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WO1999047179A1 - Compositions et methodes pour ameliorer l'apport antigenique in vivo a des cellules presentatrices de l'antigene - Google Patents

Compositions et methodes pour ameliorer l'apport antigenique in vivo a des cellules presentatrices de l'antigene Download PDF

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Publication number
WO1999047179A1
WO1999047179A1 PCT/US1999/006071 US9906071W WO9947179A1 WO 1999047179 A1 WO1999047179 A1 WO 1999047179A1 US 9906071 W US9906071 W US 9906071W WO 9947179 A1 WO9947179 A1 WO 9947179A1
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Prior art keywords
antigen
cells
factor
cell
apc
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Michael A. Perricone
Bruce L. Roberts
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Genzyme Corp
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Genzyme Corp
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Priority to AU31939/99A priority Critical patent/AU3193999A/en
Priority to JP2000536418A priority patent/JP2002506834A/ja
Priority to CA002322699A priority patent/CA2322699A1/fr
Priority to EP99913986A priority patent/EP1071470A1/fr
Publication of WO1999047179A1 publication Critical patent/WO1999047179A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2026—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001156—Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/00117—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001182—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001188—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • A61K39/001191—Melan-A/MART
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • A61K39/001192—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001194—Prostate specific antigen [PSA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001195—Prostate specific membrane antigen [PSMA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention is in the field of molecular immunology and medicine.
  • compositions and methods for enhancing site-specific in vivo delivery of tumor associated antigens are provided.
  • TAA tumor necrosis virus
  • i.p. intraperitoneal
  • i.m. intramuscular
  • i.v. intravascular
  • s.c. subcutaneous
  • i.d. intradermal
  • TAA-expressing TAAs do not suppress growth of actively growing tumors.
  • IL-2 interleukin 2
  • Antigen presenting cells a class of cells which includes dendritic cells, monocytes, macrophages, and B cells, are the presumed target for the delivery of TAAs.
  • the TAAs being investigated are, in fact, antigens recognized by cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • the APC appears to be the appropriate target for both gene therapy vectors and recombinant TAA proteins.
  • TAA transgenes have been directly delivered to isolated dendritic cells ex vivo, and the transfected dendritic cells are capable of inhibiting actively growing tumors when readministered to the mouse.
  • tumor growth inhibition is accomplished without IL-2 supplementation. It is desirable, from a clinical standpoint, to deliver the tumor antigen to the patient's APCs in vivo rather than first isolating APCs for transfection and subsequent readministration. At the current time, however, in vivo delivery of the TAA transgene to APC is not as effective in suppressing tumor cell growth as delivery of the TAA transgene to APCs ex vivo. There are a number of possible explanations as to why in vivo transfections of APCs is not as efficacious as ex vivo transfection. First, there may not be many APCs in the region where the gene therapy vector is administered.
  • the number of copies of the gene therapy vector per APC may also be too low for effective transfection.
  • the APCs at the site of administration may be at an inappropriate state of maturity for effective transfection with a TAA transgene. As a result, the TAA may not be presented to the immune system in the manner that produced effective tumor growth inhibition.
  • the present invention provides compositions and methods for enhancing in vivo site-specific delivery of tumor associated antigens.
  • this invention provides a method of recruiting antigen presenting cells to a predetermined site in a subject by administering to the subject an effective amount of an antigen presenting cell (APC) recruitment or proliferation factor to the predetermined site.
  • APC antigen presenting cell
  • the APC recruitment or proliferation factor may be a proinflammatory agent, a chemotactic agent, a growth factor or a mitogenic factor, and may be administered as a protein, a peptide or in a gene delivery vehicle.
  • the APC recruitment or proliferation factor is granulocyte-macrophage colony-stimulating factor (GM-CSF), Sepragel, IL4 or macrophage inflammatory protein 3 ⁇ .
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Sepragel Sepragel
  • IL4 macrophage inflammatory protein 3 ⁇ .
  • a growth factor, a cytokine, a co-stimulatory molecule and/or a mitogenic factor can also be administered with the APC recruitment or proliferation factor.
  • the present invention provides a method to enhance the presentation of an antigen into antigen presenting cells (APCs) in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor, and administering an effective amount of the antigen to the site.
  • the antigen is administered as a protein, a recombinant protein, or a peptide.
  • the antigen can be administered in the form of an antigne-encoding gene in a gene delivery vehicle.
  • the invention also encompasses a method to augment transduction of a transgene encoding an antigen into APCs in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor prior to the administration of an effective amount the transgene to the site.
  • the antigen is a tumor- associated antigen (TAA).
  • TAA tumor-associated antigen
  • the APC recruitment or proliferation factor is a proinflammatory agent, a chemotactic agent, a growth factor and/or a mitogenic factor.
  • the APC recruitment or proliferation factor is selected from the group consisting of granulocyte- macrophage colony stimulating factor (GM-CSF), Sepragel, interleukin 4 (IL4) and macrophage inflammatory protein 3 alpha (MIP-3 ⁇ ).
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • IL4 interleukin 4
  • MIP-3 ⁇ macrophage inflammatory protein 3 alpha
  • a growth factor, a cytokine, a co-stimulatory molecule and/or a mitogenic factor can be administered with the antigen including TAA.
  • a cell includes a plurality of cells, including mixtures thereof.
  • cancer refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition.
  • Tumor cells often express antigens which are tumor specific.
  • the term "tumor associated antigen” or “TAA” refers to an antigen that is associated with or specific to a tumor. Localized increases in dendritic cell populations can be achieved by inducing either migration or proliferation of these cells.
  • Recombinant adenoviral vectors expressing the melanoma TAAs such as gplOO (Ad2/hugpl00 v2), MARTI (Ad2/MART1), TRPl (Ad2/TRP1) and TRP2
  • Ad2/TRP2 were made at Genzyme Corporation.
