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WO1999046992A1 - Induction de l'immunite contre des autoantigenes tumoraux - Google Patents

Induction de l'immunite contre des autoantigenes tumoraux Download PDF

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Publication number
WO1999046992A1
WO1999046992A1 PCT/US1999/006039 US9906039W WO9946992A1 WO 1999046992 A1 WO1999046992 A1 WO 1999046992A1 US 9906039 W US9906039 W US 9906039W WO 9946992 A1 WO9946992 A1 WO 9946992A1
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Prior art keywords
antigen
cells
cell
self
population
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PCT/US1999/006039
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English (en)
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Johanne Kaplan
Richard J. Gregory
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Genzyme Corporation
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Application filed by Genzyme Corporation filed Critical Genzyme Corporation
Priority to EP99912716A priority Critical patent/EP1071333A4/fr
Priority to CA002322624A priority patent/CA2322624A1/fr
Priority to JP2000536244A priority patent/JP2002506618A/ja
Priority to AU31029/99A priority patent/AU758265B2/en
Publication of WO1999046992A1 publication Critical patent/WO1999046992A1/fr

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    • 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—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
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A61K40/4245—Tyrosinase or 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
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4257—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4264—Cancer antigens from embryonic or fetal origin
    • A61K40/4266—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/4269—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • A61K40/4272—Melan-A/MART
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • A61K40/4273—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4274—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
    • A61K40/4275—Prostate specific antigen [PSA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4274—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
    • A61K40/4276—Prostate specific membrane antigen [PSMA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • TECHNICAL FIELD This invention is in the field of molecular immunology and medicine.
  • the present invention provides compositions and methods for inducing an immune response to a native self-antigen in a subject.
  • T and B cells are highly antigen specific and can develop into memory cells, and therefore are the target for a successful vaccine.
  • Tumor specific T cells derived from cancer patients, will bind and lyse tumor cells. This specificity is based on their ability to recognize short amino acid sequences (epitopes) presented on the surface of the tumor cells by MHC class I and class II molecules. These epitopes are derived from the proteolytic degradation of intracellular proteins called tumor antigens encoded by genes that are either uniquely or aberrantly expressed in tumor or cancer cells.
  • a critical target of vaccines is the specialized antigen-presenting cell ("APC"), the most immunologically powerful of which is the bone marrow-derived dendritic cell (“DC”).
  • APC antigen-presenting cell
  • DC bone marrow-derived dendritic cell
  • DCs are potent antigen presenters that express high levels of co- stimulatory molecules and are capable of activating both CD4 + and CD8 ⁇ naive T lymphocytes.
  • Results obtained in several animal models have shown that DCs pulsed with defined tumor-associated peptides or with peptides eluted from the surface of tumor cells are capable of inducing an antigen-specific CTL response resulting in protection from tumor challenge and, in some instances, regression of established tumors.
  • the same type of approach has also been tested in human clinical trials with encouraging results. For example, Hsu et al.
  • Mechanisms of systemic immune tolerance include deletion of potentially autoreactive B or T cells, induction of anergy in B and T cells, and the poorly defined phenomenon of suppression of immune response by suppressor cells. Houghton and Lewis, pages 37-54 in Forni. et al.. eds. (1994)
  • immunization is carried out with a heterologous antigen or an altered antigen that is structurally distinct from the self-antigen or "native" antigen yet is still capable of inducing an immune response against the self-antigen.
  • antigens are immunogenic (seen as foreign) and serve to induce an immune response that cross-reacts with the native antigen.
  • the invention comprises using modified (altered) tumor antigens or tumor antigens derived from heterologous species to break immunological tolerance and induce a cross-reactive immune response against the corresponding native or self-antigen.
  • immunizing humans against the human melanoma antigen gpl 00 requires breaking tolerance against a self-antigen.
  • the use of the non self-antigen can provide protective immunity and tumor reduction in vivo.
  • Immunization and therapy are accomplished by any of the following methods: 1) administration of a vector encoding altered tumor antigen or antigen from a heterologous species; 2) infecting dendritic cells ex vivo or in vivo with the same vector; or 3) use of transduced dendritic cells or APCs to stimulate production of an enriched population of antigen-specific immune effector cells that can be adoptively transferred into the host.
  • Antigen presenting cells such as dendritic cells also are useful to expand a population of immune effector cells that specifically recognize and lyse the cells presenting the heterologous antigen and its native or self-counterpart.
  • the expanded immune effector cell populations and their use in prophylactical and therapeutical methods also are provided herein.
  • BRIEF DESCRIPTION OF THE FIGURES Figures 1 A through ID show the results of immunizing mice with syngeneic DCs. Five female C57BL/6 mice (represented by five different symbols in the panels) were immunized with B16 melanoma using dendritic cells transfected with Ad vector encoding homologous mouse gplOO versus heterologous human gplOO.
  • Figures 2A through 2C show induction of CTL activity following immunization with Ad2/hugpl00vl vector or Ad2/hugpl00vl -transduced DCs.
  • Spleens from groups of 3 animals were collected 15 days after i.v. administration of vehicle ( Figure 2A), Ad2/hugpl00vl-transduced DCs ( Figure 2B) or i.d. delivery of Ad2/hugpl00vl vector ( Figure 2C).
  • Pooled spleen cells from each group were re-stimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2/hugpl00v 1 and were tested for cytolytic activity after 6 days of culture.
  • Targets consisted of B16 cells and SVB6KHA fibroblasts untransduced or transduced with Ad2/hugpl00v 1 or wild type Ad2 deleted for E3
  • Figure 3 compares the effectiveness of immunization with DCs transduced with Ad vector encoding various melanoma-associated antigens. The figure shows the evaluation of the nature of the antigen.
  • Groups of 5 C57BL/6 mice were injected i.v. with 5x10 5 DCs that were either untransduced or transduced with Ad2/hugpl00vl, Ad2/mgpl00 or Ad2/mTRP-2 vector.
  • the animals were challenged 15 days later with a s.c. injection of 2x10 B16 melanoma cells.
  • Figure 4 shows the frequency of gplOO-reactive splenic T lymphocytes following immunization with Ad2/hugpl00- or Ad2/empty vector-transduced DCs.
  • Spleen cells from immunized mice were stimulated in vitro with a cytotoxic T lymphocyte peptide epitope derived from hugpl OO (open bar); or the corresponding epitope from mgplOO (solid bar).
  • An ovalbumin-derived epitope was used as a negative control (hatched bar).
  • the number of T lymphocytes that produced ⁇ -interferon upon recognition of peptide was measured by elispot.
  • MOLECULAR BIOLOGY F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1989) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I.
  • a cell includes a plurality of cells, including mixtures thereof
  • cytokine refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • cytokines which may be used alone or in combination in the practice of the present invention include, interleukin-2 (IL-2).
