EP1064354A1 - Compositions et methodes pour provoquer une reponse des cellules t par des vaccins a base de genes - Google Patents

Compositions et methodes pour provoquer une reponse des cellules t par des vaccins a base de genes

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Publication number
EP1064354A1
EP1064354A1 EP99912709A EP99912709A EP1064354A1 EP 1064354 A1 EP1064354 A1 EP 1064354A1 EP 99912709 A EP99912709 A EP 99912709A EP 99912709 A EP99912709 A EP 99912709A EP 1064354 A1 EP1064354 A1 EP 1064354A1
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Prior art keywords
antigen
cells
cell
polynucleotide
ofthe
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EP99912709A
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German (de)
English (en)
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EP1064354A4 (fr
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Bruce L. Roberts
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Genzyme Corp
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Genzyme Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464491—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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A61K39/464492—Glycoprotein 100 [Gp100]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma

Definitions

  • TECHNICAL FIELD This invention is in the field of molecular immunology and medicine.
  • the present invention provides compositions and methods for inducing CD4 and CD8 T cell responses in a subject.
  • the mammalian immune system is capable of generating responses to foreign antigens, to self antigens present on cancerous cells, and to self antigens present on normal tissues.
  • the immune system comprises two types of antigen- specific cells: B cells and T cells.
  • B cells synthesize both membrane-bound and secreted antibody.
  • T cells can be characterized phenotypically by the manner in which they recognize antigen, by their cell surface markers, and by their secreted products.
  • T cells express distinctive membrane molecules. Included among these are the T cell antigen receptor (TCR), which appears on the cell surface in association with CD3; and accessory molecules such as CD5, CD28 and CD45R. Subpopulations of T cells can be distinguished by the presence of additional membrane molecules.
  • TCR T cell antigen receptor
  • T cells that express CD4 recognize antigen associated with class II MHC molecules and generally function as helper cells whose roles include enhancement of antibody production by B cells
  • T cells that express CD8 recognize antigen associated with class I MHC molecules and generally function as cytotoxic cells.
  • Immune cells recognize discrete sites, known as epitopes or antigenic determinants, on the antigen. Epitopes are regions of an immunogen or antigen that bind to antigen-specific membrane-bound receptors on immune cells or to their soluble counterparts, such as antibodies. Both membrane-bound antibody on the surface of a B lymphocyte and secreted antibody recognize soluble antigen. Unlike B cells, which recognize soluble antigen, T cells recognize antigen only when the antigen is associated with self major histocompatibility complex (MHC) gene products on the surface of an antigen presenting cell. This antigen can be displayed together with MHC molecules on the surface of antigen-presenting cells or on virus-infected cells, cancer cells, and grafts.
  • MHC self major histocompatibility complex
  • Disease states can result from invasion by a pathogenic organisms, including bacterial, viral, and protozoan pathogens, and subsequent inefficient or ineffective immune response to the invader. Disease states can also result from the activation of self-reactive T lymphocytes, from the activation of T lymphocytes that provoke allergic reactions, or from the activation of autoreactive T lymphocytes following certain bacterial and parasitic infections, which can produce antigens that mimic human protein, rendering these protein "autoantigens”.
  • diseases include, but are not limited to, the autoimmune diseases, autoimmune disorders that occur as a secondary event to infection with certain bacteria or parasites, T cell mediated allergies, and certain skin diseases such as psoriasis and vasculitis.
  • undesired rejection of a foreign antigen can result in graft rejection or even infertility, and such rejection may be due to activation of specific T lymphocyte populations.
  • Foreign antigens include macromolecules associated with pathogens such as bacteria, viruses, and protozoans; allergens; and allografts.
  • Self antigens under normal physiological conditions, are usually non- immunogenic. However, self antigens can also be immunogenic. as is the case with autoimmune diseases. Autoimmune diseases affect approximately 5% of adults in Europe and North America, often causing chronic debilitating illnesses. Steinman ( 1993) Scientific American 269: 107- 114. Autoimmunity is characterized by activation of auto-reactive clones of T or B cells, generating humoral or cell-mediated responses against self-antigens.
  • Tumor cell antigens are also self antigens, and frequently do not elicit an immune response that results in elimination ofthe cancerous cells effective to control or eliminate the disease.
  • induction of an immune response to a self antigen is desirable.
  • these include the induction of an immune response to certain antigens as a means of contraception.
  • the introduction into an animal of an antigen has been widely used for the purposes of modulating the immune response, or lack thereof, to the antigen for a variety of purposes.
  • These include vaccination against pathogens, induction of an immune response to a cancerous cell, reduction of an allergic response, reduction of an immune response to a self antigen that occurs as a result of an autoimmune disorder, reduction of allograft rejection, and induction of an immune response to a self antigen for the purpose of contraception.
  • a critical target of vaccines is the specialized professional antigen- presenting cell ("APC"), the most immunologically powerful of which is the bone marrow-derived dendritic cell ("DC").
  • APC specialized professional antigen- presenting cell
  • DC bone marrow-derived dendritic cell
  • Presentation of antigenic peptides on the surface of APCs in the context of MHC class I molecules leads to a cellular response while presentation of antigenic peptides by MHC class II molecules provokes a humoral response.
  • Area et al. (1997) J. Immunol. 20(2):138-45, examined the interactions of CD4 + and CD8 + T cells involved in the immune response to a poorly immunogenic tumor transduced to secrete GM-CSF. The authors report CD4 + and CD8 + cells are independently sensitized during tumor growth and both have functional capacity as effector cells in adoptive transfer. Both CD4 + and CD8 + T cells were induced concurrently against a poorly immunogenic tumor.
  • nucleated cells have the capacity to present endogenously produced antigen (including, for example, self antigens as well as viral antigens) on MHC Class I molecules.
  • Antigen-presenting cells have the capacity to present endogenously produced antigen on MHC Class I molecules, and soluble exogenous (i.e.. extracellular) antigens on both MHC Class I and MHC Class II molecules, but they generally present soluble exogenous antigens on Class I molecules relatively inefficiently.
  • Extracellular antigens are internalized by antigen-presenting cells and are processed in endocytic vesicles, where they encounter and bind to MHC Class II molecules.
  • viral and cellular proteins synthesized within the cell are hydrolyzed by proteasomes in the cytoplasm into peptides. some of which are transported by transporters associated with antigen processing (TAPs) into the endoplasmic reticulum, where they encounter and bind MHC Class I molecules. Raychaudhuri and Rock (1998) Nature 16:1025-1031; and Lindauer et al. (1998) J. Mol. Med. 76:32-47.
  • This invention provides a polynucleotide encoding first antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC), and a second coding sequence encoding a second antigen that is processed and presented with an MHC Class II molecule on the APC.
  • the first and second antigens may or may not have the same amino acid sequence.
  • a polynucleotide ofthe invention may comprise: (a) a first coding sequence which comprises a nucleotide sequence encoding an antigen; and (b) a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention ofthe encoded antigen in the endoplasmic reticulum (ER).
  • a first coding sequence which comprises a nucleotide sequence encoding an antigen
  • a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention ofthe encoded antigen in the endoplasmic reticulum (ER).
  • a polynucleotide ofthe invention may comprise: (a) a first coding sequence which comprises a nucleotide sequence encoding an antigen; (b) a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention ofthe encoded antigen in the endoplasmic reticulum (ER); and (c) a third coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that directs (or promotes retention of, or targets) the encoded antigen into a non- endosomal MHC Class II pathway.
  • the encoded antigen is a naturally occurring antigen or fragment of a naturally occurring antigen.
  • the antigen is a synthetic antigen.
  • An encoded antigen can be secreted or a cell-surface protein.
  • the invention further provides methods of increasing antigen presentation on the surface of an APC, comprising introducing a polynucleotide ofthe invention into the APC under conditions which favor expression ofthe polynucleotide.
  • the invention also provides APCs produced by these methods. These APCs have enhanced presentation of antigen encoded by a polynucleotide ofthe invention in both Class I and Class II MHC molecules.
  • Polynucleotides ofthe invention are useful in a variety of methods of modulating an immune response to the antigen thereby encoded.
  • the polynucleotides ofthe invention encode tumor antigens, or synthetic antigens corresponding to tumor antigens, and are thus useful as vaccines against tumor cells expressing cell surface tumor antigen.
  • the polynucleotides ofthe invention encode self antigens on normal
  • the polynucleotides ofthe invention encode foreign (non-self) antigens, such as those associated with organisms such as pathogenic bacteria, viruses and protozoans.
  • the polynucleotides ofthe invention encode antigens present on allografts which mediate their rejection.
  • the invention further provides polypeptides encoded by polynucleotides ofthe invention, and compositions comprising the polypeptides. The polypeptides are useful in immunomodulatory methods of the invention.
  • compositions containing the polynucleotides are further provided by this invention.
  • the polynucleotides can be contained in gene delivery vehicles such as viral vectors and liposomes, and in host cells. These polynucleotides are useful diagnostically and therapeutically, as well as in methods of expressing the polynucleotide for reproduction, expression and purification ofthe antigenic products.
  • the polynucleotides can be used to modulate an immune response to an antigen in a subject. Accordingly, the invention further provides methods for modulating a cellular and/or a humoral immune response to the antigen in a subject by administering to the subject an effective amount ofthe polynucleotide.
  • the polynucleotide can be delivered as naked DNA or in a gene delivery vehicle. In one aspect, a host cell containing the polynucleotide is administered to the subject.
  • the figure is a schematic of one embodiment ofthe polynucleotide of this invention.
  • the figure shows a single transcription cassette that encodes intracellular and secreted forms ofthe same antigen.
