CA2340085A1 - Engineered antigen-presenting cells expressing an array of antigens and uses thereof - Google Patents

Abstract

The present invention provides a method for presentation of multiple disease associated antigens in antigen-presenting cells which can be used to generate a prophylactic or therapeutic immune response against the disease with which the antigens are associated. The method employs differential screening of nucleic acid sequences expressed by target and non-target cells. By identifying nucleic acid sequences preferentially expressed in a target cell population and expressing the identified sequences in antigen-presenting cells, one can stimulate an immune response directed at a target cell population without being limited to single, previously identified antigens.

Description

ENGINEERED ANTIGEN PRESENTING CELLS EXPRESSING, AN ARRAY OF ANTIGENS AND USES
THEREOF
Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state o~f the art to which this invention pertains.
TECHNICAL FIELD OF TH)E: INVENTION
The invention relates to immunotherapy involving the activation of T
lymphocytes by IO antigen-presenting cells that are genetically modified to express and present, on their _ surface an array of antigens that are differentially expressed by a target population. The antigen-presenting cells can be genetically modifed by transduction with nucleic acid sequences identified by differential screening of nucleic acid sequences expressed in a target population relative to a non-target population.
1 ~ BACKGROUND OF THE INVENTION
Dendritic cells, which are specialized antigen-presenting cells, can be used to process antigens from diseased tissue and present them to the immune system. Dendritic cells are leukocytes derived From bone marrow and are considerably more potent than other antigen-presenting cells with respect to presentation and activation of cytotoxic T
20 lymphocytes (CTLs). Publications relevant to dendritic cells include Young et al. 1996, J. Exp. i~Ied. 183:7-11; Aiavaikko et ai. 1994, Am. J. Clin. Pathol. 101:761-767;
Shunichi et aI. 1995, Cancer 75:1478-1483; Hsu et al., 1996, Nature Medicine

2:~2-~8;
Nlayordomo et al. 1995, Nature Medicine 1:1297-1302; Paglia et al. 1996, J.
Exp. Med.
183:317-322; and Boczkowski et al. 1996, J. Exp. Med. 184:46-472.
25 There is a need for a vaccine that can.boost multiple clones of CTLs to enhance the immune response and to prevent the escape of a tumor from immune selection.

Accordingly, there is a need for a system for providing antigen-presenting cells with (i) a broader spectrum of potential anti-tumor antigens .and (ii) providing the antigens in a form which the antigen-presenting cells can effectively process and present.
SUMMARY OF THE iN'VENTION
The invention provides a system for priming antigen-presenting cells with a repertoire of antigens of a specific cell type, for example, a tumor cell or a virally-infected cell.
Further, the approach need not be restricted to stimuuating an immune response against diseased tissue. It can be used to mount an immune .response against any target tissue and to ablate any target cell expressing a particular set of antigens.
I 0 The invention provides a method of producing at least one vector encoding an array of antigens for expression in an antigen-presenting cell. In one embodiment, the method _ comprises comparing first nucleic acid sequences expressed by a target cell population with second nucleic acid sequences expressed by ~z non-target cell population.
The method further comprises selecting nucleic acid sequences preferentially expressed by I~ the target cell population relative to the non-target cell population, and introducing the selected nucleic acid sequences into at least one vector capable of directing expression of the selected nucleic acid sequences in an antigen-presenting cell. In one embodiment, the antigen-presenting cell is a dendritic cell, macrophage, B cell, monocyte or f bracyte. In another embodiment, the vector further comprises a dendritic cell targeting 20 element.
In one embodiment, the first and second nucleic acid sequences are of the same tissue of origin. The selected nucleic acid sequences can number one or more. For example, the selected nucleic acid sequences can comprise at Ieast 3 different nucleic acid sequences, at least 5 different nucleic acid sequences, at least 7 different nucleic acid sequences, or 25 at Ieast 9 different nucleic acid sequences. The vector can further comprise a nucleic acid sequence encoding an immunomodulatory cofactor. The immunomodulatorv cofactor can be, for example, IL-2, IL-3, IL-8, OKT3, a.-interferon, y-interferon, or MIP-la. The vector can further encode at least one selectable marker. Examples of WO 00!09665 PCT/US99/18087 selectable markers include, but are not limited to, PLAP, GFP and neomycin resistance.
In one embodiment, the target cell is a cancer cell. In another embodiment, the target cell is an infectious agent, such as a virus, a bacteritun or a parasite:
The invention additionally provides a composition comprising at least one vector .
produced by the method described above. In one embodiment, the vector further comprises an antigen-presenting cell targeting element. The composition can further comprise an antigen-presenting cell.
The invention also provides a method of producing an antigen-presenting cell that presents an array of antigens. The method comprises; comparing first nucleic acid sequences expressed by a target cell population with second nucleic acid sequences expressed by a non-target cell population. The method further comprises selecting nucleic acid sequences preferentially expressed by the target cell population relative to the non-target cell population, and genetically modifying an antigen-presenting cell to express the selected nucleic acid sequences. Also provided is an antigen-presenting cell 1 ~ produced by the method described above or genetically modified with a vector produced by a method of the invention.
The invention provides a method of activating immune cells comprising contacting an immune cell with an antigen-presenting cell genetically modified in accordance with the invention. In one embodiment, the immune cell is a 'T cell. Examples of T
cells activated by the method include, but are not limited t~o, cytotoYic T
lymphocytes (CTLs) and helper T cells. Also provided is a method of inducing a toleragenic response comprising contacting an immune cell with an antigen-presenting cell genetically modified in accordance with the invention. In one embodiment. the immune cell is a helper T cell such as a Tm cell. In one embodiment o~f the method of activating T cells or inducing a toIeragenic response, the contacting occurs in viva.
Alternatively, the contacting can occur ex vivv. The invention also provides immune cells, such as CTLs and T~ cells, activated by a method of the invention. The activated immune cells can be provided in the form of a composition:

The invention provides a method of activating T cells in vivo comprising administering a composition comprising a vector or antigen-presenting cell of the invention to a subject. Also provided is a method of killing a target cell in viva comprising administering a composition, vector or antigen-presenting cell of the invention to a S subject. The invention also provides methods of preventing infection, treating cancer, and treating a viral infection, the methods comprising administering a composition, vector or antigen-presenting cell of the invention to a subject.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 depicts cell surface markers that can be used to identify dendritic cells. A (-) indicates a marker not present on dendritic cells that can be used in negative selection strategies. The (+), (-+-+) ~d (+++) respectively indicate increasingly useful markers present on the dendritic cell surface.
DET:~I3.ED DESCRIPTION OF T'HE I~iVE~iTIOI~I
The present invention provides a strategy for presentation of multiple antigens in antigen-presenting cells which can be used to generate a prophy lactic or therapeutic immune response against one or more target cell populations with which the antigens are associated. The strategy employs comparing and selecting nucleic acid sequences e~cpressed by target and non-target cells. By identifying nucleic acid sequences preferentially expressed in a target cell population anct expressing the identified 2fl sequences in antigen-presenting cells, one can stimulate an immune response directed at a target cell population without being limited to previously identified antigens. The use of an array of antigens can elicit a more effective immune response by directing lymphocytes to a variety of antigen targets. This multiple antigen strategy is advantageous both for targeting an immune response to more antigens associated with a 2~ given disease, and for targeting an immune response to a disease for which an effective antigen for a particular patient or for a particular disease is not known.

