WO1999046984A1

WO1999046984A1 - Compositions and methods for the frozen storage of dendritic cells

Info

Publication number: WO1999046984A1
Application number: PCT/US1999/006033
Authority: WO
Inventors: Srinivas Shankara; Charles A. Nicolette
Original assignee: Genzyme Corporation
Priority date: 1998-03-20
Filing date: 1999-03-19
Publication date: 1999-09-23
Also published as: AU3102599A

Abstract

The present invention provides compositions and methods relating to freezing and thawing viable and functional dendritic cells are provided. The method requires suspending the cells in at least 30 % (v/v) human derived-serum and/or plasma and lowering the temperature of the suspension to at least about -80 °C. The frozen suspension is thawed in a temperature range of about 34 to 41 °C, more preferably at 37 °C.

Description

COMPOSITIONS AND METHODS FOR THE FROZEN STORAGE OF

DENDRITIC CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS Tliis application claims priority under 35 U.S.C. § 119 (e) to U.S.

Provisional Application No. 60/078,929, filed March 20, 1998, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD This invention is in the field of molecular immunology and medicine. In particular, compositions and methods relating to freezing and thawing viable and functional dendritic cells are provided.

BACKGROUND In spite of numerous advances in medical research, cancer remains the second leading cause of death in the United States. In the industrialized nations, roughly one in five persons will die of cancer. Traditional modes of clinical care, such as surgical resection, radiotherapy and chemotherapy, have a significant failure rate, especially for solid tumors. Failure occurs either because the initial tumor is unresponsive, or because of recurrence due to regrowth at the original site and/or metastases.

Cellular immunotherapy is emerging as a technologically and intellectually compelling anti-cancer treatment. The generation of an immune response against tumors has been demonstrated in several animal models and has been inferred from reports of spontaneous tumor regression in man (Stotter and

Lotze (1990) Cancer Cells 2:44-55). 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. In animal models, established tumors can be eradicated by the adoptive transfer of T-cells that specifically lyse malignant cells (Beun et al.

(1994) Immunol. Today 15:11-15). 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. See Colaco, (January, 1999) Mol. Med. Today 14-17 and Hart (1997) Blood 90:3245-3287 for review of dendritic cells and their use in immunotherapy. 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. Gong J. et al. (1997) Nat. Med. 3:558-561. 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. To date, however, long-term storage of APCs, especially dendritic cells, has proven difficult. Typical protocols call for between 20-30% seinm and about 10% dimethylsulfoxide (DMSO). (See, e.g., U.S. Patent No. 5,788,963; Makino et al. (1997) Scan. J. Immunol. 45(6):618-622; and Brown et al. (1992) Biotherapy 5(4):301-308). Viability and functionality of the DCs was greatly reduced by the process. Thus, a need exists for a method to freeze and thaw DCs that does not hamper viability and functionality of the cells. This invention satisfies this need and provides related advantages as well.

DISCLOSURE OF THE INVENTION The present invention provides compositions and methods for freezing, storing and thawing dendritic cells. In contrast to prior art methods, 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. In a preferred embodiment, 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. In a further aspect, the suspension is maintained at -80°C for at least 24 hours and then transferred to liquid nitrogen for the duration of the storage. In a further embodiment, 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. In another aspect, the present invention provides a method for ex vivo generation of antigen-specific immune effector cells, comprising educating naϊve 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). 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 naϊve immune effector cells by co- culture with the DCs, identified above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 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. In this graph: -•- shows cells incubated with G9-209; -■- shows cells incubated with GP100-F9; and -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. In 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. In this graph: -•- shows cells incubated with G9-209; -■- shows cells incubated with GP100-F9; and -A- shows cells incubated with GP 100-K9.

Figure 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

FACS.

MODES FOR CARRYING OUT THE INVENTION Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Definitions

As used in the specification and claims, the singular form "a" "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The term "antigen presenting cell" as used herein, 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. Examples of suitable APCs are discussed in detail below and 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.

Dendritic cells (DCs) are potent antigen-presenting cells. DCs have been shown to provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types. The first type, which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 ("TCR/CD3") complex and an antigenic peptide presented by a major histocompatibility complex ("MHC") class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T cell activation to occur. In fact, without the second type of signals, the first type of signals can result in T cell anergy. The second type of signals, called co-stimulatory signals, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals. As used herein, 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. For instance, 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). Methods for purifying and/or generating dendritic cells from peripheral blood or bone marrow progenitors are well known in the art. See, for example, methods disclosed in Inaba et al. (1992) J. Exp. Med. 175:1157; Inaba et al. (1992) J. Exp. Med. 176:1693-1702; Romani et al. (1994) J. Exp. Med. 180:83- 93; and Sallusto et al. (1994) J. Exp. Med. 179:1109-1118. "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).

As used herein, "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.

By "substantial 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.

