs-caFc vector (D4K) through the linker program adapter and ligation to the DNA constructs encoding canine endoestatin or angioestatin. Similar to the construction of pdCs-huFc, which was described in detail in Lo e al. (Ie at the Protein Engineering (1998) 1 1: 495), the expression vector for the pdCs-caFc was constructed as follows. The Aflll-Xhol fragment encoding the canine Fc was ligated to the Xbal-Aflll fragment encoding the light chain signal peptide and the Xbal-Xbaol extracted the vector pdCs. The resulting pdCs-caFc expression vector was thus used to transfect 293 cells. Approximately 3 days post-transfection, the supernatant was purified by Protein A chromatography. The expression of dog Fc (DNA sequence set forth in SEQ ID NO: 32; amino acid sequence set forth in SEQ ID NO: 33) was confirmed by SDS-PAGE followed by Western blot analysis using an anti-rabbit Dog IgG conjugated with peroxidase, specific Fc fragment (Jackson ImmunoResearch, West Grove, PA ). Example 8. Expression of caFc-caestastatin The coding sequence for canine endoestatin (DNA sequence set forth in SEQ ID NO: 34, amino acid sequence set forth in SEQ ID NO: 35) was adapted for a HindIII-Xhol fragment for expression as an Fc fusion protein, essentially as described in Example 5. At the 3 'end, a STOP codon was introduced, for example, by PCR, immediately after the codon encodes the C-terminal lysine residue, and this was followed by the Notl restriction site. At the 5 'end, however, there was a convenient Dralll restriction site for reconstruction. A duplex oligonucleotide consisting of sticky ends Hindlll and Dralll were chemically synthesized and used to bind to the Dralll-Xhol restriction fragment encoding the remainder of the canine endoestatin cDNA. The duplex used is shown below: Hindlll 5'-AGCTT CAC ACC CAC CAG GAC TTC CAG CCG GTG CTG CAC CTG (SEQ ID NO: 36) A GTG TGG GTG GTC CTG AAG GTC GGC CAC GAC GTG-5 '(SEQ ID NO : 38) Dralll The first CAC in the duplex encodes the N-terminal histidine residue of the canine endoestatin. The Hindll l-XhoI fragment encoding the full length canine endoestatin in this manner could be linked to the HindIII-XhoI extracted from the pdCs-caFc vector (see Example 7) for expression. Stable NS / O clones expressing caFc-caEnd were selected and tested by anti-caFc ELISA, as described in Examples 2 and 3. The protein product was analyzed on SDS-PAGE and confirmed by Western blot analysis. . EXAMPLE 9. Expression of caFc-caAngioestatin The cDNA encoding full length canine angioestatin (DNA sequence set forth in SEQ ID NO: 39, amino acid sequence set forth in SEQ ID NO: 40) was adapted for expression as a protein fCF fusion essentially as in the aforementioned examples. Briefly, at the 3 'end, a STOP codon was introduced, for example, by PCR, immediately after the codon encodes the C terminal lysine residue, and this was followed by the Notl restriction site instead of an Xhol site since there was an internal Xhol restriction site in the canine angioestatin cDNA. At the 5 'end, a Hi nd f I i site was introduced into the upstream structure immediately from the N terminus of angioestatin. The Hindlll-Xhol fragment encoding full-length canine angioestatin in this manner could be linked to the Hind III-Xhol extracted from the pdCs-caFc vector (where the Notl site was introduced into the Xhol site via the linker program linkage) for expression. Stable NS / O clones expressing caFc-caAngio were selected and tested by anti-caFc ELISA, as described in Examples 2 and 3.

The protein product was analyzed on SDS-PAGE and confirmed by Western blot analysis. Example 10. Expression of muAngium muFc-K1 Angioestatin comprises the first four domains of Kringle's five of plasminogen. To determine whether any one or more of the Kringle domains are responsible for the observed antiangiogenic activity of angioestatin, it is possible to produce unique Kringle domains by themselves or combination thereof for testing. To demonstrate the utility of Fc as a fusion protein pair, the expression of the first Kringle domain of murine angioestatin (K1) was achieved in the following manner. The first Kringle domain ends in Glu-87 murine angiostatin (SEQ ID NO: 25). There was a convenient Nsil restriction site in the cDNA in its position such that after extraction by Nsil, the fourth base of the 3 'overhang was removed by T4 polymerase to create an obtuse limb. A translation STOP codon was immediately introduced downstream of Glu-87 encoding GAA through binding to the TGA CTC GAG TCA palindromic linker program (SEQ ID NO: 41), where the TGA STOP codon was followed by a site Xhol. The HindIII-XhoI fragment encoding this truncated angioestatin, ie, first Kringle only, was then ligated into the vector pdCs-muFc (DK) for expression. Elevated levels of expression were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE. Example 1 1. Expression of muAngio internal muFc-K1 A Kringle domain consists of multiple cycles, including an external cycle and an internal cycle. In the first Kringle of murine angiostatin, the internal cycle is defined by Cys 55 and Cys 79, which together form a disulfide bond at the base of the cycle. The Cys-67 of the internal cycle forms another disulfide bond with a Cys residue of the external cycle to give the Kringle structure. To test whether the internal cycle has any anti-angiogenic activity, it was expressed as an internal muFc-K1 (Kringle 1) as follows. With a DNA fragment encoding the first Kringle as annealed, a mutagenic primer having the sequence 5'GGG CCT TGG AGC TAC ACT ACA (SEQ ID NO: 42; encoded amino acids described in SEQ ID NO: 43) was used to mutagenize TGC (Cys-67) to AGC (Ser), by PCR. This ensures that Cys-67 does not form a disulfide bond when the internal Kringle 1 cycle is expressed without the external cycle. An upstream primary having the sequence 5'GCGGATCCAAGCTT AGT ACA CAT CCC AAT GAG GG (SEQ ID NO: 44; encoded amino acids described in SEQ ID NO: 45) was used to introduce a Hindill site in the structure immediately 5 'to the codon for Ser-43 (AGT). A BamHl site was also introduced immediately upstream of the Hindlll site. The BamHl site is useful for binding to the BamHl site at the extremity of the flexible Gly-Ser linker program in Example 12 below. In this manner a DNA fragment of HindIII-XhoI encoding Ser-43 via murine angioestatin Glu-87 was ligated to the vector pdCs-muFc (D4K) for expression. Elevated expression levels of internal muFc-K1 were obtained in both stable and transient expression, as analyzed by anti-muFc ELISA and SDS-PAGE.

