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WO1996033746A1 - Traitement de tumeurs avec des cellules exprimant des interferons, des facteurs de necrose des tumeurs ou d'autres cytokines - Google Patents

Traitement de tumeurs avec des cellules exprimant des interferons, des facteurs de necrose des tumeurs ou d'autres cytokines Download PDF

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Publication number
WO1996033746A1
WO1996033746A1 PCT/US1996/006054 US9606054W WO9633746A1 WO 1996033746 A1 WO1996033746 A1 WO 1996033746A1 US 9606054 W US9606054 W US 9606054W WO 9633746 A1 WO9633746 A1 WO 9633746A1
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Prior art keywords
tumor
cells
interferon
necrosis factor
csf
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PCT/US1996/006054
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English (en)
Inventor
David L. Ennist
Yawen Chiang
Suzanne Forry-Schaudies
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Genetic Therapy, Inc.
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Application filed by Genetic Therapy, Inc. filed Critical Genetic Therapy, Inc.
Priority to AU56346/96A priority Critical patent/AU5634696A/en
Publication of WO1996033746A1 publication Critical patent/WO1996033746A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • 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
    • 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 relates to the treatment of tumors . More particularly, this invention relates to the treatment of tumors by administering to the host at the site of the tumor cells (e.g., fibroblasts) , which express an interferon or a tumor necrosis factor or granulocyte macrophage colony- stimulating factor (GM-CSF) .
  • This invention also relates to treating a tumor by administering to a host a non-tumor cell engineered with a polynucleotide encoding a cytokine and a tumor cell.
  • Dranoff, et al . , Proc. Nat. Acad. Sci .. Vol. 90, pgs. 3539-3543 (1993) disclose the vaccination of mice with irradiated B16 melanoma cells engineered with a gene encoding murine granulocyte macrophage-colony stimulating factor. Such vaccination stimulates anti-tumor immunity.
  • Gansbacher, et al . , Cancer Research. Vol. 50, pgs. 7820-7825 discloses the injection of mice with CMS-5 tumor cells transduced with retroviral vectors including the Interferon- ⁇ gene.
  • the Interferon- ⁇ producing cells induced a long lasting state of T-cell immunity, as judged by rejection of CMS-5 tumor challenge and persistence of specific cytotoxic activity in the spleen cell population.
  • Ferrantini et al . , Cancer Research. Vol. 53, pgs. 1107-1112 (March 1, 1993) , teach the injection of Friend Leukemia Cells transduced with a retroviral vector including the Interferon- ⁇ l gene into mice. Mice that were given injections of such cells developed a long-lasting tumor-specific immune resistance to subsequent injection with highly metastatic Friend Leukemia Cells.
  • an immune response can be generated against a tumor, such an immune response is not an immediate response, but rather is generated over a matter of days. Thus, it would be desirable to generate an immediate anti-tumor effect independent of an anti-tumor response generated by the immune system. It also would be desirable to combine such an anti-tumor response with an anti-tumor response generated by the immune system.
  • Figure 1 is a schematic of the construction of plasmid PG1;
  • Figure 2 is the sequence of the multiple cloning site in pGl;
  • Figure 3 is a map of plasmid pGl
  • Figure 4 is a map of plasmid pBg
  • Figure 5 is a map of plasmid pN2
  • Figure 6 is a map of plasmid pGlNa
  • Figure 7 is a map of plasmid pLNSX
  • Figure 8 is a map of plasmid pSvNa
  • Figure 9 is a map of plasmid pGlXSvNa
  • Figure 10 is a map of plasmid pGlFlSvNa
  • Figure 11 is a map of plasmid pGlF2SvNa
  • Figure 12 is a map of plasmid pGlF31SvNa
  • Figure 13 is a map of plasmid pGlmF3SvNa
  • Fi-gure 14 is a graph of the survival times of mice inoculated with parental B16F10 cells, or with B16F10 cells transduced with B16F10/GlNa or B16F10/GlF2SvNa;
  • Figure 15 is a graph of the tumor volumes of mice treated with parental B16F10 cells; B16F10/GlF2SvNa cells; B16F10/GlmF3SvNa cells; or a mixture of B16F10/GlF2SvNa cells and B16F10/GlmF3SvNa cells;
  • Figure 16 is a graph of the anti-B16F10 CTL response from mice bearing a primary B16F10 tumor, and in mice inoculated with B16F10/GlF2SvNa cells or B16F10/GlmF3SvNa cells prior to secondary exposure with parental B16F10 cells;
  • Figure 17 is a graph of the survival over a 70-day period of mice inoculated with Renca tumor cells and BALB3T3/GlNa, BALB3T3/GlF2SvNa, or BALB3T3/GlmF3SvNa fibroblasts;
  • Figure 18 is a graph of the survival over a 180-day period of mice inoculated with Renca tumor cells and BALB3T3/GlNa, BALB3T3/GlF2SvNa, or BALB3T3/GlmF3SvNa fibroblasts;
  • Figure 19 is a map of pGlmGmSvNa
  • Fi-gure 20 is a graph of the tumor size in mice injected with Renca cells; Renca cells and PA317/GlNa cells; or Renca cells and PA317 GlmGmSvNa cells;
  • Figure 21 is a graph of the tumor volume in mice injected with PA317/GlNa cells and non-irradiated B16F10 cells, or with PA317/GlmGmSvNa cells and non-irradiated B16F10 cells;
  • Figure 22 is a graph of the percent survival of mice challenged with Renca cells subsequent to vaccination with irradiated Renca cells; iradiated Balb 3T3/GlNa cells and irradiated Renca cells; or irradiated Balb 3T3/GlmGmSvNa cells and irradiated Renca cells; and
  • Figure 23 is a map of plasmid pGlGm. DETAILED DESCRIPTION OF THE INVENTION
  • a method of treating a tumor in a host comprises administering to a tumor site cells, preferably non-tumor cells, which express a therapeutically effective amount of one or more agents selected from the group consisting of an interferon, or a tumor necrosis factor.
  • treating a tumor means that one provides for the inhibition, prevention, or destruction of the growth of the tumor cells.
  • treating a tumor also encompasses preventing recurrence of a tumor which has been resected.
  • the scope of the present invention is not intended to be limited to any theoretical reasoning, Applicants have found that when one administers to a host at a tumor site cells which express an interferon or a tumor necrosis factor, that one obtains a local, non-immune mediated anti-tumor effect; i.e., growth of the tumor cells is inhibited, prevented, or destroyed, and that such effect is not dependent upon an immunological response.
  • a local, non-immune mediated anti-tumor effect i.e., growth of the tumor cells is inhibited, prevented, or destroyed, and that such effect is not dependent upon an immunological response.
  • the inhibition, prevention or destruction of the growth of the tumor is not delayed until the onset of an immune response against the tumor.
  • the immediate anti-tumor effect may be a result of the destruction of the vasculature of the tumor, or of direct destruction of the tumor cells, or of inhibition of tumor cell proliferation.
  • Non-tumor cells which may be administered to the host include, but are not limited to, fibroblasts, keratinocytes, or "universal" cells engineered to be tolerated by all individuals; and retroviral producer cells capable of generating retroviral vectors, including at least one polynucleotide encoding an agent selected from the group consisting of interferons and tumor necrosis factors.