As used herein, the term "cytokine" refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation. Non-limiting examples of cytokines which may be used alone or in combination in the practice of the present invention include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12

(IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin- 1 alpha (IL-lα), interleukin- 11 (IL-11), MlP-lα, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and 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., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.

"Co-stimulatory molecules" are involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. Research accumulated over the past several years has demonstrated convincingly that resting T cells require at least two signals for induction of cytokine gene expression and proliferation (Schwartz R.H. (1990) Science

248:1349-1356; Jenkins M.K. (1992) Immunol. Today 13:69-73). One signal, the one that confers specificity, can be produced by interaction of the TCR/CD3 complex with an appropriate MHC/peptide complex. The second signal is not antigen specific and is termed the "co-stimulatory" signal. This signal was originally defined as an activity provided by bone-marrow-derived accessory cells such as macrophages and dendritic cells, the so called "professional" APCs. Several molecules have been shown to enhance co-stimulatory activity. These are heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M.F., et al. (1993) Cell 74:257-268), intracellular adhesion molecule 1 (ICAM-1) (Van

Seventer, G.A. (1990) J. Immunol. 144:4579-4586), B7-1, and B7-2/B70 (Schwartz R.H. (1992) Cell 71:1065-1068). These molecules each appear to assist co-stimulation by interacting with their cognate ligands on the T cells. Co- stimulatory molecules mediate co-stimulatory signal(s) which are necessary, under normal physiological conditions, to achieve full activation of naϊve T cells.

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. The term "co-stimulatory molecule" encompasses any single molecule or combination of molecules which, when

8 acting together with a peptide/MHC complex bound by a TCR on the surface of a T cell, provides a co-stimulatory effect which achieves activation of the T cell that binds the peptide. The term thus encompasses B7, or other co-stimulatory molecule(s) on an antigen-presenting matrix such as an APC, fragments thereof (alone, complexed with another molecule(s), or as part of a fusion protein) which, together with peptide/MHC complex, binds to a cognate ligand and results in activation of the T cell when the TCR on the surface of the T cell specifically binds the peptide. Co-stimulatory molecules are commercially available from a variety of sources, including, for example, Beckman Coulter. It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified co-stimulatory molecules (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.

As used herein, "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.

The term "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. The term "peptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc. As used herein the term "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. The term "immune effector cells" refers to cells capable of binding an antigen or which mediate an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates. Certain diseased tissue expresses specific antigens and CTLs specific for these antigens have been identified. For example, approximately 80% of melanomas express the antigen known as gplOO.

A "naϊve" immune effector cell is an immune effector cell that has never been exposed to an antigen.

As used herein, the term "educated, antigen-specific immune effector cell", is an immune effector cell as defined above, which has encountered antigen and which is specific for that antigen. An educated, antigen-specific immune

10 effector cell may be activated upon binding antigen. "Activated" implies that the cell is no longer in G₀ phase, and begins to produce cytokines characteristic of the cell type. For example, activated CD4⁺ T cells secrete IL-2 and have a higher number of high affinity IL-2 receptors on their cell surfaces relative to resting CD4⁺ T cells.

A peptide or polypeptide of the invention may be preferentially recognized by antigen-specific immune effector cells, such as B cells and T cells. In the context of 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. The term "preferentially recognized" intends that a 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.

The term "autogeneic" or "autologous" as used herein, indicates the origin of a cell. Thus, a cell being administered to an individual (the "recipient") is autogeneic if the cell was derived from that individual (the "donor") or a genetically identical individual. An autogeneic cell can also be a progeny of an autogeneic cell. The term also indicates that cells of different cell types are derived from the same donor or genetically identical donors. Thus, 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.

Similarly, the term "allogeneic" as used herein, indicates the origin of a cell. Thus, a cell being administered to individual (the "recipient") is allogeneic if the cell was derived from an individual not genetically identical to the recipient; in particular, the term relates to non-identity in expressed MHC molecules. An

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. For example, an APC is said to be allogeneic to an effector cell if they are derived from genetically non-identical donors.

The terms "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. The term "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. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, 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 ). Similarly, 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

12 obtained commercially or assembled by the sequences described in methods well .known in the art, for example, the methods described below for constructing vectors in general.

"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. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria, viruses, such as baculovirus, adenovirus, 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. 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. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors and the like. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. As used herein, "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.

13 As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

The terms "major histocompatibility complex" or "MHC" refers to a complex of genes encoding cell-surface molecules that are required for antigen presentation to T cells and for rapid graft rejection. In humans, the MHC complex is also known as the HLA complex. The proteins encoded by the MHC complex are .known as "MHC molecules" and are classified into class I and class II MHC molecules. Class I MHC molecules include membrane heterodimeric proteins made up of an α chain encoded in the MHC associated noncovalently with β2- 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 α and β chains. Class II MHC are known to participate in antigen presentation to

CD4⁺ T cells and, in humans, include HLA-DP, -DQ, and DR. Class II molecules generally bind peptides 12-20 amino acid residues in length. The term "MHC restriction" refers to a characteristic of T cells that permits them to recognize antigen only after it is processed and the resulting antigenic peptides are displayed in association with either a 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. An immune response to an antigen

14 (or epitope), 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. For example, 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.

