US20030139365A1 - Expression and export of angiogenesis inhibitors as immunofusins - Google Patents

Expression and export of angiogenesis inhibitors as immunofusins Download PDF

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US20030139365A1
US20030139365A1 US10/292,418 US29241802A US2003139365A1 US 20030139365 A1 US20030139365 A1 US 20030139365A1 US 29241802 A US29241802 A US 29241802A US 2003139365 A1 US2003139365 A1 US 2003139365A1
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Kin-Ming Lo
Yue Li
Stephen Gillies
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Merck Patent GmbH
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EMD Serono Research Center Inc
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    • C07K14/475—Growth factors; Growth regulators
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier

Definitions

  • 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 Fc region and an angiogenesis inhibitor.
  • angiostatin O'Reilly et al. (1994) Cell 79:315) and endostatin (O'Reilly et al. (1997) Cell 88:277)
  • 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.
  • angiogenesis inhibitors are non-toxic even at very high doses and that they may suppressed the growth of metastases and primary tumors may regress to a dormant microscopic state. Both inhibitors were identified as proteolytic fragments of much larger intact molecules.
  • Angiostatin was found to be a fragment of plasminogen, and endostatin a fragment of collagen XVIII.
  • mice Despite promising results in mice, it has not been possible to produce clinical grade soluble, active angiostatin and endostatin in commercial quantities using E. coli , baculoviral, yeast, and mammalian expression systems. Expression in E. coli yielded insoluble protein aggregates of undefined composition, which could not be injected into humans. Other production methods, such as baculovirus and mammalian expression systems, yielded very low levels of the recombinant proteins (O'Reilly et al. (1997) Cell 88:277).
  • angiostatin has 26 cysteine residues which form numerous disulfide bonds. Expression of angiostatin by itself may not provide the optimal environment for these numerous disulfide bonds to form correctly in the secretory pathway. Also, the recombinant endostatin protein produced in E. coli precipitated during dialysis, possibly due to the hydrophobicity of endostatin (O'Reilly et al. (1997) Cell 88:277).
  • a major hurdle with the use of angiostatin and endostatin in their present forms is that relatively large amounts of proteins have to be injected daily for weeks to months to achieve the desired clinical outcome. For example, in current mouse models, dosages of 20 mg/kg/day of endostatin are needed to demonstrate optimal efficacy (Boehm et al. (1997) Nature 390:404). Given that there is an urgent need to test endostatin and angiostatin clinically, a production method that can generate large quantities of clinical grade material is important.
  • One expression system that has been used to produce high level expression of fusion proteins in mammalian cells is a DNA construct encoding, a signal sequence, an immunoglobulin Fc region and a target protein.
  • the fusion product of this construct generally is termed an “immunofusin.”
  • target proteins have been expressed successfully as immunofusins which include: IL2, CD26, Tat, Rev, OSF-2, ⁇ IG-H3, IgE Receptor, PSMA, and gp120.
  • These expression constructs are disclosed in U.S. Pat. No. 5,541,087 and U.S. Pat. No. 5,726,044, the disclosures of which are incorporated herein by reference.
  • a major purpose of expressing recombinant fusion proteins in mammalian cells has been to attempt to confer novel or useful properties to the hybrid molecules, e.g., proper folding, increased solubility, targeting of a cytokine or toxin in vivo, Fe receptor binding, complement fixation, protein A binding, increased circulation half-life, and increased ability to cross the blood-brain barrier.
  • Examples of recombinant fusion proteins produced in mammalian cells include cytokine immunoconjugates (Gillies et al. (1992) Proc. Natl. Acad. Sci. USA 89:1428; Gillies et al. (1993) Bioconjugate Chemistry 4:230), immunoadhesins (Capon et al.
  • the present invention features methods and compositions useful in making and using fusion proteins containing an angiogenesis inhibitor protein.
  • the fusion proteins can facilitate a high level expression of biologically active angiogenesis inhibitor proteins.
  • the angiogenesis inhibitor proteins can then be cleaved from the fusion protein and combined with a pharmaceutically acceptable carrier prior to administration to a mammal, for example, a human.
  • nucleic sequences encoding, or amino acid sequences defining the angiogenesis inhibitor containing fusion proteins can be combined with a pharmaceutically acceptable carrier and administered to the mammal.
  • the invention provides nucleic acid molecules, for example, DNA or RNA molecules, encoding 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 inhibitor protein, selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, a collagen XVIII fragment having endostatin activity, and combinations thereof.
  • the nucleic acid molecule encodes, serially in a 5′ to 3′ direction, the signal sequence, the immunoglobulin Fc region and the target protein sequence.
  • the nucleic acid molecule encodes, serially in a 5′ to 3′ direction, the signal sequence, the target sequence, and immunoglobulin Fc region.
  • the immunoglobulin Fc region comprises an immunoglobulin hinge region and preferably comprises at least one immunoglobulin constant heavy region, for example, an immunoglobulin constant heavy 2 (CH 2 ) domain, an immunoglobulin constant heavy 3 (CH 3 ) domain), and depending upon the type of immunoglobulin used to generate the Fc region, optionally an immunoglobulin constant heavy region 4 (CH4) domain.
  • the immunoglobulin Fc region comprises a hinge region, a CH 2 domain and a CH 3 domain. Under certain circumstances, the immunoglobulin Fc region preferably lacks at least the CH 1 , domain.
  • the immunoglobulin Fc regions may be based on any immunoglobulin class, for example, IgA, IgD, IgE, IgG, and IgM, immunoglobulin Fc regions based on IgG are preferred.
  • nucleic acid of the invention can be incorporated in operative association into a replicable expression vector which can then be transfected into a mammalian host cell.
  • the invention provides host cells harboring such nucleic acid sequences of the invention.
  • the invention provides a fusion protein comprising an immunoglobulin Fc region linked, either directly through a polypeptide bond or by means of a polypeptide linker, to a target protein selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, a collagen XVIII fragment having endostatin activity, and combinations thereof.
  • a target protein selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, a collagen XVIII fragment having endostatin activity, and combinations thereof.
  • the target protein may be fused via its C-terminal end to an N-terminal end of the immunoglobulin Fc region.
  • the target protein is fused via its N-terminal end to a C-terminal end of the immunoglobulin Fc region.
  • the fusion protein may comprise a second target protein selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, and a collagen XVIII fragment having endostatin activity.
  • the first and second target proteins can be the same or different proteins.
  • the fusion protein comprises a first target protein of angiostatin, an immunoglobulin Fc region and a second target protein of endostatin.
  • the first and second target proteins may be linked together, either directly or by means of a polypeptide linker.
