WO1999046984A1 - Compositions et methodes pour entreposer des cellules dendritiques congelees - Google Patents

Compositions et methodes pour entreposer des cellules dendritiques congelees Download PDF

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Publication number
WO1999046984A1
WO1999046984A1 PCT/US1999/006033 US9906033W WO9946984A1 WO 1999046984 A1 WO1999046984 A1 WO 1999046984A1 US 9906033 W US9906033 W US 9906033W WO 9946984 A1 WO9946984 A1 WO 9946984A1
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Prior art keywords
cells
dcs
antigen
cell
human
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PCT/US1999/006033
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English (en)
Inventor
Srinivas Shankara
Charles A. Nicolette
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Genzyme Corporation
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Priority to AU31025/99A priority Critical patent/AU3102599A/en
Publication of WO1999046984A1 publication Critical patent/WO1999046984A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages

Definitions

  • TECHNICAL FIELD This invention is in the field of molecular immunology and medicine.
  • compositions and methods relating to freezing and thawing viable and functional dendritic cells are provided.
  • Cytotoxic T-lymphocyte (CTL) responses can be directed against antigens specifically presented by tumor cells, both in vivo and in vitro, without the need for prior .knowledge of the molecular mechanism by which the tumor arose.
  • established tumors can be eradicated by the adoptive transfer of T-cells that specifically lyse malignant cells (Beun et al.
  • an antigen In order to provoke a specific CTL response, an antigen must be presented to T cells. This is accomplished via antigen presenting cells (-APCs), a class of cells which includes dendritic cells (DCs), monocytes, macrophages, and B cells.
  • DCs were first described as the morphologically distinct Langerhans cells in the skin (Bancheraeau and Steinman (1998) Nature 392:245-252) and have since been shown to be the most efficient APC for the activation of naive T cells. Lanzavecchia A. (1993) Science 260:937-944 and Bancheraeau and Steinman (1988), supra.
  • DCs can be loaded with exogenous antigen for presentation to T cells by a number of methods including: 1) pulsing with peptides eluted from major histocompatibilty complex (MHC) class I molecules; 2) tumor-specific idiotype protein; 3) transduction with RNA encoding antigen such as RNA derived from neoplastic cells; and 4) fusing DCs with tumor cells.
  • MHC major histocompatibilty complex
  • RNA encoding antigen such as RNA derived from neoplastic cells
  • DCs presenting exogenous antigen have been shown to stimulate both CD4 + and CD8 + T cells as well as natural .killer (NK)-cell anti-tumor responses.
  • DCs also have been used to present viral and bacterial antigens to T lymphocytes. Nair S. et al. (1993) J. Virol. 67:4062. Herpes simplex peptide derivatized with a triacyl tail and delivered to DC by liposomes was more effective than virus alone. DC- generated responses to HIV proteins have been obtained in vitro. Macatonia S.E. et al. (1991) Immunol. 70:1. Therefore, DCs appear to be the appropriate vehicle for presenting antigens to provoke immune responses against a wide variety of targets.
  • the ability to store mature and precursor DCs for extended periods of time will be useful for: (1) clinical protocols in which multiple DC administrations are required; (2) providing a continuous source of autologous DCs for protracted preclinical studies; and (3) ex vivo generation of cytotoxic T lymphocytes against tumor antigens by educating naive immune effector cells, preferably from peripheral blood lymphocytes, with DCs transduced with a gene encoding the antigen of interest.
  • Typical protocols call for between 20-30% strigm and about 10% dimethylsulfoxide (DMSO). (See, e.g., U.S. Patent No. 5,788,963; Makino et al.
  • the present invention provides compositions and methods for freezing, storing and thawing dendritic cells.
  • a substantial portion of the DCs frozen as described herein retain viability and functionality when thawed.
  • Applicants have shown that DCs frozen and thawed by the methods of this invention have no effect on subsequent antigen presentation by the DCs as measured by CTL assay.
  • Applicants also have demonstrated that DCs generated by culturing monocytes in GM-CSF and IL-4 that were frozen, stored and thawed, had no effect on the expression of cell surface markers.
  • the DCs are frozen by suspending the cells in media containing at least 30% human-derived serum and/or plasma, and lowering the temperature of the suspension to at least -80°C, thereby freezing the DCs.
  • the freezing media is approximately 30% human-derived serum and/or plasma and approximately 10% of an agent that prevents ice crystal formation during freezing, e.g., DMSO.
  • the suspension is maintained at -80°C for at least 24 hours and then transferred to liquid nitrogen for the duration of the storage.
  • the suspension is thawed at a temperature in the range of 34 to 41 °C. Applicants have shown that cells frozen and stored under these conditions retain up to 88% cell viability when thawed, even up to 15 weeks in frozen storage.
  • the present invention also provides a substantially purified population of DCs produced by freezing DCs by the methods described herein, as well as compositions comprising the DCs, either in their frozen state or after being thawed.
  • the present invention provides a method for ex vivo generation of antigen-specific immune effector cells, comprising educating na ⁇ ve immune effector cells by co-culture with the frozen and thawed DCs.
  • the immune effector cells may be cytotoxic T lymphocytes (CTLs) derived from peripheral blood lymphocytes (PBLs).
  • CTLs cytotoxic T lymphocytes
  • PBLs peripheral blood lymphocytes
  • the immune effector cells and the DCs can be autologous, or allogeneic.
  • the present invention also provides a substantially purified population of immune effector cells produced by educating na ⁇ ve immune effector cells by co- culture with the DCs, identified above.
  • FIG 1 panels A to F and Figure 2, panels A through C, show FACS profiles of human dendritic cells prepared from peripheral blood monocytes.
  • the antibodies used for sorting are shown on the horizontal axis.
  • Figure 3 is a graph depicting FACS profiles on fresh and frozen/thawed human dendritic cells.
  • the markers used for sorting are shown on the horizontal axis and the percentage of cells having the marker is shown on the vertical axis.
  • Figure 4 is a graph depicting antigen-presenting activity of frozen/thawed human dendritic cells pulsed with the indicated peptides after incubation for 24 hours.
  • -•- shows cells incubated with G9-209
  • - ⁇ - shows cells incubated with GP100-F9
  • -A- shows cells incubated with GP 100-K9.
  • Figure 5 is a graph depicting antigen-presenting activity of freshly isolated human dendritic cells pulsed with the indicated peptides for 24 hours.
  • CTL assay a functional analysis (CTL assay)
  • Applicants have shown that freshly prepared DCs and DC that were frozen in 90% FCS/10% DMSO retain greater than 90% viability and functionality.
  • -•- shows cells incubated with G9-209
  • - ⁇ - shows cells incubated with GP100-F9
  • -A- shows cells incubated with GP 100-K9.
  • FIG. 6 panels A through E, graphically show CTL assay results of fresh DCs and DCs thawed at different time points (DCs were in frozen storage for over a period of 15 weeks).
  • Figure 7 shows FACS profile of thawed dendritic cell surface markers after various periods of frozen storage. The dendritic cells were infected with the Ad2CMVgpl00 virus. The vertical axis is % positive cells and the horizontal axis shows the markers identified by FACS.
