Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/49—Platelet-derived growth factor [PDGF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/303—Liver or Pancreas
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• Hepatocellular carcinoma or hepatoma
• Hepatocellular carcinoma is the most common type of primary liver cancer. It is particularly common (and an important cause of death) in parts of Africa and southeast Asia where chronic hepatitis B is endemic.
• the precise etiology of hepatocellular carcinoma (HCC) is unknown.
• the tumors are composed of malignant hepatocytes.
• There is a strong correlation with hepatitis B incidence, and incorporation of viral DNA into the host genome is believed to lead to malignant transformation.
• Chronic hepatitis C infection has also been linked to HCC, although the hepatitis C virus (an RNA virus) does not incorporate into the host genome.
• Fibrogenesis in hepatitis C patients may play a role in tumor formation.
• HCC is also a major complication of hemochromatosis.
• Other risk factors for HCC include environmental toxins (e.g., aflatoxins), alcoholism, family history, .and being male.
• Signs and symptoms of HCC include pain in the right upper abdomen, presence of tissue mass or swollen abdomen, weight loss, loss of appetite and feelings of fullness, weekness or tiredness, nausea and vomiting, jaundice, and fever.
• Serum - fetoprotein and des-g-carboxy-prothrombin are diagnostic markers. Additional diagnostic methods include ultrasound, CT scanning, magnetic resonance imaging, hepatic arteriography, and biopsy. Current treatments for HCC have achieved only limited success.
• HCC head-by-diluent ospasmodic ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
• chemoembolization using, for example, bischlorethylnitrosourea (Dakhil et al., Cancer 50:631-635, 1982) or iodized oil and doxorubicin hydrochloride (Choi et al, Radiology 182:709-713, 1992); immunotherapy (Takaya a, et al., Lancet 356(9232):802-807, 2000); and a combination of cisplatin chemotherapy with radiation (Epstein et al., Cancer 67:896-900, 1991).
• the present invention provides materials and methods for treating hepatocellular carcinoma in a mammal.
• a method of treating hepatocellular carcinoma in a mammal comprising administering to a mammal having a hepatocellular carcinoma a composition comprising a zvegf3 antagonist in combination with a pharmaceutically acceptable delivery vehicle, wherein the zvegf3 antagonist is selected from the group consisting of anti-zvegf3 antibodies, mitogenically inactive receptor-binding zvegf3 variant polypeptides, and inhibitory polynucleotides, in an amount sufficient to produce a tumor response in the mammal.
• a tumor response is measured as a complete response, a partial response', or a reduction in time to progression.
• the zvegf3 antagonist is selected from the group consisting of anti-zvegf3 antibodies and inhibitory polynucleotides.
• the antagonist is an anti-zvegf3 antibody.
• the antibody is a monoclonal antibody, such as an IgG monoclonal antibody.
• the zveg ⁇ antagonist is an antibody that specifically binds to a dimeric protein having two polypeptide chains, wherein each of the polypeptide chains consists of a sequence of amino acid residues selected from the group consisting of residues 230-345 of SEQ ID NO:2, residues 231-345 of SEQ ID NO:2, residues 232-345 of SEQ ID NO:2, residues 233-345 of SEQ ID NO:2, residues 234-345 of SEQ ID NO:2, residues 235-345 of SEQ ID NO:2, residues 236-345 of SEQ ID NO:2, residues 237-345 of SEQ ID NO:2, residues 238-345 of SEQ ID NO:2, residues 239-345 of SEQ ID NO:2, and residues 240-345 of SEQ ID NO:2.
• the zvegf3 antagonist is administered by intravenous infusion.
• a method of reducing cancer cell proliferation comprising administering to a mammal with hepatocellular carcinoma an amount of a composition comprising a zvegf3 antagonist in combination with a pharmaceutically acceptable delivery vehicle, wherein the zvegf3 antagonist is selected from the group consisting of anti-zvegf3 antibodies, mitogenically inactive receptor-binding zvegf3 variant polypeptides, and inhibitory polynucleotides, in an amount sufficient to reduce cancer cell proliferation within the hepatocellular carcinoma.
• the zvegf3 antagonist is selected from the group consisting of anti-zvegf3 antibodies and inhibitory polynucleotides.
• the antagonist is an anti-zvegf3 antibody.
• the antibody is a monoclonal antibody, such as an IgG monoclonal antibody.
• the zvegf3 antagonist is an antibody that specifically binds to a dimeric protein having two polypeptide chains, wherein each of the polypeptide chains consists of a sequence of amino acid residues selected from the group consisting of residues 230-345 of SEQ ID NO:2, residues 231-345 of SEQ ID NO:2, residues 232-345 of SEQ ID NO:2, residues 233-345 of SEQ ID NO:2, residues 234-345 of SEQ ID NO:2, residues 235-345 of SEQ ID NO:2, residues 236-345 of SEQ ID NO:2, residues 237-345 of SEQ ID NO:2, residues 238-345 of SEQ ID NO:2, residues 239-345 of SEQ ID NO:2, and residues 240-345 of SEQ ID NO:2.
• the zvegf3 antagonist is administered by intravenous infusion.
• a zvegf 3 antagonist in the preparation of a medicament for treatment of hepatocellular carcinoma in a mammal, wherein the zvegf3 antagonist is selected from the group consisting of anti-zveg£3 antibodies, mitogenically inactive receptor-binding zvegf3 variant polypeptides, and inhibitory polynucleotides.
• the medicament is formulated for intravenous infusion.
• IA - 1G are a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO:2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated in the figure by lower case letters.
• Fig. 2 is an alignment of human (SEQ ID NO:2) and mouse (SEQ ID NO:4) amino acid sequences.
• the term "antagonist" is used herein to denote a compound that reduces a biological activity of another compound.
• a "zvegf3 antagonist” is a compound that reduces the receptor-mediated biological activity (e.g., mitogenic activity) of zvegf3 on a target cell.
• Antagonists may exert their action by competing with zvegf3 for binding sites on a cell-surface receptor, by binding to zvegf3 and preventing it from binding to a cell-surface receptor, by otherwise interfering with receptor function, by reducing production of zvegf3, or by other means.
• the term “cancer” or “cancer cell” is used herein to denote a tissue or cell found in a neoplasm which possesses characteristics which differentiate it from normal tissue or tissue cells.
• Such characteristics include but are not limited to degree of anaplasia, irregularity in shape, indistinctness of cell outline, nuclear size, changes in structure of nucleus or cytoplasm, other phenotypic changes, presence of cellular proteins indicative of a cancerous or pre-cancerous state, increased number of mitoses, and ability to metastasize.
• Words pertaining to "cancer” include carcinoma, sarcoma, tumor, epithelioma, adenoma, leukemia, lymphoma, polyp, scirrus, transformation, neoplasm, and the like.
• an “inhibitory polynucleotide” is a DNA or RNA molecule that reduces or prevents expression (transcription or translation) of a second (target) polynucleotide.
• Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences.
• the term “inhibitory polynucleotide” further includes DNA and RNA molecules that encode the actual inhibitory species, such as DNA molecules that encode ribozymes.
• treatment is used broadly to denote therapeutic and prophylactic interventions that favorably alter a pathological state. Treatments include procedures that moderate or reverse the progression of, reduce the severity of, prevent, or cure a disease. In the case of cancers, treatment includes an increase in survival rate over a given time period or an increase in survival time, reduction in tumor mass, reduction in tumor metastasis, cessation of disease progression, reduction in time to progression, and the like.
• the present invention provides methods for treating hepatocellular carcinoma in a patient using zvegf3 antagonists.
• Zvegf3 is a protein that is structurally related to platelet-derived growth factor (PDGF) and the vascular endothelial growth factors (VEGF).
• PDGF platelet-derived growth factor
• VEGF vascular endothelial growth factors
• VEGF-R WIPO Publication WO 99/37671
• PDGF-C WO 00/18212; Li et al., Nature Cell Biol. 2:302-309, 2000; Cao et al., FASEB J. 16:1575-1583, 2002.
• Zvegf3/PDGF-C is a multi-domain protein with significant homology to the PDGF/VEGF family of growth factors. Representative amino acid sequences of human and mouse zvegf 3 are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively. DNAs encoding these polypeptides are shown in SEQ ID NOS:l and 3, respectively.
• zvegf3 protein is used herein to denote proteins comprising the growth factor domain of a zvegf 3 polypeptide (e.g., residues 235-345 of human zvegf3 (SEQ ID NO:2) or mouse zvegf3 (SEQ ID NO:4)), wherein the protein is mitogenic for cells expressing cell-surface PDGF ⁇ -receptor subunit.
• Zvegf3 has been found to bind to the ⁇ o. and ⁇ isoforms of PDGF receptor.
• Zvegf3 is a homodimeric protein that is naturally produced as a precursor that is proteolytically activated to release the mature protein, a di er of the growth factor domain.
• zvegf3 protein includes precursors that are activatable in vivo. Using methods known in the art, zvegf3 proteins can be prepared in a variety of forms, including glycosylated or non-glycosylated, pegylated or non-pegylated, with or without an initial methionine residue, and as fusion proteins as disclosed in more detail below.
• the zvegf3 polypeptide chain comprises a growth factor domain and a CUB domain.