The term "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". For example, where 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

15 polypeptides). It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may be procaryotic or eucaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.

An "antibody" is an immunoglobulin molecule capable of binding an antigen. As used herein, 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. The term "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. For example, with respect to a polynucleotide, an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, 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

16 characteristic such as glycosylation pattern. Although not explicitly stated for each of the inventions disclosed herein, it is to be understood that all of the above embodiments for each of the compositions disclosed below and under the appropriate conditions, are provided by this invention. Thus, 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%.

A "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. As used herein, 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)). \n "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. In the context of a disease state, 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

17 sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. 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. "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 is provided by this invention. The method 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. In one aspect, 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.

The cell suspension should be maintained at least this temperature during storage. In one aspect, the suspension is transferred to liquid nitrogen for storage or initially frozen under liquid nitrogen. In a further aspect, the cells are thawed at temperatures of about 35 to 41°C, and more preferably at 37°C. Preferably, the agent (e.g., DMSO) is removed from the thawing cells as soon as possible.

18 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.

Applicants have shown that modified DCs, frozen and stored by the methods of this invention retain the functionality upon thawing. Thus, the DCs can be modified to present endogenous self-antigen or exogenous antigen prior to freezing. For example, 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. Non-limiting examples of 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. As used herein, "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

19 oligonucleotides or genes useful for antisense suppression and ribozymes for ribozyme-mediated therapy. For example, in the present invention, 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. Generally, 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. In one embodiment, the therapeutic gene is a dominant inhibiting mutant of the wild-type immunosuppressive agent. Alternatively, 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. Depending on the genetic defect, the therapeutic gene can include the regulatory and untranslated sequences. For gene therapy in human patients, 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. Various

20 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 naϊve 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. In one aspect, the population is combined with a pharmaceutically acceptable carrier prior to use. Methods of educating naϊve 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.

The agents identified herein as effective for their intended purpose can be administered to subjects or individuals susceptible to or at risk of developing a disease, such as cancer. When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. To

21 determine patients that can be beneficially treated, a tumor regression can be assayed. Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the therapy. When delivered to an animal, the method is useful to further confirm efficacy of the agent. As an example of an animal model, groups of nude mice (Balb/c NCR nu/nu female, Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 10⁵ to about 10⁹ hyperproliferative, cancer or target cells as defined herein. When the tumor is established, 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.

The 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 intradeπnal) 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.

22 General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al. (1989)); "Oligonucleotide Synthesis" (M.J. Gait, ed. (1984)); ".Animal Cell Culture" (R.I. Freshney, ed. (1987)); the series "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Weir &

C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds. (1987)); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds. (1987)), and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds. (1994)); "Current Protocols in Immunology" (J.E. Coligan et al., eds. (1991)).

The following are brief descriptions of several techniques useful to carry out the methods of this invention.

Isolation and Expansion of Antigen Presenting Cells, Including Dendritic Cells Hart (1997), supra, describes various methods to isolate and characterize mature DCs and DC precursors. Bender et al. (1996) J. Immunol. Methods 196:121-135 describes a method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure requires 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or "priming" phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or "differentiation" phase requires the exposure to macrophage conditioned medium.

Romani et al. (1996) J. Immunol. Methods 196:137-151, describes a method to generate human dendritic cells from hematopoietic precursor cells in peripheral blood. A 3 day maturation culture is added to the initial 6-7 day culture

23 in the presence of GM-CSF and IL-4. Human plasma, rather than fetal calf serum and media approved for clinical use are optimal, additional conditions for use in this method.

The following describe additional methods. In one aspect, 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:

(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;

24 57; and CD 19 and 20. Monoclonal antibodies specific to these cell surface markers are commercially available.

More specifically, the method requires collecting an enriched collection of white cells and platelets from leukapheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE) (Abrahamsεn et al. (1991) J. Clin.

Apheresis. 6:48-53). Cell samples are placed in a special elutriation rotor. The rotor is then spun at a constant speed of, for example, 3000 rpm. Once the rotor has reached the desired speed, pressurized air is used to control the flow rate of cells. Cells in the elutriator are subjected to simultaneous centrifugation and a washout stream of buffer that is constantly increasing in flow rate. This results in fractional cell separations based largely but not exclusively on differences in cell size.

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).

When combined with a third color reagent for analysis of dead cells, propridium iodide (PI), it is possible to make positive identification of all cell

25 subpopulations (see Table 1):

TABLE 1

FACS analysis of fresh peripheral cell subpopulations

Color #1 Color #2 Color #3

Cocktail HLA-DR PI

3/14/16/19/20/56/57

Live Dendritic cells Negative Positive Negative

Live Monocytes Positive Positive Negative

Live Neutrophils Negative Negative Negative

Dead Cells Variable Variable Positive

Additional markers can be substituted for additional analysis:

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.

