WO1998052615A9 - Regulation de la reponse immunitaire a des antigenes specifiques - Google Patents

Regulation de la reponse immunitaire a des antigenes specifiques

Info

Publication number
WO1998052615A9
WO1998052615A9 PCT/US1998/010381 US9810381W WO9852615A9 WO 1998052615 A9 WO1998052615 A9 WO 1998052615A9 US 9810381 W US9810381 W US 9810381W WO 9852615 A9 WO9852615 A9 WO 9852615A9
Authority
WO
WIPO (PCT)
Prior art keywords
vector
ligand
cells
gene
cell
Prior art date
Application number
PCT/US1998/010381
Other languages
English (en)
Other versions
WO1998052615A1 (fr
Inventor
John D Mountz
David T Curiel
Huang-Ge Zhang
Original Assignee
Uab Research Foundation
John D Mountz
David T Curiel
Zhang Huang Ge
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 Uab Research Foundation, John D Mountz, David T Curiel, Zhang Huang Ge filed Critical Uab Research Foundation
Priority to US09/424,281 priority Critical patent/US6689605B1/en
Priority to AU75859/98A priority patent/AU7585998A/en
Publication of WO1998052615A1 publication Critical patent/WO1998052615A1/fr
Publication of WO1998052615A9 publication Critical patent/WO1998052615A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • This invention relates generally to gene therapy. More specifically, the invention relates to suppressing immune system response to antigens expressed on an infected host cell.
  • T-cells are one component of the immune system. T-cells can become activated to specific antigens, and function to directly destroy materials which display that antigen, and they also function to sensitize other components of the immune system to the presence of that antigen. While a properly functioning immune system is vital to the health of an organism, in some instances there is a need for the selective inhibition of an immune response to particular materials.
  • viral vectors such as adenovirus
  • adenovirus are employed in genetic therapies to introduce genetic material and products into an organism.
  • One problem encountered with the use of such viral vectors is that they can provoke an immune response in the organism.
  • This immune response can destroy the viral vector, and those host cells which are intentionally infected by the vector, as well as therapeutic gene products produced by the action of the vector. Furthermore, immune system "memory" provides a lasting response to this vector; hence, readministration of the material will be ineffective. Therefore, there is a need for a method whereby the immune response to a selected viral vector may be blocked or destroyed. Suppression of immune response is also desirable in the instances of autoimmune disease. As is known, such disease results when the immune system of an organism inappropriately recognizes an organ or tissue of that organism as being foreign, and commences an immune response against it. If this immune response can be blocked, the autoimmune disease can be controlled. Immune suppression is also needed in those instances where organs are transplanted.
  • Immune system suppressing drugs are sometimes employed in the foregoing situations; however, such drugs produce a generalized suppression of the immune system, which leaves a patient open to a number of infections. It would therefore be advantageous if immune response to a specific antigen could be suppressed and/or an immune suppressed zone of tissue created within an organism.
  • Gene therapy is limited by induction of an immune response to the virus or the gene-therapy protein product (1-4).
  • a specific T-cell response to the viral vector usually results the failure of re-expression of transgene (5-6).
  • Many efforts have been made to reduce the T-cell response to the viral vector during gene therapy, including the blockade of MHC class I and II antigen, reduce the antigenicity of the viral vector, and prevention of co-stimulation of T-cells (1,7- 11).
  • Cytokine and cytokine ligand mediated apoptosis has been shown to be an important pathway for activation-induced cell death in T-cells (16-17).
  • T-cell activation leads to upregulation of cytokine ligand and cytokine apoptosis signaling (18,19).
  • Activated macrophages express increased levels of cytokine 2 lgs
  • Ad5 vectors ligand and mediate apoptosis in the T-cells during antigen presentation, which has been thought to be a critical mean of down-modulating T-cell response (20,21).
  • the efficiency of adenovirus-mediated gene transfer has been found to be far superior to other methods for the treatment of heart, lung, and liver disease, and is capable of producing more recombinant protein (22,23).
  • the cell-mediated immune response to El a-E3 -deleted adenoviral (Ad5) vector and the limited distribution of reporter gene expression suggest that less immunogenic recombinant vectors and more homogeneous administration methods are required before Ad5 vectors can be used successfully for phenotypic modulation.
  • Neonatal intrathymic injection of the vector was able to induce long- term LacZ expression for more than 2 months after heart injection, although neutralizing as well as anti- ⁇ -Gal antibodies were detected in the sera of the animals (24).
  • Pretreatment with the anti-TCR monoclonal antibody (mAb) H57 resulted in a significant reduction in lymphocytic infiltration and a prolongation of transgene expression (25).
  • Studies with adenoviral vectors show that immune responses limit the efficacy and persistence of gene expression. HSVtk/ganciclovir therapy was more effective in nude rats and immunosuppressed Fischer rats than in immunocompetent Fischer rats (26).
  • Adenoviral transgene expression was transient in the thymus of immunocompetent mice but persistent in CD8 + T-cell-deficient and severe combined immunodeficiency (SCID) mice, implicating a role for cytotoxic T lymphocytes in viral clearance (27).
  • SCID severe combined immunodeficiency
  • Ad5 vector expressing the lacZ transgene upon delivery intra-articularly (5 x 10 8 p.f.u.), lacZ expression was observed in the articular synovium for at least
  • Second-generation adenovirus for CFTR gene was evaluated after transfer to baboon lung. This second-generation virus is deleted of El and contains a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein. Using a second-generation adenovirus, recombinant gene stability was prolonged and associated with a diminished level of perivascular inflammation as compared to first-generation vectors (29). These data suggest that second-generation adenoviral vectors provide an improved gene delivery vehicle and are useful in gene therapy for diseases such as cystic fibrosis.
  • Another mechanism of tolerance is the use of immune privileged sites. This tolerance makes use of the natural occurrence of immune privileged sites which has more recently been thought to be due to production of Fas ligand in subsequent killing of T-cells that may develop and react with antigens within these sites. Installation of adenovirus into these sites results in tolerance to adenovirus and its transgene product. This has been tested using El deleted adenovirus injected into this subretinal space which resulted in minimal cellular and humeral immune response (32). The pancreatic islet may also be an immune privileged sites since murine pancreatic islets injected ex-vivo with Ad5 resulted in high level of beta galactosidase for at least 20 weeks after re-implantation (33).
  • Adenovirus mediated gene transfer in adult mouse islets does not impair insulin secretion by the islets (34).
  • Ad lacZ injected subretinally resulted in prolonged gene expression, which was equivalent to that observed in either nude mice or after treatment with CTLA4Ig (8).
  • the present invention is more widely applicable since transgene expression is not restricted to immune privileged sites.
  • antigen presenting cells that express apoptosis inducing ligands and processed viral vector antigens are utilized to directly induce apoptosis of T-cells expressing the ligand receptor resulting in vector-specific T-cell tolerance.
  • High levels of ligand and vector antigens are induced in APCs by co-infection.
  • Fas ligand FasL
  • AdLoxpFasL+AxCANCre pre-treatment of recipient mice with the adenovirus-infected APCs that express Fas ligand resulted in induction of T-cell tolerance to the adenovirus.
  • T-cell response to the viral vector is demonstrated by decreased cytokine production, decreased cytotoxic T-cell response, inhibition of clonal expansion of CD3+ T- cells, and prolonged the expression of a marker transgene.
  • Induction of T-cell tolerance to adenovirus requires expression of FasL on the APCs, and does not occur with adenovirus infected control APCs.
  • T-cell tolerance also requires expression of Fas on the T-cells of recipient mice, since lpr/lpr mice are not tolerized.
  • the T-cell tolerance is virus antigen-specific as there is normal T-cell response to mouse cytomegalovirus (CMV) in tolerized mice.
  • the instant invention includes a method for promoting immunotolerance in a host to a gene therapy vector, including transfecting a host cell with the vector, such that the vector expresses a transgene, an antigen and a ligand. Expression of the ligand induces apoptosis in a T-cell that is raised against the antigen.
  • the instant invention also includes a method for creating an immune privileged site in a tissue of an organism, the method including providing a gene therapy vector encoding and capable of expressing a ligand, a transgene and an antigen and infecting cells of the tissue with the vector.
  • the expression of the ligand in the tissue thereby induces apoptosis in T-cells raised against the ligand so as to confer specific immunity to infected cells.
  • the instant invention also teaches a gene therapy viral vector that includes a transgene, an apoptosis ligand gene and a gene expression control means for directing product synthesis of said transgene and said ligand gene.
