EP0835130A1 - Gentherapie von soliden tumoren mit interferone allein oder mit anderen immunoeffektoren proteinen - Google Patents

Gentherapie von soliden tumoren mit interferone allein oder mit anderen immunoeffektoren proteinen

Info

Publication number
EP0835130A1
EP0835130A1 EP96921666A EP96921666A EP0835130A1 EP 0835130 A1 EP0835130 A1 EP 0835130A1 EP 96921666 A EP96921666 A EP 96921666A EP 96921666 A EP96921666 A EP 96921666A EP 0835130 A1 EP0835130 A1 EP 0835130A1
Authority
EP
European Patent Office
Prior art keywords
interferon
cells
interleukin
ifn
molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96921666A
Other languages
English (en)
French (fr)
Inventor
Sidney Pestka
Srijata Sarkar
Idhaliz Flores
Yacov Ron
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
Original Assignee
University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Medicine and Dentistry of New Jersey, Rutgers State University of New Jersey filed Critical University of Medicine and Dentistry of New Jersey
Publication of EP0835130A1 publication Critical patent/EP0835130A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer

Definitions

  • the present invention relates to vectors and compositions for gene therapy strategies against solid tumors, particularly malignant tumors.
  • IFN- ⁇ human interferon alpha
  • IFN- ⁇ produced by mononuclear leukocytes and other cells, is an important regulator of cellular growth and differentiation affecting cellular communication and signal transduction pathways as well as immunological control (Pestka et al. 1987, Ann. Rev. Biochem. 56:727-777); (Lengyl P, 1993, Proc. Natl. Acad. Sci. USA 90:5893-5895).
  • MHC major histocompatibility complex
  • gp96 a heat shock protein associated with antigen presentation
  • B7 T Cell Costimulatory Molecule The B7 T cell costimulatory molecule is expressed on activated B cells, macrophages and dendritic cells, the so-called professional antigen presenting cells (APC).
  • APC professional antigen presenting cells
  • the invention is directed to a solid tumor vaccine comprising tumor cells transfected to express interferon- ⁇ in a pharmaceutically acceptable excipient.
  • the tumor cells are also transfected to express an immunomodulatory molecule, with the proviso that the immunomodulatory molecule is not interferon- ⁇ .
  • additional tumor cells i.e. , cells not transfected with IFN- ⁇ are transfected with the immunomodulatory molecule.
  • immunomodulatory (or immuno-effector) molecules include, but are not limited to, interferon- ⁇ , interferon-/?, interferon- ⁇ , interferon- ⁇ , tumor necrosis factor- ⁇ , tumor necrosis factor-/3, interleukin-2, interleukin-7, interleukin- 12, interleukin- 15, B7-1 T cell costimulatory molecule, B7-2 T cell costimulatory molecule, immune cell adhesion molecule (ICAM) -I T cell costimulatory molecule, granulocyte colony stimulatory factor, granulocyte-macrophage colony stimulatory factor, and combinations thereof.
  • the tumor cells are from the tumor in the subject.
  • tumor cells from a corresponding or closely related tumor cell line can be used.
  • a soluble immunomodulatory molecule can be included in a vaccine of the invention.
  • soluble immunomodulatory molecules include, but are not limited to, interferon- ⁇ , interferon-jS, interferon- ⁇ , interferon- ⁇ , mmor necrosis factor- ⁇ , mmor necrosis factor-jS, interleukin-2, interleukin-7, interleukin- 12, interleukin- 15, granulocyte colony stimulatory factor, granulocyte-macrophage colony stimulatory factor, and combinations thereof.
  • the present invention is further directed to a related method for treating a solid mmor.
  • the method comprises introducing into a subject suffering a solid mmor a therapeutically effective number of mmor cells from a solid mmor, which mmor cells are transfected to express interferon- ⁇ .
  • the mmor cells are also transfected to express an immunomodulatory molecule, with the proviso that the immunomodulatory molecule is not interferon- ⁇ .
  • a therapeutically effective number of additional mmor cells transfected to express an immunomodulatory molecule can be introduced into the subject.
  • the therapeutic method of the invention comprises introducing an therapeutically effective amount of a soluble immunomodulatory molecule into the subject.
  • the mmor cells are from the mmor in the subject.
  • tumor cells from a corresponding or closely related mmor cell line can be used. More preferably, the mmor cells are introduced in proximity of the mmor in the subject.
  • the invention is directed to a method for treating a solid mmor comprising introducing into a subject suffering from a solid mmor an expression vector directed to cells of the solid tumor, which expression vector codes on expression for interferon- ⁇ .
  • the method further comprises introducing into the subject a second expression vector directed to cells of the solid mmor, which second expression vector codes on expression for an immunomodulatory molecule.
  • the expression vector directed to cells of the solid mmor additionally codes on expression for an immunomodulatory molecule.
  • the present invention is directed to a solid tumor vaccine comprising mmor cells transfected to express interferon- ⁇ and an immunomodulatory molecule, in a pharmaceutically acceptable excipient.
  • additional mmor cells transfected to express an immunomodulatory molecule with the proviso that the immunomodulatory molecule is not interferon- ⁇ .
  • immunomodulatory molecules include but are not limited to interferon- ⁇ , interferon-/?, interferon- ⁇ , interferon-r, mmor necrosis factor- ⁇ , mmor necrosis factor-/3, interleukin-2, interleukin-7, interleukin- 12, interleukin-15, B7-1 T cell costimulatory molecule, B7-2 T cell costimulatory molecule, immune cell adhesion molecule (ICAM) -I T cell costimulatory molecule, granulocyte colony stimulatory factor, granulocyte-macrophage colony stimulatory factor, and combinations thereof.
  • IAM immune cell adhesion molecule
  • the immunomodulatory molecule can be expressed by the mmor cells transfected with IFN- ⁇ , by additional mmor cells not transfected with IFN- ⁇ , but only transfected with the immunomodulatory molecule, or by addition of a soluble immunomodulatory molecule.
  • the solid mmor vaccine of this aspect of the invention further comprises a soluble immunomodulatory molecule, e.g. , interferon- ⁇ , interf eron-/3, interferon- ⁇ , interferon- ⁇ , mmor necrosis factor- ⁇ , mmor necrosis factor-/3, interleukin-2, interleukin-7, interleukin- 12, interleukin- 15, granulocyte colony stimulatory factor, granulocyte-macrophage colony stimulatory factor, and combinations thereof.
  • a soluble immunomodulatory molecule e.g. , interferon- ⁇ , interf eron-/3, interferon- ⁇ , interferon- ⁇ , mmor necrosis factor- ⁇ , mmor necrosis factor-/3, interleukin-2, interleukin-7, interleukin- 12, interleukin- 15, granulocyte colony stimulatory factor, granulocyte-macrophage colony stimulatory factor, and combinations thereof.
  • the invention provides a method for treating a solid mmor comprising introducing into a subject suffering the solid tumor a therapeutically effective number of mmor cells from a solid mmor, which mmor cells are transfected to express interferon- ⁇ and an immunomodulatory molecule, with the proviso that the immunomodulatory molecule is not interferon- ⁇ .
  • the method of the invention comprises introducing a therapeutically effective number of additional mmor cells transfected to express an immunomodulatory molecule into the subject.
  • the mmor cells can be transfected both with IFN- ⁇ and an immunomodulatory molecule, or separately with IFN- ⁇ and the immunomodulatory molecule.
  • the method of the invention may involve introducing the therapeutically effective amount of a soluble immunomodulatory molecule into the subject.
  • the mmor cells are from the mmor in the subject.
  • a related or similar tumor cell line can be used.
  • the mmor cells are introduced in proximity of the tumor in the subject.
