WO2001005821A2 - Viral il-10 for the inhibition of angiogenesis, tumorigenesis and metastasis - Google Patents

Viral il-10 for the inhibition of angiogenesis, tumorigenesis and metastasis Download PDF

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WO2001005821A2
WO2001005821A2 PCT/EP2000/006558 EP0006558W WO0105821A2 WO 2001005821 A2 WO2001005821 A2 WO 2001005821A2 EP 0006558 W EP0006558 W EP 0006558W WO 0105821 A2 WO0105821 A2 WO 0105821A2
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tumor
cells
viral
vil
administering
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WO2001005821A3 (en
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Maria Teresa Bejarano
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Maria Teresa Bejarano
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates generally to methods of using specific cytokines, more particularly, to methods of treating proliferative diseases or conditions.
  • the immune system consists of a wide range of distinct cell types, each with important roles to play. See Paul (ed. 1997) Fundamental Immunology 4th ed. , Raven Press, New York.
  • the lymphocytes occupy central stage because they are the cells that determine the specificity of immunity, and it is their response that orchestrates the effector limbs of the immune system.
  • Two broad classes of lymphocytes are recognized: the B lymphocytes, which are precursors of antibody secreting cells, and the T ( thymus-dependent ) lymphocytes.
  • T lymphocytes express important regulatory functions, such as the ability to help or inhibit the development of specific types of immune response, including antibody production and increased microbicidal activity of macrophages .
  • Other T lymphocytes are involved in direct effector functions, such as the lysis of virus infected-cells or certain neoplastic cells.
  • Lymphokines apparently mediate cellular activities in a variety of ways . They have been shown to support the proliferation, growth, and differentiation of pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. Proper and balanced interactions between the cellular components are necessary for a healthy immune response. The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents .
  • B-cells which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal)
  • T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T-cells) making up the immune network.
  • mast cell which has not been positively identified in all mammalian species
  • mast cell is a granule-containing connective tissue cell located proximal to capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts.
  • Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows: when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators, e.g., histamine, serotonin, heparin, and prostaglandins, which cause allergic reactions, e.g., anaphylaxis.
  • Synthesis Inhibitory Factor was isolated in the 1980 's. See, e.g., Mosmann, et al . , U.S. Patent No. 5,231,012. A viral counterpart, known as viral IL-10 from EBV has also been described. Much has been learned of the biology and physiology mediated by these cytokines. See, e.g., de Vries and de Waal Malefyt (1995) Interleukin-10 Austin Co., Austin, TX.
  • the discovery of new biological activities for these cytokines can contribute to new therapies for a wide range of degenerative or abnormal conditions, e.g., which directly or indirectly involve the immune system and/or hematopoietic cells.
  • the discovery and development of new clinical targets for use of these lymphokines would be highly advantageous .
  • the present invention provides new methods for their use.
  • the present invention is based, in part, upon the surprising discovery that the cytokines IL-10 and BCRF1 (also known as viral IL-10, or vIL-10), have anti-angiogenic , anti- tumorigenic, and anti-metastatic activities.
  • the present invention provides methods of inhibiting angiogenesis to a growing cellular mass comprising administering an effective amount of viral IL-10 to the location of the mass.
  • the cellular mass is a tumor; the tumor is: highly vascularized, a lymphoid tumor, Burkitt's lymphoma, Kaposi sarcoma, osteosarcoma, mammary adenocarcinoma, malignant melanoma, or prostatic adenocarcinoma .
  • the administering will often be local, topical, subcutaneous, intradermal, or tran ⁇ dermal, or, in certain embodiments, by expression of a nucleic acid encoding the viral IL-10.
  • the administering will often be in combination with another anti-neoplastic therapeutic and/or an anti-inflammatory agent.
  • the effective amount further results in a decrease in tumor metastasis or angiostasis, or in inhibiting tumorigenesis .
  • the invention also provides methods of inhibiting tumorigenesis of a growing cellular mass comprising administering an effective amount of viral IL-10 to the location of the mass.
  • the cellular mass will often be a tumor, e.g., which is highly vascularized, or a lymphoid tumor.
  • Preferred tumors will be Burkitt's lymphoma,
  • the administering will preferably be local, topical, subcutaneous, intradermal, or transdermal ; or by expression of a nucleic acid encoding the viral IL-10. Often, the administering is in combination with: another anti-neoplastic therapeutic agent; or an anti- inflammatory agent; and will preferably further result in inhibiting tumorigenesis.
  • the invention further provides methods of inhibiting metastasis of a cellular mass comprising administering an effective amount of viral IL-10 to the host.
  • the invention is based, in part, on the surprising discovery that the cytokine BCRFl, also known as viral IL-10 (vIL-10), has anti-angiogenic, anti-tumorigenie, and anti- metastatic activities.
  • the cytokines viral IL-10 (also known as BCRFl) and IL-10 have been described earlier in, e.g., de Vries and de Waal Malefyt Interleukin-10 Austin TX; US Pat. No. 5,627,155; and US Pat. No. 5,231,012, which are incorporated herein by reference for all purposes. Additional viral forms of IL-10 also exist, which may have some of the same anti-tumor activities. See Rode, et al . (1993) Virus Genes 7:111-116; and Fleming, et al . (1997) J. Virol. 71:4857- 4861.
  • vIL-10 has the same activities as the cellular form.
  • Human and viral IL-10 genes were introduced into the human Burkitt's lymphoma line DG75. Stable transfectants were monitored for tumorigenicity in SCID mice and angiogenic capacity. The two cytokines displayed concordant effects and no differences were found in the in vitro growth capacity, agarose clonability or NK-sensitivity, but the tumorigenicity in SCID mice was markedly reduced. In vivo assays for angiogenesis revealed that IL-10 inhibits the angiogenic capacity of DG75. The present observations suggest a role for this cytokine in the prevention of angiogenesis in human lymphoid malignancies, possibly even angiostatic effect.
  • the present study sought to analyze and compare the anti- tumor and the anti-angiogenic capacity of human- and viral- IL-10 using a human B-cell Burkitt's lymphoma line constitutively expressing either cytokine. It is shown that both human and viral IL-10 abolish tumorigenicity in SCID mice and evidence is provided that IL-10 exerts its anti-tumor activity by inhibiting angiogenesis in vivo.
  • IL-10 is not limited to one tumor cell type nor to syngeneic, allogeneic, or xenogenic tumor models. See, e.g., Richter, et al . (1993) Cancer Res. 53:4134-4137; Giovarelli, et al. (1995) J. Immunol. 155:3112-3123; Kundu, et al . (1996) J ⁇ Natl. Cancer Inst. 88:536-541; Huang, et al . (1996) Clin. Cancer. Res. 2:1969-1979; and Stearns, et al . (1997) Invassion Metastasis 17:62-74.
  • Angiogenesis is a biological process whereby endothelial cells divide and migrate to form new vessels.
  • microvessel density correlates with the capacity of the tumor to metastasize and is an important predictor of tumor progression. Weidner, et al.(1995) Am. J. Pathol .
  • the IL-10 effects may be multifold in vivo, they can be related to direct inhibition of IL-10 on the angiogenic process, per se, or indirectly, e.g., by affecting the angiogenic capacity or signals from tumor infiltrating cells. There is experimental evidence to support either model. Addition of IL-10 inhibited in vitro the angiogenic capacity of human prostate cancer cells. Stearns, et al. (1999) Clin. Cancer. Res. 5:189-196. The effect was related to stimulation of TIMP-1 and inhibition of MMP-2 and MMP-9 secretion by the tumor cells.
  • IL-10 secreted by the melanoma cells decreased the production of macrophage-derived angiogenic factors such as vascular endothelial growth factor (VEGF) , IL- l ⁇ , TNF-OC, IL-6, and MMP-9. Huang, et al . (1996) Clin. Cancer. Res. 2:1969-1979.
  • VEGF vascular endothelial growth factor
  • vIL-10 shares only some of the properties of its cellular counterpart. vIL-10 lacks T cell, NK cell, and mast cell stimulatory activities, but it inhibits cytokine production and promotes growth of B cells. See Hsu, et al . (1990) Science 350:830-832; Moore, et al . (1990) Science 248:1230-1234; de Waal Malefyt, et al . (1991) J.
  • the tumor incidence was higher in the anti- asialo GM1 treated mice.
  • the differences in tumorigenicity between the vector control and IL-10 sublines were maintained.
  • the antitumor effect of IL-10 was not correlated with the amount of IL-10 produced by the transfected sublines.
  • the amounts of IL-10 produced by the different sublines ranged from 13/730 U/ml/10 6 cells/36 h. These concentrations of IL-10 may then be sufficient to mediate a local antitumor effect, though not a systemic one. Injection of IL-10 transfectants could not inhibit the growth of contralaterally injected mock transfected cells.
  • IL-10 is a potent inhibitor of tumor growth. Furthermore, it is demonstrated in this report, for the first time, that this cytokine has potent angiogenic properties in vivo. Burkitt's lymphoma is considered the fastest growing tumor incidence in children in Equatorial Africa. The possible use of IL-10 in the treatment of this lymphoid tumor deserves consideration. Because of their immunosuppressive properties, human IL-10 (hIL-10) and viral IL-10 (vIL-10) are thought to play a potential pathogenic or therapeutic role in a number of human disease states such as inflammation, autoimmunity, and transplant rejection. See reviews by Ho and Moore (1994) Therapeutic Immunology 1:173-185; Bejarano, et al .
  • transfected with the IL-10 gene were less effective at establishing primary tumors and or metastasis compared to untransfected cells in ⁇ yngeneic and SCID mice.
  • IL-10 systemic administration of IL-10 was shown to inhibit tumor metastasis and stimulate anti-tumor immune response in various murine models. Zheng, et al . (1996) J . Exp . Med . 184:579-584; and Berman, et al . (1996) J. Immunol. 157:231-238. The mechanisms behind these antitumor effects are poorly understood. A role for natural killer (NK) cells (Kundu, et al. (1996) J. Natl. Cancer Inst. 88:536-541; Kundu and Fulton
  • IL-10 was shown to inhibit the metastatic activity of human prostatic adenocarcinoma cell lines via an inhibition of the production of metalloproteases (MMP) and to an increase in the production of tissue inhibitors of metalloproteases (TIMP) .
  • MMP metalloproteases
  • TIMP tissue inhibitors of metalloproteases
  • BCRFl BCRFl
  • vIL-10 (from Epstein Barr Virus) has been described previously in US Pat. No. 5,627,155, and in de Vries and de Waal Malefyt Interleukin-10 Austin TX.
  • Various agonists and antagonists of the natural ligands can be produced.
  • Various mutein agonists and antagonists can be generated, as can antibodies which block the binding of the cytokine to its natural receptor.
  • Mutein agonists will include variants with substitutions at non-essential residues, e.g., away from the receptor interaction regions.
  • Mutein antagonists will include variants which compete with natural vIL-10 to bind to the receptor.
  • Antagonist antibodies may bind to the cytokine ligand, or to the IL-10 receptor, and block vIL-10 binding to the receptor. See, e.g., Ho, et al . (1993) Proc .
  • vIL-10 and variants vIL-10 agonists will exhibit some or all of the signaling functions of the vIL-10, e.g., binding and signal transduction initiation into the appropriate cells.
  • Various IL-10 based sequences may be evaluated to determine what residues are conserved across species, suggesting what residues may be changed without dramatic effects on biological activity.
  • the present invention provides for the use of an antibody or binding composition which specifically binds to vIL-10, and neutralizes the ability of the cytokine to mediate its signal.
