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WO2002040059A2 - Procedes et compositions pour induire des reponses immunitaires a mediation cellulaire - Google Patents

Procedes et compositions pour induire des reponses immunitaires a mediation cellulaire Download PDF

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Publication number
WO2002040059A2
WO2002040059A2 PCT/US2001/045626 US0145626W WO0240059A2 WO 2002040059 A2 WO2002040059 A2 WO 2002040059A2 US 0145626 W US0145626 W US 0145626W WO 0240059 A2 WO0240059 A2 WO 0240059A2
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antigen
variant
cells
cell
psma
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WO2002040059A3 (fr
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Milcho S. Mincheff
Dmitri I. Loukinov
Seguei Zoubak
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American Foundation for Biological Research Inc
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American Foundation for Biological Research Inc
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    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • 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

  • MHC major histocompatibility complex
  • CTLs CD8 cytotoxic T-lyn phocytes
  • T cells need to be " pre-educated” by recognizing the antigen in question on the membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs) which, in addition to the antigen, provide co-stimulatory "maturation" signals to the T cells. In the absence of such signals the T cells become paralyzed and tolerant to the antigens in question.
  • APCs professional antigen-presenting cells
  • DCs dendritic cells
  • tumor cells are poor APCs, and that "professional” antigen presenting cells are essential for induction of CD4+ and CD8+ T cell responses against tumors.
  • Huang, A. Y. C, et al Science (Wash.
  • DC 264:961 (1994), reported that the in vivo priming of MHC class I-restricted responses involves a transfer of tumor antigens to a host bone marrow-derived dendritic cell ("IBM-DC") and subsequent presentation to CD8+T cell effectors.
  • IBM-DC bone marrow-derived dendritic cell
  • the tumor antigen(s) need(s) to be expressed by professional APCs. This presentation has been accomplished by in vitro exposure of dendritic cells to either tumor lysates that presumably contain tumor antigens, to purified tumor antigens, or to peptides derived from such antigens.
  • U.S. Patent 5,788,963 discloses the use of human dendritic cells to activate T cells for immuno-therapeutic response against primary and metastatic prostate cancer.
  • Human dendritic cells are isolated and exposed to PSMA-derived peptides in vitro.
  • the PSMA-derived peptides exchange with peptides already bound to MHC molecules on the dendritic cells, enabling them to stimulate killer cells which then lyse prostate cells.
  • a major problem with this technique comes from the low efficiency of peptide exchange and from the necessity to work with different immunodominant peptides for patients with different MHC phenotypes.
  • Other methods for treating cancer include administering to a patient antibodies directed against a surface antigen of the tumor cells, wherein the antibodies are conjugated with a cytotoxic agent.
  • a cytotoxic agent for example, U.S. Patent 5,227,471 discloses a method for treating prostate cancer which involves an antibody directed against the prostate-specific membrane antigen and a cytotoxic agent conjugated thereto.
  • the PSMA is expressed on normal brain cells, use of antibodies which may transverse through the blood-brain barrier and may damage normal brain cells may not be permitted.
  • the invention provides methods and compositions for inducing a cell-mediated immune response against a target antigen in a subject, without inducing a humoral immune response against that target antigen.
  • the invention provides methods for eliminating undesired cells (referred to herein as "target cells") in a subject (in vivo) or in vitro (e.g., ex vivo).
  • the method comprises administering to the subject a polynucleotide encoding a polypeptide which is a variant of an antigen that is expressed in a target cell, which target antigen is then presented within the context of MHC class I on the surface of the target cells such that a cell-mediated immune response against the cell comprising the target antigen develops; and wherein the variant of the target antigen is not secreted from, nor expressed on the extracellular side of the cell membrane of a cell in which it is synthesized.
  • the variant of the target antigen is biologically inactive, so that the survival and the function of cells in which the said antigen is expressed, is not altered.
  • the undesired cells are tumor cells, and the target antigen is a tumor antigen, e.g., an antigen that is expressed preferentially on tumor cells.
  • the undesired cells are prostate tumor cells and the target antigen is prostate specific membrane antigen (PSMA), prostate specific antigen (PSA) or prostate acidic phasphatase (PAP), h an even more preferred embodiment, the variant of the PSMA antigen comprises amino acids 44-750; 55-750; 61-750; 76-750; or 94-750 of SEQ ID NO: 2.
  • the variant of PSA comprises the amino acid sequence set forth in SEQ ID NO: 4. Yet other preferred methods involve the destruction of other carcinoma cells.
  • melanoma cells can be destroyed using the method of the invention, wherein the target antigen is a MAGE or MART-1 antigen.
  • Preferred variants of these antigens include the amino acid sequence set forth in SEQ ID NO: 10, 12, or 14.
  • Pancreatic or colorectal cancer cells can be targeted by the use of a variant of carcino- embryonic antigen (CEA) including, e.g., the amino acid sequence set forth in SEQ ID NO: 6.
  • CEA carcino- embryonic antigen
  • Breast cancer cells can be targeted by the use of a variant of the target antigen is Her2/Neu including, e.g., the amino acid sequence set forth in SEQ ID NO: 8.
  • the polynucleotide encoding a variant of the target antigen may be included in a plasmid vector.
  • a plasmid vector that contains an expression cassette encoding a variant of PSMA, e.g., comprising amino acids 44-750; 55-750; 61-750; 76-750; or 94-750 of SEQ ID NO: 2.
  • the plasmid vector is termed "XC-PSMA-plasmid," having a Designation Number 203168, deposited on August 28, 1.998 at the American Type Culture Collection, 10801 University Boulevard., Manassas, Virginia 20110.
  • the polynucleotide encoding a variant of the target antigen may also be included in a viral vector, e.g., an adenoviral vector.
  • a viral vector that is a replication-defective recombinant adenovirus comprising a nucleotide sequence encoding a variant of PSMA, e.g., comprising amino acids 44-750; 55-750; 61-750; 76-750; or 94-750 of SEQ ID NO: 2.
  • the adenoviral vector is the viral vector termed "Ad5-PSMA," having ATCC Designation Number NR2361.
  • the undesired cells are cells that are reactive against an auto-antigen, i.e., a self-antigen, and the method is used, e.g., for treating auto-immune diseases.
  • the invention provides a method for preventing attack of a graft recipient by the graft, e.g., graft- versus-host disease, by selective destruction of cells reacting against the subject.
  • the invention provides methods for treating diseases caused by, or associated with, an intracellular infectious agent, e.g., a virus.
  • Diseases include malaria, colds, rabies, leishmaniasis, tuberculosis, and borrelia infection.
  • cells presenting viral proteins can be targeted for destruction according to the invention by administering to the subject a polynucleotide encoding a variant of the foreign protein.
  • the invention provides a method for treating a subject having undesired cells, comprising (i) obtaining professional antigen presenting cells from the subject; (ii) introducing into said cells ex vivo a polynucleotide encoding a polypeptide which is a variant of a target antigen of the undesired cells, which variant is not secreted from, nor expressed on the extracellular side of the cell membrane of, a cell in which it is expressed, and which variant is biologically inactive, to obtain cells that express the variant in association with MHC class I complexes; and (iii) administering the cells obtained in (ii) to the subject; such that the number of undesired cells is reduced in the subject
  • plasmid vectors and viral vectors encoding a variant of a target antigen, e.g., a variant of the human PSMA, wherein the variant is not secreted from, nor expressed on the extracellular side of the cell membrane of, a cell in
  • Preferred plasmid and viral vectors comprise a nucleotide sequence a nucleotide sequence encoding amino acids 44-750; 55-750; 61-750; 76-750; or 94-750 of SEQ ID NO: 2.
  • the plasmid vector is "XC-PSMA" plasmid, which was deposited under the Budapest Treaty at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, on August 28, 1998, and assigned ATCC Deposit Number 203168.
  • the viral vector is the adenoviral vector "Ad5 PSMA,” which was deposited under the Budapest Treaty at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, NA 20110-2209, on August 28, 1998, and assigned ATCC Deposit Number NR 2362.
  • the present invention discloses, in particular, the construction of polynucleotides encoding variants of genetically modified forms of target antigens, e.g., tissue- specific or tumor antigens, and for the use of such constructs for immunotherapy of primary or metastatic cancer.
  • target antigens e.g., tissue- specific or tumor antigens
  • the genetic modification of the antigens leads to expression of either functionally inactive products (variants) or prevents functionally active molecules from being secreted or expressed on the membrane of transfected cells.
  • Such genetically modified antigens could also be fused to, or co-expressed with, other known immunogenic eukaryotic, prokaryotic or viral products or parts of them for the purpose of increasing the immunogenicity of the target antigens and bypassing the tolerance that may exist to them.
  • Such genetic modifications may affect the antigenicity of the expressed protein, its primary structure or the generation of peptides available for binding to cell's MHC molecules, however, the variants must remain sufficiently immunogenic such as to induce a cellular immune response.
  • the genetic modifications preferably will not significantly affect the efficiency of transcription and translation of the D ⁇ A, nor the translation of the R A.
  • the present invention discloses the development of a D ⁇ A, which leads to expression of a truncated form of the human prostate specific membrane antigen (PSMA).
  • PSMA prostate specific membrane antigen
  • PSMA is a type II protein, it lacks a hydrophobic signal sequence and therefore is not secreted by the cell that produces it. Since the construct lacks membrane and cytoplasmic sequences, the resulting protein is not expressed on the membrane, therefore does not transduce signals and is not released from the membrane. Cells transfected with the XC-PSMA plasmid retain viability and express PSMA-derived peptides.
  • the synthesized protein is not released but remains confined to the intracellular milieu, there is no production of antibodies directed against the protein and the immune response remains strictly cell-mediated.
  • the extraordinar engagement of cell- mediated immunity against particular antigen is very important especially in cases where the target antigen of interest is expressed on normal tissues that are anatomically sequestered in immuno-privileged sites such as the eye, brain, testis etc. Those tissues are inaccessible to cell mediated injury, but may readily be damaged by antibodies.
  • the present invention differs from the prior art at least in that it is believed to cause the dendritic cells to present an antigen derived from a target cell, e.g., a prostate cancer cell, on their surface through transfection with a polynucleotide of the invention, included in, e.g., a plasmid or adenovirus.
  • a polynucleotide of the invention included in, e.g., a plasmid or adenovirus.
  • the transfection may occur in vivo using injected polynucleotide.
  • the transfection may occur in vitro using purified DC precursor cells isolated from the prostate cancer patient's blood. If transfection is done in vitro, the transfected cells are injected into the patient.
  • Transfected DCs are superior to DCs, which have been primed with antigen in vitro because both their loading with antigen-derived peptide and their ability to stimulate killer cells are more efficient.
  • in vivo transfection using, e.g., a plasmid or adenovirus is less laborious and less expensive than in vitro methods.
  • the use of transfected cells avoids the necessity of identifying peptides capable of binding to different HLA phenotypes, as is required in methods which involve the addition of peptides to cells.
  • a DNA sequence that encodes a variant of a target antigen e.g., a truncated molecule of the PSMA, guarantees that the protein is not released by the transfected cells and no antibodies against the target protein that are potentially hazardous to normal brain tissue are produced.
  • the methods of the present invention bypass the normal tolerance for self-antigens. This enables the cytolysis of target cells normally shielded from immune recognition.
  • Fig.l represents the amino acid sequence of human PSMA (SEQ ID NO: 2).
  • the boxed region corresponds to the transmembrane domain.
  • Fig.2. Delayed type hypersensitivity (DTH) reaction in a prostate cancer patient at different time points following intradermal injection of either empty plasmid vector (circles), sGM-CSF (squares) or PSMA plasmid +sGM-CSF (triangles).
  • DTH Delayed type hypersensitivity
  • Fig.5. PSA (diamonds) and DTH (squares) values of patient # 5, age 58. Diagnosis about 6 months before “0 months” on the graph (T3cNoMo). Hormone therapy (Zoladex, Casodex) was initiated one month after diagnosis. Immunotherapy was initiated four months later (at "o" months in the graph). No tumor palpable at the time of prostatectomy (at 6 months on the graph). PSA became detectable at 9 months on the graph and it has been rising steadily despite immunotherapy.
  • Fig.6 PSA (diamonds) and DTH (squares) values of patient # 16, age 63. Diagnosis at one month (T2cNoMo). Several months after the 13 months date, no tumor but a small remnant of the prostate gland was palpable, no obstructive voiding symptoms.
  • Fig.8 PSA (diamonds) and DTH (squares) values of patient # 8, age 69. Diagnosed three years before the "0" month on the graph, and received TURP at that time. Tumor recurrence 3 months later. TURP three years after first TURP ("0" month on graph). Gleason pattern 3-4. Distant LN involvement. Hormone therapy (Zoladex, Casodex) started at 11 months. Immunotherapy initiated at 11 months. Hormone therapy discontinued at 16 months. Currently no obstructive voiding symptoms, no prostate palpable on DRE, no LN involvement on CAT scan.
  • Fig.9. Immunologic profile of prostate cancer patients prior to, and 15 months after, continuous IT.
  • Fig. 10. Changes in selected flow cytometry parameters in responders and non- responders prior to, and 15 months after, continuous IT.
  • Fig.l Immunologic profile of patients with advanced colorectal cancer prior to and following chemotherapy, and of healthy volunteers.
  • the invention is based at least in part on the observation that administration of a nucleic acid encoding a variant of a prostate antigen, which variant is not secreted from, nor expressed on the extracellular side of the cell membrane of, the cell it is synthesized in, to subjects having advanced stages of prostate cancer was shown to improve the disease.
  • dendritic cells transfected with a nucleic acid encoding the extracellular domain of PSMA are capable of activating CD8+ T cells in vitro, which are then capable of lysing target cells.
  • the present invention pertains generally to methods for eliminating undesired cells, e.g., in a subject.
  • the undesired cells can be tumor cells, auto-reactive cells or cells infected with a foreign particle, such as a virally infected cell.
  • the invention provides methods for inducing a cell- mediated immune response against a target antigen, without inducing a significant humoral immune response against the target antigen, hi a preferred embodiment, the invention comprises administering to a subject in need thereof, a nucleic acid encoding a variant of a target antigen, e.g., a portion of the antigen, which variant is not secreted from, or expressed on the extracellular side of the cellular membrane of, the cell it is synthesized in, which variant is preferably biologically inactive and/or prevents functionally active molecules from being secreted or expressed on the membrane of transfected cells.
  • a target antigen e.g., a portion of the antigen, which variant is not secreted from, or expressed on the extracellular side of the cellular membrane of, the cell it is synthesized in, which variant is preferably biologically inactive and/or prevents functionally active molecules from being secreted or expressed on the membrane of transfected cells.
  • the target antigen can be homologous, e.g., a polypeptide from the subject, such as a tumor antigen, or heterologous, e.g., a viral polypeptide.
  • the invention provides a method for treating a subject having tumor cells, comprising administering to the subject a pharmaceutically efficient amount of a nucleic acid encoding a variant of a tumor antigen, e.g., a portion of the extracellular domain of a tumor antigen that is normally expressed on the surface of tumor cells.
  • a polynucleotide of the invention enters professional antigen presenting cells (APCs), where the polynucleotide is expressed into the variant of a target antigen, and then fragments of this variant antigen arc presented on the surface of the APC together with MHC class I and class II complexes, resulting in activation of CD4+ and CD8+ T cells.
  • APCs professional antigen presenting cells
  • Stimulation of CD4+ T cells seems to be essential because they provide the necessary signals for dendritic cell and CD8+ T cell maturation.
  • CD8+ T cells will then lyse tumor cells expressing the target antigen.
  • CD4+ T cells helper (CD4+ T cells) and the effector (CD8+ T cells) arms of the immune system.
  • Recognition by CD4+ T cells requires that antigenic peptides be expressed in conjunction with class II MHC molecules on the DC surface. This can be achieved by in vivo or in vitro transfection of DC with plasmid or infection of DC with recombinant adenovirus, which carry the DNA encoding the extracellular fragment of PSMA.