  • the cationic lipids, GL67 and GL 89) and DNA vaccines expressing gplOO (pCFlhugplOO), TRPl (pCFlTRPl), and TRP2 (pCFlTRP2) were made at Genzyme Corporation.
  • immune effector cells refers to cells that specifically recognize an antigen present, for example on a neoplastic or tumor cell.
  • immune effector cells include, but are not limited to, B cells, monocytes, macrophages, NK cells and T cells such as cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.
  • CTLs cytotoxic T lymphocytes
  • T-lymphocytes denotes lymphocytes that are phenotypically CD3+, typically detected using an anti-CD3 monoclonal antibody in combination with a suitable labeling technique.
  • the T-lymphocytes of this invention are also generally positive for CD4, CD8, or both.
  • immune effector cells refers to immune effector cells that have not encountered antigen and is intended to by synonymous with unprimed and virgin.
  • Education refers to immune effector cells that have interacted with an antigen such that they differentiate into an antigen-specific cell.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • the proteins, peptides and polynucleotides of the present invention may be administered or applied transdermally, orally, subcutaneously, intramuscularly, intravenously, intradermally or parenterally.
  • an effective amount of the protein, peptide or polynucleotide is that amount which provokes an antigen-specific immune response in the subject.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes single-, double-stranded and triple helical molecules.
  • Oligonucleotide refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A "primer” refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil,
  • uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
  • the polynucleotides encode a peptide, a ribozyme or an antisense sequence.
  • protein protein
  • oligopeptide polypeptide
  • peptide polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • a “subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • the term "predetermined site” refers to the region of the subject into which it is intended that the methods described herein will be practices. Preferably, the predetermined site is a region that is naturally rich in antigen presenting cells such as skin.
  • priming is meant any treatment or preparation causing a desired result. For purposes of the this invention, priming a site means preparing that site for administration of transgene by first recruiting antigen presenting cells to that site.
  • antigen presentation cells includes both intact, whole cells as well as other molecules which are capable of inducing the presentation of one or more antigens, preferably with class I MHC molecules.
  • suitable APCs include, but are not limited to, whole cells such as macrophages, dendritic cells and B cells.
  • DCs Dendritic cells
  • APCs potent antigen-presenting cells
  • DCs are minor constituents of various immune organs such as spleen, thymus, lymph node, epidermis, and peripheral blood.
  • DCs represent merely about 1% of crude spleen (Steinman et al. (1979) J. Exp. Med. 149:1) or epidermal cell suspensions (Schuler et al. (1985) J. Exp. Med. 161:526; and Romani et al. (1989) J. Invest. Dermatol. 93:600), and 0.1-1% of mononuclear cells in peripheral blood (Freudenthal et al. (1990) Proc. Natl. Acad. Sci. USA 87:7698).
  • Co-stimulatory molecules are molecules involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells.
  • Co-stimulatory activity was originally defined as an activity provided by bone-marrow-derived accessory cells such as macrophages and dendritic cells, the so called “professional” APCs.
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • IMM-1 intracellular adhesion molecule 1
  • B7-1 and B7-2/B70 (Schwartz R.H. (1992) Cell 71:1065) and B7's counter-receptor CD28 or CTLA-4 on T cells
  • IAM-1 intracellular adhesion molecule 1
  • B7-1 and B7-2/B70 (Schwartz R.H. (1992) Cell 71:1065)
  • B7's counter-receptor CD28 or CTLA-4 on T cells Freeman et al. (1993) Science 262:909; Young et al. (1992) J. Clin. Invest 90: 229; and Nabavi et al. (1992) Nature 360:266).
  • Other important co-stimulatory molecules are CD40, CD54, CD80, CD86.
  • co-stimulatory molecule encompasses any single molecule or combination of molecules which, when acting together with a peptide/MHC complex bound by a TCR on the surface of a T cell, provides a co-stimulatory effect which achieves activation of the T cell that binds the peptide.
  • the term thus encompasses B7, or other co-stimulatory molecule(s) on an antigen-presenting matrix such as an APC, fragments thereof (alone, complexed with another molecule(s), or as part of a fusion protein) which, together with peptide/MHC complex, binds to a cognate ligand and results in activation of the T cell when the TCR on the surface of the T cell specifically binds the peptide.
  • Co-stimulatory molecules are commercially available from a variety of sources, including, for example, Beckman Coulter. It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified co-stimulatory molecules (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.
  • APC recruitment or proliferation factors includes both intact, whole cells as well as other molecules which are capable of recruiting antigen presenting cells.
  • APC recruitment or proliferation factors include "proinflammatory agents,” “chemotactic agents,” “growth factors” and “mitogenic agents.” Numerous mediators of dendritic cell migration and proliferation have been described in both in vitro and in vivo models.
  • suitable APC factors include molecules such as interleukin 4 (IL4), granulocyte macrophage colony stimulating factor (GM-CSF), Sepragel and macrophage inflammatory protein 3 alpha (MIP3 ⁇ ). Schering-Plough, Genzyme, Immunex.
  • IL4 interleukin 4
  • GM-CSF granulocyte macrophage colony stimulating factor
  • MIP3 ⁇ macrophage inflammatory protein 3 alpha
  • APC recruitment or proliferation factors include cytokines such as IL-2, stem cell factor (SCF), IL-3, IL-6, IL-12, G-CSF, GM-CSF, IL-l ⁇ , IL-11, MlP-l ⁇ , LIF, c-kit ligand, TPO, and flt3 ligand.
  • Cytokines are commercially available from several vendors such as, for example, Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA) and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that
  • dendritic cell chemotaxis is induced by macrophage-derived chemokine (MDC) (Godiska et al. (1997) J. Exp. Med. 185:1595:1604), formyl peptides, C5a, and chemokines such as monocyte chemotactic protein (MCP)-3 and RANTES (Morelli et al. (1996) Immunology 89:126-134; Sozzani et al. (1995) J. Immunol. 155:3292-3295).