  • the invention provides a method for treating a solid mmor comprising introducing into a subject suffering from a solid mmor an expression vector directed to cells of the solid mmor, which expression vector codes on expression for interferon- ⁇ , and introducing into the subject a second expression vector directed to cells of the solid mmor, which second expression vector codes on expression for an immunomodulatory molecule, with the proviso that the immunomodulatory molecule is not interferon- ⁇ .
  • the method for treating a solid mmor comprises introducing into a subject suffering from a solid mmor an expression vector directed to cells of the solid mmor, which expression vector codes on expression for interferon- ⁇ and an immunomodulatory molecule, with the proviso that the immunomodulatory molecule is not interferon- ⁇ -
  • the present invention is directed toward treatment and therapy of solid mmors in a subject.
  • the subject is a mammal, and more preferably, a human.
  • the subject is a mouse suffering from melanoma.
  • FIGURE 1 Cytofluorographic analysis of MHC class I and class II expression on B16 parental and IFN- ⁇ cDNA-transfected cells. Expression of MHC class I and class II molecules was determined as described in "Materials and Methods", in Example 1. Briefly, 1 x 10 6 B16 parental (panel A) or B 16. IFN- ⁇ cells (panel B) were incubated with 50 ⁇ l of culmre supernatant containing MAb 28-8-6S (anti H- 2K b ) or MAb AF6-120.1.2 (anti-I-A b ) at 4°C for 30 min.
  • MAb 28-8-6S anti H- 2K b
  • MAb AF6-120.1.2 anti-I-A b
  • FIGURE 2 Cytofluorograph in analysis of B7 expression on B7 cDNA- transfected cells. Expression of B7 molecules was determined as described in "Materials and Methods" (Example 1, infra). Briefly, 1 x IO 6 B16 parental (A), B16.B7.7 (B), or B16.IFN- ⁇ /B7.11 (C) cells were incubated with 50 ⁇ l of culmre supernatant containing MAb 1G10-G9 (anti-mouse B7) at 4°C for 30 min. After washing with medium containing 10% FBS, cell pellets were stained by incubation with goat anti-rat FITC-conjugated secondary antibody (80 ⁇ g/ml) at 4°C for 30 min.
  • FIGURE 3 Tumorigenicity of B16 parental and B16.IFN- ⁇ cells in syngeneic mice.
  • B16 parental or B16. IFN- ⁇ cells were injected s.c. into 6-8 weeks old male syngeneic C57BL/6J mice in 0.2 ml HBSS.
  • B16.pD5neo a group of mice was injected with B16 cells transfected with vector only (B16.pD5neo). Animals were monitored twice a week by palpation of the injection site over a period of 100 days.
  • Panel A Percentage of mice with a palpable mmor at various days after injection of 1 x 10 5 B16 parental or B 16. IFN- ⁇ cells.
  • Panel B Percentage of mice with a palpable mmor at various days after injection of 1 x IO 6 B16 parental, B 16. IFN- ⁇ , or B16.pD5neo cells.
  • FIGURE 4 Tumorigenicity of B16.B7, B16.IFN- ⁇ /B7 and a mixture of B16.B7 and B16.IFN- ⁇ cells.
  • mice B16.B7 and B16.
  • IFN- ⁇ cells were injected s.c. into syngeneic C57BL/6J mice at 1 x 10 6 cells/mouse. Parental B16 and B 16. IFN- ⁇ cells were also injected as controls. Mice were monitored twice a week by palpation of the injection sites. Animals with massive mmor burden were sacrificed for humane reasons. Percentage of mice with a palpable mmor at various days after injection of 1 x IO 6 B16 parental, B16.IFN- ⁇ , B16.B7, B16.IFN- ⁇ /B7, or after injection of a combination of B16. IFN- ⁇ and B16.B7.7 cells (1 x 10 6 cells each) are shown.
  • FIGURE 5 Tumorigenicity of B16 parental and cytokine-secreting cells in syngeneic C57/BL6 mice. Mice were injected with either 1 x 10 6 B16 parental (5 mice) or B 16. IFN- ⁇ (10 mice) cells subcutaneously on the flank. Tumor development was monitored twice a week by palpation of the injection site.
  • FIGURE 6 Tumorigenicity of a combination of B16.IFN- ⁇ and B16.IFN- ⁇ cells.
  • C57BL/6 mice were injected with 1 x 10 6 B16 (5 mice), B16. IFN- ⁇ (5 mice), B16. IFN- ⁇ (14 mice) and a combination of B16. IFN- ⁇ and B16. IFN- ⁇ (20 mice) injected s.c. in the flank. Tumor development was monitored as described under "Materials and Methods" in Example 2.
  • FIGURE 7 Tumorigenicity of a combination of B16.IFN- ⁇ and B16.TNF- ⁇ cells.
  • C57BL/6 mice were injected with 1 x IO 6 B16.
  • TNF- ⁇ cells (5 mice), combination of B16. IFN- ⁇ and B16.
  • TNF- ⁇ cells (1 x 10 6 each; 10 mice) and B 16.
  • TNF- ⁇ cells on the left flank (1 x IO 6 cells; 10 mice).
  • FIGURE 8 Tumorigenicity of a combination of B16 and B16.IFN- ⁇ cells in a mixed tumor transplantation assay.
  • C57BL/6 mice were injected with 1 x IO 6 B16 parental cells (5 mice), B 16.
  • IFN- ⁇ 14 mice or a combination of B16 parental and B16.
  • IFN- ⁇ cells (1 x IO 6 each; 10 mice).
  • Tumor development was monitored as described under "Materials and Methods" in Example 2.
  • FIGURE 9 Local curative potential of irradiated B16.IFN- ⁇ cells. Mice were injected with 1 x 10 5 B16 parental cells. Beginning three days after the injection of the mmor inoculum, mice were injected 3 times at weekly interval with 5 x 10* irradiated B 16.
  • IFN- ⁇ cells at the mmor site Tumor growth was compared with the control group which received only the parental cells but no irradiated B 16.
  • IFN- ⁇ cells Ten mice were injected with 1 x IO 5 B16 cells. Three days later, 5 mice were treated with irradiated B16. IFN- ⁇ cells as described above. There was no difference in IFN- ⁇ secretion between irradiated and non-irradiated cells.
  • FIGURE 10 Local curative potential of irradiated B16.IFN- ⁇ cells. The experiment was performed as described in the legend to FIGURE 9 except that the mmors were permitted to establish for 7 days prior to initiating the injections of the B 16. IFN- ⁇ cells. Thirteen mice were injected with 1 x IO 5 B16 cells. Seven of them were treated with irradiated B 16. IFN- ⁇ cells as described in the legend to FIGURE 9.
  • FIGURE 11 Plasmid pD5 used for construction of the mouse IFN- ⁇ expression vector.
  • FIGURE 12 Expression vector for cotransfection. This vector contains the neomyocis phosphotransferase gene (neo) under control of the phosphoglycerol lunase promoter (pPpk). It was used in contranfection experiments where the expression plasmid for IFN- ⁇ or other factors did not contain the neo gene.
  • neo neomyocis phosphotransferase gene
  • pPpk phosphoglycerol lunase promoter
  • the present invention provides effective vaccines for solid mmor cancers.
  • the invention represents the first time interferon- ⁇ (IFN- ⁇ ) has shown activity in abrogating mmorigenicity.
  • IFN- ⁇ interferon- ⁇
  • the present inventors have also provided a much more effective use of interferon- ⁇ , and were able to completely abrogate mmorigenicity (as opposed to merely delaying mmor growth) by transfecting mmor cells with IFN- ⁇ and another immunomodulatory molecule, specifically, B7 T cell costimulatory molecule.
  • solid mmor and “mmor” refer to a neoplasm, i.e. , tissue that grows by cellular proliferation more rapidly than normal, e.g. , more rapidly than adjoining cells, or other cells in the tissue. Neoplastic cells continue to grow after growth stimuli cease. Generally, mmors represent or form a distinct mass of tissue. A tumor may be benign or malignant (cancer). The present invention relates to both types of mmors, but is particularly valuable in the treatment of cancers. Various type of solid mmors are described in detail, infra.
  • Tumor cells are the neoplastic cells from the tumor.
  • such cells are part of the living cells in the mmor, so that they can be transfected to express either IFN- ⁇ , or IFN- ⁇ with another immunostimulatory molecule; or IFN- ⁇ with another immunostimulatory molecules.
  • mmor cells from a subject suffering from a solid mmor are obtained, e.g. , by biopsy, and these cells are transfected.
  • the invention also contemplates use of mmor cell lines that correspond to or sufficiently emulate an in vivo mmor transfected with an ⁇ or ⁇ IFN, and possibly with another immunomodulatory molecule.
  • immunomodulatory molecules refers to lymphokines, cytokines, cellular adhesion molecules associated with immune responses, i.e. , any molecule that mediates immunostimulation, immunosuppression, specific immune recognition, and the like.