  • transfection of dendritic cells with constructs encoding for known immunogenic eukaryotic, prokaryotic or viral products fused to, or co-expressed with, the variant of the target antigen (e.g., PSMA), increases the likelihood of breaking the tolerance towards said target antigen by stimulating "by-standing" CD4+ T cells reactive to said known immunogenic eukaryotic, prokaryotic or viral products.
  • the target antigen e.g., PSMA
  • Such processed peptides bind to newly synthesized class I heavy chain- beta 2- microglobulin heterodimers in the ER. See. for example, Yewdell, J. W., et al., Science 244:1072 (1989); Townsend, A., et al., Cell 62:285 (1990); and Nuchtem, J. G., et al., Nature 339:223 (1989).
  • the processed peptide is bound to the class I heavy chain-light chain dimer molecule via the class I antigen binding site/peptide cleft.
  • the complex thereby generated is a transport competent trimer as reported by Yewdell, J. W., et al., Science 244:1072 (1989); Townsend, A., et al, Cell 62:285 (1990); and Nuchtem, J. G., et al.,
  • This class I histocompatibility molecule-processed peptide complex is then expressed on the surface of the target cell where it may be ultimately recognized by T cell clonotypic receptors on CD8+ cells in conjunction with CD8 accessory molecules.
  • T cell clonotypic receptors on CD8+ cells in conjunction with CD8 accessory molecules.
  • MHC class II molecules can also present antigen that has been synthesized in a cell. This is described, e.g., in Nuchtem et al. (1990) Nature 343: 74 and Loss et al. (1993) J. Exp. Med. 178: 73.
  • an "activated lymphocyte” is one that as a result of binding of a cognate antigen peptide:MHC molecule is producing polypeptide stimulatory factors (including, for example, cytokines) at a level that is elevated relative to the lymphocyte without the bound ligand.
  • polypeptide stimulatory factors including, for example, cytokines
  • An “agretope” is the portion of an antigen or antigenic fragment which allows it to bind to an MHC molecule.
  • a T cell epitope is an agretope.
  • Antigen Presenting Cells include known APCs such as Langerhans cells, veiled cells of afferent lymphatics, dendritic cells and interdigitating cells of lymphoid organs.
  • the definition also includes mononuclear cells such as (1) lymphocytes and macrophages which take up and express polynucleotides according to the invention in skin and (2) mononuclear cells depicted on histological photographs contained herein. These cells are not tissue cells but are likely to be antigen presenting cells.
  • APCs which are known to be present in high numbers in epithelia and thymus dependent areas of the lymphoid tissues, including epidermis and the squamous mucosal epithelia of the buccal mucosa, vagina, cervix and esophagus (areas with “relatively high” concentrations of APCs).
  • skin and mucosa as used herein particularly refer to these sites of concentration of APCs.
  • professional APCs shall refer to cells whose primary purpose is antigen presentation; i.e., bone marrow derived dendritic cells.
  • Antigen specific expression refers to expression that occurs when the T cell recognizes its cognate antigen.
  • biologically inactive is used interchangeably herein with the term “functionally inactive,” which, as applied to a polypeptide, refers to a polypeptide that has essentially no biological activity, i.e., no detectable biological activity that would be deleterious for its use within the context of the invention, and will not negatively affect the subject to which it is administered, in particular, e.g., it will not affect the cells taking up the nucleic acid and expressing the encoded polypeptide. Although a polypeptide may retain some negligible biological activity, it will be considered for the purposes herein, to be biologically inactive whenever the beneficial effect of the polypeptide significantly outweighs its negative effect resulting from residual biological activity.
  • biological activity does not include antigenicity.
  • a polypeptide that is biologically inactive is still capable of stimulating a humoral or cell-mediated immxuie response.
  • cellular immune response is used interchangeably herein with “cell- mediated immune response” and refers to an immune response in which cells mediate the lysis of target cells.
  • Cognate antigen refers to an antigen, a peptide of which when associated with an MHC molecule forms a ligand that binds to a lymphocyte that recognizes it and causes triggering of signals for the effector function of the cell and/or for proliferation.
  • a CTL is "cytolytically specific for" cells expressing antigens if the CTL is capable of selectively recognizing and lysing the cells bearing the antigen.
  • a CTL is "cytolytically reactive against” cells expressing antigens if the CTL is capable of lysing the cells bearing the antigen, without regard to its ability to selectively recognize such cells.
  • a “delivery complex” shall mean a targeting means (e.g. a molecule that results in higher affinity binding of a gene, protein, polypeptide or peptide to a target cell surface and/or increased cellular or nuclear uptake by a target cell).
  • targeting means include: sterols (e.g. cholesterol), lipids (e.g. a cationic lipid, virosome or liposome), vimses (e.g. adenoviras, adeno-associated viras, and retrovirus) or target cell specific binding agents (e.g. ligands recognized by target cell specific receptors).
  • Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to intemalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the gene, protein, polypeptide or peptide is released in a functional form.
  • endogenous refers to a molecules which are naturally occurring in the cell, e.g., provided in the natural genome of the host organism or encoded thereby.
  • exogenous refers to molecules which are not naturally present in the cell, and which have been, e.g., introduced by transfection or transduction of the cell (or the parent cell thereof).
  • the term “gene” or “recombinant gene” refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence.
  • the term “intron” refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons.
  • Helper cells or “T H cells” or “T helper cells” or “T helper lymphocytes” are a functional subclass of T cells which can help to generate cytotoxic T cells and cooperate with B cells in the production of an antibody response. Helper cells usually recognize antigen in association with class II MHC molecules.
  • helper construct refers generally to a nucleic acid molecule that includes nucleotide sequences providing functions deleted from a vector which is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest.
  • AAN helper constracts are commonly used to provide transient expression of AAN rep and/or cap genes to complement missing AAN functions that are necessary for lytic AAN replication; however, helper constracts lack AAN ITRs and can neither replicate nor package themselves.
  • AAN helper constracts can be in the form of a plasmid, phage, transposon, cosmid, viras, or virion.
  • a number of AAN helper constracts have been described, such as the commonly used plasmids pAAN/Ad and pIM29 + 45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Nirol. 63:3822- 3828; and McCarty et al. (1991) J. Nirol. 65:2936-2945.
  • a number of other vectors have been described which encode Rep and/or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.
  • a "helper virus” is a virus which supplies some or all of the functions necessary for replication of a viral vector which are not encoded by a wild-type virus or which is encoded by a wild-type viras, but not by a recombinant viras.
  • An "AAN helper viras” is a viras which supplies some or all of the functions necessary for AAN (and rAAN vector) replication which are not encoded by a wild-type AAN or which are encoded by a wild-type AAN, but not by a recombinant AAN.
  • these functions are supplied in trans by viruses such as adenoviras or herpes viras during viral replication.
  • viruses such as adenoviras or herpes viras during viral replication.
  • adenoviras and the herpes viras are examples of AAN helper viruses.
  • “Host” refers to the recipient of the therapy to be practiced according to the invention.
  • the host may be any vertebrate, but will preferably be a mammal. If a mammal, the host will preferably be a human, but may also be a domestic livestock or pet animal.
  • the term "humoral immune response” refers to an immune response that involves antibodies.
  • An "immune response" to a target antigen is the development in the host of a cellular and/or antibody-mediated immune response to the cell containing the target antigen (cellular immune reaction) or to soluble target antigen (antibody mediated response).
  • cellular immune reaction cellular immune reaction
  • antibody mediated response soluble target antigen
  • Such a response comprises the individual producing CTLs and/or B cells and/or a variety of classes of T cells directed specifically to APCs expressing the target antigen.
  • “Infection,” in the context of a viral-cell interaction refers to the process wherein a viras enters the cell in a manner whereby the genetic material of the viras can be expressed in the cell.
  • a “productive infection” refers to the process wherein a viras enters the cell, is replicated, and then released from the cell.
  • “Infectious agent” refers to an agent which can infect cells, e.g., mammalian cells.
  • An agent can be a microorganism, such as bacteria or a protozoan, a viras, or a prion.
  • Intracellular infectious agent refers to a microorganism or virus, in those cases where part or all of their replicative cycle occurs within the cells of an infected individual.
  • Intracellular infectious agents include, for example, protozoa, fungi, bacteria, and viruses.
  • Intracellular pathogen refers to an agent capable of causing a disease state in a susceptible individual, in which part or all of its replicative cycle occurs within the cells of an infected individual.
  • Intracellular pathogens include, for example, protozoa, fungi, bacteria, and viruses.
  • Isolating the recombinant vector refers to the process of purifying the encapsidated vector away from components found in the biological system (most typically tissue culture) from which it was produced. Procedures for the purification of biological materials, including vectors are well-known in the art, and may be found, e.g., in Sambrook and in Ausbel (both supra). Typically, viral particles (i.e., nucleic acids encapsidated in a capsid) are purified by CsCl gradient or step gradient centrifugation (see, Sambrook). Typically, the vectors are approximately 90% pure after CsCl gradient centrifugation.
  • Mucosa refers to mucosal tissues of a host wherever they may be located in the body including, but not limited to, respiratory passages (including bronchial passages, lung epithelia and nasal epithelia), genital passages (including vaginal, penile and anal mucosa), urinary passages (e.g., urethra, bladder), the mouth, eyes and vocal cords.
  • respiratory passages including bronchial passages, lung epithelia and nasal epithelia
  • genital passages including vaginal, penile and anal mucosa
  • urinary passages e.g., urethra, bladder
  • control sequences e.g., transcriptional regulatory sequences, operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
  • naked polynucleotide(s) refers to DNA or RNA or oligonucleotides. Naked in this context means polynucleotides, which are not complexed to colloidal materials (including liposomal preparations), or contained within a vector which would cause integration of the polynucleotide into the host genome.
  • non-human animals include mammalians such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • Preferred non- human animals are selected from the rodent family including rat and mouse, most preferably mouse, though amphibians, such as members of the Xenopus genus, and chickens are also included.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • a “packaging cell” refers to a host cell which, by way of stable or transient transfection with heterologous nucleotide sequences, harbors a nucleic acid molecule comprising a viral helper constract, wherein the construct is capable of providing transient expression of viral helper functions that can be provided in trans for productive viral replication. Expression of the viral helper functions can be either constitutive, or inducible, such as when the helper functions are under the control of an inducible promoter.
  • An example of a packaging cell is a 293 cell.
  • a "packaging signal" refers to a nucleotide sequence, which when present in a nucleic acid allows the nucleic acid to be packaged into a viral particle in appropriate conditions, such as in the presence of packaging extracts or in a packaging cell.
  • Polynucleotide of the invention refers to a polynucleotide encoding a variant of a target antigen, which variant is not secreted from, or expressed on the extracellular side of the cell membrane of, the cell in which it is synthesized; and which is biologically inactive.
  • polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post- expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. "Polypeptide of the invention” refers to a polypeptide which is encoded by a polynucleotide of the invention.
  • a “producer cell” refers to a packaging cell that has been stably or transiently transfected with a viral vector-either before, subsequent to, or at the same time as transfection of the cell with the viral helper functions.
  • a producer cell contains viral sequences that are provided in cis for replication and packaging (e.g., functional ITR sequences), and viral sequences encoding helper functions missing from the viral vector and provided in trans for replication and packaging.
  • the producer cell is thus capable of encoding viral polypeptides that are required for packaging transfected viral DNA (e.g., AAN viral vectors containing a recombinant nucleotide sequence of interest) into infectious viral particles for subsequent gene delivery.
  • promoter means a D ⁇ A sequence that regulates expression of a selected D ⁇ A sequence operably linked to the promoter, and which effects expression of the selected D ⁇ A sequence in cells.
  • the term encompasses "tissue specific” promoters, i.e. promoters, which effect expression of the selected D ⁇ A sequence only in specific cells (e.g. cells of a specific tissue).
  • tissue specific promoters
  • leaky promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
  • the term also encompasses non-tissue specific promoters and promoters that constitutively express or that are inducible (i.e. expression levels can be controlled).
  • recombinant when used with reference to a cell or virus indicates that the cell or virus contains a nucleic acid whose origin is exogenous to the cell or virus type. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell.
  • a "recombinant expression cassette” is a nucleic acid constract, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid.
  • the recombinant expression cassette can be part of a plasmid, viras, or nucleic acid fragment.
  • the recombinant expression cassette includes a nucleic acid to be transcribed, and a promoter.
  • the expression cassette also includes, e.g., an origin of replication, and/or chromosome integration elements.
  • recombinant expression vector refers to a replicable unit of DNA or RNA in a form capable of being introduced into a target cell by transformation, electroporation, transduction or viral infection, and which codes for the expression of a heterologous structural coding sequence, for example, a cytokine, which is transcribed into mRNA and translated into protein under the control of elements having a regulatory role in gene expression.
  • a heterologous structural coding sequence for example, a cytokine, which is transcribed into mRNA and translated into protein under the control of elements having a regulatory role in gene expression.
  • Such vectors will preferably also contain appropriate transcription and translation control sequences, including initiation sequences operably linked to the coding sequence.
  • a "recombinant helper nucleic acid” or more simply helper nucleic acid is a nucleic acid which encodes functions which allow a nucleic acid to be encapsidated in a capsid.
  • the helper plasmid, or other nucleic acid encodes viral functions and structural proteins which allow a recombinant viral vector to be encapsidated into a capsid.
  • a recombinant AAV helper nucleic acid is a plasmid encoding AAN polypeptides, and lacking the AAN ITR regions.
  • the helper plasmid encodes the AAN genome, with the exception of the AAN ITR regions, which are replaced with adenovirus ITR sequences. This pemiits replication and encapsidation of the AAN replication defective recombinant vector, while preventing generation of wild-type AAN viras, e.g., by recombination.
  • recombinant protein refers to a polypeptide, which is produced by recombinant D ⁇ A techniques.
  • a protein that is encoded by a nucleic acid which is introduced into a cell, as opposed to being expressed from the naturally-occurring genomic D ⁇ A of the cell is a recombinant protein.
  • the phrase "a first polypeptide derived from a second polypeptide” refers to a first polypeptide that is identical to the second polypeptide but for the introduction of one or more amino acid changes therein.
  • a “recombinant virion,” such as “rAAN”, is defined herein as an infectious, replication-defective viras composed of a protein shell, encapsidating a viral vector comprising a heterologous nucleotide sequence of interest.
  • Sequences necessary for viral packaging in the context of a viral helper nucleic acid include viral sequences active in trans which encode proteins necessary for encapsidation of the viral vector into an infectious particle.
  • the packaging proteins for AAN can include capsid proteins (Npl, Np2, N ⁇ 3) and replicase proteins (Rep 78, Rep 68, Rep 40, Rep 52).
  • signal peptide is used interchangeably herein with “signal sequence” and “leading sequence” to refer to a sequence or portion of a polypeptide that is located at the ⁇ -tenninus of a polypeptide and which associates with the endoplasmic reticulum and mediates the secretion or membrane attachment of a polypeptide.
  • Skin refers to the epidermal, dermal and subcutaneous tissues of a host.
  • the term “specifically hybridizes” or “specifically detects” refers to the ability of a nucleic acid molecule of the invention to hybridize to at least approximately 6, 12, 20, 30, 50, 100, 150, 200, 300, 350, 400 or 425 consecutive nucleotides of a vertebrate.
  • target antigen used interchangeably herein with “target peptide” refers to an antigen, which is presented on the surface of a target cell in association with an MHC class I complex.
  • the target antigen can be a polypeptide, or a derivative thereof, such as a glycoprotein.
  • a “target cell” refers to a cell expressing a target antigen, which is targeted for lysis by a cell-mediated immune reaction.
  • target tissue refers to a tissue containing target cells.
  • Tc or "cytolytic T cell” or “CTL” or “cytotoxic T cell” refers to a CD8+ T cells which is capable of lysing a cell.
