JP2002515734A - Immunostimulation mediated by genetically modified dendritic cells - Google Patents

Abstract

  (57) [Summary] Compositions and methods are provided that are useful for stimulating an immune response to one or more disease-associated antigens by genetically modifying dendritic cells in vivo or ex vivo. These compositions and methods allow for the administration of lower doses of the gene delivery vehicle to achieve immunostimulatory levels comparable to those obtainable by conventional methods. Alternatively, administration of a dose of a conventional gene delivery vehicle enhances the resulting immune response.

Description

DETAILED DESCRIPTION OF THE INVENTION             Immunostimulation mediated by genetically modified dendritic cellsTECHNICAL FIELD OF THE INVENTION   The present invention relates generally to recombinant DNA technology. In particular, the present invention provides (i) Transduction in vivo, or (ii) functionally co-administering at least one disease-associated antigen. Injection of dendritic cells transduced ex vivo with an expression vector Compositions or methods useful for prophylactic or therapeutic stimulation of the animal's immune system. Related.Background of the Invention   Stimulation of the immune system against disease-related antigens is an accepted approach to disease prevention. It is a approach. Conventional technology is based on killed bacterial vaccines made from various viral pathogens. Or the use of live attenuated vaccines. With the advent of recombinant DNA technology, A new generation of safer vaccines to combat viral infections (where immunostimulants Are typically immunogenic proteins encoded by pathogens) Noh.   Despite these technologies, many viral diseases and cancers have Only few effective treatments or preventive measures have been developed. In recent years, some guru Has been used in animals to respond to HIV-encoded gene products by using gene therapy techniques. We report immunity induction. Specifically, retroviruses encoding the HIV env / rev gene Autologous fibroblasts transduced ex vivo with viral vectors were Injections into humans, non-human primates, and humans to elicit an immune response to these antigens Led. See Warner et al., 1991; Laube et al., 1993; and Zeigner et al., 1994. .   However, such an ex-vivo approach requires individual products (ie, Autologous cells) must be generated for each patient. Not practical for a chin vaccination program. This of the ex vivo approach To overcome these and other drawbacks, direct the expression of disease-specific immunogens Administering a gene delivery vehicle carrying an expression vector directly to a patient, Technology development is being attempted. WO 91/02805, WO 93/10814, WO 93/15207, WO 94 See / O6921, WO 94/21792, and WO 95/07994. Irwin et al. (1994) After intramuscular injection of recombinant retrovirus into mouse, rhesus monkey, and baboon, Immune induction against a particular immunogen was reported. Improved immunity to disease-associated antigens There is a need for compositions and methods that allow for epidemiological stimulation.Summary of the Invention   It is an object of the present invention to provide immunoprophylactic or immunization of mammals, especially animals, including humans. It is to provide compositions and methods for therapeutic treatment. The composition of the present invention , Against diseases such as cancer, or bacterial, parasitic, or viral To immunize an animal against infection, the animal is given an immunogen (ie, a disease-associated antigen). Can be used to deliver. Such immunity can be achieved by (i) gene delivery vehicles. Encoded by an expression vector that is delivered by Immunogen expressed and presented on the surface of the vesicle (APC), or (ii) APC (especially dendritic cells) transduced by such an expression vector The result of the development of a cellular immune response to the presented immunogen.   One aspect of the present invention targets dendritic cells, either in vivo or in vitro. Gene delivery vehicle. Such gene delivery vehicles include dendritic cells Expression vector directing the expression of a target element and at least one disease-associated antigen Including In one embodiment, the expression vector is a recombinant virus. Will be retained. Recombinant viruses are useful in the practice of the present invention, and DNA viruses And RNA viruses. In a preferred embodiment, the recombinant Russ is derived from either a minus-strand RNA virus or a plus-strand RNA virus. It is. Examples of positive-strand RNA viruses from which the recombinant virus can be derived include Toroviruses (eg, chicken leukemia virus, bovine leukemia virus, mouse Leukemia virus, mink cell focus forming virus, mouse sarcoma virus, Reticuloendotheliosis virus, Rous sarcoma virus, Mason-Petizer monkey virus , Baboon endogenous virus, cat endogenous retrovirus, gibbon ape leukemia virus , HIV I, HTLV I, and HTLV III), especially mouse retroviruses, togavirus Viruses, such as alphaviruses, especially Sindbis virus, Semliki Forest Ills and Venezuelan equine encephalitis virus, picornaviruses, and Ronaviruses, including retroviruses and alphaviruses Are preferred. As an example of a minus-strand RNA virus from which a recombinant virus can be derived Rhabdoviruses (eg, vesicular stomatitis virus), myxoviruses , Paramyxoviruses, orthomyxoviruses (eg, influenza Irs), and Bunia viruses. Useful in the practice of the present invention Useful DNA viruses from which the recombinant virus can be derived include adenoviruses and antigens. Includes deno-associated viruses.   In another embodiment, the gene delivery vehicle is a non-viral gene delivery vehicle. Kuru. That is, the expression vector is not carried by the virus. This Expression vectors are either DNA or RNA, and are linear or circular obtain. Particularly preferred expression vectors are eukaryotic layered vector initiation systems. is there. In one embodiment, the expression vector contains one or more polynucleotides. Forms a complex with a condensing agent. Polynucleotide flocculant is polycarbonate Thione. Preferred polycations include polylysine, polyarginine , Histones, protamine, spermidine, and spermine; Lysine is particularly preferred. In another embodiment, the expression vector is a dendritic cell target Form a complex with only the element. In yet another embodiment, the expression vector Is associated with lipids, preferably liposomes (particularly those composed of cationic lipids). Posomes).   If the gene delivery vehicle targets dendritic cells, the dendritic cell targeting element , Can be any molecule that targets the gene delivery vehicle to dendritic cells. Various implementations In an embodiment, the dendritic cell targeting element is a high affinity binding pair, a dendritic cell surface marker. Antibodies reactive against dendritic cell surface markers Embodiments selected from the group consisting of antigen binding domains are included. Preferred high parents Biotin / avidin, cytostatin / papain and phos And those selected from the group consisting of phonate / carboxypeptidase A. Good Ma New dendritic cell surface markers (to which antibodies (or antigen-binding To generate dendritic cell targeting elements) include CD11c, CD54, C D58, CD25, CD11a, CD23, CD32, CD40, CD1, CD45, MHC class I, MHC class II , Mac-1, Mac-2, and Mac-3. In another embodiment, a dendritic cell The target element is a hybrid envelope protein, where the envelope The envelope part is a virus envelope protein (for example, a retrovirus envelope). Protein) and the dendritic cell targeting element is a dendritic cell It is derived from a protein that specifically interacts with molecules displayed on the membrane.   The expression vector of the invention directs the expression of at least one disease-associated antigen. So Antigens such as are preferably cancer, hyperproliferative diseases, bacterial infections, parasitic infections, And viral infections. Described in this specification That can be treated, suppressed, or prevented using the immunostimulatory compositions and methods provided Among them are, among others, breast, colon, melanoma, lung, brain and Leukemia. Bacterial infections that can be treated, suppressed, or prevented include: This includes pneumonia, sepsis, tuberculosis, and staph infections. Can be Parasitic infections that can be treated include, among others, malaria (Plasmodium spp.) Protozoa (including P. falciparum, P. malariae, P. ovale, and P. vivax) ), Sleeping sickness (caused by trypanosoma), and And infections that cause ocular onchocerciasis. As described herein As a viral infection that can be treated, suppressed, or prevented using the compositions and methods Is a cause of hepatitis A, hepatitis B, hepatitis C, non-A non-B hepatitis, hepatitis delta, among others. Offspring (hepatitis delta agent), CMV, Epstein-Barr virus, HTLV I, HT Infections caused by LV II, and HIV I.   In one embodiment, the expression vector encodes one disease associated antigen. In other embodiments, the expression vector comprises a plurality of disease associated antigens (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more antigens). Multiple disease related If the antigens are encoded by a single expression vector, they will Or it can be associated with a different disease. Alternatively, a disease encoded by another expression vector A set of expression vectors each encoding one or more antigens associated with a disease different from the disease Compositions containing the combinations can also be prepared. One or more anti-tumors associated with different diseases Compositions encoding the original are particularly useful as vaccines.   