EP0930892A1 - Zytokin-erhoehte immuntherapie gegen gehirntumore - Google Patents

Zytokin-erhoehte immuntherapie gegen gehirntumore

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Publication number
EP0930892A1
EP0930892A1 EP97910921A EP97910921A EP0930892A1 EP 0930892 A1 EP0930892 A1 EP 0930892A1 EP 97910921 A EP97910921 A EP 97910921A EP 97910921 A EP97910921 A EP 97910921A EP 0930892 A1 EP0930892 A1 EP 0930892A1
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European Patent Office
Prior art keywords
cytokine
cells
mmor
tumor
csf
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EP97910921A
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English (en)
French (fr)
Inventor
Henry Brem
Drew M. Pardoll
Reid C. Thompson
Elizabeth M. Jaffee
Kam W. Leong
Matthew G. Ewend
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Johns Hopkins University
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Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention is in the field of treating cancer by administering a combination of systemic and local immunotherapy using cytokines. Rights of the Federal Government The United States government has certain rights in this invention by virtue of a grant by the National Cooperative Drug Discovery Group UO1- CA52857 of the National Cancer Institute of the National Institutes of Health, Bethesda, Maryland.
  • systemic chemotherapy has had minor success in the treatment of cancer of the colon- rectum, esophagus, liver, pancreas, and kidney.
  • a major problem with systemic chemotherapy for the treatment of these types of cancer is that the systemic doses required to achieve control of tumor growth frequently result in unacceptable systemic toxicity.
  • Efforts to improve delivery of chemotherapeutic agents to the tumor site have resulted in advances in organ- directed chemotherapy, as by continuous systemic infusion, for example.
  • continuous infusions of anticancer drugs generally have not shown a clear benefit over pulse or short-term infusions.
  • Implantable elastomer access ports with self-sealing silicone diaphragms have also been tried for continuous infusion, but extravasation remains a problem.
  • Portable infusion pumps are now available as delivery devices and are being evaluated for efficacy.
  • Controlled release biocompatible polymers have been used successfully for local drug delivery and have been utilized for contraception, insulin therapy, glaucoma treatment, asthma therapy, prevention of dental related disorders, and certain types of cancer chemotherapy.
  • Laser, R., and Wise, D. , eds, Medical Applications of Controlled Release. Vol. I and II, Boca Raton, CRC Press (1986) ).
  • the design and development of effective anti-tumor agents for treatment of patients with malignant neoplasms of the central nervous system have been influenced by two major factors: 1) the blood-brain barrier provides an anatomic obstruction, limiting access of drugs to these tumors; and 2) the drugs given at high systemic levels are generally cytotoxic.
  • Efforts to improve drug delivery to the tumor bed in the brain have included transient osmotic disruption of the blood brain barrier, cerebrospinal fluid perfusion, and direct infusion into a brain tumor using catheters. Each technique has had significant limitations. Disruption of the blood brain barrier increased the uptake of hydrophilic substances into normal brain, but did not significantly increase substance transfer into the tumor.
  • a therapy for treatment of patients with cancer utilizing the combination of a cytokine in a pharmaceutical acceptable carrier for systemic administration and a cytokine in a pharmaceutical acceptable carrier for local administration is described.
  • brain tumors are treated with a cytokine such as granulocyte macrophage-colony stimulating factor (GM-CSF) administered systemically, most preferably in combination with tumor antigen such as replication incompetent tumor cells, and a cytokine such as interleukin-2 (IL-2) or IL-4 administered locally, most preferably in a vehicle providing release over a period of time, such as transduced cells, again most preferably replication incompetent tumor cells, or incorporated into microparticulate vehicles such as polymeric microspheres.
  • a cytokine such as granulocyte macrophage-colony stimulating factor (GM-CSF) administered systemically, most preferably in combination with tumor antigen such as replication incompetent tumor cells, and a cytokine such as interleukin-2 (IL-2)
  • the examples demonstrate that systemic administration (vaccination) with GM-CSF transduced tumor cells or microencapsulated GM-CSF protects against growth of intracranial melanoma.
  • the examples also demonstrate that local intracranial delivery of IL-2 transduced tumor cells or microencapsulated IL-2 generates immediate anti-tumor responses within the central nervous system as well as long term memory capable of generating potent anti-tumor responses against multiple subsequent tumor challenges, including challenges outside the central nervous system.
  • the examples further demonstrate that combination immunotherapy using systemic vaccination with GM-CSF transductants and local intracranial administration of IL-2 transductants produces an anti-tumor effect that is significantly enhanced as compared to treatment with either treatment alone.
  • Figure 1 is a graph showing percent survival over time in days of experimental animal models of intracranial melanoma having received stereotactic intracranial injections of wild type B16-F10 melanoma cells in doses of 100,000 (circles), 10,000 (squares), 1,000 (triangles), and 100 (solid squares), and saline with no cells (solid circles).