  • the immunomodulatory molecules are in addition to either IFN- ⁇ or IFN- ⁇ , and include the other IFN ( ⁇ or ⁇ , respectively), and IFN-/3; cytokines such as but not limited to interleukin (IL) 1, IL-2, IL-4, IL-6, IL-7, IL-12, mmor necrosis factor (TNF) ⁇ , TNF-/3, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage CSF
  • GM-CSF GM-CSF
  • accessory molecules including members of the integrin superfamily and members of the Ig superfamily such as, but not limited to, LFA-1, LFA-3, CD22, and B7-1, B7-2, and ICAM-1 T cell costimulatory molecules (see Shevach, ibid. , pp. 531-575).
  • IFN- ⁇ and TNF- ⁇ are used as immunomodulatory molecules in addition to IFN- ⁇ .
  • B7 is used in addition to IFN- ⁇ .
  • the immunomodulatory molecules of the invention are well characterized, and generally human and murine homologs are available.
  • the present invention contemplates use of clones of such immunomodulatory molecules, or independently generating such clones by well known techniques of molecular biology, using the DNA sequence information available in the art for these molecules.
  • the present invention provides for ex vivo gene transfer of IFN- ⁇ or IFN- ⁇ , possibly with another immunomodulatory molecule, into tumor cells, followed by introduction of those tumor cells into a subject.
  • in vivo gene transfer with gene transfer targeted to mmor cells, can be employed to transpect the mmor cells with a vector or vectors capable of expressing IFN ⁇ and another immunomodulatory molecule.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, solid mmor growth.
  • a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host, e.g. , a reduction in size of a solid mmor.
  • the component or components of a therapeutic composition of the invention may be introduced parenterally, e.g. , via intravenous injection, and also including, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. More preferably, a vaccine of the invention, or an expression vector, may be introduced by injection into the tumor or into tissues surrounding the mmor.
  • a subject in whom gene therapy treatment of a solid mmor is appropriate is preferably a human, but can be any animal.
  • the compositions and methods of the present invention are particularly suited to treatment of any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc. , i.e. , for veterinary medical use.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another
  • DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the term transfection includes the revers transcription of viral RNA into DNA.
  • a cell has been "transformed” by exogenous or heterologous DNA when the transfected DNA effects a phenotypic change.
  • the transforming DNA should be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • “Heterologous" DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxy cytidine; "DNA molecules”) in either single stranded form, or a double- stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary strucmre of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g. , restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i. e. , the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • Homologous recombination refers to the insertion of a foreign DNA sequence of a vector in a chromosome.
  • the vector targets a specific chromosomal site for homologous recombination.
  • the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g. , mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ' direction) coding sequence.
  • the promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease Sl), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • a “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.
  • the term "translocation signal sequence” is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eukaryotes and prokaryotes, and are often functional in both types of organisms.
  • the present invention is based in part on experiments using gene therapy techniques for the treatment of a model melanoma in mice.
  • B16 melanoma cells were transfected with IFN- ⁇ , or IFN- ⁇ and B7.
  • B16 melanoma was chosen as the mmor model because it is a poorly immunogenic mmor that arose spontaneously in C57BL/6 mice (Fidler, 1975, Cancer Res. , 35:218-224) and thus it is more akin to human cancer than most of the mouse mmor models that have been smdied. It was found that while transfection of IFN- ⁇ alone delayed mmorigenicity, it failed to completely abrogate tumorigenicity.
  • the invention is further based on the unexpected discovery that IFN- ⁇ can be effective in abrogating solid mmorigenicity.
  • experiments were performed to explore the antimmor potential of IFN- ⁇ when autologous mmor cells transfected with cDNA encoding IFN- ⁇ are used as a mmor vaccine to induce a potent immune response against unmodified mmor cells.
  • the B16 melanoma cells were employed.
  • the experiment was further designed to investigate whether a combination of IFN- ⁇ and other cytokines such as IFN- ⁇ or TNF- ⁇ can enhance the antimmor response since the effects of IFN- ⁇ are known to be synergized by IFN- ⁇ or TNF- ⁇ (Fleischmann WR and Schwartz LA, 1986, Methods Enzymol. 79:432-440; Fiers W, 1991, FEBS Letters
  • the nucleotide sequence coding for an immunomodulatory molecule, or pharmaceutically active fragment or analog thereof can be inserted into an appropriate expression vector, i. e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a "promoter. "
  • the nucleic acid encoding an immunomodulatory molecule of the invention is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding immunomodulatory molecule and/or its flanking regions.
  • the recombinant immunomodulatory molecule of the invention is expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).
  • immunomodulatory molecules may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host mmor cells in which expression is desired.
  • Promoters which may be used to control immunomodulatory molecule gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981 , Namre 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), human cytomegalovirus (CMV) promoter, the adenovirus major late promoter, the the he ⁇ es thymidine kinase promoter (Wagner et al., 1981, Proc.
  • CMV human cytomegalovirus
  • albumin gene control region which is active in liver (Pinkert et al. , 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al. , 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al.
  • recombinant DNA expression vector for use in the invention may be prepared, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity.
  • the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus, and plasmid and cosmid DNA vectors, to name but a few.
  • Vectors are introduced into the desired host cells by methods known in the art, e.g. , ex vivo viral vectors, particularly retroviral vectors, in vivo viral vectors, particularly defective viral vectors or adeno-associated virus vectors, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g. , Wu et al. , 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al. , Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • ex vivo viral vectors particularly retroviral vectors
  • in vivo viral vectors particularly defective viral vectors or adeno-associated virus vectors
  • transfection electroporation, microinjection
  • a nucleic acid coding sequence encoding an immunomodulatory molecule may be introduced in vitro or, more preferably, in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to he ⁇ es simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • HSV he ⁇ es simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a solid tumor can be specifically targeted.
  • vectors include, but are not limited to, a defective he ⁇ es virus 1 (HSV1) vector (Kaplitt et al. , 1991, Molec. Cell. Neurosci. 2:320-330), an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (1992, J. Clin. Invest. 90:626-630), and a defective adeno-associated virus vector (Samulski et al. , 1987, J. Virol. 61 :3096-3101; Samulski et al. , 1989, J. Virol. 63:3822-3828).
  • HSV1 defective he ⁇ es virus 1
  • the gene can be introduced in a retroviral vector, e.g. , as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33: 153; Temin et al., U.S. Patent No. 4,650,764; Temin et al. , U.S. Patent No. 4,980,289; Markowitz et al., 1988, J. Virol. 62: 1120; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al. ; and Kuo et al., 1993, Blood 82:845.