  • This polynucleotide can be incorporated into a replication defective adenoviral vector deleted in the El, E3 and/E4 regions.
  • a suitable helper cell line is utilized to produce infectious virions.
  • a helper plasmid or helper virus can be co-transfected with the viral vector so that infectious, replication defective virions capable of expressing the polynucleotide are produced.
  • PCR 2 A PRACTICAL APPROACH (M.J. MacPherson. B.D. Hames and G.R. Taylor eds. (1995)), Hariow and Lane eds. (1989) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney ed. (1987)).
  • PRACTICAL APPROACH M.J. MacPherson. B.D. Hames and G.R. Taylor eds. (1995)
  • Hariow and Lane eds. (1989) ANTIBODIES, A LABORATORY MANUAL Hariow and Lane eds. (1989) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney ed. (1987)).
  • certain terms may have the following defined meanings.
  • a cell includes a plurality of cells, including mixtures thereof.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the 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, for example, single-, double-stranded and triple helical molecules, 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.
  • 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 ofthe 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 to about 2 X SSC.
  • 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
  • a polynucleotide or polynucleotide region has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "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.
  • classical MHC Class I processing pathway intends that a protein is processed within a proteasome, transported to the ER, where it associates with an MHC Class I molecule.
  • an endoplasmic reticulum retention sequence intends an amino acid sequence which, when in the same translation unit with an amino acid sequence of a peptide antigen, increases the probability that the antigen will be located in the endoplasmic reticulum (ER) of a eukaryotic cell, compared with the same antigen not in the same translation unit with the amino acid sequence.
  • Increases the probability that the antigen will be located in the ER intends that a given antigen which is in the same translation unit with an amino acid sequence of an amino acid sequence which promotes retention in the ER will be found in the ER at levels at least about 2-fold, more preferably at least about 5-fold, even more preferably at least about 10-fold or higher that the same antigen which is in a translation unit lacking the amino acid sequence which promotes retention in the ER.
  • Methods of determining whether an antigen is in the endoplasmic reticulum include, but are not limited to, a process involving disrupting the cell, fractionating the subcellular components, and determining in which subcellular compartment the protein is located by, e.g., immunoassay.
  • the term "classical MHC Class II processing pathway” intends that a protein is taken up by an antigen-presenting cell by endocytosis or phagocytosis and the protein is processed in an endosome, before associating with MHC Class II molecules. This is also referred to herein as “processing by the endosomal pathway”.
  • processing by the endosomal pathway the terms “endosome”, “endocytic compartment”, “endocytic vesicle”, and “endosomal compartment” are used interchangeably.
  • an amino acid sequence that promotes processing of a protein by a non-endosomal MHC Class II pathway is an amino acid sequence which, when in the same translation unit with an amino acid sequence of a peptide antigen, increases the probability that the antigen will be located in a subcellular compartment of a eukaryotic cell which is not an endosome, and where processing ofthe protein and subsequent association with MHC Class II molecules can occur, compared with the same antigen not in the same translation unit with the amino acid sequence.
  • antigen is well understood in the art and includes substances which are immunogenic, i.e., immunogens, as well as substances which induce immunological unresponsiveness, or anergy, i.e., anergens.
  • An antigen may comprise one or more antigenic determinants, or epitopes.
  • the terms "antigen”, “antigenic determinant”, and “epitope” are used interchangeably.
  • the term "antigen” includes naturally occurring antigens, synthetic antigens, foreign antigens, self antigens, modified self antigens, and altered antigens.
  • a “native” or “natural” antigen is a polypeptide, protein or a fragment which contains an epitope, which has been isolated from a natural biological source, and which can specifically bind to an antigen receptor, in particular a T cell antigen receptor (TCR), in a subject.
  • TCR T cell antigen receptor
  • a synthetic antigen is said to "correspond" to a native epitope if the peptide binds to the same TCR as the natural epitope.
  • peptide used interchangeably herein with “polypeptide” 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.
  • sequence motif refers to a pattern present in a group of molecules (e.g., amino acids or nucleotides).
  • a typical pattern may be identified by characteristic amino acid residues, such as hydrophobic, hydrophilic. basic, acidic, and the like.
  • a “signal sequence” is a short amino acid sequence that directs newly synthesized secretory or membrane proteins to and through cellular membranes, including, but not limited to, the endoplasmic reticulum, and an endosomal compartment (or endosome). Signal sequences can be in the amino-terminal (N- terminal), and/or the carboxy-terminal (C-terminal) portion of a polypeptide and are generally cleaved after the polypeptide has crossed the membrane.
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • Examples of gene delivery vehicles are liposomes.
  • biocompatible polymers including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, viruses, such as
  • antigen-presenting matrix intends a molecule or molecules which can present antigen in such a way that the antigen can be bound by a T-cell antigen receptor on the surface of a T cell.
  • An antigen-presenting matrix can be on the surface of an antigen-presenting cell (APC), on a vesicle preparation of an APC, or can be in the form of a synthetic matrix on a solid support such as a bead or a plate.
  • An example of a synthetic antigen- presenting matrix is purified MHC class I molecules complexed to ⁇ 2- microglobulin, or purified MHC Class II molecules, or functional portions thereof, attached to a solid support.
  • antigen presenting cell intends any cell which presents on its surface an antigen in association with a major histocompatibility complex molecule, or portion thereof, or, alternatively, one or more non-classical MHC molecules, or a portion thereof.
  • suitable APCs include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen presenting cells. Methods of making hybrid APCs have been described, see, for example,
  • DCs Dendritic cells
  • TCR/CD3 T-cell receptor/CD3
  • MHC major histocompatibility complex
  • dendritic cell is to include, but not be limited to a pulsed dendritic cell, a foster cell or a dendritic cell hybrid.
  • a "foster antigen presenting cell” is a genetically modified dendritic cell that, from the restricted association of endogeneous peptides, will take up and present exogeneous antigen on the cell surface.
  • 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 I and ⁇ chains.
  • Class II MHC are known to be present on CD4 + T cells and, in humans, include HLA-DP, -DQ, and DR. The term
  • 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 class I or class II MHC molecule. Methods of identifying and comparing MHC are well known in the art and are described in Allen et al. (1994) Human Imm. 40:25-32; Santamaria et al. (1993) Human Imm.
  • Co-stimulatory molecules are involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. Research accumulated over the past several years has demonstrated convincingly that resting T cells require at least two signals for induction of cytokine gene expression and proliferation (Schwartz, R.H. (1990) Science
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • IAM-1 intracellular adhesion molecule 1
  • One exemplary receptor-ligand pair is the B7 co-stimulatory molecule on the surface of APCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman et al. (1993) Science 262:909-911; Young et al. (1992) J. Clin. Invest. 90: 229; Nabavi et al. (1992) Nature 360:266-268).
  • 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 ofthe 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
  • 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
  • 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, 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 Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen,
  • polystyrene e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.
  • POLYHIPE® resin obtained from Aminotech, Canada
  • polyamide resin obtained from Peninsula Laboratories
  • polystyrene resin grafted with polyethylene glycol TeentaGel®, Rapp Polymere, Tubingen
  • genetically modified means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype ofthe cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous nucleotides.
  • cytokine refers to any one ofthe numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • Non-limiting examples of cytokines which may be used alone or in combination in the practice ofthe present invention include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12).
  • G-CSF granulocyte macrophage-colony stimulating factor
  • IL-H interleukin- 1 alpha
  • IL-1 1 interleukin-1 1
  • MIP-U 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
  • Vector means a self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and/or between host cells.
  • the term is intended to include vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication vectors that function primarily for the replication of nucleic acid and expression vectors that function for transcription and/or translation of the DNA or RNA. Also intended are vectors that provide more than one of the above functions.
  • PCR primers refer to primers used in "polymerase chain reaction” or "PCR,” a method for amplifying a DNA base sequence using a heat-stable polymerase such as Taq polymerase, and two oligonucleotide primers, one complementary to the (+)-strand at one end ofthe sequence to be amplified and the other complementary to the (- )-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce exponential and highly specific amplification ofthe desired sequence. (See, e.g., PCR 2: A PRACTICAL APPROACH, supra). PCR also can be used to detect the existence ofthe defined sequence in a DNA sample.
  • “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 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 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.
  • 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 ofthe immunoglobulin molecule that comprise an antigen recognition site ofthe required specificity.
  • antibody complex is the combination of antibody (as defined above) and its binding partner or ligand.
  • immunomodulatory agent is a molecule, a macromolecular complex, or a cell that modulates an immune response and encompasses an antigenic peptide ofthe invention alone or in any of a variety of formulations described herein; a polypeptide comprising an antigenic peptide of the invention; a polynucleotide encoding a peptide or polypeptide ofthe invention; an antigenic peptide ofthe 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)); an 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 ofthe antigen (or epitope) into the subject.
  • An immune response 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
  • 17 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).
  • 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, FACS, or. in the case of CTLs, ? 1 Cr-release assays, or 3 H-thymidine uptake assays.
  • 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 tissue expresses 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 G 0 phase, and begins to produce cytokines characteristic ofthe cell type. For example, activated CD4+ T cells secrete IL-2 and have a higher
  • a peptide or polypeptide ofthe 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 antigenic epitopes, is recognized, i.e., is presented on the surface of an APC together with (i.e., bound to) an MHC molecule in such a way that a T cell antigen receptor (TCR) on the surface of an antigen-specific T cell binds to the epitope wherein such binding results in activation or anergy ofthe T cell.