Example 1 2. Expression of muFc-muEndo-GlySer muAngio internal linker-K1 program The internal muFc-muEndo-K1 hybrid molecule comprises muFc-muEnd linked by a polypeptide linker program containing glycine and serine residues, to the internal cycle of the first Kringle of murine angiostatin. The DNA construction was installed as follows. There is a BspHI site at the 3 'end of the murine endoestatin cDNA. To introduce a flexible linker program of glycine and serine residues into the C-terminus of the murine endo-statin, a 540-bp HindIII-BspHI fragment encoding endoestatin was ligated to an oligonucleotide duplex coating formed by the oligonucleotides described in SEQ ID NO: 46 and SEQ ID NO: 48. The amino acid linker program encoded by SEQ ID NO: 46 is described in SEQ ID NO: 47. The HindIII-BamHI fragment encoding murine endoestatin and the The Gly-Ser linker program was subcloned into a standard cloning vector. The BamHl site was thus used to introduce the BamHi-XhoI fragment encoding the internal K1 in Example 1 1. The resulting Hindlll-Xhol fragment encoding muEndo-GlySer internal linker-K1 program, pdCs-muFc (DK) was ligated for expression. Elevated expression levels of muFc-muEndo-GlySer internal linker-K1 program were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE. Example 13. MuFc-muEndo-Glyser Expression muAngio Linker-K1 Program The hybrid molecule muFc-muEndo-K1 comprises muFc-muEndo bound by a polypeptide linker program containing glycine and serine residues, to the first Kringle of murine angiostatin. . The DNA construction was installed as follows. The BamH1 limb of the HindIII-BamHI fragment encoding muEndo-GlySer linker program (Example 12) was ligated to the HindIII-XhoI fragment encoding murine angioestatin Kringle 1 (Example 10) via the following adapter: BamHl 5'GA TCC TCA GGC C (SEQ ID NO: 49) G AGT CCG GTCGA (SEQ ID NO: 50) Hindlll The adapter has a sticky Hindlll 'tip, which in the ligation, would not regenerate the Hindlll site. In this manner, the resulting HindIII-XhoI fragment, which encodes the muEndo-GlySEr linker-Kringle 1 program, is ligated to the vector pdCs-muFc (D4K) for expression. The elevated expression levels of muFc-muEndo-GlySer program linker-K1 were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE. Example 14. Expression of muFc-muEndo-GlySer linker-muAngio program The hybrid molecule muFc-muEndo-GlySer linker-muAngio program comprises muFc-muEnd linked by a polypeptide linker program containing glycine and serine residues, to murine angioestatin. The DNA construction was installed as follows. The BamHl extremity of the HindIII-BamHI fragment encoding the muEndo-GlySer linker program (Example 1 2) was ligated to the HindIII-XhoI fragment encoding murine angioestatin via the adapter described in Example 1 3. The resulting HindIII-XhoI fragment, which encodes the muEndo-GlySEr linker-muAngio program, binds to the vector pdCs-muFc (D4K) for expression. The elevated expression levels of muFc-muEndo-GlySer linker-muAngio program were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE. Example 1 5. Expression of huAngio-huFc-Endo The hybrid molecule huAngio-huFc-huEndo comprises human angioestatin bound by a peptide bond to huFc-huEndo. The DNA construction was installed as follows. A HindIII-XhoI fragment encoding human angioestatin without a stop codon that was first generated by PCR, such that the codon by the last amino acid residue of angioestatin was immediately followed by CTCGAG from the XhoI site. The Hindlll at the 5 'end was ligated to a Xbal-Aflll fragment of the light chain signal peptide (Lo e al., Protein Engineering (1998) 1 1: 495) via an Aflll-Hindlll' adapter: Aflll 5 'TTA AGC GGC C (SEQ ID NO: 51) CG CGG GTCGA (SEQ ID NO: 52) Hindlll' The sticky Hindlll 'tip of the adapter, in ligation, would not regenerate a Hindlll site. At the 3 'end, the Xhol site was ligated to the Aflll site of the Aflll-Khol fragment encoding the huFc-hu-Endo via the following adapter Xhol'-Af lll: Xhol' 5'TC GAC TCC GGC (SEQ ID NO : 53) G AGG CCG AATT (SEQ ID NO: 54) Aflll The Xhol tip of the adapter, in ligation, would not regenerate an Xhol site. The resulting Xbal-XhoI fragment encoding the human endostatin-angioestatin-huFc-human peptide signal was cloned into the pdCs vector for expression. Elevated expression levels were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE. Example 16. Pharmacokinetics In a set of pharmacokinetic studies, mice C57 / BL6 with Lewis lung tumors implanted at 1 00-200 mm3 were injected into the terminal vein with 720 μg huFc-huAngio per mouse. The size of the tumors and the huFc-huAngio doses used in this study were chosen to stimulate the current treatment protocol described by O'Reilly (O'Reilly et al., (1996) Nature Medicine 2: 689). The blood was cultured by retro-orbital bleeding at 1/2, 1, 2, 4, 8, 24, and 48 hr post injection. Blood samples were analyzed by anti-huFc ELISA followed by Western analysis. HuFc-huAngio was found to have a circulating half-life of approximately 32 hr in the mouse and Western analysis showed that over 90% of hu-Fc-huAngio remained as an integral molecule in circulation. Pharmacokinetic studies were also repeated in Swiss mice without tumors at a dose of 200 μg / mouse. In this case it was found that huFc-huAngio has a circulation half-life of approximately 33 hr. Equivalences The invention can be incorporated into other specific forms without departing from the spirit or essential characteristics thereof. The foregoing methods are therefore considered, in all respects, to be illustrative rather than limiting to the invention described herein. The scope of the invention in this manner is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore proposed, encompassed herein.

LIST OF SEQUENCES < 110 > it, Kin-Ming Li, Yue Glll ± is, Stephen D < 120 > Expression and Exportation of Angiogenesis Inhibitors such as Immunofusins < 130 > LEX-006PC < 140 > < 141 > < 150 > US 60 / 097,883 < 151 > 1998-08-25 < 160 > 54 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 696 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (696) < 223 > Fc fragment of the gamma human Inoglobulin < 400 > 1 gag ccc aaa tet tet gac aaa act falls tgo cea cea ceg tge cea gea 48 GXu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Wing 1 5 10 15 ect gaa ctc ggg gga ceg tea gtc ttc ctc ctc ccc cea aaa ccc 96 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 aag gac acc ctc atg tc cgg ace ect gag gtc here tg gtg gtg 144 Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 gtg gao gtg age falls gaa gao ect gag gtc aag ttc aac tgg tac gtg 192 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 gac ggc gtg gag gtg cat aat gcc aag aag ceg cgg gag gag cag 240 Asp Gly Val Glu Val Kis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 tac aac age acg tac cgt gtg gtc age gtc ctc acc gtc ctg falls cag 288 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 gac tgg ctg aat ggc aag gag tac aag tge aag gtc tcc aac aaa gcc 336 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Wing 100 105 110 ctc cea gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc 384 Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys Ala Lys Gly Gln Pro 1 15 120 125 