  • both target proteins may be linked, either directly or via a polypeptide linker, to the immunoglobulin Fc region. In the latter case, the first target protein is connected to an N-terminal end of the immunoglobulin Fc region and the second target protein is connected to a C-terminal end of the immunoglobulin Fc region.
  • two fusion proteins may associate, either covalently, for example, by a disulfide or peptide bond, or non-covalently, to produce a multimeric protein.
  • two fusion proteins are associated covalently by means of one or more disulfide bonds through cysteine residues, preferably located within immunoglobulin hinge regions disposed within the immunoglobulin Fc regions of both chains.
  • the target protein comprises a plasminogen fragment having a molecular weight of approximately 40 kD and, optionally comprises, an amino acid sequence as set forth in SEQ ID NO: 3.
  • the target protein comprises a collagen XVIII fragment having an amino acid sequence set forth in SEQ ID NO: 1.
  • the target protein can be full-length angiostatin or endostatin or bioactive fragments thereof.
  • the source of the target protein in generating certain fusion proteins will depend upon the intended use of the target protein. For example, if the target protein is to be administered to a human, the target protein preferably is of human origin.
  • the invention provides methods of producing a fusion protein comprising an immunoglobulin Fc region and a target protein selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, and a collagen XVIII fragment having endostatin activity.
  • the method comprises the steps of (a) providing a mammalian cell containing a DNA molecule encoding such 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 then be harvested, refolded, if necessary, and purified using conventional purification techniques well known and used in the art.
  • the target can be cleaved from the fusion protein using conventional proteolytic enzymes and if necessary, purified prior to use.
  • the invention provides methods for treating mammals, for example, a human, in need of an angiogenesis inhibitor based therapy.
  • the angiogenesis inhibitors of the invention may be administered to a human afflicted with a tumor.
  • Treatment with the angiogenesis inhibitor may slow down or stop tumor growth and, under certain circumstances, may cause tumor regression.
  • Treatment may include administering to the mammal an amount of the angiogenesis inhibitor in an amount sufficient to slow down or stop tumor growth.
  • the angiogenesis inhibitor may 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.
  • FIGS. 1 A- 1 F are schematic illustrations of exemplary angiogenesis inhibitor fusion proteins constructed in accordance with the invention (see Examples 10-15).
  • the vertical lines represent optional disulfide bonds connecting cysteine residues (C) disposal within a hinge region of the Fc molecule.
  • the invention provides fusion proteins, referred to herein as immunofusins, which were useful in the production of commercial quantities of clinical grade angiogenesis inhibitors.
  • the angiogenesis inhibitors may be cleaved from the immunofusin protein constructs prior to use. However, it is contemplated that the immunofusins or nucleic acids encoding the immunofusins may be administered directly to mammals in need of treatment with an angiogenesis inhibitor.
  • the invention thus provides fusion proteins comprising an immunoglobulin Fc region and at least one target protein, referred to herein as an angiogenesis inhibitor.
  • the angiogenesis inhibitor preferably is selected from the group consisting of angiostatin, endostatin, a plasminogen fragment angiostatin activity, a collagen XVIII fragment having endostatin activity. It is contemplated, however, that other polypeptides having angiogenesis inhibitor activity, now known or late discovered, may be expressed as fusion proteins of the type described herein.
  • FIGS. 1 A- 1 F Six exemplary embodiments of protein constructs embodying the invention are illustrated in the drawing as FIGS. 1 A- 1 F. Because dimeric constructs are preferred, all are illustrated as dimers cross-linked by a pair of disulfide bonds between cysteines on adjacent subunits.
  • the disulfide bridges are depicted as linking together the portions of two immunoglobulin Fc regions via an immunoglobulin hinge region, and thus are characteristic of native forms of these molecules. While constructs including the hinge region of Fc are preferred and have been shown promise as therapeutic agents, the invention contemplates that the crosslinking at other positions may be chosen as desired.
  • dimers or multimers useful in the practice of the invention may be produced by non-covalent association, for example, by hydrophobic interaction.
  • FIG. 1 illustrates such constructs.
  • heterodimeric structures also are useful but, as is known to those skilled in the art, often can be difficult to purify.
  • viable constructs useful to inhibit angiogenesis in various mammalian species, including humans can be constructed comprising a mixture of homodimers and heterodimers.
  • one chain of the heterodimeric structure may comprise endostatin and the another may comprise angiostatin.
  • FIG. 1A illustrates a dimer construct produced in accordance with the procedure set forth in Example 10.
  • Each monomer of the dimer comprises an immunoglobulin Fc region 1 including a hinge region, a CH 2 domain and a CH 3 domain. Attached directly to the C terminus of the Fc region 1 is the first Kringle region of angiostatin 2, both inner and outer rings.
  • FIG. 1B shows a second embodiment of the invention (see Example 11) comprising the same Fc region as in FIG. 1A, this time having only the inner ring of Kringle one of angiostatin 3 attached to the C terminal end of the Fc region 1.
  • FIG. 1C through 1E depict various embodiments of the protein constructs of the invention, which include as a target protein plural angiogenesis inhibitors arranged in tandem and connected by a linker.
  • the target protein comprises full-length endostatin 4, a polypeptide linker 5, and the inner ring of Kringle one of angiostatin 3.
  • FIG. 1D depicts a protein comprising an Fc region the same as that of FIG. 1A and a target protein comprising a full-length endostatin 4, a polypeptide linker 5, and a full Kringle one region of angiostatin (both inner and outer rings) 2.
  • FIG. 1E differs from the construct of FIG. 1D in that the most C terminal protein domain comprises a full-length copy of angiostatin 7.
  • FIGS. 1 A- 1 E represent Fc-X type constructs, where X is the target protein, it is contemplated that X-Fc type constructs may also be useful in the practice of the invention. Furthermore, it is contemplated the useful proteins of the invention may also be depicted by the formula X-Fc-X, wherein the Xs may represent the same or different target proteins.
  • FIG. 1F depicts such a construct which comprises in an N- to C-terminal direction, full-length human angiostatin 7, a human immunoglobulin Fc region 6 including a hinge region, and full-length human endostatin domain 4.
  • angiogenesis inhibitor refers to any polypeptide chain that reduces or inhibits the formation of new blood vessels in a mammal. With regard 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 may be identified using a variety of assays well known and used in the art. Such assays include, for example, the bovine capillary endothelial cell proliferation assay, the chick chorioallantoic membrane (CAM) assay or the mouse corneal assay. However, the CAM assay is preferred (see, for example, O'Reilly et al.