  • TCR T cell antigen receptor
  • the term “preferentially recognized” intends that a polypeptide ofthe invention is substantially not recognized, as defined above, by a T cell specific for an unrelated antigen. Assays for determining whether an epitope is recognized by an antigen-specific T cell are known in the art and are described herein.
  • autogeneic indicates the origin of a cell. Thus, a cell being administered to an individual (the "recipient") is autogeneic if the cell was derived from that individual (the "donor") or a genetically identical individual. An autogeneic cell can also be a progeny of an autogeneic 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 autogeneic 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-
  • an APC is said to be allogeneic to an effector cell if they are derived from genetically non-identical donors.
  • a disease or condition related to a population of CD4 T 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 ofthe 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 ofthe disease, but which plays a key role in the pathogenesis ofthe 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 contact with educated, antigen-specific immune effector cells 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 ⁇ -thymidine inco ⁇ oration assay, or counting tumor cells.
  • "Suppressing" tumor cell growth means any or all ofthe following states: slowing, delaying, and stopping tumor growth, as well as tumor shrinkage.
  • 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 mo ⁇ hologically, 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 ofthe 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 ofthe 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 ofthe ribosome.
  • 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.
  • 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 ofthe 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)).
  • 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.
  • compositions consisting essentially ofthe 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.
  • 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.
  • An antigen for the pu ⁇ oses ofthe present invention, is a protein antigen.
  • the term "protein antigen” is used in its broadest sense and includes minimal epitopes, chimeric molecules, synthetic antigens, in addition to isolated full length proteins. The epitopes can also be derived altered antigens. While the embodiments described below are directed to tumor antigens, it should be understood, although not explicitely stated, that any protein or polypeptide which induces an immune response is intended to be within the scope of this invention.
  • antigens include, but are not limited to tumor antigens, viral
  • the antigen of this vaccine may be autologous or heterologous (i.e.. allogeneic) or a homolog from a isolated species, e.g., a murine antigen administered to a human patient.
  • the polynucleotides of this invention comprise at least two coding sequences.
  • the first sequence encodes a first antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC).
  • the second coding sequence codes for second antigen that is processed and presented with an MHC Class II molecule on the APC.
  • the first and second antigens have the same amino acid sequence.
  • the polynucleotides encode antigens comprising epitopes that are recognized by the same T cell receptor.
  • encoded antigens comprise different epitopes from the same native protein.
  • the first and second antigens share at least about 50% amino acid sequence identity with one another, as determined using alignment programs using default parameters, or are encoded by polynucleotides that hybridize to each other under stringent conditions.
  • a polynucleotide ofthe invention may comprise: (a) a first coding sequence which comprises a nucleotide sequence encoding an antigen; and
  • a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention ofthe encoded antigen in the endoplasmic reticulum (ER).
  • the polynucleotide can be constructed such that the amino acid sequence that promotes retention ofthe encoded antigen in the ER is appended to the N- terminus or the C-terminus ofthe encoded antigen.
  • the sequence that promotes retention ofthe encoded antigen in the ER can occur internally, i.e., between the N-terminus and C-terminus ofthe encoded antigen.
  • HDEF promotes retention (targets) the appended protein to the ER.
  • other sequences such as “DDEL”, “ADEL”, “SDEL”, “RDEL”, “KEEL”, “QEDL”, “HIEL”, “HTEL” and “KQDL” are known as signals for staying in endoplasmic reticulum, and polypeptides having these sequences are generally retained in the ER.
  • the amino acid sequence that promotes retention ofthe encoded antigen in the ER is selected from the group consisting of KDEL, HDEL, DDEL, ADEL, SDEL, RDEL, KEEL, QEDL, HIEL, HTEL, and KQDL
  • the amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER) is KDEL.
  • the translation unit comprises the antigen having KDEL as the carboxy-terminal amino acids.
  • the amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER) is HDEF.
  • the translation unit comprises the antigen having HDEF as the carboxy-terminal amino acids.
  • a polynucleotide ofthe invention comprises: (a) a first coding sequence which comprises a nucleotide sequence encoding an antigen; (b) a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence
  • a third coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that directs (or promotes retention of. or targets) the encoded antigen into a non- endosomal MHC Class II pathway.
  • the polynucleotide can be constructed such that the amino acid sequence that directs the encoded antigen into a non-endosomal MHC Class II pathway is appended to the N-terminus or the C-terminus ofthe encoded antigen. Alternatively, the sequence that directs the encoded antigen into a non-endosomal MHC Class II pathway is appended to the N-terminus or the C-terminus ofthe encoded antigen. Alternatively, the sequence that directs the encoded antigen into a non-endosomal
  • MHC Class II pathway can occur internally, i.e.. between the N-terminus and C- terminus ofthe encoded antigen.
  • a non-endosomal MHC Class II pathway includes, but is not limited to, a lysosome, the Golgi, and a melanosome. Amino acid sequence that direct proteins into a non-endosomal MHC Class II pathway are known in the art.
  • the encoded antigen is a naturally occurring antigen or fragment of a naturally occurring antigen.
  • the antigen is a synthetic antigen.
  • the antigen can be "self or foreign, and can be derived from any organism.
  • the antigen may be autologous or heterologous (i.e., allogeneic or a homolog from a isolated species, e.g., a murine antigen administered to a human patient).
  • Each coding sequence is operatively linked to operational and regulatory sequences which control transcription and translation ofthe coding sequences. These operational and regulatory sequences are well known to those skilled in the art.
  • the polynucleotides of this invention comprise coding regions which encode, in one embodiment, previously characterized tumor-associated antigens such as gplOO (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. 91:3515-3519; Kawakami et al.
  • CEA U.S. Patent No. 5,274,087
  • PSA prostate membrane specific antigen
  • PSMA prostate membrane specific antigen
  • tyrosinase U.S. Patent Nos. 5,530,096 and 4,898,814; Brichard et al. (1993) J. Exp. Med. 178:489-49
  • tyrosinase related proteins 1 or 2 TRP-1 and TRP-2
  • NY-ESO-1 Choen et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:1914-18
  • GA733 antigen U.S. Patent No. 5,185,254
  • an antigen encoded by a polynucleotide ofthe invention is a secreted protein, or an antigen that has been recombinantly altered to be secreted from the cell.
  • the secreted form ofthe antigen can be synthesized by splicing a signal sequence onto the sequence encoding the wild-type antigen or by removing the sequences coding for the cytoplasmic and transmembrane portions ofthe antigen and/or by removing the sequences that 1 ) direct the encoded protein to an intracellular compartment such as the cytoplasm, nucleus or mitochondia, or 2) cause the encoded protein to be inserted into cellular membranes (i.e., a transmembrane region), or 3) cause the encoded protein to be retained within cellular compartments such as a hydrophobic anchoring sequence or endoplasmic reticulum retention signal.
  • a secreted antigen can be generated by fusing a secretory signal sequence to the wild-type antigen using standard recombinant DNA methodology familiar to one of skill in the art.
  • the secretory signal sequence would typically be positioned at the N-terminus ofthe desired protein but can be placed at any position suitable to allow secretion of the antigen.
  • the figure shows a transmembrane cassette comprising DNA sequences encoding a wild-type antigen fused in frame at the 5' end with a DNA sequence encoding a secretory signal sequence such as an endoplasmic reticulum targeting sequence (ERTS).
  • ERTS endoplasmic reticulum targeting sequence
  • IVS internal ribosome entry sequence
  • Suitable secretory signal sequences include signal sequences or derivatives of signal sequences of known secretory proteins.
  • secretory proteins include, but are not limited to, certain growth factors such as fibroblast growth factors 4-6, epidermal growth factor, and lymphokines such as interleukins 2-6.
  • a secreted antigen can be generated by appending a secretory signal sequence onto the antigen and optionally removing sequences that 1 ) direct the encoded protein to an intracellular compartment such as the cytoplasm, nucleus or mitochondia, or 2) cause the encoded protein to be inserted into cellular membranes (i.e., a transmembrane region), or 3) cause the encoded protein to be retained within cellular compartments such as a hydrophobic anchoring sequence or endoplasmic reticulum retention signal.
  • the modified antigen lacks the sequences necessary for membrane anchorage, but retains the sequences required for entry into the rough endoplasmic reticulum/golgi complex and eventual secretion from the cell.
  • an antigen encoded by a polynucleotide ofthe invention is a cell-surface protein.
  • the polynucleotide encoding an antigen further comprises a sequence which encodes an amino acid sequence which causes the encoded protein to be inserted into cellular membranes (i.e., a transmembrane region).
  • 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.
  • nucleic acid sequences ofthe present invention may be characterized, isolated, synthesized and purified using no more than ordinary skill. See Sambrook et al., (1989) supra.
  • the polynucleotides ofthe present invention also can serve as primers for the detection of genes or gene transcripts that are expressed in APC, for example, to confirm transduction ofthe polynucleotides into host cells.
  • amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase. and reverse transcriptase.
  • a preferred length ofthe primer is the same as that identified for probes, above.
  • the invention further provides the isolated polynucleotide operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA.
  • a promoter of RNA transcription as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA.
  • the term "operatively linked” means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule. Examples of such promoters are SP6, T4 and T7.
  • cell-specific promoters are used for cell-specific expression ofthe inserted polynucleotide.
  • Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an
  • the present invention provides methods to enhance, or increase, presentation of a peptide on the surface of an antigen-presenting cell, comprising introducing into the cell a polynucleotide ofthe invention. Since polynucleotides ofthe invention encoding an antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC) and an antigen that is processed and presented with an MHC Class II molecule on the APC, one observes increased presentation of peptide antigens to CD4 + and to CD8 + T cells.
  • APC antigen-presenting cell
  • the invention further provides an APC produced by the method ofthe present invention.