  • Figure 2 is a graph showing percent survival over time in days of mice after systemic vaccination with a single subcutaneous injection of 10 6 irradiated B16-F10 cells engineered by gene transfer to secrete GM-CSF (solid squares), medium (circles) or 10 6 irradiated B16-F10 wild type non- cytokine producing cells (squares), challenged 14 days later with an injection of 10 2 non-irradiated wild type B16-F10 melanoma cells in the brain, (p ⁇ 0.001).
  • Figure 3 is a graph showing percent survival over time in days of mice treated with a single intracranial injection of 10 5 irradiated B16-F10 cells engineered by gene transfer to secrete IL-2 (solid squares), medium (circles) or 10 5 irradiated wild type B16-F10 non-cytokine producing cells (squares), challenged at the same time by stereotactic intracranial co- injections of 10 2 non-irradiated wild type B16-F10 cells, (p ⁇ 0.001).
  • Figure 4 is a graph showing percent survival over time in days of animals vaccinated with a subcutaneous injection of 10 6 irradiated GM-CSF producing B16-F10 melanoma cells followed two weeks later with 5 x 10 4 irradiated IL-2-secreting B16-F10 cells administered intracranially and simultaneously challenged with intracranial co-injections of 10 2 non-irradiated wild type B16-F10 cells (solid squares).
  • Control animals received GM-CSF vaccine alone followed by intracranial tumor challenge with wild type B16- F10 cells (x marks), or vaccination with medium alone followed by intracranial IL-2 therapy and tumor challenge (circles). Additional controls received vaccination with medium followed by intracranial therapy with medium and intracranial tumor challenge (squares).
  • a therapy for treatment of tumors has been developed which relies on the combination of an initial systemic "priming" of the immune system, most preferably through the combination of administration of a cytokine such as GM-CSF and tumor antigen such as replication incompetent tumor cells along with local release at the tumor site (or site of resection following tumor removal) of a cytokine such as IL-2 which enhances the immune response against the tumor cells.
  • Local release can be obtained using any of several means, but a preferred method is using microparticles to release cytokine over a period of at least days or transduced cells, most preferably replication incompetent tumor cells which are transduced with the gene encoding the cytokine to be released. The latter is shown to release for at least five days after implantation. Microparticles can be designed to release for between hours and weeks or even months, as required.
  • GM-CSF Granulocyte-macropnage colony stimulating factor.
  • Granulocyte-macrophage colony stimulating factor GM-CSF
  • GM-CSF Granulocyte-macrophage colony stimulating factor
  • Interleukin-2 is produced by CD4+ T cells, and in lesser quantities by CD8+ T cells.
  • Secreted IL-2 is a 14 to 17 kD glycoprotein encoded by a single gene on chromosome 4 in humans.
  • IL- 2 acts on the same cells that produce it, i.e., it functions as an autocrine growth factor.
  • IL-2 also acts on other T lymphocytes, including both CD4+ and CD8+ cells.
  • IL-2 induces a local inflammatory response leading to activation of both helper and cytotoxic subsets of T cells.
  • IL-2 also stimulates the growth of natural killer cells and enhances their cytolytic function.
  • Tumor necrosis factor was originally identified as a mediator of tumor necrosis present in the serum of animals exposed to bacterial lipopolysaccharide (LPS) such as endotoxin.
  • LPS lipopolysaccharide
  • the major endogenous source of TNF is the LPS-activated mononuclear phagocyte, although antigen-stimulated T cells, activated natural killer cells, and activated mast cells can also secrete this protein.
  • TNF is initially synthesized as a nonglycosylated transmembrane protein of approximately 25 kD. TNF has potent anti- tumor effects in vitro, although clinical trials of TNF in advanced cancer patients have been discontinued due to toxicity.
  • TNF- ⁇ has a diverse range of biological properties including inducing expression of a number of cytokines such as interleukin-6, interleukin-8, GM-CSF, and granulocye-colony stimulating factor, as well as causing hemorrhagic necrosis in established tumors. TNF has been reported to generate tumor suppression after tumor cell-targeted TNF- ⁇ ; gene transfer. Blankenstein, T., et al., J. Exp. Med. , 173:1047-52 (1991). iv. Interleukin-4.
  • Interleukin-4 is a helper T cell- derived cytokine of approximately 20 kD which stimulates the proliferation of mouse B cells in the presence of anti-Ig antibody (an analog of antigen) and causes enlargement of resting B cells as well as increased expression of class II MHC molecules.
  • the principal endogenous source of IL-4 is from CD4+ T lymphocytes. Activated mast cells and basophils, as well as some CD8 + T cells, are also capable of producing IL-4.
  • IFN- ⁇ Gamma interferon
  • IFN- ⁇ Gamma interferon
  • IFN- ⁇ is a homodimeric glycoprotein containing approximately 21 to 24 kD subunits. IFN- ⁇ is produced by some CD4+ helper T cells and nearly all CD8+ T cells. Transcription is directly initiated as a consequence of antigen activation and is enhanced by IL-2 and interleukin-12. IFN- ⁇ is also produced by natural killer cells. IFN- ⁇ acts as a potent activator of mononuclear phagocytes, acts directly on T and B lymphocytes to promote their differentiation and acts to stimulate the cytolytic activity of natural killer cells.