  • T1 Response(s) refers to a cellular immune response that is induced preferentially by antigens that bind to and activate certain APCs; i.e., macrophages and dendritic cells.
  • a “therapeutically efficient amount” or “pharmaceutically efficient amount” of a compound, e.g., polynucleotide of the invention refers to a dose sufficient to induce an immune response against the target antigen.
  • Titers are numerical measures of the "concentration" of a viras or viral vector compared to a reference sample, where the concentration is determined either by the activity of the virus, or by measuring the number of virases in a unit volume of buffer.
  • the titer of viral stocks are determined, e.g., by measuring the infectivity of a solution or solutions (typically serial dilutions) of the virases, e.g., on HeLa cells using the soft agar method (see, Graham & Nan Der eb (1973) Virology 52:456-467) or by monitoring resistance conferred to cells, e.g., G418 resistance encoded by the viras or vector, or by quantitating the virases by UN spectrophotometry (see, Chardonnet & Dales (1970) Virology 40:462-477).
  • the term “transfection” means the introduction of a nucleic acid, e.g., via an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • Transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous D ⁇ A or R ⁇ A.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to D ⁇ A sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • the term “treating” as used herein is intended to encompass curing as well as ameliorating at least one symptom of the condition or disease and includes prophylactic and therapeutic methods.
  • Tumor antigen or “tumor-associated antigen” refers to an antigen that is found on tumor cells, and which may be specific to certain tumors and/or specific to tumor cells relative to normal cells.
  • a tumor antigen can also be an embryonic protein that has been re- expressed by transformed cells or an autoantigens that is not truly tumor specific, but is present in mammalian tumor tissue.
  • a "variant" of a target antigen refers to all or a portion of a target antigen or modified form thereof, that (i) is sufficiently homologous to the target antigen to ensure that CD8+ T cells recognizing the variant will also recognize the target antigen; and (ii) is not secreted from, or expressed on the extracellular side of the cell membrane of, the cell in which it is expressed.
  • a preferred variant is biologically (i.e., functionally) inactive.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a form of vector that can be maintained episomally, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids” which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and vector are used interchangeably as the plasmid is the most commonly used form of vector.
  • a “viral vector” refers to a nucleic acid containing at least a portion of a viral genome sufficient for replication and packaging in the presence of an appropriate helper viras and appropriate cell line or packaging extract.
  • an “AAV vector” is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion.
  • an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the viras.
  • the ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
  • virus or “viral particle” is meant a complete virus particle, such as a wild-type (wt) viras particle (comprising a nucleic acid genome associated with a capsid protein coat), or a recombinant viras particle as described below.
  • adenoviral virion is meant a complete virus particle, such as a wild-type (wt) Ad viras particle comprising an Ad nucleic acid genome associated with an Ad capsid protein coat, or a recombinant AAV viras particle as described below.
  • single-stranded AAV nucleic acid molecules of either complementary sense, e.g., "sense” or “antisense” strands can be packaged into any one AAV virion and both strands are equally infectious.
  • Target antigens and target diseases and conditions Target antigens and target diseases and conditions
  • the methods of the invention permit the destruction of any undesirable cell, such as a cell of a subject, which has lost some of its normal characteristics or a cell infected with a foreign particle, e.g., a microorganism. Accordingly, the invention provides methods for reducing or eliminating uncontrolled cell proliferation, e.g., of malignant cells.
  • the invention provides immunotherapy methods for primary and metastatic cancer. Undesirable cells also include those, which produce an undesirable protein or other molecule.
  • autoimmxme diseases can be treated by using the method of the invention against a cell producing auto-antibodies or against a lymphocyte that is auto- reactive, i.e., which recognizes an endogenous antigen.
  • any undesirable cell may be "tagged" for destruction.
  • a polynucleotide encoding a variant of a target antigen, which is expressed in the undesirable cell is administered to the subject in need of elimination of the undesirable cells.
  • the target antigen against which a cellular immune reaction will be mounted is expressed preferentially in the undesirable cells, and/or that the undesirable cells are preferentially reached by the cytolytic cell, such as not to unnecessarily destroy non-target cells, such as normal cells.
  • Target antigens may, however, be expressed on normal tissues that are anatomically sequestered in immuno-privileged sites such as the eye, brain, testis etc., since these tissues are inaccessible to cell mediated injury, and only damaged by antibodies.
  • immuno-privileged tissues expressing the target antigen will be protected from lysis.
  • immunotherapy based on eliciting cellular responses to differentiation (tyrosinase; gplOO; TRP1; TRP2; MART-I /Melan-A; membrane-associated mucin, MUC- 1 mucin) or normal tissue-specific (PSMA, PSA) antigens constitute an example where the production of antibodies against the target is undesirable.
  • the target antigen chosen for inducing the destruction of the undesirable cells is preferably an antigen containing a least one immxmogenic peptide or T cell epitope, i.e., a peptide that can be presented to CD8+ T cells within the context of MHC class I complexes.
  • the target antigen comprises at least 2, 3, 5, 10 or more T cell epitopes, since this will allow the preparation of a variant of the target antigen to comprise more than one T cell epitope, thereby increasing the likelihood of a strong immune reaction.
  • target peptide may be preferably a peptide of which at least a portion will be targeted to the ER, and then associate with an MHC class I complex.
  • preferred target antigens include any naturally-occurring secreted or membrane protein, i.e., a protein that migrates through the ER during its synthesis.
  • preferred antigens include foreign or heterologous proteins, e.g., proteins from microorganisms.
  • the target antigen does not have to be secreted or a membrane protein, but it must merely contain at least a portion, which is capable of associating with an MHC class I complex of the target cell, and be able to be recognized within the context of MHC class I by CD8+ T cells.
  • the target antigen can thus be a eukaryotic protein, a prokaryotic protein or a viral protein.
  • a target peptide can be derived from a cytosolic protein, a secreted protein, a transmembrane protein or a nuclear protein.
  • Preferred eukaryotic proteins include tumor antigens.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2/Neu/ErbB-2.
  • Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA).
  • CEA carcinoembryonic antigen
  • the tumor-specific idiotype immxmoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor.
  • B-cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.
  • Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.
  • the antigen can be a product of a completely silent gene (e.g., MAGE or RAGE (Gaugler et al.
  • a differentiation antigen e.g., CEA and gp75 (Wang et al. (1996) J. Exp. Med. 183:1131), antigens resulting from mutations (e.g., bcr-abl in myeloid leukemias; Bosch et al. (1996) Blood 88:3522); high-density normal antigens (e.g., Her2/Neu, p53 and PRAME (Dceda et al. (1997) Immunity 6:99); or a tissue specific antigen.
  • a preferred tumor antigen is prostate specific membrane antigen (PSMA) or an alternatively spliced form thereof, which are described in U.S. patent Nos.
  • PSMA expression is an integral transmembrane protein which is restricted to prostate epithelial cells (Horoszewicz JS, Kawinski E and Murphy GP. Monoclonal antibodies to a new antigenic marker in epithelial prostate cells and seram of prostate cancer patients. Anticancer Res. 7:927;1987) and human brain tissue (Luthi Carter R, Barczak AK, Speno H, Coyle JT. Molecular characterization of human brain N acetylated alpha-linked acidic dipeptidase (NAALADase). J Pharmacol. Exp. Therap. 286:1020;1998).
  • prostate cancer can be treated, e.g., by administering to a subject having prostate cancer a polynucleotide encoding a portion of PSMA which, when expressed in cells, is not secreted, nor expressed on the extracellular side of the cellular membrane, and which is essentially biologically inactive, e.g., at least a portion of the extracellular domain.
  • dendritic cells of the patients are prepared by transfection using, e.g., a plasmid or a recombinant replication- deficient adenoviras whose DNA includes DNA encoding, e.g., a truncated fragment of the prostate specific membrane antigen.
  • Dendritic cells may be transfected in vivo by injection of the plasmid or recombinant replication-deficient adenoviras in the patient.
  • the DC may be transfected (infected) in vitro by treating isolated dendritic cell precursor cells with the plasmid or recombinant replication-deficient adenoviras. The dendritic cells are then injected into the patient.
  • the plasmid is injected intradermally, sub-cutaneously, intramuscularly, intrathecally or through the blood circulation.
  • the plasmid is first incorporated into the genome of a replication- deficient adenoviras which is injected intradermally, subcutaneously, intramuscularly, intrathecally or through the blood circulation, into a patient.
  • CD 14+ monocyte cells of a prostate cancer patient are isolated and matured into dendritic cells and transfected with either the plasmid or the adenoviras of the first two methods.
  • the DCs are then stimulated to express MHCs and are infused back into the prostate cancer patient where they stimulate autologous T-cells. These stimulated T-cells then destroy malignant and some normal prostate cells.
  • One effect of all of these treatments is to either by-pass the normal tolerance for self-antigens or the tolerance to tumor antigens. This will enable the cytolysis of target malignant prostate cells normally shielded from immune recognition.
  • the destruction of normal prostate cells in the process is not detrimental to the patient because a malignant prostate (with its mixture or normal and malignant cells) customarily is destroyed through surgery or radiation in the primary treatment for this disease.
  • normal brain cells which also express PSMA, will not be destroyed, since CTLs do not access the brain and, since no antibodies (which can access the brain) are produced according to the method of the invention.
  • prostate specific antigens include the prostatic acid phosphatase (PAP). Elevated levels of PAP in the bloodstream are considered indicative of prostate cancer, and this enzyme has been widely studied (Yam, Amer J Med (1974) 56:604. Improved methods of cancer detection using this enzyme were described by Chu et al. in PCT application WO79/00475. The structure of the enzyme has also been studied by Sharief, F. S., et al., Biochem Biophys Res Commun (1992) 184:1468-1476 and by Van Etten, R. L., et al., J Biol Chem (1991) 266:9993-9999.
  • PAP prostatic acid phosphatase
  • the nucleotide sequence encoding human PAP has been determined from a full length cDNA clone (Sharief, F. S., et al., Biochem Biophys Res Commun (1989) 180:79-86; Tailor, P. G., et al, Nucleic Acids Res (1990) 18:4928.
  • PSA prostate specific antigen
  • PSA is present in the epithelial cells comprising the prostatic ductal elements. It has been demonstrated in all primary and metastatic prostatic tumors tested and in normal benign prostate but not in non-prostatic cancer tissues or in normal tissues other than prostate.
  • the complete amino acid sequence of PSA from human seminal plasma has been determined (Watt KW et al, Proc Natl Acad Sci USA (1986) 83:3166-3170).
  • PSA consists of a single polypeptide chain with 240 amino acid residues and has a calculated molecular weight of 26,496. Carbohydrate side chains are possibly attached.
  • the cDNA encoding PSA has been produced and characterized (Lundwall A, Lilja, H, FEBS Lett (1987)
  • the target antigen is the Her-2/neu antigen, which is a member of the epidermal factor receptor family and is presumed to function as a growth receptor (Beckmann etal., 1992, Eur. J. Cancer 28:322).
  • DNA encoding a truncated form of the Her 2/neu protein lacking the transmembrane portion and the leading sequence can be constructed and included in a plasmid or viral vector(s), and administered to a subject having breast, ovary, uterine, prostate or lung cancer.
  • Another antigen that can be targeted is the melanocyte differentiation antigen
  • MART-1 which is a common melanoma antigen recognized by many CTLs from melanoma patients (Coulie et al, 1994, J. Exp. Med. 180:35; Hawakami et al., 1994, PNAS 91:3515; Bakker et al., 1994, J. Exp. Med. 179: 1005).
  • MART-1 also referred to as MART-1/Melan A
  • MART-1/Melan A is a membrane protein of 118 amino acids having a single transmembrane domain.
  • melanoma associated antigens include gplOO, tyrosinase/albino, p97 melanoma antigen, and any of the various MAGEs (melanoma associated antigen E), including MAGE 1, 2, 3, 4, etc. (Boon, T. Scientific American (March 1993):82-89; e.g., Zhai, et al., J. Immunol., 156(2):700-10 (1996); Kawakami, et al, J. Exp. Med., 180(l):347-52 (1994); and Topalian, et al, Proc. Natl. Acad. Sci. USA, 91(20):9461-5 (1994)).
  • Proteins associated with breast cancer that can be used as target antigens, include bcl-1, bcl-2, vasopressin related proteins; see, North, et al., Breast Cancer Res. Treat., 34(3):229-35 (1995); Hellemans, Br. J. Cancer, 72(2):354-60 (1995); and Hurlimann, et al., Virchows Arch., 426(2). 163-8 (1995)).
  • Antigens associated with other carcinomas include, e.g., c-myc, int-2, hst-1, ras and p53 mutants; see, Issing, et al., Anticancer Res., 13(6B):2541-51 (1993); Tjoa, et al., Prostate, 28(l):65-9 (1996); Suzich, et al., Proc. Natl. Acad. Sci. USA, 92(25): 11553-7 (1995); and Gjertsen, et al, Lancet, 346(8987): 1399-400 (1995)).
  • B cell lymphoma tumor antigens that can be used include CD19, CD20, CD37, as well as an antigen described in U.S. 6,099,846.
  • An antigen associated with renal carcinoma is RAGE (Gaugler et al. (1996) hnmunogenetics 44:323).
  • Carcinoembryonic antigen is localized to epithelia of the intestinal lumen (see, e.g., Benchimol, et al, Cell, 57:327-324, 1989).
  • the invention can be used to treat numerous types of cancers, including solid tumors as well as cancers of blood cells, lymphomas and leukemias.
  • Numerous antigens associated with any number of cancers, which can be used according to the invention, including ovarian, breast, colorectal, melanoma, pancreas, stomach, gall baldder, oesophagus, lung, gliomas, renal, thyroid, are set forth in US 6,093,399.
  • Target antigens can be from virases, e.g., virases that result in chronic infections, for example, the hepadnaviruses (including HBV), the lentiviruses (including HIV), herpesvirases (including HSV-1, HSV-2, EBV, CMV, VZV, and HHV-6), and the flaviviruses/pestivirases (including HCN). Also included, as virases that cause chronic viral infections are human retrovirases, for example, human T lymphotropic virases
  • HTLV-1 and HTLV-2 that cause T cell leukemia and myelopathies.
  • Other organisms that cause chronic infections include, for example, pathogenic protozoa, (e.g., Pneumocystis carinii, trypanosoma, malaria and Toxoplasma gondii), bacteria (e.g., mycobacteria, salmonella and listeria) and fungi (e.g. Candida and aspergillus).
  • pathogenic protozoa e.g., Pneumocystis carinii, trypanosoma, malaria and Toxoplasma gondii
  • bacteria e.g., mycobacteria, salmonella and listeria
  • fungi e.g. Candida and aspergillus.
  • the nucleotide sequences of a number of these viruses, including different species, strains, and isolates are known in the art.
  • target antigens are those which induce a T cell response, and particularly a CTL-response during infection. These may include, for example, from HB V, the core antigen, the E antigen, and the surface antigen (HBsAg). Polynucleotide sequences for HBsAg including the pre-Sl, pre-S2 and S regions from a variety of surface antigen subtypes are known in the art.
  • the present invention will find use for stimulating an immune response against a wide variety of proteins from the herpesviras family, including proteins derived from herpes simplex viras (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens derived from varicella zoster viras (VZV), Epstein-Barr viras
  • HSV herpes simplex viras
  • VZV varicella zoster viras
  • Epstein-Barr viras Epstein-Barr viras
  • EBV cytomegaloviras
  • CMV cytomegaloviras
  • antigens derived from other human herpesvirases such as HHV6 and HHV7.
  • Chee et al. Cytomegalovirases (J. K. McDougall, ed., Springer- Verlag 1990) pp. 125-169, for a review of the protein coding content of cytomegaloviras; McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a discussion of the various HSV-1 encoded proteins; U.S. Pat. No.