  • These APCs exhibit enhanced presentation of a peptide antigen by both Class I and Class II MHC molecules. These levels of antigen loading on the APC surface was not achieved by prior art methods, which enhance either Class I or Class II presentation, but not both. Accordingly, these methods, and the APCs produced thereby, are novel and non-obvious over the prior art.
  • Whether presentation of peptide on the surface of an APC is enhanced or increased can be determined by comparing the CD4+ and CD8+ T cell response to the encoded peptide presented on the surface of an APC into which has been introduced a polynucleotide ofthe invention under conditions which favor expression (i.e., transcription and translation), to the CD4+ and CD8+ T cell response to the peptide when the peptide expressed on the surface of a control APC, i.e, an APC presenting the peptide encoded by a polynucleotide lacking either the nucleotide sequences encoding an amino acid sequence that promotes
  • nucleotide sequences encoding an amino acid sequence that directs the encoded antigen into a non-endosomal MHC Class II pathway or both.
  • T cell activation can be detected by any known method, including but not limited to. tritiated thymidine inco ⁇ oration (indicative of DNA synthesis), and examination ofthe population for growth or proliferation, e.g., by identification of colonies.
  • the tetrazolium salt MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5- diphenyl tetrazolium bromide) may be added.
  • this invention also provides a process for obtaining the polynucleotides of this invention by providing the linear sequence ofthe polynucleotide. appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (procaryotic or eucaryotic) for replication and amplification. The DNA so amplified can be isolated from the cell by methods well known to those of skill in the art. A process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained.
  • RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell.
  • the DNA can be inserted by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation.
  • an appropriate gene delivery vehicle e.g., liposome, plasmid or vector
  • electroporation e.g., electroporation
  • RNA can then be isolated using methods well known to those of skill in the art, for example, as set forth in Sambrook et al. (1989) Supra.
  • mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989) Supra or extracted by nucleic-acid-binding resins following the accompanying instructions provided by manufactures.
  • the present invention also provides delivery vehicles suitable for delivery of a polynucleotide ofthe invention into cells (whether in vivo, ex vivo, or in vitro).
  • a polynucleotide ofthe invention can be contained within a cloning or
  • These vectors can in turn be manipulated to assume any of a number of forms which may, for example, facilitate delivery to and/or entry into a cell.
  • Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part ofthe chromosomal DNA. Suitable expression vectors include plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, etc. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo.
  • a nucleic acid When a nucleic acid is inserted into a suitable host cell, e.g., a procaryotic or a eucaryotic cell and the host cell replicates, the protein can be recombinantly produced.
  • suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using well known methods. See Sambrook, et al. (1989) Supra.
  • the nucleic acid can be inserted into the host cell by methods well known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See Sambrook et al. (1989) Supra for this methodology.
  • this invention also provides a host cell, e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a procaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.
  • a pharmaceutically acceptable vector such as a replication-incompetent retroviral or adenoviral vector.
  • Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression ofthe inserted polynucleotide.
  • pharmaceutically acceptable vector includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic
  • An example of such a vector is a "replication- incompetent" vector defined by its inability to produce viral proteins, precluding spread ofthe vector in the infected host cell.
  • An example of a replication- incompetent retroviral vector is LNL6. Miller et al. (1989) BioTechniques 7:980- 990. The methodology of using replication-incompetent retroviruses for retroviral-mediated gene transfer of gene markers is well established. Correll et al. (1989) PNAS USA 86:8912; Bordignon (1989) PNAS USA 86:8912-52; Culver (1991) PNAS USA 88:3155; and Rill (1991) Blood 79(l0) 2694-700.
  • genetic modifications of cells employed in the present invention are accomplished by introducing a vector containing a polynucleotide comprising sequences encoding a synthetic antigenic peptide ofthe invention.
  • a vector containing a polynucleotide comprising sequences encoding a synthetic antigenic peptide ofthe invention.
  • a variety of different gene transfer vectors, including viral as well as non- viral systems can be used.
  • a wide variety of non-viral vehicles for delivery of a polynucleotide ofthe invention are known in the art and are encompassed in the present invention.
  • a polynucleotide ofthe invention can be delivered to a cell as naked DNA. WO 97/40163.
  • a polynucleotide ofthe invention can be delivered to a cell associated in a variety of ways with a variety of substances (forms of delivery) including, but not limited to cationic lipids; biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria.
  • a delivery vehicle may take the form of a microparticle. Mixtures or conjugates of these various substances can also be used as delivery vehicles.
  • a polynucleotide ofthe invention can be associated with these various forms of delivery non-covalently or covalently.
  • Non-viral vectors i.e., cloning and expression vectors having cloned therein a polynucleotide(s) ofthe invention can be used for expression of recombinant polypeptides as well as a source of polynucleotide of the invention.
  • Cloning vectors can be used to obtain replicate copies ofthe polynucleotides they contain, or as a means of storing the polynucleotides in a
  • Expression vectors can be used to obtain polypeptides produced from the polynucleotides they contain. They may also be used where it is desirable to express polypeptides, encoded by an operably linked polynucleotide, in an individual, such as for eliciting an immune response via the polypeptide(s) encoded in the expression vector(s).
  • Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and need not be described in detail herein. For example, see Gacesa and Ramji, Vectors, John Wiley & Sons (1994).
  • Cloning and expression vectors typically contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will survive and/or grow under selective conditions.
  • Typical selection genes encode protein(s) that (a) confer resistance to antibiotics or other toxins substances, e.g., ampicillin, neomycyin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice ofthe proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art.
  • Cloning and expression vectors also typically contain a replication system recognized by the host.
  • Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self- replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
  • Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4. phage DNAs, and shuttle vectors such as
  • Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide encoding a polypeptide of interest.
  • the polynucleotide encoding the polypeptide of interest is operably linked to suitable transcriptional controlling elements, such as promoters, enhancers and terminators.
  • suitable transcriptional controlling elements such as promoters, enhancers and terminators.
  • one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
  • a polynucleotide sequence encoding a signal peptide can also be included to allow a polypeptide, encoded by an operably linked polynucleotide, to cross and/or lodge in cell membranes or be secreted from the cell.
  • a number of expression vectors suitable for expression in eukaryotic cells including yeast, avian, and mammalian cells are known in the art.
  • Examples of mammalian expression vectors contain both prokaryotic sequence to facilitate the propagation ofthe vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • Examples of mammalian expression vectors suitable for transfection of eukaryotic cells include the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pRSVneo, and pHyg derived vectors.
  • viruses such as the bovine papilloma virus (BPV-1 ), or Epstein-Barr virus (pHEB, pREP derived vectors) can be used for expression in mammalian cells.
  • expression vectors for yeast systems include YEP24, YIP5, YEP51, YEP52, YES2 and YRP17, which are cloning and expression vehicles useful for introduction of constructs into S. cerevisiae. Broach et al. (1983) Experimental Manipulation of Gene Expression, ed. M. Inouye, Academic Press, p. 83.
  • Baculovirus expression vectors for expression in insect cells include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors and pBlueBac-derived vectors.
  • Viral vectors include, but are not limited to, DNA viral vectors such as those based on adenoviruses, he ⁇ es simplex virus, poxviruses such as vaccinia virus, and parvoviruses, including adeno-associated virus; and RNA viral vectors, including, but not limited to, the retroviral vectors.
  • Retroviral vectors include
  • Replication-defective retroviral vectors harboring a polynucleotide ofthe invention as part of the retroviral genome can be used. Such vectors have been described in detail. (Miller et al. (1990) Mol. Cell Biol. 10:4239; Kolberg, R.
  • 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) Current Opinion in Biotechnology 3:533-539; Muzcyzka ( 1992) Current Top.
  • 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. 20:276-286).
  • the antibodies can be conjugated to an enzyme (e.g. HRP) giving rise to a colored
  • the actual amount of antigenic polypeptides being expressed by the APCs can be evaluated by ELISA.
  • In vivo transduction of DCs, or other APCs can be accomplished by administration of a viral vectors comprising a polynucleotide ofthe invention via different routes including intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery.
  • a viral vectors comprising a polynucleotide ofthe invention via different routes including intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery.
  • One method which can be used is cutaneous delivery of Ad vector at multiple sites using a total dose of approximately lxl0 10 -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 peptide epitope being expressed.
  • the staining procedure can be carried out on biopsy samples from the site of administration or on cells from draining lymph nodes or other organs where APCs (in particular DCs) may have migrated.
  • APCs in particular DCs
  • Condon et al. (1996) Nature Med. 2:1122-1128; Wan et al. (1991) Human Gene Therapy 8:1355-1363.
  • the amount of antigen being expressed at the site of injection or in other organs where transduced APCs may have migrated can be evaluated by ELISA on tissue homogenates.
  • APCs 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- 25. Transduced APCs can subsequently be administered to the host via an intravenous, subcutaneous, intranasal. intramuscular or intraperitoneal route of delivery.
  • DCs In vivo transduction of 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. 2:1122-1128; Raz et al. (1994) PNAS 91:9519-9523.
  • Intramuscular delivery of plasmid DNA may also be used for immunization. Rosato et al. (1997) Human Gene Therapy 8:1451-1458.
  • transduction efficiency and levels of transgene expression can be assessed as described above for viral vectors.
  • Host cells comprising polynucleotides ofthe invention The present invention further provides host cells comprising polynucleotides ofthe invention.
  • Host cells containing the polynucleotides of this invention are useful for the recombinant replication ofthe polynucleotides and for the recombinant production of peptides ofthe invention.