  • Figure 8 shows FACS profile of thawed dendritic cell surface markers after various periods of frozen storage. The dendritic cells were infected with the Ad2CMVgpl00 virus. The vertical axis shows mean fluorescence intensity and the horizontal axis shows the markers identified by FACS.
  • Figure 9 shows FACS profile of thawed uninfected dendritic cell surface markers after various periods of frozen storage.
  • the vertical axis is % positive cells and the horizontal axis shows the markers identified by FACS. Note that for the cells thawed after 9 weeks of storage, the Class I markers didn't stain.
  • Figure 10 shows FACS profile of thawed uninfected dendritic cell surface markers after various periods of frozen storage.
  • the vertical axis shows mean fluorescence intensity and the horizontal axis shows the markers identified by
  • a cell includes a plurality of cells, including mixtures thereof.
  • antigen presenting cell intends any cell which presents on its surface an antigen in association with a major histocompatibility complex molecule, or portion thereof, or, alternatively, one or more non-classical MHC molecules, or a portion thereof.
  • suitable APCs include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen presenting cells. Methods of making hybrid APCs have been described. See, for example, International Patent Application No. WO 98/46785 and WO 95/16775.
  • DCs Dendritic cells
  • TCR/CD3 T-cell receptor/CD3
  • MHC major histocompatibility complex
  • the second type of signals is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals.
  • DCs may be isolated from any number of species such as human, murine, or simian.
  • DCs are minor constituents of various immune organs such as the spleen, thymus, lymph node, epidermis, and peripheral blood.
  • DCs represent merely about 1% of crude spleen (Steinman et al. (1979) J. Exp. Med. 149:1) or epidermal cell suspensions (Schuler et al. (1985) J. Exp. Med. 161:526; and Romani et al. (1989) J. Invest. Dermatol. 93:600), and 0.1-1% of mononuclear cells in peripheral blood (Freudenthal et al. (1990) PNAS USA 87:7698).
  • “Mature” dendritic cells are those cells which have fully differentiated into cells which exhibit the characteristic morphology and function of DCs, for example an array of protrusions (dendrites) and the ability to be potent stimulators of allogeneic T cells, (van Shooten et al. (1997) Mol. Med. Today 254-260). Although no single cell-surface marker has been found that uniquely identifies DCs, fully differentiated DCs have been shown to express CD la, CD40, CD54, CD80 and CD86.
  • Dendritic cell "precursors” are cells that are capable of developing into mature DCs under specific conditions. Examples of DC precursors include, but are not limited to, monocytes and peripheral blood lymphocytes. When culture conditions include cytokines, DCs can be generated in vitro from monocytes, CD34 + stem cells and from lymphoid precursor cells (van Shooten et al., supra).
  • dendritic cell is to include, but not be limited to a DC precursor, a mature DC, a pulsed DC, a foster DC, a genetically modified DC and a DC hybrid.
  • substantially portion is meant at least 50% of the frozen dendritic cells maintain their function and are viable, preferably at least 70%, more preferably at least 80%, and even more preferably at least 88%.
  • “Function” refers to the various biological properties exhibited by dendritic cells, for example, expression of major histocompatibility complex (MHC) and co-stimulatory molecules and the ability to stimulate naive and memory T cells.
  • “Viability” refers to the cell's ability to survive and/or proliferate.
  • 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 IL-12
  • G-CSF granulocyte macrophage-colony stimulating factor
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • IL-l ⁇ interleukin- 1 alpha
  • IL-11 interleukin- 11
  • MlP-l ⁇ leukemia inhibitory factor
  • LIF leukemia inhibitory factor
  • c-kit ligand thrombopoietin
  • TPO flt3 ligand.
  • the present invention also includes culture conditions in which one or more cytokine is specifically excluded from the medium.
  • Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D Systems and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinant
  • Co-stimulatory molecules are involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. Research accumulated over the past several years has demonstrated convincingly that resting T cells require at least two signals for induction of cytokine gene expression and proliferation (Schwartz R.H. (1990) Science
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • IAM-1 intracellular adhesion molecule 1
  • One exemplary receptor-ligand pair is the B7 co-stimulatory molecule on the surface of APCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman et al. (1993) Science 262:909-911; Young et al. (1992) J. Clin. Invest. 90:229; Nabavi et al. (1992) Nature 360:266-268).
  • Other important co-stimulatory molecules are CD40, CD54, CD80, CD86.
  • co-stimulatory molecule encompasses any single molecule or combination of molecules which, when
  • the term thus encompasses B7, or other co-stimulatory molecule(s) on an antigen-presenting matrix such as an APC, fragments thereof (alone, complexed with another molecule(s), or as part of a fusion protein) which, together with peptide/MHC complex, binds to a cognate ligand and results in activation of the T cell when the TCR on the surface of the T cell specifically binds the peptide.
  • human-derived plasma and/or serum is any human blood-derived fluid (plasma) as well as any synthetic compositions having the same functional activity. Autologous and non-autologous human-derived plasma and/or serum is intended to be within the scope of this invention.
  • Human serum or plasma contain four classes of lipoproteins: chylomicrons; very low density lipoproteins (pre-beta lipoproteins); low density lipoproteins (beta lipoproteins); and high density lipoproteins (alpha lipoproteins).
  • Blood plasma is the liquid part of blood containing fibrinogen. Normal human plasma is obtained from pooled blood. The pooled blood includes approximately equal volumes of the liquid portions of the whole blood. From the pooled blood human serum is derived.
  • the serum is the clear, amber, alkaline fluid of the blood from which cellular elements have been removed by clotting.
  • the serum contains the salts, soluble proteins, and lipoproteins.
  • Methods for separating the serum from plasma are well known in the art. See, e.g., U.S. Patent No. 4,045,176.
  • antigen is well understood in the art and includes substances which are immunogenic, i.e., immunogens, as well as substances which induce immunological unresponsiveness, or anergy, i.e., anergens.
  • a "native” or “natural” antigen is a polypeptide, protein or a fragment which contains an epitope, which has been isolated from a natural biological source, and which can specifically bind to an antigen receptor, in particular a T cell antigen receptor (TCR), in a subject.
  • TCR T cell antigen receptor
  • peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds.
  • the subunit may be linked by other bonds, e.g. ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein. Throughout this specification, numbering of amino acids in a peptide or polypeptide is from amino terminus to carboxy terminus.
  • the term "genetically modified” means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous nucleotides.
  • immuno effector cells refers to cells capable of binding an antigen or which mediate an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates. Certain diseased tissue expresses specific antigens and CTLs specific for these antigens have been identified. For example, approximately 80% of melanomas express the antigen known as gplOO.
  • a "na ⁇ ve" immune effector cell is an immune effector cell that has never been exposed to an antigen.
  • the term "educated, antigen-specific immune effector cell” is an immune effector cell as defined above, which has encountered antigen and which is specific for that antigen.
  • effector cell may be activated upon binding antigen.
  • Activated implies that the cell is no longer in G 0 phase, and begins to produce cytokines characteristic of the cell type.
  • activated CD4 + T cells secrete IL-2 and have a higher number of high affinity IL-2 receptors on their cell surfaces relative to resting CD4 + T cells.
  • a peptide or polypeptide of the invention may be preferentially recognized by antigen-specific immune effector cells, such as B cells and T cells.
  • antigen-specific immune effector cells such as B cells and T cells.