  • MCP monocyte chemotactic protein
  • RANTES RANTES
  • human dendritic cell chemotaxis is not induced by IL-8, IL-10, MCP-1, and MCP-2 in vitro (Sozzani et al. (1995) supra).
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • GM-CSF Human GM-CSF
  • PRL Recombinant prolactin
  • Genzyme Corporation, Cambridge, MA is a protein with a molecular weight of about 25 kD consisting of 205 amino acids.
  • Sepragel (Genzyme Corporation, Cambridge, MA) is a cross-linked hyaluronic acid gel.
  • Recombinant adenoviral vectors expressing mGM-CSF (Ad2/cmvGMCSFF9ix) and human prolactin (Ad2/EV/PRL) were made at Genzyme (Framingham, MA).
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • a “transgene” is the term given to the polynucleotide carried by the gene delivery vehicle.
  • the term “transduction” refers to the transfer of polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes, viruses, such as baculovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adeno virus vectors, adeno-associated virus vectors and the like.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
  • the integrated DNA form is called a provirus.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a therapeutic gene.
  • Ads adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. (see, e.g., WO 95/27071) Ads are easy to
  • Ad-derived vectors particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed, (see, e.g. WO 95/00655; WO 95/11984). Wild-type AAV has high infectivity and specificity integrating into the host cells genome. (Hermonat and Muzyczka (1984) Proc.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5 ' of the start codon to enhance expression.
  • Non-viral vectors including DNA/liposome complexes, and targeted viral protein DNA complexes.
  • the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
  • Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • This invention also provides the targeting complexes for use in the methods disclosed herein.
  • Polynucleotides are inserted into vector genomes using methods well known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of
  • RNA 13 restricted polynucleotide 13 restricted polynucleotide.
  • synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA.
  • an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • Other means are well known and available in the art.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al. (1989) supra ).
  • an eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • a heterologous or homologous promoter for RNA polymerase II for RNA polymerase II
  • a downstream polyadenylation signal for RNA polymerase II
  • the start codon AUG a downstream polyadenylation signal
  • a termination codon for detachment of the ribosome.
  • “Host cell” is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also is intended to
  • the cells 14 include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • the cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.
  • an “antibody” is an immunoglobulin molecule capable of binding an antigen.
  • the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
  • pharmaceutical composition is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others.
  • compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline,
  • This invention provides a method to enhance the recruitment or increase the number of APCs to a predetermined site or location in a subject by administering to the subject an effective amount of an APC recruitment or proliferation factor and under conditions which favor the recruitment or increase in the number of APC to the site of administration.
  • the factor is formulated for slow release (i.e., in a sponge or liposome) so as to create a sustained localized concentration of the APC recruitment or proliferation factor.
  • the recruitment or proliferation factor is, in some embodiments, GM-CSF or Supragel.
  • the recruitment or proliferation factor also includes, but is not limited to, biological equivalents of these protein such as amino acid sequences having conservative amino acid substitutions and fusion proteins. This may include yet unidentified proteins which may be assayed in the animal model presented below. The biological activity of an equivalent protein can be assayed by using the animal model detailed below.
  • the recruitment or proliferation factor of this invention also can be administered as a polynucleotide or gene coding for the factor.
  • These polynucleotides can be delivered using conventional gene therapy delivery vehicles, vectors and methods as provided below. Either of these means of delivering the recruitment or proliferation factor of this invention can be further modified by the co-administration of a growth factor, a cytokine, a co-stimulatory molecule or a mitogenic molecule, prior to, subsequently to or concurrently with the recruitment or proliferation factor.
  • a host cell can be transduced with gene(s) encoding these molecules which is then administered to the subject.
  • the host cell containing the gene(s) coding for the ctyokine and/or co-stimulatory molecule is a professional antigen-
  • presenting cell such as a dendritic cell which includes, but is not limited to, a pulsed dendritic cell, a dendritic cell hybrid or an antigen-presenting foster cell.
  • CTLs cancer Immunol. Immunother. 39:15-21.
  • T cells are added to target cells previously loaded with 51 Cr and one measures the release of 51 Cr from the lysed target cells.
  • Cytokine release assay can be used as described in Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515-19. Briefly, cytotoxic T cells are added to target cells and one measures the amount of IFN ⁇ released by ELIS A. To measure the relative proportion of immune effector
  • Enzyme- Linked immunoSPOT Assay for 17 cells within a mixed population that recognize a particular target, the Enzyme- Linked immunoSPOT (ELISPOT) assay is employed as described in Czerkinsky et al. (1988) J. Immunol. Methods 110:29-36. Briefly, 96 well nitrocellulose- bottomed plates are coated with an anti-cytokine antibody, generally anti- interferon- ⁇ . Target cells and immune effector cells such as cytotoxic T cells
  • CTLs Cytokine released from the CTLs is captured by the anti-interferon- ⁇ antibody and quantitated using a standard ELISA format.
  • the present invention provides a method to enhance presentation of an antigen into antigen presenting cells (APCs) in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor, and administering an effective amount of the antigen to the site.
  • the antigen is administered as a protein, a recombinant protein, or a peptide.
  • This invention f rther provides a method to augment transduction of transgenes into APCs in vivo, by priming a predetermined site in a subject with an effective amount of an APC recruitment or proliferation factor and administering an effective amount of the transgene, which may be a tumor associated antigen.
  • the antigen of this vaccine may be an altered antigen or heterologous (i.e., allogeneic or a homolog from a isolated species, e.g., a murine antigen administered to a human patient). It may be a previously characterized tumor-associated antigens such as gpl 00 (Kawakami et al. (1997) Intern. Rev. Immunol. 14: 173- 192);
  • MUC-1 (Henderson et al. (1996) Cancer Res. 56:3763-3770); MART-1 (Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515-3519; Kawakami et al. (1997) Intern. Rev. Immunol. 14:173-192; and Ribas et al. (1997) Cancer Res. 57:2865-2869); HER-2/neu (U.S. Patent No. 5,550,214), MAGE (PCT/US92/04354); HPV 16, 18E6 and E7 (Ressing et al. ( 1996) Cancer Res.