  • HCV hepatitis A viras
  • HBV hepatitis B viras
  • HCV hepatitis C viras
  • HDV delta hepatitis viras
  • HCV hepatitis E virus
  • HGV hepatitis G viras
  • HCV genome encodes several viral proteins, including El (also known as E) and E2 (also known as E2/NSI) and an N-terminal nucleocapsid protein (termed "core") (see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV proteins, including El and E2). Each of these proteins, as well as antigenic fragments thereof, will find use in the present methods.
  • antigens derived from HBV such as the core antigen, the surface antigen, sAg, as well as the presurface sequences, pre-Sl and pre-S2 (formerly called pre-S), as well as combinations of the above, such as sAg/pre-Sl, sAg/pre-S2, sAg/pre-Sl/pre-S2,and pre- Sl/pre-S2, will find use herein.
  • Antigens derived from other virases will also find use in the claimed methods, such as without limitation, proteins from members of the families Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae (e.g., rubella viras, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies viras, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles viras, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenza viras types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III
  • antigens from the isolates H ⁇ V[IIIb], HIV[SF2], HIV[LAV], HIV[LAI], HIV[MN]); HIV-1 [CM235], HIV-1 [US4]; HIV-2; simian immunodeficiency viras (SIV) among others.
  • antigens may also be derived from human papillomaviras (HPV) and the tick-bome encephalitis virases. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991), for a description of these and other virases.
  • the gpl20 envelope proteins from any of the above HIV isolates are known and reported (see, e.g., Myers et al, Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N.M. (1992); Myers et al, Human Retrovirases and Aids, 1990, Los Alamos, N.M.: Los Alamos National Laboratory; and Modrow et al., J. Virol. (1987) 61:570-578, for a comparison of the envelope sequences of a variety of HIN isolates) and antigens derived from any of these isolates will find use in the present methods.
  • the invention is equally applicable to other immunogenic proteins derived from any of the various HIN isolates, including any of the various envelope proteins such as gpl60 and gp41, gag antigens such as p24gag and p55gag, as well as proteins derived from the pol region.
  • Influenza viras is another example of a viras for which the present invention will be particularly useful.
  • the envelope glycoproteins HA and ⁇ A of influenza A are of particular interest for generating an immune response.
  • Numerous HA subtypes of influenza A have been identified (Kawaoka et al., Virology (1990) 179:759-767; Webster et al., "Antigenic variation among type A influenza virases," p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza virases. Springer- Verlag, New York).
  • proteins derived from any of these isolates can also be used in the immunization techniques described herein.
  • the methods described herein will also find use with numerous bacterial antigens, such as those derived from organisms that cause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis, and other pathogenic states, including, without limitation, Meningococcus A, B and C, Hemophilus influenza type B (HIB), and Helicobacter pylori.
  • bacterial antigens such as those derived from organisms that cause diphtheria, cholera, tuberculosis, tetanus, pertussis, meningitis, and other pathogenic states, including, without limitation, Meningococcus A, B and C, Hemophilus influenza type B (HIB), and Helicobacter pylori.
  • parasitic antigens include those derived from organisms causing malaria and Lyme disease.
  • Target antigens that can be used according to the method of the invention are also described in the following documents: U.S. Pat. No. 5,
  • Nos. 5,494,807, 5,756,103, 5,762,938 and 5,766,599 e.g., DNA encoding antigens from rabies, Hepatitis B, JEV, YF, Dengue, measles, pseudorabies, Epstein-Barr, HSV, HIV, SIV, EHV, BHV, HCMV, canine parvoviras, equine influenza, FeLV, FHV, Hantaan, C. tetani, avian influenza, mumps,
  • U.S. Pat. Nos 5,503,834 and 5,759,841 e.g., Morbillivirus, e.g., measles F, hemagglutinin, inter alia
  • U.S. Pat. No 4,722,848 e.g., HSV tk, HSV glycoproteins, e.g., gB, gD, influenza HA, Hepatitis B, e.g., HBsAg, inter alia
  • U.S. Pat. Nos 5,514,375, 5,744,140 and 5,744,141 e.g., flavivirus structural proteins
  • 5,766,598 e.g., Lentiviras antigens such as immunodeficiency virus antigens, inter alia
  • U.S. Pat. Nos. 5,658,572 and 5,641,490 e.g., IBDV antigens, inter alia
  • WO 94/16716 e.g., cytokine and/or tumor associated antigens, inter alia
  • U.S. Pat. Nos. 5,688,920, and 5,529,780 e.g., canine herpesvirus antigens
  • PCT publication WO 96/3941 e.g., cytomegaloviras antigens
  • U.S. Pat. Nos. 5,756,101 and 5,766,597 Pasmodium antigens.
  • a cell-mediated immune response against a target antigen on a target cell of a subject is induced by the administration to the subject of a polynucleotide encoding a variant of the target antigen.
  • a variant of the target antigen is preferably (i) a polypeptide which is homologous to, and/or a portion of, the target antigen; and (ii) which is not secreted from the cell, nor expressed on the extracellular side of cellular membrane of, the cell in which it was synthesized.
  • the variant is preferably biologically inactive and/or prevented from being secreted or exposed on the extracellular side of the cell surface.
  • a variant of the target antigen can also be fused to, or co-expressed with another polypeptide, e.g., a known immunogenic eukaryotic, prokaryotic or viral product.
  • the variant is preferably sufficiently related to the target antigen to ensure that the CD8+ cell-mediated response resulting from the administration of a polynucleotide encoding the variant into a subject, is directed to the target antigen.
  • the variant polypeptide shares a strong amino acid sequence homology with at least a portion of the target antigen.
  • the variant polypeptide has at least about 70% amino acid sequence homology of identity; more preferably at least about 80%; at least about 90%; at least about 95%; and even more preferably at least about 98 or 99% homology or identity.
  • the variant polypeptide is preferably not secreted, nor expressed on the extracellular side of the cellular membrane of the cell in which it was synthesized.
  • the variant may be a cytoplasmic or cytosolic protein. It can also be a properly modified nuclear, membrane, or secreted protein. The goal is to avoid the presence of any soluble form of the variant polypeptide, resulting from being either secreted or shed from the membrane, which would induce a humoral immune response against the target antigen. Thus, the production of antibodies against the target antigen is avoided by the use of a variant that will not appear in soluble form outside the transfected cell.
  • the variant may be a form of the target polypeptide that is devoid of a signal peptide, i.e., a peptide which targets peptides being synthesized to the endoplasmic reticulum to be directed to the membrane or secreted.
  • a signal peptide i.e., a peptide which targets peptides being synthesized to the endoplasmic reticulum to be directed to the membrane or secreted.
  • signal sequences There are also several algorithms that predict the location of sequences encoding signal peptides. Examples of such algorithms can be found at http://mbcf.dfci.harvard.edu/docs/Pedro.html. Other domains of the target protein may also be deleted to keep the expressed variant in the cytosol.
  • the transmembrane and/or cytoplasmic domain(s) of the antigen may be deleted.
  • the location of such domains for known proteins is usually known, hi addition, algorithms exist for the prediction of the localization of such domains. These can also be found at http://mbcf.dfci.harvard.edu/docs/Pedro.html.
  • the variant polypeptide is biologically inactive (but it is capable of inducing a cell-mediated immxine response), to avoid the occurrence of any negative effects that might result from the expression of the variant polypeptide in cells of a subject or in in vitro cultured cells.
  • a polynucleotide encoding a variant polypeptide to a subject does not adversely affect the subject's health, such as by having a toxic effect on cells. Since the function of many target antigens, e.g., tumor-associated or tissue specific antigens, may be unknown, their synthesis and release by cells of the subject in vivo may lead to serious side effects.
  • a variant can be biologically inactive when it comprises only a portion of the target antigen, which does not include a biologically active domain of the target antigen, such as a portion of a cytoplasmic domain which associates with other proteins or DNA.
  • a biologically active domain of the target antigen such as a portion of a cytoplasmic domain which associates with other proteins or DNA.
  • a variant of a target antigen can also be created by the introduction of one or more mutations in the target antigen or portion thereof.
  • the mutation can be an insertion, deletion or substitution of one or more amino acids.
  • Mutation in polynucleotides encoding the target antigen can be made according to methods known in the art.
  • Various methods exist for screening for biologically inactive polypeptides For example, tests of biological activity and toxicity of variant polypeptides can be done in vitro as well as in mice or other laboratory animals. The particular test used will depend on the wild-type activity of the target peptide (i.e., testing for those variants which are depleted of that particular activity) .
  • a variant of a target antigen can also be created by constructing polynucleotide sequences that encode for known immunogenic prokaryotic, eukaryotic or viral products that can be fused to, or co-expressed with the target antigen.
  • immunogenic sequences are well known, and include, e.g., portions of keyhole limpet hemocyanin. Expression of such known immunogenic sequences will bypass existing tolerance to target antigen at the CD4+ T cell level by stimulating CD4+T cells reactive to said immunogenic sequences and by providing "by-stander" help.
  • preferred variants of target antigens are at least 8 amino acids long. They may also be at least 10 amino acids long, or at least 13, 14, 15 or 16 amino acids long. Other variants may also be at least about 20, 25, 30, 35, 40, 50 or 100 amino acids long. There is generally no upper limit of the size of the variant. As discussed above, the shorter the variant, the less likely it is to have a biological activity. However, the longer the variant is, the more likely it is to have one or more antigenic epitopes.
  • the variant may also be chosen such as not to include any B cell epitope.
  • B cell epitope As described below, methods for identifying B cell epitopes in polypeptides are known in the art. Thus, preferred variants are those including one or more T cell epitopes, but no B cell epitope.
  • the target antigen is a receptor-type molecule, e.g.,
  • a variant of the target antigen may include only a portion of the extracellular domain, not including a signal peptide nor a transmembrane domain, and may optionally contain one or more mutations to ensure that the variant is biologically inactive.
  • the amino acid sequence of human PSMA is represented in Figure 1.
  • the sequence is identical to SEQ ID NO: 2 of patent No. 5,538,866 by Israeli et al. (GenBank Accession No. AAB144401) and is set forth as SEQ ID NO: 2.
  • the full length nucleotide sequence of the human PSMA cDNA encoding SEQ ED NO: 2 is set forth as SEQ TD NO: 1.
  • This sequence corresponds to the nucleotide sequence set forth as SEQ ID NO: 1 in patent No. 5,538,866 by Israeli et al. (GenBank Accession No. 123794).
  • the transmembrane domain corresponds to amino acids 20 to 43 of SEQ ID NO: 2.
  • the cytoplasmic domain corresponds to amino acids 1 to 19 of SEQ ID NO: 2.
  • the extracellular domain of PSMA corresponds to amino acids 44 to 750.
  • Preferred variants of human PSMA that can be used according to the method of the invention include amino acids 44-750 of SEQ ID NO: 2 or portions thereof that are sufficient for permitting to be presented within the context of MHC class I and optionally MHC class II.
  • Vectors and plasmids encoding the PSMA portion corresponding to amino acids 44-750 are further described in the Examples.
  • PSMA variant for use in the invention corresponds to all, or a portion, of the splice variant of human PSMA corresponding to amino acids 58 to 750 of SEQ ID NO: 2, described in U.S. patent No. 5,935,818 by Israeli et al.
  • This natural splice variant does not contain a transmembrane domain or a cytoplasmic domain, and could thus be used in the instant invention without further modification.
  • a plasmid encoding the extracelluar portion of human PSMA comprises nucleotides 391-2511 of SEQ I D NO: 1.
  • Plasmids encoding a portion of the extracellular domain of human PSMA may comprise or consist of, or consist of about, nucleotides 433-2511 of SEQ ID NO: 1 (alternative splice variant); nucleotides 424-2511 of SEQ ID NO: 1 (Form B); nucleotides 442-2511 of SEQ ID NO: 1 (Form A); nucleotides 487-2511 of SEQ TD NO: 1 (Form C); or nucleotides 541-2511 of SEQ ID NO: 1 (Form D). It will be understood that shorter fragments, in particular fragments which do not extend to nucleotide 2511 are also within the scope of the invention.
  • human PSMA variants comprise a portion of the extracellular domain, such as a portion containing at least 8, 10, 12, 15, 20, 25, 30, 40, 50, 100, 200 or 300 amino acids of SEQ ID NO: 2.
  • Variants preferably consist of a portion of the extracellular domain of human PSMA, optionally fused to one or more heterologous sequences.
  • portions of the cytoplasmic domain with or without portions of the transmembrane and/or the extracellular domain are portions of the cytoplasmic domain with or without portions of the transmembrane and/or the extracellular domain.
  • a preferred variant of the human PSA antigen is a form of the antigen in which the signal sequence is deleted. It is set forth in SEQ ID NO: 4, and is encoded by the nucleotide sequence set forth in SEQ ID NO: 3.
  • a preferred variant of the human CEA antigen is a form that is deleted of its signal sequence and is set forth in SEQ ID NO: 6 and encoded by the nucleotide sequence of SEQ ID NO: 5.
  • a preferred variant of human Her2 is set forth in SEQ ED NO: 8 and is encoded by the nucleotide sequence of SEQ ED NO: 7.
  • a truncated version of MAGE- 12 that can be used according to the invention has the amino acid sequence set forth in SEQ ED NO: 10 and is encoded by the nucleotide sequence set forth in SEQ ID NO: 9.
  • a truncated version of MAGE-6 that can be used according to the invention has the amino acid sequence set forth in SEQ ID NO: 12 and is encoded by the nucleotide sequence set forth in SEQ ED NO: 11.
  • a truncated version of MART-1 that can be used according to the invention has the amino acid sequence set forth in SEQ ID NO: 14 and is encoded by the nucleotide sequence set forth in SEQ ED NO: 13.
  • variants may be derived from polypeptides which are encoded by polymorphic alleles, or are mutated forms, of the target polypeptide which are naturally- occurring in cells other than the target cells.
  • Variants may also be derived from genes, which are allogeneic or xenogeneic relative to the target antigen. It has been shown that expression of a xenogeneic form of a protein overcomes tolerance of the protein (J. Immun. (1997) 159:3113).
  • variants may be prepared from allogeneic or xenogeneic genes, provided they are sufficiently similar to the target antigen that the ensuing cellular immune response is directed against the target antigen. Allogeneic or xenogeneic genes encoding the target antigen may be publicly available. Alternatively, the genes can be isolated by known methods, e.g., hybridization under low stringency conditions or PCR using degenerate primers.
  • the invention provides a nucleic acid encoding a fusion protein of an antigen against which an immune response is desired and a peptide which enhances immune responses (immunogenic peptides).
  • immunogenic peptides are well known in the art. These can be viral or prokaryotic, and include keyhole limpet homocyanin (KLH) and tetanos toxin.
  • the immunogenic peptide is administered as a separate protein.
  • One method that can be used to increase the likelihood of a portion of a target antigen to become associated with MHC class I genes in the APCs is to target the polypeptide to proteasomes.
  • a proteasome is composed of up to 24 protein subunits that form a cylindrical complex, whose function is the degradation of cytosolic proteins that are tagged for turnover by covalent linkage to a small protein called ubiquitin.
  • a cytosolic protein becomes “ubiquitinated,” it gains access to the proteasomal enzymatic activity, resulting in the degradation of the protein. It has been reported that at least certain protein antigens require ubiquitination before they can be presented to class I-restricted cells.
  • tagging the variant polypeptide for ubiquitination increases the likelihood of the variant to be degraded, allowing fragments thereof to gain access to the exocytic pathway where the fragments can associate with MHC class I molecules and migrate to the cell surface for presentation to T cells.
  • Polypeptides can be targeted for ubiquitination by fusing them with a domain that is recognized by ubiquitination enzymes, as described, e.g., in Winston et al. (1999) Curr. Biol. 9: 1180.
  • Hsps heat shock proteins
  • the method includes increasing the amount of Hsps in antigen presenting cells, such as professional antigen presenting cells and tumor cells, e.g., by cotransfecting antigen presenting cells with a nucleic acid encoding an Hsp.