  • the term "recognized” intends that a peptide or polypeptide of the invention, comprising one or more synthetic antigenic epitopes, is recognized, i.e., is presented on the surface of an APC together with (i.e., bound to) an MHC molecule in such a way that a T cell antigen receptor (TCR) on the surface of an antigen-specific T cell binds to the epitope wherein such binding results in activation of the T cell.
  • TCR T cell antigen receptor
  • polypeptide of the invention is substantially not recognized, as defined above, by a T cell specific for an unrelated antigen.
  • Assays for determining whether an epitope is recognized by an antigen-specific T cell are known in the art and are described herein.
  • autogeneic indicates the origin of a cell.
  • a cell being administered to an individual is autogeneic if the cell was derived from that individual (the "donor") or a genetically identical individual.
  • An autogeneic cell can also be a progeny of an autogeneic cell.
  • the term also indicates that cells of different cell types are derived from the same donor or genetically identical donors.
  • an effector cell and an antigen presenting cell are said to be autogeneic if they were derived from the same donor or from an individual genetically identical to the donor, or if they are progeny of cells derived from the same donor or from an individual genetically identical to the donor.
  • allogeneic indicates the origin of a cell.
  • a cell being administered to individual is allogeneic if the cell was derived from an individual not genetically identical to the recipient; in particular, the term relates to non-identity in expressed MHC molecules.
  • 11 allogeneic cell can also be a progeny of an allogeneic cell.
  • the term also indicates that cells of different cell types are derived from genetically non- identical donors, or if they are progeny of cells derived from genetically non- identical donors.
  • an APC is said to be allogeneic to an effector cell if they are derived from genetically non-identical donors.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
  • Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes, for example, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule may also comprise modified nucleic acid molecules.
  • Oligonucleotide refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also .known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. As used herein, "expression” refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG Sambrook et al. (1989) infra ).
  • an eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • Such vectors can be
  • Under transcriptional control is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operably (operatively) linked to an element which contributes to the initiation of, or promotes, transcription. "Operably linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
  • a “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria, viruses, such as baculovirus, adenovirus, adeno-associated virus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • liposomes include natural polymers and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria
  • viruses such as baculovirus, adenovirus, adeno-associated virus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors and the like.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • MHC major histocompatibility complex
  • the proteins encoded by the MHC complex are .known as "MHC molecules” and are classified into class I and class II MHC molecules.
  • Class I MHC molecules include membrane heterodimeric proteins made up of an ⁇ chain encoded in the MHC associated noncovalently with ⁇ 2- microglobulin.
  • Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8 + T cells.
  • Class I molecules include HLA-A, -B, and -C in humans.
  • Class I molecules generally bind peptides 8-10 amino acids in length.
  • Class II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated ⁇ and ⁇ chains. Class II MHC are known to participate in antigen presentation to
  • Class II molecules generally bind peptides 12-20 amino acid residues in length.
  • MHC restriction refers to a characteristic of T cells that permits them to recognize antigen only after it is processed and the resulting antigenic peptides are displayed in association with either a self class I or class II MHC molecule. Methods of identifying and comparing MHC are well known in the art and are described in Allen et al. (1994) Human Immun. 40:25-32; Santamaria et al. (1993) Human Immun. 37:39-50 and Hurley et al. (1997) Tissue Antigens 50:401-415.
  • “Inducing an immune response in a subject” is a term well understood in the art and intends an increase in an immune response to an antigen (or epitope) that can be detected (measured) after introducing the antigen (or epitope) into the subject relative to the immune response (if any) before introduction of the antigen (or epitope) into the subject.
  • the increase is intended to be at least about 2-fold, more preferably at least about 5-fold, more preferably at least about 10-fold, more preferably at least about 100-fold, even more preferably at least about 500-fold, even more preferably at least about 1000-fold.
  • antigen-specific antibody 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 that specifically binds to an antigen (or epitope).
  • Methods of determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art.
  • antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, wherein, for example, binding of an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).
  • Immune effector cells specific for the antigen can be detected any of a variety of assays known to those skilled in the art, including, but not limited to, FACS, or, in the case of CTLs, Cr-release assays, or H-thymidine uptake assays.
  • culturing refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
  • a "subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • a “control” is an alternative subject or sample used in an experiment for comparison purpose.
  • a control can be "positive” or “negative".
  • the purpose of the experiment is to determine a correlation of an altered expression level of a gene with a particular type of cancer, it is generally preferable to use a positive control (a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that disease), and a negative control (a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease).
  • “Host cell” or “recipient cell” is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or peptides (or
  • the cells may be procaryotic or eucaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.
  • an “antibody” is an immunoglobulin molecule capable of binding an antigen.
  • the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, bi-specific antibodies, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity.
  • an “antibody complex” is the combination of antibody (as defined above) and its binding partner or ligand.
  • isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature.
  • an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment 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 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 polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof which differs from the naturally occurring counterpart in its primary sequence or for example, by its glycosylation pattern, need not be present in its isolated form since it is distinguishable from its naturally occurring counterpart by its primary sequence, or alternatively, by another
  • a non-naturally occumng 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 eucaryotic cell in which it is produced in nature.
  • An “isolated” population of cells is “substantially free” of cells and materials with which it is associated in nature. By “substantially free” or
  • substantially purified means at least 50% of the population are the desired cell type, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90%.
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent , solid support or label) or active, such as an adjuvant.
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • an effective amount is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • an effective amount of an immunomodulatory agent of the invention including a peptide of the invention, a polynucleotide of the invention, an educated, antigen- specific immune effector cell and/or an APC of the invention, is an amount that is
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • a method for freezing, storing and thawing DCs comprises suspending the DCs in media comprising at least 30% (v/v), (and more preferably at least 50%, more preferably, at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 85 or 90%) human-derived serum and/or plasma and lowering the temperature of the suspension to at least -80°C.
  • at least 10% (v/v) of an agent that will prevent formation of crystals during freezing is added to the suspension.
  • Non-limiting examples of such agents include dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, e-erythritol, D-ribitol, D-mannitol, D-sorbitol, I-inositol, D-lactose, choline chloride, amino acids and methanol, acetamide, glycerol monoacetate, and inorganic salts.
  • DMSO dimethyl sulfoxide
  • glycerol polyvinylpyrrolidone
  • polyethylene glycol albumin
  • dextran sucrose
  • ethylene glycol e-erythritol
  • D-ribitol D-ribitol
  • D-mannitol D-mannitol
  • D-sorbitol I-inositol
  • D-lactose choline chloride
  • the cell suspension should be maintained at least this temperature during storage.
  • the suspension is transferred to liquid nitrogen for storage or initially frozen under liquid nitrogen.
  • the cells are thawed at temperatures of about 35 to 41°C, and more preferably at 37°C.
  • the agent e.g., DMSO
  • the thawing cells as soon as possible.
  • the human-derived serum and/or plasma can be obtained from commercial sources or isolated and purified from the blood of human patients just prior to use. Because isolation and purification methods are well known in the art, (see, e.g., U.S. Patent No. 5,788,963), a detailed description of these methods is not reproduced herein.
  • DC precursors and mature, fully differentiated DCs frozen, stored and thawed by the methods of this invention retain viability and functionality.