  • a gene therapy viral vector that includes a transgene, an apoptosis ligand gene and a gene expression control means for directing product synthesis of said transgene and said ligand gene.
  • the use of such a vector for a gene therapy application is detailed.
  • the instant invention also discloses a gene therapy viral vector including a transgene, a viral vector gene that is expressed as an antigen on an infected host cell, a functional equivalent of a Fas ligand gene and a gene expression control means for directing product synthesis of said transgene and said Fas ligand gene.
  • Figure 1 A schematic illustrating a production method of gene therapy viral vector to inhibit an immune response to viral vector antigens and methods of using the same to produce immune privileged transduced mammalian host cells.
  • FIG. 1 Co-infection of APCs with AdloxpFasL + AxCanCre (APC-AdFasL) results in high levels of FasL capable of inducing apoptosis of A20 target cells.
  • the AdLoxpFasL is infected into APCs from lpr/lpr mice with and without AxCANCre.
  • the APCs are also electroporation transfected with pcDNA3FasL and stimulated with lipopolysaccharide (LPS) (1 ug/ml). FasL expression is determined by ability of the transfected APCs to induce apoptosis of a 51 Cr labeled, Fas sensitive cell line
  • mice Ten-week-old C57BL/6-+/+ mice are treated with 1 x 106 of the APCs co-infected with AdLoxpFasL plus AxCANCre (APC-AdFasL) or APCs co-infected with AdLoxpFasL plus AdCMVGFP (APC-AdControl) or PBS every 3 days for 5 doses.
  • mice After induction of T-cell tolerance, mice are intravenously inoculated with 1010 Ad/LacZ.
  • LacZ gene expression in the liver is analyzed by a quantitative assay (a) and In situ LacZ histochemical staining (b). The error bars indicate the mean ⁇ SEM for 3 mice analyzed separately in triplicate assay.
  • FIG. 4 Induction of tolerance to adenovirus by APC-AdFasL.
  • Ten-week-old C57BL/6-+/+ mice are injected intravenously with 1 x 106 APC-AdFasL, APC-AdControl or with PBS every 3 days for 5 doses as described above.
  • mice are challenged with AdCMVlacZ and T-cell cytotoxic response against APC + adenovirus is determined by killing of the APC cells infected with AdCMVGFP (5 pfu/cell). The percentages of viable GFP expressing APC cells are quantitated by FACS analysis. The error bars indicate the mean ⁇ SEM for 3 mice analyzed separately in triplicate assays.
  • FIG. 1 Decreased IFN-gamma and IL-2 induction by spleen cells from tolerized B6 +/+ mice. 10 6 of the APC-AdFasL or APC-AdControl cells were transferred to B6 +/+ mice. The spleen cells were incubated for 24 hours with
  • APCs that were uninfected, or infected with adenovirus, and irratiated.
  • Levels of IL-2(A) and IFN- ⁇ (B) in the supernatant was determined by ELISA.
  • APC-AdFasL or APC-AdControl cells are transferred to B6 +/+ mice.
  • the spleen cells are incubated for 24 hours with APCs that were uninfected, or infected with adenovirus, and irratiated.
  • Levels of IL-2 in the supernatant is determined by ELISA.
  • FIG. 7 IFN-gamma induction by spleen cells from B6 Ipr/lpr mice. 10 6 of the Ipr APC-AdFasL or APC-AdControl cells are transferred to B6-lpr/lpr mice. The spleen cells were incubated for 24 hours with APCs that are uninfected, or infected with adenovirus, and irratiated. Levels of IFN- ⁇ in the supernatant is determined by ELISA.
  • Figure 8. Ad/FasL APCs induces specific T-cell tolerance to adenovirus.
  • mice/group are treated with either C57BL/6-+/+ mice (5 mice/group) are treated with APC-AdFasL or APC-AdControl (M ⁇ -CV).
  • mice are challenged in vivo with either AdCMVLacZ or mouse cytomegalovirus (MCMV).
  • MCMV mouse cytomegalovirus
  • splenic T-cells are stimulated in vitro with APCs alone, or APCs infected with MCMV or AdCMVLacZ.
  • IL-2 production in the supernatants was determined by ELISA 48 hours later.
  • FIGS 9a-9e Characterization of Fas ligand expressing APCs.
  • Peritoneal resident macrophages from B6-lpr/lpr mice are isolated and cultured in RPMI- 1640-12% FCS. After short-term culture, growing macrophages are tested for MHC and B7 expression, (a)-(c) 1 x 10 6 macrophages are stained with biotin- co ⁇ jugated anti-H-2D b , anti-IA b (PharMingen) or CTLA4-Ig (Dr. Linsley: Bristol- Myers Squibb), followed by FITC-conjugated streptavidin (Southern
  • Macrophages are tranfected with a pcDNAIII expression vector (Invitrogen) containing a full length murine Fas ligand cDNA, or empty vector, using a standard DEAE-Dextran method. Transfected macrophages are selected with 0.5 mg/ml of G418 (Sigma).
  • the selected macrophages are mixed with [ 51 C]r-labeled, Fas ligand sensitive A20 cells at the indicated ratios and, after an 8 h incubation, the specific release is determined, (e)
  • the splenic T-cells are purified from 4-wk-old MRL/MpJ-+/+ and MRL/MpJ-lpr/lpr mice (Jackson Laboratory) using a T-cell enrichment column (R&D Systems).
  • 5 x 10 5 purified T-cells are cultured with 5 x 10 4 ⁇ -irradiated macrophages in round-bottom, 96-well plates for 5 d, and proliferation is determined by adding 1 mCi of [ 3 H]-thymidine (Amersham) 16 h prior to harvest.
  • APCs 4-wk-old of MRL-+/+ and -Ipr/lpr mice are injected i.v. with macrophages (2 x 10 5 ) transfected with Fas ligand or control vector every 3 d for 6 times.
  • splenic T-cells are isolated from treated mice and cultured under various stimulatory conditions, a, 5 x 10 5 T-cells are cultured with 2 x 10 5 ⁇ -irradiated total spleen cells from B6 +/+ mice, b, 5 x 10 5 T-cells are cultured with 2 x 10 5 ⁇ -irradiated total spleen cells from BALB/c mice, c, 5 x 10 5 T-cells are cultured with 5 mg/ml of anti-CD3 antibody.
  • T-cell proliferation is determined by incorporation of [ 3 H]-thymidine at indicated time points. The error bars indicate the mean ⁇ SEM for 3 mice analyzed separately in triplicate assays.
  • FIG. 11 Antigen-specific clonal deletion of the T-cells induced by Fas ligand expressing APCs in H-2D b HY reactive TCR transgenic mice, (a) Expression of H-2D b is determined as described above and analyzed by flow cytometric analysis, (b) Fas ligand activity is assayed by specific lysis of A20 target cells at the indicated E/T ratio as described in Figure 9. (c) The CD4 CD8 T-cells (2 x 10 6 ) from B6-lpr/lpr female or male mice are injected every 3 d for 3 times into female, TCR transgenic D b /HY -+/+ and -lpr mice.
  • T-cells (a) Expression of M33, CD8, and Fas on the T-cells in the PLN is determined by 3-color flow cytometric analysis. 1 x 10 6 total PLN cells are stained with biotin-conjugated M33, then with FITC-conjugated anti-CD8 and PE- conjugated anti-Fas (PharMingen). 10,000 viable lymphocytes were analyzed by FACScan. Two-color contour plots of CD8 and M33 are shown, and the percentage of M33 D8 + T-cells multiplied by the total number of spleen cells. The error bars indicate the mean ⁇ SEM for 3 mice analyzed, (c) Fas expression on the M33 + CD8 + cells. The M33 + CD8 + cells are gated and the histograms of Fas are shown. The percentage of Fas expression on the gated M33 + CD8 + T-cells is indicated.
  • NIT-1 cells are transfected with pcDNAIII vector containing Fas ligand gene (NIT-l/FL) or empty vector (NIT-1/Ctl), and selected with G418. Fas ligand activity is measured by a [ 51 Cr] release assay,
  • NIT-l/FL pcDNAIII vector containing Fas ligand gene
  • NIT-1/Ctl empty vector
  • Fas ligand activity is measured by a [ 51 Cr] release assay
  • 6-wk-old female NOD mice are i.p. injected with 5 x 10 5 NIT-l/FL or NIT-1/Ctl once.
  • Splenic T-cells are isolated
  • FIG. 14 Histologic Analysis of Insulitis. 6 wk-old female NOD mice are i.p. injected with 5 x 105 NIT-1/Ctl (A) or NIT-l/FL (B). Mice are sacrificed at 12 week of age. H&E stained paraffin sections of pancreas were examined (400x).
  • FIG. 15 Prolonged expression of Ad/Luc in muscle co-transfected with pFasL. Tongue muscle of mice (5 mice/group) were analyzed at different time points for luciferase production. There was increased production of luciferase in muscle cells injected with adenovirus plus FasL compared to muscle injected with adenovirus and control empty vector.
  • FIG. 16 Construction of p ⁇ Elsplb/FL and PJM17. Production of p ⁇ Elsplb/FasL. Shown is a 10.5 kb vector that contains Ad from 0 map units to 1 map unit, the CMV promoter, full length Fas ligand and a 0.4 kb SV40 polyA tail. This shuttle vector was combined with the 40.3 kb pJM17 vector containing the adenovirus genome - ⁇ E1 and also contains an origin replication and an ampicillan-resistant site.
  • FIG. 17 Production of p ⁇ ElsplBloxp/FasL.
  • a 10.4 kb shuttle vector containing the fragment of adenovirus from 0 map unit to 1 map unit is followed by the 0.7 kb CMV promoter. This is followed by 2 LOXP sites separated by a 2 kb stuffer fragment plus a 0.3 kb bovine growth hormone polyA tail.
  • the ftill- length 0.9 kb Fas ligand is cloned downstream from the stuffer fragment which is followed by an SV40 PolyA tail and by the 9.8 - 16.1 map units of adenovirus.
  • Vectors and methods are providing for introducing a transgene into a host using a virus-based delivery system, the vectors and methods designed to inhibit the host immune system from interfering with the specific gene therapy vector.
  • the present invention incorporates the production of apoptosis inducing ligands into antigen presenting cells through gene therapy. Normally, a host T-cell directed towards an antigen of a transfected cell encounter an antigen resulting in elimination of expression of the transfecting transgene.
  • the present invention promotes immunotolerance towards transfected host cells.
  • the term "gene” or "transgene” is a nucleic acid, either naturally occurring or synthetic which encodes a polypeptide product.