  • antigen presenting cells such as professional antigen presenting cells and tumor cells
  • a nucleic acid encoding an Hsp e.g., by cotransfecting antigen presenting cells with a nucleic acid encoding an Hsp.
  • a nucleic acid encoding a fusion protein between an Hsp and an antigen against which an immune response is desired.
  • the cellular level of other chaperones may also be increased in a cell to enhance antigen presentation within the context of MHC class I (see, infra).
  • polynucleotides encoding more than one variant.
  • variants deriving from two or more tumor antigens can be encoded by the polynucleotide for use according to the invention.
  • variants from two or more microbial antigens may be encoded by the polynucleotide of the invention.
  • These variants may be expressed as fusion proteins.
  • the two or more variant polypeptides may be from the same pathogenic intracellular microorganism or from different ones, where the cells of the host are infected with more than one microorganism.
  • Tumor- derived T cell epitopes of human tumor types i.e., melanoma, ovarian, and breast carcinoma
  • Storkus W. J., et al., Biologic Therapy of Cancer 2 nd ed. DeVita, V. T., et al., editors. J. B. Lippincott Co., Philadelphia, Pa. 66-77 (1995).
  • the second isolation method entails acid denaturation of immunoaffinity purified class I-peptide complexes.
  • the second method of peptide isolation is highly class I selective, and even class I allele specific since monoclonal antibodies directed against individual class I allotypes can be used to immunopurify class I complexes.
  • the majority of known class I-bound peptide sequence data has been acquired. See, for example, Van Bleek, G.
  • T cell epitopes Another method for identifying T cell epitopes is disclosed in U.S.6,077,519, which describes methods for isolation and use of T cell epitopes eluted from viable cells in vaccines for treating cancer patients.
  • T cell epitope can be derived from the following method, which has been used to identify B cell epitopes. This method includes the use of synthetic peptides have been used for "epitope mapping" to identify immimodominant determinants or epitopes on the surface of proteins for the development of new vaccines and diagnostics.
  • Epitope mapping employs a series of overlapping peptides corresponding to regions on the protein of interest to identify sites which participate in antibody immxmogenic determinant interaction.
  • epitope mapping employs peptides of relatively short length to precisely detect linear determinants.
  • PEPSCAN A fast method of epitope mapping known as PEPSCAN is based on the simultaneous synthesis of hundreds of overlapping peptides, of lengths of 8 to 14 amino acids, coupled to solid supports. The coupled peptides are tested for their ability to bind antibodies.
  • the PEPSCAN approach is effective in localizing linear determinants, but not for the identification of epitopes needed for mimicry of discontinuous effector sites such as the HEV receptor/coreceptor binding site (Meloen et al., Ann Biol. Clin., 1991; 49:231-242).
  • An alternative method relies on a set of nested and overlapping peptides of multiple lengths ranging from 15 to 60 residues.
  • Cytotoxic T cell assays can be conducted by methods known in the art, which include, e.g., measurement of the amount of a labeled substance released from the cells targeted for lysis by the cytotoxic T cells.
  • Dendritic cells can also be immortalized as described, e.g., in WO 94/28113.
  • Cell lines expressing specific target antigens can be prepared by transfecting an expression vector encoding the target antigen in a cell line. Expression of the target antigen can be demonstrated by Northern blot analysis and/or Western blots using an antibody reactive against the target antigen. Numerous cell lines expressing target antigens are also available from the ATCC. For example, the ATCC deposit number of cell lines from various carcinomas, e.g., bladder, colon, breast, melanoma, and renal carcinoma, are listed in U.S. patent No. 6,093,399.
  • Polynucleotides of the invention can be isolated by hybridization under stringent or low stringency conditions.
  • a target antigen such as allogeneic or xenogeneic variants
  • a nucleic acid encoding a target antigen can be used as a probe to screen a cDNA library.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1- 6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or temperature of salt concentration may be held constant while the other variable is changed.
  • a biologically inactive form of a target antigen for use as a variant in the invention, several methods can be used. For example, an analysis of the amino acid sequence of the antigen will reveal the location of specific biologically active domains and sequences, e.g., phosphorylation sites, SH2 or SH3 sites. The analysis may be performed by comparison of the amino acid sequence of the target antigen with that of other proteins, e.g., by using BLAST. Alternatively, algorithms known in the art can be used which predict the location of biologically active domains. Examples of such algorithms can be found at http://mbcf.dfci.harvard.edu/docs/Pedro.html.
  • libraries of fragments of the target antigen can be prepared and screened for those fragments, which lack biological activity.
  • a variety of techniques are known in the art for generating such libraries, including chemical synthesis.
  • a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of target antigen coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with SI nuclease; and (v) ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of homo logs of a target antigen.
  • the most widely used techniques for screening large libraries typically comprises cloning the library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial DNA fragments under conditions in which the absence of detection of a desired activity facilitates relatively easy isolation of the vector encoding the cDNA.
  • Assays used for detecting biologically inactive fragments will usually be chosen based on the biological activity of the wild type target antigen, and will be amenable to high throughput analysis as necessary to screen large numbers of sequences of a target antigen created by combinatorial mutagenesis techniques.
  • a nucleic acid of the invention can also be linked to a stretch of CpG dinucleotides.
  • Polynucleotides of the invention are polynucleotides of the invention, expression vectors, and methods of delivery
  • the polynucleotides of the invention may be either a DNA or RNA sequence.
  • the polynucleotide encoding a variant of a target antigen is preferably operably linked to all necessary transcriptional and translational regulatory elements, such as a promoter, enhancer and polyadenylation sequence. Regulatory sequences are art-recognized and are described, e.g., in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the promoter is a constitutive promoter, e.g., a strong viral promoter, e.g., CMV promoter.
  • the promoter can also be cell- or tissue-specific, that permits substantial transcription of the DNA only in predetermined cells, e.g., in professional antigen presenting cells, such as the dendritic cell-specific CD1 lc promoter described in Brocker T, J. Leuk. Biology 66:331 -335, 1999.
  • the promoter can also be an inducible promoter, e.g., a metallothionein promoter.
  • Other inducible promoters include those that are controlled by the inducible binding, or activation, of a transcription factor, e.g., as described in U.S. patent Nos.
  • Other inducible transcription systems involve steroid or other hormone-based regulation.
  • the polynucleotide of the invention together with all necessary transcriptional and translational control sequences is referred to herein as "constract of the invention” or "transgene of the invention.”
  • the polynucleotide of the invention may also be introduced into the cell in which it is to be expressed together with another DNA sequence (which may be on the same or a different DNA molecule as the polynucleotide of the invention) coding for another agent. Exemplary agents are further described below.
  • the DNA encodes a polymerase for transcribing the DNA, and may comprise recognition sites for the polymerase and the injectable preparation may include an initial quantity of the polymerase.
  • it may be preferred that the polynucleotide is translated for a limited period of time so that the polypeptide delivery is transitory. This can be achieved, e.g., by the use of an inducible promoter.
  • the polynucleotides used in the present invention may also be produced in part or in total by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Carrathers, Tetra. Letts., 22:1859-1862 (1981) or the triester method according to the method described by Matteucci et al., J. Am. Chem. Soc, 103:3185 (1981), and may be performed on commercial automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • the polynucleotide of the invention operably linked to all necessary transcriptional and translational regulation elements can be injected as naked DNA into a subject or contacted in vitro with professional antigen presenting cells (further described below).
  • the polynucleotide of the invention and necessary regulatory elements are present in a plasmid or vector.
  • the polynucleotide of the invention may be DNA, which is itself non-replicating, but is inserted into a plasmid, which may further comprise a replicator.
  • the DNA may be a sequence engineered so as not to integrate into the host cell genome.
  • Preferred vectors for use according to the invention are expression vectors, i.e., vectors that allow expression of a nucleic acid in a cell vectors.
  • Preferred expression vectors are those which contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, ⁇ SV2gpt, pSV2neo, pSV2- dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drag resistance selection in both prokaryotic and eukaryotic cells.
  • viruses such as the bovine papillomaviras (BPV-1), of Epstein- Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papillomaviras
  • pHEBo Epstein- Barr virus
  • pHEBo Epstein- Barr virus
  • pREP-derived and p205 Epstein- Barr virus
  • any means for the introduction of polynucleotides into mammals, human or non- human, may be adapted to the practice of this invention for the delivery of the various constructs of the invention into the intended recipient, hi one embodiment of the invention, the DNA constracts are delivered to cells by transfection, i.e., by delivery of "naked" DNA or in a complex with a colloidal dispersion system.
  • a colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a lipid-complexed or liposome-formulated DNA.
  • a plasmid containing a transgene bearing the desired DNA constracts may first be experimentally optimized for expression (e.g., inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al., Ann NY Acad Sci 126-139, 1995).
  • Formulation of DNA, e.g. with various lipid or liposome materials may then be effected using known methods and materials and delivered to the recipient mammal.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active.
  • Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject (see below).
  • the DNA constructs are delivered using viral vectors.
  • the transgene may be incorporated into any of a variety of viral vectors useful in gene therapy, such as recombinant retrovirases, adenoviras, adeno-associated viras (AAV), and herpes simplex viras- 1, or recombinant bacterial or eukaryotic plasmids. While various viral vectors may be used in the practice of this invention, AAV- and adenoviras-based approaches are of particular interest. Such vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. The following additional guidance on the choice and use of viral vectors may be helpful to the practitioner. As described in greater detail below, such embodiments of the subject expression constracts are specifically contemplated for use in various in vivo and ex vivo gene therapy protocols.
  • a viral gene delivery system useful in the present invention utilizes adeno virus- derived vectors.
  • Knowledge of the genetic organization of adenoviras, a 36 kB, linear and double-stranded DNA virus, allows substitution of a large piece of adenoviral DNA with foreign sequences up to 8 kB.
  • retroviras the infection of adenoviral DNA into host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal mamier without potential genotoxicity.
  • adenovirases are structurally stable, and no genome rearrangement has been detected after extensive amplification.
  • Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in the human.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and high infectivity.
  • Both ends of the viral genome contain 100-200 base pair (bp) inverted terminal repeats (ITR), which are cis elements necessary for viral DNA replication and packaging.
  • ITR inverted terminal repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (EIA and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • adenoviras The genome of an adenoviras can be manipulated such that it encodes a gene product of interest, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al, (1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell 68:143-155).
  • Suitable adenoviral vectors derived from the adenoviras strain Ad type 5 dl324 or other strains of adenoviras are well known to those skilled in the art.
  • Recombinant adenovirases can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al., (1992) cited supra), endothelial cells (Lemarchand et al., (1992) PNAS USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993) PNAS USA 90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA 89:2581-2584).
  • Adenoviras vectors have also been used in vaccine development (Grunhaus and
  • the viras particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity.
  • adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of virases can be obtained in high titers, e.g., 10 9 - 10 11 plaque-forming unit (PFU)/ml, and they are highly infective.
  • PFU plaque-forming unit
  • the life cycle of adenoviras does not require integration into the host cell genome.
  • the foreign genes delivered by adenoviras vectors are episomal, and therefore, have low genotoxicity to host cells.
  • adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkner et al., supra; and Graham et al., in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127).
  • Expression of the inserted polynucleotide of the invention can be under control of, for example, the EIA promoter, the major late promoter (MLP) and associated leader sequences, the viral E3 promoter, or exogenously added promoter sequences.
  • MLP major late promoter
  • the nature of the adenoviras vector is not believed to be crucial to the successful practice of the invention.
  • the adenoviras may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenoviras type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenoviras vector for use in the method of the present invention.
  • Adenoviras type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenoviras as a vector.
  • the typical vector according to the present invention is replication defective and will not have an adenovirus El region.
  • the position of insertion of the polynucleotide or constract on the invention also referred to as "nucleic acid of interest" in a region within the adenoviras sequences is not critical to the present invention.
  • E3 replacement vectors as described previously by Karlsson et. al. (1986) or in the E4 region where a helper cell line or helper viras complements the E4 defect.
  • helper cell line is 293 (ATCC Accession No. CRL1573).
  • This helper cell line also termed a "packaging cell line” was developed by Frank Graham (Graham et al. (1987) J. Gen. Virol. 36:59-72 and Graham (1977) J.General Virology 68:937-940) and provides EIA and E1B in trans.
  • helper cell lines may also be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenoviras. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • Adenovirases can also be cell type specific, i.e., infect only restricted types of cells and/or express a transgene only in restricted types of cells.
  • the virases comprise a gene under the transcriptional control of a transcription initiation region specifically regulated by target host cells, as described e.g., in U.S. Patent No. 5,698,443, by Henderson and Schuur, issued December 16, 1997.
  • replication competent adenovirases can be restricted to certain cells by, e.g., inserting a cell specific response element to regulate a synthesis of a protein necessary for replication, e.g., EIA or E1B.
  • DNA sequences of a number of adenoviras types are available from Genbank.
  • human adenovirus type 5 has GenBank Accession No.M73260.
  • the adenoviras DNA sequences may be obtained from any of the 42 human adenovirus types currently identified.
  • Various adenoviras strains are available from the American Type Culture Collection, Rockville, Maryland, or by request from a number of commercial and academic sources.
  • a transgene as described herein may be incorporated into any adenoviral vector and delivery protocol, by restriction digest, linker ligation or filling in of ends, and ligation.
  • Adenoviras producer cell lines can include one or more of the adenoviral genes El,
  • E2a, and E4 DNA sequence for packaging adenoviras vectors in which one or more of these genes have been mutated or deleted are described, e.g., in PCT/US95/15947 (WO 96/18418) by Kadan et al.; PCT/US95/07341 (WO 95/346671) by Kovesdi et al; PCT/FR94/00624 (WO94/28152) by Imler et al.;PCT/FR94/00851 (WO 95/02697) by Perrocaudet et al., PCT/US95/14793 (WO96/14061) by Wang et al.
  • Adeno-associated viras is a naturally occurring defective viras that requires another viras, such as an adenoviras or a herpes viras, as a helper viras for efficient replication and a productive life cycle.
  • AAV adeno-associated viras
  • AAV has not been associated with the cause of any disease.
  • AAV is not a transforming or oncogenic virus.
  • AAV integration into chromosomes of human cell lines does not cause any significant alteration in the growth properties or morphological characteristics of the cells. These properties of AAV also recommend it as a potentially useful human gene therapy vector.
  • AAV is also one of the few virases that may integrate its DNA into non-dividing cells, e.g., pulmonary epithelial cells, and exhibits a high frequency of stable integration (see for example Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al, (1989) J. Virol. 63:3822-3828; and McLaughlin et al., (1989) J. Virol. 62:1963-1973).
  • Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
  • An AAV vector such as that described in Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., (1984) PNAS USA 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993) J. Biol. Chem. 268:3781-3790).
  • the AAV-based expression vector to be used typically includes the 145 nucleotide
  • AAV inverted terminal repeats flanking a restriction site that can be used for subcloning of the transgene, either directly using the restriction site available, or by excision of the transgene with restriction enzymes followed by blunting of the ends, ligation of appropriate DNA linkers, restriction digestion, and ligation into the site between the ITRs.
  • the capacity of AAV vectors is about 4.4 kb.
  • the following proteins have been expressed using various AAV-based vectors, and a variety of promoter/enhancers: neomycin phosphotransferase, chloramphenicol acetyl transferase, Fanconi's anemia gene, cystic fibrosis transmembrane conductance regulator, and granulocyte macrophage colony- stimulating factor (Kotin, R.M., Human Gene Therapy 5:793-801, 1994, Table I).
  • a transgene incorporating the various DNA constructs of this invention can similarly be included in an AAV-based vector.
  • an AAV promoter can be used (ITR itself or AAV p5 (Flotte, et al. J. Biol.Chem. 268:3781-3790, 1993)).
  • Such a vector can be packaged into AAV virions by reported methods.
  • a human cell line such as 293 can be co-transfected with the AAV-based expression vector and another plasmid containing open reading frames encoding AAV rep and cap (which are obligatory for replication and packaging of the recombinant viral constract) under the control of endogenous AAV promoters or a heterologous promoter.