  • Methods of isolating DC precursors such as monocytes or peripheral blood lymphocytes as well as mature, fully differentiated DCs are well known in the art. Several methods of the known methods are briefly described below.
  • modified DCs frozen and stored by the methods of this invention retain the functionality upon thawing.
  • the DCs can be modified to present endogenous self-antigen or exogenous antigen prior to freezing.
  • the DCs can be pulsed with antigen, fused with antigen- presenting tumor cells, genetically modified to express antigen.
  • Foster DCs also can be used to present antigen.
  • Any peptide that can be presented in the context of an MHC molecule on the surface of a DC can be used to modify the DC prior to or subsequent to freezing.
  • antigens include self- antigens, tumor associated antigens (TAAs) and viral antigens.
  • the DCs can be genetically modified or unmodified prior to frozen storage. DCs also can be genetically modified to express any one or a combination of an antigen, a cytokine, a therapeutic gene and a co-stimulatory molecule.
  • DCs can be genetically modified by insertion of naked DNA or by insertion of the polynucleotide via a gene delivery vehicle. Exemplary gene delivery vehicles are described below. Usually, the vectors will contain at least two heterologous genes or gene sequences: (i) the gene to be transferred; and (ii) a marker gene that enables tracking of infected cells.
  • therapeutic gene can be an entire gene or only the functionally active fragment of the gene capable of compensating for the deficiency in the patient that arises from the defective endogenous gene. Therapeutic gene also encompasses antisense
  • a therapeutic gene may be one that neutralizes the immunosuppressive factor or counter its effects.
  • Therapeutic genes that encode dominant inhibitory oligonucleotides and peptides as well as genes that encode regulatory proteins and oligonucleotides also are encompassed by this invention.
  • gene therapy will involve the transfer of a single therapeutic gene although more than one gene may be necessary for the treatment of particular diseases.
  • the therapeutic gene is a dominant inhibiting mutant of the wild-type immunosuppressive agent.
  • the therapeutic gene could be a wild- type, copy of a defective gene or a functional homologue.
  • More than one gene can be administered per vector or alternatively, more than one gene can be delivered using several compatible vectors.
  • the therapeutic gene can include the regulatory and untranslated sequences.
  • the therapeutic gene will generally be of human origin although genes from other closely related species that exhibit high homology and biologically identical or equivalent function in humans may be used, if the gene product does not induce an adverse immune reaction in the recipient.
  • the therapeutic gene suitable for use in treatment will vary with the disease.
  • Nucleotide sequences for the therapeutic gene will generally be known in the art or can be obtained from various sequence databases such as GenBank.
  • the therapeutic gene itself will generally be available or can be isolated and cloned using the polymerase chain reaction PCR (Perkin-Elmer) and other standard recombinant techniques.
  • the skilled artisan will readily recognize that any therapeutic gene can be excised as a compatible restriction fragment and placed in a vector in such a manner as to allow proper expression of the therapeutic cells.
  • a marker gene can be included in the vector for the purpose of monitoring successful transduction and for selection of cells into which the DNA has been integrated, as against cells which have not integrated the DNA construct.
  • marker genes include, but are not limited to, antibiotic resistance markers, such as resistance to G418 or hygromycin. Less conveniently, negative selection may be used, including, but not limited to, where the marker is the HSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir. Alternatively, selections could be accomplished by employment of a stable cell surface marker to select for transgene expressing cells by FACS sorting.
  • the NeoR (neomycin /G418 resistance) gene is commonly used but any convenient marker gene whose sequences are not already present in the recipient cell, can be used.
  • a substantially purified population of DCs frozen and thawed by the methods of this invention are useful to present antigen and to educate na ⁇ ve immune effector cells, which in turn are administered to a subject in an effective amount to induce an immune response specific to the antigen presented by the DC.
  • the population is combined with a pharmaceutically acceptable carrier prior to use.
  • Methods of educating na ⁇ ve immune effector cells are known in the art.
  • a substantially purified population of educated immune effector cells can be combined with a pharmaceutically acceptable carrier prior to administration to a subject.
  • the DCs and/or immune effector cells can be autologous or allogeneic to the subject being treated.
  • the above therapeutic methods can be further modified by co- administration of an effective amount of a cytokine and/or co-stimulatory molecule is added to the pharmaceutical composition.
  • the cytokine and/or co- stimulatory molecule also can be in the form of a protein or they can be administered in the form of a polynucleotide coding for the molecule of interest. Methods of administration of DCs and educated immune effector cells are known in the art and therefore, not reproduced herein.
  • agents identified herein as effective for their intended purpose can be administered to subjects or individuals susceptible to or at risk of developing a disease, such as cancer.
  • a disease such as cancer.
  • the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • a tumor regression can be assayed.
  • Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the therapy.
  • the method is useful to further confirm efficacy of the agent.
  • groups of nude mice (Balb/c NCR nu/nu female, Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 10 5 to about 10 9 hyperproliferative, cancer or target cells as defined herein.
  • the agent is administered, for example, by subcutaneous injection around the tumor. Tumor measurements to determine reduction of tumor size are made in two dimensions using venier calipers twice a week. Other animal models may also be employed as appropriate.
  • Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
  • agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions. More particularly, an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parental (including subcutaneous, intramuscular, intravenous and intrade ⁇ nal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.
  • a large number of precommitted APCs already circulating in the blood are isolated.
  • Previous techniques for isolating committed APCs from human peripheral blood have involved combinations of physical procedures such as metrizamide gradients and adherence/nonadherence steps (Freudenthal et al. (1990) PNAS USA 87:7698- 7702); Percoll gradient separations (Mehta-Damani et al. (1994) J. Immunol. 153:996-1003); and fluorescence activated cell sorting techniques (Thomas R. et al. (1993) J. Immunol. 151:6840-52).
  • the APC can be precommitted or mature dendritic cells which can be isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060).
  • the white blood cell fraction can be from the peripheral blood of the mammal. This method includes the following steps:
  • step (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step; (a) into four or more subfractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore; (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4°C.
  • One way to identify the dendritic cell-enriched fraction is by fluorescence-activated cell sorting.
  • the white blood cell fraction can be treated with calcium ionophore in the presence of other cytokines, such as rhIL-12, rhGM-CSF, or rhIL-4.
  • the cells of the white blood cell fraction can be washed in buffer and suspended in Ca ⁇ /Mg " ⁇ free media prior to the separating step.
  • the white blood cell fraction can be obtained by leukapheresis.
  • the dendritic cells can be identified by the presence of at least one of the following markers: HLA-DR; HLA-DQ; or B7.2, and the simultaneous absence of the following markers: CD3; CD 14; CD 16; 56;
  • the method requires collecting an enriched collection of white cells and platelets from leukapheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE) (Abrahams ⁇ n et al. (1991) J. Clin.
  • DCs Quality control of APC and more specifically DC collection and confirmation of their successful activation in culture is dependent upon a simultaneous multi-color FACS analysis technique which monitors both monocytes and the dendritic cell subpopulation as well as possible contaminant T lymphocytes. It is based upon the fact that DCs do not express the following markers: CD3 (T cell); CD14 (monocyte); CD16, 56, 57 (NK LAK cells); CD19, 20 (B cells). At the same time, DCs do express large quantities of HLA-DR, significant HLA-DQ and B7.2 (but little or no B7.1 ) at the time they are circulating in the blood (in addition they express Leu M7 and M9, myeloid markers which are also expressed by monocytes and neutrophils).