  • the non-tumor cell is a fibroblast.
  • fibroblasts which may be employed include, but are not limited to, autologous-syngeneic fibroblasts, allogeneic fibroblasts, xenogeneic fibroblasts, autologous fibroblasts, human foreskin fibroblasts, mouse BLKcl4 fibroblasts, BALB 3T3 fibroblasts, and NIH 3T3 fibroblasts.
  • the agent is an interferon.
  • Interferons which may be expressed include, but are not limited to, interferons of the Interferon-or family, including the Interferon- A/D chimera described hereinbelow, Interferon-/3, and Interferon- ⁇ .
  • the interferon is an interferon of the Interferon- ⁇ family.
  • the interferon in Interferon- ⁇ is an interferon of the Interferon- ⁇ .
  • the agent is a tumor necrosis factor.
  • Tumor necrosis factors which may be employed include, but are not limited to, TNF- ⁇ and TNF-jS.
  • the tumor necrosis factor is TNF- ⁇ .
  • the cell which expresses the interferon or tumor necrosis factor is genetically engineered with at least one polynucleotide which encodes the interferon or tumor necrosis factor.
  • polynucleotide as used herein means a polymeric form of nucleotide of any length, and includes ribonucleotides and deoxyribonucleotides. Such term also includes single- and double-stranded DNA, as well as single- and double-stranded RNA. The term also includes modified polynucleotides such as methylated or capped polynucleotides .
  • Genes encoding the agents are obtainable through sources known to those skilled in the art (e.g., Genbank, ATCC, etc.) , and/or may be isolated from expression vehicles (e.g. , plasmids) obtainable through sources known to those skilled in the art through standard techniques (e.g., PCR) known to those skilled in the art.
  • expression vehicles e.g. , plasmids
  • standard techniques e.g., PCR
  • the polynucleotide encoding the interferon or tumor necrosis factor is contained within an appropriate expression vehicle which has been transduced into the cell.
  • expression vehicles include, but are not limited to, plasmids, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial vectors), and viral vectors.
  • the vector is a viral vector.
  • Viral vectors which may be employed include RNA viral vectors (such as retroviral vectors) , and DNA virus vectors (such as adenoviral vectors, adeno-associated virus vectors, Herpes Virus vectors, and vaccinia virus vectors) .
  • RNA virus vector such as retroviral vectors
  • DNA virus vectors such as adenoviral vectors, adeno-associated virus vectors, Herpes Virus vectors, and vaccinia virus vectors
  • the polynucleotide encoding the interferon, or tumor necrosis factor is in the form of DNA.
  • the viral vector is a retroviral vector.
  • retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • the vector is generally a replication incompetent retrovirus particle.
  • Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
  • Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env) , are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal .
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter. Alternatively, two genes may be expressed from a single promoter by the use of an Internal Ribosome Entry Site.
  • a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
  • retroviral vectors examples include, but are not limited to, Moloney Murine Leukemia Virus vectors such as those described in Miller, et al . , Biotechniques, Vol. 7, pgs. 980-990 (1989) , and in Miller, et al., Human Gene Therapy, Vol. 1, pgs. 5-14 (1990) .
  • the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
  • Preferred cloning sites are selected from the group consisting of NotI, SnaBI, Sail, and Xhol .
  • the retroviral vector includes each of these cloning sites. Such vectors are further described in U.S. Patent Application Serial No. 08/340,805, filed November 17, 1994, and in PCT Application No. W091/10728, published July 25, 1991, and incorporated herein by reference in their entireties.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of NotI, SnaBI, Sail, and Xhol located on the retroviral vector.
  • the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art .
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the vector includes one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al. , Biotechni-crues, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters) .
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the vector then is employed to transduce a packaging cell line to form a producer cell line.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ - 2 , iV-AM, PA12, T19-14X, VT- 19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines, as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990) .
  • the vector containing the polynucleotide encoding the interferon, tumor necrosis factor, or GM-CSF may transduce the packaging cells through any means known in the art.
  • Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the packaging cells thus become producer cells which generate retroviral vectors which include a polynucleotide encoding the interferon, tumor necrosis factor, or GM-CSF.
  • retroviral vectors then are transduced into cells, which may be non-tumor cells (such as, for example, fibroblasts) , or tumor cells, whereby the transduced cells will express the interferon or tumor necrosis factor.
  • the cells are administered to the host at the site of the tumor in an amount effective to inhibit, prevent, or destroy the growth of the tumor in the host.
  • Such administration may be non-systemic, and wherein the cells are not administered at a site remote from the tumor.
  • administration may be by direct, non-systemic injection of the cells to the site of the tumor.
  • the host may be a mammalian host, including human and non-human primate hosts.
  • such cells are administered to the host in an amount of at least 10 5 cells, and in general such amount does not exceed 10 10 cells.
  • the cells are administered in an amount of from about 10 ⁇ cells to about 10 ⁇ cells.
  • the exact dosage of cells which is to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, and the type and severity of the tumor to be treated.
  • Suitable pharmaceutical carriers include, li-quid carriers such as a saline solution, aqueous buffers such as phosphate buffers and Tris buffers, or these buffers containing Polybrene (Sigma Chemical, St. Louis, Missouri) .
  • li-quid carriers such as a saline solution, aqueous buffers such as phosphate buffers and Tris buffers, or these buffers containing Polybrene (Sigma Chemical, St. Louis, Missouri) .
  • a suitable pharmaceutical carrier is deemed to be apparent to those skilled in the art from the teachings contained herein.
  • Tumors which may be treated in accordance with the present invention include malignant and non-malignant tumors.
  • Malignant (including primary and metastatic) tumors which may be treated include, but are not limited to, those occurring in the adrenal glands; bladder, bone; breast; cervix; endocrine glands (including thyroid glands, the pituitary gland, and the pancreas) ; stomach; small intestine; peritoneal cavity; colon; rectum; heart; hematopoietic tissue; kidney; liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx; larynx and other head and neck cancers; ovaries; penis; prostate; skin (including melanoma, basal cell carcinoma, and s-quamous cell carcinoma) ; testicles; thymus; and uterus.
  • the method of the present invention also is particularly applicable to the treatment of surgically inaccessible tumors.
  • the non-tumor cells which express an interferon, or a tumor necrosis factor may be administered to a host whose tumor has been resected (for example, head and neck tumors) .
  • a tumor mass is removed from a host, and the cells expressing an interferon or a tumor necrosis factor are administered to the site of the tumor.
  • Such administration of the cells expressing the interferon or tumor necrosis factor or GM-CSF provides for the inhibition, prevention, or destruction of any residual tumor cells remaining in the host subse-quent to removal of the tumor mass.
  • the cell which is administered to the site of the tumor is a retroviral producer cell, such as those hereinabove described.
  • the producer cells Upon administration of the producer cells to the site of the tumor, the producer cells generate retroviral vectors including a polynucleotide encoding an interferon or tumor necrosis factor or GM-CSF.