  • nucleic acid is intended to mean natural or synthetic linear, circular and sequential arrays of nucleotides and nucleosides, e.g. cDNA, genomic DNA, mRNA, and RNA, oligonucleotides, oligonucleosides, and derivatives thereof.
  • An apoptosis ligand is any polypeptide cytokine that induces apoptosis or otherwise is lethal to a cell upon complexing the ligand.
  • the present invention is detailed with the exemplary naturally occurring ligand, Fas ligand; however, it is appreciated that other known apoptosis ligands are similarly operative.
  • ligands illustratively include: Fas ligand 2 which induces apoptosis by acting with death domain region molecules DR3, DR4 and DR5; TNF which induces apoptosis by acting with TNFRI; Granzyme B and porferin which are natural killing molecules associated with T-cells; and antibodies specific to T-cell apoptosis ligand receptors: anti-Fas, anti-DR3, anti-DR4, anti- DR5 and anti-TNFRl.
  • Fas ligand 2 which induces apoptosis by acting with death domain region molecules DR3, DR4 and DR5
  • TNF which induces apoptosis by acting with TNFRI
  • Granzyme B and porferin which are natural killing molecules associated with T-cells
  • antibodies specific to T-cell apoptosis ligand receptors anti-Fas, anti-DR3, anti-DR4, anti- DR5 and anti-TNFRl.
  • Figure 1 is a schematic illustrating a production method of gene therapy viral vector to inhibit an immune response to viral vector antigens and methods of using the same to produce immune privileged transduced mammalian host cells.
  • the method of producing an immune tolerated gene therapy vector of the present invention involves a series of steps. In selecting a virus to be modified by way of the present invention one examines a series of factors including: viral vector tropism, sites of vector expression within a host cell, ease of vector gene manipulation, required duration of expression, pathogenicity and the like.
  • the adenovirus (Ad) affords many advantages as a vector as evidenced by its popularity. Ad replicates episomally within a host cell and as such the host cell genome is unaltered resulting in no transgene expression in host cell daughters.
  • the adeno-associated virus is a smaller virus than Ad, which is capable of integrating into a host cells chromosomes, thereby affords the option of long-term expression.
  • the herpes virus (HV) is trophic for the nervous system of a host and affords the option of transducing cells of the nervous system.
  • the upstream regulatory region (URR) of the virus is excised.
  • a URR contains at least a promoter which may be regulatable, for example, by TCN or steroids, or inducible, such as in Loxp/Cre system.
  • the URR is closed into a shuttle vector plasmid.
  • the shuttle vector contains an origin of replication, and an apoptosis ligand gene expression cassette.
  • a marker gene, an enhancer, a signal sequence, or a stuffer fragment are included in the plasmid.
  • An apoptosis ligand gene or fragment thereof is excised from a source cell line and cloned into an apoptosis ligand gene expression cassette.
  • the cassette contains control elements necessary for replication within a host cell such as a promoter, a 5' untranslated region and a polyadenylation sequence.
  • the cassette is incorporated into the shuttle vector plasmid so as to stimulate apoptosis ligand expression in concert with reading of the viral URR.
  • the shuttle vector plasmid is then combined with a viral vector replication plasmid from with the pathogenic protein encoding genes have been deleted or at least inactivated.
  • the combined plasmids form a recombinant for delivering selected portions of the viral genome and an apoptosis ligand for suppressing the immune response to transfected cells presenting viral antigens thereon.
  • Figure 1 shows the transgene expression cassette as being incorporated into the vector replication cassette.
  • the transgene expression cassette is incorporated into the shuttle vector plasmid.
  • the recombinant is then alternatively introduced into a cell culture or into a mammalian host.
  • the transfection of a cell culture is carried out by a prior art method (35).
  • the transfected cells expressing viral antigens and an apoptosis ligand are identified by methods illustratively including indirect immune fluorescent assay and 51 Cr release assay.
  • the transiently transfected antigen presenting cell lines are macrophages or NIT-1 ⁇ islet pancreas cells.
  • the present invention is readily extended to be mediated by a cell-cell interaction where the apoptosis ligand is expressed on cell type one which also expresses a different ligand, the different ligand being able to activate a receptor on a second cell type.
  • the preferred situation is where the different ligand of cell type one - receptor of the second cell type interaction up-regulates a death domain molecule in the second cell type.
  • Cultured cells expressing both the ligand and viral vector antigens are then exposed to T-cells that have been sensitized to the viral vector.
  • the immune system challenged transfected cells are then assayed as to proliferation, or cytotoxicity or as to the inducement anti-viral vector antibody production.
  • the recombinant is introduced into a mammalian host by a route dictated by the targeted host cells.
  • lung tissue to be transfected with CFTR or protease inhibitor so as to treat cystic fibrosis is preferably administered intra- nasally as an aerosol suspension;
  • blood to be transfected with Factor 8 so as to treat hemophilia is preferably administered intravenously, intra peritoneally to transfect organ specific diseases of the liver, pancreas, etc., intra marrow for marrow and intra-myocardial for heart tissue
  • adjuvants are readily added to a gene therapy vector of the present invention to facilitate administration.
  • the host transfected tissues are biopsied or excreted gene product markers associated with the gene therapy are assayed to monitor the efficacy of the therapy. In vitro assays are also applicable to in vivo therapy monitoring.
  • the present invention provides for a gene therapy vector capable of delivering a complete apoptosis ligand, as well as smaller functional components of these ligands. Certain truncations of these ligands interact with death domain molecules and thereby induce T-cell apoptosis.
  • the nucleic acid sequences coding for Fas ligand, Fas ligand 2, Granzyme B and porferin can be altered by substitutions, additions, deletions or multimeric expression that provide for functionally equivalent ligands. Due to the degeneracy of nucleic acid coding sequences, other sequences which encode substantially the same amino acid sequences as those of the naturally occurring ligands may be used in the practice of the present invention.
  • nucleic acid sequences comprising all or portions of the nucleic acid sequences encoding the above ligands, which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • one or more amino acid residues within a sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged
  • (acidic) amino acids include aspartic acid and glutamic acid.
  • ligands or fragments or derivatives thereof which are differentially modified during or after translation, e.g., by glycosolation, protolytic cleavage, linkage to an antibody molecule or other cellular ligands, etc.
  • the recombinant ligand encoding nucleic acid sequences of the present invention may be engineered so as to modify processing or expression of a ligand.
  • a signal sequence may be inserted upstream of a ligand encoding sequence to permit secretion of the ligand and thereby facilitate apoptosis.
  • a ligand encoding nucleic acid sequence can be mutated in vitro or in vivo to create and/or destroy translation, initiation, and/or termination sequences or to create variations in coding regions and/or form new restriction endonuclease sites or destroy pre-existing ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to in vitro site directed mutagenesis (36), use of Tab linkers (Pharmacea), etc.
  • Fas ligand polymorphisms in the intracellular domain modify the hydrophilic regions of the ligand but do not greatly affect Fas ligand function in inducing apoptosis.
  • mutations of Fas ligand that do not affect the apoptosis inducing potential of the ligand including additions, substitutions, truncations and the like are recognized to be usable in the present invention.
  • a polynucleotide modification of Fas ligand to produce multimers of the Fas ligand is a means of increasing apoptosis potential of the Fas ligand. By extension, the same holds true for other ligands.
  • Fas ligand binds to Fas and may impede apoptosis by endocytosis of Fas without inducing apoptosis. Therefore, larger conglomerates of Fas ligand such as surface Fas ligand or Fas ligand that has been engineered to be cross-lined and produced by cells is more affective in the induction of apoptosis than the naturally occurring Fas ligand.
  • T-cells which are activated against a specific antigen, are selectively eliminated thereby preventing or reducing immune response to that antigen. T-cells are eliminated by activation-induced cell death of T-cells, which is caused by Fas-mediated apoptosis of those activated T- cells that express Fas and Fas ligand.
  • APCs antigen presenting cells
  • MHC major histocompatibility complex
  • a particular peptide/MHC complex is recognized by the specialized receptor on the surface of a T-cell thereby activating that T-cell.
  • Activated T-cells then reproduce, and the offspring proceed to initiate an immune response by attacking those materials displaying the foreign antigen, and by further activating B cells and other components of the immune system.
  • some of the activated T-cells persist so as to provide an enhanced response to further infection.