  • angiogenesis inhibitors useful in the practice of the invention include, for example, angiostatin (O'Reilly et al. (1994) Cell 79: 315-328, and U.S. Pat. Nos. 5,733,876; 5,837,682; and 5,885,795), and endostatin (O'Reilly et al. (1997) Cell 88: 277-285 and U.S. Pat. No. 5,854,205).
  • angiostatin and endostatin are specific inhibitors of endothelial cell proliferation and are capable of inhibiting tumor growth by blocking angiogenesis, the formation of new blood vessels that nourish tumors.
  • angiostatin has been identified as a proteolytic fragment of plasminogen (O'Reilly et al. (1994) Cell 79: 315-328, and U.S. Pat. Nos. 5,733,876; 5,837,682; and 5,885,795, the disclosure of which is incorporated herein by reference). Specifically, angiostatin is a 38 kDa internal fragment of plasminogen containing at least three of the Kringle regions of plasminogen. Endostatin 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).
  • endostatin is a 20 kDa C-terminal fragment of collagen XVIII.
  • angiostatin and endostatin refer not only to the full length proteins, but also to variants and bioactive fragments thereof, as well as to bioactive fragments of plasminogen and collagen XVIII, respectively.
  • bioactive fragment refers to any protein fragment of plasminogen or angiostatin that has at least 30%, more preferably at least 70%, and most preferably at least 90% of the activity of full-length angiostatin as determined by the CAM assay.
  • bioactive fragment refers to any protein fragment of collagen XVIII or endostatin that has at least 30%, more preferably at least 70% and most preferably at least 90% of the activity of full length endostatin as determined by the CAM assay.
  • variants includes specifies and allelic variants, as well as other naturally occurring or non-naturally occurring variants, for example, generated by conventional genetic engineering protocols, that are at least 70% similar or 60% identical, more preferably at least 75% similar or 65% identical, and most preferably 80% similar or 70% identical to either the naturally-occurring sequences of endostatin or angiostatin disclosed herein.
  • the candidate amino acid sequence and the reference amino acid sequence are first aligned using the dynamic programming algorithm described in Smith and Waterman (1981), J. Mol. Biol. 147:195-197, in combination with the BLOSUM62 substitution matrix described in FIG. 2 of Henikoff and Henikoff (1992), “Amino acid substitution matrices from protein blocks”, Proc. Natl. Acad Sci. USA 89:10915-10919.
  • an appropriate value for the gap insertion penalty is ⁇ 12
  • an appropriate value for the gap extension penalty is ⁇ 4.
  • Computer programs performing alignments using the algorithm of Smith-Waterman and the BLOSUM62 matrix such as the GCG program suite (Oxford Molecular Group, Oxford, England), are commercially available and widely used by those skilled in the art.
  • a percent similarity score may 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 matrix corresponding to the two aligned amino acids is zero or a negative number, the pair-wise similarity score is zero; otherwise the pair-wise similarity score is 1.0.
  • the raw similarity score is the sum of the pair-wise similarity scores of the aligned amino acids. The raw score then is normalized by dividing it by the number of amino acids in the smaller of the 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 again are compared sequentially.
  • the pair-wise identity score is zero; otherwise the pair-wise 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 of the 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. Accordingly, gap penalties are not used in this calculation, although they are used in the initial alignment.
  • each immunoglobulin heavy chain constant region is comprised of four or five domains.
  • the domains are named sequentially as follows: CH 1 -hinge-CH 2 -CH 3 (-CH 4 ).
  • the DNA sequences of the heavy chain domains have cross-homology among the immunoglobulin classes, e.g., the CH 2 domain of IgG is homologous to the CH 2 domain of IgA and IgD, and to the CH 3 domain of IgM and IgE.
  • an 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.
  • an immunoglobulin Fc region may comprise 1) a CH 1 domain, a CH 2 domain, and a CH 3 domain, 2) a CH 1 , domain and a CH 2 domain, 3) a CH 1 , domain and a CH 3 domain, 4) a CH 2 domain and a CH 3 domain, or 5) a combination of two or more domains and an immunoglobulin hinge region.
  • the Fc region used in the DNA construct includes at least an immunoglobulin hinge region a CH 2 domain and a CH 3 domain and preferably lacks at least the CH 1 , domain.
  • the currently preferred class of immunoglobulin from which the heavy chain constant region is derived is IgG (Ig ⁇ ) ( ⁇ subclasses 1, 2, 3, or 4).
  • IgG immunoglobulin
  • Other classes of immunoglobulin, IgA (Ig ⁇ ), IgD (Ig ⁇ ), IgE (Ig ⁇ ) and IgM (Ig ⁇ ) may be used.
  • IgA immunoglobulin heavy chain constant regions
  • IgA immunoglobulin heavy chain constant regions
  • IgD Ig ⁇
  • IgE Ig ⁇
  • IgM IgM
  • the choice of appropriate immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044.
  • the choice of particular immunoglobulin heavy chain constant region sequences from certain immunoglobulin classes and subclasses to achieve a particular result is considered to be within the level of skill in the art.
  • the portion of the DNA construct encoding the immunoglobulin Fc region preferably comprises at least a portion of a hinge domain, and preferably at least a portion of a CH 3 domain of Fc ⁇ or the homologous domains in any of IgA, IgD, IgE, or IgM.
  • constant region genes from species other than human e.g., mouse or rat may be used.
  • the Fc region used as a fusion partner in the immunofusin DNA construct generally may be from any mammalian species. Where it is undesirable to elicit an immune response in the host cell or animal against the Fc region, the Fc region may be derived from the same species as the host cell or animal.
  • human Fc can be used when the host animal or cell is human; likewise, murine Fc can be used where the host animal or cell will be a mouse.
  • substitution or deletion of constructs of these constant regions, in which one or more amino acid residues of the constant region domains are substituted or deleted also would be useful.
  • One of ordinary skill in the art can prepare such constructs using well known molecular biology techniques.
  • the use of human Fc ⁇ 1 as the Fc region sequence has several advantages.
  • the Fc ⁇ 1 domain may confer the effector function activities to the fusion protein.
  • the effector function activities include the biological activities such as complement fixation, antibody-directed cellular cytotoxicity, placental transfer, and increased serum half-life.
  • the Fc domain also provides for detection by anti-Fc ELISA and purification through binding to Staphylococcus aureus protein A (“Protein A”). In certain applications, however, it may be desirable to delete specific effector functions from the Fc region, such as Fc receptor binding or complement fixation.