  • retroviral vectors then transduce the tumor cells, which then produce the interferon or tumor necrosis factor in an amount which inhibits, prevents, or destroys the growth of the tumor.
  • the producer cells may be administered in an amount of from about 10 5 cells to about 10 10 cells, preferably from about 10 € cells to about 10 ⁇ cells.
  • the producer cells may be administered in combination with an acceptable pharmaceutical carrier, such as those hereinabove described.
  • the viral vector is an adenoviral vector.
  • the vector is an adenoviral vector.
  • the adenoviral vector which is employed may, in one embodiment, be an adenoviral vector which includes essentially the complete adenoviral genome. (Shenk, et al. , Curr. To . Microbiol. Immunol. , (1984); 111 (3) :1-39) .
  • the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted.
  • the vector comprises an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence encoding an interferon or tumor necrosis factor; and a promoter for expressing the DNA sequence encoding the interferon or tumor necrosis factor or GM-CSF.
  • the vector is free of at least the majority of adenoviral El and E3 DNA sequences, but is not necessarily free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins transcribed by the adenoviral major late promoter.
  • the gene in the E2a region that encodes the 72 kilodalton binding protein is mutated to produce a temperature sensitive protein that is active at 32°C, the temperature at which viral particles are produced, but is inactive at 37°C, the temperature of the animal or human host.
  • This temperature sensitive mutant is described in Ensinger, et al . , J.Virology, 10:328-339 (1972) ; Van der Vliet, et al. , J.Virology. 15:348-354 (1975) ; and Friefeld, et al., Virology, 124:380-389 (1983) ; Englehardt, et al . , Proc.Nat.Acad.Sci. , Vol. 91, pgs. 6196-6200 (June 1994) ; Yang, et al. , Nature Genetics, Vol. 7, pgs. 362-369 (July 1994) .
  • An adenoviral vector is constructed preferably first by constructing, according to standard techniques, a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter) ; a tripartite leader sequence, a multiple cloning site (which may be as herein described) ; a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome.
  • a shuttle plasmid which contains, beginning at the 5' end, the "critical left end elements," which include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an Ela enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter) ; a tripartite
  • Such DNA segment serves as a substrate for homologous recombination with a modified or mutated adenovirus, and such segment may encompass, for example, a segment of the adenovirus 5 genome no longer than from base 3329 to base 6246 of the genome.
  • the plasmid may also include a selectable marker and an origin of replication.
  • the origin of replication may be a bacterial origin of replication.
  • a desired DNA sequence encoding an interferon or tumor necrosis factor may or GM-CSF be inserted into the multiple cloning site of such plasmid for production of a vector for use in accordance with the invention.
  • the plasmid is used to produce an adenoviral vector by homologous recombination with a modified or mutated adenovirus in which at least the majority of the El and E3 adenoviral DNA sequences have been deleted.
  • homologous recombination may be effected through co-transfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells, by CaP0 4 precipitation.
  • the homologous recombination produces a recombinant adenoviral vector which includes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous recombination fra-gment, and DNA derived from the El and E3 deleted adenovirus between the homologous recombination fragment and the 3' ITR.
  • the homologous recombination fragment overlaps with nucleotides 3329 to 6246 of the adenovirus 5 genome (ATCC VR-5) .
  • a vector which includes an adenoviral 5' ITR, an adenoviral encapsidation signal; an Ela enhancer sequence; a promoter; a tripartite leader sequence; a DNA sequence encoding an interferon or tumor necrosis factor or GM-CSF; a poly A signal; adenoviral DNA free of at least the majority of the El and E3 adenoviral DNA sequences; and an adenoviral 3' ITR.
  • This vector may then be transfected into a helper cell line, such as the 293 helper cell line (ATCC No. CRL1573) , which will include the Ela and Elb DNA sequences, which are necessary for viral replication, to generate replication defective viral vector particles.
  • the vector hereinabove described may include a multiple cloning site to facilitate the insertion of the DNA sequence encoding the interferon or tumor necrosis factor or GM-CSF into the cloning vector.
  • the multiple cloning site includes "rare" restriction enzyme sites; i.e., sites which are found in eukaryotic genes at a frequency of from about one in every 10,000 to about one in every 100,000 base pairs.
  • An appropriate vector is thus formed by cutting the cloning vector by standard techniques at appropriate restriction sites in the multiple cloning site, and then ligating the DNA sequence encoding an interferon or tumor necrosis factor or GM-CSF into the cloning vector.
  • the DNA sequence encoding the interferon, tumor necrosis factor, or GM-CSF is under the control of a suitable promoter, which may be selected from those hereinabove described, or such DNA may be under the control of its own native promoter.
  • the adenovirus may be constructed by using a yeast artificial chromosome (or YAC) containing an adenoviral genome according to the method described in Ketner, et al . , PNAS. Vol. 91, pgs. 6186-6190 (1994) , in conjunction with the teachings contained herein.
  • the adenovirus yeast artificial chromosome is produced by homologous recombination in vivo between adenoviral DNA and yeast artificial chromosome plasmid vectors carrying segments of the adenoviral left and right genomic termini .
  • a DNA sequence encoding an interferon or tumor necrosis factor or GM-CSF then may be cloned into the adenoviral DNA.
  • the modified adenoviral genome then is excised from the adenovirus yeast artificial chromosome in order to be used to generate adenoviral vector particles as hereinabove described.
  • the adenoviral vector particles which include the DNA sequence encoding an interferon or tumor necrosis factor, then are transduced into non-tumor cells such as those hereinabove described, whereby such transduced cells will express the interferon or tumor necrosis factor.
  • the transduced cells then are administered to the site of the tumor in the host in amount hereinabove described.
  • a tumor is treated by administering non-tumor cells which express GM-CSF to elicit a systemic immune response against the tumor, wherein said non-tumor cells are administered at the site of the tumor or are administered at some other site in combination with such tumor cells or a tumor antigen of the type of tumor to be treated.
  • a systemic immune response may be elicited by administering such cells to a host in combination with tumor cells of the type of tumor to be treated or a tumor antigen of the type of tumor to be treated.
  • a method of treating a tumor in a host which comprises administering to a host a mixture of (a) non-tumor cells engineered with a polynucleotide encoding granulocyte macrophage colony stimulating factor (GM-CSF) ; and (b) an agent selected from the group consisting of (i) tumor cells of the type of tumor which is to be treated and (ii) at least one tumor antigen of the type of tumor which is to be treated.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Non-tumor cells engineered with a polynucleotide encoding GM-CSF, and which may be administered to a host include, but are not limited to, fibroblasts, keratinocytes, "universal" cells, and producer cells such as those hereinabove described.
  • the polynucleotide encoding GM-CSF is contained in an appropriate expression vehicle, such as those hereinabove described, which has been transduced into the non-tumor cell.
  • the expression vehicle is a retroviral vector including a polynucleotide encoding GM-CSF, which is transduced into the non-tumor cell.
  • the agent is a tumor cell.
  • the tumor cells prior to administration of the tumor cells, are treated (such as, for example, by irradiation) such that the tumor cells are incapable of forming a new tumor when administered to the host.