  • propridium iodide PI
  • Color #1 CD3 alone, CD 14 alone, etc.; Leu M7 or Leu M9; anti-Class I, etc.
  • Color #2 HLA-Dq, B7.1, B7.2, CD25 (IL2r), ICAM, LFA-3, etc.
  • the goal of FACS analysis at the time of collection is to confirm that the DCs are enriched in the expected fractions, to monitor neutrophil contamination, and to make sure that appropriate markers are expressed.
  • This rapid bulk collection of enriched DCs from human peripheral blood, suitable for clinical applications is absolutely dependent on the analytic FACS technique described above for quality control. If need be, mature DCs can be immediately separated from monocytes at this point by fluorescent sorting for "cocktail negative" cells. It may not be necessary to routinely separate DCs from monocytes because, as will be detailed below, the monocytes themselves are still capable of differentiating into DCs or functional DC-like cells in culture.
  • the DC rich/monocyte APC fractions (usually 150 through 190) can be pooled and cryopreserved for future use, or immediately placed in short term culture.
  • cytol ⁇ nes have been used successfully to amplify (or partially substitute) for the activation/conversion achieved with calcium ionophore: these cytokines include but are not limited to rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when given alone is inadequate for optimal upregulation.
  • the APCs and cells expressing one or more antigens are autologous. In another embodiment, the APCs and cells expressing the antigen are allogeneic, i.e., derived from a different subject.
  • Peptide fragments from antigens must first be bound to peptide binding receptors (major histocompatibility complex [MHC] class I and class II molecules) that display the antigenic peptides on the surface of the APCs.
  • MHC major histocompatibility complex
  • T lymphocytes produce an antigen receptor that they use to monitor the surface of APCs for the presence of foreign peptides.
  • the antigen receptors on T H cells recognize antigenic peptides bound to MHC class II molecules whereas the receptors on CTLs react with antigens displayed on class I molecules.
  • antigens can be delivered to antigen- presenting cells as protein peptide or in the form of polynucleotides encoding the
  • APCs genetically modified APCs. These include (1) the introduction into the APCs of RNA that express antigen or fragments thereof, (2) infection of APCs with recombinant vectors to induce endogenous expression of antigen, and (3) introduction of tumor antigen into the DC cytosol using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med. 184:465; Rouse et al. (1994) J. Virol. 68:5685; and Nair et al. (1992) J. Exp. Med. 175:609).
  • any method which allows for the introduction and expression of the heterologous or non-self antigen and presentation by the MHC on the surface of the APC is within the scope of this invention.
  • Antigen Pulsing is accomplished in vitro/ex vivo by exposing APCs to antigenic protein or peptide(s). The protein or peptide(s) are added to APCs at a concentration of 1-10 ⁇ m for approximately 3 hours. Paglia et al. (1996) J. Exp. Med. 183:317, has shown that APC incubated with whole protein in vitro were recognized by MHC class I-restricted CTLs, and that immunization of animals with these APCs led to the development of antigen-specific CTLs in vivo. Protein/peptide antigen can also be delivered to APC in vivo and presented by the APC.
  • Antigen is preferably delivered with adjuvant via the intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
  • This method of generating APCs bypasses the need for gene transfer into the APC and permits control of antigenic peptide densities at the cell surfaces.
  • Foster APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763). This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1, TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC class 1 -restricted CD8 + CTLs. In effect, only
  • empty MHC class I molecules are presented on the surface of these cells. Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif.
  • T2 cells are referred to herein as "foster" APCs. They can be used in conjunction with this invention to present the heterologous, altered or control antigen.
  • T2 cells with specific recombinant MHC alleles allows for redirection of the MHC restriction profile.
  • Libraries tailored to the recombinant allele will be preferentially presented by them because the anchor residues will prevent efficient binding to the endogenous allele.
  • High level expression of MHC molecules makes the APC more visible to the CTLs. Expressing the MHC allele of interest in T2 cells using a powerful MHC allele of interest in T2 cells using a powerful
  • 29 transcriptional promoter results in a more reactive APC (most likely due to a higher concentration of reactive MHC-peptide complexes on the cell surface).
  • Hybrid APCs. WO 98/58541 describes a method to fuse cells expressing an antigen with dendritic cells in a manner that the dendritic cells take up and present the antigens expressed by the antigen-expressing cells.
  • the DCs are fused with the cells in the presence of a fusing agent (e.g., polyethylene glycol or Sendai virus).
  • a fusing agent e.g., polyethylene glycol or Sendai virus.
  • the cultured fused cells are separated from unfused non-DC parental cells based on the different adherence properties of the two cell groups.
  • the unfused parental DCs do not proliferate, and so die off.
  • the present invention also provides delivery vehicles suitable for delivery of a polynucleotide of the invention into cells (whether in vivo, ex vivo, or in vitro).
  • a polynucleotide of the invention can be contained within a cloning or expression vector. These vectors (especially expression vectors) can in turn be manipulated to assume any of a number of forms which may, for example, facilitate delivery to and/or entry into a cell.
  • nucleic acid can be inserted into the host cell by methods well known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See Sambrook et al. (1989) supra for this methodology.
  • a pharmaceutically acceptable vector such as a replication-incompetent retroviral or adenoviral vector.
  • Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression of the inserted polynucleotide.
  • the term "pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells.
  • An example of such a vector is a "replication- incompetent" vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell.
  • genetic modifications of cells employed in the present invention are accomplished by introducing a vector containing a polynucleotide comprising sequences encoding a synthetic antigenic peptide of the invention.
  • a vector containing a polynucleotide comprising sequences encoding a synthetic antigenic peptide of the invention.
  • a variety of different gene transfer vectors, including viral as well as non- viral systems can be used.
  • a wide variety of non- viral vehicles for delivery of a polynucleotide of the invention are known in the art and are encompassed in the present invention.
  • a polynucleotide of the invention can be delivered to a cell as naked DNA.
  • a polynucleotide of the invention can be delivered to a cell associated in a variety of ways with a variety of substances (forms of delivery) including, but not limited to: cationic lipids; biocompatible polymers; including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria.
  • a delivery vehicle may take the form of a microparticle. Mixtures or conjugates of these various substances can also be used as delivery vehicles.
  • a polynucleotide of the invention can be associated with these various forms of delivery non-covalently or covalently.
  • Non-viral vectors i.e., cloning and expression vectors
  • Cloning vectors can be used to obtain replicate copies of the polynucleotides they contain, or as a means of storing the polynucleotides in a depository for future recovery.
  • Expression vectors can be used to obtain polypeptides produced from the polynucleotides they contain. They may also be used where it is desirable to express polypeptides, encoded by an operably linked polynucleotide, in an individual, such as for eliciting an immune response via the polypeptide(s) encoded in the expression vector(s).
  • Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and need not be described in detail herein. For example, see Gacesa and Ramji, Vectors, John Wiley & Sons (1994).
  • Cloning and expression vectors typically contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will survive and/or grow under selective conditions.
  • Typical selection genes encode protein(s) that: (a)
  • Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self- replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
  • Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE 1 , pCRl , RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
  • Bluescript e.g., pBS SK+
  • mpl8 mpl9 mpl9
  • pBR322 pMB9
  • ColE 1 e.g., pCRl
  • RP4 phage DNAs
  • shuttle vectors such as pSA3 and pAT28.
  • Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide encoding a polypeptide of interest.
  • the polynucleotide encoding the polypeptide of interest is operably linked to suitable transcriptional controlling elements, such as promoters, enhancers and terminators.
  • suitable transcriptional controlling elements such as promoters, enhancers and terminators.
  • one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
  • a polynucleotide sequence encoding a signal peptide can also be included to allow a polypeptide, encoded by an operably linked polynucleotide, to cross and/or lodge in cell membranes or be secreted from the cell.
  • a number of expression vectors suitable for expression in eukaryotic cells including yeast, avian, and mammalian cells are known in the art.
  • Examples of mammalian expression vectors contain both prokaryotic sequence to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • Examples of mammalian expression vectors contain both prokaryotic sequence to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Examples of mammalian expression vectors
  • eukaryotic cells include the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pRSVneo, and pHyg derived vectors.
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEB, pREP derived vectors) can be used for expression in mammalian cells.
  • expression vectors for yeast systems include YEP24, YIP5, YEP51, YEP52, YES2 and YRP17, which are cloning and expression vehicles useful for introduction of constructs into S.
  • Baculoviras expression vectors for expression in insect cells include pVL-derived vectors (such as pVL 1392, pVL1393 and pVL941), pAcUW-derived vectors and pBlueBac-derived vectors.
  • Viral vectors include, but are not limited to, DNA viral vectors such as those based on adenoviruses, herpes simplex virus, poxviruses such as vaccinia virus, and parvoviruses, including adeno-associated virus; and RNA viral vectors, including, but not limited to, the retroviral vectors.
  • Retroviral vectors include murine leukemia virus, and lentiviruses such as human immunodeficiency virus. Naldini et al. (1996) Science 272:263-267.
  • Replication-defective retroviral vectors harboring a polynucleotide of the invention as part of the retroviral genome can be used. Such vectors have been described in detail. (Miller et al. (1990) Mol Cell Biol. 10:4239; Kolberg, R.
  • Adenovirus and adeno-associated virus vectors useful in the genetic modifications of this invention may be produced according to methods already taught in the art. (See, e.g., Karlsson et al. (1986) EMBO J. 5:2377; Carter (1992) Current Opin. Biotech. 3:533-539; Muzcyzka (1992) Current Top. Microbiol.
  • the efficiency of transduction of DCs or other APCs can be assessed by immunofluorescence using fluorescent antibodies specific for the tumor antigen being expressed (Kim et al. (1997) J. Immunother. 20:276-286).
  • the antibodies can be conjugated to an enzyme (e.g. HRP) giving rise to a colored product upon reaction with the substrate.
  • HRP enzyme
  • the actual amount of antigenic polypeptides being expressed by the APCs can be evaluated by ELISA.
  • APCs can also be transduced in vitro/ex vivo by non-viral gene delivery methods such as electroporation, calcium phosphate precipitation or cationic lipid/plasmid DNA complexes. Arthur et al. (1997) Cancer Gene Therapy 4:17- 25. Transduced APCs can subsequently be administered to the host via an intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
  • DCs In vivo transduction of DCs, or other APCs, can potentially be accomplished by administration of cationic lipid/plasmid DNA complexes delivered via the intravenous, intramuscular, intranasal, intraperitoneal or cutaneous route of administration. Gene gun delivery or injection of naked plasmid DNA into the skin also leads to transduction of DCs. Condon et al.
  • transduction efficiency and levels of transgene expression can be assessed as described above for viral vectors.
  • the APCs prepared as described above are mixed with na ⁇ ve immune effector cells.
  • the cells may be cultured in the presence of a cytokine, for example IL2.
  • a cytokine for example IL2.
  • IL-12 potent immunostimulatory cytokines
  • the culture conditions are such that the antigen-specific immune effector cells expand (i.e. proliferate) at a much higher rate than the APCs.
  • Multiple infusions of APCs and optional cytokines can be performed to further expand the population of antigen- specific cells.
  • the immune effector cells are T cells.
  • the immune effector cells can be genetically modified by transduction with a transgene coding for example, IL-2, IL-11 or IL-13.
  • a transgene coding for example, IL-2, IL-11 or IL-13.
  • An effector cell population suitable for use in the methods of the present invention can be autogeneic or allogeneic, preferably autogeneic.
  • effector cells are allogeneic, preferably the cells are depleted of alloreactive cells before use. This can be accomplished by any known means, including, for example, by mixing the allogeneic effector cells and a recipient cell population and incubating them for a suitable time, then depleting CD69 + cells, or inactivating alloreactive cells, or inducing anergy in the alloreactive cell population.
  • Hybrid immune effector cells can also be used. Immune effector cell hybrids are known in the art and have been described in various publications. See, for example, International Patent Application Nos. WO 98/46785; and WO 95/16775.
  • the effector cell population can comprise unseparated cells, i.e., a mixed population, for example, a PBMC population, whole blood, and the like.
  • effector cell population can be manipulated by positive selection based on expression of cell surface markers, negative selection based on expression of cell surface markers, stimulation with one or more antigens in vitro or in vivo, treatment with one or more biological modifiers in vitro or in vivo, subtractive stimulation with one or more antigens or biological modifiers, or a combination of any or all of these.
  • Effector cells can obtained from a variety of sources, including but not limited to, PBMC, whole blood or fractions thereof containing mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, biopsy tissue, lymph nodes, e.g., lymph nodes draining from a tumor.
  • Suitable donors include an immunized donor, a non-immunized (na ⁇ ve) donor, treated or untreated donors.
  • a "treated” donor is one that has been exposed to one or more biological modifiers.
  • An "untreated” donor has not been exposed to one or more biological modifiers. Methods of extracting and culturing effector cells are well known.
  • effector cells can be obtained by leukapheresis, mechanical apheresis using a continuous flow cell separator.
  • lymphocytes and monocytes can be isolated from the buffy coat by any known method, including, but not limited to, separation over Ficoll-HypaqueTM gradient, separation over a Percoll gradient, or elutriation.
  • the concentration of Ficoll-HypaqueTM can be adjusted to obtain the desired population, for example, a population enriched in T cells.
  • Other methods based on affinity are known and can be used. These include, for example, fluorescence-activated cell sorting (FACS), cell adhesion, magnetic bead separation, and the like.
  • FACS fluorescence-activated cell sorting
  • Affinity-based methods may utilize antibodies, or portions thereof, which are specific for cell-surface markers and which are available from a variety of commercial sources, including, the American Type Culture Collection (Manassas, VA). Affinity-based methods can alternatively utilize ligands or ligand analogs, of cell surface receptors.
  • the effector cell population can be subjected to one or more separation protocols based on the expression of cell surface markers.