In yet another embodiment of this aspect of the invention, the expression vector is also immunomodulatory. Encode a cofactor.   Preferred embodiments of the invention are directed to various gene delivery vehicles and pharmaceuticals of the invention. Pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent. Especially preferred Some embodiments include those in which the pharmaceutical composition is in a solid form.   A second aspect of the present invention is an in vivo method for producing a genetically modified dendritic cell. Bo method. Such a method is a gene delivery vehicle targeting dendritic cells. Administering the gene delivery vehicle to the animal, wherein the gene delivery vehicle comprises a dendritic cell targeting agent. An expression vector that directs the expression of at least one disease-associated antigen. Including.   In a related aspect, a method of prevention is provided. In one embodiment, the Prevention is accomplished by administering to the animal a prophylactically effective amount of a gene delivery vehicle according to the present invention. Is achieved. In another embodiment, the prevention is at least the anti-antigen of the disease associated antigen. A gene encoding sufficient genetic information to direct expression of the gene encoding the Transduced ex vivo with a gene delivery vehicle carrying the current vector , By administering a prophylactically effective amount of the population of dendritic cells to the animal.   In yet another related aspect of the invention, therapeutic treatment of disease (but not prophylaxis). ) Are provided. In one embodiment, the therapeutic treatment is a dendritic cell. By administering to the animal a therapeutically effective amount of a gene delivery vehicle that targets the vesicles Achieved. Here, the gene delivery vehicle is at least the antigenic portion of the disease-associated antigen. Expression vector that encodes sufficient genetic information to direct expression of the gene encoding Owns In another embodiment, the therapeutic treatment is to reduce the amount of the disease-associated antigen. Both encode sufficient genetic information to direct expression of the gene encoding the antigenic portion Ex vivo transduction with a gene delivery vehicle carrying an expression vector This is accomplished by administering to the animal a therapeutically effective amount of the population of dendritic cells.   Methods for preventing or treating such animals include gene delivery vehicles, or simply Single direct injection of single or multiple ex vivo transduced dendritic cell populations Thus, it can be achieved. In another embodiment, the method comprises the gene delivery of the present invention. Vehicle or ex vivo transduced dendritic cell population To multiple sites. Preferred routes of administration are intravenous and Includes subcutaneous route. Preferred animals for prophylactic treatment by such methods include birds , Mammals, and fish. Particularly preferred mammals are humans, cows, cormorants Encompasses mammals selected from the group consisting of ma, dog, cat, pig, and sheep I do.   Yet another aspect of the invention is an ex vivo method for producing a genetically modified dendritic cell. Bo method. In one embodiment, such a method is performed from dendritic cells. Substantially composed (ie, greater than about 50% dendritic cells, more preferably about 75% Greater than about 90% dendritic cells, more preferably still greater than about 90% dendritic cells, Obtaining more than 95% of the dendritic cells). Genetically modifying this cell population by introducing a gene delivery vehicle I do. In another embodiment, the method comprises obtaining a first population of cells comprising dendritic cells. The first population of cells by introducing the gene delivery vehicle of the present invention. Genetically modifying the second population of cells substantially consisting of dendritic cells. Partially isolating from the first population of genetically modified cells. Dendritic Preferred methods for isolating a cell population substantially composed of cells include Affinity Tea chromatography and FACS.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows some cell surface markers useful for identifying dendritic cells . Since the marker shown by (-) is not present on dendritic cells, Can be used in strategies. Markers indicated by (+) are present on dendritic cells And (++) and (+++) indicate particularly useful dendritic cell markers. (+), (++) , And (+++) dendritic cell markers are also targeted by the gene delivery vehicles of the present invention. Indicate suitable targets that can be used.   The flow chart in FIG. 2 illustrates the spleen cell separation strategy using the Percoll density gradient. Is shown.   FIG. 3 shows the effect of dendritic cell therapy on CT26.p24. Balb / c mice in total 2 × 10^FiveOf tumor cells were injected. Shown in the figure on days 8, 15, and 22 Animals were treated as described. The tumor volume of the mice was measured twice a week.   FIG. 4 shows the effect of dendritic cell therapy on JC.p24. 3 in Balb / c mice × 10^FiveOf tumor cells were injected. As shown in the figure on days 8, 15, and 22 The animals were treated. The tumor volume of the mice was measured twice a week.   FIG. 5 shows the effect of dendritic cell immunization on JC.p24. Balb / c mouse with p24 Immunize twice (at one week intervals) with transduced scleral dendritic cells and then^Five Of tumor cells were injected. The tumor volume of the mice was measured twice a week.   FIG. 6 shows the dendritic cell immunity against JC.p24 after a single immunization with BM-DC.p24. Show the effect. Balb / c mice were immunized once with BM-DC.p24. One week later, this mouse 3 × 10 in total^FiveOf tumor cells were injected. Measure tumor volume twice a week in mice Specified.   FIG. 7 shows the effect of dendritic cell immunization on JC.p24. Balb / c mouse, D2SC / 1 .beta.-gal and D2SC / 1.p24 were treated on days 1 and 8. Next, this mouse 3 × 10 in total^FiveOf tumor cells were injected on day 15. Tumor volume of mice in one week Measured twice.   FIG. 8 shows the effect of dendritic cell immunization on CT26.p24. Balb / c mice for one day Eyes and days 7 were immunized with the vaccine described in the figure legend. On the fifteenth day, 2 × 10 in total^FiveOf tumor cells were injected. Measure tumor volume twice a week in mice Specified.   FIG. 9 shows a comparison between methods of introducing antigen into dendritic cells. This is a retro ui Russ vectors are compared for protein or peptide loading. Balb / c mice were immunized on days 1 and 7 with the vaccine described in the figure legend. 8 On day, a total of 2 × 10^FiveOf CT26.β-gal tumor cells were injected. of mouse Tumor volume was measured twice a week.Definition of terms   The following terms are used throughout the specification. Unless otherwise indicated, these Words are defined as follows:   "Gene delivery vehicle"Represents one or more genes or sequences of interest in a host cell. (And in preferred embodiments, expressed). This Representative examples of such vehicles include viral vectors, naked DNA or RNA Current vector, DNA or RNA expression vector associated with cationic flocculant, Liposo A DNA or RNA expression vector encapsulated in a vector and certain eukaryotic cells. Vesicles (eg, producer cells). Of the preferred embodiment of the present invention Within the scope, the gene delivery vehicle may include a target element (eg, the gene Covalently linked to the delivery vehicle, expressed on the gene delivery vehicle, or Is included as an integral part of the gene delivery vehicle, of a high affinity binding pair. Members).   "High affinity binding pairIs 10^-yK less than M_DMolecules that can bind to each other in Means a set of where y is a group consisting of 8, 9, 10, 11, 12, 13, 14, and 15 Selected from As used herein, "K_DIndicates the dissociation of reaction A + B = AB Means a number, where A and B are members of this high affinity binding pair. The minute As is understood in the field, as the affinity of two molecules increases, K_DDecreases I do. Affinity constants can be determined, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. . 51: 660-672, 1949), easily determined by various techniques, including Can be done. Representative examples of suitable affinity binding pairs include biotin / avidin, cytos Statins / papain, and phosphonate / carboxypeptidase A You.   "Target element"Means a molecule that can specifically bind to dendritic cells. Departure In the context of the description, the binding of the target element and its complement If biological effects are seen in the More than 10-fold difference between the binding of the target and non-target cells, preferably 25, 50 or 10 If there is more than a 0-fold difference, the target element will bind specifically to dendritic cells. Conceivable. Generally, the target element is a dendritic cell, K_D((Scatchard minutes mentioned above) 10)^-FiveLess than M, preferably 10^-6Less than M, more preferably 10^-7Less than M And most preferably 10^-8It is preferred that the bond be less than M. Furthermore, high affinity When a sexual binding pair is used for targeting, the target element is preferably a high affinity sex Dendritic with an affinity at least 1 log (ie 10 times) less than the affinity constant of the binding pair Binds to cells. Suitable targeting elements are preferably non-immunogenic, It is not degraded by proteolysis and is not removed by the immune system. You. Particularly preferred target elements are preferably in animals (clearin g) (in the absence of g agent) has a half-life of between 10 minutes and 1 week. Appropriate target Representative examples of the elements will be described later in more detail.   "Target dendritic cells"Refers to a method of targeting dendritic cells. Such a method is Defined as follows: gene delivery vehicles are one of many traditional modalities. After administration in a desired manner, occurs when the gene delivery vehicle lacks the element. Elements that cause increased transduction of dendritic cells compared to transduction Or has reduced transduction of dendritic cells compared to the normal or conventional route of administration. The gene delivery vehicle is administered in a particular manner or by a particular route so as to be enhanced. It is.   "Clarifying agentMeans binding and / or cross-linking to the circulating bound target element Means a molecule. Preferably, the clarifying agent is non-immunogenic and Specific to the target element and large enough to avoid rapid renal clearance. Good. Further, the clarifying agent is preferably not degraded by proteolysis and Not removed by the system. Particularly preferred fining agents include bound target elements. Binding to a different site than the affinity binding member. Also preferably binds to block the binding of the target element to its target Things. Numerous fining agents may be used in the context of the present invention, for example, Marshall et al., Brit. J. Cancer 69: 502-507, 1994.   "Expression vector"Refers to the expression of one or more genes encoding disease-associated antigens. Refers to a recombinant nucleic acid molecule (DNA or RNA) that is capable of directing. The expression vector encodes the antigen A promoter operably linked to the gene to be expressed (the expression vector Unless designed for site-specific integration adjacent to the motor ), And a polyadenylation sequence. In certain embodiments Thus, the expression vector is part of the plasmid construct. In addition to the expression vector components The plasmid construct may also include one or more of the following: Dot; one or more selectable markers; plasmid construct present as single-stranded DNA Signals (eg, M13 origin of replication); multiple cloning sites; Animal "origin of replication (eg, SV40 or adenovirus origin of replication). Other embodiments The expression vector is a recombinant viral genome and the specific Either RNA or DNA depending on the viral system. Alternatively, an expression vector Can include RNA transcribed in vitro. As used herein, An expression vector is also a vector that is transformed into a different form after introduction into a cell. Say. For example, an RNA genome carrying a recombinant retrovirus is reverse transcribed into DNA. And is integrated into the cell's genome. For the purposes of the present invention, RNA and D Both forms of NA are “expression vectors”.   `` Altered cellular components '' usually cause cells to become tumorigenic Or related to tumorigenic cells, but causes the cells to become tumorigenic Proteins that are not required or required for sex And other cell components. Prior to alteration, this cellular component is responsible for normal cell growth and And regulation, and may be essential for intracellular proteolysis, transcriptional regulation, Includes proteins that regulate cell cycle control, and cell-cell interactions. After transformation , This cellular component no longer performs its normal regulatory function and therefore the cell is unregulated Can experience invasive growth. Ras is a typical example of a degenerated cell component.^*, P53^*, Rb^*, Ubiquitin, an altered protein encoded by the Lumus oncogene^*, Mucin^*, DCC , APC, and proteins encoded by MCC genes, and receptors or Receptor-like structures such as neu, thyroid hormone receptor, platelet-derived growth Factor (PDGF) receptor, insulin receptor, epidermal growth factor (EGF) receptor And colony stimulating factor (CSF) receptors. These and other Cellular components are described in more detail below and in the cited references.   "Non-tumorigenic'' Does not cause cell transformation in nude mice, Or a degenerated cell component that does not induce tumorigenesis. Non-tumorigenic tumorigenic cell components Representative assays for distinguishing between cellular components are described in more detail below.   As used in the present invention,ImmunogenicityElicits an immune response under appropriate conditions. It refers to a degenerated cell component that can be rubbed. This response must be cellular and Humoral response. Representative methods that can be used to determine immunogenicity The essays are described in more detail below.   Numerous aspects and advantages of the present invention are set forth in the following detailed description, which provides a description of the practice of the invention. It will be apparent to those skilled in the art in light of the detailed description.DETAILED DESCRIPTION OF THE INVENTION   The present invention relates to the expression of at least one disease-associated antigen in dendritic cells and cells Surface presentation can generate a prophylactic or therapeutic immune response to a disease associated with that antigen. Based on the discovery that it can be used to In addition, genetically modified trees Genetically Modified Fibroblasts Efficiency of Immune System Stimulation Mediated by Dendritic Cells Large, several orders of magnitude higher than the efficiency of immune system stimulation mediated by muscle, muscle, and other cell types It was discovered that it could be bulky. As a result, these findings are Reducing the dosage required to achieve a protective or therapeutic result; Or gene therapy-mediated immune stimulation, either by enhancing the immune response Enables an improved approach.   Diseases suitable for treatment with an immunostimulatory strategy include HBV (WO 93/15207). HCV (see WO 93/15207), HPV (WO 92/05248, WO 90/10459, EPO 133,1 23), Epstein Barr virus (EPO 173,254; JP 1,128,788; and the United States) Patent Nos. 4,939,088 and 5,173,414), feline leukemia virus (WO 93 / 09070, EPO 377,842, WO 90/08832, and WO 93/09238), feline immunodeficiency Ils (US Pat. No. 5,037,753, WO 92/15684, WO 90/13573, and JP 4,126, 085), caused by HTLV I and II, and HIV (see WO 91/02805) Virus infection such as melanoma, cervical cancer, rectal cancer, kidney cancer, breast cancer , Ovarian cancer, prostate cancer, cancer such as leukemia; and heart disease.   Dendritic cells are a line of antigen-presenting cells that function to initiate an immune response (St. einman, R. (1991), Annu. Rev. Immunol., 9: 272-296; Research in Immunolo gy, Volume 140, International Reviews in Immunology, Volume 6, Advances in Immunol ogy, Volume 47, and Epidermal Langerhans Cell, G .; See also Shuler ). Dendritic cells are found in many non-lymphoid tissues, but also in afferent lymphatic flow. Or through the bloodstream to T-dependent regions of lymphoid organs. Non-lymphoid organs In, dendritic cells include Langerhans cells and stromal dendritic cells. Rin In fluid and blood, dendritic cells are afferent lymphoid cells (aff), respectively. erent lymph veiled cells) and blood dendritic cells. Dendritic cells are efferent It is not found in the buffer solution. In lymphoid organs, dendritic cells are Cells and interdigitating cells. In the context of the present invention When used, each of these cell types and their precursors, unless otherwise indicated Are called "dendritic cells".   Dendritic cells have a unique dendritic shape, are motile, and are unique to cell surface stimulation. Efficient clustering and activation of different T cells. Typically, run Dendritic cells in non-lymphoid organs such as gelhans cells and stromal cells are afferent Veil cells in lymph and blood (continuously spread and regress large membrane pseudopodia) Cells that contract), which migrate to lymphoid tissues, where dendritic cells or Can be isolated as clamped cells.   A partially enriched population of epithelial Langerhans cells (here, Langerhans cells) Vesicles can make up up to about 60% of the total cell population). Because α -thy-1 (monoclonal antibody) and complement and attachment This is because latinocytes can be depleted. Enriched preparation of human Langerhans cells Can be prepared by replacing α-thy-1 with an anti-CD-1 antibody. During culture, 1 Until after 3 days of culture, mouse and human Langerhans cells Are not active antigen presenting cells. After this period, the blood and lymphatic system Completely similar to dendritic cells, these cells expand and become more MHC class II and And expresses cell adhesion molecules and loses the Fc receptor. 90% from human blood A cell population comprising more than 0.1 dendritic cells is obtained, wherein less than 0.1% of leukemia without enrichment The sphere is a dendritic cell. Such enrichment is due to T cells, monocytes, and B cells plus N It can be achieved by continuous depletion of K cells, with dendritic cells ranging from 30-60% Produces an initial population of vesicles. Next, a monoclonal antibody that selectively reacts with impurities (Especially CD45R_AHigher purity by panning or FACS using (Freudenthal, P, Steinman, R.M., Proc. Nat'l. Acad. Sci. (USA) (19) 9 0) 87: 7698-7702). Low buoyant density, generally during culture, to enrich dendritic cells Non-adherent to plastic (especially after more than one day), and on other cells A selection is made for the absence of the found marker. Such methods are Depletes cell types, but does not actively select dendritic cells.   