  • Antibodies can be raised to various vIL-10 proteins, including individual or strain variants, and fragments thereof, both in their naturally occurring (full-length) forms or in their recombinant forms. Additionally, antibodies can be raised to vIL-10 polypeptides in both their native (or active) forms or in their inactive, e.g., denatured, forms, which may neutralize ligand capacity to mediate its signal. Certain antibodies may block the interaction of the ligand with its receptor. Thus, various antibodies may be generated against the receptor, which antibodies may block vIL-10 binding.
  • a number of immunogens may be selected to produce antibodies specifically reactive, or selective for binding, with vIL-10 proteins.
  • Recombinant protein is a preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein may also be used either in pure or impure form. Synthetic peptides, made using the vIL-10 protein sequences described herein, may also used as an immunogen for the production of antibodies to vIL-10 proteins.
  • Recombinant protein can be expressed and purified in eukaryotic or prokaryotic cells as described, e.g., in Coligan, et al. (eds. 1995 and periodic supplements) Current Protocols in Protein Science John Wiley & Sons, New York, NY; and
  • an immunogen preferably a purified protein
  • animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the antigen of interest.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be performed, if desired. See, e.g., Harlow and Lane Antibodies , A Laboratory Manual; or Coligan (ed. ) Current Protocols in
  • Immunology can also be performed through other methods, e.g., DNA vector immunization. See, e.g., Wang, et al. (1997) Virology 228:278-284.
  • Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art.
  • spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell.
  • Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. See, e.g., Doyle, et al . (eds. 1994 and periodic supplements) Cell and Tissue Culture: Laboratory Procedure . John Wiley and Sons, New York, NY.
  • Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
  • Antibodies or binding compositions, including binding fragments and single chain versions, against predetermined fragments of antigen can be raised by immunization of animals with conjugates of the fragments with carrier proteins as described above.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective vIL-10 protein, or screened for capacity to block vIL-10 mediated signal tranduction. These monoclonal antibodies will usually bind with at least a Kr j of about 1 mM, more usually at least about
  • 300 ⁇ M typically at least about 10 ⁇ M, more typically at least about 30 ⁇ M, preferably at least about 10 ⁇ M, and more preferably at least about 3 ⁇ M or better.
  • mAbs monoclonal antibodies
  • mammalian hosts such as mice, rodents, primates, humans, etc.
  • Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al . (eds.) Basic and Clinical Immunology (4th ed. ) Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.
  • Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see, Cabilly, U.S. Patent No. 4,816,567; and Queen, et al . (1989) Proc . Nat ' 1 Acad. Sci .
  • Antibody binding compounds, including binding fragments, of this invention can have significant diagnostic or therapeutic value. They can be useful as non-neutralizing binding compounds and can be coupled to toxins or radionuclides so that when the binding compound binds to the antigen, a cell expressing it, e.g., on its surface, is killed. Further, these binding compounds can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting. C. Other Molecules
  • Antibodies are merely one form of specific binding compositions.
  • Other binding compositions which will often have similar uses, include molecules that bind with specificity to the receptor, e.g., in a binding partner-binding partner fashion, an antibody-antigen interaction, or in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent , e.g., proteins which specifically associate with IL-10 receptor protein.
  • the molecule may be a polymer, or chemical reagent.
  • a functional analog may be a protein with structural modifications, or may be a structurally unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate binding determinants.
  • Drug screening using antibodies or vIL-10 or fragments thereof can be performed to identify compounds having binding affinity to vIL-10, or can block or simulate the natural interaction with ligand. Subsequent biological assays can then be utilized to determine if the compound has intrinsic blocking activity and is therefore an antagonist. Likewise, a compound having intrinsic stimulating activity can signal to the cells via the vIL-10 pathway and is thus an agonist in that it simulates the activity of a ligand. Mutein antagonists may be developed which maintain receptor binding but lack signaling. Structural studies of the ligands will lead to design of new variants, particularly analogs exhibiting agonist or antagonist properties on the receptor. This can be combined with previously described screening methods to isolate muteins exhibiting desired spectra of activities .
  • receptor specific binding molecules are provided, also included are small molecules identified by screening procedures. In particular, it is well known in the art how to screen for small molecules which interfere, e.g., with ligand binding to the receptor, often by specific binding to the receptor and blocking of binding by natural ligand. See, e.g., meetings on High Throughput Screening, International Business Communications, Southborough, MA 01772-1749. Such molecules may compete with natural ligands, and selectively bind to vIL- 10. III . Uses
  • IL-10 IL-10
  • cytokines may also have antimetastatic effects, preventing dispersion of the neoplastic cells .
  • Effects on various cell types may be indirect, as well as direct.
  • a statistically significant change in the parameters, e.g., numbers of cells, will typically be affected by at least about 10%, preferably 20%, 30%, 50%, 70%, 90%, or more. Effects of greater than 100%, e.g., 130%, 150%, 2X, 3X, 5X, etc., will often be desired.
  • the effects may be specific in causing angiogenesis, tumorigenesis, or metastasis, according to objective measures generally recognized in the art.
  • the present invention is useful in the treatment of medical conditions or diseases, e.g., associated with neoplastic disease. See, e.g., Bertino, et al . (eds. 1996) Encyclopedia of Cancer Academic Press; Devita, et al . (eds. 1997) Cancer: Principles & Practice of Oncology Lippincott, Williams and Wilkins; Devita (1997) Principles and Practice of Oncology Lippincott Williams and Wilkins; Cavalli, et al . (1996) Textbook of Medical Oncology Dunitz Martin Ltd; Horwich (ed. 1995) Oncology: A Multidisciplinary Textbook Lippincott- Raven; Peckham, et al . (eds. 1995) Oxford Textbook of Oncology Oxford Univ.
  • agonists or antagonists described may be combined with other treatments of the medical conditions described herein, e.g., an angiogenic or angiostatic reagent, immune suppressive therapeutic, immune adjuvant, analgesic, anti-inflammatory drug, growth factor, cytokine, vasodilator, or vasoconstrictor, depending upon the desired response.
  • Preferred combination therapies include the vIL-10 reagent with various anti-inflammatory agents, such as systemic steroids or corticosteroids .
  • Metastasis is a multistep process involving numerous tumor cell-host cell and cell-matrix associations, a number of processes. Typically, it requires escape from local origin by the neoplastic cells, often across tissue barriers. The cells become mobile, and extravasate, and invade a remote secondary site to proliferate and establishment a viable tumor mass. Such typically requires the establishment of vascularization with small capillaries to feed the growing cell mass. Thus, angiogenesis both serves to allow the neoplastic cells to escape the local environment, and once established, to grow. Thus, anti-angiogenic functions, e.g., angiostasis, may be a useful component in the minimization of growth and metastasis of a tumor.
  • anti-angiogenic functions e.g., angiostasis
  • the vIL-10 ligands would be expected to signal specifically to the cell types expressing the receptor.
  • compositions including vIL-10 the cytokine is admixed with a pharmaceutically acceptable carrier or excipient which is preferably inert.
  • a pharmaceutically acceptable carrier or excipient which is preferably inert.
  • Preparation of such pharmaceutical compositions is known in the art, see, e.g., Remington ' s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984) .
  • vIL-10 antagonist compositions can be prepared.
  • Agonists, e.g., natural ligand, or antagonists, e.g., antibodies or binding compositions are normally administered parenterally, preferably intravenously.
  • protein or peptide antagonists may be immunogenic they are preferably administered slowly, either by a conventional IV administration set or from a subcutaneous depot, e.g. as taught by Toma ⁇ i, et al., U.S. patent 4,732,863.
  • This invention further would encompass expression of recombinant DNA molecules and fragments having a DNA sequence identical to or highly homologous to the vIL-10 described herein.
  • the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA replication.
  • recombinant clones derived from the genomic sequences e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt (ed.)
  • expression may be effected by operably linking a coding segment to a heterologous promoter, e.g., by inserting a promoter upstream from an endogenous gene. See, e.g., Treco, et al . W096/29411 or USSN 08/406, 030.
  • the therapeutics When administered parenterally the therapeutics will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently nontoxic and nontherapeutic .
  • the antagonist may be administered in aqueous vehicles such as water, saline or buffered vehicles with or without various additives and/or diluting agents.
  • a suspension such as a zinc suspension, can be prepared to include the peptide .
  • a suspension can be useful for subcutaneous (SQ) , intradermal (ID) , or intramuscular (IM) injection.
  • SQ subcutaneous
  • ID intradermal
  • IM intramuscular
  • the proportion of therapeutic entity and additive can be varied over a broad range so long as both are present in effective amounts.
  • the therapeutic is preferably formulated in purified form substantially free of aggregates, other proteins, endotoxins, and the like, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20 mg/ml.
  • the endotoxin levels are less than 2.5 EU/ml .
  • an administration regimen for a therapeutic agonist or antagonist depends on several factors, including the serum or tissue turnover rate of the therapeutic, the immunogenicity of the therapeutic, or the accessibility of the target cells.
  • an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of therapeutic delivered depends in part on the particular agonist or antagonist and the severity of the condition being treated.
  • Guidance in selecting appropriate doses of antibodies is found in the literature on therapeutic uses, e.g. Bach et al . , chapter 22, in Ferrone, et al . (eds. 1985) Handbook of Monoclonal Antibodies Noges Publications, Park Ridge, NJ; and Russell, pgs . 303-357, and Smith et al . , pgs . 365-389, in Haber, et al . (eds. 1977) Antibodies in Human Diagnosis and Therapy Raven Press, New York, NY.
  • Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known in the art to affect treatment or predicted to affect treatment.
  • the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • an antibody or binding composition thereof that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing a humoral response to the reagent.
  • the total weekly dose ranges for antibodies or fragments thereof, which specifically bind to vIL-10 range generally from about 1 ng, more generally from about 10 ng, typically from about 100 ng; more typically from about 1 ⁇ g, more typically from about 10 ⁇ g, preferably from about 100 ⁇ g, and more preferably from about 1 mg per kilogram body weight. Although higher amounts may be more efficacious, the lower doses typically will have fewer adverse effects. Generally the range will be less than 100 mg, preferably less than about 50 mg, and more preferably less than about 25 mg per kilogram body weight .
  • the weekly dose ranges for antagonists range from about 10 ⁇ g, preferably at least about 50 ⁇ g, and more preferably at least about 100 ⁇ g per kilogram of body weight. Generally, the range will be less than about 1000 ⁇ g, preferably less than about 500 ⁇ g, and more preferably less than about 100 ⁇ g per kilogram of body weight. Dosages are on a schedule which effects the desired treatment and can be periodic over shorter or longer term. In general, ranges will be from at least about 10 ⁇ g to about 50 mg, preferably about 100 ⁇ g to about 10 mg per kilogram body weight, though local administration may be generally preferred.
  • Hourly dose ranges for muteins range from at least about 10 ⁇ g, generally at least about 50 ⁇ g, typically at least about 100 ⁇ g, and preferably at least 500 ⁇ g per hour. Generally the dosage will be less than about 100 mg, typically less than about 30 mg, preferably less than about 10 mg, and more preferably less than about 6 mg per hour. General ranges will be from at least about 1 ⁇ g to about 1000 ⁇ g, preferably about 10 ⁇ g to about 500 ⁇ g per hour.
  • certain tumor locations or types may involve the administration of the therapeutics in different forms.
  • genetic transformation of cells may be achieved to express the desired vIL-10 proteins at the correct local environment.
  • the present invention also provides for administration of vIL-10 compositions in combination with known therapies, e.g., steroids, particularly glucocorticoids , which alleviate the symptoms associated with excessive inflammatory responses.