  • the cells can be subjected to positive selection on the basis of expression of one or more
  • cell surface polypeptides including, but not limited to, "cluster of differentiation” cell surface markers such as CD2, CD3, CD4, CD8, TCR, CD45, CD45RO, CD45RA, CDl lb, CD26, CD27, CD28, CD29, CD30, CD31, CD40L; other markers associated with lymphocyte activation, such as the lymphocyte activation gene 3 product (LAG3), signaling lymphocyte activation molecule (SLAM),
  • LAG3 lymphocyte activation gene 3 product
  • SLAM signaling lymphocyte activation molecule
  • T1/ST2 chemokine receptors such as CCR3, CCR4, CXCR3, CCR5; homing receptors such as CD62L, CD44, CLA, CD 146, ⁇ 4 ⁇ 7, ⁇ E ⁇ 7; activation markers such as CD25, CD69 and OX40; and lipoglycans presented by CDl.
  • the effector cell population can be subjected to negative selection for depletion of non-T cells and/or particular T cell subsets. Negative selection can be performed on the basis of cell surface expression of a variety of molecules, including, but not limited to, B cell markers such as CD19, and CD20; monocyte marker CD14; the NK cell marker CD56.
  • An effector cell population can be manipulated by exposure, in vivo or in vitro, to one or more biological modifiers.
  • suitable biological modifiers include, but are not limited to, cytokines such as IL-2, IL-4, IL-10, TNF- ⁇ , IL-12, IFN- ⁇ ; non-specific modifiers such as phytohemagglutinin (PHA), phorbol esters such as phorbol myristate acetate (PAM), concanavalin-A, and ionomycin; antibodies specific for cell surface markers, such as anti-CD2, anti-CD3, anti-IL2 receptor, anti-CD28; chemokines, including, for example, lymphotactin.
  • cytokines such as IL-2, IL-4, IL-10, TNF- ⁇ , IL-12, IFN- ⁇
  • non-specific modifiers such as phytohemagglutinin (PHA), phorbol esters such as phorbol myristate acetate (PAM), concanavalin-A
  • the biological modifiers can be native factors obtained from natural sources, factors produced by recombinant DNA technology, chemically synthesized polypeptides or other molecules, or any derivative having the functional activity of the native factor. If more than one biological modifier is used, the exposure can be simultaneous or sequential.
  • the present inventors have shown that the APCs, preferably DCs, isolated as described above can be frozen and thawed and still retain both viability and functionality.
  • the isolated cells prepared as described above and in the Examples provided below are gently pelleted and resuspended in suitable freezing media.
  • the media has at least about 30%, more preferably 50%, more preferably 75% and most preferably 85% human-derived serum and/or plasma in combination with various amounts of DMSO, e.g., at least 10% but more preferably, at least 12% DMSO.
  • DMSO e.g., at least 10% but more preferably, at least 12% DMSO.
  • the most prefered combination is at least approximately 90% serum and 10% DMSO.
  • the serum and/or plasma must be derived from human sources to eliminate the possibility of presentation of antigen from non-humans. For example, the freezing of DCs in fetal calf serum will result in the DCs presenting non-human antigen on the surface of the cells. All percentages recited herein are (v/v).
  • the cells are suspended in the freezing media at a concentration between about 1 x 10 2 cells/mL to about 1 x 10 10 cells/mL, preferably between about 1 x 10 4 cells/mL and about 1 x 10 8 cells/mL and even more preferably between about 1 x 10 6 to 1 xlO 7 cells/mL.
  • the cells may then be aliquoted into freezing vials and frozen by slowly bringing down their temperature according to standard protocols.
  • the cells are stored at -80°C for approximately one day before being moved to liquid nitrogen storage.
  • the cells stored in liquid nitrogen may be stored indefinitely.
  • the cells are preferably thawed quickly, for example in a 37°C water bath. They are diluted in complete medium and pelleted by gentle centrifugation to remove the freezing media. The washed cells are resuspended in complete medium and plated into tissue culture dishes. Some of the reagents and their sources used in the media are shown in Table 2.
  • the present inventors have shown that the dendritic cells frozen as described above are viable and, in addition, retain their ability to function as antigen presenting cells. Viability can be tested by trypan blue staining.
  • fluorescence activated cell sorting (FACS) analysis for human cell surface markers may also be conducted as is know in the art and discussed below in the Examples. Non-limiting examples of markers and their sources are shown in Table 3.
  • Example 1 Freeze/Thawed DCs Pulsed with Epitope Retain Function Generation of Human Denditic Cells
  • Leukokpac (175 mLs) was diluted 1 :1 with 1 x PBS (Mg2+ and Ca2+ free). A volume of 30 mLs diluted blood was slowly layered onto 20 mLs of Ficoll-Paque (Pharmacia Biotech) in 50 mL tubes, taking care not mix the ficol with the blood. The 50 mL tubes were centrifuged at 1,400 rpm at room temperature for 40 minutes in a swinging bucket rotor with no brake. The cells from the interface were carefully aspirated. Approximately 10 mLs of cells was collected per tube.
  • the cells were washed three times with a large volume (350 mLs divided into 50 mL tubes) of PBS. The first spin was at 1,400 rpm for 10 minutes, the second and third at 1,200 rpm for 10 minutes. The washed cells were resuspending in RPMI supplemented with P/S, glutamine, 5% human AB serum (Sigma) or 10% fetal bovine serum, 10 mM HEPES and the cells counted. Approximately 1-2 x 10 9 cells were counted.
  • the cells were plated a concentration of 2-3 x 108 in T-150 flasks and incubated for at least 1 hour to allow monocytes to adhere to the flask.
  • the media were plated a concentration of 2-3 x 108 in T-150 flasks and incubated for at least 1 hour to allow monocytes to adhere to the flask.
  • the cells were resuspended in 25 mLs of complete RPMI (including 100 ng/mL GM-CSF and 20 ng/mL IL-4) in T-150 flasks. At around day 3 in culture, approximately 5 mL of fresh media including concentrated cytokines was added to the flasks.
  • DCs were obtained at approximately day 4 in culture and the maximum amount obtained by day 6-7 of culture with cytokines. The cells were collected for further studies.
  • Dendritic cells generated by using the protocol described in Example 1 were analyzed by FACS for the expression of the following markers: MHC class I and class II, B7J, B7.2, CD83, CDl lc, CD3, CD4 and CD14.
  • the cells were harvested 6 days after culture with GM-CSF and IL-4, washed once with PBC and counted. Approximately 2 x 10 5 cells were used to reaction. These cells were resuspended in 200 ⁇ l FACS buffer with 10% human AB serum and incubated 15 minutes on ice to block non-specific binding. The reactions were centrifuged at 2,500 rpm for 4 minutes in a variable speed microfuge.
  • the blocking buffer was aspirated and the cells were resuspended in 100 ⁇ l of FACS buffer. Five microliters of undiluted, FITC or PE-conjugated primary antibody were added and the reactions incubated on ice for 30 minutes. The reactions were then washed two times with 1 mL of FACS buffer and resuspended in 100 ⁇ l of FACS buffer containing 100 ⁇ l 2% paraformaldehyde. The samples were stored in the dark at 4°C until loading on the flow cytometer.
  • Mature dendritic cells and precursors were isolated as described in Example 1. The cells were frozen for 10 days in liquid nitrogen in various media. The cells were then thawed quickly in a 37°C water bath and diluted in complete media. The freezing media was removed by gentle centrifugation. Viability was determined by the percentage of cells surviving the freezing process, as measured by trypan blue staining.
  • Results demonstrate that DCs frozen in 90% serum and 10% DMSO retain greater than 90% viability.
  • the effects of different freezing media was also tested.