cga gaa cea cag gtg tac acc ctg ccc cea tea cgg gag gag atg acc 432 Arg Glu Pro Glp Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 aag aac ggc age ctg ace tge ctg gtc aaa ggc ttc tat ccc age 480 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 gac atc gcc gtg gag tgg gag age aat ggg cag ceg gag aac aac tac 528 Asp He Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 aaction acc acct ect ccc gtg ctg gac tcc gac gcc tcc tcc ctc tat 576 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 age aag ctc acc gtg gac aag age agg tgg cag ggg aac gtc ttc 624 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 tea tge tcc gtg atg cat gag get ctg falls aac eac tac acg cag aag 672 Ser Cys Ser Val Met His Glu Wing Leu His Asn His Tyr Thr Gln Lys 210 215 220 age ctc tcc ctg tcc ceg ggt aaa 696 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 < 210 > 2 < 211 > 232 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Wing 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Wing 100 105 110 Leu Pro Wing Pro He Glu Lys Thr He Ser Lys Wing Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp He Wing Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 < 210 > 3 < 211 > 549 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (1) .. (549) < 223 > endostatin < 400 > 3 falls age falls cgc gac ttc cag ceg gtg ctc falls ctg gtt gcg ctc aac 48 His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 age ccc cg ggc ggc atg cgg ggc atc cgc ggg gcc gac tcc cag 96 Ser Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Ala Asp Phe Gln 20 25 30 tge ttc cag cag gcg cgg gcg ggg ggg gg gcg ggc acc ttc cgc gcc 144 Cys Phe Gln Gln Wing Arg Wing Val Gly Leu Wing Gly Thr Phe Arg Wing 35 40 45 ttc ctg tcc teg cgc ctg cag gac ctg tac age atc gtg cgc cgt gcc 192 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser He Val Arg Arg Ala 50 55 60 gac cgc gca gcc gtg ccc atc gtc aac ctc aag gac gag etg ctg ttt 240 Asp Arg Ala Ala Val Pro He Val Asn Leu Lys Asp Glu Leu Leu Phe 65 70 75 80 ccc age tgg g'ag get ctg ttc tea ggc tet gag ggt ceg ctg aag ccc 288 Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro 85 90 95 ggg gca cgc atc ttc tcc ttt gac ggc aag gac gtc ctg agg falls ccc 336 Gly Wing Arg He Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro 100 105 110 acc tgg ccc cag aag age gtg tgg cat ggc teg gac ccc aac ggg cgc 384 Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg 115 120 125 agg ctg acc gag age tac tgt gag acg tgg cgg acg gag get ccc teg 432 Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Wing Pro Ser 130 135 140 gcc acg ggc cag gcc tcc teg ctg ctg ggg ggc agg ctc ctg ggg cag 480 Wing Thr Gly Gln Wing Being Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln 145 150 155 160 agt gcc gcg age tge cat falls gcc tac atc gtg ctc tge att gag aac 528 Be Ala Ala Be Cys His His Ala Tyr He Val Leu Cys He Glu Asn 165 170 175 age ttc atg act gcc tcc aag 549 Be Phe Met Thr Ala Ser Lys 180 < 210 > 4 < 211 > 183 < 212 > PRT < 213 > Homo sapiens < 400 > 4 His Ser fíis Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Ser Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Wing Asp Phe Gln 20 25 30 Cys Phe Gln Gln Wing Arg Wing Val Gly Leu Wing Gly Thr Phe Arg Wing 35 40 45 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser He Val Arg Arg Ala 50 55 60 Asp Arg Wing Wing Val Pro He Val Asn Leu Lys Asp Glu Leu Leu Phe 65 70 75 80 Pro Ser Trp Glu Wing Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro 85 90 95 Gly Wing Arg He Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro 100 105 110 Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg 115 120 125 Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Wing Pro Ser 130 135 140 Wing Thr Gly Gln Wing Being Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln 145 150 155 160 Be Ala Ala Be Cys His His Ala Tyr He Val Leu Cys He Glu Asn 165 170 175 Be Phe Met Thr Ala Ser Lys 180 < 210 > 5 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Front Primary for human FC-epdo < 220 > < 221 > CDS < 222 > (3) .. (29) < 400 > 5 ce ceg ggt aaa falls age falls cgc gac ttc c 30 Pro Gly Lys His Ser His Arg Asp Phe 1 5 < 210 > 6 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 400 > 6 Pro Gly Lys His Ser His Arg Asp Phe 1 5 < 210 > 7 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Front Primary for human FC-endo < 220 > < 221 > CDS < 222 > (2) . (25) < 400 > 7 c aag ctt falls age falls cgc gac ttc c 26 Lys Leu His Ser His Arg Asp Phe 1 5 < 210 > 8 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 400 > 8 Lys Leu His Ser His Arg Asp Phe 1 5 < 210 > 9 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Posterior Primary for human Fc-Endo < 400 > 9 cctcgagcta cttggaggca stcatg 26 < 210 > 10 < 211 > 1089 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > ( 1) . . (1089) < 223 > angiostatin < 400 > 10 aaa gtg tat ctc tea gag tge aag act ggg aat gga aag aac tac aga 48 Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg 1 5 10 15 ggg acg atg tcc aaa here aaa aat ggc atc acc tgt caa aaa tgg agt 96 Gly Thr Met Ser Lys Thr Lys Asn Gly He Thr Cys Gln Lys Trp Ser 20 25 30 tcc act tet ccc falls aga ect aga ttc tea ect get here cae ccc tea 144 Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Wing Thr His Pro Ser 35 40 45 gag gga ctg gag gag aac tac tge agg aat cea gac aac gat ceg cag 192 Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln 50 55 60 ggg ccc tgg tge tat aet act gat cea gaa aag aga tat gac tac tge 240 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys 65 70 75 80 gac att ctt gag tgt gaa gag tgt atg cat tge agt gga gaa aac 288 Asp He Leu Glu Clu Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn 85 90 95 tat gac ggc aaa att tcc aag acc atg tet gga ctg gaa tge cag gcc 336 Tyr Asp Gly Lys He Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Wing 100 105 110 tgg gac tet cag age cea cae get cat gga tac att ect tcc aaa ttt 384 Trp Asp Ser Gln Ser Pro His Wing His Gly Tyr He Pro Ser Lys Phe 115 120 125 cea aac aag aac ctg aag aag aat tac tgt cgt aac ccc gat agg gag 432 Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu 130 135 140 ctg cgg ect tgg tgt ttc ac acc gac ccc aac aag cgc tgg gaa ctt 480 leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu 145 150 155 160 tge gac ato ccc cgc tge here ect cea tet tet tet ggt ccc acc 528 Cys Asp He Pro Arg Cys Thr Thr Pro Pro Pro Ser Gly Pro Thr 165 170 175 tac cag tgt ctg aag gga ac ggt gaa aac tat cgc ggg aat gtg get 576 Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Wing 180 185 190 gtt acc gtt tcc ggg falls acc tgt cag falls tgg agt gca cag acc ect "624 Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro 195 200 205 falls here cat aac agg here cea gaa aac ttc ccc tge aaa aat ttg gat 672 His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp 210 215 220 gaa aac tac tge egc aat ect gac gga aaa agg gcc cea tgg tge cat 720 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala P ro Trp Cys His 225 230 235 240 here acc aac age ca gtg cgg tgg gag tac tgt aag ata ceg tcc tgt 768 Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys He Pro Ser Cys 245 250 255 gac tcc tcc cea gta tcc acg gaa cata ttg get ccc here gca cea ect 816 Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Wing Pro Thr