  • Daily dosages for glucocorticoids will range from at least about 1 mg, generally at least about 2 mg, and preferably at least about 5 mg per day. Generally, the dosage will be less than about 100 mg, typically less than about 50 mg, preferably less than about 20 mg, and more preferably at least about 10 mg per day. In general, the ranges will be from at least about 1 mg to about 100 mg, preferably from about 2 mg to 50 mg per day.
  • an effective amount means an amount sufficient to effect a desired response, or to ameliorate a symptom or sign of the neoplastic condition.
  • Typical mammalian hosts will include mice, rats, cats, dogs, and primates, including humans.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route, and dose of administration and the severity of side affects.
  • the effect will result in a change in quantitation of at least about 10%, preferably at least 20%, 30%, 50%, 70%, or even 90% or more.
  • an effective amount is in ratio to a combination of components and the effect is not limited to individual components alone.
  • an effective amount of therapeutic will modulate the symptoms typically by at least about 10%; usually by at least about 20%; preferably at least about 30%; or more preferably at least about 50%.
  • modulation of angiogenesis will mean that the vascularization of, e.g., the cellular mass, is affected. Such will result in, e.g., statistically significant and quantifiable changes in the vascularization, tumor growth, or metastasis formation. Preferably, this will prevent any angiogenesis, e.g., achieve an angiostatic condition.
  • the present invention provides reagents which will find use in therapeutic applications as described elsewhere herein, e.g., in the general description for treating disorders associated with neoplastic conditions. See, e.g., Berkow (ed. ) The Merck Manual of Diagnosis and Therapy, Merck & Co . , Rahway, N.J.; Thorn, et al . Harrison's Principles of Internal Medicine, McGraw-Hill, NY; Gilman, et al . (eds. 1990) Goodman and Gilman' s: The Pharmacological Bases of Therapeutics, 8th Ed. , Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed.
  • Antibodies to vIL-10 or receptor proteins may be used for the identification or sorting of cell populations expressing cytokine or receptor protein, e.g., specific tumor cells. Methods to sort such populations are well known in the art, see, e.g., Melamed, et al . (1990) Flow Cvtometry and Sorting Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical Flow Cvtometry Liss, New York, NY; and Robinson, et al .
  • antisense against the ligand encoding nucleic acids may function in a manner like ligand antagonists, and antisense against the receptor may function like receptor antagonists. Thus, it may be possible to block the signaling through the pathway with antisense nucleic acids.
  • nucleic acids for the receptor may serve as agonists, increasing the numbers of receptor on the cell, thereby increasing cell sensitivity to ligand, and perhaps blocking the normal apoptotic signal described.
  • the antibodies or binding compositions are useful in diagnosing diseases states which may be responsive to the methods provided.
  • Antibodies raised against a vIL-10 protein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens. Combinations of these signals may be also pursued.
  • Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis , centrifugation, crystallization, and others. See, e.g., Ausubel, et al . (1987 and periodic supplements) ; Deutscher (1990) "Guide to Protein Purification” in Methods in Enzvmology, vol. 182, and other volumes in this series; manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA; and Coligan, et al . (eds.) (1995 and periodic supplements) Current Protocols in Protein Science, John Wiley & Sons, New York, NY.
  • Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
  • appropriate segments e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
  • mice were bred and maintained in pathogen limited conditions at the Microbiology and Tumor Biology Center (MTC) , Karolinska Institutet. Four to six weeks old mice, usually littermates, or otherwise age matched within two weeks were used in all experiments.
  • MTC Microbiology and Tumor Biology Center
  • DG75 is an EBV negative BL line (Ben-Bassat, et al . (1977) Int. J. Cancer 19:27-33).
  • the hIL-10 and vIL-10 cDNAs were excised from the pCDSR 296 modified expression vector (Takebe, et al. (1988) Mol . Cell. Biol . 8:466-472) and subcloned in the pcDNA3 Neo vector (Invitrogen) .
  • Plasmid control pcDNA3 Neo
  • plasmids containing the h- or v-IL-10 cDNAs were introduced into tumor cells by electroporation using a Gene-Pulser (BioRad Laboratories, Richmond, CA) under the following conditions: 5 x 10 cells in 0.5 ml Dulbecco's phosphate buffered saline (without Ca 2+ and Mg 2+ ) were pulsed with 875 V/cm, and 975 ⁇ F . Transfectants were selected in G418 (750 ⁇ g/ml; Gibco, BRL) for establishment of lines and clones.
  • Cells were maintained in RPMI 1640 (Gibco, BRL) supplemented with 10% heat inactivated fetal calf serum, 100 mg/ml of streptomycin, 100 U/ml Penicillin and 750 ⁇ g/ml G418 (referred as complete medium) . They were cultured at 37° C/5% C ⁇ 2 ⁇
  • IL-10 production by the clones and sublines was determined by measurement of IL-10 released to the medium by specific ELISA (capture clone JES3-9D7, biotinylated detection clone JES3-12G8, PharMingen, San Diego, CA) .
  • the sensitivity of the detection for IL-10 ELISA was 20 to 40 pg/ml . Results are expressed as units of IL-10/ 1 x 10 cells/ml, per 36 h. Phenotypical analysis.
  • Anti CD23, -CD40, -CD74, -CD86, -CD95 PE or FITC conjugated anti- HLA ABC, -DR (clone L243), CDlla, - CD54, -CD58, -CD80 antibodies were purchased from Becton Dickinson (Mountain View, CA) .
  • FITC conjugated goat anti-mouse antibody was purchased from DAKO (Denmark) .
  • Murine anti-IL-10 R Ab was provided by DNAX Research Institute, Palo Alto, CA. Cells (10 ) were labeled with FITC or PE conjugated antibodies or isotype matched controls for 30 min on ice.
  • Tumorigenicity in SCID mice n Tumor cells (10 ) were inoculated subcutaneously in the right flank in a 0.2 ml volume of PBS. Tumor size was determined at least once weekly by palpations and measurements of the tumor. Mice were sacrificed when the tumors reached a size of > 15 mm in diameter. Mice without any signs of tumor growth were kept under observation for at least six weeks after inoculation. Small groups of mice, never exceeding five mice per group, were tested in several independent experiments throughout the study to minimize random fluctuations in the quantity or quality of cells inoculated. Results from several independent tests were pooled.
  • Human lung fibroblasts were grown to confluence on 35 x 10 mm Petri dishes (Lux tissue culture dishes with 2 mm grids, Lab Tek, Miles, McLean, VA) and irradiated with 6,000 rads .
  • One ml of 0.45% w/w agarose (Sea-plaque agarose, Marine colloids, FMC, Rockland, ME) was heated and diluted in RPMI-1640 supplemented with 15% FCS and added to the plates.
  • the underlayer had solidified (1 h, RT) three ml of a 0.35% w/w agarose solution in complete media were mixed with the cells at 40° C and poured on top of the agarose underlayer.
  • Corneal assays were performed in New Zealand white rabbits (Charles River, Calco, Como, Italy) , as described (Ziche, et al. (1982) J. Natl. Cancer. Inst. 69:475-482), and in accordance with the guidelines of the European Economic Community for animal care and welfare (EEC Law No. 86/609) . Briefly, cell suspensions (in a volume of 5 ⁇ l) were implanted into surgically produced corneal micropockets . The angiogenic response was measured daily with a slit lamp stereo microscope and capillary progression scored as previously reported. Ziche, et al . (1994) J. Clin. Invest. 94:2036-2044.
  • angiogenic score was calculated on the basis of the number and growth rate of newly formed capillaries calculated as: (vessel density x distance from limbus) . See Ziche, et al . (1994) J. Clin. Invest. 94:2036-2044. Corneas were removed at the end of the experiment as well as at defined intervals after surgery and/or treatment and fixed in formalin for histological examination.
  • DG75 sublines were implanted in the corneal micropocket according to the following protocol: a) angiogenic activity of equal number of cells of each sublime (IX DG75- vector cells, DG75-hIL-10 cells, DG75-vIL-10 cells); b) angiogenic activity of 2X DG75-vector cells; c) angiogenic activity of IX DG75-Vector cells + IX DG75 IL-10 transfectants .
  • IL-10 prevents tumor growth of DG75 sublines
  • BL Burkitt's lymphoma
  • This EBV negative B cell line was chosen for these studies because it has been shown previously that these cells did not express or secrete endogenous IL-10. Finke, et al . (1993) Leukemia 7:1852-1857. Lines and clones expressing IL-10 activity ranging from 13-730 U IL-10/10 6 cells/ml/36 h were isolated. IL-10 could not be detected in conditioned media from parental (referred to as wild type (WT) ) , and mock transfected DG75 (referred as vector control) lines. DG75 is tumorigenic following subcutaneous (SC) implantation into SCID mice. Ben-Bassat, et al . (1977)
  • mice (12/16) given injections of WT cells developed a tumor after three weeks.
  • the vector control cells developed tumors with similar incidence and kinetics as the WT cells, 67% of the mice (12/18) .
  • the tumor incidence was markedly reduced for all the IL-10 expressing lines.
  • the proportion of animals developing tumors was 9% (4/42) and 16% (6/38) for the h- and v- IL-10 transfectants, respectively.
  • no direct correlation was found between the tumorigenicity of the sublines and the production of IL-10, the few tumors that developed from h- or v- IL-10 transfectants came from clones that produced low, intermediate, and high levels of IL-10.
  • hIL-10 sublime and vector control DG75 cells were either mixed before injection or injected contralaterally and tumor growth was evaluated.
  • Coinjection of both cell types at the same site resulted in suppression of tumor growth of vector control cells (0/5 animals developed tumor) .
  • the hIL-10 sublime was unable to suppress the growth of contralaterally injected vector control cells, 60% (3/5) of the mice developed tumors at the site of DG75 vector control injection.
  • V. IL-10 does not affect the growth behavior in vitro
  • IL-10 is known to affect the proliferation and autonomous growth of human Epstein-Barr virus (EBV) - transformed B cells and melanoma cell lines.
  • EBV Epstein-Barr virus
  • IL-10 has been shown to downregulate the expression of MHC and costimulatory molecules.
  • Zeidler, et al . (1997) Blood 90:2390-2397; Yun Yue, et al . (1997) Int. J. Cancer 71:630-637; Salazar-Onfray, et al . (1997) J. Immunol. 159:3195-3202; Petersson, et al . (1998) J . Immunol . 161:2099-2105; and Tsuruma, et al . (1998) Cell. Immunol. 184:121-128.
  • the decreased MHC class I expression has been correlated to an increase in NK sensitivity and decrease in the metastatic potential.
  • Kundu, et al . (1996) J. Natl. Cancer Inst. 88:536-541; Zheng, et al . (1996) J . Exp . Med. 184:579-584; Zheng, et al . (1996) J. Exp. Med. 184:579- 584; and Kundu and Fulton (1997) Cell. Immunol. 180:55-61.
  • IL-10 expression did not correlate with the levels of expression of either MHC class I/II or costimulatory molecules.
  • IL-10 did not affect the expression of CD95.
  • NK cells have been shown previously to play a role in the antitumor effects of IL-10. Zheng, et al . (1996) J . Exp . Med . 184:579-584; Kundu, et al . (1996) J. Natl. Cancer Inst. 88:536- 541; and Kundu and Fulton (1997) Cell . Immunol . 180:55-61. Therefore, the tumorigenicity of the IL-10 transfected sublines were tested in SCID mice pre-treated with asialo GMl antibody to suppress NK activity. When DG75 vector cells were injected into NK-depleted mice, all mice (8/8) developed tumors two weeks after inoculation.
  • IL-10 transfectants Although the incidence of tumor formation induced by IL-10 transfectants increased when compared to untreated mice, from 10 to 33% (4/12) for hIL-10, and from 16 to 62% (5/8) for vIL-10, the differences in tumorigenicity between the vector and IL-10 transfectants were still maintained. In addition, the DG75 sublines were resistant to spontaneous or Tilorone-boosted NK activity from SCID mice in vitro.
