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WO1999046988A1 - Compositions et methodes de vaccination specifique de l'antigene - Google Patents

Compositions et methodes de vaccination specifique de l'antigene Download PDF

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Publication number
WO1999046988A1
WO1999046988A1 PCT/US1999/006034 US9906034W WO9946988A1 WO 1999046988 A1 WO1999046988 A1 WO 1999046988A1 US 9906034 W US9906034 W US 9906034W WO 9946988 A1 WO9946988 A1 WO 9946988A1
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Prior art keywords
antigen
cells
cell
subject
self
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Charles A. Nicolette
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Genzyme Corp
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Genzyme Corp
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Priority to EP99913979A priority Critical patent/EP1071325A4/fr
Priority to AU31934/99A priority patent/AU759765B2/en
Priority to JP2000536240A priority patent/JP2002506794A/ja
Priority to CA002323232A priority patent/CA2323232A1/fr
Publication of WO1999046988A1 publication Critical patent/WO1999046988A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • A61K40/4273—Glycoprotein 100 [Gp100]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55533—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • TECHNICAL FIELD This invention is in the field of molecular immunology and medicine.
  • the present invention provides compositions and methods for inducing an immune response to a native self-antigen in a subject.
  • T and B cells are highly antigen specific and can develop into memory cells, and therefore are the target for a successful vaccine.
  • Tumor specific T cells derived from cancer patients, will bind and lyse tumor cells. This specificity is based on their ability to recognize short amino acid sequences (epitopes) presented on the surface of the tumor cells by MHC class I and class II molecules. These epitopes are derived from the proteolytic degradation of intracellular proteins called tumor antigens encoded by genes that are either uniquely or aberrantly expressed in tumor or cancer cells.
  • a critical target of vaccines is the specialized antigen-presenting cell ("APC"), the most immunologically powerful of which is the bone marrow-derived dendritic cell (“DC”).
  • APC antigen-presenting cell
  • DC bone marrow-derived dendritic cell
  • Cancer vaccines have met with limited success. Since many naturally occurring neoplasms express only non-mutated self antigens, it is hypothesized that these antigens cannot serve as inducers and/or targets for a tumor destructive immune response, although immunological reactions mediated by either lymphocytes or antibodies to cultivated human tumors have been reported. Hellstrom and Hellstrom (1969) Adv. Cancer Res. 12:167-223. Mechanisms of systemic immune tolerance to self have begun to emerge, particularly from studies in transgenic mouse systems. Hanahan (1990) Ann. Rev. Cell Biol. 6:493-537. Mechanisms of systemic immune tolerance include deletion of potentially autoreactive B or T cells, induction of anergy in B and T cells, and the poorly defined phenomenon of suppression of immune response by suppressor cells. Houghton and Lewis, pages 37-54 in Forni et al. eds. (1994) CYTOKINE-INDUCED
  • This invention relates to the use of antigens for prophylactic vaccination against disease for which an immune response to the encoded antigen would be therapeutically effective in preventing or delaying the onset of the disease or to prolong a disease-free period in the subject.
  • this invention provides a substantially pure population of educated, antigen-specific immune effector cells produced by stimulating na ⁇ ve immune effector cells with antigen presenting cells (APCs) that present a self- antigen or an altered self-antigen.
  • APCs antigen presenting cells
  • this invention provides a method of inducing an immune response to a self-antigen in a subject, comprising administering to the subject an effective amount of APCs presenting the self-antigen or or an altered self-antigen.
  • the cells have been genetically modified to express and present the antigen.
  • this invention provides a method of inducing an immune response to a self-antigen in a subject, comprising administering to the subject an effective amount of the self-antigen or an altered self-antigen to the subject.
  • a polynucleotide encoding the antigen or altered antigen is administered to the subject.
  • FIGURES Figure 1 depicts the murine and human amino acid sequences for the melanoma antigen gplOO. Minimal essential epitopes are identified in bold lettering. The coding sequences can be deduced from these amino acid sequences.
  • Figure 2 depicts the coding sequence for human and murine melanoma antigen MARTI .
  • Figure 3 depicts the coding sequence for the human antigen MARTI and its deduced amino acid sequence.
  • Figure 4 (SEQ ID NO: 16 through SEQ ID NO: 17) is the coding sequence for the human tyrosinase-related-protein-2 cDNA. CTL epitopes are underlined.
  • Figure 5 shows the results of an assay of CTLs generated from normal donor PBLs with adenoviral gplOO infected dendritic cells.
  • a cell includes a plurality of cells, including mixtures thereof.
  • genetically modified means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous nucleotides. Methods and compositions useful in genetic modification are described in detail below.
  • antigen-presenting cells includes both intact, whole cells as well as other molecules which are capable of inducing the presentation of one or more antigens, preferably in association with class I MHC molecules.
  • suitable APCs include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, purified MHC class I molecules complexed to &2-microglobulin, foster antigen presenting cells, hybrid APCs and pulsed APCs.
  • Dendritic cells are potent antigen-presenting cells (APCs). It has been shown that DCs provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types.
  • the first type which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 ("TCR/CD3”) complex and an antigenic peptide presented by a major histocompatibility complex (“MHC") class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T cell activation to occur.