  • angiogenesis inhibitor immunofusins one function of the immunoglobulin Fc fusion partner is to facilitate proper folding of the angiogenesis inhibitor protein to yield active angiogenesis inhibitor protein and to impact solubility to the active moieties, at least in the extracellular medium. Since the Fc fusion partner is hydrophilic, the angiogenesis inhibitor immunofusin readily is soluble unlike, for example, the recombinant endostatin produced in E. coli (O'Reilly (1997) Cell 88:277.)
  • angiogenesis inhibitor immunofusins were secreted into media at concentrations typically of about 30 to 100 ⁇ g/ml, and could be purified readily to homogeneity by Protein A chromatography.
  • the angiogenesis inhibitor immunofusins could be cleaved and further purified using conventional purification protocols using, for example, by heparin sepharose, lysine sepharose or affinity purification.
  • fusion proteins of the invention also exhibit longer serum half-lives, presumably due to their larger molecular sizes.
  • human Fc-human angiostatin has a serum half-life of 33 hours in mouse, as compared to 4-6 hours for human angiostatin (O'Reilly et al. (1996) Nature Medicine 2:689). It is believe that angiostatin with a molecular weight of 40 kD, and endostatin with a molecular weight of 20 kD, are small enough to be cleared efficiently by renal filtration.
  • the dimeric forms of Fc-angiostatin and dimeric Fc-endostatin are 145 kD and 100 kD, respectively, because there are two immunoglobulin Fc regions attached to either two angiostatin molecules or two endostatin molecules.
  • Such a bivalent structure may exhibit a higher binding affinity to the angiostatin or endostatin receptor. If the angiogenesis inhibiting activity is receptor-mediated, the Fc fusion proteins are potentially more effective to suppress tumors than monovalent angiostatin or monovalent endostatin by themselves.
  • angiostatin and/or endostatin belong to a class of dimeric protein ligands
  • the physical constraint imposed by the Fc on angiostatin or endostatin would make the dimerization an intramolecular process, thus shifting the equilibrium in favor of the dimer and enhancing its binding to the receptor.
  • Cysteine residues can also be introduced by standard recombinant DNA technology to the monomer at appropriate places to stabilize the dimer through covalent disulfide bond formation.
  • multivalent refers to a recombinant molecule that incorporates two or more biologically active segments.
  • the protein fragments forming the multivalent molecule may be linked through a polypeptide peptide linker which attaches the constituent parts without causing a frame shift and permits each to function independently.
  • the term “bivalent” refers to a multivalent recombinant molecule having two target proteins in a fusion construct of the invention, e.g., an Fc-X molecule, where X independently is selected from angiostatin, endostatin, or a variant thereof. Since there are two X moieties fused to an immunoglobulin Fc region (which typically itself is a dimer of the heavy chain fragments including at least a portion of the hinge region and CH 3 domain, and optionally the CH 2 domain), the molecule is bivalent (see, e.g., FIG. 1A).
  • the resulting Fc dimer molecule is tetravalent.
  • the two proteins forming the Fc-X-X molecule may be linked through a peptide linker.
  • a bivalent molecule can increase the apparent binding affinity between the molecule and its receptor. For instance, if one endostatin moiety of an Fc-endostatin can bind to a receptor on a cell with a certain affinity, the second endostatin moiety of the same Fc-endostatin may bind to a second receptor on the same cell with a much higher avidity (apparent affinity). This is because of the physical proximity of the second endostatin moiety to the receptor after the first endostatin moiety is already bound. In the case of an antibody binding to an antigen, the apparent affinity is increased by at least 10 4 .
  • multimer and “multimeric” refers to the stable association of two or more polypeptide chains either covalently, for example, by means of covalent interaction, for example, by a disulfide bond or non-covalently, for example, by hydrophobic interaction.
  • the term multimer is intended to encompass both homomultimers, wherein the polypeptides are the same, as well as heteromultimers, wherein the polypeptides are different.
  • 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 immunoglobulin Fc region Fc fragment itself typically is a dimer of the heavy chain fragments including at least a portion of the hinge region and CH 3 domain, and optionally the CH 2 domain. Many protein ligands are known to bind to their receptors as a dimer. If a protein ligand X dimerizes naturally, the X moiety 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 moieties connected by associated immunoglobulin Fc region would make the dimerization an intramolecular process, greatly shifting the equilibrium in favor of the dimer and enhancing its binding to the receptor.
  • the present invention exploits conventional recombinant DNA methodologies for generating the Fe fusion proteins useful in the practice of the invention.
  • the Fc fusion constructs preferably are generated at the DNA level, and the resulting DNAs integrated into expression vectors, and expressed to produce the immunofusins.
  • the term “vector” is understood to mean any nucleic acid comprising a nucleotide sequence competent to be incorporated into a host cell and to be recombined with and integrated into the host cell genome, or to replicate autonomously as an episome.
  • 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 adeno-associated virus.
  • the term “gene expression” or “expression” of a target protein is understood to mean the transcription of a DNA sequence, translation of the mRNA transcript, and secretion of an Fc fusion protein product.
  • a useful expression vector is pdCs (Lo et al. (1988) Protein Engineering 11:495, the disclosure 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., 1985, Cell 41:521, the disclosure 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. Nat. 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 utilized for the expression and secretion of a target protein.
  • Currently 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 preferably are generated by conventional recombinant DNA methodologies.
  • the fusion proteins preferably are produced by expression in a host cell of a DNA molecule encoding a signal sequence, an immunoglobulin Fc region and a target protein (also referred to herein as an angiogenesis inhibitor).
  • Preferred constructs may encode in a 5′ to 3′ direction, the signal sequence, the immunoglobulin Fc region and the target protein.
  • the constructs may encode in a 5′ to 3′ direction, the signal sequence, the target protein and the immunoglobulin Fc region.
  • the term “signal sequence” is understood to mean a peptide segment which directs the secretion of the angiogenesis inhibitor immunofusin protein and is thereafter cleaved following translation in the host cell.
  • the signal sequence of the invention is a polynucleotide, which encodes an amino acid sequence that initiates transport of a protein across the membrane of the endoplasmic reticulum.
  • Signal sequences which will be useful in the invention include antibody light chain signal sequences, e.g., antibody 14.18 (Gillies et. al., 1989, Jour. of Immunol.
  • antibody heavy chain signal sequences e.g., the MOPC141 antibody heavy chain signal sequence (Sakano et al., 1980, Nature 286:5774), and any other signal sequences which are known in the art (see for example, Watson, 1984, Nucleic Acids Research 12:5145). Each of these references is incorporated herein by reference.
  • Signal sequences have been well characterized in the art and are known typically to contain 16 to 30 amino acid residues, and may contain greater or fewer amino acid residues.
  • a typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.
  • the central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal peptide across the membrane lipid bilayer during transport of the nascent polypeptide.