Once collected, 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.

Alternatively, others have reported that a method for upregulating (activating) dendritic cells and converting monocytes to an activated dendritic cell phenotype. This method involves the addition of calcium ionophore to the culture

26 media convert monocytes into activated dendritic cells. Adding the calcium ionophore A23187, for example, at the beginning of a 24-48 hour culture period resulted in uniform activation and dendritic cell phenotypic conversion of the pooled "monocyte plus DC" fractions: characteristically, the activated population becomes uniformly CD 14 (Leu M3) negative, and upregulates HLA-DR, HLA-

DQ, ICAM-1, B7J, and B7.2. Furthermore this activated bulk population functions as well on a small numbers basis as a further purified.

Specific combination(s) of cytolάnes 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.

In one embodiment, 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.

Presentation of Antigen to the APC

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. Palmer

E. and Cresswell (1998) Annu. Rev. Immunol. 16:323 and Germain R.N. (1996) Immunol. Rev. 151:5. T lymphocytes produce an antigen receptor that they use to monitor the surface of APCs for the presence of foreign peptides. The antigen receptors on 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. For a general review of the methods for presentation of exogenous antigen by APC, see Raychaudhuri and Rock (1998) Nature Biotechnology 16:1025.

For purposes of immunization, antigens can be delivered to antigen- presenting cells as protein peptide or in the form of polynucleotides encoding the

27 protein/peptide ex vivo or in vivo. The methods described below focus primarily on DCs which are the most potent, preferred APCs.

Several different techniques have been described to produce 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). For the purpose of this invention, 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. 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 μm for approximately 3 hours. Paglia et al. (1996) J. Exp. Med. 183:317, has shown that APC incubated with whole protein in vitro were recognized by MHC class I-restricted CTLs, and that immunization of animals with these APCs led to the development of antigen-specific CTLs in vivo. Protein/peptide antigen can also be delivered to APC in vivo and presented by the APC. Antigen is preferably delivered with adjuvant via the intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery. Grant E.P. and Rock K.L. (1992) J. Immunol. 148:13; Norbury, C. C. et al. (1995) Immunity 3:783; and Reise-Sousa C. and Germain R.N. (1995) J. Exp. Med. 182:841.

Antigen Painting. Another method which can be used is termed "painting". It has been demonstrated that glycosyl-phosphotidylinositol (GPI)-modified proteins possess the ability to reincorporate themselves back into cell membranes after purification. Hirose et al. (1995) Methods Enzymol. 250:582; Medof et al. (1984)

J. Exp. Med. 160:1558; Medof (1996) FASEB J. 10:574; and Huang et al. (1994)

28 Immunity 1:607, have exploited this property in order to create APCs of specific composition for the presentation of antigen to CTLs. Expression vectors for β2- microglobulin and the HLA-A2J allele were first devised. The proteins were expressed in Schneider S2 Drosophila melanogaster cells, .known to support GPI- modification. After purification, the proteins could be incubated together with a purified antigenic peptide which resulted in a trimolecular complex capable of efficiently inserting itself into the membranes of autologous cells. In essence, these protein mixtures were used to "paint" the APC surface, conferring the ability to stimulate a CTL clone that was specific for the antigenic peptide. Cell coating was shown to occur rapidly and to be protein concentration dependent.

This method of generating APCs bypasses the need for gene transfer into the APC and permits control of antigenic peptide densities at the cell surfaces.

Foster Antigen Presenting Cells. Foster APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763). This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1, TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC class 1 -restricted CD8⁺ CTLs. In effect, only

"empty" MHC class I molecules are presented on the surface of these cells. Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif. These T2 cells are referred to herein as "foster" APCs. They can be used in conjunction with this invention to present the heterologous, altered or control antigen.

Transduction of 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

29 transcriptional promoter (e.g., the CMV promoter) results in a more reactive APC (most likely due to a higher concentration of reactive MHC-peptide complexes on the cell surface).

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). After culturing the post fusion cell mixture in a medium (which optionally contains hypoxanthine, aminopterin and thymidine) for a period of time (e.g., 5-

12 days), 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.

Genetic Modification Methods

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.

Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, etc. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo. When a nucleic acid is inserted into a suitable host cell, e.g., a procaryotic or a eucaryotic cell and the host cell replicates, the protein can be recombinantly produced. Suitable host

30 cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using well known methods. See Sambrook, et al. (1989) supra. In addition to the use of viral vector for insertion of exogenous nucleic acid into cells, the nucleic acid can be inserted into the host cell by methods well known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See Sambrook et al. (1989) supra for this methodology. When the vectors are used for gene therapy in vivo or ex vivo, a pharmaceutically acceptable vector is preferred, 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. As used herein, 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. An example of a replication- incompetent retroviral vector is LNL6. Miller et al. (1989) BioTechniques 7:980- 990. The methodology of using replication-incompetent retroviruses for retroviral-mediated gene transfer of gene markers is well established. Correll et al. (1989) PNAS USA 86:8912; Bordignon (1989) PNAS USA 86:8912-52; Culver (1991) PNAS USA 88:3155; and Rill (1991) Blood 79(10):2694-700.

In general, 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 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. WO

31 97/40163. Alternatively, 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. Included in the non- viral vector category are prokaryotic plasmids and eukaryotic plasmids. Non-viral vectors (i.e., cloning and expression vectors) having cloned therein a polynucleotide(s) of the invention can be used for expression of recombinant polypeptides as well as a source of polynucleotide of the invention. Cloning vectors can be used to obtain replicate copies of the polynucleotides they contain, or as a means of storing the polynucleotides in a depository for future recovery. Expression vectors (and host cells containing these 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)

32 confer resistance to antibiotics or other toxins substances, e.g., ampicillin, neomycyin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art. Cloning and expression vectors also typically contain a replication system recognized by the host.

Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self- replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE 1 , pCRl , RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

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. For expression (i.e., translation), 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

33 suitable for transfection of eukaryotic cells include the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pRSVneo, and pHyg derived vectors. Alternatively, 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. Examples of expression vectors for yeast systems, include YEP24, YIP5, YEP51, YEP52, YES2 and YRP17, which are cloning and expression vehicles useful for introduction of constructs into S. cerevisiae. Broach et al. (1983) Experimental Manipulation of Gene Expression, ed. M. Inouye, Academic Press, p. 83. 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.

(1992) J. NIHRes. 4:43; Cornetta et al. (1991) Hwm. Gene Ther 2:215).

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.

Immunol. 158:97-129; GENE TARGETING: A PRACTICAL APPROACH (1992) ed. A. L. Joyner, Oxford University Press, NY). Several different approaches are feasible.

Additional references describing viral vectors which could be used in the methods of the present invention include the following: Horwitz, M.S.,

Adenoviridae and Their Replication, in Fields, B., et al. (eds.) VIROLOGY, Vol. 2,

34 Raven Press New York, pp. 1679-1721, 1990); Graham, F. et al., pp. 109-128 in METHODS IN MOLECULAR BIOLOGY, Vol. 7: GENE TRANSFER AND EXPRESSION PROTOCOLS, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller et al.

(1995) FASEB Journal 9: 190-199, Schreier (1994) Pharmaceutical Acta Helvetiae 68:145-159; Schneider and French (1993) Circulation 88:1937-1942; Curiel et al.

(1992) Human Gene Therapy 3:147-154; Graham et al, WO 95/00655 (5 January 1995); Falck-Pedersen WO 95/16772 (22 June 1995); Denefle et al. WO 95/23867 (8 September 1995); Haddada et al. WO 94/26914 (24 November 1994); Perricaudet et al. WO 95/02697 (26 January 1995); Zhang et al. WO 95/25071 (12 October 1995).

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). Alternatively, the antibodies can be conjugated to an enzyme (e.g. HRP) giving rise to a colored product upon reaction with the substrate. The actual amount of 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.

In vivo transduction of DCs, or other APCs, can potentially be accomplished by administration of cationic lipid/plasmid DNA complexes delivered via the intravenous, intramuscular, intranasal, intraperitoneal or cutaneous route of administration. Gene gun delivery or injection of naked plasmid DNA into the skin also leads to transduction of DCs. Condon et al.

(1996) Nature Med. 2:1122-1128; Raz et al. (1994) PNAS 91:9519-9523. Intramuscular delivery of plasmid DNA may also be used for immunization. Rosato et al. (1997) Human Gene Therapy 8:1451-1458.

35 The transduction efficiency and levels of transgene expression can be assessed as described above for viral vectors.

Isolation, Expansion and Education of Immune Effector Cells The APCs prepared as described above are mixed with naϊve immune effector cells. Preferably, the cells may be cultured in the presence of a cytokine, for example IL2. Because dendritic cells secrete potent immunostimulatory cytokines, such as IL-12, it may not be necessary to add supplemental cytokines during the first and successive rounds of expansion. In any event, 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.

In one embodiment, the immune effector cells are T cells. In a separate embodiment, the immune effector cells can be genetically modified by transduction with a transgene coding for example, IL-2, IL-11 or IL-13. Methods for introducing transgenes in vitro, ex vivo and in vivo are well known in the art. See Sambrook, et al. (1989) supra.