  • the rep proteins Rep68 and Rep78 prevent accumulation of the replicative form, but upon superinfection with adenoviras or herpes viras, these proteins permit replication from the ITRs (present only in the constract containing the transgene) and expression of the viral capsid proteins.
  • Methods to improve the titer of AAV can also be used to express the polynucleotide of the invention in an AAV virion.
  • Such strategies include, but are not limited to: stable expression of the ITR-flanked transgene in a cell line followed by transfection with a second plasmid to direct viral packaging; use of a cell line that expresses AAV proteins inducibly, such as temperature-sensitive inducible expression or pharmacologically inducible expression.
  • a cell can be transformed with a first AAV vector including a 5' ITR, a 3' ITR flanking a heterologous gene, and a second AAV vector which includes an inducible origin of replication, e.g., SV40 origin of replication, which is capable of being induced by an agent, such as the SV40 T antigen and which includes DNA sequences encoding the AAV rep and cap proteins.
  • an agent such as the SV40 T antigen and which includes DNA sequences encoding the AAV rep and cap proteins.
  • the second AAV vector may replicate to a high copy number, and thereby increased numbers of infectious AAV particles maybe generated (see, e.g, U.S. Patent No. 5,693,531 by Chiorini et al., issued December 2, 1997.
  • a chimeric plasmid which incorporate the Epstein Barr Nuclear Antigen (EBNA) gene , the latent origin of replication of Epstein Barr viras (oriP) and an AAV genome.
  • EBNA Epstein Barr Nuclear Antigen
  • oriP Epstein Barr viras
  • AAV genome an AAV genome.
  • an AAV packaging plasmid that allows expression of the rep gene, wherein the p5 promoter, which normally controls rep expression, is replaced with a heterologous promoter (U.S. Patent 5,658,776, by Flotte et al., issued Aug. 19, 1997). Additionally, one may increase the efficiency of AAV transduction by treating the cells with an agent that facilitates the conversion of the single stranded form to the double stranded form, as described in Wilson et al., WO96/39530. AAV stocks can be produced as described in Hermonat and Muzyczka (1984)
  • PNAS 81:6466 modified by using the pAAV/Ad described by Samulski et al. (1989) J. Virol. 63:3822.
  • Concentration and purification of the viras can be achieved by reported methods such as banding in cesium chloride gradients, as was used for the initial report of AAV vector expression in vivo (Flotte, et al. J.Biol. Chem. 268:3781-3790, 1993) or chromatographic purification, as described in O'Riordan et al., WO97/08298.
  • Methods for in vitro packaging AAV vectors are also available and have the advantage that there is no size limitation of the DNA packaged into the particles (see, U.S. Patent No. 5,688,676, by Zhou et al, issued Nov. 18, 1997). This procedure involves the preparation of cell free packaging extracts.
  • AAV technology which may be useful in the practice of the subject invention, including methods and materials for the incorporation of a transgene, the propagation and purification of the recombinant AAV vector containing the transgene, and its use in transfecting cells and mammals, see e.g. Carter et al, US Patent No. 4,797,368 (10 Jan 1989); Muzyczka et al, US Patent No. 5,139,941 (18 Aug 1992); Lebkowski et al, US Patent No. 5,173,414 (22 Dec 1992); Srivastava, US Patent No. 5,252,479 (12 Oct 1993); Lebkowski et al, US Patent No. 5,354,678 (11 Oct 1994); Shenk et al, US Patent No.
  • Hybrid Adenoviras- AAV vectors represented by an adenoviras capsid containing a nucleic acid comprising a portion of an adenoviras, and 5' and 3' ITR sequences from an AAV which flank a selected transgene under the control of a promoter. See e.g. Wilson et al, hitemational Patent Application Publication No. WO 96/13598.
  • This hybrid vector is characterized by high titer transgene delivery to a host cell and the ability to stably integrate the transgene into the host cell chromosome in the presence of the rep gene.
  • adenovirus nucleic acid sequences employed in the this vector can range from a minimum sequence amount, which requires the use of a helper viras to produce the hybrid virus particle, to only selected deletions of adenoviras genes, which deleted gene products can be supplied in the hybrid viral process by a packaging cell.
  • a hybrid virus can comprise the 5' and 3' inverted terminal repeat (ITR) sequences of an adenoviras (which function as origins of replication).
  • the left terminal sequence (5') sequence of the Ad5 genome that can be used spans bp 1 to about 360 of the conventional adenoviras genome (also referred to as map units 0-1) and includes the 5' ITR and the packaging/enhancer domain.
  • the 3' adenoviras sequences of the hybrid viras include the right terminal 3' ITR sequence which is about 580 nucleotides (about bp 35,353- end of the adenoviras, referred to as about map units 98.4-100.
  • the AAV sequences useful in the hybrid vector are viral sequences from which the rep and cap polypeptide encoding sequences are deleted and are usually the cis acting 5' and 3' ITR sequences.
  • the AAV ITR sequences are flanked by the selected adenoviras sequences and the AAV ITR sequences themselves flank a selected transgene.
  • the preparation of the hybrid vector is further described in detail in published PCT application entitled "Hybrid Adenoviras-AAV Viras and Method of Use Thereof, WO 96/13598 by Wilson et al.
  • adenoviras and hybrid adenoviras- AAV technology which may be useful in the practice of the subject invention, including methods and materials for the incorporation of a transgene, the propagation and purification of recombinant virus containing the transgene, and its use in transfecting cells and mammals, see also Wilson et al, WO 94/28938, WO 96/13597 and WO 96/26285, and references cited therein.
  • the retrovirases are a group of single-stranded RNA virases characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin (1990) Retro viridae and their Replication" In Fields, Knipe ed. Virology. New York: Raven Press).
  • the resulting DNA then stably integrates into cellular chromosomes as a proviras and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsial proteins, polymerase enzyme, and envelope components, respectively.
  • LTR long terminal repeat
  • a nucleic acid of interest is inserted into the viral genome in the place of certain viral sequences to produce a viras that is replication- defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and psi components is constructed (Mann et al. (1983) Cell 33: 153).
  • a recombinant plasmid containing a human cDNA, together with the retroviral LTR and psi sequences is introduced into this cell line (by calcium phosphate precipitation for example), the psi sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein (1988) "Retroviral Vectors", In: Rodriguez and Denhardt ed. Vectors: A Survey of Molecular Cloning Vectors and their Uses.
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al. (1975) Virology 67:242).
  • retrovirases A major prerequisite for the use of retrovirases is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type viras in the cell population.
  • packetaging cells specialized cell lines which produce only replication-defective retrovirases has increased the utility of retrovirases for gene therapy, and defective retrovirases are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271).
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a protein of the present invention, e.g., a transcriptional activator, rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper viras by standard techniques. Protocols for producing recombinant retrovirases and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M.
  • retroviral vector is a pSR MSVtkNeo (Muller et al. (1991) Mol. Cell Biol. 11 : 1785 and pSR MSV(XbaI) (Sawyers et al. (1995) J. Exp. Med. 181:307) and derivatives thereof.
  • the unique BamHI sites in both of these vectors can be removed by digesting the vectors with BamHI, filling in with Klenow and religating to produce pSMTN2 and pSMTX2, respectively, as described in PCT/US96/09948 by Clackson et al.
  • suitable packaging viras lines for preparing both ecotropic and amphotropic retroviral systems include Crip, Cre, 2 and Am.
  • Retrovirases have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis et al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988) PNAS USA 85:6460-6464; Wilson et al., (1988) PNAS USA 85:3014-3018; Armentano et al, (1990) PNAS USA 87:6141-6145; Huber et al., (1991) PNAS USA 88:8039-8043; Ferry et al, (1991) PNAS USA 88:8377-8381; Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al, (1992) PNAS USA 89:7640-7644; Kay et al
  • Herpes virus e.g., Herpes Simplex Virus (U.S. Patent No. 5,631,236 by Woo et al., issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia viras (Ridgeway (1988) Ridgeway, "Mammalian expression vectors," In: Rodriguez R L, Denhardt D T, ed.
  • Vectors A survey of molecular cloning vectors and their uses.
  • viruses include an alphaviras, a poxiviras, an arena virus, a vaccinia viras, a polio virus, and the like.
  • Chang et al. recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al. (1991) Hepatology, 14.T24A).
  • CAT chloramphenicol acetyltransferase
  • the polynucleotide is administered to the subject in need thereof.
  • the polynucleotide can be introduced into muscle tissue, tissues of skin, brain, lung, liver, spleen or blood.
  • the preparation can be injected into the vertebrate by a variety of routes, which may be intradermally, subdermally, intramuscularly, intrathecally, or intravenously, or it may be placed within cavities of the body, hi a preferred embodiment, the polynucleotide is injected intramuscularly.
  • the preparation comprising the polynucleotide is impressed into the skin. Transdermal administration is also contemplated, as is inhalation.
  • the polynucleotide is injected into a tissue of the subject that is rich in professional antigen presenting cells, e.g., dendritic cells.
  • the polynucleotide is preferably administered to the skin or mucosa, where approximately 1% to 2% of the cell population is comprised of antigen presenting cells.
  • a preferred route of administration is intradermally.
  • MHC-peptide- or HSP- peptide- complexes Due to the existence of cross-priming, i.e., the transfer of MHC -peptide- or HSP- peptide- complexes form one cell to another, it is not necessary that the nucleic acid be transfected only into professional antigen presenting cells. It is believed that the transfer of MHC-or HSP-peptides complexes occurs by small vesicles (icosomcs) derived from cell membranes that are shed from cells, which then fuse with other cells, e.g., professional antigen presenting cells. Accordingly, a nucleic acid that is taken up and expressed in a cell other than a professional antigen presenting cell, and which is, e.g., a myocyte, may be expressed and presented within MHC class I on the cell surface. By cross-priming, this peptide can be taken up by MHC class I or class II complexes on professional antigen presenting cells and thereby activate T cells.
  • cross-priming
  • a regimen of immunization of a subject can include administration of a single type of vector (viral vector or plasmid vector) or of different types of vectors.
  • the regimen may comprise administration of a single dose of vector. However, it is preferable that the administration comprises several administrations, e.g., every other day, every third day, every fifth day, once a week, one a month, one every two or three months or once every 6 months or every year.
  • a nucleic acid of the invention is administered to a subject once per three weeks for nine weeks, and then once every three months.
  • the regimen may be interrupted depending on the state of the clinical state and the response of the subject, or it may be carried on for several years.
  • the regimen includes first administering to a patient one or more doses of a plasmid DNA, e.g., XC PSMA, and then administering to the patient one or more doses of a viral vector DNA, e.g., Ad5-XC PSMA.
  • a plasmid DNA e.g., XC PSMA
  • a viral vector DNA e.g., Ad5-XC PSMA
  • a subject to be treated receives two doses of a plasmid vector and one dose of a viral vector at 3-week intervals, followed by reimmunizations at three months intervals alternating between a plasmid or a viral vector.
  • Other regimens are set forth in the Examples.
  • the interval between the first administrations may be one week.
  • the viral and/or the plasmid vector can be administered every few months, e.g., every three months, such as to boost the immune response.
  • the later DNA is preferably coadministered together with the DNA encoding the variant.
  • the coding sequence can be on the same or on different DNA molecules.
  • this factor can be administered simultaneously or consecutively to a patient.
  • the nucleic acids of the invention can be administered to a subject in conjunction with another type of treatment.
  • the method of the invention is used in conjunction with surgery.
  • a subject may receive a nucleic acid of the invention after surgery that removed a at least a portion, and preferably most of, the cancer cells.
  • the DNA or viral particles of the invention are transferred to a biologically compatible solution or pharmaceutically acceptable delivery vehicle, such as sterile saline, or other aqueous or non-aqueous isotonic sterile injection solutions or suspensions, numerous examples of which are well known in the art, including Ringer's, phosphate buffered saline, or other similar vehicles.
  • transgene as naked DNA; as lipid-, liposome-, or otherwise formulated DNA; or as a recombinant viral vector can then be carried out in vivo or in vitro (e.g., ex vivo). This can be accomplished by various means, including nebulization/inhalation or by instillation via bronchoscopy. Recently, recombinant adenoviras encoding CFTR was administered via aerosol to human subjects in a phase I clinical trial. Vector DNA and CFTR expression were clearly detected in the nose and airway of these patients with no acute toxic effects (Bellonet al., Human Gene Therapy, 8(l):15-25, 1997).
  • the DNA or recombinant viras is administered in sufficient amounts to transfect cells within the desired tissue of the recipient, and provide sufficient levels of transgene expression to provide for expression of the polynucleotide of the invention in the desired cells, preferably at a level providing therapeutic benefit without undue adverse effects.
  • Optimal dosages of DNA or virus depends on a variety of factors, as discussed previously, and may thus vary somewhat from patient to patient. Again, therapeutically effective doses of viruses are considered to be in the range of about 20 to about 50 ml of saline solution containing concentrations of from about 1 X 10 7 to about 1 X 10 10 pfu of virus/ml, e.g. from 1 X 10 8 to 1 X 10 9 pfu of virus/ml.
  • Toxicity and therapeutic efficacy of ligands and constructs or viral particles can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 5 o (the dose lethal to 50% of the population) and the ED 5 o (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED o.
  • Reagents which exhibit large therapeutic indices are preferred. While reagents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such reagents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such reagents lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the reagents of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA.
  • systemic administration injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the reagents of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution, h addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active ingredient.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the reagents for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator
  • the reagents may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the reagents may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the reagents may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the reagents may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.
  • Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration ' may be through nasal sprays or using suppositories.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the gene delivery systems for the polynucleotid or constraction of the invention can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994) PNAS 91: 3054-3057).
  • the pharmaceutical preparation of the gene therapy constract can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the polynucleotide of the invention is first administered to a professional antigen presenting cell (professional APC) or precursor thereof, which is then administered to the subject.
  • the professional APC can be from the same subject or from another subject, or different source.
  • professional APCs such as dendritic cells
  • Dendritic cells and precursors thereof can be obtained from peripheral blood mononuclear cells (PBMCs) as described in the Examples, or they can be obtained and stored as described, e.g., in WO93/20185 by Steinman et al. or in U.S. patent No. 5,788,963.
  • PBMCs peripheral blood mononuclear cells
  • professional APCs e.g., obtained from a subject
  • a polynucleotide of the invention are contacted with a polynucleotide of the invention, and then contacted ex vivo with a population of cells comprising CD8+ T cells under conditions allowing activation of CD8+ T cells.
  • the state of activation of the CD8+ T cells can be monitored by, e.g., performing cytotoxicity tests.
  • the cells are then administered to the subject in need thereof.
  • the whole population of cells can be administered, or purified portions thereof, e.g., the CD8+ T cells.
  • the activated CD8+T cells can be contacted ex vivo with a population of cells comprising target cells, such as an organ or bone marrow to be transplanted into a subject.
  • the cell population is then administered back to the subject after destruction of the target cells.
  • entry of peptides into the exocytic pathway that delivers the peptide- MHC complex to the cell surface is usually required for presentation of peptides in association with MHC class I complexes
  • non-cellular-delivery vehicles for proteins such as pH-sensitive liposomes, can over-come the requirement for endogenous synthesis in vivo (Nair, et al., J Exp Med, (1992), 175, 609-12, Nair, et al., J Virol, (1993), 67, 4062- 9).
  • the invention also provides methods in which variant polypeptides are administered to a subject in a form that facilitates their uptake by cells, e.g., liposomes.
  • small variant peptides (8-11-mer) are administered to a subject.
  • These synthetic peptides associate with class I molecules on the cell surface without the requirement for endogenous processing.
  • an appropriate APC such as a dendritic cell
  • they can then induce a primary CTL response (Stauss, et al., Proc Natl Acad Sci U S A, (1992), 89, 7871-5, Carbone, et al., J Exp Med, (1988), 167, 1767-79).