  • the cells were frozen as described above. After 30 days, the cells were thawed and checked for viability using trypan blue staining. Of all the conditions analyzed, cryopreservation of cells in 10% DMSO and 90% serum was much superior than previously established DC freezing medium of 15.4% (2M) DMSO and 20% serum or 12% DMSO and 30% serum. Nearly 100% viability was obtained using freezing media containing less DMSO. Decreasing the serum concentration from
  • Frozen and thawed DCs were also tested for their ability to present antigens. Following 51 Cr-labeling (100 ⁇ Ci/mL for 24 hours), the cells were peptide-pulsed (1 ⁇ M) DCs with 1 ⁇ M of various tumor antigens (gp-209, gplOO- f9, gp 100-k9). The cells were washed of excess peptide and incubated with gplOO-specific, HL A- A2 -restricted cytolytic effector tumor infiltrating lymphocyte (TIL) line, Hurley R1000 for 24 hours. Effecto ⁇ target ratios are indicated and 1000 to 5000 targets were used for the assay.
  • TIL tumor infiltrating lymphocyte
  • DCs prepared from a normal HLA-A2 + donor were infected with Ad2/hugpl00v2 and frozen in 90% human AB serum/10% DMSO. Aliquots of uninfected and infected DCs were thawed at 3, 6, 9, 12 and 15 weeks and assayed for their ability to be lysed by a gplOO-specific CTL clone. For each time point, uninfected and infected DCs were thawed and assayed for lysis by a gplOO- specific CTL clone +/- peptide-pulsing with GP100-F9 peptide. By comparing
  • TIL 1520 obtained from M. Nishimura, Surgery Branch, NCI. These were originally derived from a human melanoma biopsied from a patient that had been vaccinated with the G9-209 peptide epitope (its cognate antigen). This line is clonal and HLA-A2 restricted. The CTL were maintained in culture in AIM-V medium (GibcoBRL, #12055-
  • non-adherent cells were removed by washing 3x with unsupplemented RPMI.
  • the cells were then fed with complete RPMI plus lOOng/ml GM-CSF (Genzyme, #RH-CSF-C) and 20 ng/ml IL-4 (Genzyme, #2181-01) in a volume of 20 mls/T150 flask.
  • lOOng/ml GM-CSF Genzyme, #RH-CSF-C
  • 20 ng/ml IL-4 Genzyme, #2181-01
  • 3x10 DCs were infected as a bulk culture in serum-free OptiMEM medium (GibcoBRL, #31985- 070).
  • Reagent grade gradient-purified Ad2/hugpl00v2 (Genzyme) was added to the culture at an MOI of 500.
  • the culture was allowed to incubate overnight at 37°C/5%CO 2 , and then the medium was replaced with RPMI supplemented with
  • DCs were harvested from flasks or plates and pelleted by centrifugation.
  • the cells were resuspended in 90% human AB serum/10% DMSO and aliquoted to 1.5 ml Nunc cryotubes (1 ml/tube) at a cell density of 5 x 10 cells/ml.
  • the tubes were stored at -80°C for 24 hours and transferred to liquid nitrogen for extended frozen storage.
  • DCs were thawed after being stored frozen for 3, 6, 9, or 15 weeks by warming the tubes in a 37°C water bath until there was only a small ice crystal left and immediately transferred to 10 mis PBS in a 15 ml conical centrifuge tube.
  • the cells were pelleted and washed 2x with 10 mis PBS before use.
  • Peptide-pulsing of the both uninfected and infected DCs was achieved by aliquoting 5000 51 Cr-labeled cells in a volume of 1 OO ⁇ l to the appropriate wells of the 96-well V-bottom CTL assay plate (see CTL Assay, below). These wells were supplemented with 2 ⁇ l of GP100-F9 peptide (diluted in dH 2 0 from a 10 mg/ml stock in 100% DMSO) for a final concentration of lO ⁇ M. The plate was incubated at 37°C/5%CO 2 for 1 hour before the addition of the effector cells.
  • Effector cells were added to each well in AIM-N medium 10% human AB serum at the indicated E:T ratios in a volume of lOO ⁇ l for a total reaction volume of 200 ⁇ l. Medium (no effector cells) was added to the spontaneous release wells, and no additional media was added to total release
  • Dendritic cells generated by culturing monocytes in GM-CSF and IL-4 were frozen and stored either uninfected or after infecting them with adenovirus vectors encoding gplOO (Ad2CMVgpl00). Cells were thawed at different time points (stored at least up to 15 weeks, Table 5) and analyzed for the expression of cell surface markers by FACS analysis. The results (see Figures 7 through 10) indicate that long term storage of infected or uninfected DCs has no effect on the expression of cell surface markers. These results compliment the data described above that the methods and compositions of this invention for the frozen storage of DCs have no effect on antigen presentation as measured by CTL assay. It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the invention. For example, any of the above-noted compositions and/or methods can be combined

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Abstract

La présente invention concerne des compositions et des méthodes permettant de congeler et de décongeler des cellules dendritiques fonctionnelles viables. Les méthodes de cette invention consistent à mettre ces cellules en suspension dans un milieu constitué à 30 % (v/v) d'un sérum et/ou d'un plasma dérivés à partir de sang humain, puis à baisser la température de cette suspension jusqu'à -80 °C. La suspension ainsi congelée est finalement décongelée dans une gamme de températures variant entre 34 et 41 °C, idéalement à une température de 37 °C.
PCT/US1999/006033 1998-03-20 1999-03-19 Compositions et methodes pour entreposer des cellules dendritiques congelees WO1999046984A1 (fr)

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WO2002016560A1 (fr) * 2000-08-24 2002-02-28 Gerold Schuler Procede de fabrication de cellules dendritiques matures, cryoconservees, chargees d'antigenes ou non, pretes a l'emploi

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001039594A2 (fr) * 1999-12-03 2001-06-07 Dendreon Corporation Cryoconservation of de cellules dendritiques chargees d'antigenes et leurs precurseurs dans des milieux sans serum
WO2001039594A3 (fr) * 1999-12-03 2002-07-11 Dendreon Corp Cryoconservation of de cellules dendritiques chargees d'antigenes et leurs precurseurs dans des milieux sans serum
WO2002016560A1 (fr) * 2000-08-24 2002-02-28 Gerold Schuler Procede de fabrication de cellules dendritiques matures, cryoconservees, chargees d'antigenes ou non, pretes a l'emploi
DE10041515A1 (de) * 2000-08-24 2002-03-14 Gerold Schuler Verfahren zur Herstellung gebrauchsfertiger, Antigen-beladener oder -unbeladener, kryokonservierter reifer dendritischer Zellen
KR100849740B1 (ko) * 2000-08-24 2008-08-01 제롤드 슐러 항원이 로딩된 즉시 사용가능한 동결저장된 성숙한수상돌기 세포 또는 항원이 로딩되지 않은 즉시사용가능한 동결저장된 성숙한 수상돌기 세포의 제조 방법
US8236562B2 (en) 2000-08-24 2012-08-07 Argos Therapeutics, Inc. Method for producing ready to use, antigen-loaded or unloaded, cryoconserved mature dendritic cells
US8574901B2 (en) 2000-08-24 2013-11-05 Argos Therapeutics, Inc. Cryoconserved mature dendritic cells

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