  • IL-10 inhibits angiogenesis in vivo
  • DG75 vector, hIL-10 and vIL-10 lines were implanted into rabbit corneal micropockets for evaluation of their angiogenic activity.
  • angiogenesis was observed in 2/2 and 4/5 implants respectively.
  • Angiogenesis was not induced by the either hIL-10 (0/5) or vIL- 10 (0/3) implants. Because in the tumorigenicity experiments the hIL-10 sublime inhibited the growth of the vector DG75 when admixed, whether h- or v- IL-10 could suppress the angiogenic capacity of the DG75 vector when coinjected was tested. Th s, equal numbers (2,5 x 10 ) of DG75 vector control and hIL-10 or vIL-10 sublines were mixed and implanted. Coinjection of hlL- 10 or vIL-10 transfectants inhibited in all cases (0/4 for hlL- 10 and 0/3 for vIL-10 implants) the angiogenic capacity of the vector control line. Noteworthy, implants of vIL-10, whether alone or mixed with the vector, induced a marked inflammatory response .
  • vIL-10 Various antagonists of vIL-10 are made available.
  • antibodies against the cytokine itself may block the binding of ligand to its receptor, thereby serving as a direct receptor antagonist.
  • Other antagonists may function by blocking the binding of ligand to receptor, e.g., by binding to the ligand in a way to preclude the possibility of binding to the receptor.
  • Other antagonists e.g., mutein antagonists, may bind to the receptor without signaling, thereby blocking a true agonist from binding.
  • Standard mutagenesis analysis is performed, e.g., by generating many different variants at determined positions, e.g., at the positions identified above, and evaluating biological activities of the variants. This may be performed to the extent of determining positions which modify activity, or to focus on specific positions to determine the residues which can be substituted to either retain, block, or modulate biological activity.
  • analysis of natural variants can indicate what positions tolerate natural mutations. This may result from populational analysis of variation among individuals, or across strains or species. Samples from selected individuals are analyzed, e.g., by PCR analysis and sequencing. This allows evaluation of population polymorphisms. All citations herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art.

Abstract

Various methods of use of viral IL-10s are provided. In particular, the method makes use of fact that viral IL-10 is capable of inhibiting angiogenesis, tumorigenicity, and metastasis.

Description

VIRAL IL-10 USES
FIELD OF THE INVENTION
The invention relates generally to methods of using specific cytokines, more particularly, to methods of treating proliferative diseases or conditions.
BACKGROUND The immune system consists of a wide range of distinct cell types, each with important roles to play. See Paul (ed. 1997) Fundamental Immunology 4th ed. , Raven Press, New York. The lymphocytes occupy central stage because they are the cells that determine the specificity of immunity, and it is their response that orchestrates the effector limbs of the immune system. Two broad classes of lymphocytes are recognized: the B lymphocytes, which are precursors of antibody secreting cells, and the T ( thymus-dependent ) lymphocytes. T lymphocytes express important regulatory functions, such as the ability to help or inhibit the development of specific types of immune response, including antibody production and increased microbicidal activity of macrophages . Other T lymphocytes are involved in direct effector functions, such as the lysis of virus infected-cells or certain neoplastic cells.
Lymphokines apparently mediate cellular activities in a variety of ways . They have been shown to support the proliferation, growth, and differentiation of pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. Proper and balanced interactions between the cellular components are necessary for a healthy immune response. The different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents .
Cell lineages especially important to the immune response include two classes of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal) , and T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T-cells) making up the immune network. These lymphocytes interact with many other cell types.
Another important cell lineage is the mast cell (which has not been positively identified in all mammalian species), which is a granule-containing connective tissue cell located proximal to capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows: when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators, e.g., histamine, serotonin, heparin, and prostaglandins, which cause allergic reactions, e.g., anaphylaxis.
Research to better understand and treat various clinical disorders has been hampered by the general inability to maintain cells of the immune system in vitro. Immunologists have discovered that culturing these cells can be accomplished through the use of T-cell and other cell supernatants , which contain various growth factors, including many of the lymphokines . The gene encoding IL-10, originally designated Cytokine
Synthesis Inhibitory Factor (CSIF) , was isolated in the 1980 's. See, e.g., Mosmann, et al . , U.S. Patent No. 5,231,012. A viral counterpart, known as viral IL-10 from EBV has also been described. Much has been learned of the biology and physiology mediated by these cytokines. See, e.g., de Vries and de Waal Malefyt (1995) Interleukin-10 Landes Co., Austin, TX.
However, the discovery of new biological activities for these cytokines can contribute to new therapies for a wide range of degenerative or abnormal conditions, e.g., which directly or indirectly involve the immune system and/or hematopoietic cells. In particular, the discovery and development of new clinical targets for use of these lymphokines would be highly advantageous . The present invention provides new methods for their use.
SUMMARY OF THE INVENTION The present invention is based, in part, upon the surprising discovery that the cytokines IL-10 and BCRF1 (also known as viral IL-10, or vIL-10), have anti-angiogenic , anti- tumorigenic, and anti-metastatic activities.
The present invention provides methods of inhibiting angiogenesis to a growing cellular mass comprising administering an effective amount of viral IL-10 to the location of the mass. Preferably, the cellular mass is a tumor; the tumor is: highly vascularized, a lymphoid tumor, Burkitt's lymphoma, Kaposi sarcoma, osteosarcoma, mammary adenocarcinoma, malignant melanoma, or prostatic adenocarcinoma . The administering will often be local, topical, subcutaneous, intradermal, or tranεdermal, or, in certain embodiments, by expression of a nucleic acid encoding the viral IL-10. The administering will often be in combination with another anti-neoplastic therapeutic and/or an anti-inflammatory agent.
In preferred embodiments, the effective amount further results in a decrease in tumor metastasis or angiostasis, or in inhibiting tumorigenesis . The invention also provides methods of inhibiting tumorigenesis of a growing cellular mass comprising administering an effective amount of viral IL-10 to the location of the mass. For example, the cellular mass will often be a tumor, e.g., which is highly vascularized, or a lymphoid tumor. Preferred tumors will be Burkitt's lymphoma,
Kaposi sarcoma, osteosarcoma, mammary adenocarcinoma, malignant melanoma, or prostatic adenocarcinoma. The administering will preferably be local, topical, subcutaneous, intradermal, or transdermal ; or by expression of a nucleic acid encoding the viral IL-10. Often, the administering is in combination with: another anti-neoplastic therapeutic agent; or an anti- inflammatory agent; and will preferably further result in inhibiting tumorigenesis. The invention further provides methods of inhibiting metastasis of a cellular mass comprising administering an effective amount of viral IL-10 to the host.
DETAILED DESCRIPTION OF THE INVENTION
I . General
The invention is based, in part, on the surprising discovery that the cytokine BCRFl, also known as viral IL-10 (vIL-10), has anti-angiogenic, anti-tumorigenie, and anti- metastatic activities. The cytokines viral IL-10 (also known as BCRFl) and IL-10 have been described earlier in, e.g., de Vries and de Waal Malefyt Interleukin-10 Landes Company, Austin TX; US Pat. No. 5,627,155; and US Pat. No. 5,231,012, which are incorporated herein by reference for all purposes. Additional viral forms of IL-10 also exist, which may have some of the same anti-tumor activities. See Rode, et al . (1993) Virus Genes 7:111-116; and Fleming, et al . (1997) J. Virol. 71:4857- 4861.
Observations herein directed to the biology of these cytokines show that both viral IL-10 and cellular IL-10 exhibit anti-tumor effects. The gene and protein sequences are available from the GenBank/EMBL/DDBJ databases, e.g., under the accession numbers X83413 (gene U58; BCRFl; viral IL-10); X78437, U16720 (human IL-10); and M84340, M37897 (mouse IL-10). It is particularly surprising that vIL-10 has the same activities as the cellular form.
Human and viral IL-10 genes were introduced into the human Burkitt's lymphoma line DG75. Stable transfectants were monitored for tumorigenicity in SCID mice and angiogenic capacity. The two cytokines displayed concordant effects and no differences were found in the in vitro growth capacity, agarose clonability or NK-sensitivity, but the tumorigenicity in SCID mice was markedly reduced. In vivo assays for angiogenesis revealed that IL-10 inhibits the angiogenic capacity of DG75. The present observations suggest a role for this cytokine in the prevention of angiogenesis in human lymphoid malignancies, possibly even angiostatic effect. The present study sought to analyze and compare the anti- tumor and the anti-angiogenic capacity of human- and viral- IL-10 using a human B-cell Burkitt's lymphoma line constitutively expressing either cytokine. It is shown that both human and viral IL-10 abolish tumorigenicity in SCID mice and evidence is provided that IL-10 exerts its anti-tumor activity by inhibiting angiogenesis in vivo.
Constitutive expression of IL-10 in a Burkitt's lymphoma line significantly suppresses its tumorigenic potential in SCID mice. These observations are related to a direct antiangiogenic effect in vivo.
Several recent studies in murine models have shown that introduction of the IL-10 gene into tumor cells results in decreased tumorigenicity and increased mouse survival . The efficacy of IL-10 is not limited to one tumor cell type nor to syngeneic, allogeneic, or xenogenic tumor models. See, e.g., Richter, et al . (1993) Cancer Res. 53:4134-4137; Giovarelli, et al. (1995) J. Immunol. 155:3112-3123; Kundu, et al . (1996) J^ Natl. Cancer Inst. 88:536-541; Huang, et al . (1996) Clin. Cancer. Res. 2:1969-1979; and Stearns, et al . (1997) Invassion Metastasis 17:62-74.
Besides confirming previous results, they have been extended to show that experimental angiogenesis is inhibited in vivo by the IL-10 transfected lines. Angiogenesis is a biological process whereby endothelial cells divide and migrate to form new vessels. Folkman (1972) Ann . Sur . 175:409-416. This process is required in physiological conditions, but is also a necessary requirement for solid tumors to grow and metastasize. Folkman (1972) Ann . Surg . 175:409-416; and Folkman (1995) Nat. Med. 27:27-29. Thus, microvessel density correlates with the capacity of the tumor to metastasize and is an important predictor of tumor progression. Weidner, et al.(1995) Am. J. Pathol . 147:9-19. Using the rabbit corneal assay for angiogenesis, it is demonstrated that the angiogenic capacity of the DG75 line is lost after transfection with either hIL-10 or vIL-10, and that the IL-10 transfectants inhibit the angiogenic capacity of the vector control line when coinjected. It has been suggested that the antitumor effects of IL-10 are related to vascular damage (Di Carlo, et al . (1998) Eur . Cytokine. Netw. 9:61-68), and inhibition of angiogenesis (Richter, et al . (1993) Cancer Res. 53:4134-4137; Huang, et al . (1996) Clin. Cancer. Res. 2:1969-1979; and Stearns, et al .
(1999) Clin. Cancer. Res. 5:189-196). The IL-10 effects may be multifold in vivo, they can be related to direct inhibition of IL-10 on the angiogenic process, per se, or indirectly, e.g., by affecting the angiogenic capacity or signals from tumor infiltrating cells. There is experimental evidence to support either model. Addition of IL-10 inhibited in vitro the angiogenic capacity of human prostate cancer cells. Stearns, et al. (1999) Clin. Cancer. Res. 5:189-196. The effect was related to stimulation of TIMP-1 and inhibition of MMP-2 and MMP-9 secretion by the tumor cells. Richter and collaborators observed that IL-10 transfected CHO tumor cells had a significant reduced macrophage infiltration compared to that seen in parental tumors. Richter, et al . (1993) Cancer Res. 53:4134-4137. Those authors proposed that macrophages may provide tumor growth promoting activity in the form of angiogenic factors that are inhibited by IL-10. This assumption was later corroborated in another tumor model . Human melanoma cells transfected with murine IL-10 exhibited reduced growth and metastatic abilities which correlated with a decreased neovascularization of the tumors. In vitro studies demonstrated that IL-10 secreted by the melanoma cells decreased the production of macrophage-derived angiogenic factors such as vascular endothelial growth factor (VEGF) , IL- lβ, TNF-OC, IL-6, and MMP-9. Huang, et al . (1996) Clin. Cancer. Res. 2:1969-1979.