  • the signal peptide is usually cleaved within the lumen of the endoplasmic reticulum by cellular enzymes known as signal peptidases.
  • cleavage sites of the signal peptide generally follow the “( ⁇ 3, ⁇ 1) rule.”
  • a typical signal peptide has small, neutral amino acid residues in positions ⁇ 1 and ⁇ 3 and lacks proline residues in this region.
  • the signal peptidase will cleave such a signal peptide between the ⁇ 1 and +1 amino acids.
  • the portion of the DNA encoding the signal sequence may be cleaved from the amino-terminus of the immunofusin protein during secretion. This results in the secretion of a immunofusin protein consisting of the Fc region and the target protein.
  • a detailed discussion of signal peptide sequences is provided by von Heijne (1986) Nucleic Acids Res., 14:4683 the disclosure of which is incorporated herein by reference.
  • 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, one skilled in the art is capable of creating a synthetic signal peptide following the rules presented by von Heijne, referenced above, and testing for the efficacy of such a synthetic signal sequence by routine experimentation. A signal sequence may 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 secretion cassette.
  • An exemplary secretion cassette useful in the practice of the invention is a polynucleotide encoding, in a 5′ to 3′ direction, a signal sequence of an immunoglobulin light chain gene and an Fc ⁇ 1 region of the human immunoglobulin ⁇ 1 gene.
  • the Fc ⁇ 1 region of the immunoglobulin Fc ⁇ 1 gene preferably includes at least a portion of the hinge domain and at least a portion of the CH 3 domain, or alternatively at least portions of the hinge domain, CH 2 domain and CH 3 domain.
  • the DNA encoding the secretion cassette can be in its genomic configuration or its cDNA configuration.
  • the DNA sequence encodes a proteolytic cleavage site interposed between the secretion cassette and the angiogenesis inhibitor protein.
  • a cleavage site provides for the proteolytic cleavage of the encoded fusion protein thus separating the Fc domain from the angiogenesis inhibitor protein.
  • proteolytic cleavage site is understood to mean amino acid sequences which are preferentially cleaved by a proteolytic enzyme or other proteolytic cleavage agents.
  • Useful proteolytic cleavage sites include amino acids sequences which are recognized by proteolytic enzymes such as trypsin, plasmin or enterokinase K. Many cleavage site/cleavage agent pairs are known.
  • the target protein sequence is a precursor molecule to angiostatin, endostatin, or an active variant thereof
  • the desired protein product may be produced by cleavage with the endogenous proteolytic enzyme, such as elastin or plasmin or urokinase.
  • the present invention also encompasses fusion proteins containing different combinations of recombinant angiostatin and endostatin, or fragments thereof, which can be made in large quantities. Despite the demonstrated efficacy in suppressing tumor growth, the mechanism of how angiostatin and endostatin block angiogenesis is not completely known.
  • Angiostatin has several Kringle structures and endostatin has different structural motifs, each of which may be solely responsible for or assist in binding of the proteins to endothelial cells and exerting an anti-angiogenic effect.
  • this invention includes target proteins which are bioactive fragments of angiostatin, such as Kringle 1, Kringle 2, Kringle 3, and combinations thereof, and endostatin which exhibit physiologically similar behavior to naturally occurring full-length angiostatin and endostatin.
  • a bifunctional hybrid molecule or construct 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 so that in the hybrid molecule, the functions of the two subunits may be additive or synergistic.
  • Such functional hybrid proteins would allow the synergistic effect of angiostatin and endostatin to be explored in animal models.
  • a preferred bifunctional hybrid may comprise at least two different angiogenesis inhibitors linked in tandem, either directly or by means of a polypeptide linker.
  • the target sequence encodes at least a portion of angiostatin linked in frame with at least a portion of endostatin and both the angiostatin and endostatin domains exhibit anti angiogenesis activity or angiogenesis inhibition.
  • the two units may be linked by a polypeptide linker.
  • polypeptide linker is understood to mean an peptide sequence that can link two proteins together or a protein and an Fc region.
  • the polypeptide linker preferably comprises a plurality of amino acids such as glycine and/or serine.
  • the polypeptide linker comprises a series of glycine and serine peptides about 10-15 residues in length. See, for example, U.S. Pat. No. 5,258,698, the disclosure of which is incorporated herein by reference. It is contemplated however, that the optimal linker length and amino acid composition may be determined by routine experimentation.
  • the Fc region can be glycosylated and highly charged at physiological pH, thus the Fc region can help to solubilize hydrophobic proteins.
  • the present invention also provides methods for the production of angiostatin and endostatin of non-human species as Fc fusion proteins.
  • Non-human angiogenesis inhibitor fusion proteins are useful for preclinical studies of angiogenesis inhibitors because efficacy and toxicity studies of a protein drug must be performed in animal model systems before testing in humans.
  • a human protein may not work in a mouse model because the protein may elicit an immune response, and/or exhibit different pharmacokinentics skewing the test results. Therefore, the equivalent mouse protein is the best surrogate for the human protein for testing in a mouse model.
  • mice The standard Lewis lung carcinoma model in mice (O'Reilly et al. (1997) Cell 88:277) was used to compare soluble huFc-huAngiostatin, huFc-huEndostatin, muFc-muAngiostatin, muFc-muEndostatin with the insoluble proteins produced in an E. Coli expression system.
  • the soluble Fc fusion proteins were more efficacious in suppressing tumor growth in the Lewis lung model than the corresponding proteins produced in E. coli .
  • laboratory mice are inbred and their tumors are induced and not spontaneous. Therefore, efficacy in a mouse model may not correlate to probable efficacy against human tumors.
  • Preclinical studies in dogs will provide more precise information about the efficacy of these angiogenesis inhibitors on spontaneous tumors because there are numerous naturally occurring, spontaneous canine tumors.
  • the methods of producing murine (mu) Fc-mu angiostatin, muFc-mu endostatin, and canine (ca) Fc-ca angiostatin, caFc-ca endostatin 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; blood born tumors, tumor metastasis, benign tumors including hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyrogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases (diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma) retrolental fibroplasia, rubeosis, Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints' angiofibroma; and wound granulation; and excessive or abnormal stimulation of endothelial cells, intestinal adhesions, arthe
  • the DNA constructs disclosed herein can be useful in gene therapy procedures in which the endostatin or angiostatin gene is delivered into a cell by one of various means e.g., native DNA associated with a promoter or DNA within a viral vector. Once inside a cell, the angiostatin and/or endostatin gene or gene fragment 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 treating conditions mediated by angiogenesis and may have greater clinical efficacy than native angiogenesis inhibitors and other recombinant angiogenesis inhibitors because the angiogenesis inhibitor immunofusins of the present invention have a longer serum half-life than the other recombinant angiogenesis inhibitors or native angiogenesis inhibitors alone.