  • Interleukin-3 Interleukin-3
  • IL-3 also known as multilineage colony-stimulating factor
  • IL-3 is a 20 to 26 kD product of CD4+ T cells that acts on the most immature marrow progenitors and promotes the expansion of cells that differentiate into all known mature cell types.
  • IL-3 has been reported to enhance development of tumor reactive cytotoxic T cells by a CD4-dependent mechanism. Pulaski, B. A., et al., Cancer Res.
  • Interleukin-6 Interleukin-6 (IL-6) is a cytokine of approximately 26 kD that is synthesized by mononuclear phagocytes, vascular endothelial cells, fibroblasts, and other cells in response to IL-1 and, to a lesser extent, TNF. It is also made by some activated T cells. IL-6 transfected into Lewis lung carcinoma tumor cells has been reported to suppress the malignant phenotype and to confer immunotherapeutic competence against parental metastatic cells. Porgador, A. , Cancer Res., 52:3679-87 (1992). viii. Interleukin-7.
  • Interleukin-7 is a cytokine secreted by marrow stromal cells that acts on hematopoietic progenitors committed to the B lymphocyte lineage. IL-7 has been reported to induce CD42+ T cell dependent mmor rejection. Hock, H., et al., J. Exp. Med. , 174: 1291-99 (1991). ix. Granulocyte-colony stimulating factor. Granulocyte- colony stimulating factor (G-CSF) is made by the same cells that make GM- CSF. The secreted polypeptide is approximately 19 kD. G-CSF gene transfer has been reported to suppress tumorgenicity of murine adenocarcinoma.
  • cytokines are known in the art to have an anti-tumor effect and can be used in the pharmaceutical compositions described herein. Moreover, since cytokines are known to have an effect on other cytokines, one can administer the cytokine which elicits one of the cytokines described above, or directly administer one of the cytokines which is elicited. Additional cytokines are known to those skilled in the art and are described in Abbas, et al. , “Cytokines", chapter 12, pp. 239-61, Cellular and Molecular Immunology. 2nd Ed., W.B.
  • cytokines can also be administered in combination with other cytokines, antibodies, cultured immune cells that have anti-tumor reactivity including LAK cells, or chemotherapeutic agents, including radiation therapy.
  • the active compounds can be incorporated into the same vehicle as the cytokines for administration, or administered in a separate vehicle. i. Chemotherapeutic agents.
  • cytokines can be used alone, or in combination with other chemotherapeutic agents.
  • chemotherapeutic agents include cytotoxic agents such as paclitaxel, camptothecin, ternozolamide, l,3-bis(2- chloroethyl)-l-nitrosourea (BCNU), adriamycin, platinum drugs such as cisplatin, differentiating agents such as butyrate derivatives, transforming growth factor such as factot-alp a-Pseudomonas exotoxin fusion protein, and antibodies to mmor antigens, especially glioma antigens, such as monoclonal antibody 81C6.
  • cytotoxic agents such as paclitaxel, camptothecin, ternozolamide, l,3-bis(2- chloroethyl)-l-nitrosourea (BCNU), adriamycin, platinum drugs such as cisplatin, differentiating agents such as buty
  • These agents can be incorporated into polymeric matrices for delivery along with the cytokines. See, for example, Domb, et al., Polym. Prepr. , 32(2):219-220 (1991), reported incorporating the water soluble chemotherapeutic agents carboplatin, an analog of cisplatin, and 4- hydroperoxycyclophosphamide into a biodegradable polymer matrix for treating tumors, with promising results in. ii. Other pharmaceutically active compounds. In variations of these embodiments, it may be desirable to include other pharmaceutically active compounds, such as steroidal antiinflammatories which are used to reduce swelling, antibiotics, antivirals, or anti-angiogenic compounds.
  • steroidal antiinflammatories which are used to reduce swelling, antibiotics, antivirals, or anti-angiogenic compounds.
  • dexamethasone a synthetic corticosteroid used systemically to control cerebral edema
  • Other compounds which can be included are preservatives, antioxidants, and fillers, coatings or bulking agents which may also be utilized to alter polymeric release rates.
  • Cytokine Formulations The cytokines can be administered in a pharmaceutically acceptable carrier such as saline, phosphate buffered saline, cells transduced with a gene encoding the cytokine, microparticles, or other conventional vehicles. .
  • the cytokines can be encapsulated into a biocompatible polymeric matrix, most preferably biodegradable, for use in the treatment of solid tumors.
  • the cytokine is preferably released by diffusion and/or degradation over a therapeutically effective time, usually eight hours to five years, preferably one week to one year.
  • microencapsulated includes incorporated onto or into or on microspheres, microparticles, or microcapsules.