  • MHC major histocompatibility complex
  • the first type of signals can result in T cell anergy.
  • the second type of signals called co- stimulatory signals, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals.
  • dendritic cell is to include, but not be limited to a pulsed dendritic cell, a foster cell or a dendritic cell hybrid. It also is intended to encompass dendritic cells from any source, e.g., human, murine or simian.
  • 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.
  • immunogen is intended to mean full length proteins as well as peptide fragments thereof containing or comprising epitope.
  • a “native”, “natural” or “wild-type” 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.
  • An altered antigen is one having a primary sequence that is different from that of the corresponding wild-type antigen.
  • Altered antigens can be made by synthetic or recombinant methods and include, but are not limited to 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). A synthetic or altered antigen of the invention is intended to bind to the same TCR as the natural epitope.
  • a "self-antigen” also referred to herein as a native or wild-type antigen is an antigenic peptide that induces little or no immune response in the subject due to self-tolerance to the antigen.
  • An example of a self-antigen is the human melanoma antigen gplOO.
  • immune effector cells refers to cells capable of binding an antigen and 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 GP-100.
  • 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 “naive” cell e.g., a na ⁇ ve immune effector cell, is a cell that has never been exposed to an antigen.
  • the term "educated, antigen-specific immune effector cell” is an immune effector cell as defined above, which has encountered antigen and which is specific for that antigen.
  • An educated, antigen-specific immune effector cell may be activated upon binding antigen. "Activated” implies that the cell is no longer in Go phase, and begins to produce cytokines characteristic of the cell type. For example, activated CD4 + T cells secrete IL-2 and have a higher number of high affinity IL-2 receptors on their cell surfaces relative to resting CD4 + T cells.
  • a peptide or polypeptide of the invention may be preferentially recognized by antigen-specific immune effector cells, such as B cells and T cells.
  • antigen-specific immune effector cells such as B cells and T cells.
  • the term "recognized” intends that a peptide or polypeptide of the invention, comprising one or more 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 of the T cell.
  • TCR T cell antigen receptor
  • polypeptide of the 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.
  • a cell being administered to an individual is autogeneic if the cell was derived from that individual (the "donor") or a genetically identical individual.
  • An 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.
  • culturing refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (either morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
  • a "subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al. (1989) supra).
  • 7 eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • RNA polymerase II a downstream polyadenylation signal
  • start codon AUG a downstream polyadenylation signal
  • termination codon for detachment of the ribosome.
  • 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 participate in antigen presentation to CD4 + T cells and, in humans, include HLA-DP, -DQ, and DR.
  • MHC restriction refers to a characteristic of T cells that permits them to recognize antigen only after it is processed and the resulting antigenic peptides are displayed in association with either a self class I or class II MHC molecule. Methods of identifying and comparing MHC are well known in the art and are described in Allen et al. (1994) Human Immunol. 40:25-32; Santamaria et al. (1993) Human Immunol. 37:39-50; and Hurley et al. (1997)
  • an “isolated” or “purified” population of cells is substantially free of cells and materials with which it is associated in nature. By substantially free or substantially purified is meant at least 50% of the population are immune effector cells, preferably at least 70%, more preferably at least 80%, and even more
  • “Operably linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
  • 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 baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors and the like.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell.
  • the integrated DNA form is called a provirus.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene.
  • Ads Adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. (see, e.g., WO 95/27071). Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad-derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed, (see,
  • Wild-type AAV has high infectivity and specificity integrating into the host cell's genome.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI).
  • Gene delivery vehicles also include several non- viral vectors, including DNA/liposome complexes, and targeted viral protein-DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • the nucleic acid to enhance delivery to a cell, the nucleic acid, and fragment thereof.
  • 10 acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
  • cell surface antigens e.g., TCR, CD3 or CD4.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25 °C to about 37°C; hybridization buffer concentrations of about 6 X SSC to about 10 X SSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 6 X SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9 X SSC to about 2 X SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5 X SSC to about 2 X SSC.
  • Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1 X
  • SSC to about 0.1 X SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1 X SSC, 0.1 X SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%), 90%, or 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
  • 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 248:1349-1356 and Jenkins, M.K. (1992) Immunol. Today 13:69-73).
  • One signal the one that confers specificity, can be produced by interaction of the TCR CD3 complex with an appropriate MHC/peptide complex. The second signal is not antigen specific and is termed the "co-stimulatory" signal.
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS intracellular adhesion molecule 1
  • IAM-1 Intracellular adhesion molecule 1
  • the term "inducing an immune response in a subject” is a term well understood in the art and intends that an increase of at least about 2- fold, more preferably at least about 5-fold, more preferably at least about 10-fold, more preferably at least about 100-fold, even more preferably at least about 500- fold, even more preferably at least about 1000-fold or more in an immune response to an antigen (or epitope) can be detected (measured), after introducing the antigen (or epitope) into the subject, relative to the immune response (if any) before introduction of the antigen (or epitope) into the subject.
  • An immune response to an antigen includes, but is not limited to, production of an antigen-specific (or epitope-specific) antibody, and production of an immune cell expressing on its surface a molecule which specifically binds to an antigen (or epitope).
  • Methods of determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art.
  • antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, wherein, for example, binding of an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).
  • ELISA 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 molecule refers to molecules capable of antigen-specific binding, and includes antibodies, T cell antigen receptors, and MHC Class I and Class II molecules.
  • 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.
  • Tumor cell 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 H-thymidine incorporation assay, or counting tumor cells.
  • "Suppressing" tumor cell growth means any or all of the following states: slowing, delaying, and stopping tumor growth, as well as tumor shrinkage.
  • culturing 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 (morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
  • cytokine refers to any one of the 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 of the 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 (GM-CSF), interleukin- 1 alpha (IL-U ), interleukin- 11 (IL-11), MIP-U , leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand.
  • IL-2 interleukin-2
  • SCF stem cell factor
  • IL-3 interleukin 6
  • IL-12 interleukin 12
  • G-CSF granulocyte macrophage-colony stimulating
  • the present invention also includes culture conditions in which one or more cytokine is specifically excluded from the medium.
  • Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D
  • an "immunostimulatory agent” is any agent that enhances or supplements the immune response, e.g., a cytokine or co-stimulatory molecule.
  • 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 of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • an effective amount of an immunomodulatory agent of the invention including a peptide of the invention, a polynucleotide of the invention, an educated, antigen- specific immune effector cell and/or an APC of the invention, is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the
  • This invention provides cancer vaccines and methods of using the vaccines to induce a prophylactic immune response to a native self-antigen in a subject.
  • the self-antigen (usually a tumor-associated antigen or TAA) can be delivered to antigen-presenting cells as protein/peptide or, in the form of cDNA encoding the protein/peptide.
  • the self- antigens include altered forms of the antigen (defined above) that bind to the same epitope as the native or wild-type self-antigen.
  • the antigens may be homologous or heterologous to the cell on which it is presented.
  • APCs may be genetically modified ex vivo or targeted in vivo using the vectors described herein.
  • Antigen-presenting cells can consist of dendritic cells (DCs), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules.
  • the APCs may be derived from any species, e.g., murine, human or simian, and may be autologous or heterologous to a subject.
  • antigen is used in its broadest sense and includes minimal epitopes and chimeric molecules in addition to isolated full length proteins.
  • the antigen of this vaccine is homologous, heterologous (e.g., a murine antigen administered to a human patient) or an altered antigen as compared to the corresponding native self-antigen.
  • Antigens can be antigens of any type, e.g., tumor associated antigens.
  • the polynucleotides encoding TAAs of this invention can be, in one embodiment, previously characterized tumor- associated antigens such as gplOO (Kawakami et al.
  • altered polypeptides are encompassed within the term "altered antigen” as used herein.
  • altered antigen can be identified by hybridization under stringent conditions (defined above) to the sequences disclosed in the published references or known in the art.
  • the polynucleotides and polypeptides can be identified as being at least 80%, or more preferably, at least 90% or most preferably, at least 95%, identical (defined above) to the disclosed sequences using sequence alignment programs and default parameters.
  • Polypeptides and polynucleotides having the above noted sequence identity to native antigen are intended to be encompassed within the term "altered antigen.”
  • an epitope or wild-type antigenic peptide corresponding to a yet unidentified protein is also within the scope of this invention.
  • Solid-PHase Epitope REcovery (“SPHERE”, described in PCT WO 97/35035) can be used to identify tumor antigens and produce altered antigens or epitopes that can induce a prophylactic response against a self-antigen. SPHERE is described below.
  • the invention provides a method for cloning the cDNA and genomic DNA encoding such a protein by generating degenerate oligonucleotide probes or primers based on the sequence of the epitope.
  • Compositions comprising the nucleic acid and a carrier, such as a pharmaceutically acceptable carrier, a solid support or a detectable label, are further provided by this method as well as methods for detecting the sequences in a sample using methods such as Northern analysis, Southern analysis and PCR.
  • compositions comprising the oligopeptide sequence and a carrier, such as a pharmaceutically acceptable carrier, a solid support or a detectable label, are further provided by this method as well as methods for detecting the oligopeptide sequence in a sample using methods such as Western analysis and ELISA. Harlow and Lane (1989), supra.
  • this invention provides a means to elicit CD4 + and CD8 + T cells responses in a subject who is not exhibiting disease symptoms associated with the antigen.
  • the methods involve eliciting a protective immune response in normal individuals that may be environmentally at risk or genetically predisposed to disease. Since many known tumor antigens are non-mutated self-antigens, immunization against these antigens in normal subjects involves breaking tolerance to these self antigens. Assays to determine T cell response are well known in the art and any method that will compare T cell number prior to and subsequent to therapy can be utilized. In addition, the induction of co-stimulatory cytokines by the heterologous antigen could also stimulate pre-existing anergic or low affinity self-reactive CTL clones.
  • the APCs are manipulated to express the antigen or interest.
  • Methods of inducing APCs to express antigens include peptide-pulsing, or, preferably, genetic modification that results in expression of polynucleotides encoding the antigen of interest.
  • an effective amount of a gene delivery vehicle which expresses the antigen of interest is administered to the subject.
  • the gene delivery vehicle is a recombinant adenoviral vector that preferentially infects APCs such as dendritic cells.