  • Retroviral vectors are especially attractive for transfecting solid mmors, since the cells of the mmor are replicating.
  • the vector can be introduced in vitro or in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al. , 1987, Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417; see Mackey, et al. , 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031)).
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, 1989, Science 337:387-388).
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells, in this instance mmor cells, e.g. , via tumor-specific cell surface receptors represents one area of benefit.
  • Lipids may be chemically coupled to other molecules for the pu ⁇ ose of targeting (see Mackey, et. al., 1988, supra).
  • Targeted peptides e.g. , hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g. , transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g. , Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • Solid Tumors As noted above, the present invention is directed to generating endogenous immune responses against solid mmors. To date, there has been no effective IFN- ⁇ -based treatment of solid tumors, and IFN- ⁇ -based treatments of solid tumors delay, but do not appear to prevent, tumor growth.
  • the vaccines and methods of the present invention overcome these deficiencies, and are applicable to treatment of a wide range of solid mmors.
  • dysproliferative changes are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung.
  • epithelial tissues such as those in the cervix, esophagus, and lung.
  • the present invention provides for treatment of conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hype ⁇ lasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79).
  • Hype ⁇ lasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in strucmre or function.
  • endometrial hype ⁇ lasia often precedes endometrial cancer.
  • Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells.
  • Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomo ⁇ hism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder. For a review of such disorders, see Fishman et al. , 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia.
  • the present invention is advantageously suited to intervention at the stage of dysproliferative changes, before a condition proceeds to stams as a full blown mmor.
  • the present invention is employed in the treatment of a melanoma mmor.
  • Mouse IFN- ⁇ and/or the mouse B7 (T cell costimulatory molecule) cDNAs were transfected into B16 melanoma cells to smdy the effects of local constimtive expression of these molecules on the tumorigenicity and immunogenicity of this aggressive mmor.
  • Cells expressing IFN- ⁇ (B16. IFN- ⁇ ), B7 (B16.B7), B7 and IFN- ⁇ (B16.IFN- ⁇ /B7), and parental cells were injected subcutaneously (s.c.) into syngeneic C57BL/6 mice to compare their in vivo growth. IFN- ⁇ secretion significantly reduced the mmorigenicity of B16 cells.
  • IFN- ⁇ mmors appeared earlier in athymic mice than in immunocompetent mice.
  • B16.IFN- ⁇ /B7 cells which also express increased levels of MHC class I and class II molecules as compared to parental cells, had a dramatically suppressed mmorigenicity, while B16 cells expressing the B7 molecule only (B16.B7) were as tumorigenic as the parental cells.
  • B16.IFN- ⁇ /B7 cells induced specific immune responses since all of the protected mice were able to reject challenges with parental cells.
  • mice Male C57BL/6J and C57BL/6J-athymic mice, 6-8 weeks old, were obtained from The Jackson Laboratory (Bar Harbor, ME). The parental 129/SvEv wild type and IFN- ⁇ R knockout mice were described (Huang et al., 1993, Science 259: 1742-1745).
  • B16 melanoma cells are melanocytic tumor of C57BL/6 origin (Fidler, 1975, Cancer Res. 35:218-224). B16 cells were grown as a monolayer in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 500 ⁇ g/ml gentamicin sulfate in a humidified incubator supplemented with 5% CO 2 at 37 °C. Prior to use, cell monolayers were washed with phosphate-buffered saline (PBS) and detached by incubating at 37 °C with 2 mM EDTA in PBS.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS heat-inactivated fetal bovine serum
  • PBS phosphate-buffered saline
  • the eukaryotic expression vector pRc/neoCMV-MB7 contains the complete coding sequence of murine B7 subcloned downstream from the CMV promoter and the bacterial neomycin resistance gene under the control of the SV40 early promoter.
  • the coding region for the mouse B7 protein (Baskar et al. , 1993, inserted into the Ava l/Not I sites of the RC/CMV expression vector (Invitrogen).
  • plasmids pD5-IFN- ⁇ or pD5 and 2 ⁇ g of plasmid pPGKneobpA were co-transfected into 5 x lO-' Bl ⁇ cells by the calcium- phosphate precipitation method (Chen and Okayama, 1988, Mol. Cell. Biol. 7(8): 2745-2752) followed by selection and cloning in DMEM containing the neomycin analogue G418 (Geneticin; Gibco) at a concentration of 1 mg/ml. Clones were maintained in medium contaimng antibiotic G418 at 500 ⁇ g/ml.
  • the pRc/neoCMV-mB7 construct (20 ⁇ g) was transfected into B16 melanoma cells by the calcium-phosphate precipitation method. Selection of antibiotic G418 resistant clones was carried out as described. Twenty ⁇ g of plasmid pRc/neoCMV-mB7 were also co-transfected into B 16. IFN- ⁇ cells along with 1 ⁇ g of plasmid pHM24 an E. coli plasmid containing the hygromycin gene driven by a eukaryotic promoter (Koo et al. , 1994, Virology 205:345-351; Mikawa et al. , 1991, Exp. Cell Res.
  • Antibiotic G418-resistant clones were assayed for the production of IFN- ⁇ by the cytopathic effect inhibition assay with encephalomyocarditis virus (EMCV) on mouse L929 cells (Familletti et al., 1981, Methods Enzymol. Pestka S (ed.) New York, Academic Press, 78:430-435).
  • Antibiotic G418-resistant clones were seeded in 6-well tissue culmre plates at 1 x IO 6 cells per well in 1 ml DMEM containing 10% FBS without antibiotic G418.
  • IFN activity was expressed in units/ml calibrated against the NIH reference standard Gg 02-901-533 for mouse IFN- ⁇ .
  • Hybridomas and antibodies Hybridoma 28-8-6S secreting a mouse anti-H-2K b (class I) monoclonal antibody has been described (Ozato et al., 1985, Cancer Res. 52:4571-4581). Hybridoma AF120.1.2 producing a mouse anti-I-A b (class II) monoclonal antibody was obtained from the American Type Culmre Collection (Loken and Stall, 1982, J. Immunol. Meth. 50:R85). Hybridoma 1G10-G9 secreting a rat anti-mouse B7 monoclonal antibody has been described (Leo et al. , 1987, Proc. Natl. Acad. Sci. USA 84: 1374-1380).
  • Fluorescein isothiocyanate (F ⁇ TC)-labeled goat anti-mouse or anti-rat immunoglobulin (Cappel) were used as secondary antibodies in flow cytometry analysis of cell surface expression.
  • Hybridomas were seeded at 1 x 10 5 cells/ml in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 x L-glutamine, 1 x non-essential amino acids, 10% NCTC-135 medium (Gibco) and 1 x of Solution I [1.32 g oxaloacetic acid (100 mM), 80 mg bovine insulin (20 units/ml, 25 units/mg), 550 mg sodium pyruvate (50 mM) in a volume of 100 ml].
  • Culmre supematants were centrifuged at 4000 ⁇ m on a table-top centrifuge for 20 min to pellet the cells, then filtered to eliminate remaining cells and maintain sterility. Clarified supematants were maintained at -20°C until used.
  • IFN- ⁇ cells In vitro cell growth. To assess whether IFN- ⁇ production affects the growth of B 16. IFN- ⁇ cells we performed in vitro growth assays. B16 and B 16. IFN- ⁇ cells were seeded at 1 x 10 5 cells per well in 6-well tissue culmre plates and incubated at 37 °C. On days 1, 2 and 3 cells were counted with a hemocytometer after staining with trypan blue to exclude dead cells.
  • mice In vivo tumor growth.
  • C57BL/6J male mice were injected subcutaneously (s.c.) on the flank with various numbers of B16, B16.IFN- ⁇ , B16.B7, or B16.IFN- ⁇ /B7 cells in 0.2 ml of Hank's balanced saline solution (HBSS).