The vectors and antigen-presenting cells of the invention can be prepared without prior purification of individual disease-associated antigens. This is particularly useful when a given tissue or cell expresses multiple disease-associated antigens, or when a target antigen is unknown. By making use of differential screening to identify preferentially expressed nucleic acid sequences, one can avoid problems with lack of specificity as nucleic acid sequences encoding non-target antigens can be excluded.
The invention provides a composite preparation of vectors encoding antigens for expression in antigen-presenting cells, which cells can then be used to activate T cells of a subject in vivo, ex vivo, ar in vitro, without specifi~~ prior knowledge of the relevant disease-associated antigens iri that subject. A composite preparation can be used to activate a variety of T cells, directed against more than one antigen and against more than one disease or target cell population.
Definitions All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words ar phrases have the meanings specified.
As used herein, "target cell population" means one or more cells sharing at least one common feature, the elimination or protection of which is desired. Examples of a target cell include, but are not limited to, an infectious agent such as a virus, bacterium or parasite, a cell which is susceptible to autoimmune al:tack, and a disease cell such as a cancer cell, including highly metastatie cancer cells and low metastatic cancer cells. As used in the context of a target cell, "infectious agent'' includes both the infectious agent itself and cells infected by the agent. For example, when the target cell is a virus, the target cell may be the virus alone, a virally infected cell or both.
As used herein, "preferentially expressed" refers to nucleic acid sequences that are present in substantially greater amounts in a target sample as compared to a non-target sample. A substantially greater amount can.be at least about 20% more. In one embodiment, substantially greater is about 50% more. In another embodiment, substantially greater is about twice as much. In a preferred embodiment, substantially greater is at least about I O times as much. In more preferred embodiments, substantially greater is at least about 20, 50 or 100 times as much. The relationship between the target and non-target samples is selected to optimize the identif cation of sequences encoding antigens relatively specific to the target cells. For example, a target cell can be a colon carcinoma cell and a corresponding non-target cell would be a non-cancerous colon cell. In another example, the target cell can be a highly pathogenic virus and the non-target cell a less pathogenic virus. Differential screening between such target and f0 , non-target populations can yield nucleic acid sequences encoding antigens associated with a particular disease or pathogen, without requiring knowledge of the relevant antigens.
As used herein, "vector" means a construct which is capable of delivering, and preferably expressing, one or more genes) or sequence{s) of interest in a host cell.
I~ Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, DIVA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
As used herein, "expression control sequence" means a nucleic acid sequence which 20 directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducibIe promoter, or. an enhaneer.
The expression control sequence is operably linked to the; nucleic acid sequence to be transcribed.
The term "nucleic acid" refers to a deoxyribonucIeotide or ribonucIeotide polymer in 25 either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence optionally includes the complementary sequence.

As used herein, "antigen-presenting cell" or "APC;" means a cell capable of handling and presenting antigen to a lymphocyte. Examples of APCs include, but are not limited to, macrophages, Langerhans-dendritic cells, follicular dendritic cells, B
cells, monocytes, fibroblasts and fibrocytes. Dendritic cells are a preferred type of antigen presenting cell. Dendritic cells are found in many non-lymphoid tissues but can migrate via the afferent lymph or the blood stream to the T'-dependent areas of lymphoid organs.
In non-lymphoid organs, dendritic cells include Langerhans cells and interstitial dendritic cells. In the lymph and blood, they include afferent lymph veiled cells and blood dendritic cells, respectively. In lymphoid organs, they include lymphoid dendritic ID cells and interdigitating cells. As used herein, each ofthese cell types and each of their progenitors is referred to as a "dendritic cell," unless otherwise specified.
As used herein, "antigen-presenting cell targeting element" means a molecule which is capable of specifically binding an antigen-presenting cell, such as a dendritic cell. A
targeting element specifically binds a dendritic cell when a biological effect is seen in 1 ~ that cell type after binding of the targeting element and ics ccmpiement.
or, when there is greater than a IO fold difference, and preferably I=reater than a 2~, SO or 100 fold difference between the binding of the coupled targeting element to dendritic cells and non-target cells. Generally, it is preferable that the targeting element bind to antigen-presenting cells with a KD of less than 10'511~I, preferably less than 10'6 M, more 20 preferably less than I0'' M, and most preferably less than 10'8 M (as determined by Scatchaxd analysis (Scatchard, 1949, Ann. ~1.Y. Acad. Sci. ~I:660-672)).
Suitable targeting elements are preferably non-immunogenic, not degraded by proteolysis, and not scavenged by the immune system. Particularly preferred targeting elements have a half life (in the absence of a clearing agent) in an animal of between 10 minutes and 1 25 week. Examples of dendritic cell surface markers, against which antibodies (or antigen binding domains derived therefrom) can be generated to produce dendritic cell targeting elements, include, but are not limited to, those depicted in Figure 1 {e.g., CDl, CDi la, CDl lc, CD23, CD%S, CD32, CD40, CD4~, CD~4, t~D58, NiHC Class I, MHC Class II, Niac-l, Mac-2, Mac-3).

As used herein, "immunomodulatory cofactor" includes a factor which, when expressed in APCs, causes the immune response to an antigen presented by the APC to be enhanced in quality or potency from that which would have occurred in the absence of the cofactor. The quality or potency of a response may be measured by a variety of assays known in the art including, for example, in virno assays which measure cellular proliferation (e.g.,'H-thymidine uptake), and in vitro cytotoxicity assays (e.g., which measure s'Cr release; Warner et aI. 1991, AIDS Res. and Human Retroviruses 4:645-655). In alternative embodiments, an. immunomodulatory cofactor is, rather than being encoded by the expression vector, added exogenousIy before, concurrently with, or after I0~ administration of the vector. Examples of immunomodulatory cofactors include, but are not limited to cytokines and chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, iL-9, IL-10, IL-11, IL-12, IL-I3, IL-14, IL-1~~,~OKT3, a-interferon, ~i-interferon, y-interferon, MIP-la (LD-78), granulocyte~-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), tumor necrosis factors (TNFs), CD3, CD8, ICAl4i-1, ICAVI-2, LFA-l, LFA-3, and other proteins such as HLA Class I molecules, HLA Class II molecules, E.7, B7-?, /3~-microglobulin, chaperones, and MHC linked transporter proteins or a~zaIogs thereof. The choice of irnmunomodulatory cofactor is based on the therapeutic effects of the factor.
Preferred immunomodulatory cofactors include a.-interferon, y-interferon and IL-2.
l~Fucleic Acid Sequences The invention provides nucleic acid sequences encoding an array of antigens that are preferentially expressed in a target cell population and which can be used to genetically modify antigen-presenting cells (APCs). The invention further provides a method for preparing these nucleic acid sequences. The nucleic acid sequences can be prepared by comparing first nucleic acid sequences expressed by a target cell population with second nucleic acid sequences expressed by a non-target cell population. Nucleic acid sequences preferentially expressed by the target cell population relative to the non-target cell population are then selected. This method provides nucleic acid sequences that can be used to express an array of antigens in APCs without prior knowledge of the antigens WO 00!09665 PCT/US99/18087 and without compromising specificity for the target cell population. Nucleic acid sequences include DNA, RNA, and synthetic derivatives thereof.
Methods of comparing sets of nucleic acid sequences and selecting differentially expressed sequences ase known in the art. The comparing can be performed using nucleic acid sequences isolated from samples, such as from one or more cell types, or it can be performed by comparing sequence data obtained, for example, from a public database such as GenBank, EMBL, or DNA Database of Japan (DDBJ). For example, when two nucleic acid samples are to be compared, such as sequences from a target cell . population and sequences from a non-target cell population, each sample can be derived from either an isolated pool of nucleic acid sequences, database sequence information, any other source of nucleic acid sequence information, or a combination of two or more sources. Conventional techniques for differential display of mRNA and differential screening of a cDNA library are described in F. 11~f. ,Ausubel et al., eds., 1996, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., unit 5.8 and sections 5.8.6, 5.8.14.
One example of detecting differential expression of genes is described in U.S.
Patent No. 5,677,125, which relates to a method of detecting and diagnosing pre-invasive breast cancer. In this example, epithelial cells are isolated from a sample of abnormal breast tissue which exhibits histological or cytological characteristics of pre-invasive breast cancer, and mRNA is isolated from the sample. One or more abnormal breast tissue cDNA libraries is prepared from the isolated tnRNA. One or more cDNA
libraries is then prepared by similar means from a sample of normal breast tissue. The cDNA of the abnormal and normal libraries is compared to detect the expression of at least one gene in the abnormal sample which is different from that expressed in the normal sample. A similar approach can be applied to obtain an array of nucleic acid sequences preferentially expressed by a target cell population relative to a non-target cell population for introduction into APCs.