  • human and murine MUC1 coding sequences are provided under Genbank Accession No. M35093 and M64928.
  • Proteins of known antigens also are useful in the methods described herein. These proteins are encoded by polynucleotides that hybridize under stringent conditions to the sequences disclosed in the references described above or known in the art. Alternatively, the proteins are encoded by polynucleotides that are at least 80%, or more preferably, at least 90% or most preferably, at least 95%, identical to the disclosed sequences using as determined using sequence alignment programs and default parameters. "Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6 X SSC to about 10 X SSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 6 X SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9 X SSC to about 2 X SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5 X SSC
  • Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1 X SSC to about 0.1 X SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1 X SSC, 0.1 X SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or
  • sequence identity to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • the antigen can be administered as a protein, a recombinant protein, a peptide, or, alternatively, a polynucleotide in a gene delivery vehicle.
  • the invention provides a method for cloning the cDNA and genomic DNA encoding novel recruitment or proliferation factors identified by
  • compositions comprising the nucleic acid and a carrier, such as a pharmaceutically acceptable carrier, a solid support or a detectable label, are further provided by this method as well as methods for detecting the sequences in a sample using methods such as Northern analysis,
  • compositions comprising the oligopeptide sequence and a carrier, such as a pharmaceutically acceptable carrier, a solid support or a detectable label, are further provided by this method as well as methods for detecting the oligopeptide sequence in a sample using methods such as Western analysis and ELISA. Harlow and Lane (1988), supra.
  • Any conventional method e.g., subtractive library, comparative Northern and/or Western blot analysis of normal and tumor cells, Serial Analysis of Gene Expression (U.S. Patent No. 5,695,937) and Solid PHase Epitope REcovery
  • SPHERE described in PCT WO 97/35035
  • PCT WO 97/35035 can be used to identify putative antigens for use in the subject invention.
  • Expression cloning also can be used. This methodology, as described in Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515-19, can be used to identify a novel tumor-associated antigen. Briefly, a library of cDNAs corresponding to mRNAs derived from tumor cells is cloned into an expression vector and introduced into target cells which are subsequently incubated with cytotoxic T cells. One identifies pools of cDNAs that are able to stimulate the CTL and through a process of sequential dilution and re-testing of less complex pools of cDNAs one is able to derive unique cDNA sequences that are able to stimulate the CTL and thus encode the cognate tumor antigen.
  • SAGE analysis involves identifying nucleotide sequences expressed in the antigen-expressing cells. Briefly, SAGE analysis begins with providing complementary deoxyribonucleic acid (cDNA) from (1) the antigen-expressing population and (2) cells not expressing that antigen. Both cDNAs can be linked to primer sites. Sequence tags are then created, for example, using the appropriate primers to amplify the DNA. By measuring the differences in these tags between the two cell types, sequences which are aberrantly expressed in the antigen- expressing cell population can be identified.
  • cDNA complementary deoxyribonucleic acid
  • MARTI and gplOO are melanocyte differentiation antigens specifically recognized by HLA-A2 restricted tumor-infiltrating lymphocytes (TILs) derived from patients with melanoma, and appear to be involved in tumor regression (Kawakami, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:6458-62; Kawakami, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:91:3515-9). Recently, the mouse homolog of human MART-1 has been isolated.
  • the full-length open reading frame of the mouse MARTI consists of 342 bp, encoding a protein of 113 amino acid residues with a predicted molecular weight of ⁇ 13 kDa. Alignment of human and murine MARTI amino acid sequences showed 68.6% identity. The murine homologue of gplOO has also been identified. The open reading frame consists of 1,878 bp, predicting a protein of 626 amino acid residues which exhibits 75.5% identity to human gplOO.
  • SPHERE described in PCT WO 97/35035, is a method that will identify wild-type or native antigens as well as provide altered antigens.
  • SPHERE is an empirical screening method for the identification of MHC Class I-restricted CTL epitopes that utilizes peptide libraries synthesized on a solid support (e.g., plastic beads) where each bead contains approximately 200 picomoles of a unique peptide that can be released in a controlled manner.
  • the synthetic peptide library is tailored to a particular HLA restriction by fixing anchor residues that confer high-affinity binding to a particular HLA allele (e.g. , HLA-A2) but contain a
  • peptide pools containing reactive species can be determined by measuring 51 Cr-release according to standard methods known in the art.
  • cytokine production e.g., interferon- ⁇
  • proliferation e.g., incorporation of 3 H-thymidine
  • the beads corresponding to those mixtures are separated into smaller pools and distributed to new 96-well plates (e.g. , 100 beads per well).
  • the procedure requires 1% human plasma in the place of 10% fetal calf serum and involves two steps.
  • the first step or “priming” phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4.
  • the second step or “differentiation” phase requires the exposure to macrophage conditioned medium.
  • a large numbers of precommitted APCs already circulating in the blood are isolated.
  • Previous techniques for isolating committed APCs from human peripheral blood have involved combinations of physical procedures such as metrizamide gradients and adherence/nonadherence steps (Freudenthal et al. (1990) PNAS USA 87:7698- 7702); Percoll gradient separations (Mehta-Damani et al. (1994) J. Immunol. 153:996-1003); and fluorescence activated cell sorting techniques (Thomas R. et al. (1993) J. Immunol. 151:6840-52).
  • the APC can be precommitted or mature dendritic cells, which can be isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060).
  • the white blood cell fraction can be from the peripheral blood of the mammal.
  • This method includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subtractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore; (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4°C.