  • the Fas ligand can be employed to produce apoptosis of T-cells that express Fas. More specifically, it has been found that introduction of APCs, that express a Fas ligand, into an organism will induce apoptosis of T-cells that express Fas, thereby resulting in antigen specific T-cell tolerance. It has been found that an adenovirus capable of expressing the Fas ligand can be used to transfect macrophages and other APCs. This results in a highly efficient presentation of adenovirus antigens and Fas ligand on the APCs.
  • Such APCs will then confer immune tolerance to the adenovirus vector by selectively eliminating those T-cells which are capable of reacting with antigens from the adenovirus vector.
  • This novel therapeutic approach greatly enhances the utility of adenoviral based gene therapies by producing specific tolerance to the therapeutic materials. Since the therapy of the present invention is highly selective, adverse affects heretofore encountered with broad immunosuppressive approaches are eliminated.
  • adenovirus expressing the Fas ligand
  • APCs can be targeted to APCs via the mannose receptors on the APCs.
  • APCs can be transfected with Fas, in vitro, and the transfected cells introduced into the organism; or, transfection may occur in vivo, by administration of the adenovirus vector to the organism.
  • autoimmune disease occurs when an organism's immune system becomes activated toward tissue of the organism itself. Selective apoptosis of those activated T-cells which cause the autoimmune response will control autoimmune disease. Transfection of those cells which elicit the autoimmune response with the Fas gene will produce syngeneic cells which will induce tolerance to an autoimmune antigen, in T-cells via Fas mediated apoptosis.
  • the syngeneic cells may comprise APCs or they may comprise cells of the tissue provoking the autoimmune disease, in which instance these cells will then cause the APCs to present the Fas ligand and the autoimmune antigen.
  • These principles are also be employed to produce immune privileged sites within an organism. Provision of an immune privileged site facilitates organ transplant and other such tissue graft procedures. An immune privileged site also prolongs expression of an adenovirus gene product at that site. Creation of the immune privileged site is accomplished by causing cells at the site to produce the
  • Fas ligand and the presence of this ligand will protect an adenovirus from immune system attack.
  • Production of Fas ligand is accomplished by the virus used for the therapy itself, or by genes introduced into the tissue via another carrier. Fas ligand expression induces specific tolerance by apoptosis. Fas ligand expression is also induced by clonal deletion. Peripheral T-cell tolerance is maintained by activation-induced cell death of the T-cells, which is mediated by Fas-mediated apoptosis of the activated T-cells that express Fas and Fas ligand (37-41). Thus, Fas ligand expression is used to create immune-privileged sites and prevent graft rejection by inducing apoptosis in the T-cells (42-44).
  • the instant invention includes a novel immunointervention strategy for adenovirus gene therapy in which Fas ligand gene therapy is used to confer immune privilege. This response is mediated at the cell level and an immune response to cells is prevented by Fas ligand production by these cells.
  • the mouse FasL is introduced into the El A site of Ad to produce a recombinant virus which is both replicative defective and expresses high levels of Fas ligand.
  • transgene vector inhibits the immune response of the host thereto, resulting in highly efficient presentation of adenovirus antigens and Fas ligand on the macrophages.
  • the current results demonstrate that AdLoxpFasL co-infection with AxCANCre results in very high levels of FasL in a majority of infected APCs.
  • APCs can express high levels of Fas ligand without undergoing autocrine suicide. This is in contrast to low efficiency transfection of DNA into APCs using lipofectin (l%-5%) or electroporation (8%).
  • the present invention utilizes several unique technologies to allow high expressions of Fas ligand plus high expression of process adenovirus antigen on an antigen presenting cell to induce apoptosis of T-cells that react with this antigen.
  • the present invention demonstrates extremely efficient inhibition of CD3 T T-cell expansion that are potentially reactive with APC processed adenovirus antigens leading to prolongation of gene expression by challenge after tolerance with AdCMVLacZ.
  • High efficiency inhibition of adenovirus-reactive T-cells is achieved by first treatment of mice with 5 dosages of APC-AdFasL using APCs from B6-lpr/lpr mice. After admimstration every three days with 5 dosages, these APCs toleralize to antigens for up to four weeks by inhibition of APC/antigen reactive T-cells.
  • AdCMVLacZ (10 10 pfu.) intravenously one week after tolerance does not lead to a significant T-cell response since there is deletion or inhibition of all potentially reactive T-cells.
  • the absence of cytotoxic T-cells at 7 days post-infection with AdCMVLacZ correlates with a prolonged expression of LacZ in toleralized mice compared to non-toleralized mice.
  • the present invention shows that adenovirus expression Fas ligand within an antigen presenting cell used as pretreatment can be utilized to toleralize against second administration of adenovirus/gene therapy product.
  • mice are toleralized with APC-AdFas-L.
  • inventive tolerance procedure There are several independently novel features to the inventive tolerance procedure.
  • AdCMVLacZ is used to challenge mice, but the LacZ gene is not encoded in the AdLoxpFasL + AxCANCre viruses infecting the toleralizing APCs, since this would require a triple adenovirus infection, with potentially lower infection efficiency. Nevertheless, there is tolerance to readministration of AdCMVLacZ during challenge. AdCMVLacZ elicits an immune response to LacZ as well as adenovirus (46-48). These results indicate that tolerance to adenovirus alone can prolong gene therapy even in the absence of tolerance to one of the more immunogenic transgenes, LacZ.
  • Tolerance induction by APCs infected with a viral vector expressing high levels of FasL is specific for the viral vector, but not with an irrelevant virus.
  • the present toleralizing technique completely abrogates the ability of responding to the toleralizing virus used to infect the APC, but not to APC infected with an irrelevant virus. Therefore, the present invention for toleralizing to a viral vector gene therapy is widely applicable, does not result in generalizing immune- suppression and is amenable to readministration for repeated treatment without inducing an immune-suppressed state.
  • the first approach uses a method to couple the adenovirus fiber/knob to a mannosylated polylysine peptide.
  • the modified receptor is targeted to macrophages.
  • This technique is used to attach mannosylated polylysine to a modified, replicative defective adenovirus to determine adenovirus redirection to combine with high efficiency to APCs in vivo.
  • Another method for APC infection with Ad involves using the adenovirus-polylysine infection technique to deliver adenovirus-polylysine-DNA complexes to accompany gene therapy to targeted cells for cell lines that did not already express Fas ligand. This is advantageous in the creation of immunoprivileged sites in cells that do not express Fas ligand or do not undergo apoptosis after expressing Fas ligand.
  • a more stringent test of tolerance induction involves later challenges of the mice in vivo with either the Ad-APC-FL or Ad-APC, as well as control Ad without APC. This subsequent reaction elicits a strong secondary immune response in the mice that were previously immunized with adenovirus, but there is little or no response in mice that have been tolerized with Ad-APC-FL.
  • Ad-APC-FL and Ad-APC, or Ad in the subsequent administration determines if Ad-APC-FL is required with each administration of adenovirus for a specific APC, or if the initial induction of tolerance confers long-term tolerance to adenovirus.
  • This technique is used to induce tolerance to alloantigens, and that systemic administration of APC-FL does not induce significant toxicity to the liver or long and has no other apparent toxic effect on the mouse.
  • FasL by the Ad infected cell to create immune privilege sites.
  • Fas-ligand gene therapy is useful as a strategy to prevent immune response to viral vector antigens and in this embodiment of the invention, adenovirus.
  • the ability to exploit this strategy is supported by the finding that Fas ligand expression can be targeted to APC in vitro using the polylysine method for targeting Fas ligand and adenovirus. This method promotes targeted gene delivery via the receptor mediator endocytosis pathway (49-53). It is necessary in this approach to link the vector, such as adenovirus to molecular conjugates and, at the same time, preserve both the binding and endosome disruption capabilities of the virus.
  • the conjugates are preferably linked through the hexon protein.
  • the linkage is accomplished by an antibody bridge through a molecular conjugate and the viral vector. This is accomplished by conjugating a monoclonal antibody against a foreign epitope on the viral vector hexon protein to the polylysine.
  • the normal viral tropism of the vector is ablated.
  • redirection to macrophages optionally involves the mannosylated fiber-knob (53-57).
  • Regulation of the macrophage mannose receptor expression and cloning of the mannose receptor has been carried out (58- 60).
  • the first three exons of the mannose receptor gene encode: a signal sequence, the NH 2 -terminal cysteine rich domain, and the fibronectin type II repeat, while the final exons encode the transmembrane anchor and the cytoplasmic tail.