  • the non-tumor cells engineered with a polynucleotide encoding GM-CSF and the tumor cells are administered in amounts effective to inhibit, prevent, or destroy the growth of the tumor, or residual tumor, or tumor metastase ⁇ .
  • the administration of the non-tumor cells engineered with a polynucleotide encoding GM-CSF and the tumor cells elicit an immune response (such as, for example, a systemic immune response) against the tumor.
  • the non- tumor cells and the tumor cells are administered non- systemically at a subcutaneous site.
  • the cells may be delivered intradermally or subcutaneously, or intramuscularly at a site distal to the primary tumor or any metastases.
  • the non-tumor cells are administered in an amount of at least 10 s cells, and in general does not exceed 10 10 cells.
  • the non-tumor cells are administered in an amount of from about 10 6 cells to about 10 ⁇ cells.
  • the tumor cells are administered in an amount of at least 10 s cells, and in general such amount does not exceed 10 ⁇ o cells.
  • the tumor cells are administered in an amount of from about 10 6 cells to about 10 8 cells. Tumors which may be treated include those hereinabove described.
  • the non-tumor cells engineered with a polynucleotide encoding GM-CSF and the tumor cells against which an immune response is to be elicited may be administered to a host in conjunction with the cells expressing an interferon or a tumor necrosis factor, which are administered at a tumor site.
  • a method of treating a tumor in a host which comprises administering to a tumor site cells which express an agent selected from the group consisting of an interferon and a tumor necrosis factor, in conjunction with administering to a host (such as, for example, by subcutaneous non-systemic administration) a mixture of (a) non-tumor cells engineered with a polynucleotide encoding a cytokine which enhances or promotes an immunogenic response against tumor cells which are being treated, and (b) tumor cells and/or antigens of tumor cells against which an immune response is to be elicited.
  • the non- tumor cell and tumor cell and/or antigens of tumor cells may be selected from those hereinabove described.
  • Cytokines encoded by the polynucleotide with which the non-tumor cell is engineered include, but are not limited to, GM-CSF, Interleukin-2, Interleukin-4, and Interleukin-12.
  • the cytokine is GM-CSF.
  • the polynucleotide encoding the cytokine may be contained in an expression vehicle such as those hereinabove described, which is transduced into the non-tumor cell.
  • Such response is followed by an immune response against the tumor obtained through the administration of the non-tumor cell engineered with a polynucleotide encoding a cytokine and tumor cells and/or antigens of tumor cells of a tumor which is the subject of the treatment.
  • a tumor obtained through the administration of the non-tumor cell engineered with a polynucleotide encoding a cytokine and tumor cells and/or antigens of tumor cells of a tumor which is the subject of the treatment.
  • Plasmids pGlFlSvNa, pGlF2SvNa, pGlF31SvNa, and pGlmF3SvNa were derived from plasmid pGl.
  • Plasmid pGl was constructed from pLNSX ( Figure 7) (Palmer, et al. , Blood, Vol. 73, pgs. 438-445) .
  • the construction strategy for plasmid pGl is shown in Figure 1.
  • Okb EcoRI/Clal fragment containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately.
  • a linker containing seven unique cloning sites was then used to close the EcoRI/Clal fragment on itself, thus generating the plasmid pGO.
  • the plasmid pGO was used to generate the vector plasmid pGl ( Figure 3) by the insertion of the 1.6kB EcoRI fragment containing the 5' LTR into the unique EcoRI site of pGO.
  • pGl ( Figure 3) consists of a retroviral vector backbone composed of a 5' portion derived from MoMuSV, a short portion of gag in which the authentic ATG start codon has been mutated to TAG (Bender, et al . 1987) , a 54 base pair multiple cloning site (MCS) containing, from 5' to 3 ' the sites EcoRI, NotI, SnaBI, Sail, BamHI, Xhol, Malawi, Apal, and Clal and a 3' portion of MoMuLV from base pairs 7764 to 7813 (numbered as described (Van Beveren, et al. , Cold Spring Harbor, Vol. 2, pg. 567, (1985)) ( Figure 2) .
  • MCS multiple cloning site
  • the MCS was designed to generate a maximum number of uni-que insertion sites, based on a screen of non-cutting restriction enzymes of the pGl plasmid, the neo r gene, the ⁇ -galactosidase gene, the hygromycin r gene, and the SV40 promoter.
  • the structure of the 5' linker was as follows: 5' - 1/2 Ndel - SphI - NotI - SnaBI - Sail - SacII - AceI - Nrul - Bglll - III 27 bp ribosomal binding signal - Kozak consensus sequence/Ncol - first 21 bp of the lacZ open reading frame - 1/2 BamHI - 3' .
  • the structure of the 3' linker was as follows: 5' - 1/2 mutated EcoRI - last 55 bp of the lacZ open reading frame - Xhol - Hindlll - Smal - 1/2 EcoRI - 3' .
  • the restriction sites in the linkers were chosen because they are not present in the neomycin resistance gene, the S-galactosidase gene, the hygromycin resistance gene, or the SV40 promoter.
  • the 27 bp ribosomal binding signal was included in the 5' linker because it is believed to enhance mRNA stability (Hagenbuchle, et al . , Cell 13:551-563, 1978 and Lawrence and Jackson, J. Mol. Biol. 162:317-334, 1982) .
  • the Kozak consensus sequence (5' -GCCGCCACCATGG-3' ) has been shown to signal initiation of mRNA translation (Kozak, Nucl.Acids Res. 12:857-872, 1984) .
  • the Kozak consensus se-quence includes the Ncol site that marks the ATG translation initiation codon.
  • pBR322 (Bolivar et al. Gene 2:95, 1977) was digested with Ndel and EcoRI and the 2.1 kb fragment that contains the ampicillin resistance gene and the bacterial origin of replication was isolated.
  • the ligated 5' linker - lacZ - 3' linker DNA described above was ligated to the pBR322 Ndel/EcoRI vector to generate pBg.
  • pBg has utility as a shuttle plasmid because the lacZ gene can be excised and another gene inserted into any of the restriction sites that are present at the 5' and 3' ends of the lacZ gene. Because these restriction sites are reiterated in the pGl plasmid, the lacZ gene or genes that replace it in the shuttle plasmid construct can easily be moved into pGl.
  • the "backbone" vector pGlNa was constructed from pGl and pN2 (Armentano, et al., J. Virology, Vol. 61, pgs. 1647-1650 (1987)) .
  • pGlNa was constructed by cutting pN2 ( Figure 5) with EcoRI and AsUII, filling in the ends of the EcoRI/AsuII fragment containing the neo R gene, and ligating the fragment into SnaBI digested pGl to form pGlNa ( Figure 6) .
  • pGl was cut with Hindlll and Sail.
  • pSvNa ( Figure 8) , which contains the SV40 promoter from pLNSX ( Figure 7) and the neo R gene from pN2, also was cut with Hindlll and Sail, and a Hindlll-Sall fragment containing an SV40 promoter and a ⁇ - galactosidase gene was ligated into Hindlll/Sall digested pGl to form pGlXSvNa ( Figure 9) .