  • the polynucleotide encoding the polypeptide can be administered together with another agent.
  • the agent is a polynucleotide, it can be present on the same or on a separate DNA molecule from that encoding the polypeptide of the invention.
  • the method of the invention further comprises co-admistration of one or more cytokines, in particular an immune-enhancing cytokine, such as GM-CSF, IL-2, IL-4, IL-12, IL-15, IL-18 or polynucleotide encoding such.
  • an antigen must be presented to naive T cells by professional APCs.
  • the most efficient APCs in vivo are the dendritic cells. These cells are present in tissues and, when alerted by "danger signals," they mature, accumulate antigen and migrate to the regional lymph nodes. Such "danger signals" can be coadminisfrated with a polynucleotide of the invention.
  • a preferred "danger signal” is granulocyte-macrophage colony stimulating factor (GM-CSF), which has been shown to promote differentiation, proliferation, survival and migration of dendritic cells (Jones, et al., Eur J Clin Microbiol Infect Dis, (1994), 13, S47-53).
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • GM-CSF The benefit of administrating GM-CSF is also shown in the examples. Administration of recombinant human GM-CSF is well tolerated as described herein and in Armitage (1998) Blood 92:4491. Preferred doses of GM-CSF per administration include from about 10,000 LU. to about 100,000 I.U., more preferably from about 10,000 to about 40,000 LU.
  • the polynucleotide is administered together with a polynucleotide encoding a costimulatory molecule, e.g., B7 (B7-1, B7-2, and/or B7-3).
  • B7 a polynucleotide encoding a costimulatory molecule
  • Effective stimulation of resting na ⁇ ve T and memory T cells by an antigen presenting cell requires at least two separate signal, the first one of which is the antigen and the second that of a costimulatory molecule. The absence of the second signal results in T cell paralysis or death.
  • Human B7-1 is also referred to as CD80 and human B7-2 as CD86. Both of these molecules have been cloned.
  • co-administration of a gene encoding a B7 molecule is beneficial in at least certain circumstances.
  • Methods for administering polynucleotides encoding one or more B7 molecule and an antigen are also described, e.g., in U.S.
  • polynucleotides encoding MHC class I gene(s) are co- administered to the subject, such as to increase the number of MHC class I receptors on the surface of the target cell to increase the number of target antigens presented on the target cell.
  • Progression of disease has been associated with loss or decrease of class I expression by cancer cells.
  • a complete loss of HLA class I has been reported to occur in 34% of primary prostate cancers and up to 80% of lymph node metastases (Blades et al. Urology 1995,46681-686). When individual allelic expression was assessed, the minimum estimate of down regulation was up to 85% in the primary prostate cancers and almost 100% in the metastases (Blades et al., supra).
  • the sequence encoding the derivative of the target antigen and the sequence encoding the MHC class I gene(s) are not on the same nucleic acid, since these sequences are to be expressed in different cells: the sequence encoding the derivative of the target antigen is to be introduced into professional APCs, whereas the sequence encoding MHC class I genes is to be introduced into the undesirable cell.
  • Genes encoding beta2-microglobulin can also be co-administered, either together with MHC class I genes, or on their own.
  • the density of MHC class I complexes on the surface of target cells can also be increased by contacting the target cells, e.g., by administering to the subject, agents which stimulate MHC class I complex formation or MHC class I and/or beta2-microglobulin expression in target cells.
  • agents which stimulate MHC class I complex formation or MHC class I and/or beta2-microglobulin expression in target cells include IFN-gamma.
  • Additional proteins, or nucleic acids encoding such, that can be co-administered with a nucleic acid of the invention include those which are involved in the formation of peptide-MHC class I complexes and its trafficking to the cell surface, e.g., molecular chaperones. Co-administration of such molecules would enhance presentation of antigen by antigen presenting cells, e.g., dendritic cells.
  • Exemplary proteins include proteins that are associated with or are part of TAP-Class I complexes.
  • TAPs Transport Associated Proteins
  • TAP-1 and TAP2 Transport Associated Proteins
  • calnexin calreticulin tapasin
  • Erp57 protein see, e.g., Sadasvan et al. (1996) Immunity 5:103; Morrice and Powis (1998) Curr. Biol. 8: 713; Suh et al. (1996) J. Exp. Med. 184:337; Lindquist et al. (1998) EMBO J. 17:2186; and Hughes and Cresswell (1998) Curr. Biol. 8: 709).
  • Calnexin and calreticulin are both lectins that are expressed predominantly in the ER.
  • Erp57 is a chaperone with thiolreductase activity.
  • the level of expression of the endogenous proteins can also be increased.
  • the expression level of tapansin can be increased by interferon-gamma.
  • the nucleic acid of the invention is co-administered with one or more heat shock proteins (Hsps) or nucleic acids encoding such.
  • Hsps have been shown to participate in anti-tumor T cell responses, at least in part by increasing the number of peptide/MHC Class I complexes on the surface of tumor cells. This was shown, e.g., by stably transfecting clones of B16 melanoma cells, which are not effectively recognized by MHC class I-restricted CTL due to very low levels of MHC class I antigens on the surface, with DNA encoding human heat shock protein 72 (Hsp 72), such that they constitutively express Hsp 72.
  • Hsp 72 human heat shock protein 72
  • these data demonstrate that the immxme recognition of tumor cells can be substantially enhanced by the suitable expression of a molecular chaperone.
  • Hsp70 acts as a chaperone in cross-priming, i.e., in transferring an MHC class I associated peptide from one cell to another.
  • co- administration of Hsp70 or other chaperone capable of acting in this manner, or a nucleic acid encoding Hsp70 or other chaperone would enhance cross-priming, thereby enhancing delivery of the antigen of the invention to the right target cells, i.e., professional antigen presenting cells.
  • Cross-priming is in fact believed to be a principal mechanism by which plasmid DNA delivered to cells such as myocytes effectively shuttle Ag to professional antigen presenting cells to achieve CTL induction in vivo.
  • the polynucleotide of the invention may also be co-administered with a polynucleotide encoding a homing receptor, such that the APCs at the point of entry will serve as vehicles to deliver the polynucelotide to lymphatic organs and to mucosal tissues other than those at the point of entry.
  • Other agents that can be co-administered include adhesion molecules, members of the tumor necrosis factors superfamily, e.g., TNF-alpha, TNF-beta, and Fas-ligand.
  • Yet other agents for co-administration include CD40 ligand, CD54, CD50, and CD58. The amino acid sequence of these proteins is known in the art, as well as nucleotide sequences encoding these.
  • Another agent that can be co-administered to a subject or to cells in vitro include polynucleotides encoding a target antigen.
  • cells, which do not express a target cell can be modified to become target cells by introducing in these cells a polynucleotide encoding a target antigen.
  • a target antigen can further be modified to increase the number of target antigens that it expresses, or to express one or more other target antigens.
  • a target cell can be contacted with a polynucleotide encoding a target antigen, such that the polynucleotide enters the cell and is expressed in the cell (e.g., transfected).
  • a polynucleotide could be co-administered to a subject together with the polynucleotide encoding the variant of the target antigen.
  • the target cells can also be contacted with an agent, which induces the expression of the target antigen, or otherwise increases the number of target antigens produced.
  • Example 1 Preparation of a plasmid mammalian expression vector encoding the extracellular domain of PSMA
  • cDNA of PSMA extracellular fragment (2118 bp) was obtained using total mRNA from the prostate tumor cell line LNCaP.FGC - CPvL 1740 (ATCC).
  • a PSMA-specific 3'- primer was used for reverse transcription of mRNA, which was performed using RT from avian myeloblastosis viras (Boehringer).
  • the resulting cDNA was then amplified using High Fidelity PCR System (Boehringer) and the gel purified PCR product of expected length was cloned into pCR2.1 vector (Invitrogen). Two clones were selected and checked by DNA sequencing.
  • the resulting constract contains the extracellular portion of PSMA, i.e., amino acids 44-750 of SEQ TD NO: 1 (without any mutation) with the Notl- Kozak sequence at its 5' end and Sful site at its 3' end, which were introduced during the PCR amplification.
  • the above-described DNA fragment was then introduced into the mammalian expression vector pcDNA3.1 (Invitrogen) using the Notl-Sful cloning sites.
  • the vector provides human cytomegaloviras (CMV) immediate-early promoter/enhancer region permitting efficient, high-level expression of recombinant protein as well as 3' flanking region containing bovine growth hormone polyadenylation signal for efficient transcription termination and increasing the half life of the mRNA in vivo.
  • CMV human cytomegaloviras
  • NRG neomycin resistance gene
  • XC-PSMA The resulting pcDNA3.1 expression vector containing a sequence encoding the extracellular domain of human PSMA (referred to as "XC-PSMA") was deposited as Designation Number 203168 on August 28, 1998 at the American Type Culture Collection, 10801 University Boulevard., Manassas, Virginia 20110.
  • PSMA-CD86 plasmid was constructed as follows. Th full length human CD86 cDNA was amplified from human monocytes of a helathy donor and cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). The cDNA was then sub-cloned into a modified pcDNA 3.1 (invitrogen, carlsbad, CA), in which the neomycin resistance gene was deleted.
  • XC PSMA- CD86 A portion of this plasmid containing the sequence encoding human CD86, human cytomegaloviras (CMB) immediate early promoter/enhancer located immediately upstream of the CD86 sequence, and bovine growth hormone (BGH) polyadenylation signal located immediately downstream of the CD86 sequence, was inserted into XC-PSMA plasmid between the BGH polyadenylation signal of PSMA and ht eColEl origin of replication. Thus, both genes are controlled by the same regulatory sequences.
  • This plasmid is referred to herein as "XC PSMA- CD86" plasmid.
  • Example 2 Preparation of a replication-defective recombinant adenovirus Ad5-PSMA
  • Ad5-PSMA recombinant adenovirus comprising a sequence encoding the extracellular domain of PSMA under the control of the CMV promoter
  • the portion of the plasmid that contains the CMV promoter-PSMA fragment-PolyA signal was cut from pcDNA3.1 containing the extracellular domain of PSMA described above using Bglll and Smal restriction endonucleases.
  • the resulting product was purified on an agarose gel and subcloned in the Bglll-EcoRV cloning sites of the pAdBN transfer vector (Quantum Biotechnologies hie, Montreal, Canada).
  • the transfer vector was then linearized with Clal, and co-transfected with linearized Adenovirus DNA into 293A cells.
  • the recombinant replication-defective adenoviras(which lacks El and E3) was purified three times and clones that were positive for PSMA expression were selected by immxxnoblotting.
  • the positive clone was amplified in 293 cells and then purified on two successive CsCl gradients. Finally the purified viras was dialyzed against PBS-5% sucrose.
  • the replication-defective recombinant adenovirus Ad5-PSMA was deposited as Designation Number VR2362 on August 28, 1998 at the American Type Culture Collection, 10801 University Boulevard., Manassas, Virginia 20110.
  • Example 3 In vitro activation of CD8+ T cells by dendritic cells infected with Ad5-PSMA
  • PBMC Peripheral blood mononuclear cells
  • non-adherent cells are removed and adherent cells are cultured in 30 ml medium containing 2 ng/ml granulocyte macrophage colony-stimulating factor GM-CSF (obtained from Immunex, Seattle, WA) and 4 ng/ml interleukin -4 (IL-4) (obtained from Sigma).
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • IL-4 interleukin -4
  • DCs were infected with the Ad5-PSMA recombinant adenoviras at a multiplicity of infection (MOI) of 100.
  • MOI multiplicity of infection
  • 50 ⁇ l of viral suspension was inoculated into 50 ⁇ l of cell suspension (1.5 x 10 6 cells) in complete RPMI1640 medium containing 2% of autologous serum. After inoculation, the cells were incubated 90 min at 37°C in 5% CO, in complete RPMI- 1640 medium containing 2% of autologous serum, then washed three times, and incubated in RPMI- 1640 medium containing 10% of autologous seram for an additional 24h at 37°C in 5% CO 2 . Expression of PSMA was tested by immunoblotting.
  • Ad5-PSMA recombinant adenovirus and cultured with autologous T cells in complete medium (CM) for 3 days at 37°C. T cells were harvested at the end of the incubation, CD8+ T cells were purified therefrom by negative depletion with antiCD4 antibodies and complement, and their cytotoxicity was tested. The results indicate that the CD8+ T cells that had been stimulated by autologous
  • DCs infected with Ad5-PSMA were cytotoxic against the prostate tumor cell line LNCaP.FGC (also of the HLA A2+ phenotype), but not against Jurkat (T leukemia) or U937 (myelomonocytic cell line) cells, hi comparison, freshly separated T cells showed no cytotoxicity against any of the three cell lines.
  • Example 4 Improvement of prostate cancer in subjects having received a plasmid and/or recombinant adenovirus expressing the extracellular domain of human PSMA
  • GM-CSF Lewis; Immunex, Seattle, WA
  • these seven patients and a group of 2 new patients received three injections of the replication-deficient Ad5-PSMA recombinant adenovirus (also referred to as "Ad5-XC-PSMA”) vaccine (5x10 PFUs per application) at on -week intervals.
  • the plasmid was injected intradermally between the first and second toes of the right leg, or intramuscularly.
  • the viral vaccine was administered intradermally in the navel area.
  • Constant monitoring of the clinical state and the vital signs was carried out for 2 hours after vaccination. Ef stable, the subject was allowed to leave the hospital. A brief follow-up visit occurred 24 (and 48 in the case of GM-CSF inoculation) hours later.
  • Inclusion criteria All patients signed an informed consent form before admission into the study. Data from monitoring visits were shared with the patients as the study proceeded, and the patients were reminded that hey were free to withdraw from participation at any time. Only patients with advanced, hormone-resistant cancer or patients unable to find or administer hormone therapy were included into the study. Patients with a history of another malignancy or with a serious active infection or with another illness were excluded from the study.
  • Standard laboratory tests included CBC, urinanalysis, liver enzymes, antinuclear antibodies, erythrocyte sedimentation rate, PSA. Each patient had a pelvic CAT scan, chest radiograph and a cardiograph on entry and on week 20 (week 10 for the 2 patients immunized with viras only). Safety was defined as lack of untoward clinical or laboratory events, with particular attention to local and systemic reactions as well evidence of anti- nuclear antibody.
  • CD/HLA DR + ; CD4 + ; CD8 + ; CD37CD16 + CD56 + ; CD3 + ; CD1 + ; CD25 + ; CD 19 + cells as well as CD4/CD8 ratio prior and following immunotherapy was performed by flow cytometry.
  • Patients who received intradermal immunizations with plasmid had minor DTH-like reactions 24 hours following the third immunization.
  • Patients NN 8 and 9 developed a DTH reaction 24 hours following each administration of the recombinant adenovirus.
  • Patients NN I through 7 had no DTH-like reactions 24 hrs after the first immunization with the viral vector, but developed DTH after the second and third immunization. All DTH-like local reactions were mild and resolved within 72 hrs post immunization.
  • Patient N 4 had a vesicular rash after the last viral immunization which was located on the back and which resolved in the next two days with no treatment.
  • Patient N 7 had a papular urticaria-like rash with small petechiae at the center which developed 24 hrs after the last plasmid immunization and which disappeared after the discontinuation of the antibiotic therapy he was receiving. No significant changes occurred in erythrocyte sedimentation rate, CBC, serum creatinine or other blood chemistries, or urinanalysis. Serum liver chemistry values remained within normal range in all subjects.
  • Table 1 Regimen and characteristics of patients 1-7 treated with Ad5-PSMA ent Stage of Type of Additional PSA(ng/ml) CT scan LN Bone Side
  • CT scan CAT scan to obtain an estimate of the size of the prostate gland.
  • PSA prostate specific antigen and is an indication of the prognosis of the patient: a better prognosis is indicated by a lower PSA level.
  • Plsmd stands for plasmid. Patients 1, 4, 5, and 7 received plasmid XC-PSMA, whereas patients 2, 3, and 6 received plasmid XC-PSMA-CD86.