  • Compositions comprising the modified APCs with a carrier are also provided.
  • 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 APC expressing an antigen can be used to expand and isolate a population of immune effectors which, in turn, are useful for adoptive immunotherapy alone or as an adjuvant to the methods described above.
  • cytokines and/or co-stimulatory molecules or nucleic acids encoding them can be co-administered with the immune effector cells.
  • An increase in the T cell count as compared to prior to therapeutic administration is a positive indication that the immune effector cell is a therapeutic for use in the method.
  • the immune effector cells Prior to administration in vivo, the immune effector cells are screened in vitro for their ability to lyse tumor cells.
  • the antigens of this invention can be an isolated counterpart or fragment of the counterpart to the human or animal tumor- associated antigen.
  • this invention also provides a screen to identify novel heterologous or altered antigens that induces an immune response in the subject by assaying for the ability of the putative heterologous or altered antigen for the ability to cross-react with native antigen and induce an immune response in a subject.
  • the murine melanoma antigen gplOO and its biological activity in vitro and in vivo is a positive control in this assay.
  • the biological activity of the putative heterologous or altered antigen can be compared to its biological activity.
  • Dendritic cells are specialized antigen presenting cells (APCs) that are critical for eliciting T cell mediated immune responses. At least two methods have been used for the generation of human dendritic cells from hematopoietic precursor cells in peripheral blood. One approach utilizes the rare CD34+
  • the methods described in Romani et al (1996), infra and Bender et al (1996) J. Immunol. Methods 196:121-135, are used to generate both immature and mature dendritic cells from the peripheral blood mononuclear cells (PBMC) of a mammal, such as a murine, simian or human.
  • PBMC peripheral blood mononuclear cells
  • isolated PBMC are pre-treated to deplete T- and B-cells by means of an immunomagnetic technique. Lymphocyte-depleted PBMC are then cultured for 7 days in RPMI medium, supplemented with 1% autologous human plasma and
  • Dendritic cells are nonadherent when compared to their monocyte progenitors. Thus, on day 7, non-adherent cells are harvested for further processing.
  • the dendritic cells derived from PBMC in the presence of GM-CSF and IL-4 are immature, in that they can lost the nonadherence property and revert back to macrophage cell fate if the cytokine stimuli are removed from the culture.
  • the dendritic cells in an immature state are very effective in processing native protein antigens for the MHC class II restricted pathway (Romani et al. (1989) J. Exp. Med. 169:1169. Further maturation of cultured dendritic cells is accomplished by culturing for 3 days in a macrophage-conditioned medium (CM), which contains the necessary maturation factors.
  • CM macrophage-conditioned medium
  • Mature dendritic cells are less able to capture new proteins for presentation but are much better at stimulating resting T cells (both CD4 + and CD8 + ) to grow and differentiate.
  • Older previously utilized methods involve (1) isolating bone marrow precursor cells (CD34 + ) from blood and stimulating them to differentiate into APC; or (2) collecting the precommitted APCs from peripheral blood.
  • the patient In the first approach, the patient must be treated with cytokines such as GM-CSF to boost the number of circulating CD34 + stem cells in the peripheral blood.
  • 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 adherence/nonadherence steps (Freudenthal et al. (1990) PNAS 87:7698-7702);
  • CCE countercurrent centrifugal elutriation
  • cells are subject to simultaneous centrifugation and a washout stream of buffer which is constantly increasing in flow rate.
  • the constantly increasing countercurrent flow of buffer leads to fractional cell separations that are largely based on cell size.
  • 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 of the mammal.
  • This method includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more 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 or GT-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.
  • One way to identify the dendritic cell-enriched fraction is by fluorescence-activated cell sorting.
  • the white blood cell fraction can be treated with calcium ionophore in the presence of other cytokines, such as rhIL-12, rhGM-CSF, or rhIL-4.
  • the cells of the white blood cell fraction can be washed in buffer and suspended in Ca ⁇ /Mg -* " free media prior to the separating step.
  • the white blood cell fraction can be obtained
  • the dendritic cells can be identified by the presence of at least one of the following markers: HLA-DR, HLA-DQ, or B7. 2, and the simultaneous absence of the following markers: CD3, 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 leukapheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE) (Abrahamsen et al. (1991) J. Clin. Apheresis. 6:48-53).
  • CCE countercurrent centrifugal elutriation
  • Cell samples are placed in a special elutriation rotor.
  • the rotor is then spun at a constant speed of, for example, 3000 rpm. Once the rotor has reached the desired speed, pressurized air is used to control the flow rate of cells.
  • Cells in the elutriator are subjected to simultaneous centrifugation and a washout stream of buffer 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); CD14 (monocyte); CD16, 56, 57 (NK/LAK cells); CD19, 20 (B cells). At the same time, DCs do express large quantities of HLA-DR, significant HLA-DQ and B7.2 (but little or no B7.1) at the time they are circulating in the blood (in addition they express Leu M7 and M9, myeloid markers which are also expressed by monocytes and neutrophils).
  • propridium iodide When combined with a third color reagent for analysis of dead cells, propridium iodide (PI), it is possible to make positive identification of all cell subpopulations (see Table 1):
  • Color #1 CD3 alone, CD14 alone, etc.; Leu M7 or Leu M9; anti-Class I, etc.