w0 00/09665 PCTIUS99/18087 The non-target cell can be a normal cell or it can be a diseased cell, but having a different spectrum of antigens from the target cell. For example, it may be desirable to obtain antigens specif c for a particular stage in disease. Thus, the non-target cell could be a cell in a different stage of the target disease. ArA example of a non-target cell population is one or more selected from stage l and :>tage 2 cells in cervical carcinoma.
Antigen-presenting cells can be provided that present antigens specific to a desired stage, such as stage 3 cervical carcinoma. In another example, the target cell population is a highly metastatic colon cancer cell line and the non-target cell population is a low metastatic colon cancer cell line. Differential screening of nucleic acid sequences expressed by the two cell lines can be used to select sequences encoding antigens specific to highly metastatic colon cancer cells. When the non-target cell is a normal cell, differential screening eliminates or reduces the nucleic acid sequences common to normal cells, thereby avoiding an immune response directed at antigens present on normal cells. When the non-target cell is a normal cell, differential screening eliminates i~ or reduces Nucleic acid sequences common to normal cells, thereby avoiding an immune response directed at antigens present on normal cells. Examples of target!non-target cell combinations suitable for differential screening include, but are not Limited to, K.~II2L4A cells (high metastatic colon cancer line; iVlorikawa et al., 1988, Cancer Res.
48:1943)/KM12C (low metastatic colon cancer line; Trforikawa et al., 198$, Cancer Res.
48:6863); MDA-MB-231 cells (high metastatic breast cancer line; Brinkley et al., 1980, Cancer Res. 40:3118)II~ICF-7 cells (ron-metastatic breast cancer line: Yang et al., 199$, Anticancer Res. I8{lA):53-59); and MV-522 cells (high metastatic lung cancer line;
Varki et al., 1987, Int. J. Cancer 40:46)/UCP-3 cells (low metastatic lung cancer cell line; Varki et aL, 1987, supra).
Differential screening can also provide various tailored combinations of antigen-presenting cells. One combination of APCs of the present invention encompasses antigens relating to associated pathologies. Far examl>le, HIV-infected patients are often infected with CMV. Accordingly, antigen-presenting cells can be prepared expressing genes preferentially associated with HIV-infected cells, and a second preparation can be prepared in which the antigen-presenting cells express antigen preferentially expressed by C1~IV-infected cells. These two preparations can be combined and administered to a subject providing simultaneous treatment for both pathologies. Examples of other associated pathologies wherein preparations of antigen-presenting cells can be combined include HIV and Epstein-Barr virus (EBV) positive lymphoma; and HIV and hepatitis C virus (HCV).
Another example in which separate preparations can be combined provides APCs for targeting a tumor or tumors of unclear origin. In thus case, antigen-presenting cells genetically modified to express nucleic acid sequ.erxces from a spectrum of tumor types can be used to elicit a broad anti-tumor immune re:;ponse.
A third instance in which separate preparations can be combined provides for targeting a disease associated with a different spectrum of anti;;ens in different patients.
Preparations from various patients that encompass most or all of the repertoire of antigens associated with the disease in most or all patients can be combined and 1S administered to a subject without having to first determine what antigens are expressed in that individual subject's disease.
The combinations can be prepared at the nucleic acid level. In this case, preferentially expressed nucleic acids are combined and then introduced into antigen-presenting cells in vitro. Alternatively. preferentially expressed nucleic acids can be used to prepare separate antigen-presenting cells which then can be combined to provide a composite antigen preparation.
In one embodiment of the invention, mRNA preferentially expressed by the target cell population is used to obtain nucleic acid such as cDNA encoding full-length sequences.
Nucleic acid sequences so obtained can be used to identify differentially expressed 2S antigens associated with the target cell population. ~~ne can then test antigens so obtained to determine which are most effective as immunogens.

In one example of testing an antigen's immunogen:icity, periplierai blood cells are removed from a subject. Dendritic cells are then isolated and either pulsed with the antigen to be tested or genetically modified to express the test antigen. The pulsed or modified dendritic cells can then be used to stimulate the subject's T cells in vitro. The ability of the modif ed dendritic cells to stimulate T cells is indicative of immunogenicity of the test antigen.
Vectors The invention additionally provides vectors containing nucleic acid sequences selected by differential screening as described above. The sf;lected sequences can be introduced IO into at least one vector. Preferably the vector is capable of directing expression of the selected nucleic acid sequences in an antigen-presenting cell, such as a dendritic cell. A
vector can encode a single antigen or multiple antigens. When multiple antigens are encoded by a single expression vector, the antigens can be derived from the same or different cell types. Alternatively, a composition comprising more than one vector, I S each encoding one or more antigens associated with a cell type the same as or different from those encoded by other vectors, can also be prepared.
In one embodiment, the invention provides a methoc! of producing at least one vector encoding an array of antigens for expression in an antigen-presenting cell. In one embodiment, the method comprises comparing first nucleic acid sequences expressed 20 by a target cell population with second nucleic acid sequences expressed by a non-target cell population. The method further comprises selecting nucleic acid sequences preferentially expressed by the target cell population relative to the non-target cell population, and introducing the selected nucleic acid sequences into at least one vector capable of directing expression of the selected nucleic acid sequences in an antigen-25 presenting cell. In one embodiment, the antigen-presE:nting cell is a dendritic cell, macrophage, B cell, monocyte or fibrocyte. In another embodiment, the vector further ' comprises a dendritic cell targeting element.

In one embodiment of the method, the first and second nucleic acid sequences are of the same tissue of origin. The selected nucleic acid sequences can number one or more.
For example, the selected nucleic acid sequences can comprise at least 3 different nucleic acid sequences, at least 5 different nucleic acid sequences, at least 7 different nucleic acid sequences, or at least 9 different nucleic acid sequences. The vector can further comprise a nucleic acid sequence encoding an immunomodulatory cofactor. The immunomodulatory cofactor can be, for example, IL-2, IL-3, IL-8, OKT3, a-interferon, y-interferon, or MIP-la. The vector can further encode at least one selectable marker.
Examples of selectable markers include, but are not limited to, PLAP (U.S.
Patent Application Serial No. 09/006,298, filed January l 3, 1998); GFP and neomycin resistance. In one embodiment, the target cell is a cancer cell. In another embodiment, the target cell is an infectious agent, such as a virus, a bacterium or a parasite.
Vectors of the invention can be used to genetically modify APCs such as dendritic cells either in vivo or in vitro. Several ways of geneticaIfty modifying APCs are known, including transduction with a viral vector either directly ur via a retrovi:al producer cell, calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells. with liposomes containing the DNA, DEAF dextran, receptor-mediated endocytosis, electroporation, micro-injection, and many other techniques known to those of skill. See, e.g.. Methods in Enzymology, 185, Academic Press, Inc., San Diego, CA (D.V. Gc~eddel, ed.) 1990, or M.
Krieger, Gene Transfer and Expression -- A Laboratory :Manual, Stockton Press, New York, NY, 1990, and the references cited therein, as well as Berger and KimmeI, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. Molecular Cloning - A Laboratory Manual (2nd ed.) 1-3 1989; and Current Protocols in Molecular P.~ioiogy, F.;~i. Ausubel et al., eds., Greene Publishing Associates, Inc. and John WiIey & Sons, Inc., (1994 Supplement), WO 93124640; Mannino and Gouid-Fogerite 1988, Biotechniques 6(7):682-691;
IJ.S.
' Patent No. 5,279,833; WO 91/06309; and Felgner et al. Proc. Natl. Acad. Sci.
USA
84:7413-7414 I 987.