  • the white blood cell fraction can be treated with calcium ionophore in the presence of other cytokines, such as rhIL-12, rhGM-CSF, or rhIL-4.
  • the cells of the white blood cell fraction can be washed in buffer and suspended in Ca ⁇ /Mg ⁇ free media prior to the separating step.
  • the white blood cell fraction can be obtained by leukapheresis.
  • the dendritic cells can be identified by the presence of at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD 14, CD 16, 56, 57, and CD 19, 20. Monoclonal antibodies specific to these cell surface markers are commercially available.
  • the method requires collecting an enriched collection of white cells and platelets from leukapheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE) (Abrahamsen, T.G. et al. (1991) J. Clin. Apheresis. 6:48-53).
  • CCE countercurrent centrifugal elutriation
  • Cell samples are placed in a special elutriation rotor.
  • the rotor is then spun at a constant speed of, for example, 3000 rpm. Once the rotor has reached the desired speed, pressurized air is used to control the flow rate of cells.
  • Cells in the elutriator are subjected to simultaneous centrifugation and a washout stream of buffer that is constantly increasing in flow rate. This results in fractional cell separations based largely but not exclusively on differences in cell size.
  • DCs Quality control of APC and more specifically DC collection and confirmation of their successful activation in culture is dependent upon a simultaneous multi-color FACS analysis technique which monitors both monocytes and the dendritic cell subpopulation as well as possible contaminant T lymphocytes. It is based upon the fact that DCs do not express the following markers: CD3 (T cell); CD 14 (monocyte); CD 16, 56, 57 (NK LAK cells); CD 19, 20 (B cells). At the same time, DCs do express large quantities of HLA-DR, significant HLA-DQ and B7.2 (but little or no B7.1) at the time they are circulating in the blood (in addition they express Leu M7 and M9, myeloid markers which are also expressed by monocytes and neutrophils).
  • propridium iodide When combined with a third color reagent for analysis of dead cells, propridium iodide (PI), it is possible to make positive identification of all cell subpopulations (see Table 1):
  • Color #1 CD3 alone, CD 14 alone, etc.; Leu M7 or Leu M9; anti-Class I, etc.
  • Color #2 HLA-Dq, B7.1, B7.2, CD25 (IL2r), ICAM, LFA-3, etc.
  • the goal of FACS analysis at the time of collection is to confirm that the DCs are enriched in the expected fractions, to monitor neutrophil contamination, and to make sure that appropriate markers are expressed.
  • This rapid bulk collection of enriched DCs from human peripheral blood, suitable for clinical applications is absolutely dependent on the analytic FACS technique described above for quality control. If need be, mature DCs can be immediately separated from monocytes at this point by fluorescent sorting for "cocktail negative" cells.
  • the DC rich/monocyte APC fractions (usually 150 through 190) can be pooled and cryopreserved for future use, or immediately placed in short term culture.
  • cytokines include but are not limited to rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when given alone is inadequate for optimal upregulation.
  • the APCs and cells expressing one or more antigens are autologous. In another embodiment, the APCs and cells expressing the antigen are allogeneic, i.e., derived from a different subject.
  • an APC-recruitment or proliferation factor is administered to mice by subcutaneous injection.
  • the APC-recruitment or proliferation factors is administered either as a protein or as a gene therapy vector that is capable of expressing the gene for the APC-enhancing factor.
  • the effective amount can be readily determined, for instance by administering subcutaneous injections at different dosages and evaluating the subsequent inflammatory response at different times after administration.
  • the APC recruitment or proliferation factors useful in the present invention include, but are not limited to, GM-CSF, PRL, Sepragel, Ad2/cmvGMCSFF9ix, and Ad2/EV/PRL. In addition, it may be necessary to
  • the inflammatory response at the site of APC recruitment or proliferation factor administration can be evaluated by any other known method, for example, in histology sections or in samples taken by needle aspiration.
  • enhanced numbers of dendritic cells, Langerhans cells, and other antigen presenting cells can also be evaluated using either immunohistochemistry or
  • TAA Tumor- Associated Antigens
  • Tumor-associated antigens can be administered as whole molecules or, preferably, using gene delivery vehicles carrying a polynucleotide encoding
  • TAAs may also be administered to APC recruitment or proliferation factor-primed subcutaneous sites in the form of peptides or recombinant proteins.
  • gene delivery vehicles expressing a TAA will be administered after treatment with the APC recruitment or proliferation factor.
  • the TAA is administered in a recombinant adenoviral vector or cationic lipid:DNA vectors by subcutaneous injection.
  • the TAAs include, but are not limited to,
  • the successful transfer of the TAA transgene to APCs, such as dendritic cells or Langerhans cells, at the site of vector administration may be evaluated by any suitable method, for example, using a combination of fluorescence in situ hybridization (FISH) (Gussoni et al. (1996) Nature Biotechnol. 14:1012-1016); fluorescently labeled lipids or plasmid DNA (Bebok et al. (1996) J. Pharmacol.
  • FISH fluorescence in situ hybridization
  • Peptide fragments from antigens must first be bound to peptide binding receptors ((MHC) class I and class II molecules) that display the antigenic peptides on the surface of the APCs. Palmer E. and Cresswell (1998) Annu. Rev. Immunol. 16:323 and Germain R.N. (1996) Immunol. Rev. 151:5.
  • T lymphocytes produce an antigen receptor that they use to monitor the surface of APCs for the presence of foreign peptides.
  • the antigen receptors on T H cells recognize antigenic peptides bound to MHC class II molecules whereas the receptors on CTLs react with antigens displayed on class I molecules.
  • antigens can be delivered to antigen- presenting cells as protein/peptide or in the form of polynucleotides encoding the protein/peptide ex vivo or in vivo.
  • the methods described below focus primarily on DCs which are the most potent, preferred APCs.