  • C57BL/6J-athymic mice, 129/SvEv wild type and 129/SvEv mice lacking the IFN- ⁇ receptor gene (knockout mice) were injected with parental or B 16. IFN- ⁇ cells as described above. Tumor growth was followed by palpation and recorded as the average of two-dimensional caliper measurements in cm. Mice that rejected the primary injection of tumor cells were challenged s.c.
  • mice were injected s.c. with 1 x 10 6 B 16. IFN- ⁇ cells and intraperitoneally (i.p.) with a rabbit polyclonal antiserum raised against murine IFN- ⁇ (5 x IO 4 neutralization units). As controls, one group of mice was injected with non-specific rabbit serum and another group was injected only with cells. Tumor growth was followed as described above.
  • Membranes were hybridized with a probe that contained the IFN- ⁇ coding sequence labeled by random priming (Sambrook et al. , 1989, supra) with [ ⁇ - 32 P]dCTP (111 TBq/mmol; 3000 mCi/mmol). Following hybridization, blots were washed in 1 x SSC at 68 °C and autoradiographed . Results Generation and properties of B16 transfectants. After co-transfection of plasmids pD5-IFN- ⁇ and pPGKneobpA into B16 melanoma cells, thirty G418-resistant clones were screened for constimtive secretion of IFN- ⁇ into the culmre supematant.
  • IFN- ⁇ was used for the in vivo experiments. The production of IFN- ⁇ by this clone remained constant even after prolonged growth in vitro in media containing 500 ⁇ g/ml antibiotic G418. The in vitro growth of the B 16. IFN- ⁇ cells was identical to that of B16 parental cells. The expression of class I MHC antigens on parental ( Figure IA) and B 16. IFN- ⁇ ( Figure IB) cells was assessed by flow cytometry. B 16.
  • IFN- ⁇ cells showed a significant increase in the expression of MHC class I as well as class II antigens compared to the parental cells.
  • Cells co-transfected with plasmids pD5 and pPGKneobpA (B16- pD5neo) were used as a control in the animal experiments.
  • B16 cells transfected with the pD5 vector only (B16.pD5 cells) exhibited growth comparable to the B16 parental cells.
  • the expression vector pRc/neoCMV-mB7 was transfected into B16 parental cells and co-transfected into B 16. IFN- ⁇ cells along with 1 ⁇ g of plasmid pHM24 which contains the hygromycin resistance gene. After selection with antibiotic G418 and hygromycin B, respectively, clones were assayed for expression of B7 molecules by flow cytometry as described in Materials and Methods. The level of B7 expression on parental cells was also determined (Fig. 2A). A clone expressing B7 (B16.B7.7) and a clone co-expressing IFN- ⁇ and B7 (B16.IFN- ⁇ /B7.11) were selected for the in vivo experiments (Figs. 2B and 2C, respectively). The expression of B7 molecules also remained constant after prolonged in vitro culmre in the presence of the appropriate antibiotics.
  • B16.IFN- ⁇ cells Tumorigenic potential of B16.IFN- ⁇ cells in syngeneic C57BL/6J mice.
  • B16 parental cells produced a mmor in all the animals injected after latency periods of up to 15 days at doses as low as 1 x IO 5 cells. In contrast, 1 x IO 5 B 16.
  • IFN- ⁇ cells produced tumors only in 50% of the animals injected after latency periods of 50 days or more (Fig. 3A).
  • mice injected with parental cells had a visible mmor by day 12, whereas the appearance of B 16. IFN- ⁇ tumors was delayed by almost one month. B16-pD5neo cells showed the same tumorigenic properties as the parental cells (Fig. 3B). These data show that IFN- ⁇ secretion can reduce significantly the tumorigenicity of B16 melanoma cells. In addition, we also observed that mice bearing B 16. IFN- ⁇ mmors were able to survive significantly longer than the control group injected with parental cells (data not shown; Flores, 1994).
  • IFN- ⁇ antiserum Effect of in vivo injection of IFN- ⁇ antiserum on the tumorigenicity of B 16. IFN- ⁇ cells
  • mice C57BL/6J mice were injected s.c. with 1 x IO 6 B16. IFN- ⁇ cells and i.p. with 5 x IO 4 neutralization units of a rabbit polyclonal antisemm raised against murine IFN- ⁇ .
  • one group of mice was injected with non-specific rabbit semm and another group was injected only with cells. Tumor growth was monitored by palpation of the injected site twice a week.
  • the values in the table represent the number of mice with a palpable mmor 30 days after injection (numerator) over the total number of mice injected (denominator).
  • IFN- ⁇ R knockout mice IFN- ⁇ cells in mice that lack functional IFN- ⁇ receptors. Injection of 1 x 10 6 B 16. IFN- ⁇ cells into IFN- ⁇ R knockout mice resulted in development of mmor in all of the injected animals, whereas the same number of cells were completely rejected by wild type 129/SvEv mice. This effect was not due to allorejection since C57BL/6J mice and 129/SvEv mice are both of the H-2 b haplotype. B16 parental cells, in contrast, were equally tumorigenic in both wild type and IFN- ⁇ R knockout mice (Table 2).
  • mice with a deletion of the IFN- ⁇ receptor IFN- ⁇ R knockout, ko
  • syngeneic 129/SvEv parental mice C57BL/6J mice were injected with 1 x IO 6 B16.
  • the values in the table represent the number of mice that developed a tumor during an observation period of three months over the number of mice injected.
  • B16 tumors showed numerous mitoses (33-36 per five high power fields, HPF; x 400). A few mononuclear inflammatory cells were present in the per immoral tissues, but virtually no lymphoid cells could be seen within the tumors. This mo ⁇ hology remained essentially the same throughout the observation period except for the spontaneous necrosis which became more prominent as the mmors grew older.
  • IFN- ⁇ cells showed only sparse cells with rare mitoses (3 per five HPF) 12 days after inoculation. Numerous inflammatory cells including lymphocytes, macrophages and neutrophils were present in the peritumoral tissue and within the mmor. A similar pattem was still present on day 22, whereas tumors excised on days 28 and 34 were mo ⁇ hologically similar to the B16 parental controls and displayed a high mitotic activity (30 mitoses per five HPF) and little, if any, lymphocytic infiltration.
  • B16.IFN- ⁇ cells Tumorigenicity of B16.IFN- ⁇ cells in athymic mice. Following the observation that a cellular infiltrate, consisting mainly of lymphocytes, was evident in the B 16. IFN- ⁇ mmors, we assessed the role of T cells in the host response to the injected cells. Athymic mice of C57BL/6J origin and immunocompetent C57BL/6J mice were injected with 1 x 10 6 B 16. IFN- ⁇ cells and mmor appearance was monitored as described. Tumors from B 16. IFN- ⁇ cells developed faster in athymic mice than in control C57BL/6J mice.
  • T cells are indeed the effector cells that mediate the responses activated by B16. IFN- ⁇ cells.
  • B16.B7 or B16.IFN- ⁇ /B7 cells Tumorigenic potential of B16.B7 or B16.IFN- ⁇ /B7 cells in syngeneic C57BL/6J mice.
  • B16 melanoma cells alone or in combination with IFN- ⁇ , affects their mmorigenicity
  • various numbers of B 16. IFN- ⁇ , B16.B7.7, B16.IFN- ⁇ /B7.11, and parental cells were injected s.c. into C57BL/6J mice.
  • the mmorigenicity of B16.B7 cells was almost unaffected as compared to that of parental cells (Figure 4).
  • mice C57BL/6J mice were injected s.c. with 1 x IO 6 B16.IFN ⁇ /B7 cells. Mice that rejected the tumor cells were challenged on day 62 with 1 x 10 5 B16 parental cells at the contralateral site. Naive mice injected with 1 x 10 5 B16 cells served as control. Tumor appearance was monitored as described in Materials and Methods.
  • This Example shows that secretion of IFN- ⁇ by B16 melanoma cells results in a significant reduction in tumorigenicity as compared to parental cells. Most importantly, it demonstrates that tumor cells genetically-modified to express both IFN- ⁇ and B7 can induce potent protective antitumor responses. B16 cells secreting IFN- ⁇ showed increased surface expression of MHC class I and class II molecules which are required for antigen presentation to both the CD8 + and CD4 + arms of the immune system, respectively. However, the reduced tumorigenicity of B16. IFN- ⁇ cells is not only an aftermath of the upregulation in MHC antigen expression since we demonstrated that secreted IFN- ⁇ directly affects host effector mechanisms.