In murine mammary tumors, the antimetastatic and antitumor activity resulting from IL-10 gene transfer was related to enhanced production of nitric oxide (NO). Kundu, et al . (1998) Int. J. Cancer 76:713-719. However, the biological significance of NO in tumor growth and angiogenesis is controversial. Transplant of tumor samples and cell lines derived from squamous cell carcinoma induced angiogenesis in vivo which was linked to NO production, it regressed by treatment with NO synthase (NOS) inhibitors. Gallo, et al . (1998) J. Nat'l Cancer Inst. 80:587-596. It was reported recently that NO mediates angiogenesis promoted by substance P and that it controls angiogenesis by modulating the activity of VEGF released by the tumor cells. Ziche, et al . (1994) J.
Clin. Invest. 94:2036-2044; and Ziche, et al . (1997) J. Clin. Invest . 99:2625-2634. In the present study, the fact that coinjection of IL-10 transfectants inhibited the angiogenicity and tumorigenicity of the mock transfected line suggests that IL-10 may inhibit the production of angiogenic factors from the tumor. However, since the IL-10 transfectants were not angiogenic when injected alone, the possibility cannot be excluded that IL-10 affects as well angiogenic signals from surrounding cells, or that it acts directly on the endothelial cells. In fact, IL-10 was shown to inhibit TNF-oc, IL-lβ, and
LPS-induced expression of IL-6 and IL-8 in human umbilical vein endothelial cells. Chen and Manning (1996) Cytokine 8:58-65. Moreover, histological analysis of tumors from IL-10 transfected adenocarcinoma cells revealed increased ELAM-1 and MCP-1 production by endothelial cells. Di Carlo, et al . (1998) Eur. Cytokine. Netw. 9:61-68.
Despite the extensive homology between vIL-10 (identified as an EBV-encoded open reading frame (BCRF-1) and the hIL-10 gene (Vieira, et al . (1991) Proc . Natl. Acad. Sci . USA 88:1172- 1176) , vIL-10 shares only some of the properties of its cellular counterpart. vIL-10 lacks T cell, NK cell, and mast cell stimulatory activities, but it inhibits cytokine production and promotes growth of B cells. See Hsu, et al . (1990) Science 350:830-832; Moore, et al . (1990) Science 248:1230-1234; de Waal Malefyt, et al . (1991) J. Exp. Med. 174:915-924; and Bejarano and Masucci (1998) Blood 92:4256- 4262. It was observed that implantation of vIL-10, but not of hIL-10 DG75 sublime, whether alone or in combination with the DG75 vector control line, induced a marked inflammatory response. This finding suggests that hIL-10 and vIL-10 may also differ with regard to their anti-inflammatory properties, and could differ with respect to the anti-angiogenic and antitumor properties . In a recent report, Yao, et al . (1999) Blood 93:1612-1621, demonstrated that NK cells contribute to the anti-angiogenic effect of IL-12, and that they can kill endothelial cells. In the present report, the tumor incidence was higher in the anti- asialo GM1 treated mice. However, the differences in tumorigenicity between the vector control and IL-10 sublines were maintained. These results suggest that the IL-10 effect is not dependent on NK cells despite the fact that NK cells play a role in the antitumor response. In this study, the antitumor effect of IL-10 was not correlated with the amount of IL-10 produced by the transfected sublines. The amounts of IL-10 produced by the different sublines ranged from 13/730 U/ml/106 cells/36 h. These concentrations of IL-10 may then be sufficient to mediate a local antitumor effect, though not a systemic one. Injection of IL-10 transfectants could not inhibit the growth of contralaterally injected mock transfected cells.
In conclusion, it is here shown that IL-10 is a potent inhibitor of tumor growth. Furthermore, it is demonstrated in this report, for the first time, that this cytokine has potent angiogenic properties in vivo. Burkitt's lymphoma is considered the fastest growing tumor incidence in children in Equatorial Africa. The possible use of IL-10 in the treatment of this lymphoid tumor deserves consideration. Because of their immunosuppressive properties, human IL-10 (hIL-10) and viral IL-10 (vIL-10) are thought to play a potential pathogenic or therapeutic role in a number of human disease states such as inflammation, autoimmunity, and transplant rejection. See reviews by Ho and Moore (1994) Therapeutic Immunology 1:173-185; Bejarano, et al . (1992) Int . Immunol . 4:1389-1397; Katsikis, et al . (1994) J. Exp. Med. 179:1517-1527; and Pajkrt, et al . (1997) J. Immunol. 158:3971- 3977. Additionally, the immunomodulatory effects of IL-10 have been analyzed in various tumor systems. A role for IL-10 as a tumor escape mediator has been suggested as many tumors or tumor infiltrating lymphocytes express IL-10. Studies have shown that it inhibits the tumoricidal capacity of macrophages (Nabioullin, et al . (1994) J. Leukoc. Biol. 55:437-442; and Kambayashi, et al . (1995) J.
Immunol . 154:3383-3390), inhibits cytotoxicity and cytokine production by tumor specific cells (Rohrer, et al . (1995) J .
Immunol . 155:5719-5927), and blocks the presentation of tumor antigens by antigen presenting cells (APCs; Beissert, et al . (1995) J. Immunol. 154:1280-1286; and Qin, et al . (1997) J.
Immunol . 159:770-776).
However, in vivo studies of tumor rejection have produced the opposite results. Chinese hamster ovary (CHO) cells (Richter, et al . (1993) Cancer Res. 53:4134-4137), mammary adenocarcinoma cells (Giovarelli, et al . (1995) J. Immunol.
155:3112-3123; and Kundu, et al . (1996) J. Nat ' 1 Cancer Inst.
88:536-541), and melanoma cells (Zheng, et al . (1996) J . Exp .
Med. 184:579-584) transfected with the IL-10 gene were less effective at establishing primary tumors and or metastasis compared to untransfected cells in εyngeneic and SCID mice.
Moreover, systemic administration of IL-10 was shown to inhibit tumor metastasis and stimulate anti-tumor immune response in various murine models. Zheng, et al . (1996) J . Exp . Med . 184:579-584; and Berman, et al . (1996) J. Immunol. 157:231-238. The mechanisms behind these antitumor effects are poorly understood. A role for natural killer (NK) cells (Kundu, et al. (1996) J. Natl. Cancer Inst. 88:536-541; Kundu and Fulton
(1997) Cell. Immunol. 180:55-61; and Zheng, et al . (1996) J Exp. Med. 184:579-584), T cells (Berman, et al . (1996) J.
Immunol . 157:231-238; and Kundu, et al . (1996) J. Natl. Cancer Inst. 88:536-541), macrophages (Di Carlo, et al . (1998) Eur . Cytokine. Netw. 9:61-68), and nitric oxide (Kundu, et al .
(1998) Int. J. Cancer 76:713-719) have each been implicated. In addition, it has been proposed that the antitumor effects of IL-10 may be related to vascular damage (Di Carlo, et al . (1998) Eur. Cytokine. Netw. 9:61-68) or inhibition of angiogenesis (Richter, et al . (1993) Cancer Res. 53:4134-4137; and Huang, et al . (1996) Clin. Cancer. Res. 2:1969-1979). In a SCID mouse model, IL-10 was shown to inhibit the metastatic activity of human prostatic adenocarcinoma cell lines via an inhibition of the production of metalloproteases (MMP) and to an increase in the production of tissue inhibitors of metalloproteases (TIMP) . Stearns, et al . (1997) Invassion Metastasis 17:62-74. In vitro studies revealed that IL-10 inhibits angiogenesis (Stearns, et al . (1999) Clin. Cancer Res. 5:189-196), however, its direct effect in in vivo assays had not previously been demonstrated.
II. vIL-10 Agonists and Antagonists
BCRFl, or vIL-10, (from Epstein Barr Virus) has been described previously in US Pat. No. 5,627,155, and in de Vries and de Waal Malefyt Interleukin-10 Landes Company, Austin TX. Various agonists and antagonists of the natural ligands can be produced. Various mutein agonists and antagonists can be generated, as can antibodies which block the binding of the cytokine to its natural receptor. Mutein agonists will include variants with substitutions at non-essential residues, e.g., away from the receptor interaction regions. Mutein antagonists will include variants which compete with natural vIL-10 to bind to the receptor. Antagonist antibodies may bind to the cytokine ligand, or to the IL-10 receptor, and block vIL-10 binding to the receptor. See, e.g., Ho, et al . (1993) Proc .
Nat'l Acad. Sci . USA 90:11267-11271; and Liu, et al . (1994) J. Immunol . 152:1821-1829. Similar approaches may be useful in generating antagonists to other viral forms of IL-10, e.g., from the equine hepatitis virus, or the pox virus. A. vIL-10 and variants vIL-10 agonists will exhibit some or all of the signaling functions of the vIL-10, e.g., binding and signal transduction initiation into the appropriate cells. Various IL-10 based sequences may be evaluated to determine what residues are conserved across species, suggesting what residues may be changed without dramatic effects on biological activity. Alternatively, conservative substitutions, especially away from the receptor interacting surfaces, are likely to retain biological activity, thus leading to variant forms of the cytokine which will retain agonist activity. Standard methods for screening mutant or variant vIL-10 polypeptides will determine what sequences will be useful therapeutic agonists. In addition, certain nucleic acid expression methods may be applied. For example, in solid tumor contexts, it may be useful to transfect the tumors with nucleic acids which will be expressed, as appropriate. Various promoters may be operably linked to the gene, thereby allowing for regulated expression. Alternatively, antagonist activity may be tested for. Tests for ability to antagonize vIL-10 activity can be developed using assays as described before. Various ligand homologs can be created which retain substantial receptor binding capacity, but lacking signaling capability can be prepared. Small molecules may also be screened for ability to block ligand function. See generally Gilman, et al . (eds. 1990) Goodman and Gilman' s: The Pharmacological Bases of Therapeutics , 8th Ed., Pergamon Press; Remington ' s Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn, each of which is incorporated herein by reference . B. Antibodies
The present invention provides for the use of an antibody or binding composition which specifically binds to vIL-10, and neutralizes the ability of the cytokine to mediate its signal. Antibodies can be raised to various vIL-10 proteins, including individual or strain variants, and fragments thereof, both in their naturally occurring (full-length) forms or in their recombinant forms. Additionally, antibodies can be raised to vIL-10 polypeptides in both their native (or active) forms or in their inactive, e.g., denatured, forms, which may neutralize ligand capacity to mediate its signal. Certain antibodies may block the interaction of the ligand with its receptor. Thus, various antibodies may be generated against the receptor, which antibodies may block vIL-10 binding.