  • Several different techniques have been described to produce genetically modified APCs. These include: (1) the introduction into the APCs of polynucleotides that express antigen or fragments thereof; (2) infection of APCs with recombinant vectors to induce endogenous expression of antigen; and (3) introduction of tumor antigen into the DC cytosol using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med. 184:465; Rouse et al. (1994) J. Virol.
  • any method which allows for the introduction and expression of the heterologous, altered or non-self antigen and presentation by the MHC on the surface of the APC is within the scope of this invention.
  • Pulsing is accomplished in vitro/ex vivo by exposing APCs to antigenic protein or peptide(s).
  • the protein or peptide(s) are added to APCs at a concentration of 1-10 ⁇ m for approximately 3 hours.
  • Paglia et al. (1996) J. Exp. Med. 183:317, has shown that APC incubated with whole protein in vitro were recognized by MHC class I-restricted CTLs, and that immunization of animals with these APCs led to the development of antigen-specific CTLs in vivo.
  • Protein/peptide antigen can also be delivered to APC in vivo and presented by the APC.
  • Antigen is preferably delivered with adjuvant via the intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery. Grant
  • Foster APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763). This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1 ,
  • TAP2 TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC class 1 -restricted CD8 + CTLs.
  • MHC class 1 MHC class 1 -restricted CD8 + CTLs.
  • Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif.
  • T2 cells are referred to herein as "foster"
  • APCs are used in conjunction with this invention to present the heterologous, altered or control antigen.
  • T2 cells with specific recombinant MHC alleles allows for redirection of the MHC restriction profile.
  • Libraries tailored to the recombinant allele will be preferentially presented by them because the anchor residues will prevent efficient binding to the endogenous allele.
  • MHC molecules make the APC more visible to the CTLs.
  • a powerful transcriptional promoter e.g., the CMV promoter
  • results in a more reactive APC most likely due to a higher concentration of reactive MHC-peptide complexes on the cell surface.
  • Hybrid cells typically retain the phenotypic characteristics of the APCs. Thus, hybrids made with dendritic cells will express the same MHC class II proteins and other cell surface markers. Moreover, the hybrids will express those antigens expressed on the cells from which they are derived.
  • a population of APCs are collected and isolated.
  • the ratio of APCs:antigen-expressing cells is between about 1:100 and about 1000:1.
  • the fraction enriched for antigen-expressing cells is then fused to APCs, preferably dendritic cells. Fusion between the APCs and antigen- expressing cells can be carried out with any suitable method, for example using polyethylene glycol (PEG) or Sendia virus.
  • the hybrid cells are created using the procedure described by Gong et al.(1997) Nat. Med. 3(5):558-561.
  • the fused cells can be separated from the parent cells simply by allowing the culture to grow for several days.
  • the hybrid cells both survive more and, additionally, are only lightly adherent to tissue culture surfaces.
  • the parent cells are strongly adherent to the containers. Therefore, after about 5
  • the hybrid cells can be gently dislodged and transferred to new containers, while the unfused cells remained attached.
  • fused cells lack functional hypoxanthine-guaninephosphoribosyl transferase ("HGPRT") enzyme and are, therefore, resistant to treatment with the compound HAT. Accordingly, to select these cells HAT can be added to the culture media. However, unlike conventional HAT selection, hybrid cell cultures should not be exposed to the compound for more than 12 days.
  • HGPRT hypoxanthine-guaninephosphoribosyl transferase
  • introduction of the transgene or polynucleotide encoding the APC recruitment or proliferation factor or antigen is accomplished in vivo, ex vivo or in vitro by introducing a vector containing a polynucleotide or transgene encoding a heterologous or an altered antigen or APC recruitment or proliferation factor.
  • a vector containing a polynucleotide or transgene encoding a heterologous or an altered antigen or APC recruitment or proliferation factor A variety of different gene transfer vectors, including viral as well as non- viral systems can be used.
  • Viral vectors useful in the genetic modifications of this invention include, but are not limited to adenovirus, adeno-associated virus vectors, retroviral vectors and adeno-retroviral chimeric vectors.
  • Adenovirus and adeno-associated virus vectors useful in the genetic modifications of this invention may be produced according to methods already taught in the art. (see, e.g., Karlsson et al. (1986) EMBO 5:2377; Carter (1992) Curr. Op. Biotechnol. 3:533-539; Muzcyzka (1992) Current Top. Microbiol.
  • adenoviral vectors based on the human adenovirus 5 are missing essential early genes from the adenoviral genome (usually El A/El B), and are therefore unable to replicate unless grown in permissive cell lines that provide the missing gene products in trans.
  • a transgene of interest can be cloned and expressed in cells infected with the replication deficient adenovirus.
  • adeno virus-based gene transfer does not result in integration of the transgene into the host genome (less than 0.1% adeno virus- mediated transfections result in transgene incorporation into host DNA), and therefore is not stable, adenoviral vectors can be propagated in high titer and transfect non-replicating cells.
  • Human 293 cells which are human embryonic kidney cells transformed with adenovirus El A/El B genes, typify useful permissive cell lines and are commercially available from the ATCC
  • other cell lines which allow replication-deficient adenoviral vectors to propagate therein can be used, including HeLa cells.
  • adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario, Canada (see, e.g., Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996). See also, the papers
  • Retroviral vectors useful in the methods of this invention are produced recombinantly by procedures already taught in the art.
  • WO 94/29438 describes the construction of retroviral packaging plasmids and packaging cell lines.
  • the retroviral vectors useful in the methods of this invention are capable of infecting the cells described herein.
  • the techniques used to construct vectors, and transfix and infect cells are widely practiced in the art.
  • retroviral vectors are those derived from murine, avian or primate retroviruses.
  • Retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are the most commonly used because of the availability of retroviral variants that efficiently infect human cells.
  • Other suitable vectors include those based on the Gibbon Ape Leukemia Virus (GAL) or HIV.