a - WO 00/09665 PCT/US99118087 Examples of viral vectors include, but are not limited to retrovii~al vectors based on, e.g., HSV, HIV, marine retroviruses, gibbon ape Leukemia virus and other viruses such as adeno-associated viruses (AAVs) and adenoviruses. (Miller et ai. 1990, Mol Cell Biol.
10:4239; J. Kolberg 1992, NIH Res. 4:43, and Corneas et al. 1991, Hum. Gene Ther.
2:215). Widely used retroviral vectors include those based upon marine leukemia virus (MuLV), gibbon ape Leukemia virus (GaLV), ecotropic retroviruses, human immunodeficiency virus (HIV), and combinations. :>ee, e.g. Buchscher et aI.
1992, J.
Virol. 66(5}:2731-2739; 3ohann et aI~. 1992, J. Virol. 66(5):1635-1640;
Sommerfelt et aI.
1990, Virol. 176:58-59; Wilson et aI. I989, J. Virol. 63:2374-2378; Miller et al. 1991, J.
I O Virol. 65:2220-2224, and Rosenberg and Fauci 1993 in Fundamental Immunology, Third Edition, W.E. Paul (ed) Raven Press, Ltd., New York and the references therein;
Miller et al. 1990, Mol. Cell. BioI. 10:4239; R. Kolbe;rg 1992, J. NIH Res.
4:43; and Cornetta et ai. I991, Hum. Gene Ther. 2:215.
Marine vectors comprising Gibbon Ape Leukemia Virus (GaLV) envelopes can be used to transduce many mammalian cells. See, Joha,nn et al. i 992, supra. The same receptor is used by sinuan sarcoma associated virus (SSAV), a strain ofGaLV (Sommerfelt et al.
1990, supra). The construction of hybrid virions having GaL V envelope proteins has been demonstrated (Wilson et al. 1989, supra; Miller et a1. 1991, supra). Any of these vectors and methods of making retroviral clones can be applied to the present invention.
GaLV retroviraI packaging cell Lines can be used to provide infectious replication-defective hybrid virions for use in gene transfer in humans, hamsters, cows, cats, dogs, monkeys, chimpanzees, macaques, primates, and other species whose cells have host cell receptors for GaLV envelope proteins.
Additional examples of vectors include, but are not limited to adenoviral vectors, AAV
vectors, pox viral vectors (B. Moss, 1992, Current Topics in Microbiology and Immunology 158:25-38) including vaccinia, fowl pox, and canary pox, recombinant influenza viral vectors {A. Garcia-Sastre and P. Palese 1995, Biologicals 23:1 X-I78) or non-viral gene delivery techniques (F. Leedley 1994, Biotechnology 5:626-636).
AAV-based vectors can be used to transduce cells with seIecied nucleic acids, e.g., in the in l4 ' WO 00/09665 PCT/US99118087 vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures. See, West et aI. 1987, Virology 160:38-47; U.S. Patent No.
4,797,368; WO
93/24641; Kotin 1994, Human Gene Therapy 5:793-801; and Muzyczka 1994, J.
Clin.
Invest. 94:1351.
In vitro amplification techniques suitable for amplifying sequences to be subcioned into an expression vector are known. Examples of such in vitro amplification methods, including the polymerase chain reaction (PCR), Iigase chain reaction (LCR), Q~i-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), are found in Berger, Sambrook et al. 1989, Molecular Cloning - A Laboratory Manual I O (2nd Ed) I-3; and U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. gds) Academic Press Inc. ~>an Diego, CA 1990;
Arnheim &
Levinson (October l, 1990) C&EN 36-47; J. NIH Res. 1991, 3:81-94; Kwoh et al.
1989, Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al. 1990. Proc. Natl.
Acad. Sci.
USA 8 7: I 874; Lomeli et al. 1989, 3. Clin. Chem. ~~: i 826; handegren et al.
1988, IS Science 241:1077-1080; Van Brunt 1990, Biotechnology 8:291-294; Wu and Wallace 1989, Gene 4:560; Barriner et al. 1990, Gene 89:11 T; and Sooknanan and lrialek 1995, Biotechnology 13:563-564. Improved methods of cloning in vitro amplifed nucleic acids are described in U.S. Patent No. 5,426,039.
Antigen-Presenting Cells 20 Antigen-presenting cells (APCs) process antigen and present it to a lymphocyte. The invention provides APCs that present an array of antigens. The APCs are genetically modif ed to express nucleic acid sequences preferentially expressed by a target cell population relative to a non-target cell population. Examples of APCs include, but are not limited to, macrophages, Langerhans dendritic cells, follicular dendritic cells, B
25 cells, monocytes, fibroblasts and fibrocytes. in a preferred embodiment, the APC is a dendritic cell. Selection of the optimal APC can vary, however, with the antigen to be presented (Butt and Bevan 1998, J. Immunol. 160:21.30-2144).