A number of immunogens may be selected to produce antibodies specifically reactive, or selective for binding, with vIL-10 proteins. Recombinant protein is a preferred immunogen for the production of monoclonal or polyclonal antibodies. Naturally occurring protein may also be used either in pure or impure form. Synthetic peptides, made using the vIL-10 protein sequences described herein, may also used as an immunogen for the production of antibodies to vIL-10 proteins. Recombinant protein can be expressed and purified in eukaryotic or prokaryotic cells as described, e.g., in Coligan, et al. (eds. 1995 and periodic supplements) Current Protocols in Protein Science John Wiley & Sons, New York, NY; and
Ausubel, et al (eds. 1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York, NY. Naturally folded or denatured material can be used, as appropriate, for producing antibodies. Either monoclonal or polyclonal antibodies may be generated, e.g., for subsequent use in immunoassays to measure the protein, or for immunopurification methods.
Methods of producing polyclonal antibodies are well known to those of skill in the art. Typically, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the antigen of interest. For example, when appropriately high titers of antibody to the immunogen are obtained, usually after repeated immunizations, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be performed, if desired. See, e.g., Harlow and Lane Antibodies , A Laboratory Manual; or Coligan (ed. ) Current Protocols in
Immunology. Immunization can also be performed through other methods, e.g., DNA vector immunization. See, e.g., Wang, et al. (1997) Virology 228:278-284.
Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell. See, Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. See, e.g., Doyle, et al . (eds. 1994 and periodic supplements) Cell and Tissue Culture: Laboratory Procedure . John Wiley and Sons, New York, NY. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according, e.g., to the general protocol outlined by Huse, et al. (1989) Science 246:1275-1281.
Antibodies or binding compositions, including binding fragments and single chain versions, against predetermined fragments of antigen can be raised by immunization of animals with conjugates of the fragments with carrier proteins as described above. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective vIL-10 protein, or screened for capacity to block vIL-10 mediated signal tranduction. These monoclonal antibodies will usually bind with at least a Krj of about 1 mM, more usually at least about
300 μM, typically at least about 10 μM, more typically at least about 30 μM, preferably at least about 10 μM, and more preferably at least about 3 μM or better.
In some instances, it is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al . (eds.) Basic and Clinical Immunology (4th ed. ) Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed. ) Academic Press, New York, NY; and particularly in Kohler and Milstein (1975) Nature 256:495- 497, which discusses one method of generating monoclonal antibodies. Summarized briefly, this method involves injecting an animal with an immunogen. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secretes a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
Other suitable techniques involve selection of libraries of antibodies in phage or similar vectors. See, e.g., Huse, et al . (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275- 1281; and Ward, et al . (1989) Nature 341:544-546. The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see, Cabilly, U.S. Patent No. 4,816,567; and Queen, et al . (1989) Proc . Nat ' 1 Acad. Sci . USA 86:10029-10033; or made in transgenic mice, see Mendez, et al . (1997) Nature Genetics 15:146-156. Antibody binding compounds, including binding fragments, of this invention can have significant diagnostic or therapeutic value. They can be useful as non-neutralizing binding compounds and can be coupled to toxins or radionuclides so that when the binding compound binds to the antigen, a cell expressing it, e.g., on its surface, is killed. Further, these binding compounds can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting. C. Other Molecules
Antibodies are merely one form of specific binding compositions. Other binding compositions, which will often have similar uses, include molecules that bind with specificity to the receptor, e.g., in a binding partner-binding partner fashion, an antibody-antigen interaction, or in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent , e.g., proteins which specifically associate with IL-10 receptor protein. The molecule may be a polymer, or chemical reagent. A functional analog may be a protein with structural modifications, or may be a structurally unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate binding determinants.
Drug screening using antibodies or vIL-10 or fragments thereof can be performed to identify compounds having binding affinity to vIL-10, or can block or simulate the natural interaction with ligand. Subsequent biological assays can then be utilized to determine if the compound has intrinsic blocking activity and is therefore an antagonist. Likewise, a compound having intrinsic stimulating activity can signal to the cells via the vIL-10 pathway and is thus an agonist in that it simulates the activity of a ligand. Mutein antagonists may be developed which maintain receptor binding but lack signaling. Structural studies of the ligands will lead to design of new variants, particularly analogs exhibiting agonist or antagonist properties on the receptor. This can be combined with previously described screening methods to isolate muteins exhibiting desired spectra of activities .
As receptor specific binding molecules are provided, also included are small molecules identified by screening procedures. In particular, it is well known in the art how to screen for small molecules which interfere, e.g., with ligand binding to the receptor, often by specific binding to the receptor and blocking of binding by natural ligand. See, e.g., meetings on High Throughput Screening, International Business Communications, Southborough, MA 01772-1749. Such molecules may compete with natural ligands, and selectively bind to vIL- 10. III . Uses
These studies suggest that both human and viral forms of IL-10 abolish tumorigenicity in SCID mice and provide evidence that IL-10 exerts its anti-tumor activity by inhibiting angiogenesis in vivo. Moreover, the cytokines may also have antimetastatic effects, preventing dispersion of the neoplastic cells .
Effects on various cell types may be indirect, as well as direct. A statistically significant change in the parameters, e.g., numbers of cells, will typically be affected by at least about 10%, preferably 20%, 30%, 50%, 70%, 90%, or more. Effects of greater than 100%, e.g., 130%, 150%, 2X, 3X, 5X, etc., will often be desired. The effects may be specific in causing angiogenesis, tumorigenesis, or metastasis, according to objective measures generally recognized in the art.
The present invention is useful in the treatment of medical conditions or diseases, e.g., associated with neoplastic disease. See, e.g., Bertino, et al . (eds. 1996) Encyclopedia of Cancer Academic Press; Devita, et al . (eds. 1997) Cancer: Principles & Practice of Oncology Lippincott, Williams and Wilkins; Devita (1997) Principles and Practice of Oncology Lippincott Williams and Wilkins; Cavalli, et al . (1996) Textbook of Medical Oncology Dunitz Martin Ltd; Horwich (ed. 1995) Oncology: A Multidisciplinary Textbook Lippincott- Raven; Peckham, et al . (eds. 1995) Oxford Textbook of Oncology Oxford Univ. Press; Mendelsohn, et al . (1995) The Molecular Basis of Cancer Saunders , Philadelphia; and McArdle (1990) Surgical Oncology: Current Concepts and Practice Butterworth- Heinemann. Other indications where regulation of angiogenesis will be useful include, e.g., retinopathies , macular degeneration, angiofibroma, corneal graft neovascularization, and trachoma. See, e.g., Berkow (ed. ) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn, et al . Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; and Weatherall, et al . (eds.) Oxford Textbook of Medicine, Oxford University Press, Oxford. The agonists or antagonists described may be combined with other treatments of the medical conditions described herein, e.g., an angiogenic or angiostatic reagent, immune suppressive therapeutic, immune adjuvant, analgesic, anti-inflammatory drug, growth factor, cytokine, vasodilator, or vasoconstrictor, depending upon the desired response. Preferred combination therapies include the vIL-10 reagent with various anti-inflammatory agents, such as systemic steroids or corticosteroids .
Metastasis is a multistep process involving numerous tumor cell-host cell and cell-matrix associations, a number of processes. Typically, it requires escape from local origin by the neoplastic cells, often across tissue barriers. The cells become mobile, and extravasate, and invade a remote secondary site to proliferate and establishment a viable tumor mass. Such typically requires the establishment of vascularization with small capillaries to feed the growing cell mass. Thus, angiogenesis both serves to allow the neoplastic cells to escape the local environment, and once established, to grow. Thus, anti-angiogenic functions, e.g., angiostasis, may be a useful component in the minimization of growth and metastasis of a tumor.
For example, the vIL-10 ligands would be expected to signal specifically to the cell types expressing the receptor. Thus, it will be possible to block signaling, e.g., to the IL- 10 responsive subsets, by reagents which block receptor signaling, e.g., antibodies to ligand, and small drug antagonists .
Standard immunological techniques are described, e.g., in Hertzenberg, et al . (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Methods in
Enzvmology volumes 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. These will allow use of the reagents for purifying cell subpopulations, etc.
To prepare pharmaceutical or sterile compositions including vIL-10, the cytokine is admixed with a pharmaceutically acceptable carrier or excipient which is preferably inert. Preparation of such pharmaceutical compositions is known in the art, see, e.g., Remington ' s Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984) . Alternatively, vIL-10 antagonist compositions can be prepared. Agonists, e.g., natural ligand, or antagonists, e.g., antibodies or binding compositions, are normally administered parenterally, preferably intravenously. Since such protein or peptide antagonists may be immunogenic they are preferably administered slowly, either by a conventional IV administration set or from a subcutaneous depot, e.g. as taught by Tomaεi, et al., U.S. patent 4,732,863.
This invention further would encompass expression of recombinant DNA molecules and fragments having a DNA sequence identical to or highly homologous to the vIL-10 described herein. In particular, the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA replication. Alternatively, recombinant clones derived from the genomic sequences, e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt (ed.)
Encyclopedia of Immunology Academic Press, San Diego, pp. 1502- 1504; Travis (1992) Science 256:1392-1394; Kuhn, et al . (1991) Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach IRL Press, Oxford; Rosenberg (1992) J.
Clinical Oncology 10:180-199; and Cournoyer and Caskey (1993) Ann. Rev. Immunol. 11:297-329. Alternatively, expression may be effected by operably linking a coding segment to a heterologous promoter, e.g., by inserting a promoter upstream from an endogenous gene. See, e.g., Treco, et al . W096/29411 or USSN 08/406, 030.
When administered parenterally the therapeutics will be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic . The antagonist may be administered in aqueous vehicles such as water, saline or buffered vehicles with or without various additives and/or diluting agents. Alternatively, a suspension, such as a zinc suspension, can be prepared to include the peptide . Such a suspension can be useful for subcutaneous (SQ) , intradermal (ID) , or intramuscular (IM) injection. The proportion of therapeutic entity and additive can be varied over a broad range so long as both are present in effective amounts. The therapeutic is preferably formulated in purified form substantially free of aggregates, other proteins, endotoxins, and the like, at concentrations of about 5 to 30 mg/ml, preferably 10 to 20 mg/ml. Preferably, the endotoxin levels are less than 2.5 EU/ml . See, e.g., Avis, et al . (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications 2d ed. , Dekker, NY; Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms: Tablets 2d ed., Dekker, NY; Lieberman, et al . (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY; Fodor, et al . (1991) Science 251:767-773; Coligan (ed. ) Current Protocols in Immunology; Hood, et al . Immunology Be jamin/Cummings; Paul (ed. 1997) Fundamental Immunology 4th ed., Academic Press; Parce, et al . (1989) Science 246:243-247; Owicki, et al . (1990) Proc. Nat ' 1 Acad. Sci. USA 87:4007-4011; and Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York. Local, e.g., topical or transdermal, administration will often be particularly useful.
Selecting an administration regimen for a therapeutic agonist or antagonist depends on several factors, including the serum or tissue turnover rate of the therapeutic, the immunogenicity of the therapeutic, or the accessibility of the target cells. Preferably, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of therapeutic delivered depends in part on the particular agonist or antagonist and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies is found in the literature on therapeutic uses, e.g. Bach et al . , chapter 22, in Ferrone, et al . (eds. 1985) Handbook of Monoclonal Antibodies Noges Publications, Park Ridge, NJ; and Russell, pgs . 303-357, and Smith et al . , pgs . 365-389, in Haber, et al . (eds. 1977) Antibodies in Human Diagnosis and Therapy Raven Press, New York, NY.
Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known in the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Preferably, an antibody or binding composition thereof that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing a humoral response to the reagent.
The total weekly dose ranges for antibodies or fragments thereof, which specifically bind to vIL-10, range generally from about 1 ng, more generally from about 10 ng, typically from about 100 ng; more typically from about 1 μg, more typically from about 10 μg, preferably from about 100 μg, and more preferably from about 1 mg per kilogram body weight. Although higher amounts may be more efficacious, the lower doses typically will have fewer adverse effects. Generally the range will be less than 100 mg, preferably less than about 50 mg, and more preferably less than about 25 mg per kilogram body weight .