• the growth factor domain is characterized by an arrangement of cysteine residues and beta strands that is characteristic of the "cystine knot" structure of the PDGF family.
• the CUB domain shows sequence homology to CUB domains in the neuropilins (Takagi et al, Neuron 7:295-307, 1991; Soker et al., Cell 92:735-745, 1998), human bone morphogenetic protein-1 (Wozney et al., Science 242:1528-1534, 1988), porcine seminal plasma protein and bovine acidic seminal fluid protein (Romero et al., Nat. Struct. Biol. 4:783-788, 1997), and X. laevis tolloid-like protein (Lin et al, Dev. Growth Differ. 39:43-51, 1997).
• Fig. 2 An alignment of mouse and human zvegf3 polypeptide sequences is shown in Fig. 2. Analysis of the amino acid sequence shown in SEQ ID NO:2 indicates that residues 1 to 14 form a secretory peptide.
• the CUB domain extends from residue 46 to residue 163.
• a propeptide-like sequence extends from residue 164 to residue 234, and includes two potential cleavage sites at its carboxyl terminus, a dibasic site at residues 231-232 and a target site for furin or a furin-like protease at residues 231-234.
• the growth factor domain extends from residue 235 to residue 345. Those skilled in the art will recognize that domain boundaries are somewhat imprecise and can be expected to vary by up to ⁇ 5 residues from the specified positions.
• interdomain region may be truncated at its amino terminus by a like amount. See Table 1. Corresponding domains in mouse and other non-human zvegf3s can be determined by those of ordinary skill in the art from sequence alignments.
• Zvegf3 can thus be prepared in a variety of multimeric forms comprising a zvegf3 polypeptide as disclosed above.
• These zvegf3 polypeptides include zvegf3 15- 345 , zvegf 3 46-345 , zvegf3 226-345 , and zvegf3 235-345 .
• Variants and derivatives of these polypeptides can also be prepared as disclosed herein.
• Intermediate forms can also be produced.
• a growth factor domain polypeptide may have, as an amino- terminal residue, any of residues 226-240 of SEQ ID NO:2, inclusive.
• Zvegf3 proteins can be prepared as fusion proteins comprising amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, an affinity tag, or a targetting polypeptide.
• a zvegf3 protein can be prepared as a fusion with an affinity tag to facilitate purification.
• any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
• Affinity tags include, for example, a poly-histidine tract, protein A (Nilsson et al, EMBO J. 4:1075, 1985; Nilsson et al, Methods Enzymol.
• Fusion of zvegf3 to, for example, maltose binding protein or glutathione S transferase can be used to improve yield in bacterial expression systems.
• the non- zvegf3 portion of the fusion protein ordinarily will be removed prior to use. Separation of the zvegf 3 and non-zvegf3 portions of the fusion protein is facilitated by providing a specific cleavage site between the two portions. Such methods are well known in the art.
• Zvegf3 can also be fused to a targetting peptide, such as an antibody (including polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like) or other peptidic moiety that binds to a target tissue.
• a targetting peptide such as an antibody (including polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and Fab fragments, single chain antibodies, and the like) or other peptidic moiety that binds to a target tissue.
• Variations can be made in the zvegf3 amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4 to provide mitogenically inactive, receptor-binding polypeptides that act as zvegf3 antagonists.
• the term "mitogenically inactive" means that the protein does not show statistically significant activity in a standard mitogenesis assay as compared to a wild-type zvegf3 control. Such variations include amino acid substitutions, deletions, and insertions. While not wishing to be bound by theory, it is believed that residues Arg260-Trp271 of human zvegf 3 (SEQ ID NO:2) form a loop that defines the ability of the protein to bind to PDGF receptors. It is thus predicted that binding to PDGF receptors can be blocked or enhanced by mutations in this region.
• residues Leu311-His321 of SEQ ID NO:2 are predicted to form a loop (loop3) that may be mutated to block receptor binding. Peptides that mimic this region of the molecule may act as antagonists.
• Li et al. (Cytokine & Growth Factor Rev. 14:91-98, 2003) disclose that partially processed forms of zvegf3 in which only one of the two CUB domains is removed from the homodimeric molecule (termed "hemi-dimers”) may be able to bind PDGF receptors but block receptor dimerization.
• the effects of amino acid sequence changes can be predicted by computer modeling (using, e.g., the Insight ⁇ ® viewer and homology modeling tools; MSI, San Diego, CA) or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nature 369:455, 1994), and can be assessed using art-recognized mutagenesis procedures in combination with activity assays.
• Representative mutagenesis procedures include, for example, site-directed mutagenesis and alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991).
• Mutagenesis can be combined with high volume or high- throughput screening methods to detect biological activity of zvegf3 variant polypeptides, in particular biological activity in modulating cell proliferation. For example, mitogenesis assays that measure dye incorporation or H-thymidine incorporation can be carried out on large numbers of samples. Competition assays can be employed to confirm antagonist activity. .
• Zvegf3 proteins, including full-length polypeptides, fragments, and fusion proteins, as well as antagonist variants can be produced in genetically engineered host ⁇ cells according to conventional techniques.
• Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells (including cultured cells of multicellular organisms). Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, Green and Wiley and Sons, NY, 1993. See, WO 00/34474.
• a DNA sequence encoding a zvegf3 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
• the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. See, WO 00/34474.
• Zvegf3 proteins and variants can also comprise non-naturally occurring amino acid residues.
• Non-naturally occurring amino acids include, without limitation, tr ns-3-methylproline, 2,4-methanoproline, cz ' .s-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ Mo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
• coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
• the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7410-7416, 1994.
• Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci.
• Zveg ⁇ polypeptides, fragments, or variants thereof can also be prepared through chemical synthesis according to methods known in the art, including exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989.
• Zvegf3 proteins and variants thereof are purified by conventional protein purification methods, typically by a combination of chromatographic techniques. See, in general, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification: Principles and Practice, Springer- Verlag, New York, 1994. Proteins comprising a polyhistidine affinity tag (typically about 6 histidine residues) are purified by affinity chromatography on a nickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988.
• the growth factor domain itself binds to nickel resin at pH 7.0-8.0 and 25 mM Na phosphate, 0.25 M NaCl.
• Bound protein can be eluted with a descending pH gradient down to pH 5.0 or an imidazole gradient.
• Recombinant zvegf 3 growth factor domain protein can be purified using a combination of chromatography on a strong cation exchanger followed by a tandem column array comprising a strong anion exchanger followed by an immobilized metal affinity column in series.
• zvegf3 binds to various dye matrices (e.g., BLUE1, BLUE 2, ORANGE 1, ORANGE 3, and RED3 from Lexton Scientific, Signal Hill, CA) in PBS at pH 6-8, from which the bound protein can be eluted in 1-2 M NaCl in 20 mM boric acid buffer at pH 8.8. Protein eluted from RED3 may be passed over RED2 (Lexton Scientific) to remove ' • remaining contaminants. , Proteins comprising a glu-glu- tag can be purified • . by' immunoaffinity chromatography according to conventional procedures. See, for example, Grussenmeyer et al., ibid.
• Maltose binding protein fusions are purified on an amylose column according to methods known in the art.
• overexpression of zvegf3 in the livers of transgenic mice led to marked stellate cell activation and proliferation.
• ECM perisinusiodal extracellular matrix
• perivenular ECM deposition At 8 weeks of age there was an accumulation of perisinusiodal extracellular matrix (ECM) that progressed to a perivenular ECM deposition at 22 weeks and possible early stages of cirrhosis characterized by fibrotic banding and hepatic nodule formation at 33 weeks of age.
• Older animals were found to have hemagioendothelioma, nodular hepatocellular hyperplasia, and hepatocellular adenoma.
• Zvegf3 antagonists include, without limitation, anti-zvegf3 antibodies
• Antibodies used as zvegf3 antagonists include antibodies that specifically bind to a zvegf3 protein and, by so binding, reduce or prevent the binding of zvegf3 protein to the receptor and, consequently, reduce or block the receptor-mediated activity of zvegf3.
• antibodies includes polyclonal antibodies, affinity- purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
• Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody).
• humanized antibodies may retain non human residues within the human variable region, framework domains to. enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Monoclonal antibodies can also be produced in mice that have been genetically altered to produce antibodies that have a human structure. IgG class antibodies are generally preferred for use within the present invention. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Cooligan et al.
• polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zvegf3 polypeptide or a fragment thereof.
• Immunogenic polypeptides will comprise an epitope-bearing portion of a zvegf3 polypeptide (e.g., as shown in SEQ ID NO:2) or receptor.
• An "epitope" is a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002, 1984.
• Epitopes can be linear or conformational, the latter being composed of discontinuous regions of the protein that form an epitope upon folding of the protein. Linear epitopes are generally at least 6 amino acid residues in length.
• Immunogenic, epitope-bearing polypeptides contain a sequence of at least six, often at least nine, more often from 15 to about 30 contiguous amino acid residues of a zvegf3 polypeptide or receptor.
• Polypeptides comprising a larger portion of a zvegf3 protein or receptor, i.e. from 30 to 50 residues up to the entire sequence, are included.