An effector cell population suitable for use in the methods of the present invention can be autogeneic or allogeneic, preferably autogeneic. When 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. The

36 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 (naϊve) 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. For example, effector cells can be obtained by leukapheresis, mechanical apheresis using a continuous flow cell separator. For example, lymphocytes and monocytes can be isolated from the buffy coat by any known method, including, but not limited to, separation over Ficoll-Hypaque™ gradient, separation over a Percoll gradient, or elutriation. The concentration of Ficoll-Hypaque™ 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. 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. For example, the cells can be subjected to positive selection on the basis of expression of one or more

37 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),

T1/ST2; chemokine receptors such as CCR3, CCR4, CXCR3, CCR5; homing receptors such as CD62L, CD44, CLA, CD 146, α4β7, αEβ7; 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-α, IL-12, IFN-γ; 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. 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.

Methods of Freezing and Thawing Dendritic Cells

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.

38 Preferably, 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. 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 10² cells/mL to about 1 x 10¹⁰ cells/mL, preferably between about 1 x 10⁴ cells/mL and about 1 x 10⁸ cells/mL and even more preferably between about 1 x 10⁶ to 1 xlO⁷ cells/mL. The cells may then be aliquoted into freezing vials and frozen by slowly bringing down their temperature according to standard protocols. Preferably, the cells are stored at -80°C for approximately one day before being moved to liquid nitrogen storage. Typically, 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.

39 Table 2

Media Components

Media Component Source

Ficoll-Paque research grade Pharmacia Biotech

Trypsin-EDTA 0.25% Life Technologies

OptiMEM Life Technologies

RPMI 1640 Life Technologies human AB serum for MLR Sigma

Penicillin/Streptomycin Sigma

L-glutamine Sigma

HEPES 1 M Sigma human GM-CSF Immunex Biodesign human IL-4 Genzyme

TNFα Genzyme

LPS Sigma

Analysis of Frozen Cells

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. In addition, 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.

40 Table 3

Examples of Antibodies for FACs analysis

Antibody Source anti CDl lc-PE Becton-Dickinson (B-D) anti CD3-FITC B-D anti CD4-FITC B-D mouse IgG2a isotype control PE B-D mouse IgGl isotype control FITC B-D anti-CD80-PE B-D anti-HLA-DR-FITC (class II) B-D anti CD86-FITC Pharmingen anti HLA-ABC-PE (class I) Serotec anti HLA-DR, DP, DQ-FITC (13 antibody for class II) Coulter anti CD14-FITC Coulter anti CD83-PE Coulter anti CDla-PE Coulter mouse IgGl isotype control PE Coulter

mouse IgG2a isotype control FITC Coulter

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 10⁹ 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

41 was aspirated and the flask washed three times with unsupplemented RPMI to remove non-adherent cells.

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.

Characterization of purified DCs by FACS Analysis

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 10⁵ cells were used to reaction. These cells were resuspended in 200 μl 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 μl 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 μl of FACS buffer containing 100 μl 2% paraformaldehyde. The samples were stored in the dark at 4°C until loading on the flow cytometer.

As shown in Figures 1 and 2, most of the cells expressed high levels of MHC class I and class II, high levels of costimulatory molecules B7J and B7.2 and low levels of CD3 and CD4. These results indicate the cells are DCs.

The frozen cells were also compared to freshly isolated DCs. Results, shown in Figure 3, indicate that the DC frozen according to the method described have similar FACS profiles to freshly isolated DCs.

42 Survival of Frozen Dendritic Cells

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, shown in Table 4, demonstrate that DCs frozen in 90% serum and 10% DMSO retain greater than 90% viability.

Table 4

DC Type Media Survival

Precursors Gibco Freezing Media (DMEM + serum + 72% DMSO)

Precursors 90% serum + 10% glycerol 77%

Precursors 90% serum + 10% DMSO 92%

Mature 90% serum + 10% DMSO 97%

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

90% to 50% decreased the viability of cells by around 10%, which is still much superior to the standard Gibco freezing media. Results are shown in Table 5.

43 Table 5

Freezing Media % Survival

10% DMSO + 90% pooled human AB serum 97.5

10% DMSO + 90% fetal calf serum 98

15.4% (2M) DMSO + 20% serum 80

Gibco freezing media (10% DMSO + 20% serum) 76

12% DMSO + 30% fetal calf serum 88

10% DMSO + 50% serum 87

Functionality of Frozen Dendritic Cells Frozen and thawed DCs were also tested for their ability to present antigens. Following ⁵¹Cr-labeling (100 μCi/mL for 24 hours), the cells were peptide-pulsed (1 μM) DCs with 1 μM 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. Effectoπtarget ratios are indicated and 1000 to 5000 targets were used for the assay.