Dendritic cells have an unusual dendritic shape, are motile, and efficiently cluster and activate T cells that are specific for cell surface stimuli. Typically, dendritic cells in non-lymphoid organs, such as Langerhans cells and interstitial cells, become veiled cells (cells which continually extend and retract large lamellipodia) in the afferent Iymph and blood which migrate to lymphoid tissues, where they can be isolated as dendritic or interdigitating cells.
Dendritic cells initiate T-dependent responses from quiescent lymphocytes.
Once sensitized, T cells interact with other antigen presenting cells. Dendritic cell antigen processing activity is regulated. Fresh cells, i.e., cells cultured for less than a day, isolated from skin or lymphoid organs present native; proteins. After that time, they do not process antigens. in addition, dendritic cells are not actively phagocytic.
Dendritic cells can be isolated and prepared using conventional techniques such as those described in Tedder and Jansen, i 99 i, Current Protocols in Im~-nunology, John Vv'iley &
Sons, unit 7.32. Other methods for obtaining and identifying dendritic cells are described in WO 98/1 »?9; WO 98/01 X38; WO 9811 ~6I ~; and W O 98/14561. For example; human dendritic cells can be isolated from blood mononuclear cells by first enriching a peripheral cell population for dendritic cells by depletion of T
cells and adherent cells. Density gradient centrifugation of the preparation over metrizamide is used to isolate low buoyant density cells. This population has virtually no lymphocytes and contains 20-80% dendritic cells. The puray of de:ndritic cells can be determined by a variety of techniques, including hemacytometer counting, immunostaining after cytocentrifugation onto glass slides and immunofluo>:escence staining with flow cytometric analysis. Flow cytometry is preferred, however, for optimal quantitation and consistency.
Young et aL nave identified dendritic cell colony-forming units among normal human CD34~- (positive for this hematopoietic stem cell marker) bone marrow progenitors that give rise to pure dendritic cell colonies {Young et al. 199, J. Exp. Med.
182:1111-1120). Addition of c-kit-ligand to GM-CSF- and TNF-a.-supplemented suspension of CD34+ bone marrow cells expands dendritic cell colony-forming units almost 100-fold by 14 days. These colony-derived dendritic cells ane potent stimulators of T
cells.
Partially enriched populations of epidermal Langerhans cells, wherein Langerhans cells may comprise up to about 60% of the total cell population, may be readily prepared.
Keratinocytes can be depleted from marine tissue using a-thy-1 (a monoclonal antibody) and complement plus adherence. Enriched preparations of human Langerhans cells can be prepared by substituting an anti-CD 1 antibody for a-thy-1. In culture, neither mouse nor human Langerhans cells are active antigen-presenting cells until after 1-3 days in culture, after which time they enlarge, express more MHC Class II
and cell adhesion molecules, and lose Fc receptors, fully resembling blood and lymphoid dendritic cells. Cell populations containing more than 90% dendritic cells have been obtained from human blood, where, without enrichment, fewer than 0. I % of the white cells are dendritic cells. Such enrichment can be achieved by successive depletion of T
cells, monocytes, and B plus NK cells to yield an initial population ranging from 30-1 ~ 60°,% dendritic cells. Greater purity is then obtained by panning or fluorescence activated cell sorting (FACS) using a monoclonal antibody, especially to CD4~RA, that selectively reacts to contaminants (P. Freudenthal, et al. 1990, Proc. Natl.
Acad. Sci.
(USA) 87:7698-7702). To enrich for dendritic cells generally, selection for low buoyant density, non-adherence to plastic in culture (especially after one or more days), and absence of markers found on other cells is performed. Such methods deplete other cell types, but do not positively select dendritic cells.
Dendritic cells express a distinct pattern of markers on their cell membranes.
Figure 1 illustrates this pattern by indicating the presence or absence of several distinct cell surface markers. Other markers which can be used u~ positively or negatively select for dendritic cells include ICAM-I (CD34}, LFA-3 (CDSB), and CD l lb. Dendritic cells isolated from human or mouse blood, but not skin, express CD l la or LFA-1. In skin, the immunostimulatory effect of dendritic cells may be enhanced by cyrtokines, particularly by GM-CSF.
~r Dendritic cells, or other APCs, can be selected to obtain a population comprised substantially of dendritic cells, i.e., greater than about 50% dendritic cells, more preferably greater than about 75% dendritic cells, more preferably still greater than about 90% dendritic cells, with greater than about 9~5% dendritic cells being particularly preferred. The antigen-presenting cells are preferably isolated from the subject into which the activated T cells are to be active ("autologous" therapy).
Alternatively, the cells can be obtained from a donor or a cell bank (e.g., a blood bank).
The invention provides a method for preparing antigen-presenting cells that present an array of antigens. APCs can be genetically modified! using one or more-vectors of the invention. Antigen-presenting cells are transduced with vectors encoding an array of antigens. These antigens are then expressed in the cells, processed by the cells, and the relevant processed antigen fragment is routed to the cell surface where it can be presented.
The culture of cells used in conjunction with the present invention, including cell lines and cultured cells from tissue or blood samples, including dendritic cells is well known in the art. Freshney (Culture of Animal Cells, a Il~ianual of.Basic Technique, third edition WIley-Liss, New York (1994)) and the references cited therein provides a general guide to the culture of cells.
T Cells T cells can be isolated and activated in vitro by contact with an antigen-presenting cell.
Several such techniques are well-known. The expression of surface markers facilitates identification and purification of T cells. Methods oiE identification and isolation of T
cells include FACS, incubation in flasks with fixed antibodies which bind the particular cell type and panning with magnetic beads.
T cells and dendritic cells are characterized by expression of particular markers an the surface of the cell, and lack of expression of other markers. For instance, dendritic cells express MHC molecules and costimulatory molecules (e.g., B7-l and B7-2), a lack of WO OOI09665 PCT/US99/t808'7 markers specific for granulocytes, NK cells, B cells, and T cells. In the mouse, some, but not all, dendritic cells express 33D1 (dendritic cells from spleen and Peyer's patch, but not skin or thymic medulla), NLDC 145 (denalritic cells in skin and T-dependent regions of several lymphoid organs and CD 11 c) ((~D I I c also reacts with macrophage).
T cells are positive for various markers depending on the particular subtype, most notably CD4 and CDB.
CeII isolation or immunoassays for detection of cells during cell purifcation can be performed in any of several configurations, e.g., reviewed in Ivlaggio (ed.) 1980, Enzyme Immunoassay CRC Press, Boca Raton, Florida; Tijan 198, "Practice and IO Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam; Harlow and Lane, supra; Chan (ed.} 1987, Immunoassay: A Practical Guide Academic Press.
Orlando, FL;
Price and Newman (eds.) I991, Principles and Pracaice of Immunoassays Stockton Press, NY; and Ngo (ed.) I988, Non-isotopic Immunoassays Plenum Press, NY. For a 1 ~ review of immunoiogical and immunoassay procedures in general, see Stizes and Te:r (eds.) 1991. Basic and Clinical Immunology (7th ed.). For a discussion of how to make antibodies to selected antigens, see, e.g., Coligan I991, Current Protocols in Immunology WileylGreene, N.Y.; and Harlow and Lane 1989, Antibodies: A
Laboratory Manual Cold Spring Harbor Press, N.Y"; Stites et al. (eds.) Basic and 20 Clinical Immunology (~.th ed.).
Mast preferably, cells are isolated and characterized by flow cytometry methods using fluorescence activated flow cytometry (FACS). A «ride variety of flow-cytometry methods are known. For a general overview of FAGS see, for example, Abbas et al.
1991, Cellular and Molecular Immunology; W.B. Sounders Company, particularly 2~ chapter 3, and Kuby 1992, Immunology, W.H. Freeman and Company, particularly chapter 6.
!9 Methods The invention provides methods for using antigen-presenting cells that present an array of antigens. In one embodiment, the invention provides a method of activating immune cells in vivo. Examples of immune cells include, but are not limited to, T
cells, including cytotoxic T lymphocytes and helper T cells. In one embodiment, the method comprises administering to a subject a vector encoding an array of antigens preferentially expressed by a target cell population as compared with a non-target population. The vector is preferably constructed so as to be capable of directing . _ expression of the preferentially expressed antigens in an antigen-presenting cell.
Preferably, the antigen-presenting cell is a dendritic cell. The vector can be further modified, as described above, to encode an immunonaodulatory cofactor. The target cell can be, for example, a cancer cell, virus, bacterium, parasite, or other disease-associated cell. Upon administration, the vector transduces an APC in the subject, thereby genetically modifying an APC in vivo. The genetically modified APC is then available I S to contact and activate an immune cell.
In another embodiment, the method of activating immune cells comprises contacting an immune cell with an antigen-presenting cell genetically modified to express an array of antigens preferentially expressed by a target cell population as compared with a non-target population. Preferably, the antigen-presenting cell is a dendritic cell. The contacting can occur in vivo or ex vivo. Examples of eliciting an in vitro CTL
response are provided by S. Nair et aL, 1993, 3. Virol. 68:685 and F.J. Rouse et al., 199.4, J.
Virol. 68:6685. Example of stimulating an in vivo T c~elI response are provided by A.
Porgador et aL, 1996, J. lmmunol. 156(8):2918-2926; E.C. McICinney and J.W.
Streilein, 1989, J. Immunol. 143:1560; and H. Takahashi et al., 1993, Int.
Immunol.
5:849. Far in vivo contact, the APC is administered to a subject.
Alternatively; immune cells are isolated from a subject and brought into contact with the APC in vitro or ex vivo. The immune cells, such as T cells, are activated c.~x vivo and then can be reintroduced into the subject or provided to a different subject where they can then come in contact with target cells in that subject. Techniques for adoptive immunotherapy of cancer are described and reviewed in Chang, A.E. and S. Shu, 1996, Crit. Rev. Oncol. Hematol. 22(3j:213-228; and Kradin, R.K., 1993, in Therapeutic Applications of Interleukin-2, Atkins, M.B. and J.V~. Mier, eds., Marcel Dekker, Inc.
NY, pp. 217-232.
The activation of immune cells by the above methods can, in some embodiments, be used to kill target cells. Thus, the invention provides a method for killing a target cell comprising contacting an immune cell with an APC'. genetically modified in accordance with the invention. The contacting can be effected inn vivo by administering a genetically modif ed APC or by administering a vector of the invention which transducer an APC
within the subject, which then contacts an immune cell. Alternatively, the contacting can occur in vitro. Immune cells activated in vitro b;y contact with genetically modified APCs can be administered to a subject.
Alternatively, the selection of vectors or APCs can be designed to obtain a toleragenic response, for example, by contacting the APC with a T~ cell. Thus, the invention additionally provides a method of inducing a toleragenic response comprising contacting an immune cell with an APC genetically modified in accordance with the invention. Inducing a toleragenic response can be useful for such applications as treatment of autoimmune disorders and inhibiting rejection of foreign tissue, such as transplant tissue or autologous cells which have been genetically modif ed with foreign material. As with the methods for killing a target cell, the methods for inducing a toleragenic response can be effected by APCs genetically modified in vitro or in vivo.
The invention additionally provides a method of preventing disease such as infection or cancer comprising administering to a subject a composition comprising a vector or APC of the invention. Also provided is a method of treating disease such as cancer or infection comprising administering to a subject a composition comprising a vector or APC of the invention. Examples of infections include, but are not limited to, viral, bacterial and parasitic infections. Examples of cancers include, but are not limited to, melanoma, glioma, and cancers of the colon, breast, prostate, lung and Liver.