  • pGlFlSvNa ( Figure 10) includes the human Interferon- ⁇ gene under the control of the retroviral LTR.
  • pGlF2SvNa ( Figure 11) includes a Bglll chimera of the human Interferon- a A and D genes under the control of the retroviral LTR. This chimera is active in human and mouse cells. (Rehberg, et al . , J . Biol. Chem.. Vol. 251, pg. 11497 (1992)) .
  • pGlF31SvNa Figure 12
  • Figure 12 includes the human Interferon- ⁇ gene under the control of the retroviral LTR.
  • pGlmF3SvNa ( Figure 13) includes the murine Interferon- ⁇ gene under the control of the retroviral LTR.
  • the cDNA for murine IFN ⁇ was obtained by PCR from plasmid pGEMmuIFN ⁇ (Sandoz, East Hanover, New Jersey) using a 5' primer (GCA GAT CTT TCC GCA GCA GCC GCC ACC ATG AAC GCT ACA CAC TGC ATC) that added a Bglll site, a ribosome binding site and a Kozak initiation sequence and a 3' primer (GTC CTC GAG TCA GCA GCG ACT CCT TTT CC) that added a Xhol site.
  • a 5' primer (GCA GAT CTT TCC GCA GCA GCC GCC GCC ACC ATG AAC GCT ACA CAC TGC ATC) that added a Bglll site, a ribosome binding site and a Kozak initiation sequence
  • GTC CTC GAG TCA GCA GCG ACT CCT TTT CC a 3' primer
  • the resulting PCR product was inserted into the plasmid backbone of the Bglll/Xhol cut pBg plasmid.
  • the result, pMF3 was cut with NotI and Xhol and ligated into Notl/Sall digested pGlXSvNa to create pGlmF3SvNa.
  • the cDNA for human IFN ⁇ 2 (EMBL Accession No. A15695) was obtained on plasmid pL31 from Hoffman-LaRoche, Nutley, New Jersey. It was digested with Ddel and PstI to remove the IFN ⁇ 2 cDNA. It was inserted into BamHI/PstI digested pUC18 (ATCC No.
  • Plasmid pF2 was SnaBI/EcoRI digested and the chimeric IFNcr A/D cDNA was ligated into the SnaBI site of pGlXSvNa to create pGlF2SvNa.
  • the cDNA for human IFN ⁇ (EMBL Accession No.
  • V00536 was obtained by RT PCR of human tumor infiltrating lymphocyte (TIL) cell mRNA using a 5' primer (GGT ACG TAG CCG CCA CCA TGA AAT ATA CAA GTT ATA TCT TGG) that added a SnaBI site and a Kozak initiation sequence and a 3' primer (CTG TCG ACT CGA GTT ACT GGG ATG CTC TTC G) that provided a Sail site.
  • the PCR product was cloned into pGlXSvNa that had been cut with SnaBI and Sail to create pGlF31SvNa.
  • Each of plasmids pGlFlSvNa, pGlF2SvNa, pGlF3SvNa, and pGlmF3SvNa was transfected into the murine PE501 ecotropic packaging cell line. (Miller, et al . , 1990) Transient supernatants from these transfectants were used to transinfect murine PA317 (ATCC No. CRL 9078) amphotropic packaging cells . The amphotropic producers were cloned and characterized by Southern Blot analysis and retroviral titer production. Clones having the expected retroviral insert sizes that produced high retroviral titers were picked for further analysis.
  • All producer cells were negative for contamination by mycoplasmas and replication competent retroviruses .
  • the retroviruses generated from the producer cells are hereinafter referred to as GIFlSvNa, GlF2SvNa, GlF31SvNa, and GlmF3SvNa.
  • Murine CT26 (Brattain, et al . , Cancer Research. Vol. 40, pg. 2142 (1980)) colonic tumor, Renca (Murphy, et al . , J. Nat. Can. Inst .. Vol. 50, pg. 1013 (1973)) renal carcinoma, and B16F10 (Fidler, et al . , Cancer Research, Vol. 37, pg. 3945 (1977)) melanoma cells and the non-tumorigenic BLKcl4 (ATCC No. T1B81) and BALB3T3 (ATCC No.
  • fibroblast cells were transduced with GlF2SvNa, GlmF3SvNa, or GINa retroviral supernatants.
  • 2.5xl0 5 cells were transduced with retroviral supernatant in the presence of 8 ⁇ g/ml Polybrene at multiplicities of infection from 0.5 to 10.
  • the cells were characterized by Southern Blot analysis and interferon secretion. Bioassays were performed on GlF2SvNa trausduced cells using HS68 cells (ATCC No. CRL1635) and encephalomyocarditis virus (ATCC No.
  • mice were divided into three groups of 16 and one group of 15.
  • the mice of Group I (16 mice) were injected intradermally with 5 x 10 4 CT26 cells.
  • the mice of Group II (16 mice) were injected intradermally with 5 x 10* CT26 cells transduced with GINa.
  • the mice of Group III (15 mice) were injected intradermally with 5 x 10 4 CT26 cells transduced with GlF2SvNa.
  • the mice of Group IV (15 mice) were injected intradermally with CT26 cells transduced with GlmF3SvNa.
  • Tumor onset was noted and measurements were taken at regular intervals. 11 days after injection, four mice from each group were taken for histology. The tumor onset at 11 and 34 days after injection and average tumor volume at 34 days after injection are given in Table II below.
  • CT26 tumor shows an unusually high percentage of spontaneous regressions.
  • the difficulties of this model notwithstanding, none of the animals in Group III developed a tumor during the 73 day course of the study. Also, the tumors in Group IV were substantially smaller than the tumors in control Groups I and II.
  • mice were inoculated intradermally with parental B16F10 cells, B16F10 cells transduced with GINa, B16F10/GlF2SvNa clones 1, 4, or 14 or the mixed population of B16F10 cells transduced with GlF2SvNa (See Table I above) , or the mixed population of B16F10 clones transduced with GlF2SvNa. (See Table I.)
  • Ten mice in each group were injected with 10 s cells per mouse. The survival curve is shown in Figure 14.
  • mice inoculated with clone 1 of B16F10/GlF2SvNa cells which secreted approximately 15 International Units of Interferon- ⁇ A/D per 10 6 cells in 24 hours, had a survival curve that was very similar to the animals inoculated later with control parental B16F10 cells or B16F10/GlNa cells.
  • An increase in survival was seen when animals were inoculated with the mixed population of B16F10/GlF2SvNa calls (producing about 200 I.U./10 6 cells/24 hrs.) , or clones secreting greater than 160 I.U./10 6 cells/24 hrs.
  • 6 out of 27 (22.2%) were long-term (i.e., 6 months) survivors.
  • mice treated with B16F10/GlmF3SvNa cells failed to develop a palpable tumor, and tumors in the three treated groups showed a delay in the progression of the tumor volume.
  • mice 10 6 irradiated B16F10/GlF2SvNa, or mixed populations, or clones 1, 4, or 14 , of B16F10/GlmF3SvNa cells were implanted intradermally into syngeneic mice. 9 or 10 mice received each type of cell. Twelve days later, the mice were challenged with 10 5 live, non-irradiated parental B16F10 cells, and the onset and progression of the tumor was monitored. No significant differences between animals vaccinated with interferon-producing cells, naive animals, or animals vaccinated with parental B16F10 cells or B16F10/GlNa cells were observed. This was found to be true whether the animals were vaccinated once or boosted an additional two times.