The weekly dose ranges for antagonists, e.g., antibody, binding fragments, range from about 10 μg, preferably at least about 50 μg, and more preferably at least about 100 μg per kilogram of body weight. Generally, the range will be less than about 1000 μg, preferably less than about 500 μg, and more preferably less than about 100 μg per kilogram of body weight. Dosages are on a schedule which effects the desired treatment and can be periodic over shorter or longer term. In general, ranges will be from at least about 10 μg to about 50 mg, preferably about 100 μg to about 10 mg per kilogram body weight, though local administration may be generally preferred. Other agonists or antagonists of the ligands, e.g., muteins, are also contemplated. Hourly dose ranges for muteins range from at least about 10 μg, generally at least about 50 μg, typically at least about 100 μg, and preferably at least 500 μg per hour. Generally the dosage will be less than about 100 mg, typically less than about 30 mg, preferably less than about 10 mg, and more preferably less than about 6 mg per hour. General ranges will be from at least about 1 μg to about 1000 μg, preferably about 10 μg to about 500 μg per hour.
In particular contexts, e.g., certain tumor locations or types, may involve the administration of the therapeutics in different forms. In particular, genetic transformation of cells may be achieved to express the desired vIL-10 proteins at the correct local environment.
The present invention also provides for administration of vIL-10 compositions in combination with known therapies, e.g., steroids, particularly glucocorticoids , which alleviate the symptoms associated with excessive inflammatory responses. Daily dosages for glucocorticoids will range from at least about 1 mg, generally at least about 2 mg, and preferably at least about 5 mg per day. Generally, the dosage will be less than about 100 mg, typically less than about 50 mg, preferably less than about 20 mg, and more preferably at least about 10 mg per day. In general, the ranges will be from at least about 1 mg to about 100 mg, preferably from about 2 mg to 50 mg per day.
The phrase "effective amount" means an amount sufficient to effect a desired response, or to ameliorate a symptom or sign of the neoplastic condition. Typical mammalian hosts will include mice, rats, cats, dogs, and primates, including humans. An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, route, and dose of administration and the severity of side affects. Preferably, the effect will result in a change in quantitation of at least about 10%, preferably at least 20%, 30%, 50%, 70%, or even 90% or more. When in combination, an effective amount is in ratio to a combination of components and the effect is not limited to individual components alone.
An effective amount of therapeutic will modulate the symptoms typically by at least about 10%; usually by at least about 20%; preferably at least about 30%; or more preferably at least about 50%. Alternatively, modulation of angiogenesis will mean that the vascularization of, e.g., the cellular mass, is affected. Such will result in, e.g., statistically significant and quantifiable changes in the vascularization, tumor growth, or metastasis formation. Preferably, this will prevent any angiogenesis, e.g., achieve an angiostatic condition.
The present invention provides reagents which will find use in therapeutic applications as described elsewhere herein, e.g., in the general description for treating disorders associated with neoplastic conditions. See, e.g., Berkow (ed. ) The Merck Manual of Diagnosis and Therapy, Merck & Co . , Rahway, N.J.; Thorn, et al . Harrison's Principles of Internal Medicine, McGraw-Hill, NY; Gilman, et al . (eds. 1990) Goodman and Gilman' s: The Pharmacological Bases of Therapeutics, 8th Ed. , Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn; Langer (1990) Science 249:1527-1533; and Merck Index, Merck & Co . , Rahway, New Jersey. Antibodies to vIL-10 or receptor proteins may be used for the identification or sorting of cell populations expressing cytokine or receptor protein, e.g., specific tumor cells. Methods to sort such populations are well known in the art, see, e.g., Melamed, et al . (1990) Flow Cvtometry and Sorting Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical Flow Cvtometry Liss, New York, NY; and Robinson, et al . (1993) Handbook of Flow Cvtometry Methods Wiley-Liss, New York, NY. Populations of cells expressing the vIL-190 or IL-10 receptor can also be purified using magnetic beads as described, e.g., in Bieva, et al . (1989) Exp. Hematol. 17:914-920; Hernebtub, et al. (1990) Bioconi . Chem. 1:411-418; and Vaccaro (1990) Am. Biotechnol. Lab. 3:30. Such diagnostic methods may determine susceptibility to treatment using the methods described. Moreover, antisense nucleic acids may be used. For example, antisense against the ligand encoding nucleic acids may function in a manner like ligand antagonists, and antisense against the receptor may function like receptor antagonists. Thus, it may be possible to block the signaling through the pathway with antisense nucleic acids. Conversely, nucleic acids for the receptor may serve as agonists, increasing the numbers of receptor on the cell, thereby increasing cell sensitivity to ligand, and perhaps blocking the normal apoptotic signal described.
Using the assay methods described above, the antibodies or binding compositions are useful in diagnosing diseases states which may be responsive to the methods provided. Antibodies raised against a vIL-10 protein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens. Combinations of these signals may be also pursued.
The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments.
EXAMPLES I. General Methods
Some of the standard methods are described or referenced, e.g., in Maniatis, et al . (1982) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual , (2d ed. ) , vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates, Brooklyn, NY; or Ausubel, et al . (1987 and Supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York; Innis, et al . (eds.) (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, N.Y. Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis , centrifugation, crystallization, and others. See, e.g., Ausubel, et al . (1987 and periodic supplements) ; Deutscher (1990) "Guide to Protein Purification" in Methods in Enzvmology, vol. 182, and other volumes in this series; manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA; and Coligan, et al . (eds.) (1995 and periodic supplements) Current Protocols in Protein Science, John Wiley & Sons, New York, NY. Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence. See, e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed. ) Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al . (1992) QIAexpress: The High Level Expression & Protein Purification System QIAGEN, Inc., Chatsworth, CA.
Standard immunological techniques are described, e.g., in Hertzenberg, et al . (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4, Blackwell Science; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Methods in
Enzvmology volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.
FACS analyses' are described in Melamed, et al . (1990) Flow Cvtometry and Sorting Wiley-Liss, Inc., New York, NY; Shapiro (1988) Practical Flow Cvtometry Liss, New York, NY; and
Robinson, et al . (1993) Handbook of Flow Cvtometry Methods Wiley-Liss, New York, NY.
II. Specific Methods Mice.
CB-17 SCID mice were bred and maintained in pathogen limited conditions at the Microbiology and Tumor Biology Center (MTC) , Karolinska Institutet. Four to six weeks old mice, usually littermates, or otherwise age matched within two weeks were used in all experiments.
Cells and transfections .
DG75 is an EBV negative BL line (Ben-Bassat, et al . (1977) Int. J. Cancer 19:27-33). The hIL-10 and vIL-10 cDNAs were excised from the pCDSR 296 modified expression vector (Takebe, et al. (1988) Mol . Cell. Biol . 8:466-472) and subcloned in the pcDNA3 Neo vector (Invitrogen) . Plasmid control (pcDNA3 Neo) or plasmids containing the h- or v-IL-10 cDNAs were introduced into tumor cells by electroporation using a Gene-Pulser (BioRad Laboratories, Richmond, CA) under the following conditions: 5 x 10 cells in 0.5 ml Dulbecco's phosphate buffered saline (without Ca2+ and Mg2+) were pulsed with 875 V/cm, and 975 μF . Transfectants were selected in G418 (750 μg/ml; Gibco, BRL) for establishment of lines and clones. Cells were maintained in RPMI 1640 (Gibco, BRL) supplemented with 10% heat inactivated fetal calf serum, 100 mg/ml of streptomycin, 100 U/ml Penicillin and 750 μg/ml G418 (referred as complete medium) . They were cultured at 37° C/5% Cθ2 ■
IL-10 production by the clones and sublines was determined by measurement of IL-10 released to the medium by specific ELISA (capture clone JES3-9D7, biotinylated detection clone JES3-12G8, PharMingen, San Diego, CA) . The sensitivity of the detection for IL-10 ELISA was 20 to 40 pg/ml . Results are expressed as units of IL-10/ 1 x 10 cells/ml, per 36 h. Phenotypical analysis. Anti CD23, -CD40, -CD74, -CD86, -CD95 PE or FITC conjugated anti- HLA ABC, -DR (clone L243), CDlla, - CD54, -CD58, -CD80 antibodies were purchased from Becton Dickinson (Mountain View, CA) . FITC conjugated goat anti-mouse antibody was purchased from DAKO (Denmark) . Murine anti-IL-10 R Ab was provided by DNAX Research Institute, Palo Alto, CA. Cells (10 ) were labeled with FITC or PE conjugated antibodies or isotype matched controls for 30 min on ice. For indirect immunofluorescence, cells were washed and incubated with FITC- conjugated goat anti-mouse antibody (DAKO, Denmark) for 30 min on ice after labeling with the first antibody. The cells were washed with cold PBS containing 1% FCS and 0.1% NaN3. 10,000 cells were analyzed on a FACScan flow cytometer using CellQuest software from Becton Dickinson (Mountain View, CA) . Gates were established on viable cells using forward and side scatter parameters .
Tumorigenicity in SCID mice. n Tumor cells (10 ) were inoculated subcutaneously in the right flank in a 0.2 ml volume of PBS. Tumor size was determined at least once weekly by palpations and measurements of the tumor. Mice were sacrificed when the tumors reached a size of > 15 mm in diameter. Mice without any signs of tumor growth were kept under observation for at least six weeks after inoculation. Small groups of mice, never exceeding five mice per group, were tested in several independent experiments throughout the study to minimize random fluctuations in the quantity or quality of cells inoculated. Results from several independent tests were pooled.
In vivo NK cell depletion. One day prior to inoculation of tumor cells, each mouse received an intravenous dose of rabbit anti-asialo GM1 antibody (WAKO, Dallas, TX) (25 μl /mouse) in a total volume of 0.2 ml of PBS.
Proliferation assay.
Cells were seeded by doubling dilution starting at a concentration of 4 x 10 /well in flat bottom microtiter pli in a volume of 200 μl and cultured for 48 h at 37° C/5% Cθ2
3 Cultures were pulsed with 1 μCi of H TdR/well during the 1;
10 h of the incubation period, harvested onto Fiberglas
3 filters, and H TdR incorporation determined by liquid scintillation counting.
Cloning in soft agarose.
Human lung fibroblasts were grown to confluence on 35 x 10 mm Petri dishes (Lux tissue culture dishes with 2 mm grids, Lab Tek, Miles, McLean, VA) and irradiated with 6,000 rads . One ml of 0.45% w/w agarose (Sea-plaque agarose, Marine colloids, FMC, Rockland, ME) was heated and diluted in RPMI-1640 supplemented with 15% FCS and added to the plates. When the underlayer had solidified (1 h, RT) three ml of a 0.35% w/w agarose solution in complete media were mixed with the cells at 40° C and poured on top of the agarose underlayer. Triplicate plates containing 100, 1000, and 10000 cells were seeded. Cloning efficiency was estimated visually after 7-8 days of culture at 37° C in 5% Cθ2 incubator. A group of more than 20 cells was scored as a clone. Rabbit cornea assay for angiogenesis.