As shown in Figures 4 and 5, frozen/thawed DCs are as efficient as fresh DCs at presenting enxogenously added peptide. The data points represent the average of 3 independent wells. Total reaction volume was 100 μl and incubations were 24 hours. Spontaneous ⁵¹Cr release was <25% and percent specific killing was calculated by:

% specific killings = experimental CPM - spontaneous CPM total CPM - spontaneous CPM

Example 2: Frozen/Thawed DCs Adenoviral Transduced DCs

Retain Function

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

44 the activity of the unpulsed DCs to that of fresh DCs (never frozen), it was possible to quantitatively assess the levels of antigen processing and presentation both pre- and post- freezing. In the event that reduced activity of the thawed DCs was observed, it was possible to compare the peptide-pulsed to unpulsed DCs, to determine whether the block to antigen presentation was occurring at the level of antigen processing or further dowstream in the sequence of events that must take place in order to achieve T cell recognition. Also, since the assays are performed over a long period of time, the peptide-pulsed samples help to standardize assays performed on different days. Furthermore, comparison of peptide-pulsed and unpulsed DCs revealed that peptide-pulsing Ad2/hugpl 00v2-infected DCs did not result in higher levels of specific killing indicating that the level of antigen presentation from the adenoviral vector was not limiting under these reaction conditions. The results indicate that freeze/thawed, genetically modified DC are functionally equivalent to fresh DCs (Figure 6).

Cell Lines and Peptides

Effector cells used in this assay were 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-

083)/10% human AB serum (Sigma #2520) supplemented with 6000 IU recombinant human IL-2/ml (Genzyme, reagent grade). The reagent grade peptide used in this experiment (GP100-F9) was synthesized and HPLC-purified to >95% as determined by mass spectrometry (QCB, Hopkinton MA). The peptide was dissolved at 10 mg/ml in 100% DMSO and diluted as indicated with dH₂0. This peptide differs from G9-209, the natural epitope encoded by the Ad2/gpl00v2 vector, by a substitution of phenylalanine for isoleucine at position 1. GP100-F9 binds more tightly to the HLA-A2 molecule than the natural epitope and is recognized by TIL 1520.

45 DC Preparation

Normal donor monocytes were harvested by leokophoresis (Dana Farber Cancer Institute) in a volume of -175 mis. The cells were diluted 1:1 with PBS (Mg⁺², Ce⁺² free) and 30 mis layered onto 20 mis Ficoll-Paque (research grade,

Pharmacia Biotech., 17-0840-03). Cells were collected at the interface by centrifugation (IEC, Model GP8, 1400 RPM/40 min., room temperature, no brake, swinging bucket rotor). The cells were washed 3x with PBS and resuspended in RPMI (GibCo, #11875-093) supplemented with P/S, glutamine, 5% Human AB serum, and lOmM HEPES (complete medium) and rocked overnight on a Nutator in 50 ml conical tubes at 4°C. The cells were then plated at 1.5x10 cells/T-150 Flask (Coming, #430823). After 1 hour, 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. On day 3, 5 mis complete RPMI (plus cytokines) was added to each flask. On day 6, the cells were harvested and prepared for the CTL assay and frozen storage in liquid nitrogen as indicated below.

DC Infection

To maximize uniformity among the infected frozen DCs, 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%CO₂, and then the medium was replaced with RPMI supplemented with

P/S, glutamine, 5% Human AB serum (Sigma #2520), and lOmM HEPES supplemented with lOOng/ml GM-CSF and 20 ng/ml IL-4. After an additional overnight incubation at 37°C/5%CO₂, the cells were processed for the CTL Assay and freezing as described below. The uninfected DC cultures were handled identically to the infected cultures.

46 DC Freeze/Thaw

Day 6 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-Pulsed DCs

Peptide-pulsing of the both uninfected and infected DCs was achieved by aliquoting 5000 ⁵¹ Cr-labeled cells in a volume of 1 OOμl to the appropriate wells of the 96-well V-bottom CTL assay plate (see CTL Assay, below). These wells were supplemented with 2μl of GP100-F9 peptide (diluted in dH₂0 from a 10 mg/ml stock in 100% DMSO) for a final concentration of lOμM. The plate was incubated at 37°C/5%CO₂ for 1 hour before the addition of the effector cells.

CTL Assay

5 X 10⁵ fresh or previously frozen DCs were labeled for 2 hours in 450μl complete medium supplemented with 50μl (50μCi) ⁵¹Cr (NEN/Dupont, #NEZ- 0305) in separate wells of a 24-well cluster plate (total volume=0.5 ml). The DCs were transferred to a 1.5 ml eppendorf tube and washed 3 x 1ml of serum-free AIM-V medium. 5000 cells were transferred to each well of a 96-well N-bottom plate in a volume of lOOμl/well AIMN/10% human AB serum and the plate was spun at 1200 rpm for 3 minutes. Peptides were added as described in Peptide- Pulsed DCs, above. 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 lOOμl for a total reaction volume of 200μl. Medium (no effector cells) was added to the spontaneous release wells, and no additional media was added to total release