  • Renca cells transduced with GlF2SvNa were implanted intradermally into syngeneic mice. 5 to 10 mice were in each group of mice. 14 days after the mice received the transduced Renca cells, the mice were challenged with 10 s , or 5xl0 5 ; or 10 6 ; or 5xl0 6 ; or 10 7 parental Renca cells. The mice were not protected from the challenge of the parental Renca cells.
  • mice were vaccinated intradermally with 5xlO ⁇ irradiated BALB 3T3 fibroblasts transduced with GlF2SvNa plus 10 6 parental Renca cells. On day 12, the mice were challenged with 3xl0 6 parental Renca cells. No vaccine effect was observed as a result of the administration of the transduced fibroblasts.
  • the anti-B16F10 CTL response of the mouse injected with B16F10 cells after only a primary exposure to the tumor was as good or better than the responses of animals that had been exposed to B16F10 following an initial suppression of interferon-transduced B16F10 cells. If the initial suppression of the tumor cells had been due to the stimulation of the CTL, a reaction more vigorous than the primary response of the control mouse should have occurred.
  • the "natural" immune system may not be involved in interferon-mediated suppression of tumor growth.
  • the natural immune system is presented and ready to react in a naive animal, it lacks memory for a particular antigen, but it can be stimulated in a transient sense during and shortly after the presence of the agent causing the reaction.
  • mice had similar tumor incidences, days of tumor onset, progression and survival.
  • BLKcl4 and BALB3T3 fibroblast cell lines were transduced with GINa, GlF2SvNa or GlmF3SvNa as hereinabove described.
  • mice 100 C57B1/6J mice were divided into 10 groups with 10 mice in each group. Each mouse in one group of mice was inoculated intradermally with 10 s B16F10 cells alone. Each mouse in each of the other groups of mice was inoculated with a mixture of 10 5 B16F10 cells and (i) 5 x 10"; or (ii) 10 5 ; or (iii) 10 6 , (a) BLKcl4/GlNa cells; or (b) BLKcl4/GlF2SvNa cells; or (c) BLKcl4/GlmF3SvNa cells. The average survival time in days for the mice in each group was determined. The results are given in Table V below. Table V
  • mice 100 C57B1/6J mice were divided into 10 groups with 10 mice in each group. Each mouse in one group of mice was inoculated intradermally with 10 s B16F10 cells alone. Each mouse in the other groups of mice was inoculated with 10 s B16F10 cells and (i) 5 x 10 4 ; or (ii) 10 5 ; or (iii) 10 6 , (a) BALB3T3/GlNa cells; or (b) BALB3T3/GlF2SvNa cells; or (c) BALB3T3/GlmF3SvNa cells. The average survival times for each group of mice were determined. The results are given in Table VI below.
  • mice 40 BALB/c AnN mice were divided into 4 groups of 10 mice each.
  • Group I received an intradermal inoculation of 10 5 Renca cells alone.
  • Group II was inoculated with 5xl0 € BALB3T3/GlNa cells and 10 5 Renca cells at a fibroblast-to- tumor cell ratio of 50:1.
  • Group III was inoculated with 5x10* BALB3T3/GlF2SvNa cells and 10 5 Renca cells at a fibroblast-to-tumor cell ratio of 50:1.
  • Group IV was inoculated with 5xl0 6 BALB3T3/GlmF3SvNa cells and 10 s Renca cells at a fibroblast-to-tumor ratio of 50:1. Survival curves of each group were plotted and are shown in Figure 17.
  • mice were divided into i group of 15 animals and 3 groups of 10 animals.
  • the 15 animals in group I received an intraperitoneal injection of 5x10 s Renca cells alone.
  • Groups II, III and IV were inoculated intraperitoneally with 5x10 s Renca cells on day 1 followed by 3xl0 6 irradiated BALB3T3/GlNa, BALB3T3/GlF2SvNa, or BALB3T3/GlmF3SvNa fibroblasts on day 4 and survival was monitored for 6 months.
  • a murine GM-CSF expression plasmid, pXMT2-muCSF was obtained from Sandoz Pharmaceuticals (East Hanover, NJ) .
  • the mGM-CSF cDNA in pXMT2-muCSF was made by looping introns out of a phage clone containing the murine GM-CSF gene and reassembling them into a cDNA.
  • the 1.2 kb cDNA was cloned into the Xhol site of pSM using an Xhol/Sall fusion.
  • the restriction enzyme Kpnl was used to excise the 1.2 kb fra-gment containing the mGM-CSF cDNA out of pXMT2-muCSF.
  • the 1.2 kb fragment that contains the mGM-CSF (Genbank accession number X02333 and Gough et al. , EMBO J. , Vol. 4, pgs. 645-653 (1985) ) was restriction enzyme digested with StuI and Plel to generate a 619 bp fragment containing 13 bp of 5'- untranslated sequence, the 462 bp mGM-CSF open reading frame, and 149 bp of 3' -untranslated sequence. The ends of the 619 bp fragment were blunted with DNA polymerase I large (Klenow) fragment.
  • pGlmGmSvNa was digested with SnaBI, which cuts in the multicloning region.
  • the 619 bp StuI-Plel fragment containing the mGM-CSF cDNA was ligated to the SnaBI-digested pGlXSvNa using T4 DNA ligase.
  • Ligated plasmid DNA was transformed into E. coli DH5 ⁇ competent cells (Gibco/BRL, Gaithersburg, MD) and DNA from ampicillin-resistant colonies was screened by restriction enzyme digestion.
  • Plasmids that appeared to contain the 619 bp mGM-CSF insert in the forward orientation [i.e., ATG translation initiation codon adjacent to the retroviral 5' long terminal repeat (LTR)] were termed pGlmGmSvNa ( Figure 19) .
  • Vector- containing supernatants from the transfected cultures were used to transinfect the amphotropic packaging cell line PA317.
  • Transinfected PA317 were selected under G418 (800 ⁇ g/ml) and 38 single cell clones were isolated. The relative number of retroviral particles in the supernatants of the 38 clones was compared by RNA titering and the 8 clones with highest RNA titer were further analyzed by biological (G418) titering, Southern analysis, and ELISA (enzyme linked immunosorbant assay) for mGM-CSF expression (Endogen) .
  • Clone 5 had the highest G418 titer (7.2 x 10 5 cfu/ml) .
  • Clone 25 was found to express high levels of mGM-CSF (about 200 ng mGM- CSF/10 6 cells/24 hours) and therefore was chosen for studies re-quiring high mGM-CSF expression.
  • Balb/3T3 clone A31 is a murine embryonic fibroblast cell line developed from disaggregated Balb/c mouse embryos (Aaronson et al . , J. Cell. Phvsiol.. Vol. 72, pg. 141 (1968)) .