  • host cells comprising a polynucleotide ofthe invention can be used to induce an immune response in a subject in the methods described herein.
  • Host cells which are suitable for recombinant replication of the polynucleotides ofthe invention, and for the recombinant production of peptides ofthe invention can be prokaryotic or eukaryotic. Host systems are known in the art and need not be described in detail herein.
  • Prokaryotic hosts include bacterial cells, for example E. coli, B. subtilis, and mycobacteria.
  • eukaryotic hosts are yeast, insect, avian, plant, C. elegans (or nematode) and mammalian cells. These cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells are antigen presenting cells, they can be used to expand a population of immune effector cells such as tumor infiltrating lymphocytes which in turn are useful in adoptive immunotherapies. Antigen presenting cells are described in more detail below.
  • isolated host cells are APCs.
  • APCs 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.
  • the immune effector cells and/or the APCs are genetically modified.
  • genes coding for co- stimulatory molecules and/or stimulatory cytokines can be inserted prior to, concurrent to or subsequent to expansion ofthe immune effector cells.
  • the host cell containing the gene(s) coding for the cytokine 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.
  • 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.
  • APCs suitable for use in the present invention are capable of presenting exogenous peptide or protein or endogenous antigen to T cells in association with an antigen-presenting molecule, such as an MHC molecule.
  • APCs include, but are not limited to, macrophages, dendritic cells, CD40-activated B cells, antigen- specific B cells, tumor cells, virus-infected cells, and genetically modified cells.
  • APCs can obtained from a variety of sources, including but not limited to, peripheral blood mononuclear cells (PBMC), whole blood or fractions thereof containing mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, 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 exposed to one or more biological modifiers.
  • An "untreated” donor has not been exposed to one or more biological modifiers.
  • APCs can also be treated in vitro with one or more biological modifiers.
  • the APCs are generally alive but can also be irradiated, mitomycin C treated, attenuated, or chemically fixed. Further, the APCs need not be whole cells. Instead, vesicle preparations of APCs can be used.
  • APCs can be genetically modified, i.e., transfected with a recombinant polynucleotide construct such that they express a polypeptide or an RNA molecule which they would not normally express or would normally express at lower levels.
  • polynucleotides include, but are not limited to, those which encode an MHC molecule; a co-stimulatory molecule such as B7; and a peptide or polypeptide of the invention.
  • APCs which do not normally function in vivo in mammals as APCs can be modified in such a way that they function as APCs.
  • a wide variety of cells can function as APCs when appropriately modified. Examples of such cells are insect
  • antigen-presenting polypeptides such as MHC molecules, optionally also accessory molecules such as B7
  • antigen-presenting polypeptides and accessory molecules which can insert themselves into the cell membrane can be used.
  • glycosyl- phosphotidylinositol (GPI)-modified polypeptides can insert themselves into the membranes of cells.
  • Accessory molecules include, but are not limited to. co-stimulatory antibodies such as antibodies specific for CD28, CD80. or CD86; costimulatory molecules, including, but not limited to, B7.1 and B7.2; adhesion molecules such as ICAM-1 and LFA-3; and survival molecules such as Fas ligand and CD70. See, for example, PCT Publication No. WO 201400607-613.
  • Accessory molecules include, but are not limited to. co-stimulatory antibodies such as antibodies specific for CD28, CD80. or CD86; costimulatory molecules, including, but not limited to, B7.1 and B7.2; adhesion molecules such as ICAM-1 and LFA-3; and survival molecules such as Fas ligand and CD70. See, for example, PCT Publication No. WO
  • Foster antigen presenting cells are particularly useful as APCs.
  • 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.
  • MHC molecules make the APC more visible to the CTLs.
  • a powerful transcriptional promoter e.g., the CMN promoter
  • Methods for determining whether an antigen-presenting cell is capable of presenting antigen to an immune effector cell in such a manner as to effect activation ofthe immune effector cell include, for example, H-thymidine uptake by effector cells, cytokine production by effector cells, and cytolytic 3 l Cr-release assays.
  • an antigenic peptide ofthe invention is presented on an antigen-presenting cell in a Class I or Class II MHC molecule such that the peptide is bound by a TCR on a CD4+ or CD8+ T cell, but the antigen-presenting cell lacks one or more co-stimulatory molecules required for activation ofthe T cell.
  • These antigen-presenting cells induce T cell anergy (unresponsiveness), and are useful in methods described herein for reducing or suppressing an immune response. Methods for determining whether an antigen-presenting cell is capable of presenting antigen to an immune effector cell, in such a manner as to effect T cell anergy, are known in the art.
  • the second approach for isolating APCs is to collect the relatively large numbers of precommitted APCs already circulating in the blood.
  • Previous techniques for isolating committed APCs from human peripheral blood have involved combinations of physical procedures such as metrizamide gradients and
  • CCE countercurrent centrifugal elutriation
  • the APC are 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 ofthe 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 leukopheresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions 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, GM-CSF and IL-13 or GM-CSF and IL-4, (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 recombinant (rh) rhIL-12, rhGM-CSF, or rhIL-4.
  • the cells ofthe 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 leukopheresis.
  • the dendritic cells can be identified by the presence of at least one ofthe following markers: HLA-DR, HLA-DQ, or B7. 2. and the simultaneous absence of the following markers: CD3, CD14, CD16, 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 leukopheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE).
  • 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 ⁇ m.
  • 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 which 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).
  • 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 purified or recombinant human ("rh") rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when given alone is inadequate for optimal upregulation.
  • rh purified or recombinant human
  • rhIL-2 rhIL-2
  • rhIL-4 rhIL-4
  • a foster antigen presenting cell is a genetically modified dendritic cell that, from the restricted association of endogeneous peptides, will take up and present exogeneous antigen on the cell surface.
  • the human cell line 174xCEM.T2 referred to as T2 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- 1771 ) and therefore can be used for the manufacture of a foster antigen presenting cell.
  • the cell line is available from the ATCC.
  • TAP1, TAP2, LMP1, and LMP2 are required for antigen presentation to 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.
  • These T2 cells are referred to herein as "foster" APCs. They can be used in conjunction with this invention to present the antigens.
  • T2 cells with specific recombinant MHC alleles allows for redirection ofthe 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.
  • Hybrid cells also will present antigen(s) and therefore, are useful in the methods of this invention.
  • Hybrid cells typically retain the phenotypic characteristics ofthe APCs.
  • 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. The procedure for making these hybrids is described in WO 96/30030 and Gong et al. (1997) Nature Medicine 3(5):558-561.
  • 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
  • HGPRT hypoxanthine-guaninephosphoribosyl transferase
  • Insertion ofthe gene(s) into the APCs requires the making of appropriate gene delivery vehicles and methods for efficient transduction. The following are useful for in vitro and in vivo transduction with the gene(s) of interest.
  • the present invention makes use ofthe above-described APCs to stimulate production of an enriched population of antigen-specific immune effector cells. Accordingly, the present invention provides a population of cells enriched in educated, antigen-specific immune effector cells, specific for an antigen encoded by a polynucleotide ofthe invention.
  • the antigen corresponds to an antigen on the surface of tumor cells and the educated, antigen- specific immune effector cells ofthe invention suppress growth ofthe tumor cells.
  • APCs are used, 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
  • the APCs prepared as described above are mixed with naive immune effector cells.
  • the cells may be cultured in the presence of a cytokine, for example IL2.
  • a 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-11 or IL-13. Methods for introducing transgenes in vitro, ex vivo and in vivo are well known in the art.
  • An effector cell population suitable for use in the methods ofthe present invention can be autogeneic or allogeneic, preferably autogeneic.
  • 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.
  • 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
  • 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 leukapheresis, biopsy tissue, lymph nodes, e.g., lymph nodes draining from a tumor.
  • Suitable donors include an immunized donor, a non-immunized (naive) donor, treated or untreated donors.
  • a "treated” donor is one that has been exposed to one or more biological modifiers.
  • An "untreated” donor has not been exposed to one or more biological modifiers.
  • effector cells can be obtained by leukapheresis, 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.
  • the 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. These include, for example, fluorescence-activated cell sorting (FACS).
  • 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, MD). 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,
  • 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 ofthe native factor. If more than one biological modifier is used, the exposure can be simultaneous or sequential.
  • compositions comprising immune effector cells, which may be T cells, enriched in antigen-specific cells, specific for an antigen encoded by a polynucleotide ofthe invention.
  • 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 ofthe 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%,
  • the population is said to be "substantially pure " .
  • the percentage which are antigen-specific can readily be determined, for example, by a H- thymidine uptake assay in which the effector cell population (for example, a T- cell population) is challenged by an antigen-presenting cell presenting an antigen encoded by a polynucleotide ofthe invention.
  • the present invention makes use ofthe above-described 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 ofthe 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 3:261-268; Hwu et al. (1993) J. Immunol. 150:4104-4115; and Rosenburg,et al. (1985) N. Eng. j. Med. 313(23): 1485-1492.
  • the antigen-specific immune effector cell population comprises both CD4+ and CD8+ T cells.
  • the cytotoxic T cells are polyclonal T cells isolated from a site of cytotoxic T cell infiltration from a subject.
  • such cells may be isolated from a site of cytotoxic T cell infiltration from two or more subjects or human patients, in which the subjects share an MHC halotype.
  • the CTLs may be two or more cytotoxic T cell lines.
  • the CTLs may be any combination ofthe foregoing.
  • the site of cytotoxic T cell infiltration is a tumor.
  • the tumors from which cells or cell lines are obtained can be the same type of tumor in different individuals with a shared MHC halotype or different types of tumors from different subjects who share an MHC haplotype.