  • SCF stem cell factor
  • IL-3 interleukin 3
  • IL-6 interleukin 6
  • IL-12 interleukin 12
  • G-CSF granulocyte macrophage-colony stimulating factor
  • IL-l ⁇ interleukin- 1 alpha
  • IL-1 1 interleukin-1 1
  • MlP-l ⁇ leukemia inhibitory factor
  • LIF leukemia inhibitory factor
  • c-kit ligand thrombopoietin
  • TPO thrombopoietin
  • flt3 ligand flt3 ligand.
  • the present invention also includes culture conditions in which one or more cytokine is specifically excluded from the medium. Cytokines are commercially available from several vendors such as, for example. Genzyme (Framingham. MA). Genentech (South San Francisco, CA). Amgen (Thousand Oaks, CA), R&D
  • APC antigen presenting cell
  • suitable APCs include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen presenting cells or other cell type(s) expressing the necessary MHC and co- stimulatory molecules. Methods of making hybrid APCs have been described. See, for example, International Patent Application Publication Nos. WO 98/46785 and WO 95/16775.
  • Dendritic cells are potent antigen-presenting cells. It has been shown that DCs provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types.
  • the first type which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 ("TCR/CD3”) complex and an antigenic peptide presented by a major histocompatibility complex (“MHC") class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T cell activation to occur.
  • MHC major histocompatibility complex
  • the first type of signals can result in T cell anergy.
  • the second type of signals called co- stimulatory signals, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals.
  • dendritic cell is to include, but not be limited to a pulsed dendritic cell, a foster cell . a dendritic cell hybrid or a genetically modified dendritic cell.
  • Methods for generating dendritic cells from peripheral blood or bone marrow progenitors have been described (Inaba et al. (1992) J. Exp. Med. 175:1 157; Inaba et al. (1992) J. Exp. Med. 176:1693-1702; Romani et al. (1994) J. Exp. Med. 180:83-93; Sallusto et al.
  • 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,
  • 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.
  • antigen is used in its broadest sense and includes minimal epitopes and chimeric molecules in addition to isolated full length proteins.
  • An example of a self-antigen is the melanoma antigen gp 100.
  • the antigen of this vaccine is "heterologous” (i.e., allogeneic or a homologe from an isolated species, e.g., a murine antigen administered to a human patient) or an "altered antigen" as compared to the corresponding native self-antigen.
  • the heterologous or altered antigen also can be made by chemical synthesis.
  • immune effector cells refers to cells capable of binding an antigen or which mediate an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates. Certain diseased tissues express specific antigens and CTLs specific for these antigens have been identified. For example, approximately 80% of melanomas express the antigen known as gplOO.
  • immune effector molecule refers to molecules capable of antigen-specific binding, and includes antibodies, T cell antigen receptors, and MHC Class I and Class II molecules.
  • a “na ⁇ ve” immune effector cell is an immune effector cell that has never been exposed to an antigen.
  • the term "educated, antigen-specific immune effector cell” is an immune effector cell as defined above, which has encountered antigen and which is specific for that antigen.
  • An educated, antigen-specific immune effector cell may be activated upon binding antigen. "Activated” implies that the cell is no longer in Go phase, and begins to produce cytokines characteristic of the cell type.
  • activated CD4+ T cells secrete IL-2 and have a higher number of high affinity IL-2 receptors on their cell surfaces relative to resting CD4+ T cells.
  • a peptide or polypeptide of the invention may be preferentially recognized by antigen-specific immune effector cells, such as B cells and T cells.
  • antigen-specific immune effector cells such as B cells and T cells.
  • the term "recognized" intends that a peptide or polypeptide of the invention, comprising one or more synthetic antigenic epitopes.
  • TCR T cell antigen receptor
  • a cell being administered to an individual is autogeneic if the cell was derived from that individual (the "donor") or a genetically identical individual.
  • An syngeneic cell can also be a progeny of an syngeneic cell.
  • the term also indicates that cells of different cell types are derived from the same donor or genetically identical donors.
  • an effector cell and an antigen presenting cell are said to be syngeneic if they were derived from the same donor or from an individual genetically identical to the donor, or if they are progeny of cells derived from the same donor or from an individual genetically identical to the donor.
  • the term "allogeneic” as used herein indicates the origin of a cell.
  • a cell being administered to individual is allogeneic if the cell was derived from an individual not genetically identical to the recipient; in particular, the term relates to non-identity in expressed MHC molecules.
  • An allogeneic cell can also be a progeny of an allogeneic cell.
  • the term also indicates that cells of different cell types are derived from genetically non- identical donors, or if they are progeny of cells derived from genetically non- identical donors.
  • an APC is said to be allogeneic to an effector cell if they are derived from genetically non-identical donors.
  • hybrid cell refers to a cell having both antigen presenting capability and also expresses one or more specific antigens.
  • these hybrid cells are formed by fusing, in vitro, APCs with cells that are known to express the one or more antigens of interest.
  • culture refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (either morphologically, genetically, or phenotypically) to the parent cell.
  • expansion is meant any proliferation or division of cells.
  • 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.
  • 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 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.
  • Such vectors can be
  • MHC major histocompatibility complex
  • HLA complex The proteins encoded by the MHC complex are known as "MHC molecules" and are classified into class I and class II MHC molecules.
  • Class I MHC molecules include membrane heterodimeric proteins made up of an ⁇ chain encoded in the MHC associated noncovalently with ⁇ 2- microglobulin.
  • Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8 + T cells.
  • Class I molecules include HLA-A, -B. and -C in humans.
  • Class II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated ⁇ and ⁇ chains.
  • Class II MHC are known to participate in antigen presentation to CD4 + T cells and, in humans, include HLA-DP, -DQ, and DR.
  • MHC restriction refers to a characteristic of T cells that permits them to recognize antigen only after it is processed and the resulting antigenic peptides are displayed in association with either a self class I or class II MHC molecule. Methods of identifying and comparing MHC are well known in the art and are described in Allen M. et al. (1994) Human lmm. 40:25; Santamaria P. et al. (1993) Human lmm. 37:39 and Hurley C.K. et al. (1997) Tissue Antigens 50:401.
  • peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a solid support, a detectable agent or label) or active, such as an adjuvant.
  • solid phase support or “solid support” used interchangeably, is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art.
  • Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, and alumina gels.
  • solid support also includes synthetic antigen-presenting matrices, cells, and liposomes. A suitable solid phase support may be selected on the basis of desired end use and suitability for various protocols.
  • solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE® resin (obtained from
  • a "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)).
  • 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.
  • immunomodulatory agent is a molecule, a macromolecular complex, or a cell that modulates an immune response and encompasses a synthetic antigenic peptide of the invention alone or in any of a variety of formulations described herein; a polypeptide comprising a synthetic antigenic peptide of the invention; a polynucleotide encoding a peptide or polypeptide of the invention; a synthetic antigenic peptide of the invention bound to a Class I or a Class II MHC molecule on an antigen-presenting matrix, including an APC and a synthetic antigen-presenting matrix (in the presence or absence of co-stimulatory molecule(s)); a synthetic antigenic peptide of the invention covalently or non-covalently complexed to another molecule(s) or macromolecular structure; and an educated, antigen-specific immune effector cell which is specific for a peptide of the invention.