  • LN refers to lymph node metastasis.
  • MAB maximum androgen blockade with Zoladex, Casodex or Flucinome orchiectorny: always bilateral
  • Table 2 Patients who were immunized with recombinant adenovirus
  • results indicate that the progression of metastatic prostate cancer was retarded or stopped in at least some patients, as can be seen by the reduction in PSA; the a reduction of the size of the prostate gland, as estimated by the CAT scan; and/or the absence or reduction of numbers of lymph node and/or bone metastases.
  • patient 2 had less PSA after the treatment; a significant decrease in the size of his prostate gland; and he did not develop any lymph node or bone metastases, suggesting a positive effect of the treatment.
  • patient 3 had a much lower PSA level after the treatment, a significant reduction in the size of his prostate gland, and the absence of development of lymph node metastasis.
  • This example describes a phase I/II safety/dose-escalation clinical study conducted on prostate cancer patients, which showed that administration of a viral and/or plasmid vector encoding the extracellular domain of human PSMA alone, or together with a nucleic acid encoding the costimulatory molecule CD86 and/or rhuGM-CSF to subjects is safe and efficient for treating prostate cancer.
  • the five patients described in Table 1 which were still alive were included in this study. Since PSMA is a self-antigen, inducing an immune reaction requires the presence of self-reactive T lymphocytes and the breaking of self-tolerance.
  • the breaking of self- tolerance was measured by the development of a delayed type hypersensitivity (DTH) response to an injection of the nucleic acid(s).
  • DTH delayed type hypersensitivity
  • Example 4 were constructed as follows.
  • the cDNA encoding for extracellular portion of the human PSMA (XC-PSMA) was cloned into the pCR2.1 vector (Erivitrogen, Carlsbad, CA) after amplification by RT-PCR of total mRNA, that was obtained from the human prostate cancer cell line LNCaP (CRL 1740, ATCC).
  • the complete human CD86 cDNA was cloned into the pCR2.1 vector by RT-PCR of total mRNA, isolated from human monocytes of a healthy donor.
  • Both the cDNAs were sub-cloned into a modified mammalian expression vector pcDNA 3.1 (Invitrogen, Carlsbad, CA) after deletion of the neomycin resistance gene.
  • Both the XC PSMA and CD86 plasmids contain the corresponding cDNAs under the regulation of a CMV promoter and a Bovine Growth Hormone polyadenylation signal.
  • the combined PSMA/CD86 plasmid contains both the XC PSMA and CD86 cDNAs, each under a separate CMV promoter and a polyadenylation signal. Both the PSMA and the CD86 plasmids can be expressed in mammalian cells following transfection.
  • the plasmid-DNA product specifications included endotoxin content below 0.1 EU per microgram of DNA; lack of detectable amoxints of bacterial RNA, genomic DNA or ssDNAs as determined by agarose-gel electrophoresis; less than 10 ⁇ g of protein per 1 mg of plasmid DNA as determined by colorimetric assay (Bio-Rad, Hercules, CA). Prior to injection, the plasmids were diluted with sterile pyrogen-free saline.
  • Ad5-PSMA is a replication deficient recombinant adenovirus in which the replication-essential genes El and E3 are replaced with the expression cassette containing a coding sequence for an extracellular portion of the human PSMA.
  • Ad5PSMA was diluted with sterile pyrogen-free saline.
  • Soluble GM-CSF (sGM-CSF, Leukine, Sargramostim) is a recombinant human granulocyte-macrophage colony-stimulating factor produced by recombinant DNA technology in a yeast expression system (Immunex, Seattle, WA).
  • sGM-CSF a recombinant human granulocyte-macrophage colony-stimulating factor produced by recombinant DNA technology in a yeast expression system (Immunex, Seattle, WA).
  • yeast expression system Immunex, Seattle, WA
  • a second group of six patients was injected with a cocktail of the PSMA plasmid and sGM-CSF.
  • a third group of three patients received the PSMA plasmid, the CD86 plasmid and sGM-CSF at the same inoculation site.
  • a fourth group of three patients received a cocktail of the combined PSMA/CD86 plasmid with sGM-CSF.
  • PSMA plasmid Following that, all patients have been on regular boosts, at 3-week intervals, with either the PSMA/CD86 plasmid and sGM-CSF or with the Ad5PSMA virus, some of them longer than one year.
  • the 24-hour DTH reaction was recorded following each re-immunization, the aim being to maintain an intense response following each application.
  • patients' doses for the following boost have varied from 100 to 800 ug of DNA.
  • the viral dose, whenever virus was applied, has always been 5xl0 8 PFUs.
  • Constant monitoring of the clinical state and the vital signs was carried out for 2 hours after vaccination. If stable, the subject was allowed to leave the hospital. A brief follow-up visit occurred 24 hours (and 48 hours in the case of GM-CSF inoculation) later.
  • Standard laboratory tests included CBC, urinalysis, liver enzymes, antinuclear antibodies, erythrocyte sedimentation rate, PSA. Each patient had a pelvic CAT scan, chest radiograph and a cardiograph on entry and on week 20 (week 10 for the 10 patients immunized with virus only). Additionally, analysis of HLA DR + ; CD4 + ; CD8 + ; CD3 " /CD16 + CD56 + ; CD3 + ; CDllb + ; CD25 + ; CD19 + cells as well as CD4/CD8 ratio, prior to, and following immunotherapy, were performed by flow cytometry. Safety was defined as lack of untoward clinical or laboratory events, with particular attention to local and systemic reactions, as well as evidence of anti-nuclear or anti-double-stranded DNA antibody.
  • the second, and especially the third PSMA or PSMA/CD86 plasmid application led to a measurable in vivo delayed type hypersensitivity response with an infiltrate that peaked in 48 hours and in some instances exceeded 40 mm (fig.2). Using this DTH-like response, patients who were immunized against PSMA could be identified.
  • a preferred method for induction of anti-PSMA T cell immunity includes administration of a PSMA/CD86 plasmid and soluble GM-CSF.
  • this example shows that: (1) repetitive DNA and recombinant adenoviral immunizations of humans are a safe procedure; (2) tolerance to self-antigens can be broken by immunizations with DNA that encodes the antigen, inserted in either a plasmid or in a recombinant viral vector; and (3) addition of DNA encoding co-stimulatory molecules and soluble GM-CSF increases the immunization rate to 100%.
  • Example 6 Phase II study of treatment of protate cancer patients with vectors encoding PSMA
  • a phase II study to evaluate the biological effectiveness of immxmotherapy was initiated 8 months after the beginning of the first clinical trial study.
  • Patients were initially immunized twice, 3 weeks apart, with a cocktail of 200 ⁇ g PSMA/CD86 plasmid and 80,000 IU sGM-CSF.
  • 40,000 IU sGM-CSF was injected at the immunization site 6 and 24 hours later.
  • An intradermal boost with 5xl0 8 PFUs Ad5PSMA was given 3 weeks later.
  • Booster immxinizations are currently alternated between a cocktail of 100 ⁇ g PSMA plasmid plus 40,000 sGM-CSF and 5xl0 8 PFUs Ad5PSMA at 3-month intervals.
  • LNCaP cells were incubated with patients sera, washed and stained with F(ab')2 anti-human IgG and IgM.
  • F(ab')2 anti-human IgG and IgM For immxxnoblotting, after PAA gel electrophoresis in 14% gels, LNCaP cell extracts were electroblotted at 20 V overnight on nitrocellulose membranes in tris-glycine buffer with 20% methanol. Blots were blocked with 3% skimmed milk for 1 hour and incubated for an additional hour with either patients serum (dilution 1 : 1000) or with a concentrated supernatant from hybrydoma HB 11430 (control, dilution 1:1000).
  • the prostate cancer patients group is extremely heterogeneous with respect to the stage and the progression of the of disease, as well as to the type of treatment received.
  • Cancer patients have higher numbers of ⁇ EFN+ and EL-4+ T cells than healthy subjects.
  • the percentage of T cells expressing ⁇ lFN has a tendency to increase during therapy as does the percentage of IL-4+ T cells.
  • the increase of IL-4+ T-cells seems to be delayed in time and attenuated in prostatectomized patients with biochemical recurrence. The observed tendencies do not reflect the normal history of the disease since we have observed them only in patients treated by immunizations.
  • a response - R - is defined as a fall in PSA and, where applicable, reduction in tumor volume and in symptomatology such as obstructive voiding and bone pains.
  • a partial response (PR) is defined as no increase in PSA, no tumor growth, no obstructive symptomatology and decrease in bone pains where applicable.
  • a lack of response (NR) is defined as progression of disease, rise in PSA and increase in tumor volume leading to obstructive voiding symptoms and/or bone pains where applicable.
  • the patients who respond to therapy tend to have higher percentage of CD3+, CD25+ and CD4+ T cells and lower numbers of CD3-CD56+ NK cells prior to initiation of treatment relative to patients who do not respond.
  • the percentage of GDI lb+ cells in peripheral blood increases in those patients who do not respond to therapy, including sole immxmotherapy.
  • CD25+ and DR+ T cells decrease in those patients who do not respond to IT and remains relatively unchanged in responders.
  • IL-4+ T cells and ⁇ -EFN+ T cells increase following IT and the increase is higher in patients who respond to immxmotherapy.
  • dendritic cells which reside in tissues, become alerted by some form of tissue damage or pathogen products, migrate to the regional lymph node and stimulate antigen-specific T cells.
  • the type of interaction between the dendritic cells and T cells is critical for the outcome of the immune response. Both T cells and dendritic cells mature and change phenotype during this interaction and, at the end, the T cells induce the dendritic cells into programmed cell death (apoptosis) (Garside et al. Science 1998;281:96-99 and De Smedt et al. J Immunol 1998 16:4476).
  • Dendritic cells reside in healthy as well as in cancer tissues.
  • Cancer cells bear tumor-associated antigens that can serve as targets for immune recognition and attack.
  • Tumor cells secrete numerous lymphokines, such as TGF-beta, IL-10 and PGE-2, that inhibit dendritic cell maturation and function (Kiertscher et al. J Immunol 2000;164:1269).
  • lymphokines such as TGF-beta, IL-10 and PGE-2, that inhibit dendritic cell maturation and function (Kiertscher et al. J Immunol 2000;164:1269).
  • Such "incompetent" DCs may transport tumor-associated antigens to the regional lymph nodes but they fail to stimulate T cells into potent effector cells capable of rejecting tumor. As a result the antigen persists, ineffective antigen presentation continues until the system is finally exhausted. At that time disease prevails and rapidly progresses.
  • Example 7 Phase I clinical trial on colorectal cancer using naked DNA immunization against CEA
  • the plasmid encoding the human CEA is identical to that encoding PSMA described above, except that a sequence encoding the full length human CEA devoid of signal sequence was included, instead of PSMA. No CD86 was included in this plasmid.
  • DCs Following transfection of DCs with the CEA vector, DCs express peptides derived from the CEA protein in association with MHC class I and class II molecules and stimulate a CD4 and CD8 cytotoxic T cell response against colorectal cancer cells.
  • GM-CSF granulocyte- macrophage colony-stimulating factor
  • Example 5 Monitoring the immxme response and condition of the subjects was performed as described in Example 5. The statistical significance was also determined as described in Example 5.

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Abstract

L'invention concerne des procédés et des compositions destinés à traiter un patient portant des cellules indésirables, par exemple des cellules tumorales ou de cellules infectées par un virus. Le procédé consiste, par exemple, à administrer un polynucléotide codant un variant d'un antigène présent dans la cellule indésirable ('cellule cible'). Le variant n'est pas sécrété par, ni exprimé sur le côté extracellulaire de la membrane cellulaire de, la cellule dans laquelle il est exprimé. De préférence, le variant est biologiquement inactif. L'invention concerne en outre un procédé permettant d'induire une réponse immunitaire à médiation cellulaire contre la cellule indésirable sans pour autant induire une réponse immunitaire humorale. En outre, le procédé est sûr, étant donné que l'absence d'une activité biologique du variant empêche la survenue d'effets délétères chez le patient recevant ledit variant.
PCT/US2001/045626 2000-11-01 2001-11-01 Procedes et compositions pour induire des reponses immunitaires a mediation cellulaire Ceased WO2002040059A2 (fr)

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WO2004092216A1 (fr) * 2003-04-15 2004-10-28 Trangene S.A. Antigene carcino-embryonnaire depourvu de peptide signal, acides nucleiques codant pour celui-ci et une fusion dudit antigene avec un epitope de lymphocytes t, ainsi que leur utilisation pour le traitement et/ou la prophylaxie du cancer
WO2003031569A3 (fr) * 2001-10-10 2004-12-29 Centocor Inc Vaccins a base d'acides nucleiques utilisant des acides nucleiques codant pour un antigene tumoral et un acide nucleique codant pour un adjuvant cytokine
WO2005019464A1 (fr) * 2003-08-21 2005-03-03 Virax Development Pty Ltd Vecteur du poxvirus codant des antigenes specifiques de la prostate pour le traitement du cancer de la prostate
WO2011068758A1 (fr) * 2009-12-01 2011-06-09 Medimmune, Llc Méthodes et compositions améliorées pour la détection et le traitement des cancers exprimant l'antigène carcino-embryonnaire (ace)
US8114965B2 (en) 2001-10-23 2012-02-14 Psma Development Company, Llc Compositions of PSMA antibodies
US8470330B2 (en) 2001-10-23 2013-06-25 Psma Development Company, Llc PSMA antibodies and uses thereof
JP2019517521A (ja) * 2016-06-03 2019-06-24 エトゥビクス コーポレーション 前立腺がん関連抗原を使用する腫瘍ワクチン接種用の組成物及び方法
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JP2002524081A (ja) * 1998-09-04 2002-08-06 スローン − ケッタリング インスティチュート フォー キャンサー リサーチ 前立腺−特異的膜抗原に特異的な融合受容体およびその使用

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WO2003031569A3 (fr) * 2001-10-10 2004-12-29 Centocor Inc Vaccins a base d'acides nucleiques utilisant des acides nucleiques codant pour un antigene tumoral et un acide nucleique codant pour un adjuvant cytokine
US8114965B2 (en) 2001-10-23 2012-02-14 Psma Development Company, Llc Compositions of PSMA antibodies
US9695248B2 (en) 2001-10-23 2017-07-04 Psma Development Company, Llc PSMA antibodies and uses thereof
US8470330B2 (en) 2001-10-23 2013-06-25 Psma Development Company, Llc PSMA antibodies and uses thereof
WO2004092216A1 (fr) * 2003-04-15 2004-10-28 Trangene S.A. Antigene carcino-embryonnaire depourvu de peptide signal, acides nucleiques codant pour celui-ci et une fusion dudit antigene avec un epitope de lymphocytes t, ainsi que leur utilisation pour le traitement et/ou la prophylaxie du cancer
JP2007502602A (ja) * 2003-08-21 2007-02-15 バイラックス・ディベロップメント・プロプライエタリー・リミテッド 前立腺癌の治療用の前立腺特異的抗原をコードするポックスウィルスベクター
WO2005019464A1 (fr) * 2003-08-21 2005-03-03 Virax Development Pty Ltd Vecteur du poxvirus codant des antigenes specifiques de la prostate pour le traitement du cancer de la prostate
WO2011068758A1 (fr) * 2009-12-01 2011-06-09 Medimmune, Llc Méthodes et compositions améliorées pour la détection et le traitement des cancers exprimant l'antigène carcino-embryonnaire (ace)
US20200347464A1 (en) * 2014-03-18 2020-11-05 The Johns Hopkins University Psma-based molecular-genetic reporter system
US12104214B2 (en) * 2014-03-18 2024-10-01 The Johns Hopkins University PSMA-based molecular-genetic reporter system
JP2019517521A (ja) * 2016-06-03 2019-06-24 エトゥビクス コーポレーション 前立腺がん関連抗原を使用する腫瘍ワクチン接種用の組成物及び方法
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