  • CTL infiltrates can be from sites of viral infection, autoimmune inflammation, transplantation rejection, an like sites of inflammation or lymphocyte/leukocyte infiltration.
  • the APCs prepared as described above are mixed with na ⁇ ve immune effector cells.
  • the cells may be cultured in the presence of a cytokine, for example IL2.
  • a cytokine for example IL2.
  • IL12 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 hybrid cells and optional cytokines can be performed to further expand the population of antigen-specific cells.
  • the immune effector cells are T cells and are specific for tumor-specific antigens which are presented by the APCs.
  • the invention further provides peptide antigens encoded by the polynucleotides of the invention.
  • Peptide antigens encoded by polynucleotides of the invention can be produced by any known method. Isolated peptides ofthe present invention can be synthesized using an appropriate solid state synthetic procedure. Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco. Calif. (1968). A preferred method is the Merrifield process. Merrifield, Recent Progress in Hormone Res., 23:451 (1967). The antigenic activity of these peptides may conveniently be tested using, for example, the assays as described herein.
  • an isolated peptide ofthe invention may be purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • chromatography e.g., ion exchange, affinity, and sizing column chromatography
  • centrifugation e.g., centrifugation
  • differential solubility e.g., differential solubility
  • an epitope may be isolated by binding it to an affinity column
  • affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Methods Enzymol. 194:508-509), and glutathione-S-transferase can be attached to the peptides ofthe invention to allow easy purification by passage over an appropriate affinity column.
  • a DNA affinity column using DNA containing a sequence encoding the peptides ofthe invention could be used in purification.
  • 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. 57:285-320).
  • the immune effector cells described herein are selected for their ability to modulate an immune response.
  • the immune effector cells are selected both for their ability to actively lyse the cells expressing the specific antigen and for their ability to increase a humoral response to the antigen.
  • Cytolytic activity ofthe cells can be measured in various ways, including, but not limited to, tritiated thymidine inco ⁇ oration (indicative of DNA synthesis), and examination ofthe population for growth or proliferation, e.g., by identification of colonies. (See, e.g., WO 94/21287).
  • the tetrazolium salt in another embodiment, the tetrazolium salt
  • MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5-diphenyl tetrazolium bromide) may be added (Mossman (1983) J. Immunol. Methods 65:55-63: Niks and Otto (1990) J. Immunol. Methods 130:140-151).
  • Succinate dehydrogenase found in mitochondria of viable cells, converts the MTT to formazan blue. Thus, concentrated blue: color would indicate metabolically active cells.
  • protein synthesis may be shown by inco ⁇ oration of 3:, S-methionine.
  • cytotoxicity and cell killing assays such as the classical chromium release assay, may be employed to evaluate epitope-specific CTL activation.
  • Other suitable assays will be known to those of skill in the art. Stimulation of a humoral response to an antigen can be measured by specific antibody production, using any known method, including ELISA and RIA.
  • compositions ofthe invention are provided.
  • compositions containing any ofthe above- mentioned peptides, polypeptides, polynucleotides, host cells, antigen-presenting cells, immune effector cells, vectors, antibodies and fragments thereof, and an acceptable solid or liquid carrier.
  • compositions are used pharmaceutically, they are combined with a "pharmaceutically acceptable carrier" for diagnostic and therapeutic use.
  • pharmaceutically acceptable carrier for diagnostic and therapeutic use.
  • compositions also can be used for the preparation of medicaments for the diagnostic and immunomodulatory methods of the invention.
  • the present invention provides diagnostic and immunomodulatory methods using polynucleotides, and host cells (including APCs and educated immune effector cells), i.e.. immunomodulatory agents, ofthe invention.
  • the present invention provides diagnostic methods using the polynucleotides and APCs ofthe invention.
  • the methods can be used to detect the presence of an antigen-specific CD4 + and/or CD8 + T cell which binds an antigen encoded by a polynucleotide ofthe invention.
  • the diagnostic methods ofthe invention include: (1) assays to predict the efficacy of an antigen encoded by a polynucleotide ofthe invention; (2) assays to determine the precursor frequency (i.e., the presence and number of) of immune effector cells specific for an antigen encoded by a polynucleotide ofthe invention
  • Diagnostic methods ofthe invention are generally carried out under suitable conditions and for a sufficient time to allow specific binding to occur between an antigen encoded by a polynucleotide ofthe invention, usually presented by an APC ofthe invention, and an immune effector molecule, such as a TCR, on the surface of an immune effector cell, such as a CD4+ or CD8+ T cell.
  • Suitable conditions and “sufficient time” are generally conditions and times suitable for specific binding. Suitable conditions occur between about 4°C and about 40°C, preferably between about 4°C and about 37°C, in a buffered solution, and within a pH range of between 5 and 9.
  • buffered solutions are known in the art, can be used in the diagnostic methods of this invention, and include, but are not limited to, phosphate-buffered saline.
  • Sufficient time for binding and response will generally be between about 1 second and about
  • the invention provides diagnostic ssays to predict the efficacy of an antigen encoded by a polynucleotide ofthe invention, presented by an APC ofthe invention.
  • defined T cell epitopes are used to clinically characterize tumors and viral pathogens in order to determine, in advance, the predicted efficacy of an in vivo vaccine trial. This can be achieved by a simple proliferation assay of a patient ' s peripheral blood mononuclear cells using defined T cell epitopes as stimulators. Peptides which elicit a response are viable vaccine candidates for that patient.
  • assays are provided to determine the precursor frequency (i.e., the presence and number of) of resting (na ⁇ ve) immune effector cells specific for an antigen encoded by a polynucleotide ofthe invention and which therefore have the potential to become activated.
  • an antigen-presenting cell bearing on its surface an antigen encoded by a polynucleotide of the invention is used to detect the presence of immune effector
  • PBMCs are isolated from a subject with a tumor. A sample of these PBMCs is cultured together for a suitable time with the tumor cells from the same subject. A second sample of these PBMCs is cultured together for a suitable time with surrogate APCs pulsed with an antigen encoded by a polynucleotide ofthe invention which corresponds to a natural epitope(s) expressed on the surface ofthe tumor. Both tumor cells and surrogate APCs are loaded with 3 Cr.
  • the precursor frequency of immune effector cells which are specific for tumor and the precursor frequency of immune effector cells which are specific for an antigen encoded by a polynucleotide are specific for an antigen encoded by a polynucleotide.
  • Functional assays include, but are not limited to, immune effector cell proliferation, cytokine production, specific lysis of an APC.
  • the efficacy of an immunomodulatory method, including immunomodulatory methods ofthe invention, in modulating an immune response to an antigen encoded by a polynucleotide ofthe invention and/or its natural counte ⁇ art can be tested using diagnostic assays ofthe invention.
  • the method allows detection of immune effector cells, which may be activated CD4 + or CD8 + T cells, which have become activated or anergized as a result of exposure to an antigen encoded by a polynucleotide ofthe invention.
  • a sample containing cells from a subject can be tested for the presence of CD4+ or CD8+ T cells which have become activated or anergized as a result of binding to an antigen encoded by a polynucleotide ofthe invention.
  • the method comprises the steps of: (a) contacting an immobilized APC presenting an antigen encoded by a polynucleotide ofthe invention on its surface bound to Class I and Class II MHC molecules with a biological sample under suitable conditions and for a time sufficient to allow binding of an immune effector cell which bears on its surface an antigen receptor specific for the peptide. thereby immobilizing the antigen-
  • the method comprises the steps of (a) contacting an immobilized antigen-presenting matrix which presents an antigen encoded by a polynucleotide ofthe invention on its surface bound to a Class I or Class II MHC molecule with a biological sample under suitable conditions and for a time sufficient to allow binding of an immune effector cell which bears on its surface an antigen receptor specific for the peptide, thereby immobilizing the antigen-specific immune effector cell; and (b) performing a functional assay on the immobilized immune effector cell.
  • a detectably labeled molecule such as an antibody
  • the immune effector cell is bound to the immobilized APC presenting on its surface an antigen encoded by a polynucleotide ofthe invention, it can be labeled on the basis of characteristic cell surface molecules, including, but not limited to, CD4, CD8, and cell surface markers specific for activated T cells.
  • characteristic cell surface molecules including, but not limited to, CD4, CD8, and cell surface markers specific for activated T cells.
  • CD4, CD8 cell surface markers specific for activated T cells.
  • a variety of cell surface markers specific to populations of immune effector cells are known to those skilled in the art and have been described in numerous publications, see, for example, 77ze Leukocyte Antigen Facts Book, Barclay et al., eds., 1995, Academic Press. Antibodies to these markers are commercially available from, inter alia, Beckman Coulter.
  • the immobilized immune effector cell can also be characterized by presence of mRNA and/or proteins in the cytosol which are characteristic of a given T cell type in a given activated or anergic state.
  • a characteristic mRNA can be detected by any known means, including, but not limited to, a polymerase chain reaction.
  • a detectably labeled antibody to a cell surface marker can be contacted with the immobilized immune effector cell under suitable conditions and for a time sufficient to allow specific binding. If necessary or desired, the labeled cells can be physically removed from unbound label or excess unbound label can be inactivated.
  • the requirements of an antibody specific for a cell surface marker on an immune effector cell are that the antibody bind specifically and that the antibody not interfere with binding between a TCR and the immobilized synthetic antigenic peptide epitope.
  • Labels which may be employed are known to those skilled in the art and include, but are not limited to, traditional labeling materials such as fluorophores, radioactive isotopes, chromophores, and magnetic particles.