  • the intervening 26 exons encode the 8 carbohydrate-recognition domains and intervening spacer elements.
  • the mannose receptor is expressed on alveolar macrophages and a highly homologous receptor DEC-205 is expressed on dendritic cells and thymic epithelial cells (58).
  • DEC-205 is able to bind carbohydrates and mediate endocytosis. It is rapidly taken up into the coated pits forming vesicles and delivered to a multi-vesicular endosomal compartment that resembles the MHC class Il-containing vesicles.
  • the mannose receptor on macrophages and APCs provides an excellent target for modified adenovirus tropism and delivery of genes to APCs.
  • the present invention preferably utilizes adenovirus expressing Fas ligand under the regulation of a well characterized target cell lysozyme promoter or a similar target specific promoter to transfect into a target cell (61-63) efficiently present of viral vector antigens and a cytokine ligand on the target cells.
  • Example 2 Construct Fas ligand expression adenovirus vector.
  • a 10.4 kb shuttle vector containing the fragment of adenovirus from 0 map unit to 1 map unit followed by the 1.6 kb chicken ⁇ -actin promoter plus CMV enhancer. This is followed by 2 Loxp sites separated by a Neo resistant gene plus a 0.3 kb bovine growth hormone poly A tail. The full-length 0.9 kb FasL is cloned down-stream from the bovine growth hormone poly A tail which is followed by an SV40 polyA tail and by the 9.8 - 16.1 map units of adenovirus.
  • Example 3 - MCMV Virus Example 3 - MCMV Virus.
  • MCMV Virus Smith strain is obtained from the American Type Culture
  • the virus are titrated as duplicates in log 10 dilutions on subconfluent primary murine embryo fibroblasts in 12-well plates. Seven days later, monolayers are stained with neutral red and the number of plaques counted.
  • the supernatant is dispensed into aliquots, which are stored at -80 °C and used as the MCMV stock virus pool (3xl0 7 PFU/ml).
  • Example 4 Infection of antigen presenting cells for Fas ligand expression.
  • Murine B6-lpr/lpr APCs are infected with either AdLoxpFasL plus AxCANCre (APC-AdFasL) or
  • AdLoxpFasL plus AdCMVGFP (APC-AdControl) at 5 pfu/cell of each viruses for
  • Expressed murine FasL and adenoviral antigens on the surface of B6-lpr/lpr APCs are identified using indirect immune fluorescent assay (64) and the killing activity is evaluated by 51 Cr release assay (65).
  • Example 5 Analysis of FasL by APCs Infected with AdLoxpFasL plus AxCANCre.
  • Fas ligand (FasL) cytotoxicity is assayed as previously described (65).
  • FasL expression is determined by ability of the transfected APCs to induce apoptosis of a 5I Cr labeled, Fas sensitive cell line A20.
  • Target cells (lxl 0 6 ), which are sensitive to cytotoxic lysis, are incubated with 20 ⁇ Ci of [ 51 Cr]-sodium chromate in 100 ⁇ l of RPMI- 1640 containing 10% FCS at 37 °C for 1 h. After washing with medium, these cells are used as target cells. Effector cells are prepared from B6-lpr/lpr APCs infected with AdLoxpFasL plus AxCANCre as described above.
  • effector cells are then incubated with [ 51 Cr]-labeled target cells (lxlO 4 ) at different effector/target (E/T) ratios in a total volume of 200 ⁇ l of the medium. Release of 51 Cr into the supernatant is assessed 6 h later using a ⁇ -counter. The percentage of specific 51 Cr release is calculated as follows:
  • Example 6 Administration of APC-AdFasL for induction of tolerance.
  • mice Ten-week-old C57BL/6-+/+ mice are injected intravenously with 1 x 10 6 of the APCs co-infected with AdLoxpFasL plus AxCANCre (APC-AdFasL) or
  • mice are treated with AdCMVlacZ (lxlO 10 pfu i.v.) or MCMV (1x10 s pfu i.v.). After an additional 7 days, purified splenic T-cells are stimulated in vitro with APCs alone, or APCs that are incubated either with AdCMVlacZ (lxlO 10 pfu i.v.) or MCMV (1x10 s pfu i.v.). After an additional 7 days, purified splenic T-cells are stimulated in vitro with APCs alone, or APCs that are incubated either with
  • Example 8 Quantitation of ⁇ -galactosidase expression in liver. ⁇ -galactosidase activity is determined as previously described (66).
  • Freshly isolated liver tissue is homogenization for 10 s in a tissumizer in 1 ml of ⁇ -gal buffer (Tropix, Inc., Bedford MA).
  • the homogenate is centrifuged at 12,500 x g for 10 min at 4°C, and the supernatant is heated for 60 min at 48 °C to inactivate the endogenous eukaryotic ⁇ -galactosidase activity.
  • the sample is then centrifuged at 12,500 x g for 5 min, and 10 ⁇ l of the supernatant is assayed for ⁇ -galactosidase activity using the Galacto-lightTM (Tropix, Inc., Bedford MA) chemi-luminescent reporter assay.
  • the reaction is carried out for 10 min at room temperature (RT) and ⁇ -galactosidase activity is assayed using a luminomiter
  • the adenovirus shuttle vector construct is produced by cloning the enhanced GFP gene from pCA13 (Clonetech) into the Hindlll-Xbal site. This is cotransfected with pJM17 to produce recombinant AdCMVGFP.
  • AdCMVGFP is plaque purified by 3 rounds of selection. These are used to infect APC to be used as target cells for analysis of cytotoxic effector T-cells from mice treated with
  • APC AdLoxpFasL+AdCMVGFP
  • APC AdLoxpFasL+ AxCANCre
  • Effector cells are prepared from spleen, and peripheral lymph nodes of
  • Ad-immunized and non-immunized mice are then incubated with AdCMVGFP-infected target cells (lxl 0 5 ) at different effector/target (E/T) ratios in round-bottom microtiter plates in a total volume of 200 ⁇ l of the medium for 48 hours, and Green fluorescent positive APC are sorted using FACS analysis. The percentage of specific cytotoxicity was calculated as follows:
  • Example 10 Cytokine production in vitro in response to APC infected with adenovirus.
  • B6 Ipr/lpr APCs are infected with AdCMVLacZ (10 pfu/cell) for 1 hour in 1 ml of media and then diluted to additional of 10 ml of RPMI 1640 supplemented with 10% fetal bovine serum. The cells continue to culture at 37 °C for 24 hours. Before as a targeting cells, the APC is ⁇ -irradiated, and lxl 0 5 APC are mixed at different ratios of T-cells isolated from the spleen of tolerized mice.
  • the mixed cells are incubated at 96 well plate for 2 days at 37 °C.
  • the superaatants are collected and induction of IL2 and interferon gamma are determined using ELISA assay kit (R & D systems Inc., MN).
  • Example 11 Histopathological examination of tissue sections.
  • Paraffin-embedded tissue sections are deparaffinated and treated with 3%
  • DAB diaminobenzidine
  • the two-tailed Student's t-test is used for statistical analysis when two different groups of samples are compared.
  • Example 14 Co-infection of AdLoxpFasL + AxCANCre results in high levels of FasL capable of inducing apoptosis of A20 target cells.
  • the instant invention includes an AdLoxpFasL modified adenovirus to yield high titer production of the virus in 293 cells (45).
  • This technique also facilitates control of FasL expression since FasL is not expressed in the absence of co-infection with AxCANCre.
  • This technique is used to induce high FasL expression by a APC cell from Fas-mutant B6-lpr/lpr mice which could induce apoptosis of A20 target cells (Fig. 2).
  • adenovirus gene therapy in the liver is limited due to an acute inflammatory response and a chronic cytotoxic T-cell response (67).
  • AdFasL expressing APCs leads to prolongation of transgene expression delivered by adenoviral vector
  • the APC-AdFasL tolerized and APC-AdControl treated mice are inoculated with AdCMVlacZ (lxlO 10 pfu). LacZ gene expression in the liver is kinetically analyzed by quantitative measurement of LacZ protein and histochemistry staining. The levels of LacZ gene expression in the liver rapidly decreased in mice treated with APC-AdControl (Fig. 3 a).
  • LacZ expression peaked at day 7 after expression of AdCMVLacZ in both toleralized and non-toleralized mice, and rapidly decreased in non-toleralized mice compared to non-toleralized after day 7.
  • mice are toleralized in vivo as described above and challenged with AdCMVLacZ. Seven days after challenge splenic T-cells are purified and used as effect cells at different E/T ratios to kill AdCMVGFP infected APCs. There is a high cytotoxic response by T-cells from mice treated with
  • Example 17 T-cell tolerance demonstrated by decreased IFN- ⁇ and IL-2 production in vivo.
  • mice are tolerized as above with either APC-AdFasL or APC-AdControl as a control. Thirty days after tolerance induction, mice are sacrificed and spleen cells are stimulated with APC or APC infected with AdCMVlacZ. Non-infected APCs did not stimulate T-cells as determined by low IL-2 ( Figure 5 A) and LFN- ⁇ ( Figure 5B) in the supernate at 24 or 48 hours ( Figure 4). In contrast, there is high production of IL-2 and IFN- ⁇ from spleen cells from C57BL/6 which are tolerized with APC-AdControl, which do not express FasL. B6+/+ mice that are tolerized with APC-AdFasL are tolerized as indicated by low IL-2 ( Figure 5 A) and
  • Example 18 Fas Expression by Recipient T-cells is required for tolerance Induction.
  • Fas expression in recipient C57BL/6 mice is required for tolerance induction since spleen cells from B6-lpr/lpr mice produced high levels of IFN- ⁇ and IL-2 despite being tolerized with APC-AdFasL (Fig. 6 A, 6B).
  • Example 19 Decreased T-cell expansion in APC-AdFasL treated mice.
  • mice B6+/+ mice were treated with APC-AdFasL or APC-AdControl every 3 days for 5 doses, and then all treated mice were IN. challenged with AdCMVlacZ (lxlO 10 pfu). Three days later, frozen sections of spleen from naive mice (Fig. 7a), control APC treated mice (Fig. 7b), FasL APC treated mice (Fig. 7c) and were stained with anti-CD3 antibody using a standard ABC technique. There was no expansion of CD3 + T-cells in tolerized mice spleen, whereas mice treated with control APCs resulted in clonal expansion in spleen after challenge.