  • IFN- ⁇ mmor growth in vivo provide evidence for the direct role played by secreted IFN- ⁇ on host cells. This effect of IFN- ⁇ was unrelated to its well known antiproliferative activity since B 16. IFN- ⁇ cells had an in vitro growth rate similar to that of parental cells. In addition, injection of B 16. IFN- ⁇ cells into C57BL/6J athymic mice resulted in the acceleration of tumor growth. These observations indicate that T cells are primarily responsible for the reduced tumorigenicity of B 16. IFN- ⁇ cells.
  • IFN- ⁇ is also known as immune interferon for its role in the activation and regulation of immune effector mechanisms such as cytotoxic T lymphocytes (CTL), NK cells and macrophages (Trinchieri and Perussia, 1985, Immunol. Today 6:131-136; Pestka et al. , 1987, Biochem. 56:727-777), and of the expression of tumor associated antigens and molecules required for the presentation of antigenic determinants to CTL and helper T cells such as adhesion and MHC molecules (Dustin et al. , 1986, J. Immunol.
  • IFN- ⁇ -gene transfection into various mmor models has resulted in a decrease in their tumorigenicity that was correlated by some investigators with upregulation of surface MHC molecules (Gansbacher et al. , 1990 supra; Restifo et al. , 1992, supra; Porgador et al.
  • IFN- ⁇ activates other mechanisms that result in the development of potent immune responses. This smdy helps to define the role of secreted IFN- ⁇ in the observed reduced mmorigenicity of the transfectants. With the aid of the IFN- ⁇ R knockout mice we have proved that IFN- ⁇ activates immune effector cells via interactions with specific receptors on host cells. This observation was supported by histological examination that showed the presence of a cellular infiltrate at the injection site and by the effects of IFN- ⁇ antibodies in blocking the observed responses.
  • B16.IFN- ⁇ /B7 cells can provide all the elements necessary for effective activation of immune responses: MHC molecules that can present tumor epitopes to the T cell receptor, the B7 molecule which provides the costimulatory signal that is crucial for T cell activation, and IFN- ⁇ which plays a role in the stimulation of specific T effector cells (Chen et al., 1986, Eur. J. Immunol. 16:767- 770. ; Maraskovsky et al., 1989, J. Immunol. 143:1210-1214). Results from an additional experiment in which a mixmre of B16.B7 cells and B 16. IFN- ⁇ cells were injected indicated that MHC and B7 molecules must be expressed on the same cell since co-injection of cells that expressed one molecule or the other resulted only in delays in the appearance of mmors.
  • the highly aggressive murine B16 melanoma was engineered to secrete IFN- ⁇ constitutively.
  • Cells expressing IFN- ⁇ were injected into syngeneic C57BL/6 mice and the mice were momtored for mmor development.
  • Secretion of IFN- ⁇ by B16 melanoma cells completely abrogated their mmorigenicity in syngeneic mice.
  • IFN- ⁇ -secreting cells also abrogated the mmorigenicity of IFN- ⁇ -secreting and TNF- ⁇ -secreting cells when injected in combination whereas cells secreting either IFN- ⁇ or TNF- ⁇ grow progressively in mice when injected alone.
  • protected animals developed significant immunity against subsequent challenge with parental cells.
  • IFN- ⁇ -secreting cells Injection of parental cells and IFN- ⁇ -secreting cells together in a mixed mmor transplantation assay resulted in a significant reduction of mmorigenicity of the parental cells. Histopathological smdies of the tissues from the injection site of the mice inoculated with a combination of parental and B 16. IFN- ⁇ cells revealed the existence of a massive cellular infiltrate composed of lymphocytes and granulocytes at an early stage (7-11 days). In the later stages (22 days), no recognizable mmor tissue was detected. Injection of irradiated IFN- ⁇ -secreting cells in the mice carrying an established mmor completely prevented mmor development in 80% of the treated mice when injection was performed on the same side as the mmors.
  • Injection of irradiated IFN- ⁇ -secreting cells in the contralateral site showed much less effect on the established mmor.
  • Systemic antimmor effects on the established mmor can be enhanced by using a combination of irradiated IFN- ⁇ and IFN- ⁇ secreting cells as a vaccinating inoculum.
  • mice and BL6 cell lines are described in Example 1, supra.
  • Example 2 The pD5-IFN- ⁇ constmct described in Example 1 was used for transfection into B16 cells along with plasmid pPGKneobpA.
  • Plasmids pD5-IFN- ⁇ and pPGKneobpA were tranfected into B16 cells as described in ⁇ xample 1.
  • the pLNCX-TNF- ⁇ plasmid was introduced into B16 cells via retroviral gene transduction (Markowizt et al.
  • G418 resistant colonies were isolated and screened for the expression of the particular cytokine transfected. Interferon assay. G418-resistant clones were assayed for the production of IFN- ⁇ by the cytopathic effect inhibition assay with encephalomyocarditis vims (EMCV) on mouse L929 cells (Fammilletti et al., 1981, Academic Press. 78:430-435). G418- resistant clones were seeded in 6-well tissue culture plates at 1 x IO 6 cells per well in 2 mL DMEM containing 10% FBS without antibiotic G418.
  • EMCV encephalomyocarditis vims
  • IFN activity is expressed in units/mL calibrated against the NIH reference standard for mouse IFN- ⁇ / ⁇ G-002-904-511. IFN- ⁇ production of the clones transfected with pD5-IFN- ⁇ was assayed as described above and the activity was calibrated against NIH reference standard Gg02-901-533 for mouse IFN- ⁇ .
  • TNF assay Culmre supematants from the G418 resistant clones were assayed following the method of Asher et ⁇ /.(1987, J. Immunol. (38:963-974). Briefly, 1 x IO 6 cells were seeded in a 6-well dish in 2 mL of media without any G418. Supematants were collected after 24 hrs and serially diluted 2-fold in 96-well microtiter plates. L929 cells (4 x IO 4 ) were added in the presence of actinomycin D (1 ⁇ g/mL). Following ovemight incubation at 37° C, plates were stained with crystal violet to determine the endpoint of growth inhibition. TNF- ⁇ activity was expressed in units/mL against the NIH reference standard for recombinant human TNF- ⁇ .
  • cytokine-secreting cells were seeded at 1 x 10 s cells per well in 6-well tissue culmre plates and incubated at 37 °C. After 48 hours cells were counted after staining with trypan blue to exclude dead cells.
  • mice C57BL/6 male mice were injected subcutaneously (s.c.) on the flank with various numbers of cells in 0.2 mL of Hank's balanced saline solution (HBSS). Tumor growth was followed and recorded as described in Example 1, supra. As in Example 1 , mice that rejected the primary injection of mmor cells were challenged s.c. with parental B16 cells on the opposite flank and animals with massive mmor burden were sacrificed.
  • HBSS Hank's balanced saline solution
  • Tissue at the site of injection was obtained at various times and fixed in 10% phosphate-buffered formalin as described in Example 1, supra.
  • mice were injected with 1 x 10 5 B16 cells and three days later they were treated by injection with 4-5 x IO 6 irradiated (10,000 rads) B16 parental or cytokine secreting cells once a week for three consecutive weeks at the contralateral site.
  • mice were injected with 5 x 10 6 irradiated B16.
  • IFN- ⁇ cells at the mmor site 3 or 7 days after the injection of 1 x 10 5 parental mmor cells. This treatment was repeated twice at weekly intervals.
  • B16 melanoma cells were transfected with the plasmid pLNCX-IFN- ⁇ by calcium phosphate coprecipitation method (Chen, 1988, supra).
  • Antibiotic G418-resistant clones were screened for the constimtive secretion of IFN- ⁇ .
  • IFN- ⁇ was used for the in vivo experiments.
  • Cells that were transfected with the plasmid pLNCX were used as a control in the animal experiments.
  • pD5IFN- ⁇ and pPGKneobpA were screened for the secretion of IFN- ⁇ and a clone secreting IFN- ⁇ at 240 units/mL/48 hr/10 6 cells designated as B 16. IFN- ⁇ was selected for further studies. B16 clones generated by transduction of pLNCX-TNF- ⁇ plasmid were assayed for TNF- ⁇ secretion. A clone secreting TNF- ⁇ at 1500 units/mL/24 hr/10 6 cells designated as B16. TNF- ⁇ were selected for injection.
  • MHC antigens in B16 parental and cytokine-secreting cells were assessed by cytofluorographic analysis.