  • Microcapsules is used interchangeably with microspheres and microparticles, although it is understood that those skilled in the art of encapsulation will recognize the differences in formulation methods, release characteristics, and composition between these various modalities.
  • the microspheres can be directly implanted or delivered in a physiologically compatible solution such as saline.
  • Biocompatible polymers can be categorized as biodegradable and non- biodegradable. Biodegradable polymers degrade in vivo as a function of chemical composition, method of manufacture, and implant structure. Synthetic and natural polymers can be used although synthetic polymers may be preferred due to more uniform and reproducible degradation and other physical properties. Examples of synthetic polymers include poly anhydrides, poly hydroxy acids such as poly lactic acid, poly gly colic acid and copolymers thereof, polyesters, poly amides, polyorthoesters, and some polyphosphazenes. Examples of naturally occurring polymers include proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin. The ideal polymer must also be strong, yet flexible enough so that it does not crumble or fragment during use.
  • Cytokines and optionally, other drugs or additives can be encapsulated within, throughout, and/or on the surface of the implant.
  • the cytokine is released by diffusion, degradation of the polymer, or a combination thereof.
  • biodegradable polymers There are two general classes of biodegradable polymers: those degrading by bulk erosion and those degrading by surface erosion. The latter polymers are preferred where more linear release is required.
  • the time of release can be manipulated by altering chemical composition; for example, by increasing the amount of an aromatic monomer such as p-carboxyphenoxy propane (CPP) which is copolymerized with a monomer such as sebacic acid (SA).
  • Non-biodegradable polymers remain intact in vivo for extended periods of time (years). Agents loaded into the non-biodegradable polymer matrix are released by diffusion through the polymer's micropore lattice in a sustained and predictable fashion, which can be tailored to provide a rapid or a slower release rate by altering the percent cytokine loading, porosity of the matrix, and implant structure.
  • Ethylene-vinyl acetate copolymer (EVAc) is an example of a nonbiodegradable polymer that has been used as a local delivery system for proteins and other macromolecules, as reported by Langer, R. , and Folkman, J. , Nature (London), 263:797-799 (1976).
  • polyurethanes poly aery lonitriles
  • polyphosphazenes some polyphosphazenes.
  • polymer and cytokines to be released are incorporated into the delivery device, although other biocompatible, preferably biodegradable or metabolizable, materials can be included for processing purposes as well as additional therapeutic agents.
  • Buffers, acids and bases are used to adjust the pH of the composition.
  • Agents to increase the diffusion distance of agents released from the implanted polymer can also be included.
  • Fillers are water soluble or insoluble materials incorporated into the formulation to add bulk.
  • Types of fillers include sugars, starches and celluloses.
  • the amount of filler in the formulation will typically be in the range of between about 1 and about 90% by weight.
  • Spheronization enhancers facilitate the production of spherical implants. Substances such as zein, microcrystalline cellulose or microcrystalline cellulose co-processed with sodium carboxymethyl cellulose confer plasticity to the formulation as well as implant strength and integrity.
  • extrudates that are rigid, but not plastic result in the formation of dumbbell shaped implants and/or a high proportion of fines. Extrudates that are plastic, but not rigid, tend to agglomerate and form excessively large implants. A balance between rigidity and plasticity must be maintained.
  • the percent of spheronization enhancer in a formulation depends on the other excipient characteristics and is typically in the range of 10 to 90% (w/w).
  • Disintegrants are substances which, in the presence of liquid, promote the disruption of the implants.
  • the function of the disintegrant is to counteract or neutralize the effect of any binding materials used in the formulation.
  • the mechanism of disintegration involves, in large part, moisture absorption and swelling by an insoluble material.
  • disintegrants include croscarmellose sodium and crospovidone which are typically incorporated into implants in the range of 1 to 20% of total implant weight.
  • soluble fillers such as sugars (mannitol and lactose) can also be added to facilitate disintegration of the implants.
  • Surfactants may be necessary in implant formulations to enhance wettability of poorly soluble or hydrophobic materials.
  • Surfactants such as polysorbates or sodium lauryl sulfate are, if necessary, used in low concentrations, generally less than 5%.
  • Binders are adhesive materials that are incorporated in implant formulations to bind powders and maintain implant integrity. Binders may be added as dry powder or as solution. Sugars and natural and synthetic polymers may act as binders. Materials added specifically as binders are generally included in the range of about 0.5 to 15% w/w of the implant formulation. Certain materials, such as microcrystalline cellulose, also used as a spheronization enhancer, also have additional binding properties.
  • Various coatings can be applied to modify the properties of the implants. Three types of coatings are seal, gloss and enteric. The seal coat prevents excess moisture uptake by the implants during the application of aqueous based enteric coatings. The gloss coat improves the handling of the finished product.
  • Water-soluble materials such as hydroxypropyl cellulose can be used to seal coat and gloss coat implants.
  • the seal coat and gloss coat are generally sprayed onto the implants until an increase in weight between about 0.5% and about 5%, preferably about 1 % for seal coat and about 3% for a gloss coat, has been obtained.