  • Example 20 - APC-AdFasL induces specific tolerance to adenovirus.
  • T-cell tolerance induced by Ad/FasL expressing APCs is specific for adenoviral vector rather than a general immune suppression to viral infection
  • APC-AdFasL and APC-AdControl tolerized mice to an irrelevant viral infection is measured.
  • B6 +/+ mice are treated with APC-AdFasL as described above for induction of tolerance, and then challenged
  • Example 21 - Fas ligand expressing adenovirus provides both systemic immune tolerance to Ad transfected APCs and confers privilege on cells that are transfected with the Ad/FasL- ⁇ Gal.
  • APCs transfected with Fas ligand induce specific apoptosis and specific
  • T-cell tolerance to antigens both in vitro and in vivo This is observed using a macrophage cell line derived from Fas-deficient C57BL/6(B6)-/pr//pr mice that are transiently transfected with Fas ligand, and then injected into mice of a different MHC.
  • macrophages co-infected with Fas ligand and viral vector are highly efficient presenters of viral vector antigens and Fas ligand. This results in antigen-specific apoptosis of vector-reactive T-cells.
  • Transfection of Fas ligand into a ⁇ -islet cell line also confers immune privilege on the host ⁇ -islet- reactive T-cells and prevention of diabetes where the vector is adenovirus.
  • Example 22 - APCs transfected with Fas ligand induce apoptosis and specific T-cell tolerance to antigens in vitro and in vivo.
  • An APC line derived by short-term culture of peritoneal macrophages from Fas mutant B6-lpr/lpr mice does not express Fas, but expressed MHC class II IA b ,
  • FIG. 9c This cell line is transfected with a eukaryotic expression vector (pcDNAIII) containing the full-length murine Fas ligand and selected using G418.
  • APCs transfected with Fas ligand APC-FL
  • APC-CV APC-CV cells are capable of presenting alloantigen as the ⁇ -irradiated cells induced a prohferative responses in co-cultured splenic H-2 k T-cells (MRL-+/+ or MR -lpr/lpr) (Fig. 9e).
  • APC-FasL cells are capable of presenting alloantigen and induce a prohferative response if the responding T-cells are obtained from MRL-lpr/lpr mice, which do not express Fas.
  • presentation of antigen by APCs that express Fas ligand induces tolerance of the Fas-positive responding T-cells.
  • Example 23 Induction of allogeneic T-cell tolerance by Fas ligand expressing APCs. 4-wk-old of MRL-+/+ and -Ipr/lpr mice are injected i.v. with macrophages
  • splenic T-cells are isolated from treated mice and cultured under various stimulatory conditions.
  • 5 x 10 5 T-cells are cultured with 2 x 10 5 ⁇ -irradiated total spleen cells from B6 +/+ mice (Fig. 10a).
  • 5 x 10 5 T-cells are cultured with 2 x 10 5 ⁇ -irradiated total spleen cells from BALB/c mice (Fig.
  • T-cell proliferation is determined by incorporation of [ 3 H]-thymidine at 24, 48, 72 and 96 hours.
  • Example 24 Antigen-specific clonal deletion of the T-cells induced by Fas ligand expressing APCs in H-2D b /HY reactive TCR transgenic mice.
  • TCR T-cell receptor
  • CD4 CD8 " T-cells isolated from 5-month-old, female, B ⁇ -lpr/lpr mice are used as controls in which the HY antigen is not expressed.
  • the Fas ligand activity of the CD4 " CD8 " T-cells is high and specific inhibition of this release by soluble Fas-Ig fusion protein (Fig. 1 lb). Alloantigen-specific T-cell tolerance was analyzed after i.v.
  • T-cells from female, TCR transgenic +/+ mice treated with Fas ligand " HY 1" male cells exhibited a decreased prohferative response upon stimulation with either H-2D b /HY spleen cells or crosslinking with the M33 anti-clonotypic TCR antibody, but not anti-CD3.
  • Fas ligand-positive cells derived from H-2D b female mice had no effect on the H-sD b HY reactivity of recipient T-cells in TCR transgenic female mice.
  • Example 25 Tolerance induction due to Fas-mediated deletion of H-2D b /HY Reactive CD8 + T-cells.
  • Clonal deletion of H-2D b /HY cells is examined by analyzing the numbers of H-2D b /HY reactive CD8 + T-cells in the female TCR transgenic mice using the anti-clonotypic mAb M33. Tolerance induction is carried out as described above and the numbers of M33 + CD8 ⁇ T-cells in the peripheral lymph node (Fig. 12a) and spleen (Fig. 12b) cells are determined. In untreated, female, transgenic +/+ and Ipr/lpr mice, approx.
  • Fas ligand expressing APCs is associated with Fas ligand-mediated clonal deletion of antigen-specific T-cells that recognize the antigen presented by the APCs.
  • Time-course analysis of the deletion of M33 + CD8 + T-cells in the spleen showed that the depletion commenced as early as 24 h after treatment in the female TCR transgenic +/+ mice that received Fas ligand-positive H-2D b /HY cells and continued during the 10-d period of the treatment.
  • Fas-Ig effectively inhibited the deletion in the TCR transgenic +/+ mice, which further supports the role of Fas ligand expression on the APCs in clonal deletion.
  • Fas expression also is analyzed in M33 + CD8 + PLN T-cells in the female TCR transgenic Ipr/lpr mice did not express Fas antigen regardless of treatment. Fas expression on M33 + CD8 T T-cells expressed low levels of Fas (14%), whereas additional treatment with Fas-Ig led to the majority of M33 + CD8 + T-cells are deleted by Fas ligand expressing APCs.
  • Example 26 Inhibition of insulitis in NOD mice using a synegeic ⁇ -islet cell line that expresses Fas ligand to induce T-cell tolerance.
  • NIT-1 The syngeneic ⁇ cell line, NIT-1, is used as the APC for Fas ligand expression. NIT-1 cells do not express Fas antigen and do not undergo either anti-Fas antibody or Fas ligand mediated apoptosis (data not shown). This cell line is transfected with an expression vector containing Fas ligand mediated apoptosis (data not shown). This cell ine is transfected with an expression vector containing Fas ligand (pcDNAIII) as described in Example 9. Fas ligand transfected, but not control, cells expressed functional Fas ligand (Fig. 13a).
  • NIT-1 cells There are increased T-cell prohferative and cytotoxic responses in
  • NOD mice treated with control NIT-1 cells (Fig. 13b,c).
  • NOD mice treated with Fas ligand expressing NIT-1 cells only exhibit a minimal increase in response compared with the untreated control. 100% of NOD mice that received no treatment or treatment with NIT-l/CV developed insulitis, and 100% of islets from each individual mouse are involved. In contrast, only 1 of 3 mice receiving
  • NIT-1 /FasL developed minor insulitis, with only 10% of islets involved (Fig. 14).
  • Example 27 Inhibition of Insulitis in Nod mice using NIT-1 - AdFasL as a syngenic ⁇ islet cell to induce T-cell tolerance to an Ad vector.
  • Example 25 The procedure of Example 25 is repeated with the expression vector of
  • Example 14 substituted therein.
  • NIT-1 - Ad control treated mice develop insulitis involving 100% of islet cells of individual mice.
  • NIT-1 - AdFasL treated mice did not develop insulitis.
  • Example 28 Transfection with Fas ligand and adenovirus results in high expression of ⁇ -Gal in macrophages.
  • the polylysine method is used for targeting Fas ligand and Ad to APC via the receptor-mediated endocytosis pathway (49-51, 68, 69). It is important to link Ad to molecular conjugates, and at the same time preserve both the binding and endosome disruption capabilities of the virus.
  • the linkage is accomplished by conjugating a molecular antibody against a foreign epitope on the adenovirus hexon protein to the polylysine-protein complex.
  • a chimeric adenovirus containing a foreign epitope in the surface region of its hexon protein is constructed.
  • the loop region of the hexon protein is a useful foreign epitope expression region.
  • Example 29 Creation of an immune-privileged site for prolonged expression of the adenovirus gene product using co-expression of FasL and adenovirus in muscle.
  • 10 9 adenovirus is co-injected into mouse muscle tissue with 5 ⁇ g of purified FasL DNA under the regulation of the CMV promoter (pFasL), or with identical control plasmid DNA which does not express Fas ligand.
  • FasL production by adenovirus confers a high level of specific immunity to the adenovirus, prevent immune elimination of cells expressing the adenovirus, and result in prolonged expression of the adenovirus gene product.
  • Example 30 Modification of viral tropism to allow high efficiency targeting to macrophages.
  • Ad/FasL in addition to the in vitro infection and tolerance induction by Ad/FasL, in vivo infection by an Ad/FasL virus is operative.
  • a FasL Tg mouse which overexpresses FasL specifically in T-cells without cytotoxicity is used (70).
  • Similar techniques direct Ad/FasL for high transfection of APCs in vivo (macrophages) by targeting adenovirus to the macrophage mannose receptor. This is accomplished using a synthetic molecular conjugate consisting of a mannosylated polylysine protein combined with the adenovirus fiber/knob protein.
  • a mannosylated polylysine has been demonstrated to bind to the macrophage mannose receptor and lead to high efficiency transfection of DNA complexes into islet cells (71, 72).
  • Modification of adenovirus tropism uses the methods detailed in U.S. Provisional Patent Application 60/054,112 for modification of the adenovirus knob/fiber protein to include a 10 amino acid polypeptide capable of binding E-selectin and targeting adenovirus to inflamed sites in the synovium and also using an anti-adenovirus sFv/TL-2 fusion protein to direct adenovirus tropism to T-cells.
  • Example 31 Production of an adenovirus-infected, Fas ligand expressing macrophage for induction of tolerance to adenovirus.