  • GAL Gibbon Ape Leukemia Virus
  • the viral gag, pol and env sequences are removed from the virus, creating room for insertion of foreign DNA sequences. Genes encoded by the foreign DNA are usually expressed under the control of the MoMLV
  • Such a construct can be packed into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line.
  • the gag-pol and env proteins produced by the cell assemble with the vector RNA to produce infectious virions that are secreted into the culture medium.
  • the virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences.
  • Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced.
  • the packaging cell line harbors an integrated provirus.
  • the provirus has been crippled so that, although it produces all the proteins required to assemble infectious viruses, its own RNA cannot be packaged into virus. Instead, RNA produced from the recombinant virus is packaged. The virus stock released from the packaging cells thus contains only recombinant virus.
  • the range of host cells that may be infected by a retro virus or retroviral vector is determined by the viral envelope protein.
  • the recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable production of the foreign gene product.
  • murine ecotropic env of MoMLV allows infection of rodent cells
  • amphotropic env allows infection of rodent, avian and some primate cells, including human cells.
  • Amphotropic packaging cell lines for use with MoMLV systems are known in the art and commercially available and include, but are not limited to, PA12 and PA317. Miller et al. (1985) Mol. Cell. Biol.
  • Xenotropic vector systems exist which also allow infection of human cells.
  • VSV-G vesicular stomatitis virus
  • the vectors will contain at least two heterologous genes or gene sequences: (i) the therapeutic gene to be transferred; and (ii) a marker gene that enables tracking of infected cells.
  • therapeutic gene can be an entire gene or only the functionally active fragment of the gene capable of compensating for the deficiency in the patient that arises from the defective endogenous gene.
  • Therapeutic gene also encompasses antisense oligonucleotides or genes useful for antisense suppression and ribozymes for ribozyme-mediated therapy.
  • a therapeutic gene may be one that neutralizes the immunosuppressive factor or counter its effects.
  • therapeutic genes that encode dominant inhibitory oligonucleotides and peptides as well as genes that encode regulatory proteins and oligonucleotides also are encompassed by this invention.
  • gene therapy will involve the transfer of a single therapeutic gene although more than one gene may be necessary for the treatment of particular diseases.
  • the therapeutic gene is a dominant inhibiting mutant of the wild-type immunosuppressive agent.
  • the therapeutic gene could be a wild- type, copy of a defective gene or a functional homologue.
  • More than one gene can be administered per vector or alternatively, more than one gene can be delivered using several compatible vectors.
  • the therapeutic gene can include the regulatory and untranslated sequences.
  • the therapeutic gene will generally be of human origin although genes from other closely related species
  • the therapeutic gene suitable for use in treatment will vary with the disease. Nucleotide sequences for the therapeutic gene will generally be known in the art or can be obtained from various sequence databases such as GenBank. The therapeutic gene itself will generally be available or can be isolated and cloned using the polymerase chain reaction PCR (Perkin-Elmer) and other standard recombinant techniques. The skilled artisan will readily recognize that any therapeutic gene can be excised as a compatible restriction fragment and placed in a vector in such a manner as to allow proper expression of the therapeutic gene in hematopoietic cells.
  • a marker gene can be included in the vector for the purpose of monitoring successful transduction and for selection of cells into which the DNA has been integrated, as against cells which have not integrated the DNA construct.
  • Various marker genes include, but are not limited to, antibiotic resistance markers, such as resistance to G418 or hygromycin. Less conveniently, negative selection may be used, including, but not limited to, where the marker is the HSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir.
  • selections could be accomplished by employment of a stable cell surface marker to select for transgene expressing cells by FACS sorting.
  • NeoR neomycin /G418 resistance
  • the viral vector can be modified to incorporate chimeric envelope proteins or nonviral membrane proteins into retroviral particles to improve particle stability and expand the host range or to permit cell type-specific targeting during infection.
  • the production of retroviral vectors that have altered host range is taught, for example, in WO 92/14829 and WO 93/14188.
  • Retroviral vectors that can target specific cell types in vivo are also taught, for example, in Kasahara et al.
  • MoMLV Moloney leukemia virus
  • EPO human erythropoietin
  • This hybrid virus shows tissue tropism for human red blood progenitor cells that bear the receptor for EPO, and is therefore useful in gene therapy of sickle cell anemia and thalassemia.
  • Retroviral vectors capable of specifically targeting infection of cells are preferred for in vivo gene therapy.
  • the viral constructs can be prepared in a variety of conventional ways. Numerous vectors are now available which provide the desired features, such as long terminal repeats, marker genes, and restriction sites, which may be further modified by techniques known in the art.
  • the constructs may encode a signal peptide sequence to ensure that cell surface or secreted proteins encoded by genes are properly processed post-translationally and expressed on the cell surface if appropriate.
  • the foreign gene(s) is under the control of a cell specific promoter.
  • the introduced gene may be put under the control of a promoter that will cause the gene to be expressed constitutively, only under specific physiologic conditions, or in particular cell types.
  • the retroviral LTR (long terminal repeat) is active in most hematopoietic cells in vivo and will generally be relied upon for transcription of the inserted sequences and their constitutive expression (Ohashi et al. (1992) Proc. Natl. Acad. Sci. USA 89:11332; Correll et al. (1992) Blood 80:331).
  • Other suitable promoters include the human cytomegalovirus (CMV) immediate early promoter and the U3 region promoter of the Moloney Murine Sarcoma Virus (MMSV), Rous Sarcoma Virus (RSV) or Spleen Focus Forming Virus (SFFV).
  • promoters examples include Granzyme A for expression in T-cells and NK cells, the CD34 promoter for expression in stem and progenitor
  • Inducible promoters may be used for gene expression under certain physiologic conditions.
  • an electrophile response element may be used to induce expression of a chemoresistance gene in response to electrophilic molecules.