Wing Pro Pro 260 265 270 gag cta acc ect gtg gtc cag gac tge tac cat ggt gat gga cag age 864 Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser 275 280 285 tac cga ggc here tcc tcc acc acc acc gga aag aag tgt cag tet 912 Tyr Arg Gly Thr Be Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser 290 295 300 tgg tea tet atg ac cea falls cgg falls cag aag acc cea gaa aac tac 960 Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr 305 310 315 320 cea aat get ggc ctg here atg aac tac tge agg aat cea gat gcc gat 1008 Pro Asn Wing Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Wing Asp 325 330 335 aaa ggc ccc tgg tgt ttt acc here gac ccc age gtc agg tgg gag tac 1056 Lys Gly Pro Trp Cys P he Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr 340 345 350 tge aac ctg aaa aaa tge tea gga here gaa gcg 1089 Cys Asn Leu Lys Cys Ser Gly Thr Glu Ala 355 360 < 210 > 11 < 211 > 363 < 212 > PRT < 213 > Homo sapiens < 400 > 11 Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg 1 5 10 15 Gly Thr Met Ser Lys Thr Lys Asn Gly He Thr Cys Gln Lys Trp Ser 20 25 30 Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser 35 40 45 Glu Gly Lelu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Pro Gln 50 55 60 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys 65 70 75 80 Asp He Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn 85 90 95 Tyr Asp Gly Lys He Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala 100 105 110 Trp Asp Ser Gln Ser Pro Hie Ala His Gly Tyr He Pro Ser Lys Phe 115 120 125 Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu 130 135 140 Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu 145 150 155 160 Cys Asp He Pro Arg Cys Thr Thr Pro Pro Ser Ser Gly Pro Thr 165 170 175 Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Wing 180 185 190 Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Wing Gln Thr Pro 195 200 205 Hls Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp 210 215 220 Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Wing Pro Trp Cys His 225 230 235 240 Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys He Pro Ser Cys 245 250 255 Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro 260 265 270 Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser 275 280 285 Tyr Arg Gly Thr Be Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser 290 295 300 Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr 305 310 315 320 Pro Asn Wing Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Wing Asp 325 330 335 Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr 340 345 350 Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Wing 355 360 < 210 > 12 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Front Primary for human Fc-Angio < 220 > < 221 > CDS < 222 > (3) .. (29) < 400 > 12 ce ceg ggt aag aaa gtg tat ctc tea gag 29 Pro Gly Lys Lys Val Tyr Leu Ser Glu 1 5 < 210 > 13 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 400 > 13 Pro Gly Lys Lys Val Tyr Leu Ser 'Glu 1 5 < 210 > 14 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Front Primary for Fc-Apg¡o humano < 220 > < 221 > CDS < 222 > (2) .. (28) < 400 > 14 c ccc aag ctt aaa gtg tat ctc tea gag 28 Pro Lys Leu Lys Val Tyr Leu Ser Glu 1 5 < 210 > 15 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 400 > 15 Pro Lys Leu Lys Val Tyr Leu Ser Glu 1 5 < 210 > 16 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Posterior Primary for human Fc-Angio < 400 > 16 cccctcgagc tacgcttctg ttcctgagca 30 < 210 > 17 < 211 > 552 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > CDS < 222 > (1) . . (552) < 223 > endostatin < 400 > 17 cat act cat cag gac ttt cag cea gtg ctc falls ctg gtg gca ctg aac 48 His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 ace ccc ctg tet gga ggc atg cgt ggt atc cgt gga gca gat ttc cag 96 Thr Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Ala Asp Phe Gln 20 25 30 tge ttc cag cage gee cga gcc gtg ggg ctg teg ggc acc ttc cgg get 144 Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ser Gly Thr Phe Arg Ala 35 40 45 ttc ctg tcc tet agg ctg cag gat ctc tat age atc gtg cgc cgt get 192 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser He Val Arg Arg Ala 50 55 60 gac cgg ggg tet gtg ccc atc gtc aac ctg aag gac gag gtg cta tet 240 Asp Arg Gly Ser Val Pro He Val Asn Leu Lys Asp Glu Val Leu Ser 65 70 75 80 ccc age tgg gac tcc ctg ttt tet ggc tcc cag ggi ca gtg ca ccc 288 Pro Ser Trp Asp Being Leu Phe Being Gly Being Gln Gly Gln Val Gln Pro 85 90 95 ggg gcc cgc atc ttt ttt ttt gac ggc aga gat gtc ctg aga cae cea 336 Gly Wing Arg He Phe Ser Phe Asp Gly Arg Asp Val Leu Arg His Pro 100 105 110 gcc tgg ceg cag aag age gta tgg cao ggc teg gac ccc agt ggg cgg 384 Wing Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg 115 120 125 agg ctg atg gag agt tac tgt gag here tgg cga act gaa act act gg? 432 Arg Leu Met Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Thr Thr Gly 130 135 140 get here ggt cag gcc tcc tcc ctg ctg tea ggc agg ctc ctg gaa cag 480 Ala Thr Gly Gln Ala Ser Ser Leu Leu Ser Gly Arg Leu Leu Glu Gln 145 150 155 160 aaa get gcg age tge falls aac age tac atc gtc ctg tge att gag aat 528 Lys Ala Ala Ser Cys His Asn Ser Tyr He Val Leu Cys He Glu Asn 165 170 175 age ttc atg acc tet ttc tcc aaa 552 Ser Phe Met Thr Ser Phe Ser Lys 180 < 210 > 18 < 211 > 184 < 212 > PRT < 213 > Mus museulus < 400 > 18 His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 Thr Pro Leu Ser Gly Gly Met Arg Gly He Arg Gly Wing Asp Phe Gln 20 25 30 Cys Phe Gln Gln Wing Arg Wing Val Gly Leu Be Gly Thr Phe Arg Wing 35 40 45 Phe Leu Be Ser Arg Leu Gln Asp Leu Tyr Ser He Val Arg Arg Ala 50 55 60 Asp Arg Gly Ser Val Pro He Val Asn Leu Lys Asp Glu Val Leu Ser 65 70 75 80 Pro Ser Trp Asp Ser Leu Phe Ser Gly Ser Gln Gly Gln Val Gln Pro 85 90 95 Gly Wing Arg He Phe Ser Phe Asp Gly Arg Asp Val Leu Arg Hi3 Pro 100 105 110 Wing Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg 115 120 125 Arg Leu Met Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Thr Thr Gly 130 135 140 Wing Thr Gly Gln Wing Being Ser Leu Leu Ser Gly Arg Leu Leu Glu Gln 145 150 155 160 Lys Ala Ala Ser Cys His Asn Ser Tyr He Val Leu Cys He Glu Asn 165 170 175 Ser Phe Met Thr Ser Phe Ser Lys 180 < 210 > 19 < 211 > 699 < 212 > DNA < 213 > Mus ptusculus < 220 > < 221 > CDS < 222 > (1) .. (699) < 223 > Fc < 400 > 19 gag ccc aga ggg ccc here atc aag ccc tgt ect cea tge aaa tge cea 48 Glu Pro Arg Gly Pro Thr He Lys Pro Cys Pro Pro Cys Lys Cys Pro 1 5 10 15 gca ect aac ctc ttg ggt gga cea tcc gtc ttc atc ttc ect cea aag 96 Wing Pro Asn Leu Leu Gly Gly Pro Ser Val Phe He Phe Pro Pro Lys 20 25 30 atc aag gat gta ctc atg atc tcc ctg age ccc ata gtc here tgt gtg 144 He Lys Asp Val Leu Met He Ser Leu Ser Pro He Val Thr Cys Val 35 40 45 gtg gtg gat gtg age gag gat gac cea gat gtc cag atc age tgg ttt 192 Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln He Ser Trp Phe 50 55 60 gtg aae aac gtg gaa gta falls ac ac g ca g ac cat aga gag 240 Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu 65 70 75 80 gat tac aac agt act ctc cgg gtg gtc agt gcc ctc ccc atc cag cae 288 Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro He Gln His 85 90 95 cag gac tg atg agg ggc aag gag ttc aaa tge aag gtc aae aac aaa 336 Gl Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 100 105 110 gac ctc cea gcg ccc atc gag aga acc atc tea aaa ccc aaa ggg tea 384 Asp Leu Pro Pro Wing He Glu Arg Thr He Ser Lye Pro Lys Gly Ser 115 120 125 gta aga get cea cag gta tat gtc ttg ect cea gaa gaa gag atg 432 Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met 130 135 140 act aag aaa cag gtc act ctg acc tge atg gtc here gac ttc atg ect 480 Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro 145 150 155 160 gaa gac att tac gtg gag tgg acc aac aac ggg aaa ac gag cta aac 528 Glu Asp He Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn 165 170 175 tac aag aac act gaa ce gtc ctg gac tet gat ggt tet tac ttc atg 576 Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met 180 185 190 tac age aag ctg aga gtg gaa aag aag aac tgg gtg gaa aga aat age 624 Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser 195 200 205 tac tcc tgt tea gtg gtc falls gag ggt ctg falls aat cae falls acg act 672 Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr 210 215 220 aag age ttc tcc cgg acc ceg ggt aaa 699 Lys Ser Phe Ser Arg Thr Pro Gly Lys 225 230 < 210 > 20 < 211 > 233 < 212 > PRT < 213 > Mus musculue < 400 > 20 Glu Pro Arg Gly Pro Thr He Lys Pro Cys Pro Pro Cys Lys Cys Pro 1 5 10 15 Wing Pro Asn Leu Leu Gly Gly Pro Ser Val Phe He Phe Pro Pro Lys 20 25 30 He Lys Asp Val Leu Met He Ser Leu Ser Pro He Val Thr Cys Val 35 40 45 Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln He Ser Trp Phe 50 55 60 Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu 65 70 75 80 Asp Tyr Asn Be Thr Leu Arg Val Val Be Ala Leu Pro He Gln His 85 90 95 Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 100 105 110 Asp Leu Pro Ala Pro He Glu Arg Thr He Ser Lys Pro Lys Gly Ser 115 120 125 Val Arg Wing Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met 130 135 140 Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro 145 150 155 160 Glu Asp He Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn 165 170 175 Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met 180 185 190 Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser 195 200 205 Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr 210 215 220 Lys Ser Phe Ser Arg Thr Pro Gly Lys 225 230 < 210 > 21 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Front Primary for mouse Fc-Epdo < 220 > < 221 > CDS < 222 > (2) .. (28) < 400 > 21 c cec aag ctt cat act cat cag gac ttt c 29 Pro Lys Leu His Thr His Gln Asp Phe 1 5 < 210 > 22 < 211 > 9 < 212 > PRT < 213 > Artificial Sequence < 400 > 22 Pro Lys Leu His Thr His Gln Asp Phe 1 5 < 210 > 23 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Posterior Primary for mouse Fc-Endo < 400 > 23 cccctcgagc tatttggaga aagaggtc 28 < 210 > 24 < 211 > 1086 < 212 > DNA < 213 > Mus musculus < 220 > < 221 > CDS < 222 > (1) .. (1086) < 223 > Angiostatin < 400 > 24 gtg tat ctg tea gaa tgt aag acc ggc atc ggc aac ggc tac aga gga 48 Val Tyr Leu Ser Glu Cys Lys Thr Gly He Gly Asn Gly Tyr Arg Gly 1 5 10 15 ace atg tcc agg aaag agt ggt gtt gcc tgt caa aag tgg ggt gcc 96 Thr Met Ser Arg Thr Lys Ser Gly Val Wing Cys Gln Lys Trp Gly Wing 20 25 30 acg ttc ccc falls gta ccc aac tac tet ccc agt here cat ccc aat gag 144 Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser Thr His Pro Asn Glu 35 40 45 gga cta gaa gag aac tac tgt agg aac cea gac aat gat gaa ca ggg 192 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly 50 55 60 ect tgg tge tac act here gat ceg gac aag aga tat gac tac tge aac 240 Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn 65 70 75 80 att cet gaa tgt gaa gag gag tge atg tac tge agt gga gaa aag tat 288 He Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr 85 90 95 gag ggc aaa atc tcc aag acc atg tet gga ctt gac tge cag gcc tgg 336 Glu Gly Lys He Ser Lys Thr Met Ser Gly Leu Asp Cys Gln Wing Trp 100 105 110 gat tet cag age cea cat get cat gga tac atc ect gcc aaa ttt cea 384 Asp Ser Gln Ser Pro His Wing His Gly Tyr He Pro Wing Lys Phe Pro 115 120 125 age aag aae ctg aag aat tat tge cae aac ect gac ggg gag cea 432 Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro Asp Gly Glu Pro 130 135 140 agg ccc tgg tge ttc here here gac ccc acc aaa egc tgg gaa tac tgt 480 Arg Pro Trp Cys Phe Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr Cys 145 150 155 160 gac atc ccc cgc tge here ccc ceg ccc cea ccc age cea acc tac 528 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Pro Pro Thr Tyr 165 170 175 cag tgt ctg aaa gga aga ggt gaa aat tac cga ggg acc gt g tet gtc 576 Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val 180 185 190 acc gtg tet ggg aaa acc tgt cag cgc tgg agt gag caa acc ect cat 624 Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His 195 200 205 agg falls aac agg ac cea gaa aat ttc ccc tge aaa aat ctg gaa gag 672 Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220 aac tac tge cgg aac cea gat gga gaa act get ccc tgg tat action 720 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Wing Pro Trp Cys Tyr Thr 225 230 235 240 act gac age cag ctg agg tgg gag tac tgt gag att cea tcc tge gag 768 Thr Asp Ser Gln Leu Arg Trp Glu Tyr Cys Glu He Pro Ser Cys Glu 245 250 255 tcc tea gca tea cea gac cag tea gat tcc tea gtt cea gag gag 816 Being Ser Ala Ala Pro Pro Asp Gln As As Ser Ser Val Pro Pro Glu Glu 260 265 270 caá here ect gtg cag gaa tge tac cag age gat ggg cag age tat 864 Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr 275 280 285 c 99gt here teg tcc act acc atc here ggg aag aag tge cag tcc tgg 912 Arg Gly Thr Be Ser Thr Thr He Thr Gly Lys Lys Cys Gln Ser Trp 290 295 300 gca get atg ttt cea falls agg cat teg aag acc cea gag aac ttc cea 960 Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro 305 310 315 320 gat get ggc ttg gag atg aac tac tge agg aac ceg gat ggt gac aag 1008 Asp Wing Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys 325 330 335 ggc ect tgg tge tac acc act gac ceg age gtc agg tgg gaa tac tge 1056 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys 340 345 350 aac ctg aag cgg tge tea gag gga ggg ggg 1086 Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly 355 360 < 210 > 25 < 211 > 362 < 212 > PRT < 213 > Mus usculus < 400 > 25 Val Tyr Leu Ser Glu Cys Lys Thr Gly He Gly Asn Gly Tyr Arg Gly 1 5 10 15 Thr Met Ser Arg Thr Lys Ser Gly Val Wing Cys Gln Lys Trp Gly Wing 20 25 30 Thr Phe Pro His Val Pro Asn Tyr Ser Pro Thr His Pro Asn Glu 35 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn 65 70 75 80 He Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr 85 90 95 Glu Gly Lys He Ser Lys Thr Met Ser Gly Leu Asp Cys Gln Ala Trp 100 105 110 Asp Ser Gln Pro Pro His Wing His Gly Tyr He Pro Wing Lys Phe Pro 115 120 125 Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr Cys 145 150 155 160 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Pro Pro Ser Pro Thr Tyr 165 170 175 G__a Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val 180 185 190 Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His 195 200 205 Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asp Ser Gln Leu Arg Trp Glu Tyr Cys Glu He Pro Ser Cys Glu 245 250 255 Being Ser Wing Being Pro Asp Gln Being Asp Being Ser Val Pro Pro Glu Glu 260 265 270 Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr 275 280 285 Arg Gly Thr Ser Ser Thr Thr He Thr Gly Lys Lys Cys Gln Ser Trp 290 295 300 Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro 305 '310 315 320 Aep Wing Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys 325 330 335 Gly Pro Trp Cye Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys 340 345 350 Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly 355 360 < 210 > 26 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Front Primary for Fc -Angio mouse < 220 > < 221 > CDS < 222 > (2) .. (49) < 400 > 26 c ccc aag ctt gtg tat ctg tea gaa tgt aag 31 Pro Lys Leu Val Tyr Leu Ser Glu Cys Lys 1 5 10 < 210 > 27 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 400 > 27 Pro Lys Leu Val Tyr Leu Ser Glu Cys Lys 1 5 10 < 210 > 28 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Posterior Primary for mouse Fc-Angio < 400 > 28 cccctcgagc taccctcctg tctctgagca 30 < 210 > 29 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Front Primary for canine Fc < 220 > < 221 > CDS < 222 > (3) . (29) < 400 > 29 ce tta age gaa aat gga aga gtt ect cgc 29 Leu Ser Glu Asn Gly Arg Val Pro Arg 1 5 < 210 > 30 < _11 > 9 < 212 > PRT < 213 > Artificial Sequence < 400 > 30 Leu Ser Glu Asn Gly Arg Val Pro Arg 1 5 < 210 > 31 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Posterior Primary for canine Fc < 400 > 31 cctcgagtca tttacccggg gaatgggaga gggatttctg 40 < 210 > 32 < 211 > 702 < 212 > DNA < 213 > Canis familiaris < 220 > < 221 > CDS < 222 > (1) . . (702) < 223 > Fc < 400 > 32 gaa aat gga aga gtt ect cgc cea ect gat t? T ccc aaa tge cea gcc 48 Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Wing 1 5 10 15 ect gaa atg ctg gga ggg ect teg gtc ttc atc ttt ccc ceg aaa ccc 96 Pro Glu Met Leu Gly Gly Pro Ser Val Phe He Phe Pro Pro Lys Pro 20 25 30 aag gac acc ctc ttg att gce cga ac ect gag gtc here tgt gtg gtg 144 Lys Asp Thr Leu Leu He Wing Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 gtg gat ctg gga cea gaa gac ect gag gtg cag atc age tgg ttc gtg 192 Val Asp Leu Gly Pro Glu Asp Pro Glu Val Gln He Ser Trp Phe Val 50 55 60 gac ggt aag cag atg cag ac gcc aag act cag cet cgt gag gag cag 240 Asp Gly Lys Gln Met Gln Thr Wing Lys Thr Gln Pro Arg Glu Glu Gln 65 70 75 80 ttc aat ggc acc itt gtg gtc gtc agt gtc ctc ccc att ggg falls cag 288 Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro He Gly His Gln 85 90 95 gac tgg ctc aag ggg aag cag ttc acg tge aaa gtc aac aac aac aaa gcc 336 Asp Trp Leu Lys Gly Ly3 Gln Phe Thr Cys Lys Val Asn Asn Lys Wing 100 105 110 ctc cea tcc ceg atc gag agg acc atc tcc aag gce aga ggg cag gcc 384 Leu Pro Ser Pro He Glu Arg Thr He Ser Lys Wing Arg Gly Gln Wing 115 120 125 cat cag eco agt gtg tat gtc ctg ceg cea tcc cgg gag gag ttg age 432 His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser 130 135 140 aag aac here gtc age ttg here tge ctg atc aaa gac ttc tte cea ect 480 Lys Asn Thr Val Ser Leu Thr Cys Leu He Lys Asp Phe Phe Pro Pro 145 150 155 160 gac att gat gtg gag tgg cag age aat gga cag gag ect gag age 528 Asp He Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser 165 170 175 aag tac cgc acg acc ccg ccc cag ctg gac gag gac ggg tcc tac ttc 576 Lys Tyr Arg Thr Pro Pro Gln Leu Asp Glu Asp Gly Se r Tyr Phe 180 185 190 ctg tac age aag ctc tet gtg gao aag age cgc tgg cag cgg gga gac 624 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp 195 200 205 acc ttc ata tgt gcg gtg atg cat gaa get cta falls aac falls tac here 672 Thr Phe He Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 cag aaa tcc ctc tcc cat tet ceg ggt aaa 702 Gln Lys Ser Leu Ser His Ser Pro Gly Lys 225 230 < 210 > 33 < 211 > 234 < 212 > PRT < 213 > Canis fa iliaris < 400 > 33 Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Wing 10 15 Pro Glu Met Leu Gly Gly Pro Ser Val Phe He Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Leu He Wing Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Leu Gly Pro Glu Asp Pro Glu Val Gln He Ser Trp Phe Val 50 55 60 Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln 65 70 75 80 Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro He Gly His Gln 85 90 95 Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Wing 100 105 110 Leu Pro Ser Pro He Glu Arg Thr He Ser Lys Ala Arg Gly Gln Wing 115 120 125 His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser 130 135 140 Lys Asn Thr Val Ser Leu Thr Cys Leu He Lys Asp Phe Phe Pro Pro 145 150 155 160 Asp He Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser 165 170 175 Lys Tyr Arg Thr Thr Ero Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe 180 185 190 Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp 195 200 205 Thr Phß He Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser His Ser Pro Gly Lys 225 230 < 210 > 34 < 211 > 552 < 212 > DNA < 213 > Canis familiaris < 220 > < 221 > CDS < 222 > (1) .. (552) < 223 > Endostatine < 400 > 34 falls acc cag gac ttc cag ceg gtg ctg -cae ctg gtg gcc ctg aac 48 His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn 1 5 10 15 age ceg cag ceg ggc ggc atg cga ggc atc egg gga gcg gac ttc cag 96 Ser Pro Gln Pro Gly Gly Met Arg Gly He Arg Gly Wing Asp Phe Gln 20 25 30 tge ttc cag cag gcg cgc gce gcg ggg ctg gcc ggc acc ttc cgg gcc 144 Cys Phe Gln Gln Ala Arg Ala Ala Gly Leu Wing Gly Thr Phe Arg Wing 35 40 45 ttc ctg teg teg cgg ctg cag gac ctc tac age atc gtg cgc cgc gcc 192 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser He Val Arg Arg Ala 50 55 60 gac cgc acc ggg gtg ccc gtc gtc aac ctc agg gac gag gtg ctc ttc 240 Asp Arg Thr Gly Val Pro Val Val Asn Leu Arg Asp Glu Val Leu Phe 65 70 75 80 ccc age tgg gag gcc tta ttc teg ggc tcc gag ggc cag ctg aag ccc 288 Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Gln Leu Lys Pro 85 90 95 ggg gcc cgc atc ttc tet ttc gac ggc aga gat gtc ctg cag drops ccc 336 Gly Wing Arg He Phe Ser Phe Asp Gly Arg Asp Val Leu Gln His Pro 100 105 110 gcc tgg ccc cgg aag age gtg ggg tgg gcc tcc gcc ccc age ggg cgc 384 Wing Trp Pro Arg Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg 115 120 125 cgc ctg ace gac age tac tge gag acg tgg cgg acg gag gcc ceg gcg 432 Arg Leu Thr Asp Ser Tyr Cys Glu Thr Trp Arg Thr Glu Wing Pro Wing 130 135 140 gcc ace ggg cag gcg teg teg ctg ctg gcg ggc agg ctg ctg gag cag 480 Wing Thr Gly Gln Wing Being Ser Leu Leu Wing Gly Arg Leu Leu Glu Gln 145 150 155 160 gag gcc gcg age tge cgc falls gcc ttc gtg gtg ctc tge atc gag aac 528 Glu Wing Wing Cy3 Arg His Wing Phe Val Val Leu Cys He Glu Asn 165 170 175 age gtc atg acc tcc tcc acc 552 Ser Val Met Thr Ser Phe Ser Lys 180 < 210 > 35 < 211 > 184 < 212 > PRT < 213 > Canis familiaris < 400 > 35 His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Aen 1 5 10 15 Ser Pro Gln Pro Gly Gly Met Arg Gly He Arg Gly Wing Asp Phe Gln 20 25 30 Cys Phe Gln Gln Wing Arg Wing Wing Gly Leu Wing Gly Thr Phe Arg Wing 35 40 45 Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser He Val Arg Arg Ala 50 55 60 Asp Arg Thr Gly Val Pro Val Val Asn Leu Arg Asp Glu Val Leu Phe 65 70 75 80 Pro Ser Trp Glu Wing Leu Phe Ser Gly Ser Glu Gly Gln Leu Lys Pro 85 90 95 Gly Wing Arg He Phe Ser Phe Asp Gly Arg Asp Val Leu Gln His Pro 100 105 110 Wing Trp Pro Arg Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg 115 120 125 Arg Leu Thr Asp Ser Tyr Cys Glu Thr Trp Arg Thr Glu Wing Pro Wing 130 135 140 Wing Thr Gly Gln Wing Being Ser Leu Leu Wing Gly Arg Leu Leu Glu Gln 145 150 155 160 Glu Ala Ala Ser Cys Arg His Wing Phe Val Val Leu Cys He Glu Asn 165 170 175 Ser Val Met Thr Ser Phe Ser Lys 180 < 210 > 36 < 211 > 41 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Linker HinD lll Dra lll: upper filament < 220 > < 221 > CDS < 222 > (3) .. (41) < 400 > 36 ag ctt falls acc falls oag gac ttc cag ceg gtg ctg falls ctg 41 Leu His Thr His Gln Asp Phe Gln Pro Val Leu His Leu 1 5 10 < 210 > 37 < 211 > 13 < 212 > PRT < 213 > Artificial Sequence < 400 > 37 Leu His Thr His Gln Asp Phe Gln Pro Val Leu His Leu 1 5 10 < 210 > 38 < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Hindlll / Dralll linker: inner filament < 400 > 38 gtgcagcacc ggctggaagt cctggtgggt gtga 34 < 210 > 39 < 211 > 1077 < 212 > DNA < 213 > Canis familiaris < 220 > < 221 > CDS < 222 > (1) . . (1077) < 223 > angiostatin < 400 > 39 ata tat ctt tea gag tge aag act gga aat ggg aaa acc tac agg ggg 48 He Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Thr Tyr Arg Gly 1 5 10 15 acc atg gcc aaa acg aag aat gat gtt gcc tgt caa aaa tgg agt gac 96 Thr Met Wing Lys Thr Lys Asn Asp Val Wing Cys Gln Lys Trp Ser Asp 20 25 30 aat tet ceg falls aaa ect aac tat acg ect gag aag falls ccc ttg gag 144 Asn Pro Pro His Lys Pro Asn Tyr Thr Pro Glu Lys His Pro Leu Glu 35"40 45 ggg ctg gag gag aac tat tge agg aac ect gac aac gac gag aac ggg 192 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly 50 55 60 ccc tgg tge tac acc acac aac cea gac gtg agg ttc gac tac tge aac 240 Pro Trp Cys Tyr Thr Thr Asn Pro Asp Val Arg Phe Asp Tyr Cys Asn 65 70 75 80 att cea gaa tgt gaa gag gag tgt atg cat tge agt ggg gaa aat tat 288 He Pro Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr 85 90 95 gag ggc aaa att tce aag ac aag tet gga ctc gag tge ca gcc tgg 336 Glu Gly Lys He Ser Lys Thr Lys Ser Gly Leu Glu Cys Gln Wing Trp 100 105 110 aac tet caa acc cat cat get cat gga tat att ect tcc aaa ttt cea 384 Asn Ser Gln Thr Pro His Wing His Gly Tyr He Pro Ser Lys Phe Pro 115 120 125 age aag aac ttg aag t a t t c t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t tgg tgt ttc acc atg gat eco aac aaa cgc tgg gaa ttc tgt 480 Arg Pro Trp Cys Phe Thr Met Asp Pro Asn Lys Arg Trp Glu Phe Cys 145 150 155 160 gac att ccc cgc tgt here here cea ccc ect teg ggc cea acg tac 528 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Pro Gly Pro Thr Tyr 165 170 175 cag tgt ctg aag ggc aga ggg gag age tac cga ggg aag gtg tcc gtc 576 Gln Cys Leu Lys Gly Arg Gly Glu Ser Tyr Arg Gly Lys Val Ser Val 180 185 190 act gtc tet gga cat here tgt cag falls tgg agt gaa cag acc cet falls 624 Thr Val Ser Gly His Thr Cys Gln His Trp Ser Glu Gln Thr Pro His 195 200 205 aag falls aac agg ac cea gaa aac ttc ect tge aaa aat ttg gat gaa 672 Lys His Asn Arg Thr Pro Glu Asn Phß Pro Cys Lys Asn Leu Asp Glu 210 215 220 aac tac tgt cgc aac ect gat gga gaa here get cea tgg tge tac here 720 Asn Tyr Cys Arg Asn. Pro Asp Gly Glu Thr Wing Pro Trp Cys Tyr Thr 225 230 235 240 acc aac agt gag gtg agg tgg gaa falls tge cag att ceg tcc tgt gag 768 Thr Asn Ser Glu Val Arg Trp Glu His Cys Gln He Pro Ser Cys Glu 245 250 255 tcc tet cea ata acc here gaa tat ttg gat gcc cea get tea gtg cea 816 Ser Ser Pro He Thr Thr Glu Tyr Leu Asp Wing Pro Wing Ser Val Pro 260 265 270 ect gaa caa act ect gtg gtc oag gag tge tac cae ggc aat ggg cag 864 Pro Glu Gln Tlir Pro Val Val Gln Glu Cys Tyr His Gly Asn Gly Gln 275 280 285 agt tat cga ggc here teact act act atc here gga aga aaa tgt cag 912 Ser Tyr Arg Gly Thr Ser Ser Thr Thr He Thr Gly Arg Lys Cys Gln 290 295 300 tet tgg tea tet atg here cea cae cga cat gag aag acc cea gaa cae 960 Ser Trp Ser Ser Met Thr Pro His Arg His Glu Lys Thr Pro Glu His 305 310 315 320 ttc ceg gag get ggc ctg here atg aac tac tge agg aat ccc gac gcc 1008 Phe Pro Glu Wing Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Wing 325 330 335 gac aaa age ect tgg tgt tac acc acc gac cec tet gtg cgc tgg gag 1056 Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu 340 345 350 ttc tgt aac ttg aga aaa tge 1077 Phe Cys Asn e *? Arg Lys Cys 355 < 210 > 40 < 211 > 359 < 212 > PRT < 213 > Canis familiaris < 400 > 40 He Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Thr Tyr Arg Gly 1 5 10 15 Thr Met Wing Lys Thr Lys Asn Asp Val Wing Cys Gln Lys Trp Ser Asp 20 25 30 Asn Ser Pro His Lys Pro Asn Tyr Thr Pro Glu Lys His Pro Leu Glu 35 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asn Pro Asp Val Arg Phe Asp Tyr Cys Asn 65 70 75 80 He Pro Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr 85 90 95 Glu Gly Lys He Ser Lys Thr Lys Ser Gly Leu Glu Cys Gln Ala Trp 100 105 110 Asn Ser Gln Thr Pro His Wing His Gly Tyr He Pro Ser Lys Phe Pro 115 120 125 Ser Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Met Asp Pro Asn Lys Arg Trp Glu Phe Cys 145 150 155 160 Asp He Pro Arg Cys Thr Thr Pro Pro Pro Pro Ser Gly Pro Thr Tyr 165 170 175 Gln Cys Leu Lys Gly Arg Gly Glu Ser Tyr Arg Gly Lys Val Ser Val 180 185 190 Thr Val Ser Gly His Thr Cys Gln His Trp Ser Glu Gln Thr Pro His 195 200 205 Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asn Ser Glu Val Arg Trp Glu His Cys Gln He Pro Ser Cys Glu 245 250 255 Ser Ser Pro Thr Thr Glu Tyr Leu Asp Wing Pro Wing Ser Val Pro 260 265 270 Pro Glu Gln Thr Pro Val Val Gln Glu Cys Tyr His Gly Asn Gly Gln 275 280 285 Ser Tyr Arg Gly Thr Ser Ser Thr Thr He Thr Gly Arg Lys Cys Gln 290 295 300 Ser Trp Ser Ser Met Thr Pro His Arg His Glu Lys Thr Pro Glu His 305 310 315 320 Phe Pro Glu Wing Gly Leu Thr Met Asn Tyr Cy3 Arg Asn Pro Asp Wing 325 330 335 Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu 340 345 350 Phe Cys Asn Leu Arg Lys Cys 355 < 210 > 41 < 211 > 12 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: palindromic linker, where the TGA stop codon is followed by an Xhol site < 400 > 41 tgactcgagt ca 12 < 210 > 42 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Mutagenic Primary for murine angiostatin < 220 > < 221 > CDS < 222 > (1). (21) < 400 > 42 ggg ect tgg age tac act 21 Gly Pro Trp Ser Tyr Thr Thr 1 5 < 210 > 43 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 400 > 43 Gly Pro Trp Ser Tyr Thr Thr 1 5 < 210 > 44 < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: first used to introduce Hind lll in murine angiostatin < 220 > < 221 > CDS < 222 > (9) (32) < 400 > 44 gcggatcc aag ctt agt here cat ccc aat gag gg 34 Lys Leu Ser Thr His Pro Asn Glu 1 5 < 210 > 45 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 400 > 45 Lys Leu Ser Thr His Pro Asn Glu 1 5 < 210 > 46 < 211 > 59 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: linker BspHI / BamHI: upper filament < 220 > < 221 > CDS < 222 > (2) . (58) < 400 > 46 c atg acc tet tcc tcc aaa teg age ggg ggc age ggg ggc gga ggc age 49 Met Thr Ser Phe Ser Lys Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 ggc ggg ggc 59 Gly Gly Gly < 210 > 47 < 211 > 19 < 212 > PRT < 213 > Artificial Sequence < 400 > 47 Met Thr Ser Phe Ser Lys Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly < 210 > 48 < 211 > 59 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: linker BspHI / BamHI: lower filament < 400 > 48 gatccgcccc cgccgctgcc tccgcccecg ctgcccccgc tcgatttgga gaaagaggt 59 < 210 > 49 < 211 > 12 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: linker BamHI / Hind lll: upper filament < 220 > < 221 > CDS < 222 > (3). (H) < 400 > 49 ga tcc tea ggc c 12 Ser Ser Gly 1 < 210 > 50 < 211 > 12 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: linker BamHI / HInd III: f i lower lament < 400 > 50 agctggcctg ag 12 < 210 > 51 < 211 > 10 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Artificial Sequence Description: Aflll linker Hind lll: upper filament < 220 > < 221 > CDS < 222 > (1). . (9) < 400 > 51 tta age ggc c 10 Leu Ser Gly 1 < 210 > 52 < 211 > 10 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: linker Afl ll / Hínd I lower filament < 400 > 52 agctgggcgc 10 < 210 > 53 < 211 > 11 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Xhol / Afl II linker: upper filament < 220 > < 221 > CDS < 222 > (3) .. (l?) < 400 > 53 tc gac tcc ggc 11 Asp Ser Gly 1 < 210 > 54 < 211 > 11 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: linker Xhol Afl II: lower filament < 400 > 54 ttaagecgga g 11