  • the APC line of Example 22 expresses high level of MHC class I and II antigen, B7 and Fas ligand. This macrophage cell line express high levels of H-
  • This cell lines does not express Fas, exhibits low levels of Fas ligand activity, and has been transfected with a CMV promoter/FasL construct to produce a stable transfected macrophage cell line which expresses FasL.
  • This cell line can also be infected with Ad by known techniques to allow expression of adenovirus antigens and gene products.
  • Example 32 Analysis of Tolerance to Ad/Fas Ligand.
  • Tolerance to adenovirus will analyzed using a macrophage cell line that stably expresses Fas ligand (APC-FL) such as that of Example 22 and are infected with the adenovirus by intravenous (i.v.) or intranasal (i.n.) injection to induce tolerance. Tolerance is analyzed at d 2, 7, 14, 28, and 56 h after injection of 5 x 10 6 Ad-APC-FL. Mice are bled by retro-orbital sinus puncture for analysis of antibody titer to adenovirus.
  • APC-FL Fas ligand
  • T-cells tolerance are evaluated by [ 3 H]-thymidine incorporation to measure the T-cell prohferative response, BrdU incorporation, and flow cytometric analysis of BrdU-positive T-cells to determine the frequency of prohferative T-cells, and 7AAD three color flow cytometric analysis to determine apoptosis of the T-cells.
  • the level of IL-2 in the culture supernatants is also measured to determine T-cell activation.
  • a similar technique is used to test to determine if cytotoxic CD8 + T- cells are toleralized or deleted from the spleen in vivo. The CD8 + T-cells are tested for their ability to lyse chromium-labeled Ad-APC.
  • T-cells are isolated as described in reference to Fig. 10.
  • a suitable effector: target (E:T) ratio of CD8 + T-cells to chromium-labeled, adenovirus-pulsed macrophage target cells is thereby obtained.
  • E:T target
  • Example 34 Construction of an adenovirus producing Fas ligand.
  • a full length 1114bp murine Fas ligand cDNA clone is obtained by conventional methods (73-75).
  • this Fas ligand clone is used to produce the Ad/FasL vector (Fig. 16).
  • this clone has undergone recombination with the adenovirus genome in 293 cells. This construct and variations of this construct are used in the present invention.
  • the Fas ligand cDNA clone is introduced into the p ⁇ Elsplb shuttle vector. To produce recombinant adenovirus, this DNA is co-transfected into weakly Fas + 293 cells.
  • transfections are carried out using 3 different transfection methods including: lipofectin, dotap, and the calcium chloride precipitation method.
  • lipofectin the majority of the transfected 293 cells undergo apoptosis within 24 h, whereas minimal apoptosis occurs after transfection of 293 cells with the control shuttle vector.
  • Example 35 Production of p ⁇ ElsplBloxp/FasL:.
  • AdLOXP/FasL recombinant virus A Fas ligand expressing recombinant adenovirus, denoted as AdLOXP/FasL recombinant virus is shown in Fig. 17.
  • the p ⁇ ElsplBloxp/Fas shuttle vector is co-transfected with pJM17 to produce the AdLOXP/FasL.
  • the AdLOXP/FasL is co-infected with the Ad/CRE recombinant adenovirus.
  • the CRE excises the LOXP sites placing FasL under the control of the CMV promoter resulting in high levels of expression of FasL.
  • AdLOXP/FasL does not induce toxicity in the 293 cells.
  • the AdLOXP/FasL adenovirus is combined with the Ad/CRE recombinant adenovirus.
  • the CRE protein has been well studied and is demonstrated to be able to excise the LOXP sites which in the present invention construct results in the production of FasL under the CMV promoter. This system was first heavily utilized for production of transgenic mice. It has applied by several investigators for adenovirus recombination (73-75). These viruses can be co-infected into any cell, such as macrophages used herein with high efficiency.
  • Macrophages are transfected with the recombinant adenovirus. Lac z expression is confirmed by ⁇ -galactosidase staining as described in Example 8.
  • mice are analyzed at different time courses for expression of the lacZ marker gene in the lung and liver. Fas ligand expression is confirmed by ability of the transfected macrophages to induce apoptosis of 51 Cr labeled and Fas sensitive cell line A20 as per Example 5. These experiments are carried out with and without the presence of a soluble Fas (sFas) capable of neutralizing Fas ligand activity to demonstrate that cytotoxicity is specific for Fas ligand.
  • sFas soluble Fas
  • Example 37 Treatment of a lung disease with AdCF FasL transfected into APCs.
  • the CF gene is ligated into the EcoRV site of the Ad shuttle vector of
  • FIG 16 so as to be under the control of the regulatory element.
  • the CF modified vector, Ad Shuttle CF is co-transfected with pJM17 to produce recombinant AdCF.
  • FasL this is co-infected with the AdLOXP FasL and AxCanCre.
  • These three viruses will be co-administered intra-nasally into the airways of 6 week old, female bleomycin - TPF mice.
  • the mice are challenged with AdCF.
  • the mice so treated are tolerant of the CF gene therapy vector 7 days after challenge.
  • Example 38 Treatment of a lung disease with AdPI/FasL transfected into APCs.
  • the protease inhibitor (PI) gene is ligated into the EcoRV site of the Ad shuttle vector of Figure 16 so as to be under the control of the regulatory element.
  • the PI modified vector, Ad Shuttle PI is co-transfected with pJM17 to produce recombinant AdPI.
  • FasL this is co-infected with the AdLOXP FasL and AxCanCre.
  • Example 39 Treatment of hemophilia with AdF8/FasL transfected in APCs.
  • the factor VIII gene is ligated into the EcoRV site of the pJM 17 vector of Figure 16 so as to be under the control of the regulatory element.
  • the PI modified vector, Ad Shuttle Factor VLTJ is co-transfected with pJM 17 to produce recombinant Ad Factor VIII.
  • FasL this is co-infected with the Ad LOXP FasL and AxCanCre.
  • Example 40 Determine the expression of Fas ligand and Ad/ ⁇ -gal in vivo at different time points after infection in vivo in tolerized and non-tolerized mice.
  • Detailed analysis of expression of Fas ligand RNA and protein, viral RNA and protein, and ⁇ -gal is carried out at different time point and under different conditions of tolerance induction involves analysis of tissue sections using immunohistochemical staining for Fas, ⁇ -Gal. Tissue sections are also analyzed for in-situ expression by RT-PCR and for apoptosis by the tunel method. The phenotype of T-cell and macrophages in lymphoid organs and lung is determined by flow cytometry analysis.
  • Fas ligand expression by single cell suspension is determined by 1) Cr release assay of Fas apoptosis sensitive target cells, 2) frequency analysis by single cell Fas ligand PCR.
  • Fas apoptosis signaling To abolish cell surface Fas expression, it is sufficient to prevent Fas apoptosis signaling, since it is well established that Fas expression does not necessarily correlate with Fas apoptosis signaling (76-81).
  • the analysis of Fas- apoptosis signaling and inhibition of this by IL-l ⁇ converting enzyme family members and also inhibitors of HCP are useful in testing abolition. The will be accomplished by incorporating both Fas and known inhibitory proteins of Fas apoptosis into modified Ad virus.
  • a specific construct capable of expressing Fas ligand safely and at the same time protect the Fas ligand expressing cell from autocrine-mediated apoptosis includes both FasL and fragments of IL-l ⁇ or repeats of the peptide sequence the CPP32/Yama inhibitor DEVD-CHO, the ICE inhibitor YVAD-CHO which inhibit ICE and CPP32 and prevent Fas-mediated apoptosis in different cells, and Crm A, which block the cas pase pathway (81).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'un des problèmes majeurs rencontrés lors de la thérapie génique au moyen d'adénovirus est la réponse immunitaire induite par les lymphocytes T, déclenchée par inoculation d'un adénovirus qui conduit à une clairance rapide du virus et à une perte d'expression transgénique. Selon la présente invention, on empêche la réponse immunitaire à un virus par prétraitement avec une cellule présentant un antigène (APC) infecté par un adénovirus, un virus adénoassocié ou un herpès virus exprimant un ligand Fas avec tolérance induite des lymphocytes T. L'administration de AdCMVLacZ après tolérance se traduit par une expression prolongée de LacZ chez des animaux dans lesquels on a induit une tolérance comparativement à des animaux témoins traités. Chez des animaux témoins, mais non chez des animaux dans lesquels on a induit une tolérance, on a observé une prolifération de lymphocytes T CD3+ dans la rate, en réponse à un traitement AdCMVLacZ. L'induction de la tolérance est également indiquée par une production décroissante d'interféron-η et IL-2 par des lymphocytes T périphériques isolés à partir d'animaux témoins après stimulation par des APC infecté par adénovirus. La tolérance des lymphocytes T est spécifique au virus du fait que les réponses des lymphocytes T à un virus étranger, un cytomégalovirus de souris (MCMV), restent sans obstacles. La présente invention utilise une tolérance des lymphocytes T spécifique à un virus, qui est induite par des APC qui coexpriment un ligand Fas et des antigènes de virus. La présente invention implique de nouveaux procédés et vecteurs destinés à induire une tolérance à une thérapie génique à vecteur viral et prolonge l'expression d'un transgène chez un hôte viral.
PCT/US1998/010381 1997-05-22 1998-05-22 Regulation de la reponse immunitaire a des antigenes specifiques WO1998052615A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US09/424,281 US6689605B1 (en) 1997-05-22 1998-05-22 Controlling immune response to specific antigens
AU75859/98A AU7585998A (en) 1997-05-22 1998-05-22 Controlling immune response to specific antigens