Dendritic cells express a discernable pattern of markers on their cell membrane. FIG. By indicating the presence or absence of several different cell surface markers This pattern is illustrated. For positive or negative selection of dendritic cells Other markers that may be used for ICAM-1 (CD54), LFA-3 (CD58), and C D11b. Isolated from human or mouse blood but not from skin Non-dendritic cells express CD11a and LFA-1. In the skin, dendritic cells The immunostimulatory effect can be enhanced by cytokines, especially GM-CSF.   Dendritic cells initiate a T-dependent response from quiescent lymphocytes. Once sensitization The T cells then interact with other antigen presenting cells. Dendritic cell antigen processing Activity is regulated. Fresh cells isolated from skin or lymphoid organs Only cells cultured for less than one day) present the native protein. This period After a while, the cells do not process the antigen. In addition, dendritic cells actively eat Not active.   Gene therapy-mediated immunostimulation can be achieved in various ways. For example, disease-associated antigens Or a modified form thereof (hereinafter collectively referred to as "antigen") An expression vector can be delivered to dendritic cells to mount an immune response against that antigen . Antigen expression can be transient or stable over time. Exemption If the epidemic response is due to a virulence factor (eg, viral, bacterial, or neoplastic, or If not stimulated by other diseased autologous cells). Preferably, a modified form of the antigen (with reduced pathogenicity compared to the native antigen, Yet stimulates an immune response to fear).   Certain diseases caused by viral infections (eg, triggered by HIV) Induced AIDS), the immunostimulatory expression product encoded by the expression vector Is either an HLA class I-restricted immune response or a class II-restricted immune response or It is a form that induces both. For HIV, the preferred antigen for immunostimulation is , Preferably from an envelope protein selected from gp160, gp120 and gp41 This is to reduce pathogenicity (especially reducing the potential for syncytia) In order to avoid the expression of epitopes that lead to a disease enhancing immune response, To remove some but strain-specific epitopes, or some strain-specific epitopes Have been modified (to present loops). Other HIs that can be expressed for this purpose V genes or gene combinations include gag, pol, rev, vif, nef, prot, gag / pol, gag prot and the like. In addition to this, licenses from other desired antigens Epidemiological moieties can be expressed. The immunogenic portion of the desired antigen may vary in length. And preferably at least 9 amino acids, and may include the entire protein . As one skilled in the art will appreciate, this and similar immunostimulation approaches Can be used to treat a number of diseases.   In a further embodiment, the expression vector comprises one or more immunizations of a disease associated antigen. Directs the expression of at least one gene of interest that encodes the intrinsic portion. In this specification When used in at least one gene, the gene of interest Encode the product. "Disease-related" antigens affect cells (or organs) To affect cells or to be generally associated with a disease state An antigen that is not required. A wide variety of "disease-related" antigens are known and These include immunogenic non-tumorigenic altered cell components that are usually associated with tumor cells. (See WO 93/10814). Tumor-associated antigens corresponding to the cellular immune response (eg, , MAGE-1, MAGE-3, MART-1, and gp100) are also used herein as "disease". Disease-associated antigens ". "Disease-related" antigens can be found in various eukaryotes, prokaryotes, It is also understood that it encompasses all or part of a viral pathogen. Sa See WO 93/10814 for further details.   Nucleic acid molecules encoding the above products, as well as advantageous for use in the present invention Other nucleic acid molecules are available from various sources (eg, American Type Culture Or a depository institution such as a collection) or British Bio-Technology Limited (Cowley, Oxford England) and can be readily obtained from commercial sources. Alternatively, a cDNA sequence for use in the present invention expresses or comprises that sequence It can be obtained from cells, for example, by RT PCR from isolated mRNA. In the present invention Nucleic acid molecules suitable for use also include, in whole or in part, e.g., Applied Bios ystems Inc. DNA synthesizer (eg ABI DNA synthesizer model 392 (Foster City, CA) Can be synthesized above.   A.Gene delivery vehicle   Gene delivery vehicles (“GDVs”) are used to transfer nucleic acid molecules, specifically expression vectors. A composition that can be delivered to a eukaryotic cell. Representative examples of gene delivery vehicles include: Recombinant viral vectors (eg, retroviruses; see WO 89/09271, and Alphaviruses such as Sindbis; see WO 95/07994), other recombinants and Non-recombinant virus system (eg, adenovirus; see WO 93/19191), one or more Nucleic acid associated with a coagulant (see WO 93/03709), nucleic acid associated with liposomes Molecules (Wang et al., PNAS 84: 7851, 1987), modified bacteriophages, and Include bacteria. Regardless of the form, GDV is at least present in the target cells. Also carry an expression vector that directs the expression of one disease associated antigen.   In one embodiment, GDV is astrovirus, corona Virus, orthomyxovirus, papovavirus, paramyxovirus, Rubovirus, picornavirus, poxvirus, retrovirus, togau Recombinant virus derived from viruses such as irs, or adenoids . In a preferred embodiment, the recombinant viral vector is a recombinant retrovirus It is a vector. Retroviruses GDV are, for example, B, C and D retroviruses. A wide variety of retroviruses, including ills, and spumaviru (spumaviru s) and can easily be constructed from lentiviruses (RNA Tumor Viruses, 2nd edition, C old Spring Harbor Laboratory, 1985). Such retroviruses are Depositary institutions such as Cantype Culture Collection (ATCC, Rockville, MD) Or readily available from collections or generally available from known sources Can be isolated using various techniques. Numerous reto that can be used in the practice of the present invention GDV is disclosed in U.S. Patent Nos. 5,219,740 and 4,777,127, EP 345,242 and And WO 91/02805.   A particularly preferred recombinant retrovirus is chicken leukemia virus (ATCC No. VR -535 and VR-247), bovine leukemia virus (VR-1315), mouse leukemia virus ( MLV), mink cell focus forming virus (Koch et al., J. Vir. 49: 828, 1984; And Oliff et al. Vir. 48: 542, 1983), mouse sarcoma virus (ATCC number VR-844, 4 5010 and 45016), Reticuloendotheliosis virus (ATCC numbers VR-994, VR-770 and 45011) , Rous sarcoma virus, Mason-Petizer monkey virus, baboon endogenous virus , Cat endogenous retrovirus (e.g., RD114), gibbon ape leukemia virus (GA LV), human immunodeficiency virus (HIV), HTLV I, HTLV III, and retroviral vectors. Retrovirus containing mouse or rat gL30 sequence used as a vector Originated from As a particularly preferred strain of MLV capable of producing a recombinant retrovirus Is Is mentioned. A particularly preferred non-mouse retrovirus is Rous sarcoma virus. You. Preferred Rous sarcoma viruses include Is mentioned.   Any of the disclosures provided herein and standard recombinant techniques, if any, Retroviruses are easily used to create or construct retroviral GDV Can be done. In addition, parts of the retrovirus GDV are derived from different retroviruses Can be For example, the recombinant retrovirus is LTR derived from mouse sarcoma virus. , Rous sarcoma virus-derived tRNA binding site, MLV-derived packaging signal , And a second strand origin of synthesis from chicken leukemia virus. These recombination Use retroviral vectors to transfer these into the appropriate packaging cell line. By introducing the recombinant retroviral vector of Torovirus vector particles can be generated (see WO 92/05266). In addition, host Site-specific integration of the recombinant retroviral genome into specific regions of vesicle DNA Directed recombinant retroviruses can be produced. Such site-specific integration Is mediated by a chimeric integrase incorporated into the retroviral particle. Can be See, for example, WO 96/06727. Recombinant viral vectors lack replication Preferably, the virus is a recombinant virus.   In another embodiment, the GDV is from a togavirus. Preferred togaoil Examples of the virus include alphaviruses, particularly alphaviruses described in WO 95/07994, which are related to sharing. Viruses. Typical alphavirus is Sindbis virus . Briefly, recombinant Sindbis expression vectors are typically Sindbis 5 'sequence capable of initiating viral transcription, encoding a Sindbis nonstructural protein Nucleotide sequence, modified or inactivated viral junction region, Sindbis RN A polymerase recognition sequence, at least one gene of interest, and polyadenyl Includes tracts. Corresponding regions from other alphaviruses are substituted for the above regions. Can be used instead.   In a preferred embodiment, the recombinant alphavirus vector has a structural protein It does not code for quality and the gene of interest is located downstream of the viral junction region. No. In a vector having two viral junction regions, the gene of interest is the second virus And may be located downstream of the junction region. In such cases, the vector is And a polylinker located between the target gene and the gene of interest.   