• the amino acid sequence of the epitope-bearing polypeptide is selected to provide substantial solubility in aqueous solvents, that is the sequence includes relatively hydrophilic residues, and hydrophobic residues are substantially avoided. See Figs. IA - 1G. Such regions include residues 43-48, 96-101, 97-102, 260-265, and 330-335 of SEQ ID NO:2. As noted above, it is generally preferred to use somewhat longer peptides as immunogens, such as a peptide comprising residues 80-104, 195-225, 299-314, and 299-326 of SEQ ID NO:2. The latter peptide can be prepared with an additional N-terminal Cys residue to facilitate coupling. The immunogenicity of.
• a polypeptide immunogen may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
• Polypeptides useful for immunization also include fusion polypeptides, such as fusions of a zvegf3 polypeptide or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.
• KLH keyhole limpet hemocyanin
• BSA bovine serum albumin
• tetanus toxoid tetanus toxoid
• Alternative techniques for generating or selecting antibodies include in vitro exposure of lymphocytes to a polypeptide immunogen, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled polypeptide).
• Techniques for creating and screening such random peptide display libraries are known in the art (e.g., Ladner et al., US Patent No. 5,223,409; Ladner et al., US Patent No. 4,946,778; Ladner et al., US Patent No. 5,403,484 and Ladner et al., US Patent No.
• Random peptide display libraries can be screened using the zvegf3 sequences disclosed herein to identify proteins which bind to zvegf3.
• Antibodies are determined to be specifically binding if they bind to their intended target (e.g., zvegf3 protein or receptor) with an affinity at least 10-fold greater than the binding affinity to control (e.g., non-zvegf3 or non-receptor) polypeptide or protein.
• a "non-zvegf3 polypeptide” includes the related molecules VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF, PDGF-A, and PDGF-B, but excludes zvegf3 polypeptides from non-human species.
• antibodies specific for human zvegf3 may also bind to zvegf 3 from other species.
• the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949). Methods for screening and isolating specific antibodies are well known in the art. See, for example, Paul (ed.), Fundamental Immunology, Raven Press, 1993; Getzoff et al., Adv. in Immunol.
• Binding affinity can also be determined using a commercially available biosensor instrument (BIACORE, Pharmacia Biosensor, Piscataway, NJ), wherein protein is immobilized onto the surface of a receptor chip. See, Karlsson, J. Immunol. Methods 145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-563, 1993. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
• Antibodies are considered to be "isolated” when they are prepared essentially free of other antibodies of different specificity. Use of isolated antibodies facilitates targeting of specific epitopes or portions of zvegf 3, such as the growth factor domain.
• a variety of assays known to those skilled in the art can be utilized to detect antibodies that specifically bind to zvegf3 proteins or receptors. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988.
• neutralizing antibodies denotes an antibody that inhibits at least 50% of the biological activity of the cognate antigen when the antibody is added at a 1000-fold molar access. Those of skill in the art will recognize that greater neutralizing activity is sometimes desirable, and antibodies that provide 50% inhibition at a 100-fold or 10-fold molar access may be advantageously employed.
• Zvegf3 antagonists further include antisense polynucleotides, which can be used to inhibit zvegf3 gene transcription and thereby inhibit cell activation and/or proliferation in vivo.
• Polynucleotides that are complementary to a segment of a zvegf3- encoding polynucleotide e.g., a polynucleotide as set forth in SEQ ID NO:l
• Antisense polynucleotides can be targetted to specific tissues using a gene therapy approach with specific vectors and/or promoters, such as viral delivery systems as disclosed in more detail below.
• Ribozymes can also be used as zvegf3 antagonists within the present invention.
• Ribozymes are RNA molecules that contain a catalytic center and a target RNA binding portion.
• the term includes RNA enzymes, self-splicing RNAs, self- cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
• a ribozyme selectively binds to a target RNA molecule through complementary base pairing, bringing the catalytic center into close proximity with the target sequence. The ribozyme then cleaves the target RNA and is released, after which it is able to bind and cleave additional molecules.
• Ribozyme -gene A nucleic acid molecule that encodes a ribozyme is termed a "ribozyme -gene.” Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example, Draper and Macejak, U.S. Patent No. 5,496,698; McSwiggen, U.S. Patent No. 5,525,468; Chowrira and McSwiggen, U.S. Patent No. 5,631,359; and Robertson and Goldberg, U.S. Patent No. 5,225,337).
• An expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
• expression vectors can be constructed in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of mRNA molecules that encode a zvegf3 polypeptide.
• an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see, for example, Altaian et al., U.S. Patent No.
• An external guide sequence generally comprises a ten- to fifteen-nucleotide sequence complementary to zvegf3 mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine.
• the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 '-side of the base-paired region.
• the growth factor domain of zvegf3 has been found to be the active
• zvegf3 antagonists are formulated for parenteral, particularly intravenous or intraperitoneal, delivery according to conventional methods.
• pharmaceutical formulations will include a zvegf3 antagonist in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, 5% human serum albumin, or the like.
• a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water, 5% human serum albumin, or the like.
• Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
• Liposomes may be used as carriers according to known procedures. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed., 1995.
• a zvegf3 antagonist will normally be formulated and packaged in unit dose form.
• Antibodies will typically be formulated at concentrations of about 1 mg/ml to about 10 mg/ml.
• a "therapeutically effective amount" of a thereapeutic agent or composition is that amount that produces a statistically significant effect, such as a statistically significant increase in survival rate over a given time period, increase in survival time, reduction in tumor mass, reduction in tumor metastasis, reduction in disease progression, reduction in time to progression, and the like.
• the exact dose will be determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art.
• the therapeutic formulations will generally be administered over the period required to achieve a beneficial effect, commonly up to several months. Dosing is daily or intermittently over the period of treatment. Intravenous administration ordinarily will be by infusion over a typical period of one to several hours.
• Sustained release formulations can also be employed.
• a large loading dose may be administered initially, followed by smaller, periodic, maintenance doses.
• Other mitogenic factors including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF), have been implicated in the initiation or progression of hepatocellular carcinoma. It may therefore be advantageous to combine a zvegf3 inhibitor with one or more inhibitors of these other factors.
• a zvegf3 antagonist can be administered in combination with known therapies for hepatocellular carcinoma, including surgery, chemotherapy, radiofrequency ablation, laser or microwave therapy, percutaneous ethanol injection, cryosurgery, immunotherapy, or combinations thereof.
• a zvegf3 antagonist can be administered in combination with conventional chemotherapeutic agents such as cisplatin (Epstein et al., Cancer 67:896-900, 1991), doxorubicin hydrochloride (Choi et al, Radiology 182:709-713, 1992), 5- fluorodeoxyuridine (Ensminger et al., Cancer Treat Rep. 65:393-400, 1981), dichloromethotrexate (Ensminger et al., ibid.), or bischlorethylnitrosourea (Dakhil et al., Cancer 50:631-635, 1982).
• conventional chemotherapeutic agents such as cisplatin (Epstein et al., Cancer 67:896-900, 1991), doxorubicin hydrochloride (Choi et al, Radiology 182:709-713, 1992), 5- fluorodeoxyuridine (Ensminger et al
• Regional selectivity of chemotherapy may be enhanced and systemic toxicity reduced through chemoembolization (e.g., Choi et al., ibid.; Dakhil et al., ibid.) or hepatic arterial infusion (e.g., Ensminger et al., ibid.).
• Administration of multiple therapeutic agents can be simultaneous or sequential.
• Chemoembolization, cryosurgery, percutaneous ethanol injection, and radiofrequency ablation are of particular interest in the treatment of smaller ( ⁇ 5 cm), localized tumors that are unresectable due to location or the presence of other medical conditions.
• administration of zvegf 3 antagonists alone or in combination with other therapy will result in a tumor response.
• tumor response means a reduction or elimination of all measurable lesions or metastases.
• Disease is generally considered measurable if it comprises bidimensionally measurable lesions with clearly defined margins by medical photograph or X-ray, computerized axial tomography (CT), magnetic resonance imaging (MRI), or palpation.
• CT computerized axial tomography
• MRI magnetic resonance imaging
• Evaluable disease means the disease comprises unidimensionally measurable lesions, masses with margins not clearly defined, lesion with both diameters less than 0.5 cm, lesions on scan with either diameter smaller than the distance between cuts, palpable lesions with diameter less than 2 cm, or bone disease.
• Non-evaluable disease includes pleural effusions, ascites, and disease documented by indirect evidence. Previously radiated lesions which have not progressed are also generally considered non-evaluable. Criteria for objective status are required for protocols to assess solid tumor response. Representative criteria includes the following: (1) Complete Response (CR), defined as complete disappearance of all measurable and evaluable disease. No new lesions. No disease related symptoms.
• CR Complete Response
• Partial Response defined as greater than or equal to 50% decrease from baseline in the sum of products of perpendicular diameters of all measurable lesions. No progression of evaluable disease. No new lesions. Applies to patients with at least one measurable lesion; (3) Progression, defined as 50% or an increase of 10 cm 2 in the sum of products of measurable lesions over the smallest sum observed using same techniques as baseline, or clear worsening of any evaluable disease, or reappearance of any lesion which had disappeared, or appearance of any new lesion, or failure to return for evaluation due to death or deteriorating condition (unless unrelated to this cancer); (4) Stable or No Response, defined as not qualifying for CR, PR, or Progression.