  • Enzyme labels include, but are not limited to, luciferase; a green fluorescent protein (GFP), for example, a GFP from Aequorea victoria, or any of a variety of GFP known in the art; ⁇ -galactosidase, chloramphenicol acetyl transferase. See, for example, Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, and periodic updates). Any assay which detects the label, either by directly or indirectly, is suitable for use in the present invention. Assays include colorimetric, fluorimetric, or luminescent assays, radioimmunoassays or other immunological assays.
  • the invention provides methods of modulating an immune response in an individual to an antigen encoded by a polynucleotide ofthe invention, and thus to the corresponding natural epitope.
  • Immunomodulatory methods ofthe invention include methods that result in induction or increase, as well as methods that result in suppression or reduction, of an immune response in a subject, and comprise administering to the subject an effective amount of a polynucleotide, or an APC, or an immune effector cell, of the invention in formulations and/or under conditions that result in the desired effect on an immune response (or lack thereof) to the peptide antigen.
  • Immunomodulatory methods ofthe invention include vaccine methods, adoptive immunotherapy, and methods to induce T cell unresponsiveness, or anergy.
  • CTL activation can be detected by any known method, including but not limited to, tritiated thymidine inco ⁇ oration (indicative of DNA synthesis), and examination ofthe population for growth or proliferation, e.g., by identification of colonies.
  • the tetrazolium salt MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5- diphenyl tetrazolium bromide) may be added.
  • Tumor cells or APCs ofthe invention, are radiolabeled as targets with about 200 ⁇ Ci of Na? 3 l CrO 4 for 60 minutes at 37° C, followed by washing. T cells and target cells ( ⁇ 1 x 10 4 /well) are then combined at various effector-to-target ratios in 96-well, U-bottom plates. The plates are centrifuged at 100 x g for 5 minutes to initiate cell contact, and are incubated for 4-16 hours at 37°C with 5% CO 2 .
  • an APC ofthe invention will vary, depending on the desired result.
  • peptides presented on an APC by Class I and Class II MHC molecules, together with the appropriate co-stimulatory molecules, will result in induction of an immune response to the peptide.
  • An anergic (or unresponsive) state may be induced in T lymphocytes by presentation of an antigen by an APC ofthe invention which contains appropriate MHC molecules on its surface, but which lacks the appropriate co-stimulatory molecules.
  • Polynucleotides of the invention can be administered in a gene delivery vehicle or by inserting into a host cell which in turn recombinantly transcribes, translates and processed the encoded polypeptide.
  • 59 polynucleotide of the invention in a pharmaceutically acceptable carrier can be combined with appropriate and effective amount of an adjuvant, cytokine or co- stimulatory molecule for an effective vaccine regimen.
  • the host cell is an APC, such as a dendritic cell.
  • the host cell can be further modified by inserting of a polynucleotide coding for an effective amount of either or both of a cytokine a co-stimulatory molecule.
  • the methods of this invention can be further modified by co-administering an effective amount of a cytokine or co-stimulatory molecule to the subject.
  • the agents provided herein as effective for their intended pu ⁇ ose can be administered to subjects having a disease to be treated with an immunomodulatory method ofthe invention or to individuals susceptible to or at risk of developing such a disease.
  • 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.
  • Therapeutic amounts can be empirically determined and will vary with the pathology or condition being treated, the subject being treated and the efficacy and toxicity ofthe therapy.
  • the amount of a polynucleotide or APC or immune effector cell ofthe invention will vary depending, in part, on its intended effect, and is ultimately at the discretion ofthe medical or veterinary practitioner.
  • the factors to be considered include the condition being treated, the route of adminitstration, and nature ofthe formulation, the mammal's body weight, surface area, age, and general condition and the particular peptide to be administered.
  • Cells can be administered once, followed by monitoring of the clinical response, such as diminution of disease symptoms or tumor mass. Administration may be repeated on a monthly basis, for example, or as appropriate.
  • an appropriate administrative regimen would be at the discretion of the physician or veterinary practitioner.
  • 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
  • compositions used for therapy the pu ⁇ ose 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 ofthe 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. More particularly, an agent ofthe 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), parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age ofthe recipient, and the disease or condition being treated.
  • immunomodulatory methods ofthe present invention comprise vaccines for cancer treatment.
  • Cancer cells contain many new antigens potentially recognizable by the immune system.
  • custom anticancer vaccines can be generated for affected individuals by isolating TILs from patients with solid tumors, determining their MHC restriction, and assaying these CTLs against the appropriate library for reactive epitopes. These vaccines will be both treatments for affected individuals as well as preventive therapy against recurrence (or establishment ofthe disease in patients which present with a familial genetic predisposition to it).
  • Polynucleotides and APCs ofthe present invention are also useful in methods to induce (or increase, or enhance) an immune response to a pathogenic organism. These include pathogenic viruses, bacteria, and protozoans.
  • Viral infections are ideal candidates for immunotherapy. Immunological responses to viral pathogens are sometimes ineffective as in the case ofthe lentiviruses such as HIV which causes AIDS. The high rates of spontaneous mutation make these viruses elusive to the immune system. However, a saturating profile of CTL epitopes presented on infected cells will identify shared antigens among different serotypes in essential genes that are largely intolerant to mutation which would allow the design of more effective vaccines.
  • the expanded populations of antigen-specific immune effector cells and APCs of the present invention find use in adoptive immunotherapy regimes and as vaccines.
  • 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 parental 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.
  • Synthetic antigenic peptide epitopes of the present invention are useful in methods to induce T cell unresponsiveness, or anergy. Disorders which can be treated using these methods include autoimmune disorders, allergies, and allograft rejection.
  • Autoimmune disorders are diseases in which the body's immune system responds against self tissues. They include most forms of arthritis, ulcerative colitis, and multiple sclerosis.
  • Polynucleotides ofthe invention encoding antigens corresponding to endogenous elements that are recognized as foreign can be used in the development of treatments using gene therapy or other approaches.
  • synthetic CTL epitopes which can act as "suicide substrates" for CTLs that mediate autoimmunity. can be designed as described above. That is to say, peptides which have a high affinity for the MHC allele but fail to activate the TCR could effectively mask the cellular immune response against cells presenting the antigen in question.
  • T cell activation as measured by interleukin-2 production and proliferation in vitro requires both antigenic and co-stimulatory signals engendered by cell to cell interactions among antigen-specific T cells and antigen presenting cells.
  • Various interactions of these CD2 proteins on the T-cell surface with CD58 (LFA-3) proteins and antigen-presenting cells, those of CDl la/CD 18 (LFA-1) proteins with CD54 (ICAM-1) proteins and those of CD5 proteins with CD72 proteins can impart such a co-stimulatory signal in vitro.
  • Cytokines derived from antigen-presenting cells e.g., interleukin- 1 and interleukin-6) can also provide co-stimulatory signals that result in T-cell activation in vitro.
  • 63 delivery of both antigenic and co-stimulatory signals leads to stable transcription ofthe interleukin-2 gene and other pivotal T cell-activation genes.
  • the foregoing co-stimulatory signals depend on protein kinase C and calcium.
  • Potent antigen presenting cells express CD80 (B7 and BB1) and other related surface proteins and many T cells express B7 binding proteins, namely CD28 and CTLA-4 proteins. Binding of CD80 by CD28 and CDLA-4 stimulates a T cell co- stimulatory pathway that is independent of protein kinase C and calcium leading to vigorous T cell proliferation.
  • the stimulation of B cells also depends on the interaction between the specific antigen and the cell-surface immunoglobulin.
  • T cell derived cytokines e.g., interleukins 1 and 4
  • T cells and B cells through specific pairs of receptors and co-receptors, or both, provide the signal or signals essential for B cell stimulation.
  • T-ceM stimulating or anergy producing amount (or therapeutically effective amount as described above) of an immunotherapeutic antigen-superantigen polymer according to the invention is contacted with the target cells.
  • T-cell anergy effective amount is intended an amount which is effective in producing a statistically significant inhibition of a cellular activity mediated by a TCR. This may be assessed in vitro using T-cell activation tests. Typically, T-cell anergy or activation is assayed by tritiated thymidine inco ⁇ oration in response to specific antigen.
  • T cell anergy can be induced is to present to a T cell an APC which presents an antigen in MHC Class I and Class II molecules, but which lack co-stimulatory molecules necessary to activate a T cell.
  • a cell other than a normal antigen presenting cell (APC), which has been transfected with MHC antigen to which a selected T cell clone is restricted, can be used.
  • Resting T cells are provided with an appropriate peptide recognized by the resting T cells in the context of the MHC transfected into a cellular host other than an APC.
  • the MHC is expressed as a result of introduction into a mammalian cell other than an antigen presenting cell of genes constitutively expressing the ⁇ and ⁇ chains ofthe MHC class II, or an MHC Class I molecule together with invariant chain. Importantly, these cells do not provide other proteins, either cell surface
  • the T cells to be tested can be cultured together with an APC which presents an antigen encoded by a polynucleotide ofthe invention in MHC Class I and Class II molecules together with co-stimulatory molecules necessary to activate the T cell.
  • the cultures are incubated for about 48 hours, then pulsed with tritiated thymidine and inco ⁇ oration measured about 18 hours later.
  • the absence of inco ⁇ oration above control levels, where the T-cells are presented with antigen presenting cells which do not stimulate the T cells, either due to using an MHC to which the T cells are not restricted or using a peptide to which the T cells are not sensitive, is indicative of an absence of activation.
  • the cells and compositions of this invention are useful as cancer vaccines and in adoptive immunotherapy.
  • the ability of autologous antigen-pulsed dendritic cells to induce a clinically relevant immune response has previously been reported. Hsu et al. (1996) Nature Med. 2(l):52-58.