  • Enteric coatings consist of polymers which are insoluble in the low pH (less than 3.0) of the stomach, but are soluble in the elevated pH (greater than 4.0) of the small intestine.
  • Polymers such as Eudragit * , RohmTech, Inc., Maiden, MA, and Aquateric * , FMC Corp. , Philadelphia, PA, can be used and are layered as thin membranes onto the implants from aqueous solution or suspension.
  • the enteric coat is generally sprayed to a weight increase of about one to about 30%, preferably about 10 to about 15%, and can contain coating adjuvants such as plasticizers, surfactants, separating agents that reduce the tackiness of the implants during coating, and coating permeability adjusters.
  • coating adjuvants such as plasticizers, surfactants, separating agents that reduce the tackiness of the implants during coating, and coating permeability adjusters.
  • Other types of coatings having various dissolution or erosion properties can be used to further modify implant behavior. Such coatings are readily
  • Controlled release devices are typically prepared in one of several ways.
  • the polymer can be melted, mixed with the substance to be delivered, and then solidified by cooling.
  • Such melt fabrication processes require polymers having a melting point that is below the temperature at which the substance to be delivered and polymer degrade or become reactive.
  • the device can be prepared by solvent casting, where the polymer is dissolved in a solvent, and the substance to be delivered dissolved or dispersed in the polymer solution. The solvent is then evaporated, leaving the substance in the polymeric matrix. Solvent casting requires that the polymer be soluble in organic solvents and that the agents to be encapsulated be soluble or dispersible in the solvent. Similar devices can be made by phase separation or emulsification or even spray drying techniques. In still other methods, a powder of the polymer is mixed with the cytokine and then compressed to form an implant.
  • Methods of producing implants also include granulation, extrusion, and spheronization.
  • a dry powder blend is produced including the desired excipients and microspheres.
  • the dry powder is granulated with water or other non-solvents for microspheres such as oils and passed through an extruder forming "strings" or "fibers" of wet massed material as it passes through the extruder screen.
  • the extrudate strings are placed in a spheronizer which forms spherical particles by breakage of the strings and repeated contact between the particles, the spheronizer walls and the rotating spheronizer base plate.
  • the implants are dried and screened to remove aggregates and fines.
  • Cytokine secreting Genetically engineered cells Cytokine secreting cells can be genetically engineered by methods known in the art to deliver cytokines systemically or locally. Cells can either be transformed or transduced, with bacterial or viral DNA vectors, respectively. In a preferred embodiment the cells are mmor cells. To prevent replication and preserve cytokine production, the transduced cells can be irradiated prior to in vivo injection.
  • the advantage of the tumor cells is that the patient is exposed to antigen in combination with cytokine stimulating the immune response to the cytokine.
  • mmor cells engineered to secrete IL-4 are described in Golumbek, P. T., et al., Science, 254:713-6 (1991) and Yu, J. S., et al., Cancer Res. 53:3125-8 (1993).
  • IL-2 gene transfer into mmor cells is described in Gansbacher, B., et al., J. Exp. Med. , 172:1217-24 (1990).
  • Tumor cells engineered to secrete GM-CSF are described in Dranoff, G. , et al., Proc. Natl. Acad. Sci.
  • TNF gene transfer is described in Blankenstein, T. , et al., J. Exp. Med., 73:1047-52 (1991).
  • IL-6 transfection into lung carcinoma mmor cells is described in Progador, A, et al., Cancer Res. , 52:3679-87 (1992).
  • ⁇ lFN cDNA transduced into non-immunogenic sarcoma is described in Restifo, N., J. Exp. Med. , 175:1423-28 (1992).
  • G-CSF gene transfer is described in Colombo, M., et al., Cancer Res., 52:4853-57 (1991).
  • cytokines including IL-3 and IL-7 can be secreted from cells by applying the methods used for the cytokines described above or by the general procedures for genetically modifying nonimmunogenic murine fibrosarcoma described in Karp, S. E. , et al., /. Immunol. , 150:896-908 (1993) and Jaffee, E. et al., J. Immunotherapy, in press (1995).
  • the publications cited above describe methods of genetically engineering cells to secrete cytokines.
  • cancer vaccines expressing two or more cytokines from engineered vectors containing genes encoding two different cytokines or by sequential recombinant retrovirus-mediated genetic transductions can be prepared.
  • Suitable pharmaceutical vehicles are known to those skilled in the art.
  • Inducers can also be used to produce cells which secrete cytokines. Inducers may be used alone in some cases or in combination with transforming agents, and include mmor necrosis factor (TNF), endotoxin, and other agents known to those skilled in the art.
  • TNF mmor necrosis factor
  • the cultured cells are exposed to an amount effective to activate the cells, as determined by cytokine expression, immunoglobulin secretion, and/or other indicators such as proliferation or alteration of cell surface properties or markers.