Other recombinant togavirus vectors that can be used in the present invention include Sem Lyki Forest Virus (ATCC VR-67; ATCC VR-1247), Middelburg Virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan euma Encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532) And U.S. Patent Nos. 5,091,309, 5,217,879, and WO 92 / 10578. The recombinant Sindbis expression vector described above, And many similar vector constructs are described essentially in WO 95/07994. Thus, it can be easily prepared.   In another embodiment, the recombinant viral vector is a recombinant adenovirus vector. It is a tar. Such expression vectors are compatible with the disclosure provided herein. Easily prepared and used (Berkner, Biotechniques 6: 616, 1988, and Ro senfeld et al., Science 252: 431, 1991, WO 93/07283, WO 93/06223, and WO 93 /. 07282).   Other viral vectors suitable for use in the present invention include, for example, Derived vectors.   In another embodiment of the invention, the GDV comprises a DNA or RNA expression vector. Representative examples of such GDV include plasmids, cosmids, and linear double stranded And single-stranded polynucleotides. Such vectors are used in dendritic cells. Genetic information required to express disease-related antigens in The open reading frame encoding the disease-associated antigen, transcription termination Signal, and polyadenylation signal. Such polynucleotides One advantage of the system is that large quantities of a given gene delivery vehicle can be easily produced. is there. In vitro transcription may be performed using any of a variety of techniques known in the art. Can produce large amounts of RNA. Similarly, large amounts of DNA are easily cultured in bacteria. Can be   In a related embodiment, the GDV is DNA associated with an aggregating agent (eg, a polycation). Or an RNA expression vector. Polycations are negatively charged phosphate bone Aggregate the expression vector by masking the case, making the molecule more compact Fold it into shape. As a polycation useful in this embodiment of the invention Is, inter alia, polylysine, polyarginine, histone, protamine, spermi Gin, spermine, and other highly basic proteins or polypeptides No. These polycations are modified to incorporate the target element And / or reduce the immunogenicity of the nucleic acid / polycation complex. example For example, the polycation may include one or more polyalkylene glycols (eg, polyethylene Glycol). Alternatively (or in addition), The ON can bind to one or more polysaccharides. Simply put, chemical bonds are With the formation of a covalent bond between the ON and the polyalkylene or polysaccharide. like that Many suitable methods for making bonds are known in the art. For example, share See WO 96/21036 according to.   In another embodiment, the GDV is a liposome-associated DNA or RNA expression vector It is. Liposomes are small lipid vesicles that contain an aqueous core surrounded by a lipid bilayer. Compartment is typically composed of a few hundred angstroms Slightly stretched structure. Under appropriate conditions, the liposome is Membrane of endocytic vesicles in cells that have taken up liposomes And release its contents into the cytoplasm. But interaction with the cell surface Before, the liposome membrane sequesters its contents from, for example, degrading enzymes in plasma. Acts as a relatively impermeable barrier to protect. In addition, liposomes have a synthetic structure Thus, specially designed liposomes incorporating desired characteristics can be produced (St. ryer, L., Biochemistry, 236-240, 1975 (WH Freeman, San Francisco, C A); Szoka et al., Biochim. Biophys. Acta 600: 1, 1980; Bayer et al., Biochim. Bioh ys. Acta. 550: 464, 1979; Rivnay et al., Meth. Enzymol. 149: 119, 1987; Wang et al. , PNAS 84: 7851, 1987; Plant et al., Anal. Biochem. 176: 420, 1989, and United States Patent No. 4,762,915). Liposomes are useful for practicing the present invention. A variety of nucleic acid molecules can be encapsulated, including rasmids, and other expression vectors.   GDV (including GDV bound to one or more different dendritic cell target element types) Can be further modified. One particularly useful modification is the immunogenicity of the resulting composition. Of compounds that bind to polycations and gene delivery vehicles Is included. See WO 96/21036 for sharing.   B.Gene of interest   GDV useful in the practice of the invention described above may include one or more genes of interest, Or at least one antigen associated with or characteristic of the disease to be prevented. Includes or contains the encoding nucleic acid molecule. Such antigens include: Antigens derived from altered cell components, and antigens from foreign organisms or other pathogens You. A variety of other disease-associated antigens can be used in the gene delivery vehicles of the present invention. See, for example, WO 91/02805, WO 96/20414, and U.S. Patent No. 5,580,859. When.   In another embodiment of the invention, an immunogen of an antigen from a foreign organism or other pathogen Expression vectors are provided that direct the expression of the sex moiety. These antigens are also described herein. It is called "disease-associated antigen" in the text. Representative examples of such antigens include bacteria (Eg, E. coli, streptococci, staphylococci, mycobacteria, etc.), fungi, parasites Insects and viruses (eg, influenza virus, HIV, hepatitis A virus) Virus, hepatitis B virus and hepatitis C virus ("HAV", "HBV" and And "HAC"), human papillomavirus ("HPV"), Epstein-Barr virus Virus (“EBV”), herpes simplex virus (“HSV”), hantavirus, HTLV I, From HTLV II, cytomegalovirus ("CMV"), and feline leukemia virus Antigens. An "immunogenic moiety" as used herein, is a suitable Each of which can cause an immune response (ie, cellular or humoral) under conditions Refers to a part of an antigen. The "portions" can be of various sizes, but are preferably less Both are 9 amino acids long and can include the entire antigen.   In one embodiment of the invention, the expression vector comprises at least one disease-related vector. In addition to the serial antigen, it encodes a gene encoding an immunomodulatory cofactor. Immune regulation A factor means that when expressed in dendritic cells in addition to a disease-associated antigen, The quality or strength of the immune response to the antigen is determined by the immunity It is a factor that enhances the response. The quality or strength of the response depends on species known to those of skill in the art. Can be measured by various assays, including, for example, cell proliferation In vitro assays to measure (eg,^ThreeH-thymidine incorporation), and in vitro Cytotoxicity assays (eg,⁵¹Assay to measure Cr release) (Waener et al., AIDS  Res. and Human Retroviruses 7: 645-655, 1991). Another implementation In an embodiment, the immunomodulatory cofactor is a genetic Either before, simultaneously with, or after the child delivery vehicle is administered Is added from outside.   Immunomodulators can be active both in vivo and ex vivo. like this Representative examples of various immunomodulators include, for example, cytokines, for example, , And MHC (Powis Nature 354: 528, 1991). Choosing which immunomodulator to use The choice is based on the therapeutic effect of the factor. Preferred immunomodulators include α-in Examples include terferon, γ-interferon, and IL-2.   Nucleic acid molecules encoding disease-associated antigens can be obtained from a variety of sources, such as American Thai. From contractors such as the Culture Collection (ATCC, Rockville, MD) Or a business such as British Bio-Technology Limited (Cowley, Oxford, England) It can be easily obtained from commercial sources. Alternatively, encode such an antigen cDNA is produced from cells known to express or contain the corresponding gene. (U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,800,159). No. PCR Technology: Prlnciples and Applications for DNA Amplificatio n, Erlich (ed.), Stockton Press, 1989). Alternatively, a known distribution Sequence, or a gene encoding an antigen of known amino acid sequence, such as Appl ied Biosystems Inc. DNA synthesizer (eg, APB DNA synthesizer model 392 (Foster City , CA).   C.Target element   As mentioned above, one aspect of the present invention is to incorporate a gene delivery vehicle into dendritic cells. Provide compositions and methods for targeting either in vivo or in vitro . In the context of the present invention, a target element is a molecule present on the surface of a dendritic cell. Is a molecule that has an affinity for A wide variety of target elements are involved in the practice of the present invention. Can be used to specifically direct gene delivery vehicles to dendritic cells.   One embodiment of the present invention provides a method for targeting a gene delivery vehicle to dendritic cells. For the use of high affinity binding pairs. In other embodiments using targeting elements , The targeting element is covalently linked to the gene delivery vehicle via a multifunctional binder It is. See generally WO 92/05266 for sharing.   As will be apparent to those skilled in the art, other targeting mechanisms that can target dendritic cells are Within the scope of the invention.   D.GDV production   Once the GDV has been designed, it is useful for binding to the desired target element. And / or must be produced in sufficient quantities for administration to animals. GDV If it is a recombinant viral vector, it is produced using a packaging system. Can be Various viral vector packaging systems are described below. this is One or more essential functions of the parent virus are deleted, resulting in some functions (eg, Genomic replication) is deleted, but the packaging signal and one or more It retains the ability to express gene products from nucleic acid molecules. Virus vector Typical examples of packaging systems are retroviral vectors, alphavirus vectors. And those for adenovirus vectors.   