  • 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 possible synergistic effects of two different angiogenesis inhibitors connected by the fused Fc region or a flexible polypeptide linker.
  • compositions of the present invention may be provided to an animal by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., parenterally or orally).
  • parenterally such as by intravenous, subcutaneous, ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intranasal or by aerosol administration
  • the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution.
  • the carrier or vehicle is physiologically acceptable so that in addition to delivery of the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance.
  • the fluid medium for the agent thus can comprise normal physiologic saline (e.g., 9.85% aqueous NaCl, 0.15 M, pH 7-7.4).
  • Preferred dosages of the immunofusins per administration are within the range of 50 ng/m 2 to 1 g/m 2 , more preferably 5 ⁇ g/m 2 to 200 mg/m 2 , and most preferably 0.1 mg/m 2 to 50 mg/m 2 .
  • Preferred dosages of nucleic acids encoding the immunofusins per administration are within the range of 1 ⁇ g/m 2 to 100 mg/m 2 , more preferably 20 ⁇ g/m 2 to 10 mg/m 2 , and most preferably 400 ⁇ g/m 2 to 4 mg/m 2 . It is contemplated, however, that the optimal modes of administration, and dosages may be determined by routine experimentation well within the level of skill in the art.
  • Human endostatin was expressed as a human Fc-human endostatin (huFc-huEndo) fusion protein according to the teachings of Lo et al. (1998) Protein Engineering 11:495.
  • Fc refers to the Fc fragment of the human immunoglobulin gamma (DNA sequence set forth in SEQ ID NO: 1; amino acid sequence set forth in SEQ ID NO: 2).
  • Polymerase chain reactions PCR was used to adapt the endostatin cDNA (SEQ ID NO: 3; whose amino acid sequence is disclosed in SEQ ID NO: 4), for expression in an Fc-Endo fusion protein.
  • the forward primer was either 5′-CC CCG GGT AAA CAC AGC CAC CGC GAC TTC C (SEQ ID NO: 5; encoded amino acids disclosed in SEQ ID NO: 6) or 5′-C AAG CTT CAC AGC CAC CGC GAC TTC C (SEQ ID NO: 7; encoded amino acids disclosed in SEQ ID NO: 8), where the XmaI site or the HindIII site was followed by sequence encoding the N-terminus of endostatin.
  • the primer with the XmaI site adapted the endostatin cDNA for ligation to the XmaI site at the end of the CH 3 domain of the IgGFc region.
  • the primer with the HindIII site adapted the endostatin cDNA for ligation to the HindIII site of the pdCs-Fc(D 4 K) vector, which contains the enterokinase recognition site Asp 4 -Lys (LaVallie et al. (1993) J. Biol. Chem. 268:23311-23317) at the junction of the fusion protein.
  • the reverse primer was 5′-C CTC GAG CTA CTT GGA GGC AGT CAT G (SEQ ID NO: 9), which was designed to put a translation STOP codon (anticodon, CTA) immediately after the C-terminus of endostatin, and this was followed by an XhoI site.
  • the PCR products were cloned and sequenced, and the XmaI-XhoI fragment was ligated to the resulting XmaI and XhoI digested pdCs-Fc vector.
  • the HindIII-XhoI fragment encoding endostatin was ligated into appropriately digested pdCs-huFc(D 4 K) vector.
  • Stable clones expressing Fc-endo or Fc(D 4 K)-endostatin were obtained by electroporation of NS/0 cells followed by selection in growth medium containing 100 nM methotrexate.
  • Protein expression level was assayed by anti-human Fc ELISA (Example 3) and confirmed by SDS-PAGE, which showed a protein product of ⁇ 52 kD. The best producing clones were subcloned by limiting dilutions.
  • the plasmid was introduced into human kidney 293 cells by co-precipitation of plasmid DNA with calcium phosphate (Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual, Cold Spring Harbor, N.Y.) or by lipofection using LipofectAMINE Plus (Life Technologies, Gaithersburg, Md.) according to supplier's protocol.
  • plasmid DNA was introduced into the mouse myeloma NS/0 cells by electroporation.
  • NS/0 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
  • About 5 ⁇ 10 6 cells were washed once with PBS and resuspended in 0.5 ml PBS.
  • Ten ⁇ g of linearized plasmid DNA then was incubated with the cells in a Gene Pulser Cuvette (0.4 cm electrode gap, BioRad, Hercules, Calif.) on ice for 10 min.
  • Electroporation was performed using a Gene Pulser (BioRad, Hercules, Calif.) with settings at 0.25 V and 500 ⁇ F. Cells were allowed to recover for 10 min. on ice, after which they were resuspended in growth medium and then plated onto two 96 well plates. Stably transfected clones were selected by growth in the presence of 100 nM methotrexate (MTX), which was introduced two days post-transfection. The cells were fed every 3 days for three more times, and MTX-resistant clones appeared in 2 to 3 weeks. Supernatants from clones were assayed by anti-Fc ELISA to identify high producers. High producing clones were isolated and propagated in growth medium containing 100 nM MTX.
  • MTX methotrexate
  • the anti-human Fc (huFc) ELISA was used to measure the amount of human Fc-containing proteins.
  • the anti-murine Fc (muFc) and anti-canine Fc (caFc) antibodies were used in ELISAs to measure the amount of murine Fc-and canineFc-containing proteins, respectively.
  • the procedure for the anti-huFc ELISA is described in detail herein below.
  • ELISA plates were coated with AffiniPure Goat anti-Human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) at 5 ⁇ g/ml in PBS, and 100 ⁇ l/well in 96-well plates (Nunc-Immuno plate MaxiSorpTM, Nalge Nunc International, Rochester, N.Y. ). Coated plates were covered and incubated at 4° C. overnight. Plates then were washed 4 times with 0.05% Tween 20 in PBS, and blocked with 1% BSA/1% Goat Serum in PBS, 200 ⁇ l/well. After incubation with the blocking buffer at 37° C. for 2 hours, the plates were washed 4 times with 0.05% Tween in PBS and tapped dry on paper towels.
  • Test samples were diluted to the proper 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.
  • serial dilutions were made in the sample buffer to give a standard curve ranging from 125 ng/ml to 3.9 ng/ml.
  • the diluted samples and standards were added to the plate, 100 ⁇ l/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.