• Antibodies are preferably administered parenterally, generally by intravenous infusion (including hepatic arterial infusion) over the course of treatment. Administration may also be intraperitoneal. Antibodies are generally administered in the range of about 0.1 to about 20 mg/kg of patient weight, commonly about 0.5 to about 10 mg/kg, and often about 1 to about 5 mg/kg. In this regard, it is preferred to use antibodies having a circulating half-life of at least 12 hours, preferably at least 4 days, more preferably up to 14-21 days. Chimeric and humanized antibodies are expected to have circulatory half-lives of up to four and up to 14-21 days, respectively.
• Antibodies can also be delivered by, slow-release delivery,' systems, pumps, and other known delivery systems- for continuous infusion. Dosing regimens may be varied to provide the desired. circulating levels of a particular antibody based on its pharmacokinetics. Thus, doses will be calculated so that the desired circulating level of therapeutic agent is maintained. The actual dose and treatment regimen will be determined by the physician, taking into account the nature of the cancer (primary or metastatic), number and size of tumors, other therapies, and patient characteristics. In view of the life- threatening nature of hepatocellular carcinoma, large doses with significant side effects may be employed.
• zvegf3 antagonists The dosing regimen for a given antagonist will be determined by a number of factors including potency, pharmacokinetics, and the physicochemical nature of the antagonist.
• non-peptidic zvegf3 antagonists may be administered enterally.
• Therapeutic polynucleotides such as antisense polynucleotides, can be delivered to patients or test animals by way of viral delivery systems.
• Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno- associated virus (AAV).
• Adenovirus 'a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acids. For review, see Becker et al., Meth. Cell Biol. 43:161-189, 1994; and Douglas and Curiel, Science & Medicine 4:44-53, 1997.
• the adenovirus system offers several advantages. Adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
• adenovirus By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
• adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein.
• An alternative method of gene delivery comprises removing cells from the body and introducing a vector into the cells as a naked DNA plasmid.
• DNA vectors are introduced into host cells by methods known in the art, including transfection, electroporation, microinjection, - transduction, cell - fusion, DEAE .dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, Wu et l., J. Biol. Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994..
• Activity of zvegf3 antagonists can be measured in vitro using assays (including cell-based assays) designed to measure zvegf3 activity. Antagonists will reduce the effects of zvegf3 within the assay.
• Ligand-receptor binding can be assayed by a variety of methods well known in the art, including receptor competition assays (Bowen-Pope and Ross, Methods Enzymol. 109:69-100, 1985) and through the use of soluble receptors, including receptors produced as IgG fusion proteins (U.S. Patent No. 5,750,375).
• Receptor binding assays can be performed on cell lines that contain known cell-surface receptors for evaluation.
• the receptors can be naturally present in the cell, or can be recombinant receptors expressed by genetically engineered cells.
• Mitogenic activity can be measured using known assays, including 3 H-thymidine incorporation assays (as disclosed by, e.g., Raines and Ross, Methods Enzymol. 109:749-773, 1985 and Wahl et al., Mol. Cell Biol. 8:5016-5025, 1988), dye incorporation assays (as disclosed by, for example, Mosman, J. Immunol. Meth. 65:55-63, 1983 and Raz et al., Acta Trop. 68:139-147, 1997) or cell counts.
• Suitable itogenesis assays measure incorporation of 3 H-thymidine into (1) 20% confluent cultures to look for the ability of zvegf3 proteins to further stimulate proliferating cells, and (2) quiescent cells held at confluence for 48 hours to look for the ability of zvegf3 proteins to overcome contact-induced growth inhibition.
• Suitable dye incorporation assays include measurement of the incorporation of the dye Alamar blue (Raz et al., ibid.) into target cells. See also, Gospodarowicz et al., J. Cell. Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol.
• zvegf3 antagonists can be studied in non- human animals by administration of exogenous compounds, by expression of zvegf3 antisense polynucleotides, and by suppression of endogenous zvegf3 expression through knock-out techniques. Viral delivery systems (disclosed above) can be employed. Zvegf3 antagonists can be administered or expressed individually, in combination with other zvegf3 antagonists, or in combination other compounds, including other growth factor antagonists.
• Test animals are monitored for changes in such parameters as clinical signs, body weight, blood cell counts, clinical chemistry, histopathology, tumor size and progression, and the like.
• Animal models that can be used in assessing treatments for hepatocellular carcinoma include transgenic mice (see, e.g., Singh and Kuman, Rev. Med. Virol. 13:243-253, 2003), diethylnitrosamine perfusion in rats (e.g., Wang et al., World J. Gastroenteroh 9:930-935, 2003;' Hemmings and Strickland; Cellular Physiology' and Biochemistry 12:345-352, 2002), tumor implantation: (e.g.,' Qian et al., World J.
• Example 1 A human salivary gland library was screened for a full-length clone of zvegf3 by PCR. This library was an arrayed library representing 9.6 X 10 5 clones made in the vector pZP5x.
• the vector pZP5x is the same as vector pZP-9 (deposited with American Type Culture Collection, 10801 University Boulevard., Manassas, VA under Accession Number 98668), but contains a cytomegalovirus promoter instead of a metallothionein promoter between the Asp718 and BarnHI sites.
• the plasmid thus comprises a dihyrofolate reductase gene under control of the SV40 early promoter and S V40 polyadenylation site, and a cloning site to insert the gene of interest under control of the CMV promoter and the human growth hormone (hGH) gene polyadenylation site.
• the working plate containing 80 pools of 12,000 colonies each was screened by PCR using oligonucleotide primers ZC19,045 (SEQ ID NO:6) and ZC19,047 (SEQ ID NO:7) with an annealing temperature of 60°C for 35 cycles. There were two strong positives, pools 58 (T-8 F1-F12) and 77 (T-7 H1-H12).
• the corresponding pools in the transfer plate were then screened by PCR using the same conditions. Two positives were obtained at the transfer level. The positives were T-7 Hll and T-8 F10. 5' RACE reactions were done on the transfer plate pools, and the fragments were sequenced to check zvegf3 sequence and determine if a full-length clone was present.
• oligonucleotide primers ZC12,700 (SEQ ID NO:8) and ZC19,045 (SEQ ID NO:6) were used at an annealing temperature of 61°C for 5 cycles, then 55°C for 30 cycles. Sequencing showed that the pool T-7 Hll had a frameshift.
• Pool F10 from transfer plate 8 was plated and filter lifted using nylon membranes (Hybond-NTM; Amersham Corporation, Arlington Heights, IL). Approximately 1200 colonies per plate on each of 5 filters were lifted for a total of approximately 6000 colonies. The filters were marked with a hot needle for orientation, then denatured for 6 minutes in 0.5 M NaOH and 1.5 M Tris-HCl, pH 7.2. The filters were then neutralized in 1.5 M NaCl and 0.5 M Tris-HCl, pH 7.2 for 6 minutes. The DNA.
• the probe was generated using an approximately 400-bp fragment produced by digestion of a clone comprising a zvegf3 expressed sequence tag with EcoRI and Bgi ⁇ followed by gel-purification using a spin column containing a silica gel membrane (QIAquickTM Gel Extraction Kit; Qiagen, Inc., Valencia, CA).
• the probe was radioactively labeled with 32 P by random priming using a commercially available kit (RediprimeTM II, Amersham Corp., Arlington Heights, IL) and purified using a push column.
• ExpressHybTM solution was used for the hybridizing solution for the filters. Hybridization took place overnight at 65°C.
• Blots were rinsed 2X in 65°C solution 1 (2X SSC, 0.1% SDS), then washed 4 times in solution 1 at 65°C. The filters were exposed to film overnight at -80°C. There were 14 positives on the filters. 85 clones were picked from the positive areas and screened by PCR using oligonucleotide primers ZC19 . 045 (SEQ ID NO:6) and ZC19,047 (SEQ ID NO:7) and an annealing temperature of 60°C. Thirteen positives were obtained and streaked out for individual clones. Twenty-four colonies were picked and checked by PCR as previously described. Six positives were obtained, two of which were sequenced. Both sequences were the same and full length.
• Example 2 A PCR panel was screened for mouse zvegf3 DNA. The panel contained 8 cDNA samples from brain, bone marrow, 15-day embryo, testis, salivary gland, placenta, 15-day embryo (Clontech Laboratories), and 17-day embryo (Clontech Laboratories) libraries. PCR mixtures contained oligonucleotide primers ZC21,222 (SEQ ID NO:9) and ZC21.224 (SEQ ID NO: 10). The reaction was run at an annealing temperature of 66°C with an extension time of 2 minutes for a total of 35 cycles using Ex TaqTM DNA polymerase (PanVera, Madison, WI) plus antibody.
• Ex TaqTM DNA polymerase PanVera, Madison, WI
• DNA samples found to be positive for zvegf3 by PCR and confirmed by sequencing included mouse 15-day embryo library total pool cDNA, mouse 15-day embryo (Clontech Laboratories) and 17-day embryo (both obtained from Clontech Laboratories), mouse salivary gland library total pool cDNA, and mouse testis library total pool cDNA. Fragments of about 600 bp from each of the mouse 15-day embryo library total pool cDNA, mouse 15-day embryo cDNA, and mouse 17-day embryo cDNA PCR products were sequenced. Sequence from the mouse 17-day embryo cDNA and mouse 15-day embryo library total pool cDNA products confirmed the fragments to be mouse zvegf3 DNA.