  • Color #2 HLA-Dq, B7.1, B7.2, CD25 (IL2r), ICAM, LFA-3, etc.
  • the goal of FACS analysis at the time of collection is to confirm that the DCs are enriched in the expected fractions, to monitor neutrophil contamination, and to make sure that appropriate markers are expressed.
  • This rapid bulk collection of enriched DCs from human peripheral blood, suitable for clinical applications is absolutely dependent on the analytic FACS technique described above for quality control. If need be, mature DCs can be immediately separated from monocytes at this point by fluorescent sorting for "cocktail negative" cells. It may not be necessary to routinely separate DCs from monocytes because, as will be detailed below, the monocytes themselves are still capable of differentiating into DCs or functional DC-like cells in culture.
  • cytokines include but are not limited to rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when given alone is inadequate for optimal upregulation.
  • the APCs and cells expressing one or more antigens are autologous. In another embodiment, the APCs and cells expressing the antigen are allogeneic, i.e., derived from a different subject.
  • Any conventional method e.g., subtractive library, comparative Northern and/or Western blot analysis of normal and tumor cells, SAGE (U.S. Patent No.
  • SAGE analysis can be employed to identify the antigens recognized by expanded immune effector cells such as CTLs. SAGE analysis involves identifying nucleotide sequences expressed in the antigen-expressing cells.
  • SAGE analysis begins with providing complementary deoxyribonucleic acid (cDNA) from (1) the antigen-expressing population and (2) cells not expressing that antigen. Both cDNAs can be linked to primer sites. Sequence tags are then created, for example, using the appropriate primers to amplify the DNA. By measuring the differences in these tags between the two cell types, sequences which are over expressed in the antigen-expressing cell population can be identified.
  • cDNA complementary deoxyribonucleic acid
  • Muteins of the antigen as well as allogeneic and antigens from a different species, of previously characterized antigens are useful in the subject invention.
  • Examples of known antigens from which muteins and altered antigens can be derived include, but are not limited to MARTI and gplOO, The full-length open reading frame of the mouse MARTI consists of 342 bp, encoding a protein of 113
  • the murine homologue of gplOO has also been identified.
  • the open reading frame consists of 1,878 bp, predicting a protein of 626 amino acid residues which exhibits 75.5% identity to human gplOO.
  • SPHERE described in PCT WO 97/35035, is a method that will identify wild-type or native antigens as well as provide altered antigens.
  • SPHERE is an empirical screening method for the identification of MHC Class I-restricted CTL epitopes that utilizes peptide libraries synthesized on a solid support (e.g., plastic beads) where each bead contains approximately 200 picomoles of a unique peptide that can be released in a controlled manner.
  • the synthetic peptide library is tailored to a particular HLA restriction by fixing anchor residues that confer high-affinity binding to a particular HLA allele (e.g., HLA-A2) but contain a variable TCR epitope repertoire by randomizing the remaining positions.
  • peptide pools containing reactive species can be determined by measuring 51 Cr-release according to standard methods known in the art.
  • cytokine production e.g., interferon- ⁇
  • proliferation e.g., proliferation
  • the beads corresponding to those mixtures are separated into smaller pools and distributed to new 96-well plates (e.g., 100 beads per well). An additional percentage of peptide is released from each pool and reassayed for activity by one of the methods listed above.
  • the beads corresponding those peptide mixtures are redistributed at 1 bead per well of a new 96-well plate. Once again, an additional percentage of peptide is released and assayed for reactivity in order to isolate the single beads containing the reactive library peptides.
  • the sequence of the peptides on individual beads can be determined by sequencing residual peptide bound to the beads by, for example, N-terminal Edman degradation or other analytical techniques known to those of skill in the art.
  • Peptide fragments from antigens must first be bound to peptide binding receptors ((MHC) class I and class II molecules) that display the antigenic peptides on the surface of the APCs.
  • MHC peptide binding receptors
  • T lymphocytes produce an antigen receptor that they use to monitor the surface of APCs for the presence of foreign peptides.
  • the antigen receptors on T H cells recognize antigenic peptides bound to MHC class II molecules whereas the receptors on CTLs react with antigens displayed on class I molecules.
  • antigens can be delivered to antigen- presenting cells as protein/peptide or in the form of polynucleotides encoding the protein/peptide ex vivo or in vivo.
  • the methods described below focus primarily on DCs which are the most potent, preferred APCs.
  • APCs genetically modified APCs. These include: (1) the introduction into the APCs of polynucleotides that express antigen or fragments thereof; (2) infection of APCs with recombinant vectors to induce endogenous expression of antigen; and (3)
  • Pulsing is accomplished in vitro/ex vivo by exposing APCs to antigenic protein or peptide(s).
  • the protein or peptide(s) are added to APCs at a concentration of 1-10 Tm for approximately 3 hours.
  • Paglia et al. (1996) J. Exp. Med. 183:317, has shown that APC incubated with whole protein in vitro were recognized by MHC class I-restricted CTLs, and that immunization of animals with these APCs led to the development of antigen-specific CTLs in vivo.
  • Protein peptide antigen can also be delivered to APC in vivo and presented by the APC.