Claims (31)

1. CLAIMS 1. A DNA molecule encoding a fusion protein comprising: (a) a signal sequence; (b) an immunoglobulin Fc region; and (c) an objective protein sequence that is selected from the group consisting of angioestatin, endoestatin, a fragment of plasminogen having angioestatin activity, a fragment of collagen XVIII having endoestatin activity, and combinations thereof. The DNA according to claim 1, characterized in that said signal sequence, said immunoglobulin Fc region and said target protein sequence are serially encoded in a 5 'to 3' direction. The DNA according to claim 1, characterized in that said signal sequence, said target sequence, and said immunoglobulin Fc region are serially encoded in a 5 'to 37 direction. The DNA according to claim 1, characterized in that said Fc region of immunoglobulin comprises an immunoglobulin joint region. The DNA according to claim 1, characterized in that said Fc region of immunoglobulin comprises an immunoglobulin joint region and an immunoglobulin constant heavy chain domain. 6. The DNA according to claim 1, characterized in that said Fc region of immunoglobulin comprises a region of articulation and a CH3 domain. The DNA according to claim 1, characterized in that said Fc region of immunoglobulin lacks at least one CHi domain. The DNA according to claim 1, characterized in that said Fc region of immunoglobulin encodes at least a portion of the immunoglobulin range. 9. A reproducible expression vector for transfecting a mammalian cell, said vector comprising the DNA of claim 1. 1 0. A mammalian cell that hosts the DNA of claim 1. eleven . A fusion protein comprising an immunoglobulin Fc region, a target protein that is selected from the group consisting of angioestatin, endoestatin, a fragment of plasminogen having angioestatin activity, a fragment of collagen XVIII having endoestatin activity, and combinations of the same. The fusion protein according to claim 1, characterized in that said plasminogen fragment has a molecular weight of about 40 kD and comprises an amino acid sequence set forth in SEQ ID NO. 3. The fusion protein according to claim 1, characterized in that said target protein comprises the amino acid sequence set forth in SEQ ID NO: 3. The fusion protein according to claim 1, characterized in that said fragment of collagen XVIII comprises the amino acid sequence set forth in SEQ ID NO: 1. 15. The fusion protein according to claim 1, characterized in that said target protein comprises at least two molecules that are selected from the group consisting of angioestatin, endoestatin, a fragment of plasminogen, and a fragment of collagen XVIII, wherein said two molecules they are linked by a polypeptide linker program. 16. The fusion protein according to claim 1, characterized in that said target protein is linked to an N-terminal extremity of said immunoglobulin Fc region. 7. The fusion protein according to claim 11, characterized in that said target protein is linked to a C-terminal extremity of said immunoglobulin Fc region. 18. A multimeric protein comprising at least two fusion proteins of claim 1 linked via a disulfide bond. 19. The multimeric protein according to claim 18, characterized in that the target protein of said at least one fusion protein is angioestatin and the target protein of said at least one fusion protein is endoestatin. 20. The multimeric protein according to claim 18, characterized in that the target protein of both said fusion proteins is angioestatin. twenty-one . The multimeric protein according to claim 18, characterized in that the target protein of both said fusion proteins is endo-statin. 22. The fusion protein according to claim 1, characterized in that it further comprises a second target protein selected from the group consisting of angioestatin, endoestatin, a fragment of plasminogen having angioestatin activity, and a fragment of collagen XVIII having endoestatin activity. 23. The fusion protein according to claim 22, characterized in that said second target protein is linked by a polypeptide linker program to said first target protein. 24. The fusion protein according to claim 22, characterized in that said first target protein is connected to an N-terminal extremity of said immunoglobulin Fc region and said second target protein is connected to a C-terminal extremity of said immunoglobulin Fc region. . 25. A multimeric fusion protein comprising at least two fusion proteins according to claim 1, characterized in that said fusion proteins are linked by a polypeptide linkage. 26. A method of producing a fusion protein, the method comprising the steps of: (a) providing the mammalian cell according to claim 10; and (b) culturing the mammalian cell to produce said fusion protein. 27. The method according to claim 26 comprising the additional step of collecting said fusion protein. The method according to claim 26 comprising the additional step of separating said immunoglobulin Fc region from said target protein. 29. A method of treating an angiogenesis-mediated condition comprising the step of administering the DNA according to claim 1 to a mammal in need of an angiogenesis inhibitor. 30. A method of treating an angiogenesis-mediated condition comprising the step of administering the vector according to claim 9 to a mammal in need of an angiogenesis inhibitor. 31 A method of treating a condition mitigated by the administration of angioestatin or endoestatin comprising the step of administering an effective amount of the fusion protein according to claim 1 to a mammal having said condition. • - - 88 SUMMARY The described ones are nucleotide sequences, for example, DNA or RNA sequences, which encode an immunoglobulin Fc-angiogenesis inhibitory fusion protein. The angiogenesis inhibitors can be angioestatin, endo-statin, a fragment of plasminogen having angio-statin activity or a fragment of collagen XVIII having endo-statin activity. The nucleotide sequences can be inserted into a suitable expression vector and expressed in mammalian cells. A family of fusion proteins inhibiting Fc-0 immunoglobulin angiogenesis that can be produced by the expression of such nucleotide sequences is also disclosed. Methods using such nucleotide sequences and fusion proteins to treat conditions mediated by angiogenesis are also discussed. 5 0