  • NIH/3T3 is a line of contact-inhibited mouse fibroblasts developed from disaggregated NIH Swiss mouse embryos. Both cell lines were grown in culture and transduced with 10 ml of GlmGmSvNa.5 supernatant. Transduced cells were selected in G418 and single cell clones were isolated.
  • Balb/3T3 transduced cells (Balb/GlmGmSvNa) , 60 clones were isolated, and after a preliminary test of mGM-CSF expression by ELISA (Kurstak, Enzyme Immunodia-gnosis, Acadmic Press, Orlando Florida) , 25 clones were further expanded and tested by ELISA for mGM-CSF expression.
  • the Balb/GlmGmSvNa mixed cell population (the G418-selected, transduced Balb3T3 cells prior to cloning) secreted 190 ng mGM-CSF/10 6 cells/24 hours, whereas the clones secreted 60-833 ng/10 6 cells/24 hours.
  • Balb/GlmGmSvNa.5 secretes approximately 240 ng mGM-CSF/10 6 cells/24 hours and has been characterized by Southern and Northern analyses.
  • 26 NIH3T3/GlmGmSvNa clones were isolated and tested for mGM-CSF expression by ELISA.
  • NIH3T3/GlmGmSvNa clone 1 secretes approximately 240 ng mGM-CSF/10 6 cells/24 hours and was chosen for further studies because this level of mGM-CSF production closely matches that of PA317/GlmGmSvNa.25, and Balb/GlmGmSvNa.5.
  • All transduced clones used for in vivo studies were found to have intact proviral DNA when analyzed by Southern analysis.
  • Northern analyses showed long and short proviral messages of the expected sizes.
  • PA317/GlmGmSvNa.25 cells were mixed with the murine renal carcinoma cell line Renca (Murphy, et al . , J. Nat. Cancer Inst. , Vol. 50, pgs. 1013-1025 (1975)) .
  • Renca murine renal carcinoma cell line
  • PA317/GlNa.40 or PA317/GlmGmSvNa.25 producer cells and nonirradiated Renca (1x10 s cells per mouse) were mixed at either 1:1 or 10:1 ratios and implanted intradermally in the right flanks of Balb/cJ mice (Jackson Laboratories, Bar Harbor, Maine) .
  • NIH3T3 fibroblasts were derived from NIH Swiss mice and therefore are allogeneic to C57B1/6 mice.
  • the following experiment tests whether allogeneic fibroblasts that secrete mGM-CSF are capable of functioning in a tumor vaccine setting, or whether they would be rapidly rejected. This question is pertinent to a clinical setting in which autologous fibroblasts may not be available.
  • Mixtures of mGM-CSF-secreting, allogeneic fibroblasts and irradiated B16F10 tumor were tested for their ability to elicit systemic immunity to wild-type tumor challenge.
  • Fibroblast type ( 10 6 ) Tumor cell ( 10 6 ) post - challenge
  • the example describes experiments performed to test the efficacy of syngeneic murine GM-CSF secreting fibroblast- tumor cell mixtures as tumor vaccines.
  • lOBalb/cAnN mice were injected intradermally in the flanks with 10 6 irradiated Renca cells; 20 mice were injected with 10 6 irradiated Balb 3T3/GlNa cells and 10 6 irradiated Renca cells; 20 mice were injected with a mixed population of 10 6 irradiated Balb 3T3/GlmGmSvNa cells and 10* irradiated Renca cells; and 20 mice received 10 6 irradiated Balb 3T3/GlmGmSvNa.5 (clone 5) cells.
  • mice were injected intradermally with (i) 10* irradiated Renca cells; or (ii) 10* irradiated Renca cells and 10* irradiated Balb/GlmGmSvNa.5 cells; or (ii) 10* irradiated Renca cells and 10* irradiated Balb/GlNa cells .
  • 11 days after vaccination the mice received Renca cells intradermally in amounts of 1x10 s ; or 5x10 s ; or 1x10*; or 5x10*; or IxlO 7 cells.
  • the number of mice in each group with tumors at 57 days post- challenge is given in Table IX below.
  • Tumor Fibroblast 10 5 5X1 10' 5xl0 10 7 0 5
  • mice receiving a GM-CSF vaccination reject a IxlO 7 Renca cell challenge, where as none of the control animals remain tumor-free at 57 days post- challenge .
  • the human GM-CSF cDNA was originally derived by RTPCR of RNA from human Mo T cell leukemia cells (ATCC CRL 8066) because these cells are known to express GM-CSF.
  • the GM-CSF coding sequence was subsequently cloned into retroviral vectors to create pGlGmSvNa and pGlNaSvGm.
  • pGlGmSvNa and pGlNaSvGm were constructed as follows: PCR of the human GM-CSF from Mo cell reverse transcribed RNA is carried out using the following primers: 5' -GCA CAG CTT TCC GCA GCA GCC GCC ACC ATG TGG CTG CAG AGC CTG which includes a Bglll site (bp3-8) , a ribosome binding site (bp4- 18; see also reference to ribosome binding site in the pBg description) , a Kozak sequence (bpl9-30) , and GM-CSF open reading frame sequence from the ATG translation initiation codon (bp 28-45) ; and 5' -GGC AAG CTT GTC GAC TCA CTC CTG GAC TGG CTC where bp 4-9 are a Hindlll site, bp 10-16 are a Sail site, and bp 17-33 are the antisense se-quence to
  • pGlNaSvX is a plasmid derived from pGlNaSvBg (Mc Lachlin, et al . , Virology, Vol. 195, pgs. 1-5 (1993)) , in which the lacZ gene was excised by digestion of pGlNaSvBg with Bglll and Hindlll.
  • Ligated plasmid DNA was transformed into E.
  • Plasmids that give the expected restriction fragments are termed pGlNaSvCm.
  • the dideoxy sequence of the GM-CSF cDNA in one such clone is determined and aligned to the expected sequence of human GM-CSF (Wong et al . , Science, Vol. 228, pgs. 810-814 (1985)) to confirm correctness.
  • pGlGmSvNa is restriction enzyme digested with Bglll and end-filled with Klenow.
  • the cut plasmid is then digested with Sail to isolate the fragment containing the GM-CSF open reading frame. This fragment is ligated to pGlXSvNa that has been digested with SnaBI and Sail (both cut in the polycloning "X" region) . Ligated plasmid is transfomred into DH5 ⁇ and clones are screened as described above for pGlNaSvGm. The human GM-CSF coding sequence used in the construct pGIGm ( Figure 23) was subcloned from the construct pGlNaSvGm.
  • the GM-CSF open reading frame was excised from pGlNaSvGm using restriction enzymes Bglll and Hindlll. This fragment was ligated into pGl that had been digested with restriction enzymes BamHI and Hindlll (these enzymes cleave pGl in the multicloning region) .
  • Ligated plasmid DNA was transformed into E.Coli DH5 ⁇ competent cells and DNA from ampicillin- resistant colonies was screened by restriction enzyme digestion. Plasmids that appeared by restriction mapping to contain the GM-CSF insert were termed pGIGm.
  • the dideoxy sequences of the cloning junctions i.e. the regions where the fragments were joined by ligation) were determined and found to be correct.
  • the sequence of the GM-CSF cDNA in pGlNaSvGm had previously been determined to be correct.