  • Antigen is preferably delivered with adjuvant via the intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
  • Foster APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763). This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1, TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC class 1 -restricted CD8 + CTLs. In effect, only "empty" MHC class I molecules are presented on the surface of these cells.
  • T2 cells Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif.
  • These T2 cells are referred to herein as "foster" APCs. They can be used in conjunction with this invention to present the heterologous, altered or control antigen.
  • T2 cells with specific recombinant MHC alleles allows for redirection of the MHC restriction profile.
  • Libraries tailored to the recombinant allele will be preferentially presented by them because the anchor residues will prevent efficient binding to the endogenous allele.
  • Hybrid APCs WO 98/58541 describes a method to fuse cells expressing an antigen with dendritic cells in a manner that the dendritic cells take up and present the antigens expressed by the antigen-expressing cells.
  • the DCs are fused with the cells in the presence of a fusing agent (e.g., polyethylene glycol or Sendai virus).
  • a fusing agent e.g., polyethylene glycol or Sendai virus.
  • a medium which optionally contains hypoxanthine, aminopterin and thymidine
  • the cultured fused cells are separated from unfused non-DC parental cells based on the different adherence properties of the two cell groups.
  • the unfused parental DCs do not proliferate, and so die off.
  • isolated peptides of the 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 of the invention may be purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any 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 comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
  • affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991)
  • isolated nucleic acid sequences that encode the novel antigenic peptides described herein.
  • isolated means: an RNA or DNA polymer, portion of genomic nucleic acid, cDNA, or synthetic nucleic acid which, by virtue of its origin or manipulation: (i) is not associated with all of a nucleic acid with which it is associated in nature (e.g. is present in a host cell as a portion of an expression vector); or (ii) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature.
  • isolated it is further meant a nucleic acid sequence: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.
  • PCR polymerase chain reaction
  • nucleic acid sequences of the present invention may be characterized, isolated, synthesized and purified using no more than ordinary skill.
  • the antigens and fragments thereof may be recombinantly produced and isolated for use in the subject invention using methods well known in the art and described below. See Sambrook et al., (1989) supra.
  • the antigen is delivered to the antigen presenting cell (APC) by a gene delivery vehicle, preferably an adenoviral vector.
  • APC antigen presenting cell
  • the APC is genetically modified to express the antigen.
  • genetic modifications of cells employed in the present invention are accomplished by introducing a vector containing a polypeptide or transgene encoding a
  • Viral vectors useful in the genetic modifications of this invention include, but are not limited to adenovirus, adeno-associated virus vectors, retroviral vectors and adeno-retroviral chimeric vectors.
  • a recombinant adenoviral vector is used to genetically modify the DC cells.
  • 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. Microbiol. Immunol. 158:97-129; GENE TARGETING: A PRACTICAL APPROACH (1992) ed. A. L. Joyner, Oxford University Press, NY). Several different approaches are feasible. Preferred is the helper-independent replication deficient human adenovirus system.
  • adenovirus vectors and other viral vectors which could be used in the methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields et al.
  • adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario (see, e.g., Microbix
  • chimeric adenoviral vectors for example as described in PCT/US97/21494, the disclosure of which is hereby incorporated by reference.
  • a "chimeric adenoviral vector” is an adenoviral vector that comprises polynucleotides from more than one adenovirus serotype.
  • the chimeric vectors contain an adenoviral backbone, for example, Ad 2/CFTR-l and Ad 2 FTR2 and others described in U. S. Patent No. 5,670,488, issued September 23, 1997.
  • Such vectors may include deletion of the El region, partial or complete deletion of the E4 region, and deletions within, for example, the E2 and E3 regions.
  • Construction of the chimeric adenoviral vectors can be based on adenovirus DNA sequence information widely available in the field, e.g., nucleic acid sequence databases such as GenBank.
  • Preparation of replication-defective chimeric adenoviral vector stocks can be accomplished using cell lines that complement viral genes deleted from the vector, e.g., 293 or A549 (available from the ATCC) cells containing the deleted adenovirus El genomic sequences.
  • complement viral genes deleted from the vector e.g., 293 or A549 (available from the ATCC) cells containing the deleted adenovirus El genomic sequences.
  • HER3 cells human embryonic retinoblasts transformed by Ad 12
  • the viruses can be recovered by freeze-thawing and subsequently purified using cesium chloride centrifugation.
  • virus purification can be performed using chromatographic techniques, e.g., as set forth in International Application No. PCT/US96/13872, filed August 30, 1996.
  • Titers of replication-defective chimeric adenoviral vector stocks can be determined by plaque formation in a complementing cell line, e.g., 293 cells.
  • Endpoint dilution using an antibody to the adenoviral hexon protein may be used to quantitate virus production or infection efficiency of target cells (Armentano et al. (1995) Hum. Gene Ther. 6:1343-53.
  • the chimeric adenoviral vectors of the invention may contain any expression control sequences such as a promoter or enhancer, a polyadenylation element, and any other regulatory elements that may be used to modulate or increase expression, all of which are operably linked in order to allow expression of the transgene.
  • any expression control sequences, or regulatory elements, which facilitate expression of the transgene is within the scope of the invention.