• the mouse 15-day embryo library was screened for full-length zvegf3 DNA.
• This library was an arrayed library representing 9.6 X 10 5 clones in the pCMV»SPORT 2 vector (Life Technologies, Gaithersburg, MD).
• the working plate containing 80 pools of 12,000 colonies each, was screened by PCR using oligonucleotide primers ZC21 . 223 (SEQ ID NO:ll) and ZC21 . 224 (SEQ ID NO:10) with an annealing temperature of 66°C for 35 cycles. Eighteen positives were obtained. Fragments from four pools (A2, A10, B2, and C4)were sequenced; all were confirmed to encode zvegf3.
• the DNA was fixed to the filters using a a UV crosslinker (Stratalinker®, Stratagene, La Jolla, CA) at 1200 joules.
• a probe was generated by PCR using oligonucleotide primers ZC21.223 (SEQ ID NO: 11) and ZC21 . 224 (SEQ ID NO: 10), and a mouse 15-day embryo template at an annealing temperature of 66°C for 35 cycles.
• the PCR fragment was gel purified using a spin column containing a silica gel membrane (QIAquickTM Gel Extraction Kit; Qiagen, Inc., Valencia, CA).
• the DNA was radioactively labeled with 32 P using a commercially available kit (RediprimeTM II random-prime labeling system; Amersham Corp., Arlington Heights, IL) according to the manufacturer's specifications.
• the probe was purified using a commercially available push column (NucTrap® column; Stratagene, La Jolla, CA; see U.S. Patent No. 5,336,412).
• the filters were prewashed at 65°C in prewash buffer consisting of 0.25X SSC, 0.25% SDS, and ImM EDTA. The solution was changed a total of three times over a 45-minute period to remove cell debris.
• Filters were prehybridized overnight at 65°C in 25 ml of a hybridization solution (ExpressHybTM Hybridization Solution; Clontech Laboratories, Inc., Palo Alto, CA), then hybridized overnight at 65° C in the same solution. Filters were rinsed twice at 65 °C in pre-wash buffer (0.25X SSC, 0.25% SDS, and ImM EDTA), then washed twice in pre-wash buffer at 65°C. Filters were exposed to film for 2 days at -80°C. There were 10 positives on the filters.
• Example 3 A mammalian cell expression vector for the growth factor domain of zvegf 3 was constructed by joining the zvegf 3 fragment to a sequence encoding an optimized t-PA secretory signal sequence (U.S. Patent No. 5,641,655) in the linearized pZMPll vector downstream of the CMV promoter.
• the plasmid pZMPll is a mammalian expression vector containing an expression cassette having the CMV immediate early promoter, a consensus intron from the variable region of mouse immunoglobulin heavy chain locus, Kozak sequences, multiple restriction sites for insertion of coding sequences, a stop codon, and a human growth hormone terminator.
• the plasmid also contains an IRES element from poliovirus, the extracellular domain coding sequence of CD8 truncated at the C-terminal end of the transmembrane domain, an E. coli origin of replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHFR gene, the SV40 terminator, and the URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae.
• the resulting vector was designated pZMPll/zv3GF-otPA.
• BHK 570 cells were transfected with pZMPll/zv3GF-otPA by liposome- mediated transfection using a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(spe ⁇ mnecarboxamido)ethyl]-N,N-dimethyl-l-propaniminium- trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine in membrane- filtered water (LipofectamineTM; Life Technologies) and cultured according to conventional procedures.
• BHK cell-conditioned media was adjusted to 20 mM MES at pH 5.5.
• a column of cation exchange resin (Poros® HS 50; PerSeptive Biosystems, Framingham, MA) (2-cm diameter; 50 ml bed volume) was equilibrated in 20 mM MES, 150 mM NaCl, pH 5.5. The adjusted media was pumped into the column at 20 ml/minute. When loading was completed, the column was washed, first in 20 mM MES, 150 mM NaCl, pH 5.5, then with the same composition buffer at pH 6.0. Once the baseline was back to zero absorbance, the column was eluted with a 10-column- volume gradient to 20 mM MES, 1 M NaCl, pH 6.0.
• the zvegf3 growth factor domain eluted at 25 to 50 mS conductivity during the evolving gradient. Reducing SDS -PAGE revealed a distinct band, at 20 kD, which was confirmed as.zvegf3 by Western blotting.
• This material was pooled and prepared for loading to a tandem column array comprising a strong anion exchange resin (Poros® HQ 50; PerSeptive Biosystems) followed by an immobilized metal (nickel) affinity column in series. The system of columns was equilibrated in 20 mM MOPS buffer at pH 7.0. The vegf3 pool was in-line diluted at 1:10 (v/v) with the MOPS equilibration buffer while loading.
• the column series was washed with 20 mM MOPS pH 7.0 buffer until baseline absorbance was obtained.
• the nickel column was then disconnected fron the anion exchanger and washed with 20 mM MOPS pH 7.0 containing 150 mM NaCl.
• the column was then eluted with a 1 -column-volume gradient between the last washing buffer and the same buffer containing 20 mM imidazole at pH 7.0.
• the fractions containing the zvegf3 growth factor domain were pooled and concentrated using a 5 kD cuttoff membrane in preparation for buffer exchange and polishing on a size exclusion column equilibrated in PBS.
• Example 4 To make transgenic animals expressing zvegf3 genes requires adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl, 2-4 months, (Taconic Farms)).
• the donors are acclimated for 1 week, then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Co., St. Louis, MO) I.P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma Chemical Co.)) I.P. to induce superovulation.
• Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating. Fertilized eggs are collected under a surgical scope (Leica MZ12 Stereo
• the oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma Chemical Co.). Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (Table 2; all reagents available from Sigma Chemical Co.) that has been incubated with 5% CO2, 5%
• the eggs are stored in a 37°C/5% CO2 incubator until microinjection.
• a rat insulin II intron (ca. 200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru I site.
• the vector comprises a mouse metallothionein (MT-1) promoter (ca. 750 bp) and human growth hormone (hGH) untranslated region and polyadenylation signal (ca. 650 bp) flanked by 10 kb of MT-1 5' flanking sequence and 7 kb of MT-1 3' flanking sequence.
• the cDNA is inserted between the insulin II and hGH sequences. 10-20 micrograms of plasmid DNA is linearized, gel-purified, and resuspended in 10 mM Tris pH 7.4, 0.25 mM EDTA pH 8.0, at a final concentration of
• Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO2-equilibrated mineral oil.
• the DNA is drawn into an injection needle (pulled from a 0.75 mm ID, 1 mm OD borosilicate glass capillary) and injected into individual eggs. Each egg is penetrated with the injection needle into one or both of the haploid pronuclei. Picoliters of DNA are injected into the pronuclei, and the injection needle is withdrawn without coming into, contact with the nucleoli. The procedure is repeated until all the eggs are injected.
• microinjected eggs are transferred into an organ tissue-culture dish with pregassed W640 medium for storage overnight in a 37°C/5% CO 2 incubator.
• 2-cell embryos are transferred into pseudopregnant recipients.
• the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
• Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope.
• a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
• the reproductive organs are exteriorized onto a small surgical drape.
• the fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
• a baby serrefine Robot, Rockville, MD
• 12-17 healthy 2-cell embryos from the previous day's injection are transferred into the recipient.
• the swollen ampulla is located, and holding the oviduct between the ampulla and the bursa, a nick in the oviduct is made with a 28 g needle close to the bursa, making sure not to tear the ampulla or the bursa.
• the pipette is transferred into the nick in the oviduct, and the embryos are blown in, allowing the first air bubble to escape the pipette.
• the fat pad is gently pushed into the peritoneum, and the reproductive organs are allowed to slide in.
• the peritoneal wall is closed with one suture, and the skin is closed with a wound clip.
• the mice recuperate on a 37°C slide warmer for a minimum of 4 hours.
• the recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning.
• the weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
• Genomic DNA is prepared from the tail snips using a commercially available kit (DNeasyTM 96 Tissue Kit; Qiagen, Valencia, CA) following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to the human growth hormone (hGH) 3' UTR portion of the transgenic vector. The use of a region unique to the human sequence (identified from an alignment of the human and mouse growth hormone 3' UTR DNA sequences) ensures that the PCR reaction does not amplify the mouse sequence. Primers ZC17,251 (SEQ ID NO: 13) and ZC17,252 (SEQ ID NO: 14) amplify a 368-base-pair fragment of hGH.
• hGH human growth hormone
• primers ZC17,156 SEQ ID NO:15
• ZC17,157 SEQ ID NO:16
• DNA from animals positive for the transgene will generate wo bands, a 368-base-pair band corresponding to the hGH 3' UTR fragment and a band of variable size corresponding to the cDNA insert.
• a partial hepatectomy is performed.
• a surgical prep is made of the upper abdomen directly below the xiphoid process.
• sterile technique a small 1.5-2 cm incision is made below the sternum, and the left lateral lobe of the liver is exteriorized.
• 4-0 silk a tie is made around the lower lobe securing it outside the body cavity.
• An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid, Wayne, N.J.) is placed proximal to the first tie.
• a distal cut is made from the Dexon tie, and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish.
• the excised liver section is transferred to a 14-ml polypropylene round bottom tube, snap- frozen in liquid nitrogen, and stored on dry ice.