  • IFN- ⁇ is known to have growth inhibitory effect on a variety of cell lines
  • secretion of IFN- ⁇ by B16 cells on their in vitro growth rate was tested.
  • B16 parental and B 16. IFN- ⁇ cells were plated at a concentration of 1 x 10 5 cells per well in 6- well plates and the live cells were counted after 48 hours by trypan blue exclusion.
  • B16. IFN- ⁇ cells grew at one third the rate of B16 parental cells. Addition of up to 10,000 units/mL of IFN- ⁇ to B16 cells exogenously, however, did not affect the growth of B16 cells.
  • B 16. IFN- ⁇ and B 16. TNF- ⁇ cells did not show any significant change in their growth rate as compared to the parental cells.
  • IFN- ⁇ and B16.IFN- ⁇ are highly synergistic (Fleishmann, 1986 supra) we injected a combination of B 16. IFN- ⁇ and B 16. IFN- ⁇ cells into syngeneic C57BL/6 mice. Groups of mice injected with either B 16. IFN- ⁇ or B 16. IFN- ⁇ cells served as controls. Eighty percent of the mice injected with B16. IFN- ⁇ cells developed mmor with a latency period of 20 days while only 5 % of the mice injected with a combination of B 16. IFN- ⁇ and B 16.
  • IFN- ⁇ cells developed mmor with a latency period of 50 days. Injection of B 16. IFN- ⁇ and B 16. IFN- ⁇ cells on the contralateral sites did not result in a significant effect on the growth of B 16. IFN- ⁇ cells (data not shown). None of the mice injected with B 16. IFN- ⁇ cells developed tumor during the 80 days observation period ( Figure 6).
  • mice Balkwill et al. (1986, supra) showed that a combination of TNF- ⁇ with either IFN- ⁇ or IFN- ⁇ resulted in an antimmor effect greater than either cytokine alone, although the effects were more marked with a combination of IFN- ⁇ and TNF- ⁇ than with IFN- ⁇ and TNF- ⁇ . Based on this observation, it remained uncertain whether a combination of B 16. IFN- ⁇ and B 16. TNF- ⁇ cells can induce the rej ection of cytokine- secreting cells when injected in syngeneic mice. B 16. TNF- ⁇ cells developed mmors in mice, with 100% of the mice exhibiting mmors within 23 days, whereas none of the mice injected with a combination of B16.
  • TNF- ⁇ cells developed mmor during a 65 days observation period. Injection of B 16. IFN- ⁇ and B 16. TNF- ⁇ at contralateral sites however, generated a smaller, but nevertheless significant effect on the growth of B 16. TNF- ⁇ cells ( Figure 7).
  • IFN- ⁇ and IFN- ⁇ exert multiple antimmor effects that may be categorized either into the direct antiproliferative activity against the mmor or into activity indirectly mediated through the host immune system, including the enhancement of MHC class I and class II antigen expression, activation of macrophages and natural killer cells, generation of cytotoxic T lymphocytes, and induction of tumor associated antigens (Balkwill FR, 1989, "Cytokines in Tumor Therapy, In Interferons, Oxford University Press: New York, pp. 8-57).
  • mice that rejected the cytokine-secreting cells were challenged with 1 x IO 5 B16 cells on the contralateral sites. Naive mice injected with 1 x IO 5 B16 cells served as controls. Mice immunized with B16. IFN- ⁇ cells showed 40% protection while mice immunized with a combination of B16. IFN- ⁇ and B16. IFN- ⁇ or B16. IFN- ⁇ and B16. TNF- ⁇ showed 60% -67% and 80% protective immunity, respectively, against subsequent challenge with parental cells. Results are shown in Table 4.
  • mice were injected with live cytokine-secreting cells and challenged with 1 x 10 5 B16 parental cells at the contralateral site as described in the text.
  • IFN- ⁇ were injected, a totally necrotic mmor nodule with a marked cellular infiltrate composed of lymphocytes and granulocytes was visible on days 7 and 14, while samples taken on day 21 failed to show any mmor at all.
  • B 16. IFN- ⁇ and B 16. IFN- ⁇ cells were injected on contralateral sides, on both sides necrotic mmor nodules with massive cellular infiltrate comprising both lymphocytes and granulocytes were visible on days 7 and 14. Samples taken from the mice injected with a combination of B 16. IFN- ⁇ and B 16. TNF- ⁇ cells revealed the existence of prominent necrosis and a marked infiltrate consisting of lymphocytes and granulocytes on day 7 and 14.
  • IFN- ⁇ or B16 parental cells into athymic mice (nu/nu). C57BL/6 mice injected with parental and B 16. IFN- ⁇ cells served as control. During a two month observation period athymic mice injected with B 16. IFN- ⁇ cells failed to develop tumor while all athymic mice injected with B16 parental cells developed tumors within one week (data not shown). Inability of B 16. IFN- ⁇ cells to grow in both immunocompetent and T cell deficient mice suggests several possibilities.
  • IFN- ⁇ cells in vivo may be related to the direct antiproliferative properties of IFN- ⁇ . Moreover, rejection of B16. IFN- ⁇ cells may not be mediated by T cells, but rather by NK cells or macrophages. Furthermore, it is conceivable that B 16. IFN- ⁇ cells completely lost their tumorigenic potential in vivo.
  • mice bearing 3-day old tumors were injected contralateral ly with 4-5 x 10 6 irradiated B 16.
  • IFN- ⁇ cells three times at weekly interval.
  • a control group was treated with irradiated B16 parental cells. Tumor growth was blocked in 26% of the mice treated with B 16. IFN- ⁇ cells and in only 14% of the mice that received irradiated parental cells. However, this difference was not statistically significant.
  • the tumor was cured in 35 % of the mice when treated with a combination of irradiated B16. IFN- ⁇ and B 16. IFN- ⁇ cells. This treatment conferred significant protection compared to the treatment with parental cells only (P ⁇ 0.05). Cumulative data from 4 different experiments are shown in Table 5.
  • mice bearing tumor bearing tumor with irradiated parental and cytokine secreting cells.
  • mice with tumor/ % mice with % mice mice with tumor/ % mice with % mice
  • IFN- ⁇ has several important antimmor properties ranging from growth arrest to the induction of immunologically important molecules such as MHC class I antigens and gp96 (Gutterman, 1994, Proc. Natl. Acad. Sci. USA, 91 : 1198-1205), (Pestka S. et al. ,1987, Ann. Rev. Biochem. 56:727-777), (Lengyl, 1993, Proc. Natl. Acad. Sci.
  • mice that rejected the cytokine-secreting cells showed significant protective immunity against subsequent challenge with parental cells.
  • IFN- ⁇ cells locally caused regression of mmor in 80% of the mice, although injection of B 16.
  • IFN- ⁇ cells on the contralateral site showed much less effect on the mmor growth. This effect on mmors can be enhanced by the use of a combination of irradiated B 16. IFN- ⁇ and B 16. IFN- ⁇ cells.
  • IFN- ⁇ at the mmor site is due both to the direct antiproliferative effect of IFN- ⁇ and to the induction of some host immune responses.
  • IFN- ⁇ is known to have antiangiogenic activity.
  • the interferons like most other cytokines, are produced by the body to act in an autocrine and paracrine fashion. When used as a systemic therapy, certain toxic effects are seen. This is the first report of the use of IFN- ⁇ in a gene therapy model with a solid mmor. Comparative smdy of different cytokines in MBT and B16.F10 cells reported by Saito et al. (1994, Cancer Res. 54:3516-20) and Dranoff et al.(l993, Proc. Natl. Acad. Sci. U.S.A. 90:3539-43) did not include this important cytokine.
  • IFN- ⁇ exerts its antimmor effect
  • our results demonstrate that the use of irradiated B 16. IFN- ⁇ cells as a mmor vaccine can effectively inhibit the mmor growth when used locally.
  • the results of this Example demonstrate that it may be possible to eliminate an established mmor by optimization of dosages and combination of cytokines.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Microbiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP96921666A 1995-06-19 1996-06-18 Gentherapie von soliden tumoren mit interferone allein oder mit anderen immunoeffektoren proteinen Withdrawn EP0835130A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US30295P 1995-06-19 1995-06-19
US302 1995-06-19
PCT/US1996/010502 WO1997000085A1 (en) 1995-06-19 1996-06-18 Gene therapy of solid tumors with interferons alone or with other immuno-effector proteins