  • pGIGm does not contain a selectable marker such as the neomycin resistance gene
  • 30 micrograms of pGIGm were cotransfected with 3 micrograms of pSV 2 Neo (Powels, e al., Cloning Vectors. Elsevier, pg. VIII-B-b-6-1-9 (1986)) into the PA317 producer cell line. Standard calcium phosphate transfection methods were used (Chen, et al . , 1988) . Transfected cells were diluted into media containing C .6mg/ml G418. Sixty-six PA317/GlGm clones were isolated and the cell supernatants collected.
  • the relative number of retroviral particles in the clone supernatants was compared by RNA titering and the 9 clones with highest RNA titer were further analyzed by ELISA for human GM-CSF (R&D Systems) .
  • Standard biological titering methods cannot be employed for retroviral vectors that do not include a selectable marker such as neomycin. Instead, a Southern analysis titering method was used to titer GlGm clones the were positive by ELISA.
  • PA317/GlGm clone 55 was chosen for further studies based on RNA titer, ELISA, and Southern titer data.
  • the approximate titer of PA317/GlGm.55 is 1x10* cfu/ml.
  • PA317/GlGm.55 makes approximately 44 ng GM-CSF/10* cells/24 hours as measured by ELISA.
  • the amount of biologically active human GM-CSF in PA317/GlGm.55 supernatant was quantitated utilizing the GM-CSF-sensitive cell line M07e (Avanzi et al. , J.Cell.Phvsiol.. Vol. 145, 458-464 (1990)) .
  • the bioassay (IIT Research Institute, Chicago, 111.) demonstrated that PA317/GlGm.55 produced approximately 57 ng GM-CSF/10* cells/24 hours.
  • Southern analysis of PA317/GlGm.55 DNA has demonstrated only one proviral band of the expected size. The retroviral long message of the expected size is made by PA317/GlGm.55, as tested by Northern analysis.
  • NIH 3T3 fibroblasts are transduced with GlGm.55 supernatant and diluted for cloning.
  • Supernatants from clones are assayed by ELISA for expression of GM-CSF.
  • Clones expressing GM-CSF are assayed for the presence of the intact provirus by Southern.
  • a clone expressing high levels of GM-CSF and bearing the intact GlGm provirus is chosen for clinical applications.
  • the same number of irradiated fibroblasts or universal cells which express GM-CSF, which are engineered by transduction of the retrovirus GIGmSvNa into the fibroblasts or universal cells are injected in a similar manner directly into the tumor site in order to suppress the tumor by stimulating a systemic response from the immune system.
  • the injections with the universal cells can be repeated at 1 to 3 week intervals.
  • the preferred method is to resect surgically the tumor, treat with conventional chemo- and radiation therapies then to suppress the local regrowth with irradiated universal PA317/GlFlSvNa cells (10 s to 10 10 , preferably 10* to 10 ⁇ ) .
  • the tumor is homogenized to a single cell suspension by standard methods and mixed with an equal number of universal PA317/GIGmSvNa cells (10 s to 10 10 , preferably 10* to 10 ⁇ ) .
  • the cell mixture is then irradiated prior to vaccination intramuscularly, intradermally or subcutaneously at a site distal to the tumor to stimulate a systemic immune response. It is anticipated that the combination therapy with IFN ⁇ 2 and IFN ⁇ will result in an increase in patient survival greater than the use of either therapy by itself.

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Abstract

La présente invention concerne un procédé de traitement d'une tumeur chez un hôte. Ce traitement comprend l'administration, à l'emplacement de la tumeur, de cellules non tumorales qui expriment un ou plusieurs agents choisis dans le groupe comprenant un interféron, un facteur de nécrose de tumeurs et le GM-CSF. Quand l'agent est un interféron ou un facteur de nécrose de tumeurs, un tel procédé donne une réaction antitumorale locale, à médiation non immunitaire. La cellule qui exprime l'interféron ou le facteur de nécrose de tumeurs, et qui est administrée à l'emplacement de la tumeur, peut aussi être administrée, dans un mode de réalisation, en conjonction avec: a) une cellule non tumorale qui est fabriquée avec un polynucléotide codant une cytokine, et b) des cellules tumorales du type de tumeur qui fait l'objet du traitement, afin de donner une réaction immédiate à médiation non immunitaire, suivie d'une réaction immunitaire dirigée contre la tumeur.
PCT/US1996/006054 1995-04-28 1996-04-24 Traitement de tumeurs avec des cellules exprimant des interferons, des facteurs de necrose des tumeurs ou d'autres cytokines WO1996033746A1 (fr)

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AU56346/96A AU5634696A (en) 1995-04-28 1996-04-24 Treatment of tumors with cells expressing interferons, tumor necrosis factors, or other cytokines

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US43182895A 1995-04-28 1995-04-28
US08/431,828 1995-04-28

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WO1996033746A1 true WO1996033746A1 (fr) 1996-10-31

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO1999038954A1 (fr) * 1998-02-02 1999-08-05 Johns Hopkins University School Of Medicine Lignee cellulaire universelle immunomodulatrice exprimant les cytokines a fonction temoin, compositions correspondantes et procedes de fabrication et d'utilisation
US6579522B1 (en) 2000-06-27 2003-06-17 Genvec, Inc. Replication deficient adenoviral TNF vector
US7214368B2 (en) 2001-11-02 2007-05-08 Genvec, Inc. Therapeutic regimen for treating cancer comprising the administration of adenoviral vectors comprising a TNF-α transgene

Non-Patent Citations (5)

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Title
CANCER RESEARCH, 01 January 1994, Vol. 54, TAHARA et al., "Fibroblasts Genetically Engineered to Secrete Interleukin 12 Can Suppress Tumor Growth and Induce Antitumor Immunity to a Murine Melanoma in Vivo", pages 182-189. *
CANCER RESEARCH, 15 August 1990, Vol. 50, OGURA et al., "Implantation of Genetically Manipulated Fibroblasts Into Mice as Antitumor Alpha-Interferon Therapy", pages 5102-5106. *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999038954A1 (fr) * 1998-02-02 1999-08-05 Johns Hopkins University School Of Medicine Lignee cellulaire universelle immunomodulatrice exprimant les cytokines a fonction temoin, compositions correspondantes et procedes de fabrication et d'utilisation
US6464973B1 (en) 1998-02-02 2002-10-15 Johns Hopkins University, School Of Medicine Universal GM-CSF expressing bystander human K562 cell line
US7390483B2 (en) 1998-02-02 2008-06-24 Johns Hopkins University School Of Medicine Universal GM-CSF expressing bystander human K562 cell line
US8012469B2 (en) 1998-02-02 2011-09-06 Johns Hopkins University School Of Medicine Universal GM-CSF expressing bystander human K562 cell line
US6579522B1 (en) 2000-06-27 2003-06-17 Genvec, Inc. Replication deficient adenoviral TNF vector
US7214368B2 (en) 2001-11-02 2007-05-08 Genvec, Inc. Therapeutic regimen for treating cancer comprising the administration of adenoviral vectors comprising a TNF-α transgene

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