  • cells are initially exposed to a small amount to "prime" the cells, then to a subsequent dose to elicit greater activation of the cells.
  • Cells can be administered in medium or washed and administered in saline. Alternatively, cells can be encapsulated in a polymeric matrix and administered. II. Administration to Patients
  • the cytokines described herein or their functionally equivalent derivatives can be administered alone or in combination with, either before, simultaneously, or subsequent to, treatment using other chemotherapeutic or radiation therapy or surgery.
  • a preferred embodiment is systemic administration of a first cytokine such as GM-CSF, most preferably in combination with mmor antigen in the form of replication incompetent mmor cells, followed by local administration during surgery or by injection of either a biocompatible polymeric matrix loaded with the selected cytokine or cytokine secreting cells, using dosages determined as described herein.
  • Local administration can be at a site adjacent to the mmor to be removed, or in the tumor, or in the place where the mmor was removed.
  • the dosages for functionally equivalent derivatives can be extrapolated from the in vivo data.
  • An effective amount is an amount sufficient to induce an anti-tumor effect as measured either by a longer survival time or decrease in mmor size.
  • Local administration can also be accomplished using an infusion pump, for example, of the type used for delivering insulin or chemotherapeutic agents to specific organs or tumors.
  • the polymeric implants or cytokine secreting cells are implanted at the site of a mmor, two weeks after systemic administration of the polymeric implants or cytokine secreting cells, for example, by subcutaneous injection at a distant site.
  • biodegradable polymers preferably they are less than about 100 to 200 microns in diameter and injected by means of a catheter or syringe; it is not necessary to remove the implant following release of the immunotherapeutic agent.
  • the cytokines can also be combined with other therapeutic modalities, including radiotherapy, chemotherapeutic agents administered systemically or locally, and other immunotherapy.
  • the cytokines are preferably administered to brain cancer patients.
  • the cytokine combination therapy can also be applied to all cancer patients including those having breast cancer, lung cancer, and colon cancer.
  • the treatment will provide an immediate response at the site of the local administration as well as provide long term memory protection against cancer at the site of local administration and at a site distant from the site of local administration.
  • the immunotherapy described herein is therefore useful in preventing or treating metastases. III. Examples.
  • Example 1 Systemic vaccination with GM-CSF-transduced tumor cells Tumor cell lines and animals.
  • B16-F10 melanoma cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum and penicillin/ streptomycin. Animals used for all experiments were 6 to 12 week old C57BL/6 female mice obtained from Harlan (Indianapolis, IN).
  • B16-F10 melanoma cells were transduced with murine GM-CSF gene by using the replication-defective MFG retroviral vector as previously described in Dranoff, G., et al., Proc. Natl. Acad. Sci.
  • mice were anesthetized with an intraperitoneal injection of 0.1 ml of a stock solution containing ketamine hydrochloride 25 mg/ml, xylazine 2.5 mg/ml, and 14.25% ethyl alcohol diluted 1:3 in 0.9% NaCl solution.
  • the surgical site was shaved and prepared with 70% ethyl alcohol and prepodyne solution. After a midline incision, a 1 mm burr hole center 2 mm posterior to the coronal sumre and 2 mm lateral to the sagittal sumre was made.
  • Animals were then placed in a stereotactic frame and cells were delivered by a 26 gauge needle to a depth of 3 mm over a period of 3 minutes.
  • the total volume of injected cells was 10 ⁇ l.
  • the needle was removed, the site irrigated with sterile 0.9% NaCl solution, and the skin was sutured closed with 4.0 vicryl.
  • mice Development of an intracranial B16-F10 melanoma model in C57BL/6 mice.
  • animals were divided into five groups of at least eight animals and received stereotactic intracranial injections of either 10 2 , 10 3 , 10 4 , or 10 5 wild type B16-F10 melanoma cells into the left parietal region by the method described above.
  • Control animals received similar intracranial injections of 0.9% NaCl solution. Animals were assessed daily for survival.
  • brains were removed at the time of death and fixed in 10% formalin for at least 5 days, sectioned, embedded in paraffin, and stained with hematoxylin and eosin.
  • Vaccination studies Cytokine-secreting B16-F10 melanoma cells were evaluated for their vaccination properties and ability to protect against intracranial challenge with wild type B16-F10 melanoma.
  • animals were treated with a single subcutaneous flank injection of 10 6 irradiated GM-CSF secreting B16-F10 cells using a tuberculin syringe with a 27 gauge needle.
  • the total amount of GM-CSF secreted by the transduced cells in vitro prior to injection was between 50 and 60 ng per 10 6 cells per day.
  • the transduced cells were irradiated prior to in vivo injection.
  • Results Figure 2 shows the results of the systemic vaccination studies performed on animals receiving single subcutaneous injections of 10 6 irradiated B16-F10 cells engineered by gene transfer to secrete GM-CSF.
  • GM-CSF secreting cells administered in this manner were found to protect against intracranial challenge with the wild type mmor.