  • HRP horse radish peroxidase
  • a substrate solution was prepared by dissolving 30 mg (1 tablet) of o-phenylenediamine dihydrochloride (OPD) into 15 ml of 0.025 M citric acid/0.05 M Na 2 HPO 4 buffer, pH 5, containing 0.03% of freshly added H 2 O 2 .
  • OPD o-phenylenediamine dihydrochloride
  • the substrate solution was added to the plate at 100 ⁇ l/well. The color was allowed to develop for 30 min. at room temperature in the dark. The developing time can be subject to change, depending on lot to lot variability of the coated plates, the secondary antibody, etc.
  • the reaction was stopped by adding 4N H 2 SO 4 , 100 ⁇ /well.
  • the plate was read by a plate reader, which was set at both 490 and 650 nm, and programmed to subtract the background OD at 650 nm from the OD at 490 nm.
  • the ELISA plate was coated with AffiniPure Rabbit anti-dog IgG, Fe Fragment specific (Jackson ImmunoResearch, West Grove, Pa.) at 5 ⁇ g/ml in PBS, and 100 ⁇ l/well; and the secondary antibody was horse radish peroxidase-conjugated AffiniPure rabbit anti-dog IgG, Fc fragment specific (Jackson ImmunoResearch, West Grove, Pa.), used at 1 in 5000 dilution.
  • Human angiostatin (DNA sequence set forth in SEQ ID NO: 10; amino acid sequence set forth in SEQ ID NO: 11) was expressed as a human Fc-human angiostatin (huFc-huAngio) fusion protein essentially as described in Example 1.
  • PCR was used to adapt the angiostatin cDNA (SEQ ID NO: 3), for expression in the pdCs-huFc or pdCs-huFc(D 4 K) vectors.
  • the respective forward primers were 5′-CC CCG GGT AAG AAA GTG TAT CTC TCA GAG (SEQ ID NO 12; encoded amino acids disclosed in SEQ ID NO: 13), and 5′-C CCC AAG CTT AAA GTG TAT CTC TCA GAG (SEQ ID NO: 14; encoded amino acids disclosed in SEQ ID NO: 15), where the XmaI site or the HindIII site was followed by sequence encoding the N-terminus of angiostatin.
  • the reverse primer was 5′-CCC CTC GAG CTA CGC TTC TGT TCC TGA GCA (SEQ ID NO: 16), which was designed to put a translation STOP codon (anticodon, CTA) immediately after the C-terminus of angiostatin, and this was followed by an XhoI site.
  • the PCR products were cloned and sequenced, and the resulting XmaI-XhoI fragment and the HindIII-XhoI fragment encoding angiostatin were ligated to the pdCs-huFc and the pdCs-huFc(D 4 K) vectors, respectively.
  • Stable NS/0 clones expressing huFc-huAngio and huFc(D 4 K)-huAngio were selected and assayed as described in Examples 2 and 3.
  • Murine endostatin (DNA sequence set forth in SEQ ID NO: 17; amino acid sequence set forth in SEQ ID NO: 18) and murine Fe (DNA sequence set forth in SEQ ID NO: 19; encoded amino acids set forth in SEQ ID NO: 20) were expressed as a murine F-murine endostatin (muFc-muEndo) fusion protein essentially as described in Example 1.
  • PCR was used to adapt the endostatin cDNA (SEQ ID NO: 4), for expression in the pdCs-muFc(D 4 K) vector.
  • the forward primer was 5′-C CCC AAG CTT CAT ACT CAT CAG GAC TTT C (SEQ ID NO: 21; encoded amino acids disclosed in SEQ ID NO: 22), where the HindIII site was followed by sequence encoding the N-terminus of endostatin.
  • the reverse primer was 5′-CCC CTC GAG CTA TTT GGA GAA AGA GGT C (SEQ ID NO: 23), which was designed to put a translation STOP codon (anticodon, CTA) immediately after the C-terminus of endostatin, and this was followed by an XhoI site.
  • Murine angiostatin (DNA sequence set forth in-SEQ ID NO: 24; amino acid sequence set forth in SEQ ID NO: 25) was expressed as a murine Fc-murine angiostatin (muFc-muAngio) fusion protein essentially as described in Example 1.
  • PCR was used to adapt the angiostatin cDNA (SEQ ID NO: 6) for expression in the pdCs-Fc(D 4 K) vector.
  • the forward 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 disclosed in SEQ ID NO: 27), where the HindIII site was followed by sequence encoding the N-terminus of angiostatin.
  • the reverse primer was 5′-CCC CTC GAG CTA CCC TCC TGT CTC TGA GCA (SEQ ID NO: 28), which was designed to put a translation STOP codon (anticodon, CTA) immediately after the C-terminus of angiostatin, and this was followed by an XhoI site (CTCGAG).
  • the PCR product was cloned and sequenced, and the HindIII-XhoI fragment encoding angiostatin was ligated to the pdCs-muFc(D 4 K) vector.
  • Stable NS/0 clones expressing muFc(D 4 K)-muAngio were selected and assayed (anti-muFc ELISA) as described in Examples 2 and 3.
  • Canine peripheral blood monocytic cells isolated from dog's blood were used to prepare mRNA.
  • PCR was performed to amplify the canine Fc (Kazuhiko et al., (1992) JP 1992040894-A1) using the forward primer 5′-CC TTA AGC GAA AAT GGA AGA GTT CCT CGC (SEQ ID NO: 29; encoded amino acids disclosed in SEQ ID NO: 30), in which an AfIII site was introduced immediately upstream of the sequence encoding the hinge region of the canine Fc, and the reverse primer 5′-C CTC GAG TCA TTT ACC CGG GGA ATG GGA GAG GGA TTT CTG (SEQ ID NO: 31), in which an XhoI site was introduced after the translation STOP codon (anticodon, TCA) of the canine Fc.
  • the reverse primer also introduced a silent mutation to create a XmaI restriction site, which facilitates the construction of the pdCs-caFc(D 4 K) vector through a linker-adaptor and ligation to DNA constructs encoding canine endostatin or angiostatin.
  • the expression vector for the pdCs-caFc was constructed as follows.
  • the AfIII-XhoI fragment encoding the canine Fc was ligated to the XbaI-AfIII fragment encoding the light chain signal peptide and the XbaI-XhoI digested pdCs vector.
  • the resulting pdCs-caFc expression vector then was used to transfect 293 cells. About 3 days post-transfection, the supernatant was purified by Protein A chromatography.
  • dog Fc DNA sequence set forth in SEQ ID NO: 32; amino acid sequence set forth in SEQ ID NO: 33
  • SDS-PAGE followed by Western blot analysis using a peroxidase-conjugated Rabbit anti-Dog IgG, Fc fragment specific (Jackson ImmunoResearch, West Grove, Pa.).
  • the coding sequence for canine endostatin (DNA sequence set forth in SEQ ID NO: 34; amino acid sequence set forth in SEQ ID NO: 35) was adapted to a HindIII-XhoI fragment for expression as a Fc fusion protein, essentially as described in Example 5.
  • a STOP codon was introduced, for example, by PCR, immediately after the codon encoding the C-terminal lysine residue, and this was followed by the NotI restriction site.
  • oligonucleotide duplex consisting of a HindIII and a DraIII sticky ends was chemically synthesized and used to ligate to the DraIII-XhoI restriction fragment which encodes the rest of the canine endostatin cDNA.
  • the duplex used is shown below:
  • the first CAC in the duplex encodes the N-terminal histidine residue of the canine endostatin.
  • the HindIII-XhoI fragment encoding the full-length canine endostatin thus could be ligated to the HindIII-XhoI digested pdCs-caFc vector (see Example 7) for expression.
  • Stable NS/0 clones expressing caFc-caEndo were selected and assayed 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.
  • the cDNA encoding the full length canine angiostatin (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 caFc fusion protein essentially as in the aforementioned examples. Briefly, at the 3′ end, a STOP codon was introduced, for example, by PCR, immediately after the codon encoding the C-terminal lysine residue and this was followed by a NotI restriction site instead of an XhoI site, since there was an internal XhoI restriction site in the cDNA of the canine angiostatin. At the 5′ end, a HindIII site was introduced in-frame immediately upstream of the N-terminus of angiostatin.
  • HindIII-NotI fragment encoding the full length canine angiostatin then was ligated to the HindIII-NotI digested pdCs-caFc vector (where the NotI site was introduced at the XhoI site through linker ligation) for expression.
  • Stable NS/0 clones expressing caFc-caAngio were selected and assayed 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.
  • Angiostatin comprises the first four of the five Kringle domains of plasminogen. To determine if any one or several Kringle domains are responsible for the observed anti-angiogenic activity of angiostatin, it is possible to produce single Kringle domains by themselves or combination thereof for testing. To demonstrate the utility of Fc as a fusion protein partner, the expression of the first Kringle domain of murine angiostatin (K1) was achieved in the following way. The first Kringle domain ends at Glu-87 of murine angiostatin (SEQ ID NO: 25).
  • a Kringle domain consists of multiple loops, including an outer loop and an inner loop.
  • the inner loop is defined by Cys 55 and Cys 79, which together form a disulfide bond at the base of the loop.
  • the Cys-67 of the inner loop forms another disulfide bond with a Cys residue of the outer loop to give the Kringle structure.
  • muFc-inner K1 muFc-inner K1
  • a mutagenic primer having the sequence 5′GGG CCT TGG AGC TAC ACT ACA (SEQ ID NO: 42; encoded amino acids disclosed in SEQ ID NO: 43) was used to mutagenize TGC (Cys-67) to AGC (Ser), by PCR. This ensures that the Cys-67 does not form a disulfide bond when the inner loop of Kringle 1 is expressed without the outer loop.
  • An upstream primer having the sequence 5′GCGGATCCAAGCTT AGT ACA CAT CCC AAT GAG GG was used to introduce a HindIII site in frame immediately 5′ to the codon for Ser-43 (AGT).
  • a BamHI site was also introduced immediately upstream of the HindIII site. The BamHI site is useful for ligating to the BamHI site at the end of the flexible Gly-Ser linker shown in Example 12 below.
  • a HindIII-XhoI DNA fragment encoding Ser-43 through Glu-87 of murine angiostatin was ligated to the pdCs-muFc(D 4 K) vector for expression. High levels of expression of muFc-innerK1 were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE.
  • the hybrid molecule muFc-muEndo-innerK1 comprises muFc-muEndo joined by a polypeptide linker containing glycine and serine residues, to the inner loop of the first Kringle of murine angiostatin.
  • the DNA construct was assembled as follows.
  • the hybrid molecule muFc-muEndo-K1 comprises muFc-muEndo joined by a polypeptide linker containing glycine and serine residues, to the first Kringle of murine angiostatin.
  • the DNA construct was assembled as follows.
  • the adaptor has a HindIII′ sticky end, which upon ligation, would not regenerate the HindIII site.
  • the resulting HindIII-XhoI fragment which encodes the muEndo-GlySer linker-Kringle 1
  • High levels of expression of muFc-muEndo-GlySer linker-K1 were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE.
  • the hybrid molecule muFc-muEndo-GlySer linker-muAngio comprises muFc-muEndo joined by a polypeptide linker containing glycine and serine residues, to murine angiostatin.
  • the DNA construct was assembled essentially as follows. The BamHI end of the HindIII-BamHI fragment encoding the muEndo-GlySer linker (Example 12) was ligated to the HindIII-XhoI fragment encoding murine angiostatin via the adaptor described in Example 13.
  • the resulting HindIII-XhoI fragment which encodes the muEndo-GlySer linker-muAngio, was ligated to the pdCs-muFc(D 4 K) vector for expression. High levels of expression of muFc-muEndo-GlySer linker-muAngio were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE.
  • the hybrid molecule huAngio-huFc-huEndo comprises human angiostatin joined by a peptide bond to huFc-huEndo.
  • the DNA construct was assembled as follows. A HindIII-XhoI fragment which encodes human angiostatin without a STOP codon was first generated by PCR, so that the codon for the last amino acid residue of angiostatin was followed immediately by CTCGAG of the XhoI site.
  • HindIII at the 5′ end was ligated to an XbaI-AfIII fragment of the light chain signal peptide (Lo et al., Protein Engineering (1998) 11:495) via a AfIII-HindIII′ adaptor: AfIII 5′ TTA AGC GGC C (SEQ ID NO:51) CG CGG GTCGA (SEQ ID NO:52) HindIII′
  • HindIII′ sticky end of the adaptor upon ligation, would not regenerate a HindIII site.
  • the XhoI site was ligated to the AfIII site of the AfIII-XhoI fragment encoding the huFc-hu-Endo via the following XhoI′-AfIII adaptor: XhoI′ 5′ TC GAC TCC GGC (SEQ ID NO:53) G AGG CCG AATT (SEQ ID NO:54) AfIII
  • the XhoI sticky end of the adaptor upon ligation, would not regenerate a XhoI site.
  • the resulting XbaI-XhoI fragment encoding the signal peptide-human angiostatin-huFc-human endostatin was cloned into the pdCs vector for expression. High levels of expression of were obtained in both transient and stable expression, as analyzed by anti-muFc ELISA and SDS-PAGE.

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