Compositions The invention provides compositions which are useful for treating and preventing disease, such as cancer or infection. In one emboaliment, the composition is a pharmaceutical composition. The composition can comprise a therapeutically or prophyiactically effective amount of a vector and/or antigen-presenting cell of the invention, as described above. The composition can optionally include a carrier, such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention. Most typically, quality controls (microbiology, clonogenic assays, viability tests), are performed and the cells are reinfused back to the patient.
preceded by the administration of diphenhydramine and hydracorti;>one. See, for example :Vi.
Korbling, et aI. 1986, Blood 6 i:~29-X32 and Haas et al. 1990, EYp. Hematol. 1 Q:94-98.
1 ~ Formulations suitable far parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and carriers include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Intravenous or intraperitoneal administration is a preferred method of administration.
Methods Of Administration In one embodiment of the invention, a patient infected with a virus such as HIV-1 or suffering from a cancer such as a melanoma can be treated by administering genetically modified antigen-presenting cells, or by using genetically modified antigen-presenting cells to activate a population of T cells against the infection or cancer, and introducing the T cells back into the patient. Thus, the present invention provides a method of producing cytotoxic T cells in vitro, ex vivo or in vavo. In another embodiment, the patient is treated by administering at least one vector encoding an array of antigens, wherein the vector includes a dendritic cell target element. The patient's own dendritic cells can then be genetically modified in vivo.
S T cells such as CD8+ CTLs activated in vitro can be introduced into a subject where they are cytotoxic against target cells bearing antigenic peptides corresponding to those the T cells are activated to recognize on class I MHC molecules. These target cells are typically cancer cells, or infected cells which express unique antigenic peptides on their MHC class I surfaces.
Similarly, helper T cells (e.g., CD4+ T cells), which recognize antigenic peptides in the context of MHC class II, are also stimulated by genetically modified antigen-presenting cells (e.g., dendritic cells), which can comprise antigenic peptides both in the context.of class I and class li MHC. These helper T cells also stimulate an immune response against a target cell. As with cytotoxic T cells, helper T cells are stimulated with the I5 genetically modified antigen-presenting cells in vitro ar in vivo. In one embodiment, a toieragenic response is generated by administration of genetically modified APCs via the stimulation of Ta,, cells.
Arrays of antigens are preferably associated with diseases selected from the group consisting of cancer, a hyperproliferative disease, a bacterial infection, a parasitic infection, and a viral infection. Diseases suitable for treatment using an immunostimulation strategy include: viral infections, such as those caused by HBV (see WO 93lIS207), HCV (see WO 93/15207), HPV (se:e WO 9210524$, WO 90/10459, EF'O 133,123), Epstein-Barr Virus (see EPO 173,254; JP 1,128,788; and U.S.
Patent Nos. 4,939,088 and S,I72,414), Feline Leukemia Virus (see W0 93/09070, EPO
2S 377,842, WO 90108832, and WO 93109238), Feline Immunodeficiency Virus (U.S.
Patent No. 5,037,753, WO 92/15684, WO 90/13573, and JP 4,126,085), HTLV I and II, ' and HIV (see WO 91/02805); cancers, such as melanoma, cervical carcinoma, colon WO 00/09bb5 PCT/US99/I8087 carcinoma, renal carcinoma, breast cancer, ovarian cancer, prostate cancer, leukemias;
and heart disease.
Bacterial infections that may be treated include, bui; are not limited to, pneumonia, sepsis, tuberculosis, and Staphylococcus infections, among others.
Parasitic infections that can be treated include, but are not limited to, malaria (caused by protozoa of the genus Plasmodium, and include P. ~alciparum, P. malariae, P.
ovate, and P. vivax), sleeping sickness (caused by trypanosomes), and river blindness.
Viral infections that can be treated include, but are not limited to, those caused by hepatitis A, hepatitis B, hepatitis C, non-A, non-B hepatitis, hepatitis delta agent, CMV, I O Epstein-Barr virus, HTLV I, HTLV II, FeLV, FIV, and HIV I.
Treatment includes prophylaxis and therapy. Prophylaxis or treatment can be accomplished by a single direct injection at a single time point or multiple time paints.
Administration can also be nearly simultaneous to multiple sites.
Patients or subjects include mammals, such as human, bovine, equine, canine, feline, 1 ~ porcine, and ovine animals.
In one embodiment, T cells or antigen-presenting cells are administered directly to the subject to produce T cells active against a target cancerous, infected, or other cell type.
Administration of these is by any of the routes normally used For introducing a cell into ultimate contact with a mammal's blood or tissue cells. In another embodiment, at least 20 one vector encoding an array of antigens is administered.
Compositions are typically administered in vivo via ;parenteral (e.g, intravenous, subcutaneous, and intramuscular} or other traditional direct routes, such as buccallsublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarteriai, intraperitoneal, int~raocular, or intranasal routes or 25 directly into a specific tissue, such as the liver, bone marrow, or into the tumor in the case of cancer therapy. Non-parenteral routes are discussed further in WO
96120732.