  • Such sequences or elements may be capable of generating tissue- specific expression or be susceptible to induction by exogenous agents or stimuli.
  • Retroviral Vectors The antigen presenting cells described herein can also be genetically modified with retroviral vectors produced recombinantly by procedures already taught in the art.
  • WO 94/29438 describes the construction of retroviral packaging plasmids and packaging cell lines.
  • the retroviral vectors useful in the methods of this invention are capable of infecting the cells described herein.
  • the techniques used to construct vectors, and transfect and infect cells are widely practiced in the art.
  • retroviral vectors are those derived from murine, avian or primate retro viruses.
  • Retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are the most commonly used because of the availability of retroviral variants that efficiently infect human cells.
  • Other suitable vectors include those based on the
  • GLV Gibbon Ape Leukemia Virus
  • the range of host cells that may be infected by a retrovirus or retroviral vector is determined by the viral envelope protein.
  • the recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the
  • murine ecotropic env of MoMLV allows infection of rodent cells
  • amphotropic env allows infection of rodent, avian and some primate cells, including human cells.
  • Amphotropic packaging cell lines for use with MoMLV systems are known in the art and commercially available and include, but are not limited to, PA12 and PA317. Miller et al. (1985) Mol. Cell. Biol. 5:431-437; Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902; and Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464.
  • Xenotropic vector systems exist which also allow infection of human cells.
  • the host range of retroviral vectors has been altered by substituting the env protein of the base virus with that of a second virus.
  • the resulting, "pseudotyped" virus has the host range of the virus donating the envelope protein and expressed by the packaging cell line.
  • VSV-G G-glycoprotein from vesicular stomatitis virus
  • the vectors will contain at least two heterologous genes or gene sequences: (i) the therapeutic gene to be transferred; and (ii) a marker gene that enables tracking of infected cells.
  • the viral vector can be modified to incorporate chimeric envelope proteins or nonviral membrane proteins into retroviral particles to improve particle stability and expand the host range or to permit cell type-specific targeting during infection.
  • the production of retroviral vectors that have altered host range is taught, for example, in WO 92/14829 and WO 93/14188.
  • Retroviral vectors that can target specific cell types in vivo are also taught, for example, in Kasahara et al. (1994) Science 266:1373-1376.
  • Kasahara et al. describe the construction of a Moloney leukemia virus (MoMLV) having a chimeric envelope protein consisting of human erythropoietin (EPO) fused with the viral envelope protein.
  • This hybrid virus shows tissue tropism for human red blood progenitor cells that bear the
  • Retroviral vectors capable of specifically targeting infection of cells are preferred for in vivo gene therapy.
  • the vector construct is introduced into a packaging cell line which will generate infectious virions.
  • Packaging cell lines capable of generating high titers of replication-defective recombinant viruses are known in the art, see for example, WO 94/29438.
  • Viral particles are harvested from the cell supernatant and purified for in vivo infection using methods known in the art such as by filtration of supernatants 48 hours post transfection.
  • the viral titer is determined by infection of a constant number of appropriate cells (depending on the retrovirus) with titrations of viral supernatants.
  • the transduction efficiency can be assayed 48 hours later by a variety of methods, including Southern blotting.
  • PCR can be performed to detect the marker gene or other virally transduced sequences. Generally, periodic blood samples are taken and PCR conveniently performed using e.g. NeoR probes if the NeoR gene is used as marker. The presence of virally transduced sequences in bone marrow cells or mature hematopoietic cells is evidence of successful reconstitution by the transduced cells.
  • PCR techniques and reagents are well known in the art, See, generally, PCR PROTOCOLS, A GUIDE TO METHODS AND
  • the actual amount of antigen being expressed by the APCs can be evaluated by ELISA.
  • Transduced APCs can subsequently be administered to the host via an intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
  • In vivo transduction of DCs, or other APCs can potentially be accomplished by administration of Ad (or other viral vectors) via different routes including intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery.
  • the preferred method 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 TAA 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.
  • the amount of TAA 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.
  • DCs can also be transduced in vitro/ex vivo by non-viral gene delivery methods such as electroporation, calcium phosphate precipitation or cationic lipid/plasmid DNA complexes.
  • 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.
  • Intramuscular delivery of plasmid DNA may also be used for immunization.
  • transduction efficiency and levels of transgene expression can be assessed as described above for viral vectors.
  • the present invention makes use of these APCs to stimulate production of an enriched population of antigen-specific immune effector cells.
  • the antigen- specific immune effector cells are expanded at the expense of the APCs, which die in the culture.
  • the process by which na ⁇ ve immune effector cells become educated by other cells is described essentially in Coulie (1997) Molec. Med. Today 3:261-268.
  • the antigen-specific immune effector cells are CTLs.
  • the cytotoxic T cells are polyclonal T cells isolated from a site of cytotoxic T cell infiltration from a subject. Alternatively, 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. In yet another embodiment, the CTLs may be any combination of the 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.
  • 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
  • IL2 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 and are specific for tumor-specific antigens which are presented by the APCs.
  • compositions This invention also provides compositions containing any of the above- mentioned proteins, muteins, polypeptides, nucleic acid molecules, vectors, cells antibodies and fragments thereof, and an acceptable solid or liquid carrier. When the compositions are used pharmaceutically, they are combined with a "pharmaceutically acceptable carrier" for diagnostic and therapeutic use. These compositions also can be used for the preparation of medicaments for the diagnosis and treatment of diseases such as cancer.
  • compositions of this invention will prevent or delay onset of disease.
  • Mice immunized against heterologous human gplOO developed a protective immune response and were resistant to a lethal subcutaneous challenge of B 16 melanoma cells (syngeneic tumor cell line that expresses gplOO).
  • mice immunized with homologous mouse gplOO failed to mount a protective immune response against B 16 melanoma cells and developed tumors at the site of B16 cell injection.
  • T lymphocytes and can be reconstituted with human PBLs. It may be possible to immunize such mice with test antigen to induce a response in adoptively transferred human PBLs and evaluate protection against challenge with a human tumor cell line (Mosier et al, 1988; Parney et al, 1997; Albert et al., 1997). Another possibility is immunization of HLA-A2.1 transgenic mice to reproduce the immune reactivity of HLA-A2 individuals.
  • the expanded populations of antigen-specific immune effector cells 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.
  • the adoptive immunotherapy methods are allogeneic.
  • cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells.
  • cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells.
  • cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells.
  • cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells.
  • cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells.
  • cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells.
  • cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells.
  • 40 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 of the 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 purpose can be administered to subjects or individuals susceptible to or at risk of developing a disease, such as cancer.
  • a disease such as cancer.
  • the agent When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • a tumor regression can be assayed.
  • Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the therapy. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
  • mice groups of nude mice (Balb/c NCR nu/nu female, Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 10 5 to about 10 9 hyperproliferative, cancer or target cells as defined herein.
  • the agent is administered, for example, by subcutaneous injection around the tumor. Tumor measurements to determine reduction of tumor size are made in two dimensions using venier calipers twice a week. Other animal models may also be employed as appropriate.
  • Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or
  • agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including nasal, topical (including transdermal, aerosol, buccal and sublingual), 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 of the recipient, and the disease being treated.
  • compositions and methods described herein are particularly useful in providing or inducing a prophylactic immune response in an animal.
  • Animals in a pre-disease state or in a disease free interval, i.e., having or pre-disposed to a condition subject to immune surveillance, are most suitably treated by the methods and compositions described herein.
  • Such conditions involve the activation of an immune response in a diseased state or period.
  • Dendritic cells were derived from peripheral blood of a HLA-A2+ human donor using standard GM-CSF/IL-4 culture technique. After six days, the cells were infected with an adenovirus (serotype 2) construct encoding human gp 100.
  • adenovirus serotype 2 construct encoding human gp 100.

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  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (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)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des méthodes et des compositions permettant d'induire une réponse immunitaire prophylactique à un autoantigène chez un sujet.
PCT/US1999/006034 1998-03-20 1999-03-19 Compositions et methodes de vaccination specifique de l'antigene Ceased WO1999046988A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP99913979A EP1071325A4 (fr) 1998-03-20 1999-03-19 Compositions et methodes de vaccination specifique de l'antigene
AU31934/99A AU759765B2 (en) 1998-03-20 1999-03-19 Compositions and methods for antigen-specific vaccination
JP2000536240A JP2002506794A (ja) 1998-03-20 1999-03-19 抗原特異性予防接種のための組成物および方法
CA002323232A CA2323232A1 (fr) 1998-03-20 1999-03-19 Compositions et methodes de vaccination specifique de l'antigene

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7889098P 1998-03-20 1998-03-20
US60/078,890 1998-03-20

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WO1999046988A1 true WO1999046988A1 (fr) 1999-09-23

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JP (1) JP2002506794A (fr)
AU (1) AU759765B2 (fr)
CA (1) CA2323232A1 (fr)
WO (1) WO1999046988A1 (fr)

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WO2001030847A1 (fr) * 1999-10-22 2001-05-03 Aventis Pasteur Limited Molecule gp100 modifiee et ses applications
US7198920B1 (en) 1999-01-29 2007-04-03 Corika Corporation HER-2/neu fusion proteins
US7229623B1 (en) 2000-08-03 2007-06-12 Corixa Corporation Her-2/neu fusion proteins

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7198920B1 (en) 1999-01-29 2007-04-03 Corika Corporation HER-2/neu fusion proteins
US7375091B2 (en) 1999-01-29 2008-05-20 Corixa Corporation HER-2/neu fusion proteins
US8568733B2 (en) 1999-01-29 2013-10-29 Glaxosmithkline Biologicals Sa HER-2/neu fusion proteins
WO2001030847A1 (fr) * 1999-10-22 2001-05-03 Aventis Pasteur Limited Molecule gp100 modifiee et ses applications
US7229623B1 (en) 2000-08-03 2007-06-12 Corixa Corporation Her-2/neu fusion proteins

Also Published As

Publication number Publication date
CA2323232A1 (fr) 1999-09-23
AU759765B2 (en) 2003-05-01
JP2002506794A (ja) 2002-03-05
EP1071325A1 (fr) 2001-01-31
AU3193499A (en) 1999-10-11
EP1071325A4 (fr) 2004-12-22

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