  • Median survival for animals treated with the GM-CSF vaccine and then challenged 2 weeks later with wild type B16 in the brain was 27 days, compared to 19 and 17 days for control animals treated with a vaccine of irradiated, wild type cells (which did not secrete GM-CSF) or medium, respectively (p ⁇ 0.0001).
  • Example 2 Systemic vaccination with microencapsulated GM-CSF
  • mice Thirty-two C57BL/6 mice were divided into four groups of eight and given flank vaccinations with either: 1) saline, 2) 2500 ng bolus GM-CSF and 1 x 10 6 irradiated B16 melanoma cells, 3) 2500 ng GM-CSF microspheres and 1 x 10 6 irradiated melanoma cells, or 4) 1 x 10 6 irradiated melanoma cells transduced to produce GM-CSF.
  • Results The median survival of the GM-CSF microsphere group and the transduced GM-CSF secreting group was 27.5 days each.
  • This study demonstrates that a significant anti-tumor immune response is generated using a systemic vaccination with GM-CSF microspheres. This response is comparable to that generated by GM-CSF transduced mmor cells and represents a practical method for delivering GM-CSF in treatments employing GM-CSF immunotherapy.
  • Example 3 Local intracranial delivery of IL-2-transduced tumor cells
  • Animals received intracranial stereotactic co-injections of either 10 3 , 10 4 , 10 5 , or 10 6 irradiated cytokine-producing B16-F10 melanoma cells, which produce 80 ng of IL-2 per 10 6 cells every twenty-four hours as determined by ELISA.
  • Control animals received co-injections of irradiated non-IL-2 producing wild type B16-F10 at the equivalent doses of 10 3 , 10 4 , 10 5 , or 10 6 cells.
  • IL-2 transduced cells were found to be highly effective in treating intracranial mmor when they were delivered directly to the brain. Initial studies showed that the IL-2 effect was dose-dependent. The results are shown in Figure 3. Doses of 0.08 ng per day of IL-2 (10 3 IL-2 producing cells) showed no enhanced survival compared to controls, whereas a dose of 0.8 ng per day (10 4 IL-2 producing cells) showed a trend toward prolonged survival that did not reach statistical significance. When tested at a dose of 8.0 ng per day (10 5 IL-2 producing cells), a significant prolongation in survival was seen.
  • inflammation was notably absent in the brain parenchyma of the treated animals. Lymphocytes were observed, however, in the cerebrospinal fluid spaces of the IL-2 treated animals.
  • Example 4 Intracranial delivery of microencapsulated interleukin-2 produces long term memory and protects against re- challenges
  • IL-2 was incorporated into biodegradable sustained release microspheres having an average diameter of 4 ⁇ m. These microspheres were stereotactically injected into the brains of mice to deliver 100 ng of IL-2 over a 7 day period. Safety testing showed no evidence of clinical or histologic toxicity from this dose over a 120 day period.
  • a lethal challenge of B16 melanoma was stereotactically co-injected into the left parietal lobe of 25 mice with either saline, empty microspheres, or the IL-2 containing microspheres.
  • 9L glioma cells were co-injected into the left parietal lobe of rats.
  • mice were co-injected intracranially with IL-2 microspheres and melanoma cells which had been irradiated to prevent replication. Thirty days later these animals and a group of control mice having no previous treatment were challenged intracranially with the lethal dose of live melanoma with no additional IL-2 therapy. Results Seventy-five percent of the IL-2 treated mice (6 of 8) were alive with no sign of mmor 90 days after initial treatment while median survival of untreated controls was 18 days. No animal survived more than 21 days. The empty microspheres had no effect on survival. In a similar mouse melanoma experiment, no impact on survival from either a bolus injection of
  • IL-2 to the mmor site or systemic IL-2 administration was observed.
  • microsphere delivery of IL-2 prolonged median survival in rats to 55 days compared to 25 days for the controls.
  • Previous IL-2 vaccination extended median survival by 45 percent, with 2 to 5 animals alive at 160 days after mmor challenge with all controls dead by 23 days.
  • Kalyanasundaram S., et al., Proceed. Intern. Symp. Control. Rel. Biomater. ,
  • Example 5 Intracranial delivery of IL-2 transduced cells produces long term memory and protects against re-challenges
  • mice The melanoma challenge was fatal to all previously untreated mice, the median survival was 17 days.
  • mice rechallenged with media all lived an additional 70 days.
  • mice Twenty-four C57B16 mice were injected intracranially with irradiated,
  • IL-2 secreting B16-F10 melanoma cells and 12 C57B16 mice were injected with medium, as controls. All animals simultaneously received a co-injection of wild type B16-F10 cells which were uniformly fatal to untreated mice.
  • the 8 (of 9) long- term survivors who rejected the first flank challenge, along with 8 new control animals, were challenged a second time in the flank with a larger bolus of 50,000 wild type B16-F10 melanoma cells, 126 days after the first challenge. Seventeen days later, 8 of 8 control mice had developed tumors whereas only 2 of 8 of the long term survivors had a flank mmor (p 0.01).
  • Example 7 Local intracranial delivery of IL-4-transduced tumor cells Poorly immunogenic B16-F10 melanoma cells were transfected with the IL-4 gene to deliver IL-4 at the site of a mmor in the central nervous system.
  • the transfected B16-F10 cells were irradiated to prevent replication and injected intracranially in eight C57BL/6 mice along with non-irradiated, non-transfected B16-F10 (wild type) melanoma cells.
  • Example 8 Combination therapy: Systemic vaccination with GM-CSF transductants and local intracranial administration of IL-2 transductants
  • Control animals received either the subcutaneous GM- CSF vaccine and intracranial treatment with medium, or subcutaneous vaccination with medium and intracranial treatment with 5 x 10 4 IL-2 producing cells. Additional controls received vaccinations of medium alone followed by intracranial treatment with medium along with an intracranial mmor challenge. Results
  • the GM-CSF vaccine generates a cohort of CD4 + and CD8 + T-cells specific for a mmor antigen, which activity of the cells is enhanced by the subsequent local IL-2 therapy at the mmor site.
  • the results correspond with the theory that while the blood-brain barrier poses an obstacle to delivery of chemotherapeutic agents to the brain, it functions as an avenue of entry into the central nervous system for immunologically active cells. Wekerly, H., et al., TINS, 27:1-7 (1986).
  • Example 9 Systemic vaccination with GM-CSF transductants and local intracranial administration of IL-2 transductants produces long term memory protection against re-challenges
  • mice Forty-nine C57B16 mice were vaccinated in the flank with either GM-CSF producing B16-F10 cells or medium followed by intracranial delivery of IL-2 or medium 2 weeks later. Every animal received an intracranial co-injection of wild-type melanoma at a dose uniformly fatal to untreated animals. Initial survivors and a new control group were rechallenged with a second intracranial dose of wild-type B16-F10 melanoma 134 days after their first challenge.
  • IL-2 interleukin-2
  • IL-2 was incorporated into biodegradable sustained release microspheres (average diameter 4 ⁇ m) which were stereotactically injected into the brains of mice to deliver 100 ng IL-2 over a 7 day period.
  • Safety testing showed no evidence of clinical or histologic toxicity from this dose over a 120 day period.
  • mice were co-injected intracranially with IL-2 microspheres and melanoma cells (see example 10) which had been irradiated to prevent replication. 30 days later these animals and a group of control mice (no previous treatment) were challenged intracranially with the lethal dose of live melanoma with no additional IL-2 therapy. Results Previous IL-2 vaccination extended median survival by 45 % and generated 2/5 animals alive at 160 days after mmor challenge with all controls dead by 23 days. These results suggest that brain interstitial immunotherapy with IL-2 delivered by microspheres is an effective and safe treatment against brain metastases and may prevent mmor recurrence.
  • Example 12 Combination therapy with locally delivered IL-2 and chemotherapeutic agents
  • a combination therapy consisting of immunotherapy, using non- replicating genetically engineered mmor cells that produce IL-2, and local delivery of chemotherapy was tested to determine if the component therapies acted synergistically against an intracranial mmor challenge in a murine brain mmor model.
  • B16-F10 melanoma cells were transduced with murine IL-2 using a replication-defective MFG retroviral vector.
  • Chemotherapeutic agents, BCNU and carboplatin were incorporated into controlled-release polymers.
  • animals received polymer implants containing 4.0% BCNU, 1 % carboplatin, or placebo.
  • Example 13 IL-2 inhibited tumor growth in murine model of hepatic melanoma metastasis Methods
  • Useful immunotherapeutic strategies must demonstrate success in tumors within the liver, an organ with unique immunologic properties and a common site of metastases in humans.
  • the anti-tumor effects of local or paracrine IL-2 delivery was evaluated using the non-immunogenic B16-F10 melanoma implanted into the liver of C57B1/6 mice. Irradiated autologous mmor cells transfected with the IL-2 gene was used as the source of IL-2.
  • mice without any mmor were significantly less in those receiving the highest IL-2 dose (p ⁇ 0.05) and, in those livers in which mmor was present, the mean mmor volume was markedly reduced (2 mm 3 versus 884 mm 3 , p ⁇ 0.05). This effect was not seen when IL-2 was administered at a distant site nor in those mice receiving irradiated wild type controls. The effect was dose-dependent, with maximum inhibition seen at the highest IL-2 dose. Histologic examination revealed a marked infiltrate at the site within the livers with IL-2 secreting cells, comprised of both lymphocytes and Kupffer cells. In summary, these results demonstrate that local delivery of IL-2 inhibited mmor growth in this murine model of hepatic melanoma metastasis. This strategy has potential application for the development of immunotherapy of liver tumors.
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WO2020123716A1 (en) 2018-12-11 2020-06-18 Obsidian Therapeutics, Inc. Membrane bound il12 compositions and methods for tunable regulation
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