47 wells. The plate was then spun at 1200 rpm for 3 minutes and returned to the incubator for 4 hours. After the incubation, 100ml of 1% triton X-100 was added to the total release wells (total volume=200μl) and the plate was spun at 1000 rpm for 10 minutes. 50μl of supernatant was removed from each well and added to a Wallac 96-well plate (Wallac, polyethylene terepthalate, # 1450-401) containing

150μl scintillation fluid (Wallac Optiphase Supermix, #sc/9235/21). This plate was sealed with an adhesive plate sealer and incubated at room temperature overnight. The radioactivity was measured using a Wallac 1450 MicroBeta Trilux plate reader (Model #1450-021). All reactions were performed in triplicate, and each graphed data point represents the average of the replicates. Percent specific killing was calculated according to the formula: 100 X (Experimental CPM- Spontaneous CPM)/(Total CPM-Spontaneous CPM).

Summary Human DCs were prepared by culturing HL A-A2⁺ normal donor monocytes with GM-CSF and IL-4 for six days. Half of the DCs were transduced with Ad2/hugpl00v2. The other half were untreated. Both fresh untransduced and transduced DCs were assayed for recognition by gplOO-specific TIL 1520 in a standard ⁵¹Cr-release assay. The remainder of the cells were frozen in 90% pooled human AB serum 10% DMSO at 5 x 10⁶ cells/aliquot and stored in liquid nitrogen. These samples were thawed and assayed according to the following schedule which parallels the dosing schedule.

Table 6

SAMPLE # ASSAY

SCHEDULE

1 FRESH

2 WEEK 3

3 WEEK 6

4 WEEK 9

6 WEEK 15

48 The frozen samples were thawed and immediately put into culture medium (AIM-N/10% human AB serum) containing 100 μCi/ml ⁵¹Cr for 2 hours. The cells were then washed 3X to remove unincorporated label. One half of each DC population (i.e., transduced and untransduced) was peptide-pulsed at lOμg/mL during ⁵¹Cr labeling and all samples are then incubated with TIL 1520 at effectoπtarget ratios of 10:1 and 5:1 using 5000 labeled viable DCs per reaction. Comparison of the levels of lysis between the fresh and frozen DCs will, to a first approximation, allow determination of quantitative or qualitative differences that may have resulted from long-term frozen storage. Comparison of peptide-pulsed to unpulsed DCs will tell us, in the event of reduced lysis of thawed DCs, whether the block to antigen presentation is occurring at the level of antigen processing or further downstream. Additionally, the peptide-pulsed, uninfected DCs allows standardization of assay results performed on different days. As shown in Figure 6, Applicants did not see any decrease in the ability of DCs to present antigen (over a period of time in frozen storage).

Example 3: Long Term Storage of Freezon DCs Has Minimal or No Effect on Expression of Cell Surface Markers

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

49 with known therapies or compositions. Other aspects, advantages and modification within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

50

Claims

1. A method for freezing dendritic cells (DCs) comprising suspending the DCs in media comprising at least 30% human-derived serum and reducing the temperature of the suspension to below -80┬░C, thereby freezing dendritic cells.

2. The method of claim 1 , wherein the media is approximately 90% human-derivied serum and approximately 10% of an an agent that prevents ice crystal formation. .

3. The method of claim 1 , further comprising maintaing the suspension at -80┬░C for at least 24 hours and then transferring the suspension to liquid nitrogen.

4. The method of claim 1 , further comprising thawing the cells at a temperature of about 34-41 ┬░C.

5. The method of claim 1 , wherein the DCs are mature DCs or dendritic cell precursors.

6. The method of claim 5, wherein the precursors are monocytes or peripheral blood lymphocytes.

7. A substantially purified population of DCs produced according to the method of claim 1 or 4.

8. The method of claim 1 , wherein the DCs express at least one antigen on the surface of the DCs.

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9. The method of claim 8, wherein at least one antigen is a tumor- associated antigen (TAA).

10. The method of claim 1 , wherein the DCs are transduced with a gene coding for at least one antigen.

11. The method of claim 10, wherein at least one antigen is a tumor- associated antigen (TAA).

12. The method of claim 1 , wherein the DCs are pulsed with at least one antigen.

13. The method of claim 6, wherein the DCs are pulsed with at least one antigen.

14. The method of claim 12, wherein at least one antigen is a tumor- associated antigen (TAA).

15. The method of claim 1 , wherein the DCs are hybrid DCs that express at least one tumor-associated antigen (TAA)

16. A method for ex vivo generation of antigen-specific immune effector cells, comprising educating na╧ève immune effector cells by co-culture with the DCs cells of claim 4.

17. The method of claim 16, wherein the immune effector cells are cytotoxic T lymphocytes (CTLs).

18. The method of claim 16, wherein the immune effector cells and the DCs are autologous.

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19. The method of claim 16, wherein the immune effector cells and DCs are allogeneic.

20. The method of claim 1 , wherein the dentritic cells are of human or murine origin.

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