  • modulate an immune response includes inducing (increasing, eliciting) an immune response; and reducing (suppressing) an immune response.
  • An immunomodulatory method is one that modulates an immune response in a subject.
  • the term "inducing an immune response in a subject” is a term well understood in the art and intends that an increase of at least about 2- fold, more preferably at least about 5-fold, more preferably at least about 10-fold, more preferably at least about 100-fold, even more preferably at least about 500- fold, even more preferably at least about 1000-fold or more in an immune response to an antigen (or epitope) can be detected (measured), after introducing the antigen (or epitope) into the subject, relative to the immune response (if any) before introduction of the antigen (or epitope) into the subject.
  • An immune response to an antigen includes, but is not limited to, production of an antigen-specific (or epitope-specific) antibody, and production of an immune cell expressing on its surface a molecule which specifically binds to an antigen (or epitope).
  • Methods of determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art.
  • antigen- specific antibody can be detected using any of a variety of immunoassays known in the art. including, but not limited to, ELISA. wherein, for example, binding of
  • an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably-labeled second antibody (e.g.. enzyme-labeled mouse anti-human Ig antibody).
  • a detectably-labeled second antibody e.g.. enzyme-labeled mouse anti-human Ig antibody.
  • Immune effector cells specific for the antigen can be detected any of a variety of assays known to those skilled in the art, including, but not limited to, 3 Cr-release assays, H-thymidine uptake assays or induction of cytokine release.
  • a disease or condition related to a population of CD4 + or CD8 + T cells is one which can be related to a population of CD4 + or CD8+ T cells, such that these cells are primarily responsible for the pathogenesis of the disease; it is also one in which the presence of CD4 + or CD8 + T cells is an indicia of a disease state; it is also one in which the presence of a population CD4 + or CD8 + T cells is not the primary cause of the disease, but which plays a key role in the pathogenesis of the disease; it is also one in which a population of CD4 + or CD8 T cells mediates an undesired rejection of a foreign antigen.
  • a condition related to a population of CD4 + or CD8 + T cells include, but are not limited to, autoimmune disorders, graft rejection, immunoregulatory disorders, and anaphylactic disorders.
  • Neoplastic cells As used herein, the terms “neoplastic cells”, “neoplasia”, “tumor”, “tumor cells”, “cancer” and “cancer cells”, (used interchangeably) refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division). Neoplastic cells can be malignant or benign.
  • “Suppressing” tumor growth indicates a growth state that is curtailed when compared to growth without treatment or prevention as described herein.
  • Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3 H-thymidine incorporation assay, or counting tumor cells.
  • “Suppressing” tumor cell growth means any or all of the following states: slowing, delaying, and stopping tumor growth, as well as tumor shrinkage.
  • “Host cell” or “recipient 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 peptides (or
  • the cells may be procaryotic or eucaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.
  • isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide. polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature.
  • an isolated polynucleotide is one that is separated from the 5" and 3' sequences with which it is normally associated in the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
  • a "concentrated” “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated” or less than “separated” than that of its naturally occurring counterpart.
  • a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide.
  • a protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.
  • compositions and methods are intended to mean that the compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define 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, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • compositions and methods of this invention provide protective immunity against growth of tumor cells in vivo and a means to inhibit the growth of tumors in vivo.
  • the methods also induce tumor reduction of established tumors in vivo.
  • heterologous/altered antigens can be delivered to antigen-presenting cells as protein/peptide or in the form of polynucleotide encoding the protein/peptide.
  • Antigen-presenting cells include but are not limited to dendritic cells (DCS), monocytes/macrophages, B-lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules.
  • DCS dendritic cells
  • monocytes/macrophages monocytes/macrophages
  • B-lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules.
  • DCS dendritic cells
  • monocytes/macrophages monocytes/macrophages
  • B-lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules.
  • the methods described below focus primarily on DCS which are the most potent, preferred APCs.
  • This invention also provides an isolated novel heterologous or altered antigen that is capable of inducing an immune response against a self-antigen in a subject, an isolated nucleic acid encoding the antigen, as well as vectors and host cells containing the
  • MAAs Melanoma- associated antigens
  • CTLs cytotoxic T lymphocytes
  • Dritic cells derived from bone marrow displayed surface markers characteristic of DCs and were functionally active in vitro as determined in a mixed lymphocyte reaction and as indicated by their ability to induce primary antigen-specific proliferation of syngeneic T lymphocytes.
  • the DCs were efficiently transduced with adenovirus type 2 (Ad2) based vectors while remaining phenotypically and functionally intact.
  • polypeptides and the polynucleotides encoding antigens of this invention can be, in one embodiment, the heterologous counterpart or an altered antigen of previously characterized tumor-associated antigens such as MUC-1 (Henderson et al. (1996) Cancer Res. 56:3763); MART-1 (Kawakami et al. (1994) Proc. Natl. Acad. Sci. 91:3515; Kawakami et al. (1997) Intern. Rev.
  • an heterologous or altered antigen corresponding to an epitope or wild-type antigenic peptide corresponding to a yet unidentified protein is also within the scope of this invention.
  • a common strategy in the search for tumor antigens is to isolate tumor-specific T-cells and attempt to identify the antigens recognized by these cells.
  • specific CTLs have been derived from lymphocytic infiltrates present at the tumor site. Weidmann et al., supra. These TILs are unique cell population that can be traced back to sites of disease when they are labeled with indium and adoptively transferred. Alternatively, large libraries of putative antigens can be produced and tested.
  • Solid-PHase Epitope REcovery (“SPHERE”, described in PCT WO 97/35035) described below, can be used to identify tumor antigens and altered antigens corresponding to self antigens. After identification and cloning of an altered antigen, the antigen or epitope can be expressed and purified for presentation to APC using the methods disclosed herein. In a further embodiment, the full-length native antigen can be selectively modified to encode or present the altered epitope using methods known in the art, e.g. PCR directed mutagenesis. Sambrook et al.. supra.
  • This invention further provides methods to elicit CD4 + and CD8 + T cells responses in a subject.
  • the induction of this immune response also is a means to assay a positive response to the therapy.
  • the presence of a large number of T- cells in tumor has been correlated with a prognostically favorable outcome in some cases (Whiteside and Purani (1994) Cancer Immunol. Immunother. 39:15). Woolley et al. (1995) Immmunology 84:55, has shown that implantation of polyurethane sponges containing irradiated tumor cells can efficiently trap anti- tumor CTLs (4-times greater than lymph fluid, 50-times greater than spleen or peripheral blood).
  • TILs Following activation with T-cell cytokines in the presence of their appropriately presented recognition antigen, TILs proliferate in culture and acquire potent anti-tumor cytolytic properties. Weidmann et al. (1994) Cancer Immunol. Immunother. 39:1. Assays to determine T cell response are well known in the art and any method that will compare T cell number and activity prior to and subsequent to therapy can be utilized. In addition, the induction of co- stimulatory cytokines by the heterologous/altered antigen could also stimulate pre-existing anergic or low affinity self-reactive CTL clones.
  • induction of cytotoxic T lymphocytes capable of lysing host tumor cells indicates that the antigenic peptide and/or nucleic acid of the screen is a potential therapeutic agent.
  • the methods of this invention can be further modified by co-administering more than one heterologous/altered antigen and/or an effective amount of a cytokine or co-stimulatory molecule or other transgene to the subject.
  • the antigen is administered to the subject either as a nucleic acid coding for the peptide/protein or by administering APC presenting the antigen.
  • the APC is a dendritic cell which includes, but is not limited to a pulsed or genetically modified dendritic cell.
  • the APC may be a foster antigen presenting cell. Methods of presenting the antigen to the APC are described herein.
  • the APC can be further genetically modified to co-express a cytokine alone, or in combination with a co-stimulatory molecule or other transgene.
  • the APC expressing a heterologous and/or altered antigen also can be used to expand and isolate a population of immune effectors which, in turn, are useful for adoptive immunotherapy alone or as an adjuvant to the methods described above.
  • cytokines and/or co-stimulatory molecules or nucleic acids encoding them can be co-administered with the immune effector cells.
  • the immune effector cells can be genetically modified to express a foreign nucleic acid encoding a cytokine or co-stimulatory molecule. Prior to administration in vivo, the immune effector cells are screened in vitro for their ability to lyse tumor cells.
  • the invention provides a method for cloning the cDNA and genomic DNA encoding such protein by generating degenerate oligonucleotides probes or primers based on the sequence of the epitope.
  • 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, Southern analysis and PCR.
  • 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 ( 1989) supra.
  • SAGE analysis can be employed to identify the antigens recognized by expanded immune effector cells such as CTLs. SAGE analysis involves identifying nucleotide sequences expressed in the antigen-expressing cells.
  • 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 over expressed in the antigen-expressing cell population can be identified.
  • cDNA complementary deoxyribonucleic acid
  • Expression cloning methodology as described in Kawakami et al. (1994) PNAS 91:3515, also can be used to identify a novel tumor-associated antigen. Briefly, in this method, 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.
  • An antigen identification method SPHERE is described in PCT WO 97/35035. Briefly, an empirical screening method for the identification of MHC Class I-restricted CTL epitopes is described 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 variable TCR epitope repertoire by randomizing the remaining positions. Roughly speaking. 50 96-well plates with 10,000 beads per well will accommodate a library with a complexity
  • the eluted peptides can be further pooled to yield wells with any desired complexity. Based on experiments with soluble libraries, it should be possible to screen 10 7 peptides in 96-well plates (10.000 peptides per well) with as few as 2 X 10 6 CTL cells. After cleaving a percentage of the peptides from the beads and incubating them with 51 Cr-labeled APCs (e.g., T2 cells) and the CTL line(s). peptide pools containing reactive species can be determined by measuring 5 l Cr-release according to standard methods known in the art.
  • cytokine production e.g., interferon- ⁇
  • proliferation e.g., incorporation of 3 H-thymidine
  • assays may be used. After identifying reactive 10,000-peptide mixtures, the beads corresponding to those mixtures are separated into smaller pools and distributed to new 96-well plates (e.g., 100 beads per well). An additional percentage of peptide is released from each pool and re-assayed for activity by one of the methods listed above. Upon identification of reactive 100-peptide pools, the beads corresponding to those peptide mixtures are redistributed at 1 bead per well of a new 96-well plate.
  • sequence of the peptides on individual beads can be determined by sequencing residual peptide bound to the beads by. for example, N-terminal
  • the epitope or antigen After isolation of the epitope or antigen, it can be expressed and purified using methods known in the art.
  • MARTI and gp 100 are melanocyte differentiation antigens specifically recognized by HLA-A2 restricted tumor-
  • TILs infiltrating lymphocytes
  • PNAS USA 91 :6458 and Kawakami Y. et al. (1994) PNAS USA 91 :91 :3515.
  • the mouse homologue 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 1 13 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 homologe 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.
  • Additional antigens include, but are not limited to HER-2/neu (U.S. Patent No. 5,550,214); MAGE (PCT/US92/04354); HPV16, 18E6 and E7 (Ressing et al. (1996) Cancer Res. 56(1):582; Restifo (1996) Current Opinion in Immunol. 8:658; Stern (1996) Adv. Cancer Res. 69:175; Tindle et al. (1995) Clin. Exp. Immunol. 101:265; and van Driel et al. (1996) Annals of Medicine 28:471);
  • CEA U.S. Patent No. 5,274,087
  • PSA Longwall A. (1989) Biochem. Biophys. Research Communications 161:1151
  • PSMA prostate membrane specific antigen
  • tyrosinase U.S. Patent Nos. 5,530,096 and 4,898,814, and Brichard et al. (1993) J. Exp. Med. 178:489)
  • tyrosinase related proteins 1 or 2 TRP-1 and TRP-2
  • NY-ESO-1 Choen et al.
  • APCs including DCs
  • At least two methods have been used for the generation of human dendritic cells from hematopoietic precusor cells in peripheral blood or bone marrow.
  • One approach utilizes the rare CD34+ precursor cells and stimulate them with GM-CSF plus TNF- ⁇ .
  • the other method makes use of the more abundant CD34- precursor population, such as adherent peripheral blood monocytes, and stimulate them with GM-CSF plus IL-4 (see. for example, Sallusto et al. (1994). supra).
  • the method described in Romani et al (1996), supra; and Bender et al (1996), supra is used to generate both immature and mature dendritic cells from the peripheral blood mononuclear cells (PBMC) of a mammal, such as a murine. simian or human.
  • PBMC peripheral blood mononuclear cells
  • isolated PBMC are pre-treated to deplete T- and B-cells by means of an immunomagnetic technique.
  • Lymphocyte-depleted PBMC are then cultured for 7 days in RPMI medium, supplemented with 1% autologous human plasma and GM-CSF/IL-4, to generate dendritic cells. Dendritic cells are nonadherence as opposed to their monocyte progenitor. Thus, on day 7, non-adherent cells are harvested for further processing.
  • the dendritic cells derived from PBMC in the presence of GM-CSF and IL-4 are immature, in that they can lost the nonadherence property and revert back to macrophage cell fate if the cytokine stimuli are removed from the culture.
  • the dendritic cells in an immature state are very effective in processing native protein antigens for the MHC class II restricted pathway (Romani et al. (1989) J. Exp. Med. 169:1 169. Further maturation of cultured dendritic cells is accomplished by culturing for 3 days in a macrophage-conditioned medium (CM), which contains the necessary maturation factors.
  • CM macrophage-conditioned medium
  • Mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells (both CD4+ and CD8+) to grow and differentiate.
  • Mature dendritic cells can be identified by their change in morphology, such as the formation of more motile cytoplasmic processes; by their nonadherence; by the presence of at least one of the following markers: CD83, CD68, HLA-DR or CD86; or by the loss of Fc receptors such as CD115 (reviewed in Steinman ( ⁇ 99 ⁇ ) Annu. Rev. Immunol. 9:271.) More specifically, the method requires collecting an enriched collection of white cells and platelets from leukapheresis that is then further fractionated by
  • CCE countercurrent centrifugal elutriation
  • 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); CD14 (monocyte); CD16, 56, 57 (NK/LAK cells); CD19, 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.
  • 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. It may not be necessary to routinely separate DCs from monocytes because, as will be detailed below, the monocytes themselves are still capable of differentiating into DCs or functional DC-like cells in culture.
  • 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 G-CSF, GM- CSF, IL-2, and IL-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.
  • Peptide fragments from antigens must first be bound to peptide binding receptors (major histocompatibility complex 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 CD4 + T cells recognize antigenic peptides bound to MHC class II molecules whereas the receptors on CD8 + T cells 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.
  • APCs 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.
  • Antigen Pulsing 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 E.P. and Rock K.L. (1992) J. Immunol. 148: 13; Norbury, C. C. et al. (1995) Immunity 3:783; and Reise-Sousa C. and Germain R.N. (1995) J. Exp. Med.
  • the proteins were expressed in Schneider S2 Drosophila melanogaster cells, known to support GPI- modification. After purification, the proteins could be incubated together with a purified antigenic peptide which resulted in a trimolecular complex capable of efficiently inserting itself into the membranes of autologous cells. In essence, these protein mixtures were used to "paint" the APC surface, conferring the ability to stimulate a CTL clone that was specific for the antigenic peptide. Cell coating was shown to occur rapidly and to be protein concentration dependent. This method of generating APCs bypasses the need for gene transfer into the APC and permits control of antigenic peptide densities at the cell surfaces.
  • 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, LMP1, and LMP2, which are required for antigen presentation to MHC class 1 -restricted CD8 + CTLs. In effect, only "empty" MHC class I molecules are presented on the surface of these cells. Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif. These T2 cells are referred to herein as "foster"
  • 29 APCs They can be 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.
  • the present invention makes use of these APCs to stimulate production of an enriched population of antigen-specific immune effector cells.
  • the antigen-specific immune effector cells are expanded at the expense of the APCs, which die in the culture.
  • the process by which na ⁇ ve immune effector cells become educated by other cells is described essentially in Coulie (1997) Molec. Med. Today 3: 261.
  • the substantially pure populationof educated, antigen-specific immune effector cells produced by this method are useful to cause tumor regression.
  • the APCs e.g., DCs
  • the APCs presenting the heterologous/altered antigen are mixed with na ⁇ ve immune effector cells.
  • the cells may be cultured in the presence of a cytokine, for example IL2.
  • cytokine for example IL2.
  • IL-12 potent immunostimulatory cytokines
  • the culture conditions are such that the antigen-specific immune effector cells expand (i.e., proliferate) at a much higher rate than the APCs.
  • Multiple infusions of APCs and optional cytokines can be performed to further expand the population of antigen-specific cells.
  • the immune effector cells are T cells.
  • the immune effector cells can be genetically modified by transduction with a transgene coding for example, IL-2, IL-1 1 or IL-13.
  • a transgene coding for example, IL-2, IL-1 1 or IL-13.
  • An effector cell population suitable for use in the methods of the present invention can be autologous or allogeneic, preferably autologous.
  • effector cells are allogeneic, preferably the cells are depleted of alloreactive cells before use. This can be accomplished by any known means, including, for example, by mixing the allogeneic effector cells and a recipient cell population and incubating them for a suitable time, then depleting CD69 + cells, or inactivating alloreactive cells, or inducing anergy in the alloreactive cell population.
  • Hybrid immune effector cells can also be used. Immune effector cell hybrids are known in the art and have been described in various publications. See, for example, International Patent Application Nos. WO 98/46785 and WO
  • the effector cell population can comprise unseparated cells, i.e., a mixed population, for example, a PBMC population, whole blood, and the like.
  • the effector cell population can be manipulated by positive selection based on expression of cell surface markers, negative selection based on expression of cell surface markers, stimulation with one or more antigens in vitro or in vivo, treatment with one or more biological modifiers in vitro or in vivo, subtractive stimulation with one or more antigens or biological modifiers, or a combination of any or all of these.
  • Effector cells can obtained from a variety of sources, including but not limited to, PBMC, whole blood or fractions thereof containing mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukopharesis, biopsy tissue, lymph nodes, e.g., lymph nodes draining from a tumor.
  • Suitable donors include an immunized donor, a non-immunized (na ⁇ ve) donor, treated or untreated donors.
  • a "treated" donor is one that has been
  • effector cells can be obtained by leukopharesis, mechanical apheresis using a continuous flow cell separator.
  • lymphocytes and monocytes can be isolated from the buffy coat by any known method, including, but not limited to, separation over Ficoll-HypaqueTM gradient, separation over a Percoll gradient, or elutriation.
  • concentration of Ficoll-HypaqueTM can be adjusted to obtain the desired population, for example, a population enriched in T cells. Other methods based on affinity are known and can be used.
  • Affinity-based methods may utilize antibodies, or portions thereof, which are specific for cell-surface markers and which are available from a variety of commercial sources, including, the American Type Culture Collection (Manassas, VA). Affinity-based methods can alternatively utilize ligands or ligand analogs, of cell surface receptors.
  • the effector cell population can be subjected to one or more separation protocols based on the expression of cell surface markers.
  • the cells can be subjected to positive selection on the basis of expression of one or more cell surface polypeptides, including, but not limited to. "cluster of differentiation" cell surface markers such as CD2, CD3, CD4, CD8, TCR, CD45, CD45RO, CD45RA, CD1 lb, CD26, CD27, CD28, CD29, CD30, CD31, CD40L; other markers associated with lymphocyte activation, such as the lymphocyte activation gene 3 product (LAG3), signaling lymphocyte activation molecule (SLAM), T1/ST2; chemokine receptors such as CCR3, CCR4.
  • LAG3 lymphocyte activation gene 3 product
  • SLAM signaling lymphocyte activation molecule
  • the effector cell population can be subjected to negative selection for depletion of non-T cells and/or particular T cell subsets. Negative selection can be performed on the basis of cell surface expression of a variety of molecules, including, but not limited to.
  • B cell markers such as CD 19, and CD20; monocyte marker CD 14; the NK cell marker CD56.
  • An effector cell population can be manipulated by exposure, in vivo or in vitro, to one or more biological modifiers.
  • suitable biological modifiers include, but are not limited to, cytokines such as IL-2, IL-4, IL-10, TNF- ⁇ . IL-12. IFN- ⁇ ; non-specific modifiers such as phytohemagglutinin (PHA), phorbol esters such as phorbol myristate acetate (PMA). concanavalin-A, and ionomycin; antibodies specific for cell surface markers, such as anti-CD2, anti-CD3, anti-IL2 receptor, anti-CD28; chemokines, including, for example, lymphotactin.
  • cytokines such as IL-2, IL-4, IL-10, TNF- ⁇ . IL-12. IFN- ⁇
  • non-specific modifiers such as phytohemagglutinin (PHA), phorbol esters such as phorbol myristate acetate (PMA). concanavalin-A
  • the biological modifiers can be native factors obtained from natural sources, factors produced by recombinant DNA technology, chemically synthesized polypeptides or other molecules, or any derivative having the functional activity of the native factor. If more than one biological modifier is used, the exposure can be simultaneous or sequential.
  • the present invention provides compositions comprising immune effector cells, which may be T cells, enriched in antigen-specific cells, specific for a peptide of the invention. By “enriched” is meant that a cell population is at least about 50-fold, more preferably at least about 500-fold, and even more preferably at least about 5000-fold or more, enriched from an original naive cell population.
  • the proportion of the enriched cell population which comprises antigen-specific cells can vary substantially, from less than 10% up to 100% antigen-specific cells. If the cell population comprises at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%, antigen-specific immune effector cells, specific for a peptide of the invention, then the population is said to be "substantially pure".
  • the percentage which are antigen-specific can readily be determined, for example, by a 3 H-thymidine uptake assay or cytokine release assay in which the effector cell population (for example, a T-cell population) is challenged by an antigen-presenting matrix presenting an antigenic peptide of the invention. Assaying Antigen-Specificity
  • DCs will be used to present antigen to autologous peripheral blood lymphocytes.
  • the DCs can be pulsed or transduced.
  • TAA-specific cells can be measured by several methods including proliferation or cytokine production (e.g. TNF- ⁇ , interferon- ⁇ ) upon exposure to TAA or lysis of TAA-expressing target cells as assessed by release of various intracellular labels/markers such as 51 Chromium or lactose dehydrogenase (LDH).
  • proliferation or cytokine production e.g. TNF- ⁇ , interferon- ⁇
  • lysis of TAA-expressing target cells as assessed by release of various intracellular labels/markers such as 51 Chromium or lactose dehydrogenase (LDH).
  • the antigen that induces the strongest response (in particular cytolytic activity) against the native human antigen would then be selected for immunization purposes.
  • the methods of this invention also can use newly identified antigens which can be identified as exemplified below.
  • heterologous/altered antigens and peptides of the present invention can be synthesized using an appropriate solid state synthetic procedure.
  • an isolated peptide of the invention may be purified by standard methods including chromatography (e.g.. ion exchange, affinity, and sizing column chromatography), centrifugation. differential solubility, or by any combination thereof
  • an epitope may be isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
  • affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Methods Enzymol. 194:508), and glutathione-S-transferase can be attached to the peptides of the invention to allow easy purification by passage over an appropriate affinity column.
  • Isolated peptides can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
  • antigenic peptides that are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, crosslinking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285).
  • isolated nucleic acid sequences that encode the novel antigenic peptides described herein.
  • isolated means: an RNA or DNA polymer, portion of genomic nucleic acid, cDNA, or synthetic nucleic acid which, by virtue of its origin or manipulation: (i) is not associated with all of a nucleic acid with which it is associated in nature (e.g. is present in a host cell as a portion of an expression vector); or (ii) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature.
  • isolated it is further meant a nucleic acid sequence: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.
  • PCR polymerase chain reaction
  • nucleic acid sequences of the present invention may be characterized, isolated, synthesized and purified using no more than ordinary skill. See Sambrook et al. ( 1989) supra.
  • compositions containing any of the above- mentioned proteins, muteins, polypeptides, nucleic acid molecules, vectors, cells antibodies and fragments thereof, and an acceptable solid or liquid carrier.
  • compositions are used pharmaceutically, they are combined with a
  • compositions for diagnostic and therapeutic use. These compositions also can be used for the preparation of medicaments for the diagnosis and treatment of diseases such as cancer.
  • the murine B 16 melanoma model was used.
  • C57BL/6 mice were immunized with bone marrow-derived DCs transduced with an Ad vector encoding either human gpl OO (Ad/hugplOO) or mouse gplOO (Ad/mgplOO).
  • Mice immunized against heterologous human gplOO developed a protective immune response and were resistant to a lethal subcutaneous challenge of B 16 melanoma cells (syngeneic tumor cell line that expresses gplOO).
  • mice immunized with homologous mouse gplOO failed to mount a protective immune response against B16 melanoma cells and developed tumors at the site of B16 cell injection.
  • This finding illustrates the difficulty in breaking tolerance against a self antigen (mouse gplOO).
  • the corresponding heterologous antigen from a different species is likely to contain several Class I and Class II- associated epitopes that will be recognized as foreign and elicit CD8 + and CD4 + T cell responses, respectively.
  • the induction of cross-reactive CTLs that recognize both the heterologous and homologous self-antigen can then lead to lysis of host tumor cells.
  • T lymphocytes and can be reconstituted with human PBLs. It may be possible to immunize such mice with test antigen to induce a response in adoptively transferred human PBLs and evaluate protection against challenge with a human tumor cell line (Mosier et al. (1988) Nature 335:256; Parney et al. (1997) Human Gene Therapy 8:1073; and Albert et al. (1997) J. Immunol. 159:1393).
  • HLA-A2.1 transgenic mice Another possibility is immunization of HLA-A2.1 transgenic mice to reproduce the immune reactivity of HLA-A2 individuals (Wentworth et al. (1996) Eur. J. Immunol. 26:97).
  • genetic modifications of cells employed in the present invention are accomplished by introducing a vector containing a polypeptide or transgene encoding a heterologous or an altered antigen.
  • 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.
  • APC and immune effector cells can be modified using the methods described below or by any other appropriate method known in the art.
  • 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)
  • adenoviral vectors based on the human adenovirus 5 are missing essential early genes from the adenoviral genome (usually E1A/E1B). 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.
  • adenovirus-based gene transfer does not result in integration of the transgene into the host genome (less than 0.1% adenovirus-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 E1A/E1B genes, typify useful permissive cell lines.
  • adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario (see, e.g.. Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996). See also, the papers by Vile et al. (1997) Nature Biotechnology 15:840; and Feng et al. (1997) Nature Biotechnology, 15:866, describing the construction and use of adeno-retroviral chimeric vectors that can be employed for genetic modifications.
  • APCs can be transduced with viral vectors encoding a relevant antigen.
  • the most common viral vectors include recombinant poxviruses such as vaccinia and fowlpox virus (Bronte et al. (1997) PNAS 94:3183: and Kim et al. (1997) J. Immunother. 20:276) and, preferentially, adenovirus (Arthur et al. (1997) J. Immunol. 159:1393; Wan et al. (1997) Human Gene Therapy 8:1355; and Huang et al. (1995) J. Virol. 69:2257). Retrovirus also may be used for transduction of human APCs (Marin et al. (1996) J. Virol. 70:2957).
  • adenovirus (Ad) vector at a multiplicity of infection (MOI) of 500 for 16-24 hours in a minimal volume of serum-free medium reliably gives rise to transgene expression in 90-100% of DCs.
  • MOI multiplicity of infection
  • the efficiency of transduction of DCs or other APCs can be assessed by immunofluorescence using fluorescent antibodies specific for the tumor antigen being expressed (Kim et al. (1997) J. Immunother. 20:276).
  • the antibodies can be conjugated to an enzyme (e.g. HRP) giving rise to a colored product upon reaction with the substrate.
  • the actual amount of antigen being expressed by the APCs can be evaluated by ELISA.
  • Transduced APCs can subsequently be administered to the host via an intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
  • In vivo transduction of DCs, or other APCs can be accomplished by administration of Ad (or other viral vectors) via different routes including intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery.
  • Ad or other viral vectors
  • the preferred method is cutaneous delivery of Ad vector at multiple sites using a total dose of approximately lxl ⁇ '°-lx 10 12 i.u.
  • Levels of in vivo transduction can be roughly assessed by co-staining with antibodies directed against APC marker(s) and the antigen being expressed.
  • the staining procedure can be carried out on biopsy samples from the site of administration or on cells from draining lymph
  • DCs can also be transduced in vitro/ex vivo by non-viral gene delivery methods such as electroporation, calcium phosphate precipitation or cationic lipid/plasmid DNA complexes (Arthur et al. (1997) Cancer Gene Therapy 4:17).
  • Transduced APCs can subsequently be administered to the host via an intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
  • DCs. or other APCs can potentially be accomplished by administration of cationic lipid/plasmid DNA complexes delivered via the intravenous, intramuscular, intranasal, intraperitoneal or cutaneous route of administration.
  • Gene gun delivery or injection of naked plasmid DNA into the skin also leads to transduction of DCs (Condon et al. (1996) Nature Med. 2:1122; and Raz et al. (1994) PNAS 91:9519).
  • Intramuscular delivery of plasmid DNA may also be used for immunization (Rosato et al. (1997) Human Gene Therapy 8:1451. The transduction efficiency and levels of transgene expression can be assessed as described herein.
  • Dendritic cells derived from peripheral blood of a subject such as a human patient are transduced with adenovirus vector encoding the tumor antigen using a multiplicity of infection of 200-500. Approximately 24 hours after infection, the transfected dendritic cells (10 xlO 7 cells) are administered to the patient iv or subcutaneously. The process is repeated 3-4 weeks later with up to 6 administrations of dendritic cells. Since it is possible to freeze dendritic cells and administer thawed cells, the subject does not have to be leukopharesed each time.
  • the agents identified herein as effective for their intended purpose can be administered to subjects having tumors or to individuals susceptible to or at risk of developing a tumor by inducing an immune response against the tumor.
  • 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.
  • Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment.
  • 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 nasal, topical (including transdermal, aerosol, buccal and sublingual), 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.
  • tumors expressing the antigen can be eradicated using the methods and compositions described herein.
  • Adoptive immunotherapy methods involve, in one aspect, administering to a subject a substantially pure population of educated, antigen-specific immune effector cells made by culturing na ⁇ ve immune effector cells with APCs as described above.
  • the APCs are dendritic cells.
  • the adoptive immunotherapy methods described herein are autologous.
  • the APCs are made using cells isolated from a single subject.
  • the expanded population also employs T cells isolated from that subject.
  • the expanded population of antigen-specific cells is administered to the same patient.
  • APCs or immune effector cells are administered with an effective amount of a stimulatory cytokine, such as IL-2 or a co- stimulatory molecule.
  • a stimulatory cytokine such as IL-2 or a co- stimulatory molecule.
  • mice Female C57BL/6 mice were purchased from Taconic (Germantown, NY) and were used at 8-12 weeks of age. Syngeneic SV40-transformed fibroblasts
  • B16.F 10 melanoma cell line syngeneic to C57BL/6 mice was obtained from the National Cancer Institute (Bethesda, MD).
  • B16.F10 cells (1.5-2xl0 4 cells) were resuspended in phosphate-buffered saline (PBS) and delivered to the abdomen subcutaneously (s.c.) in a 100 ⁇ l volume. Tumor size was measured with electronic digital calipers 3 times per week starting around day
  • Tumors >3 mm in size were scored as positive.
  • Ad vectors used were derived from Ad serotype 2 from which the El region was deleted and replaced with an expression cassette containing a cytomegalovirus (CMV) promoter driving expression of the transgene.
  • CMV cytomegalovirus
  • the vector encoding ⁇ -Galactosidase (Ad2/ ⁇ Gal-4) and human gplOO (Ad2/hugpl00vl) contained intact E3 and E4 regions (Armentano D. (1997) J. Virol. 71:2408 and Zhai Y. (1996) J. Immunol. 156:7001.
  • the vector encoding murine gpl OO (Ad2/mgpl00) or vector lacking a transgene (Ad2/empty vector), possessed an intact E3 region with an E4 region modified by removal of all open reading frames and replacement with the E4 open reading frame 6 and protein IX moved from its original location (Armentano D. (1985) Human Gene Therapy 6: 1343).
  • Ad vector encoding murine tyrosinase-related protein 2 (Ad2/mTRP-2) contained an intact E4 region but was deleted for E3. The E2 region was left intact in all vectors.
  • Adenoviral particles were gradient-purified as previously described (Armentano D. (1985) Human Gene Therapy 6:1343) and titers were determined by end-point dilution on 293 cells using fluorescent isothiocyanate (FITC)- conjugated anti-hexon antibody (Rich D.P. (1993) Human Gene Therapy 4:461).
  • FITC fluorescent isothiocyanate
  • DCs Dendritic cells
  • Bone marrow cells were then treated with a cocktail of antibodies (Pharmingen, San Diego, CA) directed against CD8 (clone 53-6.7), CD4 (clone GK1.5), CD45R/B220 (clone RA3-6B2), Ly-6G/Gr-1 (clone RB6-8C5) and la (clone KH74) followed by rabbit complement (Accurate Chemical and Scientific Corporation, Westbury. N.Y.) to deplete lymphocytes, granulocytes and Ia + cells. The remaining cells were cultured for 6 days in 6-well plates in RPMI-1640 medium (Gibco, Grand Island,
  • DCs were first incubated with unlabeled antibodies (Pharmingen) specific for the major histocompatibility complex (MHC) Class I (clone AF6-88.5) and Class II (clone AF6-120.1) molecules, the co-stimulatory molecules B7.1 (CD80; clone IG 10) and B7.2 (CD86; clone GL-
  • MHC major histocompatibility complex
  • B7.1 CD80; clone IG 10
  • B7.2 CD86; clone GL-
  • Transduction of DCs with Ad vector was conducted in 6- well plates with 4xl0 6 DCs/well in a 3 ml volume of RPMI-1640 medium containing 10% FCS and 100 ng/ml GM-CSF.
  • Virus was added to the wells at a multiplicity of infection (MOI) of 500 and the DCs were collected after 18-24 hours of incubation.
  • MOI multiplicity of infection
  • transduced DCs were washed and resuspended in a 100 ⁇ l volume of PBS and delivered either s.c. to the abdomen or intravenously (i.v.) into the tail vein as specified in the text.
  • Cytotoxic T cell assay To evaluate levels of cytotoxic T lymphocyte (CTL) activity, spleen cells from mice in the same treatment group (3 mice/group) were pooled and stimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2 vector at an MOI of 100 for 24 hours. Cells were cultured in 24-well plates containing 5x10 6 spleen cells and 0.8-1.5x10 5 stimulator fibroblasts per well in a 2 ml volume. Cytolytic activity was assayed after 6 days of incubation. Target cells consisted of B16 melanoma cells and fibroblasts untransduced or transduced with virus at
  • Targets were treated with 100 U/ml recombinant mouse ⁇ -interferon (Genzyme) for 24 hours labeled with ''Chromium (51-Cr;
  • the amount of 51 Cr spontaneously released was obtained by incubating target cells in medium alone and the total amount of 5 l Cr incorporated was determined by adding 1% Triton X-100 in distilled water. The percentage lysis was calculated as follows:
  • Ad2/hugpl00- or Ad2/empty vector-transduced DCs (4 mice/group) were pool and stimulated with H-2 b -restricted CTL epitopes derived from human gplOO
  • KVPRNQDWL murine gplOO
  • EVSRNQDWL murine gplOO
  • ovalbumin ovalbumin as a negative control
  • DCs Dendritic cells derived from mouse bone marrow exhibited the veiled dendrite morphology typical of DCs and displayed a characteristic set of DC surface markers (Crowley M. (1989) Cell. Immunol. 118: 108) as determined by FACS analysis (Table 2).
  • the cells expressed high levels of the major histocompatibility (MHC) Class I and Class II molecules, the co-stimulatory molecules B7.1 and B7.2, the ICAM-I adhesion molecule, the integrin CDl lc and the CD 13 myeloid surface marker.
  • MHC major histocompatibility
  • Ad2- based vectors was achieved reproducibly with an efficiency of 90% or greater. Transduction did not affect the distribution of DC surface markers significantly except for a reproducible increase in levels of MHC Class I molecules (Table 2).
  • Results shown are the percentage of bone marrow-derived DCs staining positive for each marker.
  • DCs were untransduced or transduced with Ad2/ ⁇ Gal-4.
  • DCs Induction of tumor-specific cytotoxic T lymphocyte response by transduced dendritic cells
  • CTL cytotoxic T lymphocyte
  • MAA melanoma-associated antigen
  • mice 46 transduced DCs (5x10 3 ) were administered intravenously (i.v.) to C57BL/6 mice and. 15 days later, spleens were collected for assessment of CTL activity. Separate groups of mice were also treated with vehicle as a negative control or with the Ad2/hugpl00vl vector itself for comparison. The vector was delivered under conditions previously determined to be optimal for immunization (3 x 10 9 i.u, intradermally).
  • effector splenocytes were tested for cytolytic activity against 3 Cr-labeled target fibroblasts that were either untransduced or transduced with Ad2/hugpl00vl or wild-type (WT) E3-deleted Ad (Ad2 ⁇ 2.9).
  • WT wild-type E3-deleted Ad
  • mice treated with vehicle failed to develop any significant CTL activity against any of the targets ( Figure 2 A).
  • CTLs from mice immunized with transduced DCs and, to a lesser extent, with Ad vector were both able to lyse B 16 tumor cells indicating that the CTLs raised against human gplOO also recognized the endogenous mouse gplOO expressed by the tumor cells ( Figures 2B and 2C).
  • mice Groups of 5 C57BL/6 mice were immunized against the gplOO melanoma antigen with an intravenous injection of 5 x 10 ? bone marrow-derived dendritic cells (DCs) transfected with adenovirus vector encoding mouse gpl OO (Ad2/mgp 100 DCs) or human gpl 00 (Ad2/hugpl 00 DCs). Uninfected DCs served as a negative control. Two weeks after immunization, the mice were challenged with a subcutaneous injection of 2 x 10 B16 melanoma cells and tumor growth was monitored over time.
  • DCs bone marrow-derived dendritic cells

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Abstract

L'invention concerne des méthodes et des compositions permettant de rompre la tolérance à un autoantigène, en particulier dans le contexte d'un antigène associé aux tumeurs. Dans un mode de réalisation de cette invention, des antigènes tumoraux modifiés ou des antigènes tumoraux dérivés d'espèces hétérologues sont utilisés pour rompre la tolérance immunologique, et pour induire une réponse immunitaire à réaction croisée contre l'autoantigène naturel correspondant.
PCT/US1999/006039 1998-03-20 1999-03-19 Induction de l'immunite contre des autoantigenes tumoraux WO1999046992A1 (fr)

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EP99912716A EP1071333A4 (fr) 1998-03-20 1999-03-19 Induction de l'immunite contre des autoantigenes tumoraux
CA002322624A CA2322624A1 (fr) 1998-03-20 1999-03-19 Induction de l'immunite contre des autoantigenes tumoraux
JP2000536244A JP2002506618A (ja) 1998-03-20 1999-03-19 腫瘍自己抗原に対する免疫誘導
AU31029/99A AU758265B2 (en) 1998-03-20 1999-03-19 Induction of immunity against tumor self-antigens

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WO2002070711A1 (fr) * 2001-03-03 2002-09-12 Glaxo Group Limited Vaccin
WO2003103706A2 (fr) * 2002-06-11 2003-12-18 Glaxosmithkline Biologicals S.A. Compositions immunogenes
WO2002072796A3 (fr) * 2001-03-12 2004-01-08 Cellcure Aps Lignees cellulaires de lymphocytes t humaines normales continues, comprenant un recepteur immun recombine avec specificite antigenique definie
US7786278B2 (en) 2002-04-09 2010-08-31 Sanofi Pasteur Limited Modified CEA nucleic acid and expression vectors
US7851212B2 (en) 2000-05-10 2010-12-14 Sanofi Pasteur Limited Immunogenic polypeptides encoded by MAGE minigenes and uses thereof
US8562970B2 (en) 2003-10-08 2013-10-22 Sanofi Pasteur Limited Modified CEA/B7 vector
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030847A1 (fr) * 1999-10-22 2001-05-03 Aventis Pasteur Limited Molecule gp100 modifiee et ses applications
US7851212B2 (en) 2000-05-10 2010-12-14 Sanofi Pasteur Limited Immunogenic polypeptides encoded by MAGE minigenes and uses thereof
WO2002070711A1 (fr) * 2001-03-03 2002-09-12 Glaxo Group Limited Vaccin
WO2002072796A3 (fr) * 2001-03-12 2004-01-08 Cellcure Aps Lignees cellulaires de lymphocytes t humaines normales continues, comprenant un recepteur immun recombine avec specificite antigenique definie
US7786278B2 (en) 2002-04-09 2010-08-31 Sanofi Pasteur Limited Modified CEA nucleic acid and expression vectors
WO2003103706A2 (fr) * 2002-06-11 2003-12-18 Glaxosmithkline Biologicals S.A. Compositions immunogenes
WO2003103706A3 (fr) * 2002-06-11 2004-02-19 Glaxosmithkline Biolog Sa Compositions immunogenes
US8562970B2 (en) 2003-10-08 2013-10-22 Sanofi Pasteur Limited Modified CEA/B7 vector
US8895017B2 (en) 2010-06-07 2014-11-25 Pfizer Inc. HER-2 peptides and vaccines

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