  • the therapeutic benefit may be further increased by targeting the gene product to the appropriate cellular location, for example the nucleus, by attaching the appropriate localizing sequences.
  • the vector construct is introduced into a packaging cell line which will generate infectious virions.
  • Packaging cell lines capable of generating high titers of replication-defective recombinant viruses are known in the art, see for example, WO 94/29438.
  • Viral particles are harvested from the cell supernatant and purified for in vivo infection using methods known in the art such as by filtration of supernatants 48 hours post transfection.
  • the viral titer is determined by infection of a constant number of appropriate cells (depending on the retrovirus) with titrations of viral supernatants.
  • the transduction efficiency can be assayed 48 hours later by a variety of methods, including Southern blotting.
  • PCR can be performed to detect the marker gene or other virally transduced sequences. Generally, periodic blood samples are taken and PCR conveniently performed using it NeoR probes if the NeoR gene is used as marker. The presence of virally transduced sequences in bone marrow cells or mature hematopoietic cells is evidence of successful reconstitution by the transduced cells. PCR techniques and reagents are well known in the art, See, generally, PCR PROTOCOLS, A GUIDE TO METHODS AND
  • Non- viral vectors such as plasmid vectors useful in the genetic modifications of this invention, can be produced according to methods taught in the art. References describing the construction of non- viral vectors include the
  • the agents identified herein as effective for their intended purpose can be administered to subjects or individuals susceptible to or at risk of developing a disease, such as cancer.
  • a disease such as cancer.
  • the agent When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • a tumor regression can be assayed.
  • Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the therapy. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
  • an adenoviral vector encoding a tumor antigen can be employed in vivo for direct immunization as described in Zhai et al. (1996) J. Immunol. 156:700-10.
  • C57BL/6 mice are injected subcutaneously at two sites (1.5 x 10 9 IU per site) with an adenoviral vector encoding an antigen presenting cell recruitment or proliferation factor such as IL4.
  • an adenoviral vector encoding a tumor antigen such as gplOO is injected into these two pre-treated site (1.5 x 10 9 IU per site).
  • animals are challenged with a lethal dose (2 x 10 4 ) of murine B16F10 melanoma tumor cells by subcutaneous injection. Animals are scored for survival and tumor measurements are taken.
  • Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
  • agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parental (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
  • Enhancement of antigen delivery to antigen presenting cells is tested in murine tumor models where TAAs have been identified.
  • TAAs For example, B16-F10 melanoma tumor growth in the C57BL/6 mouse strain can be used since the TAAs
  • MARTI and gplOO have been identified and offer some protective effect against tumor growth (Zhai et al. (1997) J. Immunother. 20:15-25).
  • An alternative model is the K1735 melanoma cells from the C3H murine strain, where a specific CTL response against tumor antigens has been shown (Chen et al. (1993), supra).
  • an APC recruitment or proliferation factor such as IL-
  • APC 4 is injected subcutaneously in the form of a recombinant protein formulated in either cationic lipids, or in a sponge, to allow for sustained slow release of the APC recruitment or proliferation factor at a localized site.
  • an adenoviral vector encoding an antigen such as the melanoma antigen gpl 00 is injected into the pretreated site to maximize the likelihood that the injected viral particles will contact, and cause gene transfer to APCs.
  • the genetically modified APCs will present tumor antigen derived peptides to the immune system, thereby provoking a potent anti-tumor cell immune response.

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Abstract

La présente invention concerne des compositions et des méthodes permettant d'améliorer l'apport ciblé in vivo d'antigènes associés aux tumeurs. Dans un aspect de cette invention, une méthode permet donc de mobiliser des cellules présentatrices de l'antigène (cellules APC) vers un site prédéterminé chez un sujet. L'invention concerne enfin des méthodes destinées à favoriser la transduction d'un transgène in vivo.
PCT/US1999/006071 1998-03-20 1999-03-19 Compositions et methodes pour ameliorer l'apport antigenique in vivo a des cellules presentatrices de l'antigene Ceased WO1999047179A1 (fr)

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AU31939/99A AU3193999A (en) 1998-03-20 1999-03-19 Compositions and methods for enhanced antigen delivery to antigen presenting cells (in vivo)
JP2000536418A JP2002506834A (ja) 1998-03-20 1999-03-19 生体内の抗原提示細胞への増強された抗原搬送の組成と方法
CA002322699A CA2322699A1 (fr) 1998-03-20 1999-03-19 Compositions et methodes pour ameliorer l'apport antigenique in vivo a des cellules presentatrices de l'antigene
EP99913986A EP1071470A1 (fr) 1998-03-20 1999-03-19 Compositions et methodes pour ameliorer l'apport antigenique in vivo a des cellules presentatrices de l'antigene

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DISIS M L, ET AL.: "GRANULOCYTE-MARCROPHAGE COLONY STIMULATING FACTOR: AN EFFECTIVE ADJUVANT FOR PROTEIN AND PEPTIDE-BASED VACCINES", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 88, no. 01, 1 July 1996 (1996-07-01), US, pages 202 - 210, XP002921134, ISSN: 0006-4971 *
LATTIME E C, ET AL.: "PHARMACOLOGY/THERAPEUTICS (PRECLINICAL AND CLINICAL) 3: GENE THERAPY", AMERICAN ASSOCIATION FOR CANCER RESEARCH. PROCEEDINGS OF THE ANNUAL MEETING, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 38, 1 March 1997 (1997-03-01), US, pages 11, XP002921136, ISSN: 0197-016X *
RIBAS A, ET AL.: "GENETIC IMMUNIZATION FOR THE MELANOMA ANTIGEN MART 1/MELAN-A USING RECOMBINANT ADENOVIRUS-TRANSDUCED MURINE DENDRITIC CELLS", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 57, no. 14, 1 July 1997 (1997-07-01), US, pages 2865 - 2869, XP002921135, ISSN: 0008-5472 *

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