Publications (1)

Publication Number Publication Date
EP0835130A1 true EP0835130A1 (de) 1998-04-15

Family

ID=21690885

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96921666A Withdrawn EP0835130A1 (de) 1995-06-19 1996-06-18 Gentherapie von soliden tumoren mit interferone allein oder mit anderen immunoeffektoren proteinen

Country Status (3)

Country Link
EP (1) EP0835130A1 (de)
AU (1) AU6282896A (de)
WO (1) WO1997000085A1 (de)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0834323A1 (de) * 1996-09-30 1998-04-08 Introgene B.V. Cytokin-Gentherapie für die Behandlung von malignen Tumoren
FR2760023B1 (fr) * 1997-02-21 2004-05-07 Commissariat Energie Atomique Cellules eucaryotes exprimant a leur surface au moins une isoforme d'hla-g et leurs applications
US6051428A (en) * 1997-03-21 2000-04-18 Sloan-Kettering Institute For Cancer Research Rapid production of autologous tumor vaccines
US6969609B1 (en) * 1998-12-09 2005-11-29 The United States Of America As Represented By The Department Of Health And Human Serivces Recombinant vector expressing multiple costimulatory molecules and uses thereof
EP1032428B1 (de) * 1997-11-20 2003-06-18 Vical Incorporated Behandlung von krebs durch verwendung von zytokin-exprimierender polynukleotiden und zusammensetzungen dafür
US7795020B2 (en) 1998-01-14 2010-09-14 Morphogenesis, Inc. Tumor cell vaccines
US7348015B2 (en) * 1998-01-14 2008-03-25 Morphogenesis, Inc. Antigen modified cancer cell vaccines for cancer therapy
EP1064383A2 (de) * 1998-03-19 2001-01-03 Valentis Inc. Interferon-alpha plasmide und applicationssysteme, und methode für ihre herstellung und ihre verwendungen
AUPQ755300A0 (en) * 2000-05-17 2000-06-08 Monash University Immune potentiating compositions
AU785241B2 (en) * 2000-05-17 2006-11-23 Cancure Limited Immune potentiating compositions
DK1346036T3 (da) 2000-11-29 2010-07-19 Univ Rochester Virusfri virusamplicon hjælpepartikler samt anvendelser deraf
US8092791B2 (en) 2001-05-23 2012-01-10 University Of Rochester Method of producing herpes simplex virus amplicons, resulting amplicons, and their use
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
US7674456B2 (en) 2004-06-14 2010-03-09 Charles Wiseman Breast cancer cell lines and uses thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5833975A (en) * 1989-03-08 1998-11-10 Virogenetics Corporation Canarypox virus expressing cytokine and/or tumor-associated antigen DNA sequence
US5662896A (en) * 1988-03-21 1997-09-02 Chiron Viagene, Inc. Compositions and methods for cancer immunotherapy
EP1216710B1 (de) * 1991-10-04 2006-04-05 Whitehead Institute For Biomedical Research Regulierung der systemischen Immunantworten mittels Zytokinen und Antigenen
ATE248914T1 (de) * 1993-02-17 2003-09-15 Sloan Kettering Inst Cancer Allogenes vakzin und synthesemethode für selbiges
NZ263550A (en) * 1993-03-19 1996-12-20 Boehringer Ingelheim Int Preparing cancer vaccines containing autologous tumour cells characterised in that tumour cells or firbroblasts are cultivated and transfected ex vivo with a composition of dna and a dna binding molecule conjugate

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9700085A1 *

Also Published As

Publication number Publication date
AU6282896A (en) 1997-01-15
WO1997000085A1 (en) 1997-01-03

Similar Documents

Publication Publication Date Title
Seino et al. Antitumor effect of locally produced CD95 ligand
Armstrong et al. Antitumor effects of granulocyte-macrophage colony-stimulating factor production by melanoma cells
Baskar et al. Major histocompatibility complex class II+ B7-1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice.
US6464973B1 (en) Universal GM-CSF expressing bystander human K562 cell line
Laning et al. Inhibition of in vivo tumor growth by the beta chemokine, TCA3.
Zatloukal et al. Elicitation of a systemic and protective anti-melanoma immune response by an IL-2-based vaccine. Assessment of critical cellular and molecular parameters.
Cayeux et al. Tumor cells cotransfected with interleukin‐7 and B7. 1 genes induce CD25 and CD28 on tumor‐infiltrating T lymphocytes and are strong vaccines
Schultz et al. Long-lasting anti-metastatic efficiency of interleukin 12-encoding plasmid DNA
JP4723722B2 (ja) ワクチン用アジュバントとしてのｍｈｃクラスｉｉリガンドの使用および癌治療におけるｌａｇ−３の使用
Chong et al. Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte–macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity
Visse et al. Regression of intracerebral rat glioma isografts by therapeutic subcutaneous immunization with interferon-γ, interleukin-7, or B7-1-transfected tumor cells
JP3837169B2 (ja) Ｂ７−形質移入細胞の投与による腫瘍細胞成長の阻害方法
EP0835130A1 (de) Gentherapie von soliden tumoren mit interferone allein oder mit anderen immunoeffektoren proteinen
KR19990014875A (ko) 주효세포 조절을 위한 유전자 요법
Graf et al. IL-6 secretion by a rat T9 glioma clone induces a neutrophil-dependent antitumor response with resultant cellular, antiglioma immunity
JP2975117B2 (ja) 腫瘍疾患の治療のための生ワクチン
Lebel‐Binay et al. Experimental gene therapy of cancer using tumor cells engineered to secrete interleukin‐13
Zhang et al. Tumour necrosis factor‐α (TNF‐α) transgene‐expressing dendritic cells (DCs) undergo augmented cellular maturation and induce more robust T‐cell activation and anti‐tumour immunity than DCs generated in recombinant TNF‐α
Shi et al. Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF
Hara et al. Rejection of mouse melanoma elicited by local secretion of interleukin‐2: implicating macrophages without T cells or natural killer cells in tumor rejection
Chong et al. Expression of co-stimulatory molecules by tumor cells decreases tumorigenicity but may also reduce systemic antitumor immunity
Pizzoferrato et al. Enhanced immunogenicity of B cell lymphoma genetically engineered to express both B7-1 and interleukin-12
Cao et al. Cytokine gene transfer in cancer therapy
Björkdahl et al. Vaccination with B16 melanoma cells expressing a secreted form of interleukin-1β induces tumor growth inhibition and an enhanced immunity against the wild-type B16 tumor
Li et al. Potent systemic antitumor immunity induced by vaccination with chemotactic-prostate tumor associated antigen gene-modified tumor cell and blockade of B7-H1

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19980119

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20000313

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20001123