When such a recombinant retroviral vector is used, virus particles are used. It is preferable to utilize the packaging cell line that produces. Where at least ga The codon at the 5 'end of the g / pol gene is To minimize the possibility of homologous recombination with the sequence encoding the structural protein For this reason, it is modified using the degenerate nature of the genetic code. Present in packaging cells Events between the vector and the packaged recombinant retroviral genome Additional techniques to reduce the likelihood of sharing are described in WO 95/30763 and WO 96 / 07749.   Packaging cell lines suitable for use with the above recombinant retroviral vectors Can be readily prepared using known techniques (WO 95/30763 and WO 92/05266 See also) and producer cell lines for the production of recombinant vector particles Can be utilized to produce   In a further embodiment of the present invention, an alphavirus packaging cell line Is provided. Specifically, the alphavirus packaging cell lines include: Provided to Viral structural proteins (one or more stably integrated expression vectors) Transfected), transfected, transduced Vector RNA transcript, or produced in the cell, is encapsidated in the cytoplasm. And release infectious packaged vector particles through the cell membrane As such, it produces an alphavirus vector producing cell line.   Packaging Cell Line Suitable for Use of the Alphavirus Vector Constructs Above Can be easily prepared (see WO 95/07994).   In a further embodiment of the present invention, the adenovirus packaging cell line is Provided. Adenovirus vectors are derived from nuclear replicating viruses and Can be constructed to be replication defective. One or more nucleic acid molecules is an adenovirus Vectors can be carried for delivery to target cells (Ballay et al., EMBO J. Am. 4: 3861, 1985; Thummel et al. Mol. Appl. Genetics 1: 435, 1982 and WO 92/0 See 5266).   In another embodiment of the invention, the targeted gene delivery vehicle comprises a gene delivery system. Includes one or more fusigenic proteins to assist. Fusible tongue A typical example of protein is the ecotropic mouse retrovirus envelope protein. Protein, other retroviruses modified so that normal receptor recognition is not possible Lus envelope protein, herpes simplex virus fusion protein gH and gL-derived fusogenic protein, Epstein-Barr virus fusogenic protein, rubella Viral proteins, malaria parasite fusion proteins, and have fusion properties Includes other proteins known in the art.   E. FIG.Purification of gene delivery vehicle   Once GDV is produced, they preferably bind to the desired target element. Purified before combining. Further, the composition comprising the targeted GDV is preferably administered Before being purified again. The technology used for purification depends on the type of GDV being purified Depends on. For example, there are various techniques known in the art. This is GDV , An enveloped recombinant viral vector, nucleic acid, or liposome. (See US Pat. No. 5,447,859 for an example of a purification procedure) Thing).   In addition, when GDV is a nucleic acid, there are a variety of techniques known in the art. This This includes, for example, CsCl-ethidium bromide gradient, ion exchange chromatography Gel filtration chromatography and precipitation with polyethylene glycol. Including purification. Further descriptions of nucleic acid purification can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, 1989) Provided.   When GDV is a liposome, various purification methods known to those skilled in the art may be utilized, And by Mannino and Gould-Fogerite (BioTechniqucs 6: 682, 1988) be written. Briefly, liposome preparations typically involve one or more purified phospholipids. Mixing a solution of cholesterol and cholesterol in an organic solvent, and drying the solvent Until evaporation. Next, the buffered aqueous solution containing GDV is The mixture is added to the membrane and the mixture is sonicated to give a homogenous dispersion of liposomes. Is prepared. In certain embodiments, dialysis, gel filtration, or ultracentrifugation is then Used to separate entrapped components from intact liposomes   F.Prescription   After purification of the composition comprising GDV, the preparation is preferably formulated into a pharmaceutical composition You. Such compositions may be in liquid or dry form. Preferred liquid composition Means that the desired product is suspended in a pharmaceutically acceptable carrier and / or diluent. And aqueous solutions that may further include stabilizers and other excipients. There The compositions of the present invention are prepared in a solid formulation intended to be resuspended immediately before use. Can be made. Dry formulations include those that have been lyophilized or freeze-dried.   When GDV is a virus with a lipid envelope, a preferred dry formulation is Including some or all of the following: one or more pharmaceutically acceptable carriers and And / or diluents: saccharides; high molecular weight structural additives; buffer components; water; And one or more amino acids. Some or all combinations of these ingredients Retains the activity of the targeted GDV during freezing and lyophilization or drying by evaporation Acts as follows. The pharmaceutically acceptable carrier or diluent of the present invention may be used It is non-toxic to recipients at different dosages and concentrations. Injectable dissolution Representative examples of carriers or diluents for liquids are, for example, water, isotonic saline (Ie phosphate buffered saline or Tris buffered saline, preferably Buffered to physiological pH), mannitol, dextrose, glycerol And ethanol, as well as polypeptides or human serum albumin Contains protein.   Viruses with lipid envelopes (particularly recombinant retroviruses and The preferred lyophilized composition for vaccinia) is 10 mg / mL mannitol, 1 m Those containing g / mL HSA, 20 mM Tris (pH 7.2), and 150 mM NaCl are particularly preferred (WO 95/10601). Such compositions can be stored at -70 ° C for at least 6 months. It is fixed. The pharmaceutical composition of the present invention may also comprise cell division, and consequently administered GDV. It may further include factors that stimulate the uptake and uptake of. Stores recombinant virus Particularly preferred methods and compositions for doing so are WO 95/10601, WO 95/07994, and And WO 96/21014. GDV in recombinant or liquid form After preparation of the composition when it is irs, the recombinant virus is preferably administered per dose. Approximately 10 ng to 1 mg of the substance (about 10 times the amount of the substance shown as a co-purified contaminant) Consists of Preferably, the composition is administered in a dosage of about 0.1-1.0 mL of the aqueous solution . This may include one or more additional pharmaceutically acceptable excipients, stabilizers, or diluents. It may or may not contain an agent.   I.Administration   Compositions of the Invention (Ex Vivo Modified Dendritic Cells or GD for Direct Administration) V) is typically parenteral in vivo (eg, intravenous, subcutaneous, and intramuscular) ) Or other conventional direct routes (eg, cheek / sublingual, rectal, buccal, nasal, Topical (eg, transdermal and ocular), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal Route) or a specific tissue (eg, liver, bone marrow) or cancer In the case of treatment, it is administered directly to the tumor. Non-parenteral route Are further discussed in WO 96/20732.   Preferably, the composition is administered to the animal by the desired route, and then the animal Tested for biological response. Such tests may be used in immunological screening Assays (eg, CTL assays, antibody assays). Tests performed Is the product produced by the nucleic acid molecule introduced by the targeted GDV and the treatment or Depends on the disease to be prevented. Based on the results of such tests, the If the GDV titer is more than administered, the desired effect is further enhanced Can be adjusted to   By any of the routes of administration described herein or other routes known in the art. Administration may simply be with a needle, catheter, or related device, at one time or Can be achieved by direct administration at multiple time points. In addition, at a given time The “administration” of the gene delivery vehicle (or ex vivo transduced cells therefor) Administration to one or more areas or by one or more routes. The present invention In certain embodiments of the one or more doses are administered directly below in the manner indicated Is: intravenous, 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹less than cfu Upper dose; intra-arterial, 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹c fu or more dose; intramuscular, 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 1 0¹¹cfu or higher dose (10^TenOr 10¹¹cfu dose is preferred); intradermal, 10^Three,Ten^Four ,Ten^Five,Ten⁶,Ten⁷, Ten⁸,Ten⁹,Ten^TenOr 10¹¹Dose greater than cfu; lung, 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷, Ten⁸,Ten⁹,Ten^TenOr 10¹¹cfu or higher dose; subcutaneous, 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷ ,Ten⁸,Ten⁹,Ten^TenOr 10¹¹cfu or higher dose (10⁸,Ten^TenOr 10¹¹cfu Dose is preferred); stroma, 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^Ten,Also Is 10¹¹cfu or higher dose (10⁸,Ten⁹,Ten^TenOr 10¹¹cfu dose is preferred) In lymphoid organs (eg, spleen, tonsils, or lymph nodes), 10^Three,Ten^Four,Ten^Five,Ten⁶ ,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹dose over cfu; 10 in tumor^Three,Ten^Four,Ten^Five ,Ten⁶,Ten⁷,Ten⁸,Ten⁹,Ten^TenOr 10¹¹cfu or higher dose (10⁸,Ten⁹,Ten^Ten, Or 10¹¹cfu dose is preferred); intraperitoneal, 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷,Ten⁸ ,Ten⁹,Ten^TenOr 10¹¹cfu or higher dose (10⁸,Ten⁹,Ten^TenOr 10¹¹cfu Dose is preferred); and 10^Three,Ten^Four,Ten^Five,Ten⁶,Ten⁷,Ten⁸,Ten⁹ ,Ten^TenOr 10¹¹Dosage over cfu. For simplicity, "cfu" also Ruth particles. Thus, one cfu is equivalent to one non-viral particle.   