• the surgical site is closed with suture and wound clips, and the animal's cage is placed on a 37°C heating pad for 24 hours post-operatively. The animal is checked daily post-operatively, and the wound clips are removed 7-10 days after surgery.
• RNA solution hybridization assay or real-time PCR on commercially available PCR equipment (ABI PrismTM 7700; PE Applied Biosystems, Inc., Foster City, CA) following the manufacturer's instructions.
• An adenovirus vector was prepared using a liver-specific albumin gene enhancer and basal promoter (designated "AEO promoter").
• the albumin promoter construct (designated pAEO) was constructed by inserting a 2.2 kb Notl/EcoRV fragment from pALBdelta2L (Pinkert et al., Genes Dev.
• Nrul/Notl DNA segment comprising the rat insulin II intron, an Fsel Pmel Ascl polylinker, and the human growth hormone poly A sequence into a commercially available phagemid vector (pBluescript® KS(+); Stratagene, La Jolla, CA).
• a commercially available phagemid vector pBluescript® KS(+); Stratagene, La Jolla, CA.
• the plasmid is digested with Notl to liberate the expression cassette.
• An additional adenovirus vector was constructed using an epithelial cell- specific keratin gene (K14) promoter (Vassar et al., Proc. Natl. Acad. Sci. USA 86:1563-1567, 1989).
• the 1038-bp open reading frame encoding full-length human zvegf3 was amplified by PCR so as to introduce an optimized initiation codon and flanking 5' Pmel and 3' Ascl sites using the primers ZC20,180 (SEQ ID NO:17) and ZC20,181 (SEQ ID NO: 18).
• the resulting PmeVAscl fragment was subcloned int ⁇ the polylinker of pK_FO114, a basal keratmocyte-restricted transgenic vector comprising the human keratin 14 (K14) promoter (an approximately 2.3 Kb fragment amplified from human genomic DNA [obtained from Clontech Laboratories, Inc.] based on the sequence of Staggers et al., "Sequence of the promoter for the epidermal keratin gene, K14", GenBank accession #U11076, 1994), followed by a heterologous intron (a 294- bp BstXVPstl fragment from pIRESlhyg (Clontech Laboratories, Inc.; see, Huang and Gorman, Nucleic Acids Res.
• K14 human keratin 14
• a heterologous intron a 294- bp BstXVPstl fragment from pIRESlhyg (Clontech
• the transgene insert was separated from the plasmid backbone by Notl digestion and agarose gel purification, and fertilized ova from matings of B6C3FlTac mice or inbred FVB/ ⁇ Tac mice were microinjected and implanted into pseudopregnant females essentially as described by Malik et al., Molec. Cell. Biol. 15:2349-2358, 1995.
• Transgenic founders were identified by PCR on genomic tail D ⁇ A using primers specific for the human growth hormone poly A signal (ZC 17,252, SEQ ID NO: 14; and ZC17,251, SEQ ID NO: 13) to amplify a 368-b ⁇ diagnostic product.
• Transgenic lines were initiated by breeding founders with C57BL/6Tac or FVB/NTac mice.
• Transgenic mice were generated essentially as disclosed above using MT- 1, K14, and AEO promoters.
• Four MT-l/zvegf3 transgenic mice were generated. In one animal (female) approximately 800 molecules zvegf3 mRNA/cell were produced in the liver after zinc induction. This animal had enlargement of the liver and spleen.
• H & E and trichrome stains indicated a definite increase in number of liver sinusoidal (stellate) cells and increased perisinusoidal extracellular matrix (ECM) deposition at 8 weeks of age. There was a persistent increase in number of stellate cells and in the amount and thickness of perisinusoidal ECM as well as some perivenular ECM deposition by 16 weeks. At 22 weeks similar changes were seen, but with an increase in incidence and severity of perivenular fibrosis (steato fibrosis). Changes similar to those at 16 and 22 weeks were observed at 33 .weeks, however some animals had fibrotic banding and multiple, encapsulated areas' in- which hepatocytes appeared enlarged and vacuolated.
• Example 5 Five AEO human zvegf3 transgenic mice (N7 generation) were sacrificed and necropsied, and tissues were collected in 10% buffered formalin. Additional liver samples were fixed in Carnoy's and zinc tris fixatives for immunohistochemistry.
• One section had an area of thrombosis and coagulative necrosis, the latter indicative of infarction possibly caused by the thrombosis.
• the spleen had moderate lymphoreticular hyperplasia, which correlated with the gross observation of being enlarged.
• Example 6 Two male transgenic mice, N4 generation, were sacrificed at 52 weeks of age and necropsied, and a routine physioscreen was conducted. Microscopically, the liver was the primary target organ, with a multitude of changes that included telangiectasis, vascular dilatation, sinusoidal cell proliferation, nodular hepatocytic hyperplasia, and sinusoidal fibrosis. In at least one of the two animals, the liver also had areas of hemorrhage, infarction, bile duct hyperplasia, cyst formation, and lymphoid cell infiltrates. The microscopic observations in the remaining tissues examined were considered incidental findings common in one-year old mice.
• the progressive hepatic fibrosis in these two animals resulted in the disruption of the architecture of the vascular system causing severe hypertension as evidenced by the dilated vessels and the telangiectasis within the sinusoids. Due to the extensive loss of normal hepatic parenchyma in the areas of telangiectasis, the severity of the sinusoidal cell proliferation was difficult to assess, although there were areas of obvious increased numbers of these cells in the absence of hepatocyte loss.
• Example 7 A single male transgenic AEO/zvegf3 mouse, N4 generation, was sacrificed at 57 weeks of age and necropsied, and a routine physioscreen was conducted. At necropsy the liver was noted to be " misshapen, dark, and nodular. Microscopically, ⁇ the liver had areas of moderate diffuse perisinusoidal (stellate) cell hyperplasia as well as "• moderate sinusoidal dilation, myxomatous change, perivascular fibrosis, and nodular hepatocellular hyperplasia. Also present in one or more of the sections examined were a cyst, a hepatocellular adenoma, and a focal area of necrosis.
• Example 8 Four AEO/zvegf3 transgenic mice (N4) were sacrificed at approximately 62 weeks of age and necropsied, and tissues were collected and preserved in 10% BNF (buffered neutral formalin). At necropsy, the liver in each of these mice was observed to be enlarged and fibrotic in appearance. All tissues were trimmed and processed, slides were prepared, and sections were stained with hematoxylin and eosin for routine microscopic examination. In addition, sections of liver were stained with Masson's trichrome and Sirius Red, and immunohistochemistry (IHC) was done for the detection of smooth muscle actin, desmin, and Type I collagen. Animal No.
• IHC immunohistochemistry
• SM actin alpha smooth muscle actin
• SM actin and desmin were most prominent within the neoplasm and were possibly reflective of angiogenesis.
• Type I collagen was not as prominent within the tumor but was more prominent in the areas of fibrosis and nodular hyperplasia.
• Other prominent changes in the kidney and lung were similar in both distribution and severity to those described above for Animal No. 24811. All microscopic changes observed in the remaining tissues were also considered incidental findings and unrelated to the transgene.
• SM actin, desmin, and Type I collagen there were minimal amounts of SM actin, desmin, and Type I collagen within the tumor, but increased amounts of all three of these were present in the other sections of the liver, the latter two most prominent in the areas of increased sinusoidal cell hyperplasia and fibrosis. Microscopic changes in the kidney and lung were also similar to those observed in the two previous animals. All other microscopic changes observed in the remaining tissues were considered incidental findings and not directly related to the transgene.
• the microscopic findings in the tissues examined from this animal were very similar to those observed in the three male mice described above.
• the liver had severe diffuse sinusoidal hyperplasia, fibrosis, and hepatocellular nodular hyperplasia as well as vascular and sinusoidal dilatation, bile duct hyperplasia, and focal areas of myxomatous-like matrix accumulation.
• Increased staining for desmin and Type I collagen was also prominent in these areas and was closely associated with the fibrosis.
• the hepatocytic nodule and tumor formation is similar to that ascribed to hepatic fibrosis and cirrhosis in man. Further, the impact of the resulting fibrosis and the sinusoidal cell accumulation on the vascular system is evident from the vascular and sinusoidal dilatation as well as the development of the vascular neoplasms and associated preneoplastic changes. Interestingly, the results of the IHC staining for ⁇ smooth muscle actin, which appeared to be primarily associated with the angiogenic changes and not the sinusoidal cells or the fibrotic areas, would suggest that the cells involved in matrix and collagen production may have differentiated into more specialized cells that have lost their actin and therefore can no longer.be termed myofibroblasts.
• hepatic changes were observed: increased numbers or hyperplasia of perisinusoidal (stellate) cells, sinusoidal and vascular dilatation, telangiectasis, cyst formation, perivenular and intra-sinusoidal fibrosis, nodular hepatocellular hyperplasia, and hepatocellular adenoma.
• telangiectasis a myxomatous change characterized by the presence of spindle-shaped cells embedded in a mucinous matrix.
• One animal had a hepatic tumor composed of plump, endothelial-like cells associated with areas of vascular and sinusoidal dilatation.
• This tumor was characterized as a hemangioendothelioma, and its formation was possibly related to the fibrosis and associated vascular changes.
• the gross and correlating microscopic changes observed in the livers from these older transgenic mice were typical of the end stage of a chronic, progressive fibrosis.
• the progressive increase in collagen deposition and subsequent fibrosis observed is similar to hepatic fibrosis and cirrhosis in man, and is commonly thought to contribute to the nodular hepatocellular hyperplasia and tumor formation, including a progression from hyperplasia to adenoma to carcinoma.
• Example 9 For construction of adenovirus vectors, the protein coding region of human zvegf3 was amplified by PCR using primers that added Pmel and Ascl restriction sites at the 5' and 3' termini, respectively.
• PCR primers ZC20,180 (SEQ ID NO:17) and ZC20,181 (SEQ ID NO: 18) were used with a full-length zvegf 3 cDNA template in a PCR reaction as follows: incubation at 95°C for 5 minutes; followed by 15 cycles at 95°C for 1 min., 61°C for 1 min., and 72°C for 1.5 min.; followed by 72°C for 7 min.; followed by a 4°C soak.
• the reaction product was loaded onto a 1.2 % low-melting- temperature agarose gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA).
• the zvegf 3 PCR product was excised from the gel and purified using a commercially available kit comprising a silica gel mambrane spin column (QIAquickTM PCR Purification Kit and gel cleanup kit; Qiagen, Inc.) as per kit instructions.
• the zvegf3 product was then digested with Pmel and Ascl, phenol/chloroform extracted, EtOH precipitated, and rehydrated in 20 ml TE (Tris/EDTA pH 8).
• the 1038 bp zvegf3 fragment was then ligated into the Pmel-Ascl sites of the transgenic vector pTG12-8 (also known as pHB12-8; see Example 4) and transformed into E. coli DH10BTM competent cells by electroporation.
• Clones containing zvegf3 were identified by plasmid DNA miniprep followed by digestion with Pmel and Ascl. A positive clone was sequenced to insure that there were no deletions or other anomalies in the construct. The sequence of zvegf3 cDNA was confirmed.
• DNA was prepared using a commercially available kit (Maxi Kit, Qiagen, Inc.), and the 1038bp zvegf3 cDNA was released from the pTG12-8 vector using Pmel and Ascl enzymes.
• the cDNA was isolated on a 1% low melting temperature agarose gel and was excised from the gel. The gel slice was melted at 70°C, and the DNA was extracted twice with an equal volume of Tris-buffered phenol and precipitated with EtOH. The DNA was resuspended in 10 ⁇ l H 2 O.
• the zvegf3 cDNA was cloned into the EcoRV-AscI sites of a modified pAdTrack-CMV (He, T-C.
• This construct contains the green fluorescent protein (GFP) marker gene.
• GFP green fluorescent protein
• the CMV promoter driving GFP expression was replaced with the SV40 promoter, and the SV40 polyadenylation signal was replaced with the human growth hormone polyadenylation signal.
• the native polylinker was replaced with Fsel, EcoRV, and Ascl sites.
• This modified form of pAdTrack-CMV was named pZyTrack. Ligation was performed using a commercially available DNA ligation and screening kit (Fast-LinkTM kit; Epicentre Technologies, Madison, WI).
• Clones containing zvegfS were identified by digestion of mini prep DNA with Fsel and Ascl. In order to linearize the plasmid, approximately 5 ⁇ g of the resulting pZyTrack zvegf3 plasmid was digested with Pmel. Approximately 1 ⁇ g of the linearized plasmid was cotransformed with 200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli BJ5183 cells (He et al., ibid.).
• the co- transformation was done using commercially available electroporation equipment (Gene Pulser®; Bio-Rad Laboratories, Hercules, CA) at 2.5 kV, 200 ohms and 25 ⁇ Fa.
• Gene Pulser® Bio-Rad Laboratories, Hercules, CA
• the entire co-transformation mixture was plated on 4 LB plates containing 25 ⁇ g/ml kanamycin.
• the smallest colonies were picked and expanded in LB/kanamycin, and recombinant adenovirus DNA was identified by standard DNA miniprep procedures. Digestion of the recombinant adenovirus DNA with Fsel and Ascl confirmed the presence of the zvegf3 insert.
• the recombinant adenovirus miniprep DNA was transformed into E.
• DNA was prepared using a commercially available DNA purification kit (obtained from Qiagen, Inc.) according to kit instructions. Approximately 5 ⁇ g of recombinant adenoviral DNA was digested with
• Pad enzyme (New England Biolabs) for 3 hours at 37°C in a reaction volume of 100 ⁇ l containing 20-30U of Pad.
• the digested DNA was extracted twice with an equal volume of phenol/chloroform and precipitated with ethanol.
• the DNA pellet was resuspended in 10 ⁇ l distilled water.
• a T25 flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada), inoculated the day before and grown to 60-70% confluence, were transfected with the Pad-digested DNA.
• the Pad-digested DNA was diluted up to a total volume of 50 ⁇ l with sterile HBS (150 mM NaCl, 20 mM HEPES).
• DOTAP N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethyl-ammonium salts
• the media was removed from the 293A cells and washed with 5 ml serum-free minimum essential medium (MEM) alpha containing 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, and 25mM HEPES buffer (reagents obtained from Life Technologies, Gaithersburg, MD). 5 ml of serum-free MEM was added, and the cells were held at 37°C. The DNA/lipid mixture was added drop-wise to the flask of cells, mixed gently, and incubated at 37°C for 4 hours. The media containing the DNA lipid mixture was then aspirated off and replaced with 5 ml complete MEM containing 5% fetal bovine serum.
• MEM minimum essential medium
• the transfected cells were monitored for GFP expression and formation of foci (viral plaques). Seven days after transfection of 293A cells with the recombinant adenoviral DNA, the cells expressed GFP and started to form foci.
• the crude viral lysate was collected using a cell scraper to collect all of the 293A cells. The lysate was transferred to a 50-ml conical tube. To release most of the virus particles from the cells, three freeze/thaw cycles were done in a dry ice/ethanol bath and a 37° waterbath. The crude lysate was amplified (Primary (1°) amplification) to obtain a working stock of zvegf3 rAdV lysate.
• the supernatant was transferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volume of 20% PEG8000/2.5 M NaCl solution was added. The bottles were shaken overnight on ice. The bottles were centrifuged at 20,000 X G for 15 minutes, and the supernatant was discarded into a bleach solution. Using a sterile cell scraper, the white, virus/PEG precipitate from 2 bottles was resuspended in 2.5 ml PBS. The resulting virus solution was placed in 2-ml microcentrifuge tubes and centrifuged at 14,000 X G in the microcentrifuge for 10 minutes to remove any additional cell debris.
• the supernatant from the 2-ml microcentrifuge tubes was transferred into a 15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/ml with CsCl.
• the volume of the virus solution was estimated, and 0.55 g/ml of CsCl was added.
• the CsCl was dissolved, and 1 ml of this solution weighed 1.34 g.
• the solution was transferred to 3.2-ml, polycarbonate, thick-walled centrifuge tubes and spun at 348,000 X G for 3-4 hours at 25°C.
• the virus formed a white band. Using wide-bore pipette tips, the virus band was collected.
• the virus recovered from the gradient had a large amount of CsCl, which had to be removed before the virus was used on cells.
• Commercially available ion- exchange columns (PD-10 columns prepacked with Sephadex® G-25M; Pharmacia Biotech, Piscataway, NJ) were used to desalt the virus preparation. The column was equilibrated with 20 ml of PBS. The virus was loaded onto and allowed to run into the column. 5 ml of PBS was added to the column, and fractions of 8-10 drops were collected. The optical density of a 1:50 dilution of each fraction was determined at 260 nm on a spectrophotometer. A clear absorbance peak was present between fractions 7- 12.
• TCID 50 formulation used was as per Quantum Biotechnologies, Inc., above.
• the titer (T) was determined from a plate where virus was diluted from 10 " to 10 " , and read 5 days after the infection. At each dilution a ratio (R) of positive wells for CPE per the total number of wells was determined.
• R ratio of positive wells for CPE per the total number of wells was determined.
• Example 10 Polyclonal anti-peptide antibodies were prepared by immunizing two female New Zealand white rabbits with the peptides huzvegf3-l (residues 80-104 of SEQ ID NO:2), huzvegf3-2 (residues 299-314 of SEQ ID NO:2), huzvegf3-3 (residues 299-326 of SEQ ID NO:2 with an N-terminal cys residue), or huzvegf3-4 (residues 195- 225 of SEQ ID NO:2 with a C-terminal cys residue).
• the peptides were synthesized using an Applied Biosystems Model 431 A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions.
• the peptides huzvegf3-l, huzvegf3-3, and huzvegf3-4 were then conjugated to the carrier protein maleimide-activated keyhole limpet hemocyanin (KLH) through cysteine residues (Pierce Chemical Co., Rockford, IL).
• KLH keyhole limpet hemocyanin
• the peptide huzvegf3-2 was conjugated to the carrier protein KLH using gluteraldehyde.
• the rabbits were each given an initial intraperitoneal (IP) injection of 200 ⁇ g of conjugated peptide in Complete Freund's Adjuvant (Pierce Chemical Co.) followed by booster IP injections of 100 ⁇ g conjugated peptide in Incomplete Freund's Adjuvant every three weeks. Seven to ten days after the administration of the third booster injection, the animals were bled and the serum was collected. The rabbits were then boosted and bled every three weeks.
• IP intraperitoneal
• the huzvegf3 peptide-specific antibodies were affinity purified from the rabbit serum using a CNBr-Sepharose® 4B peptide column (Pharmacia Biotech) that was prepared using 10 mg of the ; respective, .peptides per gram CNBr-Sepharose®,- followed' by dialysis in , PBS overnight.
• Peptide specific-huzvegf3 antibodies were characterized by an ELISA. titer check using 1 ⁇ g/ml of the appropriate peptide as an antibody target.
• the huzvegf3-l peptide-specific antibodies had a lower limit of detection (LLD) of 500 pg/ml by ELISA on the appropriate antibody target and recognized full-length recombinant protein (MBP-fusion) by ELISA.
• the huzvegf3-2 peptide-specific antibodies had an LLD of 1 ng/ml by ELISA.
• the huzvegf3-3 peptide- specific antibodies had an LLD of 50 pg/ml by ELISA and recognized recombinant protein by Western Blot analysis.
• the huzvegf3-4 peptide-specific antibodies had an LLD of 50 pg/ml by ELISA and recognized recombinant protein by Western Blot analysis.
• Example 11 Polyclonal antisera, designated "E2243", was raised in a rabbit by immunization with a full-length human zvegf3 polypeptide fused to E. coli maltose binding protein (MBP) and affinity purified using the fusion protein. Specificity of the antisera was examined in a Western blot format in which samples of various zvegf3 proteins were reduced and electrophoresed on a polyacrylamide gel.
• E. coli maltose binding protein MBP
• the proteins used were: recombinant human zvegf3 growth factor domain, recombinant human zvegf3 full-length, and recombinant human zvegf3 full-length fused to MBP, each at concentrations of 13.9, 41.7, and 125 ng/lane; and conditioned media from HaCat cells expressing full-length human zvegf3.
• the electrophoresed proteins were then transferred to a nitrocellulose membrane, rinsed, and blocked by overnight incubation in buffer containing 2.5% non-fat dry milk.
• the primary antibody (E2243 antisera) was diluted to 300 ng ml and added to the nitrocellulose blot, which was then incubated for 1 hour at room temperature with shaking.
• the blot was then rinsed, secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase) was added, and the blot was incubated for 1 hour at room temperature with shaking. The blot was then rinsed, developed with commercially available substrates, and exposed to film for 10 seconds.
• the Western blot showed that the E2243 antisera recognized all samples of full-length zvegf3 (fused and unfused) and the zvegf3 in the conditioned media, but did not recognize any of the samples of isolated zvegf3 growth factor domain.
• Example 12 Mouse hybridomas producing monoclonal antibodies (MAbs) specific for recombinant human zvegf 3 growth factor domain (GFD) protein were generated using purified, untagged, recombinant human zvegf3 GFD produced in BHK cells (huzvegf3- i .GFDTBHK). v, Ten-BALB/c mice were each injected IP on. day- 1 with 20 ⁇ g of huzvegf3- GFD-BHK mixed ,1:1 (v/v) in- complete Freund's . adjuvant.
• MAbs monoclonal antibodies specific for recombinant human zvegf 3 growth factor domain (GFD) protein were generated using purified, untagged, recombinant human zvegf3 GFD produced in BHK cells (huzvegf3- i .GFDTBHK).
• v Ten-BALB/c mice were each injected IP on. day- 1 with 20 ⁇ g of
• mice were subsequently injected IP with 10 ⁇ g of huzveg ⁇ .-GFDrBHK mixed 1:1, in incomplete Freund's adjuvant, on days 15, 29, 41, 57, 71, 89 and 115.
• splenocytes and lymphocytes from enlarged lymph nodes of two mice with the highest anti-huzvegf3 antibody titer (as determined in a biotinylated huzvegf3-GFD capture ELISA; see below) were fused at a 2.76:1 ratio with the X63-Ag8.653 mouse myeloma cell line (Kearney et al., J. Immunol.
• the fusion mixture was plated into 24 96-well plates at an average density of 1.2 x 10 total cells/well in Iscove's modified Dulbecco's medium (IMDM; Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal clone I serum (HyClone Laboratories, Inc., Logan, UT), 10% hybridoma cloning supplement (BM Condimed® HI; Roche Diagnostics Corp., Indianapolis, IN), 2 mM L-glutamine (Life Technologies, Inc.), 100 U/mL penicillin G sodium (Life Technologies, Inc.), and 100 ⁇ g/mL streptomycin sulfate (Life Technologies, Inc.).
• IMDM Iscove's modified Dulbecco's medium
• Wells were fed on days 4 and 7 by aspiration and replacement of approximately three-fourths of the media contents in each well. This fusion was designated HH1.
• Anti-huzvegf3 mAbs of the IgG class were detected on days 9/10 post- fusion using a biotinylated huzvegf3-GFD capture ELISA.
• Wells of plates (Immulon® II; Dynex Technologies, Chantilly, VA) were coated with 1 ⁇ g mL of goat anti-mouse IgG (obtained from Kirkegaard & Perry Laboratories, Gaithersburg, MD) in 0.05 M carbonate/bicarbonate buffer (Sigma, St. Louis, MO), 50 ⁇ L/well.
• Human zvegf3-GFD protein was biotinylated with sulfo-NHS-LC-biotin (Pierce Chemical Company, Rockford, IL) according to the manufacturer's instructions for 45 minutes at RT. The reaction was stopped with 2M glycine. Biotinylated protein was diluted to 1 ⁇ g/mL in PTB buffer. The plates were washed four times with PBST, biotinylated huzvegf3-GFD was added at 100 ⁇ L/well, and the plates were incubated at RT for 1 hour.
• Optical density was read on an ELISA plate reader (Molecular Devices, Sunnyvale, CA) at wavelengths 450 nm (LI) and 650 nm (L2). Final OD measurement was determined by the formula L1-L2.
• 78 master wells contained antibody that was capable of capturing biotinylated huzvegf3-GFD. These master wells were expanded for hybridoma cryopreservation and additional supernatant generation. ELISA analysis of the expanded master well supematants demonstrated that 27 of the original 78 master wells retained antibody specific for huzvegf3-GFD, with 20 of 27 possessing significant reactivity with the antigen.
• Each of the clones was subsequently adapted to growth in a production medium formulation consisting essentially of Dulbecco's modified Eagle's medium + 2.5% fetal clone I serum and various supplements. Supematants were again serially diluted and tested by ELISA on huzvegf3-GFD to identify the highest titered and next highest titered clones (designated the primary and secondary clones, respectively). MAb produced by each set of clones was then evaluated for IgG subclass using the Mouse Hybridoma Subtyping Kit (Cat. #1183117; Roche Diagnostics Corp.). Clones HHl-24, -40, -57 and -76 were all found to produce an IgG ! antibody. Clones HH1-58 and -78 produced an IgG 2b antibody. All antibodies possessed a K light chain.
• Example 13 A study was undertaken to test whether adenovirally delivered zvegf3 stimulated cell proliferation as determined by incorporation of bromodeoxyuridine (BrdU) into tissues. Mice (male, C57B1, 7 weeks old) were divided into three groups. On day
• Each mouse' was given two intfaperitoneal doses of 3 mg of freshly made BrdU solution at approximately 24 and 12 hours prior to sacrifice.
• days 2, 4,.6, 8, and 10 two mice from each treatment group and one or two untreated mice were sacrificed, and tissues and blood were harvested. Samples were analyzed for complete blood count (CBC) and serum chemistry, and slides were prepared for manual differential blood and marrow progenitor cell analysis.
• CBC complete blood count
• serum chemistry serum chemistry
• livers of the zvegf3 adenovirus-treated mice tended to look more pale than animals treated with the parental virus. Proliferation of sinusoidal cells was observed in liver. Visual inspection suggested that these cells were stellate cells and/or fibroblasts. Spleen color was the same in both groups. Most of the animals that received the zvegf3 adenovirus had paler femur shafts, with the marrow lighter in color.
• Peripheral blood CBCs showed a possible difference in platelet counts, but not in RBC or WBC counts between zvegf3 and parental vims-treated animals.
• the zvegf3 group had lower platelet counts on days 2, 4, 6, and 8, but not on day 10.
• the mean platelet volume (average size of individual platelets) in the zvegf3 group also tended to be greater, consistent with a relative increase in the larger, immature platelet population.
• BrdU labeling showed increased cell proliferation in kidney, mainly in the medulla and to a lesser extent in the cortex.
• Proliferating cells appeared to be interstitial cells, which may have included fibroblasts and/or mesangial cells.
• Example 14 Human zvegf 3 growth factor domain protein produced in BHK cells was tested for the ability to stimulate production of TGF- ⁇ in stellate cells.
• Rat hepatic , stellate cells obtained from Dr. Nelson Fausto, University of Washington
• Rat hepatic , stellate cells obtained from Dr. Nelson Fausto, University of Washington
• DMEM growth- medium (Life Technologies, Inc.) supplemented with pyruvate and 10% serum (HyClone Laboratories, Inc.).
• the medium was changed to serum-free medium by substituting 0.1% BSA (Fraction V; Sigma, St. Louis, MO) for serum.

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