  • Using this clinical study as a guide it is possible to administer an effective amount ofthe APCs as described herein to a subject to induce an anti-tumor immune response.
  • purified pulsed dendritic cells were administered by subcutaneous injection on days 2, 28 and 56 and then 5 to 6 months later.
  • patients received subcutaneous injections with either keyhole limpet hemocyanin or idiotype protein in saline at a site separate from intraveneous injection ofthe pulsed DCs.
  • the expanded populations of antigen-specific immune effector cells ofthe present invention also find use in adoptive immunotherapy regimes and as vaccines.
  • 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 naive 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 parental 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.
  • the adoptive immunotherapy methods are allogeneic.
  • cells from two or more patients are used to generate the APCs, and stimulate production ofthe immune effector cells.
  • cells from other healthy or diseased subjects can be used to generate antigen-specific cells in instances where it is not possible to obtain autologous T cells and/or dendritic cells from the subject providing the biopsy.
  • the expanded population can be administered to any one ofthe subjects from whom cells were isolated, or to another subject entirely.
  • 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.
  • the agents identified herein as effective for their intended pu ⁇ ose can be administered to subjects having tumors or individuals susceptible to or at risk of a 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 ofthe therapy.
  • the method is useful to further confirm efficacy of the agent.
  • 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 pu ⁇ ose 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.
  • 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.
  • 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. Using the "phage method" (Scott and Smith (1990) Science 249:386-390; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6387-6382; Devlin et al. (1990)
  • SPHERE can be used to identify antigens by creating a library of molecules, preferably peptides, and attaching one type of molecule to a solid support via a releasable linker. At least a portion ofthe molecules bound to each support can be released and it can be determined if the antigen-specific immune effector cells recognized the peptide.
  • this invention also provides a screen to identify novel wild-type antigens that can be further modified and used to induce a cellular and a humoral immune response in the subject.
  • the antigens and their biological activity in vitro and in vivo are positive controls.
  • the biological activity ofthe isolated antigen and its secreted form can be compared to their biological activity.
  • the invention provides a method for cloning the cDNA and genomic DNA encoding such a protein by generating degenerate oligonucleotides probes or primers based on the sequence ofthe 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. Hariow and Lane (1989), supra.
  • Any conventional method e.g., expression cloning methodology as described in Kawakami et al. (1994) Proc. Natl. Acad. Sci. 91:3515-19, 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.
  • SAGE U.S. Patent No. 5.695,937
  • SPHERE described in PCT WO 97/35035
  • SAGE analysis can be employed to identify the antigens recognized by expanded immune effector cells such as CTLs, by 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 tag sets between the two cell types, sequences which are aberrantly expressed in the antigen-expressing cell population can be identified.
  • cDNA complementary deoxyribonucleic acid
  • Solid PHase Epitope REcovery (“SPHERE") which is described in PCT WO 97/35035. Briefly, roughly speaking, peptide libraries are loaded onto beads and inserted into 96-well plates. The plates with 1000 beads per well will accommodate 10 6 beads; ten 96-well plates with 100 beads per well will accommodate lO 3 beads. In order to minimize both the number of CTL cells required per screen and the amount of manual manipulations, the eluted peptides can be further pooled to yield wells with any desired complexity. For example, based on experiments with soluble libraries, it should be possible to screen 10 7 peptides in 96-well plates
  • MART-1 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 et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:6458-62; Kawakami et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:91 :3515-9).
  • TILs tumor-infiltrating lymphocytes
  • the full-length open -eading frame ofthe mouse MART-1 consists of 342 bp, encoding a protein of 113 amino acid residues with a predicted molecular weight of ⁇ 13 kDa.
  • the described method for the identification of CD8 + MHC Class I restricted CTL epitopes can be applied to the identification of CD4 + MHC Class II restricted helper T-cell (Th) epitopes.
  • Th restricted helper T-cell
  • Class II allele-specific libraries are synthesized such that haplotype-specific
  • the overall length of the peptides will be 12-20 amino acid residues, and previously described methods may be employed to limit library complexity.
  • the screening process is identical to that described for MHC Class I-associated epitopes except that B lymphoblastoid cell lines (B-LCL) are used for antigen presentation rather than
  • T2 cells T2 cells.
  • B-LCLs that are defective in antigen processing (Mellins et al. (1991) J. Exp. Med. 174:1607- 1615); thus allowing specific presentation of exogenously added antigen, are employed.
  • the libraries are screened for reactivity with isolated CD4 + MHC Class II allele-specific Th cells. Reactivity may be measured by 3 H-thymidine inco ⁇ oration according to the method of Mellins, et al. supra., or by any ofthe methods previously described for MHC Class I-associated epitope screening.
  • polypeptides and proteins can be made for presentation on the APC of this invention.
  • the transcription cassette shown in the Figure is contained within an adenoviral vector such that expression ofthe genes leads to the production of intracellular and secreted forms ofthe same protein.
  • the adenoviral vector can be employed either in vivo for direct immunization as described in Zhai et al. (1996)
  • 71 dendritic cells derived from a host are transduced with the adenoviral vector and the genetically modified APCs are subsequently infused back into the host as described in Ribas et al. (1997) Cancer Research 57:2865-9.
  • C57BL/6 mice are injected subcutaneously at two sites (1.5E9 IU per site) with the adenoviral vector encoding the wild type and secreted forms of the same antigen.
  • Two weeks later, animals are challenged with a lethal dose (2 x 10 4 ) of murine B16F10 melanoma tumor cells by subcutaenous injection. Animals are scored for survival and tumor measurements are taken.
  • bone marrow cells are harvested from C57BL/6 mice and cultured in vitro with GM-CSF and IL4 to derive dendritic cells.
  • the DCs are infected at an MOI of 500 with the adenoviral vector overnight, and the next day the DCs (5 x 10 5 ) are washed and infused into recipient C57BL/6 mice via tail vein injection.
  • animals are challenged with a lethal dose of murine B16F10 melanoma tumor cells via subcutaneous injection as above and tumor size and survival are recorded as a function of time.

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  • Gastroenterology & Hepatology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un polynucléotide codant pour un antigène traité et présenté avec une molécule MHC de la classe I sur une cellule présentatrice de l'antigène (CPA), ce polynucléotide codant également pour un antigène traité et présenté avec une molécule MHC de la classe II sur cette cellule CPA. L'invention concerne également des compositions renfermant ces polynucléotides, des méthodes destinées à augmenter la présentation d'un peptide sur la surface d'une cellule CPA, et des cellules CPA produites selon ces méthodes. L'invention concerne enfin des méthodes diagnostiques et immunorégulatrices utilisant les polynucléotides et cellules CPA susmentionnés, ainsi que des cellules immunitaires effectrices.
EP99912709A 1998-03-20 1999-03-19 Compositions et methodes pour provoquer une reponse des cellules t par des vaccins a base de genes Withdrawn EP1064354A4 (fr)

Applications Claiming Priority (3)

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US7872598P 1998-03-20 1998-03-20
US78725P 1998-03-20
PCT/US1999/006030 WO1999047641A1 (fr) 1998-03-20 1999-03-19 Compositions et methodes pour provoquer une reponse des cellules t par des vaccins a base de genes

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US6800730B1 (en) * 1998-10-02 2004-10-05 Ludwig Institute For Cancer Research Isolated peptides which bind to MHC class II molecules, and uses thereof
JP4776852B2 (ja) 2000-01-28 2011-09-21 アメリカ合衆国 癌抗原ｎｙｅｓｏ−１由来の新規ｍｈｃクラスｉｉ拘束ｔ細胞エピトープ
US20040071726A1 (en) * 2000-09-12 2004-04-15 Chicz Roman M Peptide epitopes recognized by antigen specific cd4lymphocytes
AU2002347346A1 (en) * 2001-12-07 2003-06-17 University Of Liverpool Dna vaccine
US7048922B2 (en) 2002-05-29 2006-05-23 Demao Yang Stimulation of hematopoiesis by ex vivo activated immune cells
WO2005084387A2 (fr) * 2004-03-02 2005-09-15 Tsuneya Ohno Procedes et compositions ayant trait a des vaccins de cellules hybrides de traitement et prevention du cancer
KR20180006945A (ko) 2015-05-13 2018-01-19 아게누스 인코포레이티드 암 치료 및 예방용 백신
JP2021522239A (ja) 2018-04-26 2021-08-30 アジェナス インコーポレイテッド 熱ショックタンパク質結合ペプチド組成物およびその使用方法

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WO1994004171A1 (fr) * 1992-08-11 1994-03-03 President And Fellows Of Harvard College Peptides immunomodulateurs

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Publication number Priority date Publication date Assignee Title
EP0671926B1 (fr) * 1992-08-11 2002-11-13 President And Fellows Of Harvard College Peptides immunomodulateurs

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WO1994004171A1 (fr) * 1992-08-11 1994-03-03 President And Fellows Of Harvard College Peptides immunomodulateurs

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SUHRBIER A: "Multi-epitope DNA vaccines." IMMUNOLOGY AND CELL BIOLOGY, vol. 75, no. 4, 1997, pages 402-408, XP001068789 ISSN: 0818-9641 *
TINE ET AL: "NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for plasmodium falciparum malaria" INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON, US, vol. 9, no. 64, 1 September 1996 (1996-09-01), pages 3833-44, XP002077342 ISSN: 0019-9567 *
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EP1064354A4 (fr) 2002-06-12
AU3102299A (en) 1999-10-11
WO1999047641A1 (fr) 1999-09-23
JP2002506633A (ja) 2002-03-05
CA2322659A1 (fr) 1999-09-23

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