Other routes and methods for administration include non-parenteral routes (eg, WO 96/20732), as well as administration via multiple sites (shared WO 96/20731).                                  Example   The following examples include more fully illustrating the invention. In addition, these The examples provide preferred embodiments of the present invention and limit its scope. Not intended. For many procedures described in the examples below, or alternative procedures as appropriate Standard methods are described, for example, in "Molecular Cloning", 2nd edition (Sambrook et al., Col. d Spring Harbor Laboratory Press, 1987) and “Current Protocols in Mol ecular Biology ”(edited by Ausubel et al., Greene Associates / Wiley Interscience, NY , 1990) in a widely reorganized manual of molecular biology.                                 Example 1 After direct administration of the gene delivery vehicle Identification of dendritic cells as important antigen presenting cells   As described above, an expression vector encoding at least one disease-associated antigen vector Direct injection of recombinant retroviruses with New However, the desired disease-associated antigen may be transduced cells (eg, fibroblasts) (this Which is then administered to the patient) compared to the ex vivo approach expressed by Thus, direct administration of a gene delivery vehicle to a patient can be performed at or near the injection site. Cell transduction and tissue delivery (eg, after intravenous or subcutaneous administration) The transduced cells present in the delivery vehicle (which can be transported to lymphoid tissue) . Previously, expression vectors were delivered directly by injection of a gene delivery vehicle. It was unknown which cells were responsible for presenting the encoded disease-associated antigen. Direct to animals Transduction displaying antigen encoded by an expression vector delivered by direct injection The identification of the incoming cells is described below.   Expression vectors delivered by the recombinant retrovirus injected directly into the animal Previous biolocalization studies designed to track the use of whole-organ PCR Rovirus DNA was detected at the injection site (Sajjadi et al., 1994; Kamantigue et al.) , 1995). In addition, proviral DNA is often detected in secondary lymphoid organs. Was issued. The results show that these animals are not immune to the immunogen encoded by the provirus. Was consistent with the observation that increased the successful immune response.   In this study, four expression vectors delivered using a recombinant retrovirus were used. (The HIV gp160 / 120 envelope, bacterial β-galactosidase, Encoding either watrioalbumin or luciferase) Then, cells involved in antigen presentation and immunity induction after direct administration were identified. Four The cell population was tested. The first cell population contains the expression vector that was identified by PCR. Including virus copies (Group I). The second subset of cells is coded Is a subset of cells expressing the expressed protein (Group II). β-gal tissue These cells were analyzed using chemical and immunohistochemical assays. The third sa Buset (Group III) is a group that can present antigenic peptides to the immune system. Consists of a subpopulation of cells. Group III cells were isolated in a T cell assay. Identified by its ability to induce an epidemic response. Finally, Group IV is an expression vector Antigen-protein from the corresponding gene Different groups that do not express proteins or peptides but still present antigen Consists of cells. Examples of Group IV cells include primary transduced cells or fragments thereof. Contains phagocytic cells for uptake. Such cells are then re-processed to Present antigen peptides in association with their own MHC molecules.Retro virus vector   HIV gp160 / 120, bacterial beta-galactosidase, luciferase, and chicken Non-replicating amphotropicoma having an expression vector encoding ryoalbumin A male recombinant retrovirus was used. Retroviral vector backbone and high titer Virus preparation (> 1 × 10⁷cfu / mL) in WO 89/09271, WO 92/05266, and It was prepared as described in WO 96/21014. pUG-1 vector (Moore et al., 1993) ), A 1.2 kb BamHI to BglII fragment encoding chicken ovalbumin cDNA The fragment was cloned into the BamHI site of pSP73 (Promega, Madison WI). The 1.2 kb XhoI to ClaI fragment was recovered and the corresponding N2-HIV gp160 / 120 With a 3.8 kb XhoI to ClaI fragment. Aliquot of virus preparation Tested for replication competent retrovirus (RCR) and contained RCR Decided not to.Animal and vector immunity   Harlan Sprague-Dawley 7-8 week old female BALB / c (H-2^d) Mouse or C57BL / 6 (H-2^b) Mice were used for all experiments. On days 1, 4, and 7, mice receive: 100 μl / injection, a total of 200 μl / mouse, muscle in both gastrocnemius or tibialis anterior Intra (i.m.) or intradermally at the tail fin with 100 μl of the retroviral preparation did. By harvesting the spleen and mixing with appropriately irradiated spleen cells In vitro CTL culture was started.Immunohistochemistry   Antibodies to the following lymphocyte markers can be used in culture supernatant or purified immunoglobulin Used as: CD4 (rat IgG); CD8 (rat IgM); macrophage (rat IgG); and B220 (rat (IgM)). CD45 (30R11.1, rat IgG; Pharmingen , San Diego, CA), rat Ig (polyclonal; Jackson ImmunoResearch Labs., Inc., West Grove, PA) and rabbit Ig (polyclonal; Jackson ImmunoRese) arch, PA) was also used. Normal rabbit serum, Used as a negative control.   Carefully remove muscle at injection site, soak in OCT, and quickly freeze in liquid nitrogen And stored at -70 ° C until use. Cryostat sections at 5-6 μm Cut, air dried and fixed with acetone for 5 minutes. Sections were taken from the previously titrated Tr Incubate for 60 minutes with the optimal concentration of antibody diluted in is buffered saline (pH 7.4) And washed with Tris-buffered saline, biotinylated anti-rat or rabbit Incubate with IgG and wash, then bind streptavidin Incubation with alkaline phosphatase (Jackson ImmunoResearch, PA) I did it. The bound alkaline phosphatase is converted to the substrate Fastviolet (Su rh, J. Exp. Med., Vol. 176: 495-505). Sections with Meyer's Hematoxyli Counterstained lightly and photographed under a Zeiss Axioscope microscope. back To minimize grounding, Airfuge ultracentrifuges (Beckman Instruments, I nc., Palo Alto, CA), spun at 148,000 xg for 30 minutes and discarded the pellet Later, the antibody was used.Isolation of infiltrating cells   Injection site is visualized via fluorescence emission from co-injected latex beads After that, they were excised with surgical scissors and diced. Small pieces of muscle obtained (average Size 0.5cm^Three) Was digested with trypsin for 1 hour at 37 ° C with occasional shaking . At the end of the digestion, fetal calf serum is added and the remaining muscle pieces are removed by gravity. separated. Wash the invading cells with media and luciferase and B3.Z assay After the measurement.Spleen cell fractionation by Percoll density gradient (see Figure 2)   Percoll stock against 1 part 10x phosphate buffered saline (PBS) Prepared by mixing 9 parts Percoll. "1.030" and "1.075" Working Percoll solution was added to 75% PBS + 25% Percoll stock solution and And 40% PBS + 60% Percoll stock solution. Na Spleen cells from an eaves or immunized animal are transformed into B + T cells by the following method. , Macrophages, and dendritic cells. Spleen with Hanks balanced salt solution Occasionally shake at room temperature with DNAse (Calbiochem, 3800 units / mL) in (HBSS) After crushing for 1 hour while moving, it was filtered through a nylon mesh to remove debris. . The resulting cells were pooled and centrifuged at 1500 rpm for 5 minutes to give "1.075 "Re-suspended in Percoll solution. “1.075” Parker with pooled spleen cells Solution is carefully layered over the "1.030" Percoll solution, and the gradient is run for 20 minutes. And spun at 1200 rpm. After Percoll centrifugation, low-density cells and The high-density cells in the let were collected separately and thoroughly washed. High density cells Most B cells except erythrocytes, T cells and plasma cells. Low density thin The vesicles, as determined by FACS, contain several B cells (including plasma cells), dendritic cells, Included monocytes, and macrophages, but not T cells. In the low density fraction Most B cells are harvested in complete medium (RPMI1640, 10% fetal calf serum) in tissue culture media. After a 2 hour incubation in the plate, remove by careful washing. Was. Remaining adherent cells (including dendritic cells, monocytes, and macrophages) Overnight. Mature macrophages remain attached even after overnight culture. Since it was the only cell to attach, it was isolated from all other cell types.X-gal (5-bromo-4-chloro-3-indosyl bD-galactopyranoside) staining   Cultures were fixed with 3% formaldehyde for 5 minutes at room temperature, then washed with PBS. Was. Cells were harvested at 1 mg / mL X-gal / mL, 5 mM potassium ferrocyanide, 5 mM ferricisate. Potassium anide, and 2 mM MgCl_TwoLayer. Plate at 37 ° C overnight After incubation, microscopically test for the presence of blue cells expressing LacZ. Tested.In vitro CTL induction and cytotoxicity assays   Five mice were recombined with a recombinant plasmid harboring an expression vector encoding HIV gp160 / 120. Immunization was performed by administering torovirus. B + T cells, dendritic cells, and Macrophage fractions were prepared. Five non-immunized littermates were treated in a similar manner Processed. The cell fraction was mixed with the irradiated stimulant at a ratio of 50: 1, and The culture was continued for a while.