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4742697P 1997-05-22 1997-05-22
US60/047,426 1997-05-22

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/424,281 A-371-Of-International US6689605B1 (en) 1997-05-22 1998-05-22 Controlling immune response to specific antigens
US10/776,119 Division US20040157792A1 (en) 1997-05-22 2004-02-10 Controlling immune response to specific antigens

Publications (2)

Publication Number Publication Date
WO1998052615A1 WO1998052615A1 (fr) 1998-11-26
WO1998052615A9 true WO1998052615A9 (fr) 1999-04-01

Family

ID=21948905

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/010381 WO1998052615A1 (fr) 1997-05-22 1998-05-22 Regulation de la reponse immunitaire a des antigenes specifiques

Country Status (2)

Country Link
AU (1) AU7585998A (fr)
WO (1) WO1998052615A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002541855A (ja) * 1999-04-21 2002-12-10 ジェンザイム・コーポレーション 免疫調節分子コード核酸を含むアデノウイルスベクター
EP1180156A1 (fr) * 1999-05-27 2002-02-20 Genzyme Corporation Methodes d'induction de tolerance a des vecteurs adenoviraux et a des produits transgeniques
WO2020041791A1 (fr) * 2018-08-24 2020-02-27 Locana, Inc. Compositions de thérapie génique immunomodulatrice fasl et procédés d'utilisation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585362A (en) * 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
US5670488A (en) * 1992-12-03 1997-09-23 Genzyme Corporation Adenovirus vector for gene therapy
AU688428B2 (en) * 1993-11-09 1998-03-12 Johns Hopkins University, The Generation of high titers of recombinant AAV vectors

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9528088B2 (en) 2002-06-28 2016-12-27 Life Technologies Corporation Methods for eliminating at least a substantial portion of a clonal antigen-specific memory T cell subpopulation

Also Published As

Publication number Publication date
AU7585998A (en) 1998-12-11
WO1998052615A1 (fr) 1998-11-26

Similar Documents

Publication Publication Date Title
JP4588296B2 (ja) キメラワクチン
US20070172504A1 (en) In vivo cell surface engineering
EP0551401B2 (fr) Procedes et compositions pour la therapie et la potentialisation genetiques de l'immunite antitumorale
KR101195400B1 (ko) 암배아 항원 융합물 및 이의 용도
US20030215427A1 (en) CE7-specific redirected immune cells
ES2321246T3 (es) Nuevos vectores de expresion que contienen genes de ligandos de moleculas accesorias y su uso para inmunomodulacion y tratamiento de tumores malignos y enfermedades autoinmunes.
US20100292309A1 (en) Inducing immune-mediated tumor cell death
CN108070608B (zh) 靶向CD19-CD28-tEGFR的嵌合抗原受体及其用途
NZ263550A (en) 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
CN108864286B (zh) 靶向cd19的嵌合抗原受体及联合表达抗pd1抗体可变区的方法和及其用途
JP2008507282A (ja) 自己免疫および自己抗原に関連する疾患の特異的阻害
US20160317631A1 (en) Increased t-cell tumor infiltration and eradication of metastases by mutant light
CA3031725A1 (fr) Cellules exprimant la lmp-1 et leurs methodes d'utilisation
US7777004B2 (en) Polypeptides comprising Fas activation and NKG2D-ligand domains
US6689605B1 (en) Controlling immune response to specific antigens
WO2023217192A1 (fr) Préparation d'une cellule immunitaire de récepteur antigénique chimérique construite sur la base d'une protéine précurseur de msln et son utilisation
WO1998052615A9 (fr) Regulation de la reponse immunitaire a des antigenes specifiques
JP2003513619A (ja) 自己免疫疾患および免疫病因を含む疾患を治療するための遺伝子導入ベクター
WO2001079495A1 (fr) Induction de l'apoptose de cellule tumorale par transfert de gene du ligand entre trail et apo-2
JP2023545241A (ja) キメラｃｄ４０リガンド及びコロナウイルスワクチンを使用する免疫の増強
JP2001521743A (ja) 組換えベクターに対する免疫応答の阻害方法
US7064111B1 (en) Use of soluble costimulatory factor for tumor immuno-gene therapy
WO2005017163A2 (fr) Inactivation phenotypique de proteines de surface cellulaire
Castro et al. Gene therapy strategies for intracranial tumours: glioma and pituitary adenomas
JP2002508976A (ja) 遺伝子療法において使用するための、免疫原性が低下したヌクレオチド発現システム

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1-52 AND 54/57, DESCRIPTION, REPLACED BY NEW PAGES 1-48 AND 50-53; PAGE 53 RENUMBERED AS PAGE49; PAGES 58-65, CLAIMS, REPLACED BY NEW PAGES 54-60; PAGES 66-69, AMENDED CLAIMS, RENUMBERED AS PAGES 61-64; PAGES 1/17-17/17, DRAWINGS, REPLACED BY NEW PAGES 1/13-13/13; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 09424281

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998550621

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA