WO2008055354A1 - Compositions de cellules dendritiques chargées en arn pour déclencher l'aide des lymphocytes t cd4+ et procédés apparentés - Google Patents

Compositions de cellules dendritiques chargées en arn pour déclencher l'aide des lymphocytes t cd4+ et procédés apparentés Download PDF

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WO2008055354A1
WO2008055354A1 PCT/CA2007/002015 CA2007002015W WO2008055354A1 WO 2008055354 A1 WO2008055354 A1 WO 2008055354A1 CA 2007002015 W CA2007002015 W CA 2007002015W WO 2008055354 A1 WO2008055354 A1 WO 2008055354A1
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cells
antigen
apcs
rna
lysate
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Rafick-Pierre Sekaly
Bader Yassine-Diab
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Université de Montréal
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • A61K39/12—Viral 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
    • A61K39/46—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
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • 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
    • A61K39/464838—Viral antigens
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the generation of RNA-loaded antigen pulsed antigen presenting cells (APCs) and their use in immunotherapy and to educate immune effector cells.
  • APCs antigen pulsed antigen presenting cells
  • Antigen presenting cells are capable of processing and presenting one or more antigens in the form of peptide-MHC complexes on their cell surface to T cells, resulting in T-cell activation and initiation of an immune response against the antigen or antigens being presented.
  • APCs that present antigen to T cells include intact whole cells such as dendritic cells, macrophages and B-cells, as well as artificial antigen presenting cells.
  • Dendritic cells (DCs) are the most potent APCs involved in adaptive immunity. They coordinate the initiation of immune responses by naive T cells and B cells and induce antigen-specific cytotoxic T lymphocyte (CTL) responses.
  • CTL cytotoxic T lymphocyte
  • MHC The major histocompatibility complex
  • MHC molecules genes encoding cell-surface glycoproteins, termed "MHC molecules," that are required for antigen presentation to T cells.
  • HLA human leukocyte antigen
  • MHC Class I molecules include HLA-A, B, and C molecules in humans.
  • Peptide fragments of proteins synthesized and degraded in the cytosol are transported into the endoplasmic reticulum where they are bound to MHC class I molecules and presented at the cell surface to CD8 + T cells.
  • MHC Class I molecules are expressed by nearly all nucleated mammalian cells.
  • MHC class Il molecules function in antigen presentation to CD4 + T cells and, in humans, include HLA-DP, -DQ, and -DR molecules.
  • Exogenous antigens e.g., pathogens, proteins, peptides, etc.
  • APCs are taken up by APCs into intracellular vesicles, degraded into peptide fragments, bound by MHC Class Il molecules and transported to the cell surface for presentation to CD4 + T cells.
  • Antigen presenting cells and/or immune effector cells can be used to induce or enhance an immune response in a patient.
  • One approach to immunotherapy involves loading dendritic cells ex vivo with one or more antigens and administering the antigen loaded dendritic cells to a subject in order to elicit an immune response to the loaded antigens.
  • DCs have been loaded (pulsed) with antigen ex vivo either by culturing DCs with one or more antigens of interest or by transfecting DCs with a nucleic acid encoding an antigen of interest. See, for example, Gilboa and Vieweg (2004) Immunol. Rev. 199:251-263.
  • the loading method determines whether antigen will be presented by MHC class I or class Il molecules.
  • Dendritic cells take up exogenous antigen in the medium, and process and present the antigen in the form of a peptide:MHC Class Il complex to CD4 + T helper cells. In addition, there may be some exchange of exogenous peptides with peptides complexed with MHC class I or class Il molecules at the cell surface. Activated CD4 + T helper cells can then enhance an immune response through interactions with CD8 + T cells, macrophages, granulocytes and NK cells and secretion of effector cytokines, such as IFN- ⁇ . In contrast, dendritic cells transfected with a nucleic acid encoding an antigen express the antigen in the cytosol and present the antigen in the form of a peptide: MHC Class I complex to CD8 + T cells.
  • Substantial quantities of cell lysates, cell extracts or peptides are required in order to load a significant number of APCs.
  • an appropriate quantity of lysate, extract, protein or peptide is either difficult or expensive to obtain.
  • a limited quantity of tumor or pathogen cells may be available.
  • minute quantities of one or more antigen encoding nucleic acids can be easily amplified and used as a template for in vitro transcription in order to produce sufficient quantities of antigen encoding RNA for transfection of APCs.
  • loading a DC with an antigen encoding nucleic acid produces a DC which can stimulate CD8 + T cells, but not CD4 + T cells.
  • APCs antigen-presenting cells
  • the APCs are loaded with RNA prior to being pulsed with the extract or lysate.
  • the APCs are pulsed with the extract or lysate prior to being loaded with RNA.
  • the APCs are simultaneously loaded with RNA and pulsed with the extract or lysate.
  • the lysate or extract is made from RNA-loaded APCs.
  • the lysate or extract is made from the population of first APCs.
  • the APC is a dendritic cell.
  • medicaments and immunotherapeutic compositions comprising the RNA-loaded lysate/extracted-pulsed APCs of the inventions.
  • the invention provides an RNA-loaded antigen-pulsed APC.
  • APCs are useful for inducing a desired immune response in vivo and for stimulating T cells in vitro.
  • the present invention provides a method for preparing antigen-loaded antigen presenting cells (APCs), comprising: loading a population of APCs with one or more first RNAs encoding one or more first antigens, and pulsing the population of APCs with an extract or lysate of cells or virions which comprise one of more second antigens, wherein the population of APCs takes up the second antigen(s).
  • APCs antigen-loaded antigen presenting cells
  • the above-mentioned population of APCs is loaded with the RNA prior to pulsing with the extract or lysate. In another embodiment, the above-mentioned population of APCs is pulsed with the extract or lysate prior to loading with the RNA. In yet another embodiment, the above-mentioned population of APCs is simultaneously loaded with RNA and pulsed with extract or lysate.
  • the above-mentioned first antigen(s) is(are) presented by MHC class I molecules and the second antigen(s) is(are) presented by MHC class Il molecules. In an embodiment, the above-mentioned first antigen(s) and the above-mentioned second antigen(s) are the same.
  • the above-mentioned extract or lysate is prepared from cells transfected with one or more nucleic acids encoding the second antigen(s).
  • the above-mentioned nucleic acids are one or more second RNAs.
  • the above-mentioned first RNAs are the same as the above-mentioned second RNAs.
  • the above-mentioned cells are APCs.
  • the above-mentioned one or more of first RNAs encode an antigen from or derived from a tumor.
  • the above-mentioned tumor is renal cell cancer, melanoma, prostate cancer or chronic lymphocytic leukemia.
  • the above-mentioned one or more of the first RNAs encode an antigen derived from a pathogen.
  • the above-mentioned pathogen is selected from the group consisting of Helicobacter pylori, Salmonella, Shigella, Enterobacter, Campylobacter, Mycobacterium leprae, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella species, Leptospira interrogans, Staphylococcus aureus, Streptococcus, Clostridum, Candida albicans, Plasmodium, Leishmania, and Trypanosoma.
  • the above- mentioned pathogen is a bacterium or a virus.
  • the above-mentioned virus is selected from the group consisting of hepatitis B virus, hepatitis C virus (HCV) 1 human papilloma virus (HPV), cytomegalovirus (CMV), human T cell lymphotrophic virus (HTLV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), coronavirus, varicella-zoster virus, Epstein-Barr virus (EBV), influenza virus, poliomyelitis virus, measles virus, mumps virus, and rubella virus.
  • HCV hepatitis C virus
  • HPV human papilloma virus
  • CMV human T cell lymphotrophic virus
  • HSV-1 herpes simplex virus type 1
  • HSV-2 herpes simplex virus type 2
  • coronavirus varicella-zoster virus
  • Epstein-Barr virus (EBV) influenza
  • the above-mentioned virus is a retrovirus.
  • the above-mentioned retrovirus is human immunodeficiency virus (HIV).
  • the above-mentioned APCs are dendritic cells (DCs), B cells, macrophages, or artificial antigen presenting cells.
  • the above-mentioned lysate or extract is made by freezing and thawing the cells or virions. In another embodiment, the above-mentioned lysate or extract is made by sonicating the cells or virions.
  • the ratio of the number of APC pulsed with the lysate or extract (pulsed cells) to the number of cells or virions represented in the amount of lysate or extract used for the pulse (lysate/extract) is between 1 :0.01 to 1 :10,000. In a further embodiment, the above-mentioned ratio of pulsed cells to lysate/extract is between 1 :0.2 to 1 :10. In yet a further embodiment, the above-mentioned ratio of pulsed cells to lysate/extract is about 1 :1. In an embodiment, the above-mentioned extract or lysate is made from tumor cells.
  • the above-mentioned extract or lysate is made from pathogen cells or pathogen-infected cells. In another embodiment, the above-mentioned extract or lysate is made from virions. In yet another embodiment, the above-mentioned extract or lysate is made from RNA-loaded APCs. In a further embodiment, the above-mentioned extract or lysate is prepared from an aliquot of said population of RNA-loaded APCs. In another aspect, the present invention provides an APC made by the above-mentioned method. In another aspect, the present invention provides an RNA-loaded antigen-pulsed APC. In an embodiment, the above- mentioned APC is a dendritic cell.
  • the present invention provides a use of the above-mentioned APC for the preparation of a medicament for inducing an immune response to a tumor, pathogen or pathogen-infected cell.
  • the present invention provides a use of the above-mentioned APC for inducing an immune response to a tumor, pathogen or pathogen-infected cell.
  • the present invention provides a use of the above- mentioned APC for inducing T-cell activation/proliferation in a subject.
  • the present invention provides a use of the above-mentioned APC for the preparation of a medicament for inducing T-cell activation/proliferation in a subject.
  • the present invention provides a method for inducing T-cell activation/proliferation in a subject comprising contacting T cells from said subject with the above-mentioned APC.
  • the present invention provides a method for inducing T-cell proliferation, comprising contacting T cells with above-mentioned APC.
  • the present invention provides a method for inducing an immune response, comprising administering an effective amount of the above-mentioned APCs to a subject.
  • the above-mentioned immune response is to a tumor or a pathogen.
  • the above-mentioned pathogen is HIV.
  • the present invention provides a method for preparing antigen-loaded antigen-presenting cells (APCs), comprising: a. providing a population of APCs loaded with one or more first RNAs encoding one or more first antigens, and b. pulsing the population of APCs with an extract or lysate of cells or virions which comprise one of more second antigens, wherein the population of APCs takes up the second antigen(s).
  • APCs antigen-loaded antigen-presenting cells
  • Figure 1 shows the percentage of autologous CD4 + T cells and CD8 + T cells in PBMCs that proliferate (as indicated by low CarboxyFluoroscein Succinimidyl Ester (CFSE) staining) in response to dendritic cells loaded with RNA-encoding CMV antigens or a control antigen (GFP) and pulsed with a lysate of DCs loaded with these RNAs.
  • CFSE CarboxyFluoroscein Succinimidyl Ester
  • Figure 2 shows the specificities of proliferating CD4 + and CD8 + T cells produced in response to restimulation of CFSE low cells by CMV peptide pools.
  • Figure 3 shows the percentage of autologous CD4 + T cells and CD8 + T cells in PBMCs that proliferate (CFSE l0W ) in response to dendritic cells loaded with RNA encoding HIV Gag and pulsed with a lysate of DCs loaded with RNA encoding with either a negative control antigen (GFP), CMV or HIV Nef.
  • FFP negative control antigen
  • a cell includes a plurality of cells, including mixtures thereof.
  • compositions and methods include the recited elements, but do not exclude additional elements.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, 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.
  • Polypeptides or proteins that "consist essentially of a given amino acid sequence are defined herein to contain no more than three, preferably no more than two, and most preferably no more than one additional amino acid at the amino and/or carboxy terminus of the protein or polypeptide.
  • Nucleic acids or polynucleotides that "consist essentially of a given nucleic acid sequence are defined herein to contain no more than ten, preferably no more than six, more preferably no more than three, and most preferably no more than one additional nucleotide at the 5' or 3' terminus of the nucleic acid sequence.
  • 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.
  • Amplification refers to nucleic-acid-amplification procedures using primers and nucleic acid polymerase that generate multiple copies of a target nucleic acid sequence.
  • amplification reactions include, but are not limited to, the polymerase chain reaction (PCR, see U.S. 4,682,195, 4,683,202 and 4,965,188), PCR: THE POLYMERASE CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston (1994), RT-PCR (see U.S. 5,322,770 and 5,310,652) the ligase chain reaction (LCR, see EP 0 320 308), NASBA or similar reactions such as TMA described in U.S. 5,399,491 and gap LCR (GLCR, see U.S. 5,427,202).
  • RNA may first be copied into a complementary DNA strand using a reverse transcriptase (see U.S. 5,322,770 and 5,310,652).
  • antigen encompasses polypeptides, proteins and peptides that consist of or comprise at least one epitope, which when presented as an MHC/peptide complex, can specifically bind to a particular T-cell antigen receptor (TCR).
  • TCR T-cell antigen receptor
  • antigen includes substances which are immunogenic (i.e., a substance that can induce antibody production) as well as substances consisting of or comprising one or more epitopes (antigenic determinants).
  • any antigen is envisioned for use in the present invention and thus includes, but is not limited to, a self-antigen (whether normal or disease-related), an infectious antigen (e.g., a microbial antigen, viral antigen, etc.), or some other foreign antigen (e.g., a food component, pollen, etc.).
  • infectious antigen e.g., a microbial antigen, viral antigen, etc.
  • foreign antigen e.g., a food component, pollen, etc.
  • TAA tumor-associated antigen
  • examples of well-known TAAs include gplOO, survivin, MART and MAGE.
  • antigen presenting cell refers to specialized cells that can process antigens into peptide fragments and display those pepetide fragments on the cell surface together with molecules required for T cell activation.
  • APCs include dendritic cells, macrophages, B cells and artificial antigen-presenting cells.
  • artificial antigen-presenting cell refers to a living cell (other than dendritic cells, macrophages and B cells) which has been engineered to express MHC Class I and/or Il molecules and/or other molecules required for costimulating CD4+ and CD8+ T cells.
  • Artificial antigen presenting cells can include but are not limited to genetically engineered insect cells, mouse fibroblasts and human leukemia cell lines.
  • culturing refers to the in vitro maintenance, differentiation, and/or propagation of cells in a suitable liquid medium.
  • Dendritic cell refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, Steinman (1991 ) Ann. Rev. Immunol. 9:271-296. Dendritic cells constitute the most potent and preferred APCs in the organism. Dendritic cells can be isolated from a mammal or differentiated from CD14+ monocytes or CD34+ hematopoietic stem cells isolated from a mammal. The maturation state of DCs can be followed by monitoring the change of the surface markers on the DCs during this process. Some of the typical cell surface markers characteristic of the different stages of maturation of the dendritic cells are summarized in Table I, below. However, the surface markers can vary depending upon the maturation process. Table I
  • an "effective amount” is an amount sufficient to effect beneficial or desired results, such as enhanced immune response, treatment, prevention or amelioration of a medical condition (disease, infection, etc).
  • An effective amount can be administered in one or more administrations, applications or dosages. Suitable dosages will vary depending on body weight, age, health, disease or condition to be treated and route of administration.
  • enriched is meant a composition comprising cells present in a greater percentage of total cells than is found in the tissues where they are present in an organism.
  • the enriched cultures and preparations of antigen-loaded DCs made by the methods of the invention are present in a higher percentage of total cells as compared to their percentage in the tissues where they are present in an organism (e.g., blood, skin, lymph nodes, etc.).
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences or nucleotide sequences can be determined using E. Meyers and W.
  • nucleic acid and protein sequences described herein, such as the CD40L sequences can be used as a "query sequence" to perform a search of public databases to identify, for example, other family members or related sequences. Searches of this kind can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol., 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res., 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Immuno response broadly refers to the antigen-specific responses of lymphocytes to foreign substances.
  • An immune response of this invention can be humoral (via antibody activity) and/or cell-mediated (via T-cell activation).
  • immune effector cells refers to cells capable of binding an antigen and mediating an immune response. These cells include, but are not limited to B cells, monocytes, macrophages, NK cells and T cells, including cytotoxic T lymphocytes (CTLs), CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.
  • CTLs cytotoxic T lymphocytes
  • a "naive" effector T cell is an immune effector cell that has never encountered (nor been activated by) its specific antigen. Activation of naive immune effector cells requires both recognition of the peptide:MHC complex and the simultaneous delivery of a costimulatory signal by a dendritic cell or an artificial APC in order to proliferate and differentiate into antigen- specific armed effector T cells.
  • the term "educated, antigen-specific immune effector cell” refers to an immune effector cell that has previously encountered an antigen. In contrast to its naive counterpart, activation of an educated, antigen-specific, immune-effector cell does not require a costimulatory signal. Recognition of the peptide:MHC complex is sufficient.
  • Activated when used in reference to a T cell, implies that the cell is no longer in G 0 phase, and begins to produce one or more of cytotoxins, cytokines and other related membrane-associated proteins characteristic of the cell type (e.g., CD8 + or CD4 + ), and is capable of recognizing and binding any target cell that displays the particular peptide: MHC complex on its surface, and releasing its effector molecules.
  • cytotoxins e.g., CD8 + or CD4 +
  • the term "inducing an immune response in a subject” is a term understood in the art and refers to an increase of at least about twofold, or alternatively at least about fivefold, or alternatively at least about tenfold, or alternatively at least about 100-fold, or alternatively at least about 500-fold, or alternatively at least about 1000-fold or more in an immune response to an antigen (or epitope) which can be detected or measured, after introducing the APCs or educated, antigen-specific immune effector cells into the subject, relative to the immune response (if any) before introduction of the APCS or effector cells 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.
  • an 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).
  • isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with them in nature.
  • an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
  • a "concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated” or less than “separated” than that of its naturally occurring counterpart.
  • a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide.
  • a protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.
  • a mammalian cell, such as a dendritic cell is isolated if it is removed from the anatomical site from which it is found in an organism.
  • loading and “pulsing” have been used interchangeably in the art to refer to uptake by an APC of either an antigen or a nucleic acid
  • loading of antigen presenting cells (APCs)
  • APCs antigen presenting cells
  • pulses refers to contacting an APC with a lysate or extract of a cell or virion that contains one or more antigens.
  • lysate refers to material produced by the lysis of a cell or virion.
  • the lysate can be produced by any means, including freezing/thawing, sonication, induction of apoptosis, etc.
  • the lysate can be further purified after lysis to produce an extract.
  • extract refers to a fraction of the lysate. Non-limiting examples of such fractions include the pellet or supernatant of a centrifuged lysate, isolated organelles, apoptotic bodies, fractions isolated by size (e.g., by sucrose gradient) or affinity, etc.
  • mRNA means a translatable RNA.
  • the mRNA will contain a ribosome binding site and start codon.
  • the mRNA will also contain a 5' cap, stop codon and polyA tail.
  • Phathogen refers to any disease causing organism or virus, and also to attenuated derivatives thereof.
  • peptide refers to five or more amino acids covalently joined by peptide bonds.
  • 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 pharmaceutical carrier suitable for delivery of APCs or immune-effector cells, such as a phosphate-buffered saline solution, culture medium, serum, plasma, etc.
  • a preferred pharmaceutical carrier is 85% heat-inactivated autologous serum, 10% DMSO and 5% dextrose.
  • the compositions can also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin REMINGTON'S PHARM. SCI., 18th Ed. (Mack Publ. Co., Easton (1990)).
  • polynucleotide refers 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-stranded, 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 of the present invention may also comprise modified nucleic acid molecules.
  • mRNA refers to an RNA that can be translated in a dendritic cell. Such mRNAs typically are capped and have a ribosome binding site (Kozak sequence) and a translational initiation codon.
  • RNA refers to polymeric forms of ribonucleotides of any length, wherein the ribonucleotides or ribonucleotide analogs are joined together by phosphodiester bonds.
  • RNA includes, for example, single-stranded, double-stranded and triple helical molecules, primary transcripts, mRNA, tRNA, rRNA, in vitro transcripts, in vitro synthesized RNA, branched polyribonucleotides, isolated RNA of any sequence, and the like.
  • subject refers to a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • tumor or “cancer” is meant the abnormal presence of cells which exhibit relatively autonomous growth, such that a tumor cell exhibits an aberrant growth phenotype characterized by a significant loss of cell proliferation control.
  • Tumor cells can be benign or malignant.
  • the tumor affects cells of the bladder, blood, brain, breast, colon, digestive tract, lung, ovaries, pancreas, prostate gland, or skin.
  • the definition of a tumor cell, as used herein, includes not only a primary tumor cell, but also any cell derived from a tumor cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. Cancer includes both solid tumors and liquid tumors (e.g, hematologic malignancies).
  • mRNA-tranfected DCs elicit potent CD8 T-cell responses but fail to induce CD4 T-cell responses due to preferential processing of the proteins produced by transfected mRNA in the MHC class I pathway and absence (or minimal) processing through the MHC class Il pathway.
  • the absence of CD4 T cell help might negatively affect the activation and the differentiation of CD8 T cells, specifically by reducing their proliferation as well as their expression of IL-2 and cytolytic (CD107a, Granzyme B) mediators.
  • the invention is based on the discovery that both CD4 + T cell and CD8 + T cell antigen-specific responses can be stimulated by antigen-presenting cells (APCs) loaded with RNA and also pulsed with a lysate or extract of cells which express one or more antigens of interest. Accordingly, a method is provided for preparing antigen-loaded antigen-presenting cells (APCs), comprising: loading a population of APCs with one or more first RNAs encoding one or more first antigens, and pulsing the population of APCs with an extract or lysate of cells or virions which comprise one of more second antigens, wherein the population of APCs takes up the second antigen(s).
  • APCs antigen-presenting cells
  • the population of APCs is pulsed with the extract or lysate prior to loading with the RNA.
  • the APC is a dendritic cell (DC).
  • DCs can be pulsed with lysate or extract when immature or mature; however, uptake of the lysate or extract will be more efficient when the DC is immature.
  • the APCs are simultaneously loaded with RNA and pulsed with extract or lysate.
  • the APCs are loaded with the RNA prior to pulsing with the extract or lysate.
  • the invention provides a method for preparing antigen- loaded antigen-presenting cells (APCs), comprising: a. providing a population of APCs loaded with one or more first RNAs encoding one or more first antigens, and b. pulsing the population of APCs with an extract or lysate of cells or virions which comprise one of more second antigens, wherein the population of APCs takes up the second antigen(s).
  • the RNA-loaded, lysate/extract-pulsed APCs present the first antigen(s) in complex with MHC class I molecules and the second antigen(s) in complex with MHC class Il molecules.
  • the first and second antigens are the same.
  • Antigen presenting cells are capable of stimulating T cells, and include professional antigen presenting cells such as dendritic cells, macrophages and B cells, as well as artificial antigen-presenting cells.
  • the APCs are human. Most preferably, the APCs are dendritic cells. Methods for isolating APCs or differentiating APCs from precursors are known in the art.
  • Cell-based artificial APCs are known to those in the art. See, for example, Kim et al. (2004) Nature Biotechnology 22:403-410. Artificial APCs include, but are not limited to a murine fibroblast which expresses B7.1 (CD80), ICAM-1 (CD54), leukocyte function antigen (LFA)-3 (CD58), HLA molecule A2.1 and human ⁇ 2 -microglobulin (Panicolaou et al. (2003) Blood 102:2498-2505); and a human leukemia cell line K562 precursor (which is negative for HLA-A, HLA-B and HLA-DR, and positive for HLA-C).
  • the K562 precursor is made into an artificial APC (K32) by transfection with CD32 and coating with anti-CD3 and nati-CD28 mouse IgG monoclonal antibodies.
  • artificial APCs express a ligand for CD28 receptor (e.g, CD80 or an anti-CD28 mAb) MHC Class I and Class Il molecules, and preferably, ICAM-1 , and optionally, LFA-3, CD40L and B7RP-1.
  • ligand for CD28 receptor e.g, CD80 or an anti-CD28 mAb
  • MHC Class I and Class Il molecules preferably, ICAM-1 , and optionally, LFA-3, CD40L and B7RP-1.
  • artificial APCs are prepared from cells autologous to the patient to be treated.
  • a population of APCs is loaded with RNA encoding one or more antigens, resulting in the expression of the antigen(s) within the APC.
  • the translated antigen can then be processed intracellular ⁇ into fragments, and the fragments can then be bound and presented by MHC class I molecules.
  • the encoded antigen is a short peptide that does not require fragmentation in order to be presented by MHC molecules.
  • APCs may be transfected with a homogenous population of RNA (i.e., the RNAs encode the same antigen(s)), or with a heterogeneous population of RNAs.
  • the RNA may encode a single antigen or a plurality of antigens.
  • a plurality of antigens may be encoded on a single RNA or multiple RNAs.
  • a single RNA can encode one or more open reading frames, or portions of one or more open reading frames.
  • the APCs are cotransfected with antigen encoding RNA and an RNA encoding CD40 Ligand (CD40L), from or derived from a mammal, preferably a human.
  • CD40L CD40 Ligand
  • the antigens can be from or derived from any cell or pathogen or other compositions (e.g., natural or synthetic) to which one wishes to elicit an immune response. Because practicing the invention does not require identifying a particular antigen of a cell or pathogen, RNA derived or isolated from essentially any type of cell or pathogen to which an immune response is desired can be used in the methods of the invention. Alternatively, a nucleic acid, preferably, an RNA, encoding one or more preselected antigen(s) may be used for loading APCs or other cells from which the extract or lysate can be made. Preferred antigens are from or derived from tumors or pathogens (e.g. bacteria, parasites, viruses).
  • tumors or pathogens e.g. bacteria, parasites, viruses.
  • Antigens that are "from" a tumor or a pathogen have the same amino acid sequence as a naturally occurring antigen in a tumor or pathogen.
  • Naturally occurring antigens can be either wild type or mutant.
  • RNA encoding of a naturally occurring antigen may be identical to RNA since it naturally occurs in the cell, or may differ from the naturally occurring RNA, as long as it encodes the naturally occurring protein.
  • RNA encoding of a naturally occurring antigen may have a degenerate coding sequence, altered ribosome binding site, altered 3' or 5' noncoding sequences, etc.
  • RNA encoding antigens from a cell or virus need not be isolated directly from that cell or virus, but can be made using recombinant techniques.
  • RNA from a cell or virus could be made by isolating RNA from the cell or virus, reverse transcribing the RNA to make a cDNA template, optionally amplifying and/or cloning the cDNA, and transcribing RNA in vitro for RNA loading from the cDNA.
  • the loaded RNA is synthetically manufactured.
  • the loaded RNA is isolated from a cell or virus without a cloning or amplification step.
  • antigens "derived" from a tumor or a pathogen are not identical to the naturally occurring antigen (whether wild type or mutant), and can represent a fusion protein comprising a naturally occurring antigen or fragment thereof, a fragment of a naturally occurring antigen which comprises at least one epitope, a consensus sequence of a family of naturally occurring antigens, or an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, 98% or 99% sequence identity to either a naturally occurring antigen or a fragment of a naturally occurring antigen, wherein the fragment comprises or consists of an epitope of the naturally occurring antigen.
  • the antigen can be a fusion protein comprising a portion of sequence from a first polypeptide (e.g., a first antigen) linked to a portion of sequence from a second polypeptide (e.g., a second antigen, a trafficking sequence, etc.) by means of a peptide bond.
  • a first polypeptide e.g., a first antigen
  • a second polypeptide e.g., a second antigen, a trafficking sequence, etc.
  • Recombinant techniques further allow for the ready modification of the amino acid sequence of polypeptide or protein antigens, by substitution, deletion, addition, or inversion of amino acid sequences.
  • the antigen(s) is(are) from or derived from any tumor.
  • Tumors include, but are not limited to, hematologic malignancies, renal cell cancer, melanoma, breast cancer, prostate cancer, testicular cancer, bladder cancer, ovarian cancer, cervical cancer, stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, colon cancer, neuroblastoma, glioblastoma, retinoblastoma, leukemia (including chronic lymphocytic leukemia), and myeloma (including multiple myeloma), lymphoma, hepatoma, adenoma, sarcoma, carcinoma, blastoma, etc.
  • the antigen is from or derived from a tumor selected from the group consisting of renal cell cancer, melanoma, prostate cancer or chronic lymphocytic leukemia.
  • the RNA loaded into the APC may represent total RNA from the cancer cell, polyA + RNA, fractionated RNA or RNA encoding one or more preselected or defined antigens.
  • defined antigens of interest from tumors include, but are not limited to telomerase, prostate specific antigen (PSA), MART1 , survivin, and MAGE and various angiogenesis factors.
  • pathogen refers to any virus or organism involved in the etiology of a disease and also to attenuated derivatives thereof. Pathogens include, but are not limited to bacterial, protozoan, fungal and viral pathogens such as Helicobacter (such as Helicobacter pylori, Salmonella, Shigella, Enterobacter, Campylobacter), various mycobacteria (such as Mycobacterium leprae, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella species, Leptospira interrogans), Staphylococcus, such as S.
  • Helicobacter such as Helicobacter pylori, Salmonella, Shigella, Enterobacter, Campylobacter
  • mycobacteria such as Mycobacterium leprae, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella species, Leptospira interrogans
  • the pathogen is a retrovirus, and preferably, the retrovirus is HIV.
  • the antigen-encoding RNA may encode one or more open reading frames, or portions thereof, of one or more HIV polypeptides.
  • the RNA encodes antigen representing each or at least most variants of HIV present in the infected individual.
  • Preferred HIV polypeptides are gag, rev, nef, vpr and env and fragments or epitopes thereof.
  • Preferred fragments of vpr are encoded by nucleotides 5105-5320 (encoding vpr amino acid residues 1-72); 5138-5320 (encoding vpr amino acid residues 12-72); 5105-5165 (encoding vpr amino acid residues 1-20) and 5138-5165 (encoding vpr amino acid residues 12-20); and corresponding fragments of other HIV variants. Additional preferred vpr fragments are residues 25-40, 29-37, 30-38, 31-39, 31-50, 34-42, 41-49, 52-62, 53-63, 55-70, 59-67, and 62-70.
  • HIV Molecular Immunology Database available on the internet http site: (//hiv.lanl.gov/content/immunology/), or in the HIV Molecular Immunology Compendium (HIV Molecular Immunology 2006/2007, Editors: Bette T. M. Korber, Christian Brander, Barton F. Haynes, Richard Koup, John P. Moore, Bruce D. Walker, and David I. Watkins. Publisher: Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, New Mexico. LA-UR 07-4752), the contents of which are incorporated by reference.
  • Tumor antigens or pathogen antigens can, but do not need to be specifically or preferentially expressed in the tumor cell or pathogen as compared to expression in other types of cells.
  • Antigens specifically expressed in a tumor or pathogen or pathogen-infected cell are not normally expressed at the same time point in a subject's other cells.
  • Antigens preferentially expressed in a tumor or pathogen or pathogen-infected cell are expressed at a 50% or higher level in the tumor, pathogen or pathogen-infected cell as compared to their expression in a subject's other cells.
  • RNA encoding the antigen or antigens of interest can be from various sources. Methods for isolating and producing RNA are known in the art. For example, the RNA may be isolated or purified directly from a cell or virion, transcribed from a cloned and/or amplified DNA template, or made synthetically. In one embodiment, total RNA isolated from a cell or virus is used for RNA loading. In another embodiment, polyA ⁇ RNA is isolated from a cell or virus and used for RNA loading. In still another embodiment, RNA is isolated from a cell or virion and reverse transcribed to produce a cDNA.
  • the cDNA can be cloned into a vector containing a promoter for transcription, or can be amplified using primers that incorporate a promoter.
  • RNA for loading can then be produced by in vitro transcription of the cloned and/or amplified cDNA.
  • RNA is transcribed from a recombinant DNA template encoding a naturally occurring antigen, a variant having at least 70% sequence identity to a naturally occurring antigen, a consensus antigen, etc.
  • the RNA is produced synthetically instead of by an RNA polymerase.
  • RNA-loaded APCs refers to any method by which APCs uptake exogenous RNA.
  • the antigen presenting cell is a dendritic cell.
  • Transfection methods suitable for introducing RNA into an APC include, but are not limited to electroporation, lipofection, transfection mediated by cationic agents, such as cationic peptides or cationic lipids, gene gun, nanoparticle transfection, etc., and simply adding RNA to a medium containing the APC.
  • the APCs are loaded with RNA by electroporation.
  • DCs may be loaded with RNA when they are immature or mature.
  • monocytes or CD34 + stem cells can be loaded with RNA and then differentiated into RNA loaded APCs. Methods for loading RNA into APCs, dendritic cells, monocytes and CD34 + stem cells are known in the art.
  • the antigen-encoding RNA is cotransfected with a RNA encoding CD40L.
  • immature DCs are matured by culture in presence of GM-CSF, TNF ⁇ , interferon gamma (IFN ⁇ ), PGE 2 and optionally IL-4 for approximately 8-36 hours.
  • the mature DCs are then cotransfected (preferably by electroporation) with antigen- encoding RNA and RNA encoding CD40L, and then cultured in a medium. At approximately 4- 24 hours post transfection, the mature DCs are pulsed with the lysate or extract.
  • RNA-loaded APCs can be pulsed with an extract or lysate of cells or virions of interest which comprise one of more second antigens, wherein the RNA-loaded APC takes up the second antigen(s).
  • the APC will then present the antigens encoded by the loaded RNA in complex with MHC class I molecules, and will present the antigens taken up from the lysate or extract in complex with MHC class Il molecules.
  • Methods for pulsing APCs with an extract or lysate of cells of interest are known in the art.
  • the RNA-loaded APCs are pulsed simply culturing the APCs together with the extract or lysate.
  • immature DCs which are specialized in antigen capture and presentation, are pulsed with extract or lysate prior to or during the maturation process.
  • the lysate or extract may be added to APCs in a cell culture medium, serum, heat inactivated serum, plasma, etc.
  • the APCs are cultured with the lysate for 10 minutes to 48 hours, more preferably for 30 minutes to 30 hours, and most preferably for 2-24 hours.
  • mature DC are cultured with extract or lysate for about 2 hours (mature DC).
  • immature DC are cultured with extract of lysate for about 24 hours.
  • the ratio of the number of APC pulsed with the lysate or extract (Pulsed cells) to the number of cells or virions represented in the amount of lysate or extract used for the pulse (lysate/extract) is not critical. In one embodiment, the ratio of pulsed cells:lysate/extract is between 1 :0.01 to 1 :10,000. Preferably, the ratio is between 1 :0.1 to 1 :10. More preferably, the ratio is about 1 :1.
  • the extract or lysate of cells or virions can be made from any cell or virion that expresses an antigen to which one wishes to elicit an immune response.
  • the lysate or extract may be from a solid tumor, liquid tumor, tumor cells, a pathogen, pathogen infected cells, virions, or cells that have been transfected or transformed with one or more nucleic acids encoding one or more antigens of interest.
  • the APCs and the cells or virion from which a lysate or extract is made are isolated from the same subject, or are prepared or derived therefrom.
  • the APC is preferably isolated from the subject or is differentiated from cells isolated from the subject, and the cell lysate is made from the tumor, tumor cells, or cells of the patient that have been engineered to express the antigen of interest (e.g., APCs from the patient loaded with RNA from or derived from a tumor or pathogen isolated from the same subject).
  • the RNA loaded into the population of APCs encodes one or more antigens present in the lysate or extract from the cells or virion of interest, such that the first and second antigen(s) are the same.
  • the cells from which the lysate or extract is made are APCs loaded with one or more second RNAs encoding the second antigen(s), and the RNA-loaded APC takes up and presents one or more of said second antigens from the extract or lysate of the cells of interest via the MHC class Il pathway.
  • the RNA loaded into the APC population i.e., the first RNAs
  • the RNA loaded into the APC population is the same as the RNA (i.e., the second RNAs) loaded into the APCs from which the lysate or extract is made, such that that the first and second RNAs are the same.
  • the lysate or extract is made from an aliquot of the first population of RNA-loaded APCs provided in the first step of the method.
  • One aliquot of the RNA-loaded APCs can be used to make the cell lysate or extract, while another aliquot of the same RNA-loaded APCs can be pulsed with the lysate or extract.
  • the extract or lysate should be prepared at a time after RNA transfection sufficient for allowing for translation of the transfected antigen-encoding RNA.
  • preparation of the extract or lysate of RNA-loaded APCs is initiated at approximately 30 minutes to 48 hours after RNA loading and most preferably at about 2-24 hours after RNA loading, and most preferably at about 4 hours after RNA loading.
  • the tissue can be treated to produce a single cell suspension or small cell clusters.
  • the tissue can be washed on or more times with dissection medium (HBSS + 30 ⁇ g/ml catalase + 6.6 ⁇ g/ml desferoxamine + 25 ⁇ g/ml N-acetyl cysteine + 94 ⁇ g/ml cystine-2HCL + 1.25 ⁇ g/ml superoxide dismutase + 110 ⁇ g/ml sodium pyruvate + 2.4 ⁇ g/ml HEPES + 0.36% glucose + 800 ⁇ M MgCI 2 + 100 units/ml Fungi-BactTM), minced, passed through meshes of decreasing pore size (e.g., 0.38 mm, 0.14 mm and 0.21 mm).
  • dissection medium HBSS + 30 ⁇ g/ml catalase + 6.6 ⁇ g/ml desferoxamine + 25 ⁇ g/ml N-acetyl cysteine + 94 ⁇
  • Cells can then be lysed by one or more freeze thaw cycles or other methods.
  • the lysate is centrifuged to remove large particles (e.g., 1900 rpm for 10 min.) and the supernatant is filtered using a 0.2 ⁇ m filter.
  • the extract may then be loaded into APCs or stored (preferably frozen, most preferably frozen at -8O 0 C or lower) for later loading.
  • the lysate or extract is made without the use of chemicals or enzymes that are likely to result in a decrease of viability or function of APCs contacted with the lysate or extract.
  • the lysate or extract can be purified to remove such chemicals.
  • the lysate can be prepared by sonicating cells or virions.
  • the lysate is prepared by sonicating heat-treated virions.
  • the lysate can be prepared by freezing and thawing the cells or virions of interest. Any number of freeze/thaw cycles may be used.
  • the lysate is made using 1 , 2, 3 or 4 freeze thaw cycles.
  • an extract of the lysate is made by purifying subcellular compartments where MHC class I and MHC class Il molecules interface with peptides.
  • the subcellular compartments or organelles can be purified by sucrose gradient. Methods for purifying subcellular compartments and organelles are known to those of skill in the art. See, for example, Ramachandra et al., J Immunol. 1999; 162:3263-72; and Voo et al., Cancer Res. 2006; 66:5919-26, the contents of which are incorporated by reference.
  • a lysate or extract can be subjected to proteolytic cleavage of proteins using agents including, but not limited to, pepsin, cyanogen bromide, trypsin, chymotrypsin, etc. Most preferably, the lysate or extract is made from a population of RNA loaded APCs.
  • the invention provides a method for preparing antigen-loaded antigen presenting cell (APCs), comprising: providing a first population of RNA-loaded APCs, wherein the loaded RNA encodes one or more antigens; providing a lysate or extract of a second population of RNA-loaded APCs, wherein the loaded RNA encodes said one or more antigens; and pulsing the first population of RNA-loaded APCs with the extract or lysate, wherein the first population of RNA-loaded APCs take up antigens present in the extract or lysate.
  • the second population of RNA-loaded APCs is an aliquot of the second population of RNA-loaded APCs.
  • the APCs are dendritic cells.
  • Dendritic cells may be loaded with RNA when they are immature or mature. Methods for maturing dendritic cells are known in the art. If DCs are loaded with RNA when immature, they may pulsed with lysate or extract when immature or matured prior to pulsing with extract or lysate. Immature RNA-loaded and lysate/extracted loaded DCs may be formulated for administration to a subject with or without a maturation step. However, mature DCs are currently preferred to immature DCs for immunotherapy.
  • GM-CSF-R GM-CSF Receptor
  • mature DCs have been shown to be superior in inducing T-cell responses in vitro and in vivo.
  • the loaded APCs produced by the methods of the invention, and/or T cells educated from these loaded DCs have many applications, including diagnostic uses, immunotherapy and vaccination.
  • the APCs may be further processed, used to stimulate T cells ex vivo, administered to a subject, or frozen for subsequent use.
  • the RNA-loaded, lysate/extract pulsed APCs can be used to stimulate CD4 + T cells or CD8 + T cells or a combination thereof.
  • the T cells can be naive T cells or antigen- experienced T cells.
  • T cells of interest include effector T cells, memory T cells, or effector/memory T cells. Effector/memory T cells produce I FNy, IL-2, and can kill target cells. Effector T cells produce IFN ⁇ and can kill target cells, but do not produce IL-2. Memory T cells produce IFN ⁇ and IL-2, but do not kill target cells.
  • the RNA-loaded, extract/lysate-pulsed APCs of the invention efficiently present antigen to both CD4 + T cells and CD8 + T cells. Accordingly, the invention provides isolated RNA-loaded, lysate- or extract-pulsed APCs. Medication and vaccines comprising such APCs are also provided, as well as a method of enhancing/inducing an immune response by administering the APCs of the invention.
  • the APCs loaded and pulsed according to the methods described herein are useful for raising an immune response (e.g., a CD4 + and/or CD8 + T cell response) to the antigen(s).
  • an immune response e.g., a CD4 + and/or CD8 + T cell response
  • the invention provides a method of raising an immune response in a subject by administering to the subject an effective amount of the RNA-loaded, lysate/extract-pulsed APCs of the invention.
  • the loaded APCs may be allogeneic or autologous to the subject.
  • the invention further provides a method of stimulating immune effector cells (e.g., CD4 + and/or CD8 + T cells), consisting in culturing said cells in the presence of RNA- loaded lysate/extract-pulsed APCs produced by the methods of invention to produce stimulated immune effector cells.
  • the invention provides a method of enhancing immunity in a subject consisting in administering to the subject an effective amount of such stimulated immune effector cells.
  • the invention provides a method for inducing T cell proliferation, comprising contacting T cells with the RNA-loaded, antigen-pulsed APCs produced by the methods of the invention.
  • the invention provides and RNA-loaded antigen-pulsed APC.
  • the above-mentioned antigen pulsing is performed by contacting said APC with an extract or lysate.
  • compositions described herein are useful to raise an immune response in a subject by administering to the subject an effective amount of the enriched population of APCs, e.g., DCs, B cells, macrophages, artificial antigen presenting cells or educated immune effector cells.
  • the cells can be allogeneic or autologous. They can be administered to a subject to raise or induce an immune response in a subject consisting in administering to the subject an effective amount of the enriched populations as described above.
  • the cells can be allogeneic or autologous to the subject. They can also be used to educate immune effector cells such as T cells by culturing the immune effector cells in the presence and at the expense of the APCs of this invention.
  • the educated effector cells can also be used to enhance immunity in a subject by delivering to the subject an effective amount of these cells.
  • the RNA loaded and antigen-pulsed APCs of the invention can be used to activate CD8 + T cells and/or and CD4 + helper T cells in vitro.
  • the APCs are introduced into a mammal to activate the T cells in vivo.
  • the invention further provides a vaccine comprising the RNA-loaded antigen-pulsed APCs described above.
  • the loaded antigen-presenting cells will be in a buffer/excipient suitable for therapeutic administration to a patient.
  • the vaccine may further comprise an adjuvant or factors for the stimulation of antigen-presenting cells or T cells.
  • CD8 + T cells educated in vitro can be introduced into a mammal where they are cytotoxic against target cells bearing antigenic peptides corresponding to those the T cells are activated to recognize on class I MHC molecules.
  • target cells are typically cancer cells, or pathogen-infected cells which express unique antigenic peptides on their MHC class I surfaces.
  • CD4 + helper T cells which recognize antigenic peptides in the context of MHC class II, can also be stimulated by the APCs of the invention, which comprise antigenic peptides both in the context of class I and class Il MHC. Helper T cells also stimulate an immune response against a target cell. As with cytotoxic T cells, helper T cells are stimulated with the antigen-loaded APCs in vitro or in vivo.
  • the APCs and T cells can be isolated from the mammal into which the APCs and/or activated T cells are to be administered.
  • the cells can be allogeneic provided from a donor or stored in a cell bank (e.g., a blood bank).
  • RNA 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 the manufacturer.
  • a tumor lysate can be produced by sonicating tumor cells in a mammalian cell culture medium such as Opti-MEM or a buffer such as phosphate buffered saline.
  • pathogen-derived RNA can be produced by sonicating pathogens or cells containing a pathogen.
  • RNA can be prepared by employing conventional RNA purification methods such as guanidinium isothiocyanate methods, QiagenTM nucleic acid purification columns, oligo dT chromatography methods for isolating polyA + RNA, etc. Other methods for disrupting cells are also suitable, provided that the method does not completely degrade the RNA. It is not necessary that the RNA be provided to the APC in a purified form.
  • RNA sample i.e., RNA isolated from a cell or virion, or an in vitro transcribed (IVT) RNA sample
  • IVT in vitro transcribed RNA sample
  • Tumor- or pathogen-specific RNA can also be produced by employing conventional techniques for subtractive hybridization. For example, an RNA sample from tumor cells and non-tumor cells can be used in the subtractive hybridization method to obtain tumor- specific RNA.
  • Total RNA or polyA+ RNA can be isolated from lysates of cells or virions that express an antigen of interest and used directly for loading APCs. This RNA can be fractionated, amplified and/or subcloned. For example, tumor specific or pathogen specific RNA could be fractionated from total RNA or polyA ⁇ RNA by subtractive hybridization.
  • IVT in vitro transcribed RNA
  • RNA from cells (including tissues) or viruses can be reverse transcribed into cDNA, which can then be amplified by PCR or any other amplification method to provide an essentially unlimited supply of cDNA corresponding to the tumor or pathogen RNA antigen.
  • Methods for RT-PCR of RNA extracted from any cell (e.g., a tumor cell or pathogen cell) or virus, and in vitro transcription are disclosed in International patent publications WO 2005/052128, WO 2006/031870 and WO 2006/042177, the contents of which are incorporated by reference.
  • the cDNA can optionally be cloned into a replicable vector, either with or without prior amplification.
  • vectors are viruses, such as baculovirus and retrovirus, bacteriophage, adenovirus, adeno-associated virus, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art.
  • the APC is transfected with an mRNA.
  • the vector may contain a promoter for in vitro transcription, or the promoter could be included in the amplification primer.
  • the amplification primer contains a promoter, and the amplified cDNA is not cloned into a vector, but used directly as a template for in vitro transcription.
  • a DNA encoding an antigen of interest could be cloned without first isolating RNA, and that DNA could be used as a template for in vitro transcription from its wild type promoter or a heterologous promoter.
  • the IVT RNA can be synthesized from a cloned DNA sequence encoding a tumor or pathogen polypeptide antigen. Methods for identifying such antigens are known in the art; for example, several melanoma peptide antigens have been identified. RNA transcribed in vitro from cDNA encoding identified peptide antigens can serve as tumor- or pathogen-specific RNA in the invention.
  • the template In order to transcribe a template DNA sequence (e.g., a PCR product), the template should contain a RNA polymerase binding site or promoter. Furthermore, for efficient translation in eukaryotic cells, the transcribed RNA preferably contains a 5' Cap, a Kozak sequence including an ATG translational initiation codon, and a polyadenylated sequence at it 3' end. Preferably, the transcribed RNA will also contain a translational stop codon (UAA, UAG or UGA).
  • UAA translational stop codon
  • transcriptional and translational signals such as the promoter, Kozak sequence, start and stop codons may be present in the template of the of the target pathogen nucleic acid, and could be amplified during PCR or other amplification reaction. Alternatively, these sequences can be included in forward and reverse primers used at any stage of amplification. In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression. Preferably, the RNA is capped and polyadenylated during or after in vitro transcription.
  • Promoters suitable for in vitro transcription and methods of in vitro transcription are known to those skilled in the art.
  • preferred promoters are those corresponding to commercially available RNA polymerases, such as the T7 promoter and the SP6 promoter.
  • Other useful promoters are known to those skilled in the art.
  • the promoter allows for efficient transcription using commercially available in vitro transcription kits. Methods of in vitro transcription and translation are known to those skilled in the art (see, for example, U.S. 2003/0194759).
  • a DNA template is transcribed using a bacteriophage RNA polymerase in the presence of all four ribonucleoside triphosphates and a cap dinucleotide such as m 7 G(5')ppp(5')G or a cap analog, such as ARCA.
  • the promoter and translational initiation signals can be included in a primer used during primary amplification or in a nested primer used in a subsequent round.
  • the annealing target for the primers used in an optional secondary round of amplification can be the same as that used in the primary amplification reaction, or it can be a site internal ("nested") to the primary amplicon.
  • the forward primer used in the primary or secondary (preferably secondary) round of amplification will comprise a 5' nonhybridizing region (an "overhang") encoding a promoter (e.g., T7 promoter), and a 3' region complimentary to the antisense strand of the primary amplicon.
  • the forward primer will also contain a Kozak sequence, preferably an optimized Kozak sequence, e.g., bases 5' CCACCATGG, wherein the underlined ATG is the translational initiation codon.
  • the Kozak sequence including the ATG start codon may either be in the 5' overhang region of the forward primer, or in the 3' annealing region of the primer, depending upon whether such sequences are present in the primary amplicon.
  • the primary amplicon contains a less than optimal Kozak sequence, it can be optimized by using a forward primer with an optimal, although not completely complementary, Kozak sequence.
  • the 3' portion of forward primer will be essentially complimentary to the sequence immediately downstream of initiator ATG codon or the most 5' coding sequence amplified during primary amplification.
  • the reverse primer preferably contains a 5' polyT overhang at its 5' end which will serve as a template for polyadenylation during in vitro transcription.
  • the 3' half of the reverse primer will be complimentary to the sense sequence isolated in the primary amplification reaction. If an appropriate translational stop codon is not present in the primary amplicon, it can be included in the 5' overhang portion of the reverse primer.
  • the secondary amplicon obtained by amplification using primers such as those described above can then serve as a template in an in vitro transcription reaction in the presence of m 7 G cap or an analogue thereof, such as ARCA (see U.S. patent publication 2003/0194759).
  • APCs are transfected with RNA encoding HIV antigens.
  • HIV nucleic acids are be isolated or derived from a biological sample from an individual infected with HIV who will be treated with a vaccine of the invention.
  • Biological samples useful for the isolation of HIV nucleic acids, or nucleic acids from other pathogens include, but are not limited to blood, plasma, serum, peripheral blood mononuclear cells (PBMC), seminal fluid, vaginal secretions, ocular lens fluid, cerebral spinal fluid, saliva, milk, ascites fluid, synovial fluid, peritoneal fluid, amniotic fluid, tissue, tissue culture and the like.
  • PBMC peripheral blood mononuclear cells
  • virions containing HIV genomic RNA are isolated from blood or serum.
  • the genomic RNA obtained from the virions can be subjected to reverse transcription to produce an HIV cDNA.
  • Methods of isolating HIV virions from infected patients, as well as methods of making cDNA are known to those skilled in the art.
  • Reverse transcription of HIV RNA can be primed using random hexamers, polyT primers or specific oligonucleotide primers.
  • the HIV cDNA can then be used as a template in the above method of strain-independent amplification of HIV sequences. These same methods can be used to prepare a cDNA from the mRNA or genomic RNA of other pathogens.
  • the primary amplicon is subjected to a second round of amplification using nested primers containing transcription and translation signals suitable for either expression in vitro and/or expression in vitro.
  • the DNA is subjected to a second round of amplification using a forward nested primer containing: 1) a promoter suitable for in vitro transcription, such as the T7 or SP6 promoter; and 2) an optimized Kozak sequence including an ATG codon, in combination with a reverse nested primer containing a the complement of a translational stop codon and a polyT tail.
  • the secondary amplicon resulting from the nested round of amplification can be used as a template for in vitro transcription and 5' capping.
  • the resulting mRNA can then be used in a nucleic acid vaccine, or to load antigen presenting cells, which can then be used as a vaccine (or immunogenic composition).
  • pathogen RNA is reverse transcribed into single- stranded cDNA using a reverse transcriptase, appropriate reaction buffers, and random hexamers.
  • the single-stranded cDNA is then amplified by PCR into double-stranded DNA in a primary PCR reaction using multiplex primers.
  • Product from a primary PCR reaction is taken into a second round or nested PCR amplification.
  • 5' primer(s) contains overhang with T7 RNA polymerase binding sequences and 3' primer contains overhang with poly T stretches.
  • the modifications introduced by overhanging regions in a nested round of PCR enable transcription of the PCR product in vitro and successful translation upon delivery into the DCs.
  • the process can be interrupted after either step of PCR amplification, and PCR products can be stored frozen for further processing.
  • Purification of cDNA material is performed using the QIAquick ® PCR Purification Kit components (QIAquick ® Columns, PB buffer, PE buffer, and EB buffer). The double-stranded DNA is used as a template in a standard transcription reaction. Clean-up of the in wfro-transcribed RNA is performed using components in the RNeasy ® Kit (RNeasy ® Column, RLT buffer, and RPE buffer). The RNA is eluted in nuclease-free water. If necessary, ethanol precipitation is performed to concentrate the RNA.
  • RNA is re-suspended in nuclease-free water and passed through a 0.8/0.2 ⁇ m polyethersulfone (PES) filter, then dispensed into 0.5 ml_ safe-lock polypropylene tubes, and cryopreserved at ⁇ -150 0 C for long-term storage.
  • PES polyethersulfone
  • the HIV DNA is a cDNA prepared by reverse transcription of HIV genomic RNA or HIV mRNA.
  • the forward promoter primer comprises a T7 or SP6 promoter.
  • the forward primer further comprises an optimized Kozak sequence 3' to the promoter.
  • dendritic cells are cotransfected with antigen encoding RNA and an RNA encoding CD40L, or a variant or derivative thereof.
  • the CD40L can be any mammalian CD40L, and is preferably human or murine.
  • U.S. Patent No. 5,981 ,724 discloses DNA sequences encoding human CD40 Ligand (CD40L) as well as vectors, and transformed host cells for the purpose of producing CD40L polypeptides.
  • U.S. Patent No. 5,962,406 discloses DNA sequences encoding soluble forms of human CD40L.
  • Exemplary sequences of mammalian homologs to CD40L have the following Genbank accession numbers: NM_204733 (Gallus gallus (chicken)); DQ054533 (Ovis aries (sheep)); Z48469 (Bos taurus (cow)); AY333790 (Canis familiaris (dog)); Macaca nemestrina (pig-tailed macaque)); AF344844 (Callithrix jacchus (white-tufted-ear marmoset)); AF34481 (Cercicebus torquatus atys (sooty mangabey)); AF344860 (Aotus trivirgatus (douroucouli)); AF344859 Macaca mulatta (rhesus monkey)); AF116582 (Rattus nevegicus (Norway rat)); and AF079105 (Felus catus (cat)).
  • Genbank accession numbers for the nucleotide and amino acid sequences of human CD40L are NM_000074 and NP_000065, respectively.
  • Genbank accession numbers for the nucleotide and amino acid sequences of mouse CD40L are NM_011616 and NPJ335746, respectively.
  • the open reading frame for human CD40L is represented by nucleotides 73 to 855 of the nucleotide sequence disclosed in NM_000074, while the TGA stop codon at position 856 to 858.
  • any of the CD40L polynucleotide sequences of the invention a silent mutation (a variant due to codon degeneracy) of the 102 nd codon in the CD40L sequence (nucleotides 376 to 378 of NMJD00074), changing the "AAA" codon to an "AAG” codon, both of which code for Lys may be used.
  • mRNA encoding N-terminal truncated CD40L is useful in the methods of the invention.
  • a truncated CD40L fragment corresponding to or consisting essentially of amino acid residues 21 to 261 of the amino acid sequence disclosed in NP_000065 is preferred, and results in higher levels of IL-12 secretion of transfected DCs in comparison to transfection with mRNA encoding the wild type CD40L protein.
  • the truncated CD40L polypeptide is encoded by an RNA corresponding to residues 133-855 of the nucleotide sequence disclosed in NM_000074, or to variants which differ do to codon degeneracy.
  • RNA corresponding to a cDNA sequence refers to a RNA sequence having the same sequence as the cDNA sequence, except that the nucleotides are ribonucleotides instead of deoxyribonucletides, as thymine (T) base in DNA is replaced by uracil (U) base in RNA.
  • T thymine
  • U uracil
  • the RNAs are capped and polyadenylated.
  • CD40L mRNA can be made by in vitro transcription of a CD40L expression cassette.
  • the CD40L expression cassette contains a promoter suitable for in vitro transcription, such as the T7 promoter or SP6 promoter.
  • CD40L RNA can be transcribed from plasmid template pCR2.1 CD40L WT, pCR2.1 or pCR2.1 CD40L ⁇ XE-MET#1 (CD40L ⁇ XE minus MET#1), as disclosed in International Application WO 2006/042177 and U.S. patent application 11/400,774, the contents of which are incorporated by reference.
  • the cDNA corresponding to the mRNA transcribed from pCR2.1 CD40L ⁇ XE-MET#1 is represented by residues 133-855 of the nucleotide sequence disclosed in NM_000074, which encodes the truncated polypeptide containing amino acid residues 21-261 of NP_000065.
  • Transfection of DCs with an RNA encoding the above- mentioned truncated CD40L polypeptide results in high levels of IL-12 expression.
  • the preferred CD40L RNA contains an ARCA cap analog and polyA tail. mRNA stability and/or translational efficiency can also be increased by including 3'UTRs and or 5'UTRs in the mRNA.
  • 3'UTRs include those from human CD40, ⁇ -actin and rotavirus gene 6.
  • Preferred examples of 5'UTRs include CD40L, and the translational enhancers in the 5'UTRs of Hsp70, VEGF, spleen necrosis virus RU5, and tobacco etch virus.
  • Antigen-presenting cells such as dendritic cells, B cells and macrophages
  • the APC or its precursor is autologous to a subject to be treated with the loaded APCs of the invention or effectors cells stimulated by such loaded APCs.
  • the APCs are obtained from a donor that is HLA matched to the subject to be treated.
  • tissue sources for such cells include bone marrow cells, peripheral blood progenitor cells (PBPCs), peripheral blood stem cells (PBSCs), and cord blood cells.
  • PBPCs peripheral blood progenitor cells
  • PBSCs peripheral blood stem cells
  • cord blood cells Preferably, the tissue source is a peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the tissue source can be fresh or frozen.
  • PBMCs can be isolated from a subject by obtaining peripheral blood, or by leukapheresis to obtain lymphocytes present in the peripheral blood (the leukapheresis product).
  • PBMCs can be purified from dilute anticoagulated blood or the leukapheresis product by Ficoll-Hypaque centrifugation and/or elutriation.
  • B cells typically represent about 15-20% of PBMCs and can be further purified by panning, affinity columns, FACS, etc., using agents that bind to B cell markers such as surface immunoglobulin molecules.
  • U.S. Patent Publication No. 2004/0038398 discloses methods for the preparation of substantially purified populations of DCs and monocytes from the peripheral blood of mammals. Myeloid cells are isolated from the mammal and DCs are separated from this population to yield an isolated subpopulation of monocytes. DCs are then enriched by negative selection with anti-CD2 antibodies to remove T cells.
  • Monocytes represent about 5-10% of PBMCs, and can be further purified by binding to the CD14 surface marker, adherence to plastic, or elutriation. Because macrophages and dendritic cells are present as a lower percentage of PBMCs in comparison to the percentage of monocytes, dendritic cells and macrophages are often differentiated from the more abundant monocytes or from CD34 + stem cells.
  • hematopoietic factor to be administered may be determined by one skilled in the art by monitoring the cell differential of individuals to whom the factor is being administered. Typically, dosages of factors such as G-CSF and GM-CSF will be similar to the dosage used to treat individuals recovering from treatment with cytotoxic agents.
  • GM-CSF or G- CSF can be administered for 4 to 7 days at standard doses prior to removal of source tissue to increase the proportion of dendritic cell precursors.
  • U.S. Patent No. 6,475,483 teaches that dosages of G-CSF of 300 micrograms daily for 5 to 13 days and dosages of GM-CSF of 400 micrograms daily for 4 to 19 days result in significant yields of dendritic cells.
  • CD34 + cells can be obtained from a variety of sources, including cord blood, bone marrow explants, and G- CSF mobilized peripheral blood. Purification of CD34 + cells can be accomplished by antibody affinity procedures. For example, CD34 + stem cells can be further purified by panning the bone marrow cells or other sources with antibodies which bind unwanted cells, such as CD4 + and CD8 + (T cells), CD45 + (panB cells) and GR-1. See Inaba, et al. (1992) J. Exp. Med. 176:1693- 1702; Paczesny et al. (2004) J Exp Med. 199: 1503-11 ; Ho, et al. (1995) Stem Cells 13(suppl. 3):IOO-1O5; Brenner (1993) Journal of Hematotherapy 2:7-17; and Yu, et al. (1995) PNAS 92:699-703.
  • CD34 + stem cells can be differentiated into dendritic cells by incubating the cells with the appropriate cytokines.
  • Inaba et al. (1994) supra described the in vitro differentiation of murine stem cells into dendritic cells by incubating the stem cells with between 1 and 200 ng/ml murine GM-CSF in standard RPMI growth medium for approximately 5-7 days.
  • IL-4 can be added in similar ranges.
  • Dendritic cells can then be isolated by florescence-activated cell sorting (FACS) or by other standard methods.
  • FACS florescence-activated cell sorting
  • Human CD34 + hematopoietic stem cells are preferably differentiated in vitro by culturing the cells with human GM-CSF and TNF- ⁇ . See for example, Szabolcs, et al. (1995) 154:5851-5861.
  • SCF or another proliferation ligand e.g., F1t3 is added to facilitate differentiation of human DCs.
  • WO 95/28479 discloses a process for preparing dendritic cells by isolating peripheral blood cells and enriching for CD34 + blood precursor cells, followed by expansion with a combination of hematopoietic growth factors and cytokines.
  • European Patent Publication EP-B-O 633930 teaches the production of human dendritic cells by culturing human CD34 + hematopoietic cells (i) with GM-CSF, (ii) with TNF- ⁇ and IL-3, or (iii) with GM-CSF and TNF- ⁇ to induce the formation of CDIa + dendritic cells.
  • U.S. Patent Publication No. 2004/0146492 teaches a process for producing dendritic cells by transforming CD34 + hematopoietic stem cells with a nucleic acid encoding an antigen of interest followed by differentiation of the stem cells into dendritic cells by culture in medium containing GM-CSF and optionally with TNF- ⁇ , SCF, Flt3L and/or IL-4.
  • PBMCs peripheral blood mononuclear cells
  • Monocytes can be purified from PBMCs by methods known to those of skill in the art, including, but not limited to, Ficoll-HypaqueTM gradient, elutriation, panning, antibody coated magnetic beads, FACs, and adherence to plastic.
  • Methods for differentiating monocytes into immature dendritic cells include culture in medium containing GM-CSF and IL-4 or GM-CSF and IL-13. Monocytes cultured in GM-CSF in the absence of effective amounts of IL-4 or IL-13 will differentiate into macrophages. See, for example, WO 97/29182 and International applications WO 2006/031870 and WO 2006/042177, the contents of which are incorporated by reference.
  • immature dendritic cells are prepared from CD14+ peripheral blood monocytic cells (PBMCs) by culture in the presence of GM-CSF and IL-4 for about 4-7 days, preferably about 5-6 days, to produce immature DCs.
  • PBMCs peripheral blood monocytic cells
  • Jonuleit et al. disclose maturation of immature DCs by overnight culture in medium containing GM-CSF and IL-4, plus a "cytokine cocktail" comprising TNF- ⁇ , IL-1 ⁇ , IL-6 and PGE 2 .
  • Preferred concentrations of cytokines in the cocktail are 10 ng/ml TNF- ⁇ , 10 ng/ml IL-1 ⁇ , 100 ng/ml IL-6 and 1 ⁇ g/ml PGE 2 , however these levels may be increased or decreased.
  • EP-A-O 922 758 discloses the production of mature dendritic cells from immature dendritic cells derived from monocytes by culture in medium containing IFN- ⁇ .
  • U.S. Patent Publication No. 2004/0152191 discloses the maturation of dendritic cells by culture with RU 41740.
  • CD40L base process and the "Post-Maturation Electroporation (PME) CD40L process” for DC maturation are described in International Application WO 2006/042177, the contents of which are incorporated by reference.
  • immature DC are transfected with CD40L mRNA and antigen-encoding mRNA, and then treated with IFN- ⁇ (1000 U/ml) or TNF- ⁇ (10 ng/ml) or a combination of IFN- ⁇ and PGE 2 (1 ⁇ g/ml).
  • IFN- ⁇ 1000 U/ml
  • TNF- ⁇ 10 ng/ml
  • PGE 2 1 ⁇ g/ml
  • monocyte derived immature DCs are matured (typically after 4-7 days after beginning culture of monocytes with GM-CSF and IL-4) by overnight culture (12-30 hours, preferably about 18 hrs) with TNF- ⁇ (10 ng/ml), IFN- ⁇ (1000 U/ml) and PGE 2 (1 ⁇ g/ml).
  • DCs are harvested and electroporated with antigen-encoding RNA and CD40L mRNA and cultured in X-VIVO 15 media containing 800 U/ml GM-CSF and 500 U/ml IL-4 for 4 hrs or more hours prior to removing and aliquot for pulsing with a lysate or extract of cells or virions.
  • cytokine concentrations for culturing and differentiating stem cells, monocytes, macrophages and dendritic cells are approximate and can be adjusted.
  • Different suppliers and different lots of cytokine from the same supplier vary in the activity of the cytokine. Somone skilled in the art can easily titrate each cytokine, which is used to determine the optimal dose for any particular cytokine.
  • Methods for loading antigen presenting cells with RNA are known to those of skill in the art, and include, but are not limited to electroporation, passive uptake, lipofection, microinjection, cationic reagents, viral transduction, CaPO 4 and the like. See, for example, PCT/US05/22705 and U.S. Ser. No. 60/583,579; U.S. Pub. No. 2003/0143743; U.S. Pub. No. 20050008622; U.S. Pub. No. 20040235175; and U.S. Pub. No. 20040214333, the contents of which are incorporated by reference.
  • the antigen presenting cells are loaded with both CD40L mRNA and RNA encoding one or more antigens.
  • the mRNA is capped and polyadenylated.
  • the cap is ARCA.
  • Preferred polyA tail lengths are in the range of 50-1000 nucleotides, more preferably 64-900 nucleotides, and most preferably 101-600 nucleotides.
  • Dendritic cells can be loaded with RNA in vitro when mature or immature. Loaded immature dendritic cells can be matured in vitro prior to vaccination or in vivo (with or without an exogenous maturation stimulus) following vaccination.
  • RNA in 500 ⁇ l of Opti-MEM can be mixed with a cationic lipid at a concentration of 10 to 100 ⁇ g/ml, and incubated at room temperature for 20 to 30 minutes.
  • Suitable lipids for transfection include LIPOFECTIN.TM. (1 :1 (w/w) DOTMA:DOPE), LIPOFECTAMINE.TM. (3:1 (w/w) DOSPA:DOPE), DODAC:DOPE (1:1), CHOLDOPE (1:1), DMEDA, CHOL, DDAB, DMEDA, DODAC, DOPE, DORI, DORIE, DOSPA, DOTAP, and DOTMA.
  • RNA-lipid complex is then added to 1-3x10 6 cells, preferably 2x10 6 , antigen-presenting cells in a total volume of approximately 2 ml (e.g., in Opti-MEM), and incubated at 37 0 C. for 2 to 4 hours.
  • the RNA can be introduced into the antigen presenting cells by employing conventional techniques, such as electroporation or calcium phosphate transfection with 1-5x10 6 cells and 5 to 50 ⁇ g of RNA. Typically, 5-20 ⁇ g of polyA + RNA or 25-50 ⁇ g of total RNA is used.
  • APC are washed twice in Opti-MEM medium (GIBCO, Grand Island, N.Y.). Cells were resuspended in Opti-MEM medium at 2-5 x 10 6 cells/ml, and added to 15 ml polypropylene tubes (Falcon).
  • the cationic lipid DOTAP (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) is used to deliver RNA into cells (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89: 7915-7918).
  • RNA in 250-500 ⁇ l Opti-MEM medium
  • DOTAP in 250-500 ⁇ l Opti-MEM medium
  • the RNA to DOTAP ratio can be varied between 2:1 to 1 :2.
  • the complex is added to the APC (2-5 x 10 6 cells) in a total volume of 2 ml and incubated at 37 0 C in a water-bath with occasional agitation for 2 or more hours, and then washed and either cultured, formulated for administration or cyropreservation, or used to stimulate T cells.
  • the APC is transfected by electroporation.
  • Dendritic cells can be transfected when immature or mature.
  • mature dendritic cells are transfected.
  • monocytes are cultured at 1 x 10 6 cells/ml for six days in AIM V medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4 to generate immature DCs.
  • AIM V medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4 to generate immature DCs.
  • a maturation formulation in AIM V medium is added directly to the immature DC to give a final concentration of 10 ng/ml TNF- ⁇ , 1000 U/ml IFN- ⁇ , and 1 ⁇ g/ml PGE 2 .
  • the cells are cultured overnight and mature DC are harvested and co-electroporated with 1 ⁇ g of antigen encoding RNA and 4 ⁇ g of CD40L RNA per 10 6 cells.
  • Post-electroporation the cells are cultured at 1 x 10 6 cells/ml in AIM V medium supplemented with 800U/ml GM-CSF and 500 U/ml IL-4.
  • These cells can be cultured, preferably for at least 30 minutes, and, optionally, an aliquot may be removed for the preparation of a cell lysate or extract.
  • the remaining cells can then be pulsed with the lysate or extract made from the aliquot, or can be pulsed with a different lysate or extract of cells or virions.
  • APCs Methods of loading APCs with antigens are known to those with skill in the art.
  • the APC is simply cultured in the presence of antigen.
  • the APCs can then take up and process the antigen on the cell surface in association with MHC class Il molecules.
  • an APC culture containing between 10 6 to 10 7 cells is washed with culture medium.
  • Culture medium containing about 50 ⁇ g/ml of lysate from cells or virions is added, and the cells are cultured, preferably overnight and preferably on a rotator.
  • T cells are isolated from mammals so that they can be educated (or activated) by the mature, modified DC in vitro.
  • Ficoll- Hypaque density gradient centrifugation is used to separate PBMC from red blood cells and neutrophils according to established procedures.
  • PBMCs can be washed with modified AIM-V (which consists of AIM-V (GIBCO) with 2 mM glutamine, 10 ⁇ g/ml gentamicin sulfate, 50 ⁇ g/ml streptomycin) supplemented with 1% fetal bovine serum (FBS).
  • modified AIM-V which consists of AIM-V (GIBCO) with 2 mM glutamine, 10 ⁇ g/ml gentamicin sulfate, 50 ⁇ g/ml streptomycin
  • T cells are enriched from PBMCs by negative or positive selection with appropriate monoclonal antibodies coupled to columns or magnetic beads according to standard techniques. An aliquot of cells can be analyzed for cell surface phenotype including, but not limited to such cell surface markers as CD4, CD8, CD3 and CD14. T cells can be washed and resuspended at a concentration of about 5 X 10 5 cells per ml of AIM-V modified as above and containing 5% FBS and 100 U/ml recombinant IL-2 (rlL-2) (supplemented AIM-V).
  • CD4-PE40 a recombinant protein consisting of the HIV-l-binding CD4 domain linked to the translocation and ADP-ribosylation domains of Pseudomonas aeruginosa exotoxin A
  • CD4-PE40 has been shown to inhibit p24 production in HIV- infected cell cultures and to selectively kill H I V- 1 -infected cells.
  • an antibody directed againt the CD3 molecule such as the OKT3 monoclonal antibody (Ortho Diagnostics) can be added to a concentration of 10 ng/ml and the cells are plated in 24 well plates with 0.5 ml per well.
  • the cells can be cultured at a temperature of about 37°C in a humidified incubator with 5% CO 2 .
  • Cell surface markers can be used to isolate the cells necessary to practice the methods of this invention.
  • human stem cells typically express CD34 antigen while DCs express MHC molecules and costimulatory molecules (e.g., B7-1 and B7-2), a lack of markers specific for granulocytes, NK cells, B cells, and T cells. The expression of surface markers facilitates identification and purification of these cells.
  • identification and isolation include FACS, column chromatography, panning with magnetic beads, western blots, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
  • Cell isolation or immunoassays for detection of cells during cell purification can be performed in any of several configurations, e.g., those reviewed in Maggio (ed.) (1980) Enzyme Immunoassay, CRC Press, Boca Raton, FIa.; Tijan (1985) "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press, Orlando, FIa.; Price and Newman (eds.) (1991 ) Principles and Practice of Immunoassays, Stockton Press, NY; and Ngo (ed.) (1988) Non-isotopic Immunoassays, Plenum Press, NY.
  • Cells can be isolated and characterized by flow cytometry methods and FACS analysis.
  • flow-cytometry methods A wide variety of flow-cytometry methods are known.
  • fluorescence activated flow cytometry see, for example, Abbas et al. (1991 ) Cellular and Molecular immunology W.B. Saunders Company, particularly chapter 3, and Kuby (1992) Immunology W. H. Freeman and Company, particularly chapter 6.
  • Labeling agents which can be used to label cell antigen include, but are not limited to monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any known method, such as immunoblotting, western blot analysis, tracking of radioactive or bioluminescent markers, capillary electrophoresis, or other methods which track a molecule based upon size, charge or affinity.
  • the immunogenicity of the antigen-presenting cells or educated T cells produced by the methods of the invention can be determined by well known methodologies including, but not limited to the following:
  • Cytotoxic T cells can kill cells that present the particular peptide:MHC class I complex that they specifically recognize.
  • CTL function is typically determined by measuring the release of radioactive isotope by a target cell (e.g., an antigen loaded APC, tumor cell, pathogen cell, etc.).
  • target cells e.g., an antigen loaded APC, tumor cell, pathogen cell, etc.
  • target cells are incubated with 100 ⁇ Ci of Na 2 51 CrO 4 for approximately 90 minutes at 37°C. Excess 51 Cr is washed away and 5000 labeled targets are incubated with various ratios of CD8 + cells for one or more specific time points (e.g., 4 hours).
  • Non-specific lysis can be reduced by the addition of unpulsed T2 cells at 25,000 cells per well. 51 Cr released by lysed target cells is measured in the supernatant by scintillation counting. Total release is calculated by addition of 1% Triton X-100 to the targets while spontaneous release is calculated by addition of media alone. Percent lysis is calculated using the formula (sample cpm released- spontaneous cpm)/(total cpm released- spontaneous cpm released) (Ware et al. (1983) J. Immunol. 131 :1312).
  • Cytokine-release assay Analysis of the types and quantities of cytokines secreted by T cells upon contacting modified APCs can be a measure of functional activity. Cytokines can be measured by ELISA or ELISpot assays to determine the rate and total amount of cytokine production (Fujihashi et al. (1993) J. Immunol. Meth. 160:181 ; Tanquay and Killion (1994) Lymphokine Cytokine Res. 13:259).
  • PVDF membrane ELISpot plates (Millipore, Ballerica, MA) are coated with 5 ⁇ g/mL monoclonal anti-IFN- ⁇ or anti-IL-2 capture antibody (BD Pharmingen, San Diego, CA) and incubated at 4°C for 24 hours. After incubation, plates are washed with PBS/0.05% Tween 20, and blocked with 5% human AB serum/RPMI 1640 medium for 1 hour.
  • PBMCs, T-cells, or CD8 enriched T cells are plated at 1xl ⁇ 5 cells/well and mRNA transfected, antigen-loaded DC targets at 1x10 4 cells/well for a 10:1 effectortarget ratio, and incubated at 37 0 C, 5% CO 2 for a minimum of 16 hours. Following incubation, plates are washed 6 times, and anti-IFN- ⁇ detection antibody (BD Pharmingen) or anti-IL-2 detection antibody (BD Pharmingen) is added to the appropriate plates at 1 ⁇ g/ml for 2 hours. After six more washes, Streptavidin-HRP (BD Pharmingen) is added to each well for 1 hour.
  • BD Pharmingen anti-IFN- ⁇ detection antibody
  • BD Pharmingen anti-IL-2 detection antibody
  • ELISA ELISA plates
  • ELISA capture antibody specific for a marker or other antigen or epitope of interest
  • coating buffer for 24 hours at 4 0 C.
  • Plates can be blocked with 200 ⁇ i per well 10% FCS/PBS for one hour prior to the addition of standards (BD Pharmingen) and supernatant samples, in duplicate, at 100 ⁇ l per well and incubated at room temperature for 2 hours. Plates are washed and anti-capture antibody detection antibody added, incubated for one hour, the plates washed and solutions replaced with IOO ⁇ l of streptavidin-HRP and further incubated for one hour at room temperature. Plates are washed again and color development substrates applied for 10- 20 minutes, followed by cessation of color development with stop solution. Plate analysis can be undertaken using Bio-Tek instruments ELx ⁇ OO plate reader with KC junior software (Winooski, VT).
  • compositions of the invention can be assayed for the ability to elicit reactive T-cell populations from normal donor or patient-derived PBMC.
  • elicited T cells can be tested for lytic activity, cytokine-release, polyclonality, and cross- reactivity to the antigenic epitope. Parkhurst et al. (1996) Immunol. 157:2539.
  • CD8 + T cells can be activated to become CTL by coculture with antigen-loaded dendritic cells.
  • CD8 + cells can be purified using the CD8 + T Cell Isolation kit Il (Miltenyi Biotec, Auburn, CA) from non-adherent cells harvested from the monocyte adherence step.
  • Mature dendritic cells loaded with antigen e.g., by transfection with antigen encoding mRNA and/or pulsing with peptides, proteins, lysates, etc.
  • CD8+ purified T cells at 10:1 CD8 + :DC.
  • Co-cultures can be performed in R-10 media (10% FBS, RPMI-1640 supplemented with 10 mM HEPES pH 7.4, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM sodium glutamate, 55 ⁇ M ⁇ -mercaptoethanol) or other media.
  • R-10 media 10% FBS, RPMI-1640 supplemented with 10 mM HEPES pH 7.4, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM sodium glutamate, 55 ⁇ M ⁇ -mercaptoethanol
  • the cells can be cultured in media supplemented with 0.2 U/ml IL-2 (R&D Systems, Minneapolis, MN) and then aliquoted into 24-well tissue culture dishes at 1 ml (1x10 6 CD8 + cells / well).
  • the CD8 + T cells can be harvested, counted and re-cultured with fresh DC stimulators at 10:1 in media supplemented with 5 U/ml IL-2. The cells are then cultured for about one week and then restimulated with fresh DC and 20 U/ml IL-2. CTL assays can be performed 3 or 7 days following the third stimulation.
  • Transgenic animal models can be assessed in vivo by vaccinating HLA transgenic mice with the compositions of the invention and determining the nature and magnitude of the induced immune response.
  • the hu-PBL-SCID mouse model allows reconstitution of a human immune system in a mouse by adoptive transfer of human PBL. These animals may be vaccinated with the compositions and analyzed for immune response as previously mentioned in Shirai et al. (1995) J. Immunol. 154:2733; Mosier et al. (1993) Proc. Natl. Acad. Sci. USA 90:2443.
  • T cells will proliferate in response to reactive compositions. Proliferation can be monitored quantitatively by measuring, for example, 3 H-thymidine uptake. Caruso et al. (1997) Cytometry 27:71. In a preferred method, T-cell proliferation is measured using carboxy-flouroscein diacetate succinimidyl ester (CFSE).
  • CFSE carboxy-flouroscein diacetate succinimidyl ester consists of a fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties.
  • Staining with fluorescence labeled antibodies for cell surface molecules, such as CD4 and CD8, and intracellular markers allows the examination of the proliferation of specific lymphocyte subsets as well as the characterization of the phenotypic and functional properties of proliferating cells using flow cytometry.
  • the concomitant use of propidium iodide (Pl) facilitates the assessment of cell viability.
  • CFSE flow kits are available through Renovar, Inc. (Madison, Wl) and other sources.
  • a non-human primate (chimpanzee) model system can be used to monitor in vivo immunogenicities of HLA-restricted ligands. It has been demonstrated that chimpanzees share overlapping MHC-ligand specificities with human MHC molecules, thus allowing one to test HLA-restricted ligands for relative in vivo immunogenicity (Bertoni et al. (1998) Immunol. 161 :4447).
  • TCR Signal Transduction Events Several intracellular signal- transduction events (e.g., phosphorylation) are associated with successful TCR engagement by MHC-ligand complexes. The qualitative and quantitative analyses of these events have been correlated with the relative abilities of compositions to activate effector cells through TCR engagement (Salazar et al. (2000) Tnt. J. Cancer 85:829; Isakov et al. (1995) J. Exp. Med. 181 :375).
  • Dendritic cells and/or T cells produced by the methods of this invention can be administered directly to the subject to produce an immune response. Administration can be carried out by methods known in the art to successfully deliver a cell into ultimate contact with a subject's blood or tissue cells.
  • the cells are administered in any suitable manner, often with pharmaceutically acceptable carriers. Suitable methods of administering cells in the context of the present invention to a subject are available, and although more than one route can be used to administer a particular cell composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Routes of APC administration employed clinically include, but are not limited to intravenous (IV), subcutaneous (SC), intradermal (ID), and intralymphatic.
  • Preferred administration routes include, but are not limited to intradermal, subcutaneous, intratumoral and intravenous administration.
  • compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention. Most typically, quality controls (microbiology, clonogenic assays, viability tests) are performed and the cells are reinfused back to the subject, preceded by the administration of diphenhydramine and hydrocortisone. See, for example, Korbling et al. (1986) Blood 67:529-532 and Haas et al. (1990) Exp. Hematol. 18:94-98.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranodal and subcutaneous routes, and carriers can include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Methods of formulating and administering antigen presenting cells to patients are known in the art. See, for example: Fay et al.
  • the cell dose (e.g., APCs or immune effector cells) administered to a subject is in an effective amount, that is, effective to achieve the desired beneficial therapeutic response in the subject over time, or to prevent/inhibit growth of cancer cells, or to prevent/inhibit infection.
  • the optimal immunization interval for dendritic cell vaccines can be determined by one of skill in the art. Generally at least about 10 4 to 10 6 and typically, between 1 X 10 8 and 1 X 10 10 cells are administered to a 70 kg patient. Administration can be accomplished via single or divided doses. Someone skilled in the art can determine whether repeated administration is necessary and the frequency of repeated administration. Weekly, bimonthly or monthly administration is preferred.
  • the DCs and/or T cells are administered at weeks 0, 2, 4 and 8, then at months 4, 8, 12, 18, 24 and 30.
  • patients will be vaccinated 5 times with between 1x10 6 to 1x10 7 viable RNA-loaded DCs or other APC per dose.
  • the dose level selected for vaccination is expected to be safe and well-tolerated.
  • the cells of this invention can supplement other treatments for a condition by known conventional therapy, including cytotoxic agents, nucleotide analogues and biologic response modifiers.
  • biological response modifiers are optionally added for treatment by the DCs or activated T cells of the invention.
  • the cells are optionally administered with an adjuvant, immunomodulating agent, or cytokine, including but not limited to, GM-CSF, IL-12, IL-7, IL-15, IL-2, all-trans retinoic acid, denileukin diftitox, anti-CTLA-4 antibodies, TLR agonists, such as CpG oligonucleotides, etc.
  • 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-l alpha (IL-l ⁇ ), interleukin-1 L (IL-11), MIP-11 , 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 factor
  • Cytokines are commercially available from many vendors, including Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D Systems (Minneapolis, MN) and Immunex (Seattle, WA). Although not always explicitly stated, it is intended that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) be used within the spirit and scope of the invention.
  • immune responses in vaccinated individuals can be monitored by following the maturation profiles of CD4+ and CD8+ T cells.
  • restoration of HIV-specific effector cell function can be determined by the presence of cells expressing the phenotype of effector T-cells, CD45 RA + CCR7 " and secreting elevated levels of IFN- ⁇ and granzyme B.
  • Restoration of HIV-specific proliferative responses can be determined by the cells' capacity to produce IL-2 and to become CFSE l0W following stimulation with dendritic cells transfected with HIV-RNAs
  • Maturation of specific T cells induced by the vaccine can be measured using surface and intracellular markers using flow cytometry assay.
  • CD8 + T cells will be monitored by staining for surface markers including ⁇ TCR, CD45RA, CCR7 and CD107 or intracellular molecules such as granzyme B or IFN- ⁇ .
  • CD3, CD4, CCR7 and IL-2 can be used to monitor CD4 + T cells.
  • Such assays can be used to monitor immune response following incubation with peptides encompassing the autologous HIV sequences from the patient. Comparison of the cellular immune responses at baseline and monthly prior to each new vaccination enables determination of the vaccine's impact on the breadth of the cellular immune response. The breadth of the immune response can also be measured using the CFSE proliferation assay.
  • Ficoll-Paque was purchased from Wisent Laboratories. PBS was purchased from GIBCO, and X-VIVOTM 15 was purchased from Cambrex (East Rutherford, Ni). RPMI 1640 medium Fetal Bovine Serum (FBS) and AB Human serum were purchased from Sigma-Aldrish (Oakville, ON). Trypan Blue and CFSE were purchased from Invitrogen. Viaspan was purchased from Dupont Pharma Labs (Wilmington, DE). GM-CSF, IL-4, TNF- ⁇ , IL-l ⁇ , IFN- ⁇ and PGE 2 were purchased from R&D Systems (Minneapolis, MN).
  • CD40L mRNA was prepared according to the method described in International patent publication WO 2006/042177, the contents of which are incorporated by reference. Briefly, CD40L WT PCR 2.1 plasmid was linearized using Spel restriction enzyme and purified by phenol/chloroform extraction followed by ethanol precipitation. The linear template was resuspended in water and transcribed in vitro using mMessage mMachine T7 Ultra kits (Ambion) following the manufacturer's directions. An aliquot of RNA was saved for final analysis prior to proceeding to polyadenylation reaction. Polyadenylated RNA was purified using RNeasyTM column (QIAGEN) following protocol for RNA cleanup. RNA was eluted in water and stored in individual size aliquots below -15O 0 C.
  • Human PBMCs were isolated from Leukapheresis collections from high or low CMV responding donors or from HIV patients. PBMCs were prepared by Ficoll-Paque density centrifugation and washed four times in PBS at room temperature. 2 x 10 8 PBMCs were re- suspended in 30 ml X-VIVO 15 medium and allowed to adhere to 150 cm 3 plastic flasks for 2 hours at 37 ° C.
  • Non-adherent cells were removed and the remaining cells were cultured in X- VIVO 15 medium, supplemented with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml), for 5 days at 37 ° C, 5% CO 2 , and then matured by the addition of TNF- ⁇ (10 ng/ml), IFN- ⁇ (1000 U/ml) and PGE 2 (1 ⁇ g/ml), culturing for additional 18-24 hours.
  • GM-CSF 1000 U/ml
  • IL-4 1000 U/ml
  • the mature DCs were electroporated with CD40L mRNA in combination with the mRNA encoding GFP (negative control), CMV or HIV antigens and cultured in X-VIVO 15 in presence of GM-CSF (800 U/ml) and IL-4 (500 U/ml) for 4 hrs at 37 0 C, 5% CO 2 .
  • This maturation and electroporation were performed as described in International patent publication WO 2006/042177, the contents of which are incorporated by reference.
  • Example 3 CMV RNA-loaded, Lysate Pulsed DCs Induce Specific CD8 + and CD4 + T-cell Proliferative Responses
  • PBMCs were isolated from two donors showing a high or low immune response to CMV, differentiated into mature dendritic cells, and electroporated with CD40L RNA together with either a control RNA encoding a control protein (Green Fluorescent Protein (GFP)) or RNA encoding CMV pp65 antigens.
  • GFP Green Fluorescent Protein
  • RNA-loaded DCs were removed for preparation of a cell lysate. Briefly, 10 6 RNA-loaded DC were transferred to 1.5 ml conical bottom centrifuge tube and centrifuged at 2000 rpm for 2 minutes.
  • the supernatant was removed with a P1000 micropipette and the cells were resuspended in 1.0 ml of ice-cold PBS and centrifuged at 2000 rpm for 2 minutes. All of the supernatant was carefully removed with a P1000 micropipette and the tube was placed in a -8O 0 C freezer for a minimum of 1 hour. The frozen cell pellet was resuspended in RPMI so that the final concentration of cells was equivalent to 1x10 6 cells per ml. The tube containing the cells was then vortexed on the highest setting for 1 minute to produce the cell lysate.
  • FIG. 1 clearly shows that DCs loaded with pp65 CMV RNA but not pulsed with lysate (CMV NS) are able to induce strong CD8 + proliferation (20.2%) compared to the negative DC GFP control (GFP NS) (1% backround) but fail to induce any CD4 + proliferation (3.6% backround obtained with either GFP or CMV DCs, Figure 1 , first and fourth pairs of columns).
  • Figure 1 also shows that DCs loaded with RNA encoding a negative control (GFP RNA) and combined with a lysate made from DC loaded with the negative control GFP RNA (Lysate GFP) had no or very little ability to stimulate the proliferation of CD4 + or CD8 + T cells (Figure 1 , first and second pairs of columns).
  • Example 4 HIV RNA-loaded, Lysate-Pulsed DCs Induce Specific CD8 and CD4 Proliferative Responses
  • HIV RNA was isolated from the plasma of an HIV-infected patient. After low- speed centrifugation to clarify the plasma, it was diluted in lysis buffer and purified on a Nucleospin® purification column according to the manufacturer's instructions (Macherey- Nagel). RNA was eluted in nuclease free water and stored at -86 0 C until further use. HIV gag and nef mRNA were reverse transcribed, amplified by PCR and transcribed in vitro as described in International patent publication WO 2006/031870, the contents of which are incorporated by reference.
  • Mature DCs were prepared from the PBMCs of an HIV-infected donor as described in Example 2. The mature DCs were harvested, washed in PBS, re-suspended in chilled Viaspan ® (Barr Laboratories) at 4x10 7 /ml in 0.5 ml or 2.5x10 7 VmI in 0.2 ml and placed on ice. The DCs were then electroporated with in vitro transcribed mRNA (1 or 2 ⁇ g/10 6 cells) encoding HIV gag or nef antigens (or PBS or control RNA encoding GFP or CMV antigens) and CD40L mRNA (4 ⁇ g/10 6 cells).
  • DCs were washed in X-VIVO 15 medium, re-suspended in X-VIVO 15 supplemented with GM-CSF (800 U/ml) and IL-4 (500 U/ml) at 1x10 6 cells/ml and cultured for 4 hours at 37 0 C in low adherence plates (BD Biosciences, Franklin Lakes, NJ).
  • GM-CSF 800 U/ml
  • IL-4 500 U/ml
  • RNA-loaded DCs were used to stimulate autologous PBMC and for the preparation of a lysate for pulsing DCs in order to provide the CD4 help.
  • DCs loaded with RNA encoding HIV Gag were combined autologous PBMCs and either no lysate (NS), lysate made from DCs loaded with a negative control (PBS) (Lysate CTR), a lysate made from DCs loaded with a control RNA-encoding CMV (Lysate CMV) or a lysate made from DCs loaded with RNA encoding Nef (Lysate Nef).
  • N no lysate
  • PBS negative control
  • Lysate CTR a lysate made from DCs loaded with a control RNA-encoding CMV
  • Nef a lysate made from DCs loaded with RNA encoding Nef
  • the increase in CD8 + T-cell proliferation may be due to an exchange of peptides bound to MHC class I molecules at the cell surface with CMV or Nef specific peptides present in the lysate and/or to the presence of CD4 help. (This can be directly assessed by using specific-class I tetramers recognizing the peptides presented by the APCs or by the lysates or by restimulation of the proliferating cells by the corresponding antigens.) In contrast, the same DCs, cocultured with autologous PBMCs in the presence of a lysate made from DCs loaded with RNA-encoding Nef stimulated significantly increased levels of CD4 + T-cell proliferation (Figure 3, fourth pair of columns)
  • Example 6 CFSE Proliferation Assay for Analysis of T-CeII Subsets Stimulated by DCs Loaded with HIV RNA and Pulsed with Lysates
  • a nine-color reagent cocktail has been developed for detailed analysis of proliferation of antigen-specific T-cell responses.
  • This 9-color polychromatic flow cytometry include the following markers:
  • CFSE The dilution of the CFSE dye enables the identification of T cells that have proliferated following stimulation.
  • Anti-CD3 This antibody enables the limiting of the analysis to the T-cell population.
  • Anti CD8 ⁇ and anti-CD4 These antibodies are used respectively to restrict the analyses on CD8 and CD4 T cells.
  • Anti-CD45RA and anti-CCR7 The combination of these 2 markers permits the identification of different subsets of effector and memory CD4 and CD8 T cells having different functional properties.
  • Anti-CD27 The expression pattern of CD27 reflects different stages of differentiation. Lack of CD27 is associated with highly-differentiated CD8 T cells having strong effector functions (high cytolytic capacity) but poor proliferative capacity.
  • Anti-CD28 The expression pattern of CD28 reflects different stages of differentiation. Lack of CD28 is associated with highly differentiated CD4 and CD8 T cells having strong effector functions (high cytolytic capacity) but poor proliferative capacity. [0155] Anti-CD127: Expression of CD127 identifies long-term memory T cells (predominantly central memory T cells and IL-2-secreting cells).
  • Example 7 Lysate-peptides loading on class Il molecules presented by autologous DCs:
  • the first method directly examines the HLA-DR-restricted presentation of an appropriate antigen by using an antibody that is specific for these HLA-DR-peptide complexes.
  • a large number of antibodies to human MHC class Il molecules in complexes with peptides has been reported by several laboratories to detect specific class ll-peptide complexes such HLA-DR2-MBP (8 5 -99) ; -DR7-MBP (8 5_99) and -DR11- MBP(85.99), or HLA-DR4-CII( 2 63- 2 7 2 ), etc.).
  • Mature DC are generated from DR2 and DR7 HLA-typed patients, electroporated with the MBP mRNA and then the lysate is extracted. MBP-electroporated DCs are incubated either with PBS (negative control), in presence of native peptide (positive control), or the lysate and stained with the appropriate antibody specific for the complexes HLA-DR-MBP (8 5-99). DC is then analyzed by flow cytometry and the loading efficacy can be calculated.
  • the second assay uses a tagged promiscuous peptide.
  • HLA-DR alleles present the immunodominant HA( 3 o6-3i8) peptide of haemagglutinin of the influenza virus or TT(83o-8 4 3) peptide of tetanus toxoid to T cells.
  • HA immunodominant HA( 3 o6-3i8) peptide of haemagglutinin of the influenza virus or TT(83o-8 4 3) peptide of tetanus toxoid to T cells.
  • HA or TT electroporated DC are incubated either with PBS (negative control), in presence of native peptides (positive control), or the lysate and stained with specific anti-Tag antibody. DC is then analyzed by flow cytometry and the loading efficacy can be calculated.

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Abstract

Cette invention concerne des procédés permettant de préparer et d'utiliser des cellules immunostimulatrices pour améliorer une réponse immunitaire protectrice puissante et des compositions apparentées. Il a été découvert que les réponses à la fois des lymphocytes T CD4+ helpers et des lymphocytes T cytotoxiques CD8+ peuvent être déclenchées par stimulation de lymphocytes T CD4+ et CD8+ par des cellules présentant un antigène (APC), qui sont chargées à la fois avec un ARN codant pour un antigène ainsi qu'un lysat ou un extrait de cellules ou de virions comprenant un antigène. L'invention concerne un procédé permettant de préparer des cellules présentant un antigène chargé avec un antigène (APC), qui comprend les étapes consistant à : charger une population d'APC avec un ou plusieurs premiers ARN codant pour un ou plusieurs premiers antigènes, et activer la population des APC avec un extrait ou un lysat de cellules ou de virions qui comprennent un ou plusieurs seconds antigènes, la population d'APC absorbant le ou les seconds antigènes. De préférence, le lysat ou l'extrait est composé d'APC chargés avec un ARN. L'invention concerne également un APC activé par un antigène chargé avec un ARN.
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110002899A1 (en) * 2008-03-07 2011-01-06 Eric Lagasse Lymph Nodes as a Site for Regeneration
WO2011033090A1 (fr) * 2009-09-18 2011-03-24 Forskarpatent I Syd Ab Cellules spécifiques de l'antigène présentant des antigènes tolérogènes et utilisations, compositions, procédés et systèmes associés
WO2013167156A1 (fr) * 2012-05-11 2013-11-14 Otto-Von-Guericke-Universität Magdeburg Transplantation de monocytes modifiés
US8609605B2 (en) 2009-03-17 2013-12-17 Cardiovax, Llc Immunomodulatory methods for treatment of atherosclerosis via inhibition of CD4+ T cell response to APOB100
US8728806B2 (en) 2008-12-06 2014-05-20 The Board Of Regents, The University Of Texas System Methods and compositions related to Th-1 dendritic cells
US9125850B2 (en) 2009-11-14 2015-09-08 Cardio Vax, Llc Immunomodulatory methods and systems for treatment and/or prevention of atherosclerosis
US9205139B2 (en) 2010-11-12 2015-12-08 Cardiovax, Llc Immunomodulatory methods and systems for treatment and/or prevention of aneurysms
US9205141B2 (en) 2010-11-12 2015-12-08 Cardio Vax, Llc Immunomodulatory methods and systems for treatment and/or prevention of hypertension
CN106929473A (zh) * 2017-03-08 2017-07-07 广州可伴咨询中心（普通合伙） 用于hpv‑ctl细胞的体外培养试剂盒
WO2020018413A1 (fr) * 2018-07-15 2020-01-23 Enochian BioPharma, Inc. Procédés et compositions utilisant des cellules dendritiques recombinées pour une thérapie anticancéreuse
WO2020257309A1 (fr) * 2019-06-17 2020-12-24 Enochian BioPharma, Inc. Vaccin contre le vih à base de lymphocytes t allogéniques pour induire une immunité cellulaire et humorale
CN118068017A (zh) * 2024-04-18 2024-05-24 中国疾病预防控制中心传染病预防控制所 鉴定幽门螺杆菌抗原肽的方法及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034638A1 (fr) * 1994-06-14 1995-12-21 The Board Of Trustees Of Leland Stanford Junior University Procedes pour l'activation de lymphocytes t in vivo par des cellules dendritiques a impulsions antigeniques
US20060057130A1 (en) * 1996-04-30 2006-03-16 Duke University Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995034638A1 (fr) * 1994-06-14 1995-12-21 The Board Of Trustees Of Leland Stanford Junior University Procedes pour l'activation de lymphocytes t in vivo par des cellules dendritiques a impulsions antigeniques
US20060057130A1 (en) * 1996-04-30 2006-03-16 Duke University Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BOCZKOWSKI D. ET AL.: "Dendritic cells pulsed with RNA are potent antigen presenting cells in vitro and in vivo", J. EXP. MED., vol. 184, 1996, pages 465 - 472, XP002921123, DOI: doi:10.1084/jem.184.2.465 *
CELLUZI C.M. ET AL.: "Peptide pulsed dendritic cell induce antigen-specific CTL-mediated protective tumour immunity", J. EXP. MED., vol. 183, 1996, pages 283 - 287 *
DECKER W.K. ET AL.: "Double loading of dendritic cell MHC class I and MHC class II with an AML antigen repertoire enhances correlates of T-cell immunity in vitro via amplification", VACCINE, vol. 24, April 2006 (2006-04-01), pages 3203 - 3216, XP028010608, DOI: doi:10.1016/j.vaccine.2006.01.029 *
LIU Y. ET AL.: "Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8 cytotoxic T-cell activation and anti-tumour activity", CANCER GENE THERAPY, vol. 9, 2002, pages 202 - 208 *

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US9125891B2 (en) * 2008-03-07 2015-09-08 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Lymph nodes as a site for regeneration
US9993537B2 (en) 2008-12-06 2018-06-12 Board Of Regents, The University Of Texas System Methods and compositions related to Th-1 dendritic cells
US8728806B2 (en) 2008-12-06 2014-05-20 The Board Of Regents, The University Of Texas System Methods and compositions related to Th-1 dendritic cells
US9333248B2 (en) 2008-12-06 2016-05-10 Board Of Regents, The University Of Texas System Methods and composition related to Th-1 dendritic cells
US8609605B2 (en) 2009-03-17 2013-12-17 Cardiovax, Llc Immunomodulatory methods for treatment of atherosclerosis via inhibition of CD4+ T cell response to APOB100
WO2011033090A1 (fr) * 2009-09-18 2011-03-24 Forskarpatent I Syd Ab Cellules spécifiques de l'antigène présentant des antigènes tolérogènes et utilisations, compositions, procédés et systèmes associés
US9125850B2 (en) 2009-11-14 2015-09-08 Cardio Vax, Llc Immunomodulatory methods and systems for treatment and/or prevention of atherosclerosis
US9205141B2 (en) 2010-11-12 2015-12-08 Cardio Vax, Llc Immunomodulatory methods and systems for treatment and/or prevention of hypertension
US9205139B2 (en) 2010-11-12 2015-12-08 Cardiovax, Llc Immunomodulatory methods and systems for treatment and/or prevention of aneurysms
WO2013167156A1 (fr) * 2012-05-11 2013-11-14 Otto-Von-Guericke-Universität Magdeburg Transplantation de monocytes modifiés
CN106929473A (zh) * 2017-03-08 2017-07-07 广州可伴咨询中心（普通合伙） 用于hpv‑ctl细胞的体外培养试剂盒
WO2020018413A1 (fr) * 2018-07-15 2020-01-23 Enochian BioPharma, Inc. Procédés et compositions utilisant des cellules dendritiques recombinées pour une thérapie anticancéreuse
CN112703004A (zh) * 2018-07-15 2021-04-23 伊诺奇安生物制药公司 使用重组树突状细胞用于癌症治疗的方法和组合物
JP2021531765A (ja) * 2018-07-15 2021-11-25 エノキアン バイオファーマ インコーポレイテッド がん治療のための組換え樹状細胞を使用する方法及び組成物
US11413338B2 (en) 2018-07-15 2022-08-16 Enochian BioPharma, Inc. Methods and compositions using recombinant dendritic cells for cancer therapy
WO2020257309A1 (fr) * 2019-06-17 2020-12-24 Enochian BioPharma, Inc. Vaccin contre le vih à base de lymphocytes t allogéniques pour induire une immunité cellulaire et humorale
CN114269356A (zh) * 2019-06-17 2022-04-01 伊诺奇安生物制药公司 诱导细胞免疫和体液免疫的基于同种异体t细胞的hiv疫苗
CN118068017A (zh) * 2024-04-18 2024-05-24 中国疾病预防控制中心传染病预防控制所 鉴定幽门螺杆菌抗原肽的方法及应用

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