WO 00/09665 PCT/US99/t808~
The cells or vectors are administered in any suitable manner, often with pharmaceutically acceptable carriers: Suitable methods of administering cells in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular cell composition, a particular route can often provide a more immediate and more effective reaction than another route.
The dose of cells (e.g., activated T cells, or dendritic cells) administrated to a patient, in the context of the present invention should be sufficient to effect a benef cial therapeutic response in the patient over time, or to inhibit growth of cancer cells, or to inhibit infection. Thus, cells are administered to a patient: in an amount sufficient to elicit an effective immune response to the specific antigens andlor to alleviate;
reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection. An amount adequate to accomplish this is defined as a. "therapeutically effective dose."
The dose will be determined by the activity of the 'T cell or antigen-presenting cell produced and the condition of the patient, as well ~~s the body weighs or surface areas of 1 ~ the patient to be treated. The size of the dose also will be determined by the existence.
nature, and extent of any adverse side effects that accompany the administration of a particular cell in a particular patient. In determining the effective amount of the cell to be administered in the treatment or prophylaxis of diseases such as AIDS or cancer (e.g., metastatic melanoma, prostate cancer, etc.), the physician needs to evaluate circulating plasma levels, CTL toxicity, progression of the disease, and the production of immune response against any introduced cell type.
Generally at least about I O'~ to 106 and typically, between I x 108 and 1 x I0'° cells are infused intravenously or intraperitoneally into a 70 kg patient over roughly minutes. Intravenous infusion is preferred. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and I
hour following infusion arid saved for analysis. Ceil reim~usions are repeated roughly every month for a total of IO-12 treatments in a one year,period. After the first treatment, infusions can be performed on an outpatient basis at the discretion of the clinician. If WO 00!09665 PCTNS99/38087 the reinfusion is given as an outpatient, the participant is monitored for at least 4 hours following the therapy.
For administration, cells of the present invention can be administered at a rate determined by the LD-50 (or other measure of toxicity) of the cell type, and the side-s effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accom~Iished via single or divided doses.
The cells of this invention can supplement other treatments for a condition by known conventional therapy, including cytotoxic agents, nucleotide analogues and biologic _ response modif ers. Similarly, biological response nnodifiers are optionally added for treatment. For example, the cells axe optionally administered with an adjuvant, or cytokine such as GM-CSF, IL-12 or IL-2.
Administration by many of the routes of administration described herein or otherwise known in the art may be accomplished simply by direct administration using a needle, catheter or related device, at a single time point or at multiple time points.
In addition, I5 an "administration" of a gene delivery vehicle (or ex vivo transduced cells, for that matter) at a given time point includes administration to one or more areas, or by one or more routes. In certain embodiments of the invention, one or more dosages is administered directly in the indicated manner: intravenously at dosage greater than or equal to 103, 1 O5, 10', 109, 10'° or I0" cfu; intraarterially at dosages greater than or equal to 10', 10',10', 109, I0'° or 10" cfu; intrasmuss:ularly at dosages greater than or equal to 10'; I05, I0', I09, 10'° or 10" cfu, with dosages of 10'° or 10" cfu being preferred; intradermally at dosages greater than or equal to 103, I05, 10', 109, 10'° or 10"
cfu; pulmonarily at dosages greater than or equal to 10', 105, 10', 109, 10'° or 10" cfu;
subcutaneously at dosages greater than or equal to 103, 105, 10', 109, 10'° or 10" cfu, with dosages of 109, 10'° or 10" cfu being preferred; .interstitially at dosages greater than er equal to 103, I05, IO', 108, 10g, 10'° or I O" cfw; with dosages of 108, 109, 10'° or i 0" cfu being preferred; into a lymphoid organ such ~~.s the spleen, a tonsil, or a lymph node at dosages greater than or equal to 10', I05, I0', 108, 109, 10'°
or I O" cfu; into a tumor at dosages greater than or equal to 103, 10a, 105, I05, 10', 108, I09, 10'° or 10" cfu, WO 00/09b65 PCT/US99/18087 with dosages of i O8, 109, 10'° or 10" cfu being preferred; and into the afferent Iymph at dosages greater than or equal to 10', 105, 10', 10g, 1.09, l0t° or IO"
cfu. Far purposes of the convenience, "cfu" shall also refer to non-viral particles, such that one efu is equivalent to one-non-viral particle.
EXAMPLES
The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and' using the same. The examples are not intended in any way to otherwise limit the scope of the inventic>n.
Example 1: Preparation of dendritic cells genericaliv modified to present an array of antigens Dendxitic cells are isolated according to Basic Protocol I described in Tedder and Jansen, 1997, Current Protocols in Immunology, John Wiley and Sons, 7.32. I.
Cell lines from a matched pair of target and non-target cell types are obtained. A
matched pair of cell types includes, for example, cell lines derived from the same tissue of origin, I S such as prostate, and differing in a targeted feature. An example of a target cell is a prostate cancer cell. An example of a matched pair of cell types is prostate cells differing in their metastatic potential. Nucleic acid sequences preferentially expressed in prostate cancer tissue are described in United States patent application serial number 60/088,877, filed June I i, 1998, the entire contents of which are incorporated herein by reference. The dendritic cells are then transduced with the preferentially expressed nucleic acid sequences using conventional techniques, such as those described in WO
97124447, the entire contents of which are incorporated herein by refexence.
Successful txansduction of the human dendritic cells can be confirmed by in vitro T cell priming (Albert et al., 1998, Nature 392:86-89; Nair et al., 1998, Nature Biotechnology 16(4):364-369; Antigen Processing and Presentation, in Current Protocols in Immunology; Wiley, New York, 1998). Candidate immunogenic tumor-associated antigen sequence arrays can be determined by screening tmansduced dendritie cells with responding T cells obtained from peripheral blood err dLN of tumor-beaming patients.

Once a pattern of reactivity is found in.a particular tumor type, the relative efficacy of these antigens can be evaluated using marine homoiogs and syngeneic marine tumor models that express one or more of these antigens. Example 2 describes one method for evaluating e~cacy.
Example 2: Dendritic cell-based immunother~y for the treatment of metastatic tumors in combination with systemic ProIeukin~ IL-2 Genetically modified dendridc cells of the invention can be used to treat tumors, alone or in combination with other therapies. This example shows how to evaluate the contribution of dendritic cells genetically modified using marine homologs of nucleic I O acid sequences identified by the methods of Example I to systemic Proleukin IL-2 immunotherapy on marine syngeneic tumors. The C57Bl6 derived B i 6-1 10 lung metastasis model, CT-26 marine colon model or the marine 3LL lung model can be .
used to generate the most effective treatment regimen. These tumor models are poorly immunogenic and are differentially responsive to single agent Proleuitin therapy, as 1.~ measured by both survival and tumor load.
The research strategy can be used to define the optimal mixture of dendritic cells and systemic Proleukin iL-2, e.g. a lower amount of IL-2 to maintain a high objective response rate while lowering toxicities, and to develop a dendritic cell plus systemic IL-2 regimen for application to a lung and/or colon marine syngeneic tumor model that is 20 resistant to single agent IL-2 therapy.
Experimental Design:
Marine splenic dendritic cells are isolated and characterized as described by Girolomoni et al. i 990, J. Immunol. 145(9):2820-26. First, spleens from either naive or day 7 tumor bearing mice are removed, minced, and digested in Hank's Balanced Salt Solution 25 (HBSS) with 40 mg coilagenase (Sigma, St. Louis, MO) for 1 hour. Cells are then filtered over I00 Lun nylon mesh; and washed. Red blood cells are lysed with 0.8~%
Ammonium chloride, 0.1 % KHCOZ, 0.004% EDTA (ACK Lysis buffer); pellet w _ ~ WQ 00/09665 PCT/US99/18087 resuspended to 3 x 10' cellslml in 1.035 Percoll (lPharmacia biotech) and underlay with an equal volume of I .075 gJml Percoll. The suspension is centrifuged at 2200 rpm for 20 minutes at 4°C. The band is harvested at interface, washed twice with HBSS, and resuspended to S x 106 cells/ml in complete culture medium and incubated at 37°C for S 90 min. Nonadherent cells are removed and discarded. Fresh complete culture medium is added, and culture continued for 18-24 hr at 37°C. Gentle pipetting dislodges splenic dendritic cells.
CeIIs harvested from the spleen cell cultures are further enriched by overlaying 2 mls of S x 1061m1 cells onto a 3 ml layer of 14.5% Metrizamide-CM solution in a 1 S
ml I O centrifuge tube. The gradient is centrifuged at 2000 rpm for I S min at 4°C, the band harvested, and washed, counted, and resuspended to S x I06 ceils/mI in complete medium.
An aliquot can be removed for phenotyping using the following 3-color staining protocol ("DC" refers to dendritic cells):
I S Tube 1 CD3/Isotype Isotype control Tube 2 CD31CD41CD8 CD4/CD8 T cells Tube 3 CD4S/I-AbICD86 CD vs monocytes Tube 4 CD4SICD801CD40 DC vs monocytes Tube S CD4S/CD 1 I b (Mac- I )ICD DC vs monocytes 11 c 20 Tube 6 CD4SICD4SR(B22))/CD44 DC vs B cells Bone marrow derived dendritic cells (BM-DC) are isolated from erythrocyte depleted mouse bone marrow cells cultured for seven days in complete medium supplemented with 10 ng/m1 GM-CSF and 10 ng/ml IL-4. On day seven, BM-DC are harvested by 2S gentle pipetting and further enriched by 14.5% (by weight) metrizamide (Sigma, St.
Louis, MO) complete medium gradients. The Iow density interface containing the BM-DC are collected by gentle pipette aspiration. The BM-DC are washed twice with complete medium, enumerated (purity >90%, positive co-expression of MHC Class II, CD40, CD80, CD86 and CD 1 I c).

i.