Corneal assays were performed in New Zealand white rabbits (Charles River, Calco, Como, Italy) , as described (Ziche, et al. (1982) J. Natl. Cancer. Inst. 69:475-482), and in accordance with the guidelines of the European Economic Community for animal care and welfare (EEC Law No. 86/609) . Briefly, cell suspensions (in a volume of 5 μl) were implanted into surgically produced corneal micropockets . The angiogenic response was measured daily with a slit lamp stereo microscope and capillary progression scored as previously reported. Ziche, et al . (1994) J. Clin. Invest. 94:2036-2044. The number of tissue samples inducing angiogenesis over the total number of implants performed was recorded during each observation. Implants not producing a neovascular growth within 10 days were considered negative. Implants showing an inflammatory reaction were evaluated and scored separately. The potency of angiogenic activity (angiogenic score) was calculated on the basis of the number and growth rate of newly formed capillaries calculated as: (vessel density x distance from limbus) . See Ziche, et al . (1994) J. Clin. Invest. 94:2036-2044. Corneas were removed at the end of the experiment as well as at defined intervals after surgery and/or treatment and fixed in formalin for histological examination. DG75 sublines were implanted in the corneal micropocket according to the following protocol: a) angiogenic activity of equal number of cells of each sublime (IX DG75- vector cells, DG75-hIL-10 cells, DG75-vIL-10 cells); b) angiogenic activity of 2X DG75-vector cells; c) angiogenic activity of IX DG75-Vector cells + IX DG75 IL-10 transfectants .
III. IL-10 prevents tumor growth of DG75 sublines
The genes encoding human and viral IL-10 were transduced into the Burkitt's lymphoma (BL) line DG75. This EBV negative B cell line was chosen for these studies because it has been shown previously that these cells did not express or secrete endogenous IL-10. Finke, et al . (1993) Leukemia 7:1852-1857. Lines and clones expressing IL-10 activity ranging from 13-730 U IL-10/106 cells/ml/36 h were isolated. IL-10 could not be detected in conditioned media from parental (referred to as wild type (WT) ) , and mock transfected DG75 (referred as vector control) lines. DG75 is tumorigenic following subcutaneous (SC) implantation into SCID mice. Ben-Bassat, et al . (1977)
Int. J. Cancer 19:27-33. Preliminary experiments were performed with the WT cell line in order to assess the dose of cells required to induce tumors. On the basis of these
7 experiments, a dose of 10 cells was selected because two weeks after subcutaneous inoculation 75% of the mice had palpable tumors at the site of injection. The tumorigenicity of the transfected sublines and clones was compared to that of WT and vector control. To avoid clonal variability, a sublime and multiple different clones of either h- or v- IL-10 transfectants with different IL-10 activities were tested.
Seventy five per cent of the mice (12/16) given injections of WT cells developed a tumor after three weeks. The vector control cells developed tumors with similar incidence and kinetics as the WT cells, 67% of the mice (12/18) . On the other hand, the tumor incidence was markedly reduced for all the IL-10 expressing lines. The proportion of animals developing tumors was 9% (4/42) and 16% (6/38) for the h- and v- IL-10 transfectants, respectively. However, no direct correlation was found between the tumorigenicity of the sublines and the production of IL-10, the few tumors that developed from h- or v- IL-10 transfectants came from clones that produced low, intermediate, and high levels of IL-10. When tumors developed, they grew with similar kinetics as the WT or vector control sublines.
IV. The effect of IL-10 is local
To address the question of whether the tumor suppressing effect of IL-10 acted locally or systemically, the same number of hIL-10 sublime and vector control DG75 cells were either mixed before injection or injected contralaterally and tumor growth was evaluated. Coinjection of both cell types at the same site resulted in suppression of tumor growth of vector control cells (0/5 animals developed tumor) . However, the hIL-10 sublime was unable to suppress the growth of contralaterally injected vector control cells, 60% (3/5) of the mice developed tumors at the site of DG75 vector control injection.
V. IL-10 does not affect the growth behavior in vitro
IL-10 is known to affect the proliferation and autonomous growth of human Epstein-Barr virus (EBV) - transformed B cells and melanoma cell lines. Stuart, et al . (1995) Oncogene
11:1711-1719; Beatty, et al . (1997) J. Immunol. 158:4045-4051; Bejarano and Masucci (1998) Blood 92:4256-4262; and Yun Yue, et al. (1997) Int. J. Cancer 71:630-637. Whether the capability of DG75 IL-10 transfectants to form tumors was related to a direct effect of IL-10 on cell proliferation and/or anchorage dependency (tested as agarose clonability) was tested. The proliferative capacity of the IL-10 transfectants was identical to that of WT or vector control. The proliferative curves of all clones tested overlap. Addition of exogenous IL-10 to the cell cultures did not affect the proliferation of any of the sublines. Although the agarose clonability varied from experiment to experiment (37-75%) , the expression of IL-10 did not affect significantly the cloning efficiency of the sublines .
VI. IL-10 effect on phenotype
In B cells and in various tumors, IL-10 has been shown to downregulate the expression of MHC and costimulatory molecules. Zeidler, et al . (1997) Blood 90:2390-2397; Yun Yue, et al . (1997) Int. J. Cancer 71:630-637; Salazar-Onfray, et al . (1997) J. Immunol. 159:3195-3202; Petersson, et al . (1998) J . Immunol . 161:2099-2105; and Tsuruma, et al . (1998) Cell. Immunol. 184:121-128. In some cases, the decreased MHC class I expression has been correlated to an increase in NK sensitivity and decrease in the metastatic potential. Kundu, et al . (1996) J. Natl. Cancer Inst. 88:536-541; Zheng, et al . (1996) J . Exp . Med. 184:579-584; Zheng, et al . (1996) J. Exp. Med. 184:579- 584; and Kundu and Fulton (1997) Cell. Immunol. 180:55-61. In the present report, IL-10 expression did not correlate with the levels of expression of either MHC class I/II or costimulatory molecules. However, it was observed that all IL-10 transfectants had a tendency to grow as single cells, while the WT line and the vector control line grew in small loose clumps. In addition, IL-10 did not affect the expression of CD95.
VII. The effect of IL-10 is not dependent on NK activity
NK cells have been shown previously to play a role in the antitumor effects of IL-10. Zheng, et al . (1996) J . Exp . Med . 184:579-584; Kundu, et al . (1996) J. Natl. Cancer Inst. 88:536- 541; and Kundu and Fulton (1997) Cell . Immunol . 180:55-61. Therefore, the tumorigenicity of the IL-10 transfected sublines were tested in SCID mice pre-treated with asialo GMl antibody to suppress NK activity. When DG75 vector cells were injected into NK-depleted mice, all mice (8/8) developed tumors two weeks after inoculation. Although the incidence of tumor formation induced by IL-10 transfectants increased when compared to untreated mice, from 10 to 33% (4/12) for hIL-10, and from 16 to 62% (5/8) for vIL-10, the differences in tumorigenicity between the vector and IL-10 transfectants were still maintained. In addition, the DG75 sublines were resistant to spontaneous or Tilorone-boosted NK activity from SCID mice in vitro.
VIII. IL-10 inhibits angiogenesis in vivo
Given the fact that the IL-10 transfectants were not able to form solid tumors and that this effect could not ascribed to any in vitro phenotypic or behavioral change nor to an increase in NK sensitivity, the angiogenic capacity of the sublines was investigated. To this end, DG75 vector, hIL-10 and vIL-10 lines were implanted into rabbit corneal micropockets for evaluation of their angiogenic activity. Four days after inoculation of two different doses of DG75 vector, 4 x 10 , and 2,5 x 10 cells, angiogenesis was observed in 2/2 and 4/5 implants respectively. On the basis of these results, the dose
5 of 2,5 x 10 cells was selected for further experiments.
Angiogenesis was not induced by the either hIL-10 (0/5) or vIL- 10 (0/3) implants. Because in the tumorigenicity experiments the hIL-10 sublime inhibited the growth of the vector DG75 when admixed, whether h- or v- IL-10 could suppress the angiogenic capacity of the DG75 vector when coinjected was tested. Th s, equal numbers (2,5 x 10 ) of DG75 vector control and hIL-10 or vIL-10 sublines were mixed and implanted. Coinjection of hlL- 10 or vIL-10 transfectants inhibited in all cases (0/4 for hlL- 10 and 0/3 for vIL-10 implants) the angiogenic capacity of the vector control line. Noteworthy, implants of vIL-10, whether alone or mixed with the vector, induced a marked inflammatory response .
IX. vIL-10 Antagonists
Various antagonists of vIL-10 are made available. For example, antibodies against the cytokine itself may block the binding of ligand to its receptor, thereby serving as a direct receptor antagonist. Other antagonists may function by blocking the binding of ligand to receptor, e.g., by binding to the ligand in a way to preclude the possibility of binding to the receptor. Other antagonists, e.g., mutein antagonists, may bind to the receptor without signaling, thereby blocking a true agonist from binding.
Information on the criticality of particular residues is determined using standard procedures and analysis. Standard mutagenesis analysis is performed, e.g., by generating many different variants at determined positions, e.g., at the positions identified above, and evaluating biological activities of the variants. This may be performed to the extent of determining positions which modify activity, or to focus on specific positions to determine the residues which can be substituted to either retain, block, or modulate biological activity.
Alternatively, analysis of natural variants can indicate what positions tolerate natural mutations. This may result from populational analysis of variation among individuals, or across strains or species. Samples from selected individuals are analyzed, e.g., by PCR analysis and sequencing. This allows evaluation of population polymorphisms. All citations herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled; and the invention is not to be limited by the specific embodiments that have been presented herein by way of example .

Claims

WHAT IS CLAIMED IS:
1. A method of inhibiting angiogenesis to a growing cellular mass comprising administering an effective amount of viral IL-10 to the location of said mass.
2. The method of Claim 1, wherein said cellular mass is a tumor .
3. The method of Claim 2, wherein said tumor is highly vascularized.
4. The method of Claim 2, wherein said tumor is a lymphoid tumor.
5. The method of Claim 2, wherein said tumor is: a) Burkitt's lymphoma; b) Kaposi sarcoma; c) osteosarcoma; d) mammary adenocarcinoma; e) malignant melanoma; or f) prostatic adenocarcinoma.
6. The method of Claim 5, wherein said tumor is: a) Burkitt's lymphoma; or b) Kaposi sarcoma.
7. The method of Claim 1, wherein said administering is local, topical, subcutaneous, intradermal, or transdermal .
8. The method of Claim 1, wherein said administering is by expression of a nucleic acid encoding said viral IL-10.
9. The method of Claim 1, wherein said administering is in combination with another anti-neoplastic therapeutic agent.
10. The method of Claim 1, wherein said administering is in combination with an anti-inflammatory agent.
11. The method of Claim 1, wherein said effective amount further results in a decrease in tumor metastasis.
12. The method of Claim 1, wherein said effective amount further results in: a) angiostasis; or b) inhibiting tumorigenesis.
13. A method of inhibiting tumorigenesis of a growing cellular mass comprising administering an effective amount of viral IL-10 to the location of said mass.
14. The method of Claim 13, wherein said cellular mass is a tumor.
15. The method of Claim 13, wherein said tumor is: a) highly vascularized; or b) a lymphoid tumor.
16. The method of Claim 13, wherein said tumor is: a) Burkitt's lymphoma; b) Kaposi sarcoma; c) osteosarcoma; d) mammary adenocarcinoma; e) malignant melanoma; or f) prostatic adenocarcinoma.
17. The method of Claim 13, wherein said administering is: a) local, topical, subcutaneous, intradermal, or transdermal ; or b) by expression of a nucleic acid encoding said viral
IL-10.
18. The method of Claim 13, wherein said administering is in combination with: a) another anti-neoplastic therapeutic agent; or b) an anti-inflammatory agent.
19. The method of Claim 13, wherein said effective amount further results in inhibiting tumorigenesis.
20. A method of inhibiting metastasis of a cellular mass comprising administering an effective amount of viral IL-10 to the host.
PCT/EP2000/006558 1999-07-16 2000-07-11 Viral il-10 for the inhibition of angiogenesis, tumorigenesis and metastasis WO2001005821A2 (en)

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