Genetically modified dendritic cells prepared as described in Example 1 and/or 2 are then injected either before (protective) or after (therapeutic) tumor challenge. For protection, an intravenous {iv) tumor challenge is introduced 1 week following the last immunization (days -15, -8, 0). For therapy, immunizations are given at days 4, 8, and 12 post-iv tumor challenge; with or without Proleukin.
Proleukin therapy is administered as follows:
For protection: 10 mg/kg iv for 5 days starting with second immunization.
For therapy: 10 mg/kg/day iv for 5 or 10 days starti~.lg on day 2 post-iv tumor challenge.
Experiment #I : Single agent Proleukin therapy (10 mice/group):
B I 6-F 10: 50-85,000 cells iv DO
Proleukin: iv twice per day for seven days, beginning at Day 3 Sacrifice and count lung metastases Group 1 B 16 PBS control 2 B I 6 2.5 mg/kg Proleukin

3 B i 6 3.5 mg/kg Proleukin

4 B 16 5 mg/kg Proleukin Experiment #2: Take best Proleukin regimen {~2/I0 dead mice with >50%
reduction in lung metastases).
Group 1 B 16 PBS

2 B 16 Proleukin 3 B 16 modified DC alone 4 B 16 modif ed DC + Proieukin

5 B 16 DC- alone

6 B 16 DC- + proleukin

7 B 16 irradiated B 16 alone (50,000/iv) WO 00/09665 PCTIUS99l18087

8 B I 6 irradiated B I 6 + Proleukin Experiment #3: Take most active DC regimen {double single agent Proleukin lung metastases reduction) Group 1 B 16 PBS

2 B 16 Proleukin 3 B I6 modified DC alone 4 B I6 modif ed DC + Proleu~kin 5 B 16 modified DC + 75% Proleukan 6 B 16 modified DC + 50% Proleukin I O 7 B 16 modified DC + 25% Proleukin The potency of the genetically modified dendritic cells (DC) can be evaluated with a short term culture immune function assay, based on the antigen-induced expression of CD69. Briefly, whole blood andler spleen cells from naive, tumor-bearing, and DC-immunized mice are prepared as single cell preparations, depleted of macrophages by I ~ adherence, and resuspended to 5 x I 0 6 cells/ml. Five hundred microliters of various DC sources {x-irradiated) are titrated into 12 x 75 mm polypropylene tubes containing 0.5 ml of the monocyte-depleted responding cells. T'he mixed cells are cultured for 24 hours at 37°C, with a 6 hr timepoint taken for analysis. The cultures contain Brefeldin for the last 4 hours of culture. The cultures are then surfaced-stained with 20 CD3/CD4/CD69 or CD3/CD8/CD69. Duplicate cultures are permeabilized, and stained with CD3/CD4/IFNy, CD3/CD4/IL-4, CD3/CD8lIFNy, and CD3/CD8/IL-4. Reagents for TNFa and GM-CSF are used to further defne the CD8 reactivity to the genetically modified DC.
DC loaded with baculovirus-produced mouse gp 100 (cloned out from B 16F I O) are used 25 as a positive control, Serum from the immunized mice can be obtained, pelleted, heat-inactivated, and stared at -70°C for future analysis of anti-tumor binding activity, cytokinelchemokine content, sIL-2R, and other inflammatory molecules.

The foregoing detailed description provides exemplary information about the invention.
Those skilled in the art will appreciate that modifications can be made without diverging from the spirit and purpose of the invention,

Claims (40)

What is claimed is:

1. A method of producing at least one vector encoding an array of antigens for expression in an antigen-presenting cell comprising:
(a) comparing first nucleic acid sequences expressed by a target cell population with second nucleic acid sequences expressed by a non-target cell population;
(b) selecting nucleic acid sequences preferentially expressed by the target cell population relative to the non-target cell population; and (c) introducing the selected nucleic acid sequences into at least one vector capable of directing expression of the selected nucleic acid sequences in an antigen-presenting cell.

2. The method of claim 1, wherein the antigen-presenting cell is a dendritic cell, macrophage, B cell, monocyte or fibrocyte.

3. The method of claim 1, wherein the vector further comprises an antigen-presenting cell targeting element.

4. The method of claim 1, wherein the first and second nucleic acid sequences are of the same tissue of origin.

5. The method of claim 1, wherein the selected nucleic acid sequences comprise at least 5 different nucleic acid sequences.

6. The method of claim 1, wherein the selected nucleic acid sequences comprise at least 7 different nucleic acid sequences.

7. The method of claim 1, wherein the selected nucleic acid sequences comprise at least 9 different nucleic acid sequences.

8. The method of claim 1, wherein the vector further comprises a nucleic acid sequence encoding an immunomodulatory cofactor.

9. The method of claim 8, wherein the immunomodulatory cofactor is IL-2, IL-3, IL-8, OKT3, .alpha.-interferon, .gamma.-interferon, or MIP- 1.alpha..

10. The method of claim 1, wherein the vector further encodes at least one selectable marker.

11. The method of claim 10, wherein the selectable marker is PLAP, GFP or neomycin resistance.

12. The method of claim 1, wherein the target cell is a cancer cell.

13. The method of claim 1, wherein the target cell is a virus, a bacterium or a parasite.

14. A composition comprising at least one vector produced by the method of claim 1.

15. The composition of claim 14, wherein the vector further comprises an antigen-presenting cell targeting element.

i 6. The composition of claim 14, further comprising an antigen-presenting cell.

17. A method of producing an antigen-presenting cell that presents an array of antigens comprising:
(a) comparing first nucleic acid sequences expressed by a target cell population with second nucleic acid sequences expressed by a non-target cell population;

(b) selecting at least one nucleic acid sequence preferentially expressed by the target cell population relative to the non-target cell population; and (c) genetically modifying an antigen-presenting cell to express the selected nucleic acid sequences.

18. The method of claim 17, wherein the antigen-presenting cell is a dendritic cell, macrophage, B cell, monocyte or fibrocyte.

19. The method of claim 17, wherein the first and second nucleic acid sequences are of the same tissue of origin.

20. The method of claim 17, wherein the selected nucleic acid sequences comprise at least 5 different nucleic acid sequences.

21. The method of claim 17, wherein the selected nucleic acid sequences comprise at least 7 different nucleic acid sequences.

22. The method of claim 17, wherein the selected nucleic acid sequences comprise at least 9 different nucleic acid sequences.

23. The method of claim 1, wherein the selected nucleic acid sequence further encodes at least one selectable marker.

24. The method of claim 23, wherein the selectable marker is PLAP, GFP or neomycin resistance.

25. The method of claim 17, wherein the target cell is a cancer cell.

26. The method of claim 17, wherein the target cell is a virus, a bacterium or a parasite.

27. An antigen-presenting cell produced by the method of any one of claims 17-26.

28. A method of activating T cells comprising contacting a T cell with an antigen-presenting cell of claim 27.

29. The method of claim 28, wherein the T cell is a cytotoxic T lymphocyte.

30. A method of inducing a toleragenic response comprising contacting a T cell with an antigen-presenting cell of claim 27.

31. The method of claim 30, wherein the T cell is a T H2 cell.

32. The method of claim 28 or 30, wherein the contacting occurs in vivo.

33. The method of claim 28 or 30, wherein the contacting occurs ex vivo.

34. The method of claim 32 or 33, wherein the activating is in the presence of an immunomodulatory cofactor.

35. The method of claim 34, wherein the immunomodulatory cofactor is IL-2, IL-3.
IL-8, OKT3, .alpha.-interferon, .gamma.-interferon, or MIP-1.alpha..

36. A method of activating T cells in vivo comprising administering the composition of claim 14 to a subject.

37. A method of killing a target cell in vivo comprising administering the composition of claim 14 or the antigen-presenting cell of claim 27 to a subject.

38. A method of preventing infection comprising administering the composition of claim 14 or the antigen-presenting cell of claim 27 to a subject.

39. A method of treating cancer comprising administering to a subject the composition of claim 14 or the antigen-presenting cell of claim 27, wherein the target cell is a cancer cell.

40. A method of treating an infection comprising administering to a subject the composition of claim 14 or the antigen-presenting cell of claim 27, wherein the target cell is an infectious agent.