⁵¹Cr release assay (Warner et al., (1991), AIDS Res. And Human Retrov iruses, Volume 7: 645-655), and the percentage of lysis was calculated.Ex vivo transduction and adoptive transfer of dendritic cells and monocytes   Naive mice were sacrificed, the low density fraction was isolated as described above, and GM-C The cells were transferred to a medium containing SF (25 units / mL) and IL-4 (100 units / mL). DA / KT And DA / β-gal producer cell lines with a pore size of 0.45 microns. The seeds were seeded in lance wells. Dendritic cells and producer cells in transwells The vesicles were co-cultured for 3 days. The collected cells were thoroughly washed. Naive Stabilize these mice with these transduced dendritic cells or HIV gp160 / 120 Transduced fibroblasts were injected intraperitoneally. Spleen from the recipient mouse Restimulated in vitro after harvesting 7 days later, and⁵¹Do a Cr release assay Was.B3.Z Assay   B3.Z cells are H-2K^bT cells that recognize chicken ovalbumin for molecules It is an hybridoma. This is a cell fused to the minimal promoter of the human IL-2 gene. Contains a plasmid containing the bacterial LacZ gene and therefore induces LacZ upon antigen stimulation I can do it. Antigen presenting cells are cells transfected with the ovalbumin gene Or fractionated spleen cells from mice immunized with ovalbumin expression vector Is one of 2 × 10^FiveB3.Z cells and 1 × 10⁷Individual containing antigen presenting cells Incubate the culture overnight, and then evaluate the level of B3.Z activation Was stained with X-gal. The number of antigen-presenting cells in each fraction was counted by the ovalbumin gene. Estimated from a standard curve generated from a culture containing a known number of transfected cells. Was.result   Gene that defines the cell type to be transduced and is based on the recombinant retrovirus Immunize the injection site to induce immunity after direct administration of the delivery vehicle to the muscle. Tested by histochemistry to identify cells transfected in vivo. Mice were recombinantly harbored with an expression vector encoding chicken ovalbumin. Immunized with Torovirus. After three injections, the mice were sacrificed for frozen sections. And injection site with antibodies to various leukocyte markers and ovalbumin And analyzed by immunohistochemistry.   Many infiltrating cells were found in the endomesium near the injection site. Ma Injection of F4 / 80 positive monocytes / macrophages The site was identified as the most prominent infiltrating cell. Plus, a remarkable number of CD4 positives Bu cells and sometimes B cells infiltrated the injection site. CD8 positive cells I was not issued. Two dendritic cell-specific antibodies against different epitopes (NLDC-145 And N148) could detect a significant number of dendritic cells at the injection site. Of prescription buffer Injected mice show comparable lymphocyte infiltration and no needle damage from injection It was suggested that it was sufficient to cause the infiltration of the lymphocyte. Injection skin inner diameter path (equivalent Injection site), the injection site should be the same phenotype. Showed equivalent infiltration by vesicles.   Some cells infiltrate ovalbumin protein by immunohistochemistry Was found to be expressed. Double staining with anti-ovalbumin and F4 / 80 antibody Indicates that some, but not all, ovalbumin-positive cells are macrophages It was shown to be co-stained with the marker (F4 / 80). Depending on the expression vector, To confirm injection site expression of the loaded protein, infiltrate cells into Lucifera Or ovalbumin-encoding gene delivery vehicle. Was. First, cells were analyzed by the NBT assay, most of them It was confirmed that the cell line was a macrophage / monocyte cell line. The invading cells then Luciferase or B3.Z assay. Luciferase assays are Most luciferase activity is concentrated in infiltrating cells, but not in the rest of the muscle. Revealed that there is no. In addition, the B3.Z assay shows that infiltrating cells Ovalbumin was presented. Has normal rabbit serum Parallel sections or mice treated with the HIV gp160 / 120-encoding gene delivery vehicle. No staining was found with anti-ovalbumin antibody at the injection site of the mouse. This These results indicate that some fractions of infiltrating cells at the injection site were actually transduced. Expressing antigenic proteins that can be processed to stimulate an immune response And   These experiments show that inflammation caused by needle injury promotes leukocyte infiltration Leukocytes may then be transfected by the gene delivery vehicle. Is These transfected leukocytes then induce an immune response Transplanted into a secondary lymphoid organ to do so. To confirm this, a provirus DNA integration and protein expression were determined by different leukocyte populations in lymph nodes and spleen Was analyzed.   Previous studies of biolocalization using DNA isolated from whole organs Led to the detection of proviral DNA in the secondary lymphoid organs of S. japonica (Sajjadi et al., Supra; Ka). mantigue et al., supra). Due to the limited sensitivity of whole organ PCR, Staining was performed on fractionated spleen cells. Purify spleen cells from immunized mice And separates into B cell, T cell, macrophage, and dendritic cell fractions did. In mice immunized with the gene encoding HIV gp160 / 120 or β-gal In addition to a marker gene that confers resistance to neomycin, Thus, the encoded neo marker is used for the fractionation of dendritic cells and macrophage cells. Was detected.   Since expression of the proviral DNA was detected in the leukocyte cell fraction, Protein expression from these transfected cells And analyzed. BALB / c mice receive a gene delivery vehicle that directs β-gal expression Three injections were made. The spleens were pooled and fractionated as above, followed by X- Gal staining was performed. Unfractionated spleen and dendritic cell fractions consistent with PCR data Showed β-gal staining. Most of the transduced cells start in clusters Which is clonally derived from the first transferato cells Suggest that. Here, 0.2% of dendritic cells are transfected, Corresponds to approximately 2,000 cells per mouse. Furthermore, the macrophage fraction and Cells from the inguinal and popliteal lymph nodes of these mice showed some X-gal staining However, the number was greatly reduced compared to the dendritic cell fraction. The high density fraction is B Included cells and T cells and were negative for X-gal staining.   The phenotype of the transduced cells was further analyzed. B cells, T cells, and The clophage fractions are representative of those of CD45R / B220, Thy-1.2, and F4 / 80. Homogeneous after fractionation as judged by marker profile Was done. In contrast, they attach to plastic immediately after cell isolation, but after overnight culture. Cells that do not adhere to the plastic are stained based on some dendritic cell antibody staining. These cells contain most of the dendritic cells and are therefore customarily referred to as dendritic cell fractions. Also showed that some CD45R / B220 positive B cells (about 20%) and F4 / 80 positive Live monocytes / macrophages (about 30%). More adherent mature macropha Were separated by persistent adhesion to the plastic, so "Monocyte / macrophage cells in the cell fraction appeared to be monocytes. Therefore Determines the percentage of transgenic pure dendritic cells and monocytes in the "dendritic cell" fraction did. In addition, some F4 / 80 positive cells transduced at the injection site It is clear that these cells are trafficking the spleen Were determined. The mouse-derived spleen cells transduced with the β-gal gene were fractionated, and The "dendritic cell" fraction was thus obtained. Cells are sorted using two-color FACS and CD45R / B220 Positive, F4 / 80 positive, or double negative cells (ie, B cells, monocytes, or dendritic cells) were collected. Each population was then stained with X-gal. And β-gal activity was found at 0.035% in monocytes and 0.025% in dendritic cells. In the cell fraction, it was not found. These results show that spleen-derived spleen dendritic cells and And both monocytes / macrophages can be transduced, and It shows that protein can be expressed.   "Dendritic cells" from the immunized animal are recognized by the immune system and thus Such cells can be used to prime naive spleens in vitro A study was performed to determine whether antigen stimulation was possible. Free one group of mice It was infected and expressed the HIV envelope gp160 / 120. These spleen cells are Fractionated into loops: B + T cells; macrophages; and the fraction of "dendritic cells". Naive littermates were similarly processed. 6 separate cell glues obtained The mix was mixed to reconstitute the spleen cell population. Add irradiated stimulant and invite The CTL culture was started in (b). Immunized with cells expressing the HIV envelope gp160 / 120 Unfractionated immunized spleen and spleen cells from mice were Included as The first four combinations from the immunized mice (including the B + T cell fraction) M), but could induce an excellent immune response. Interestingly, the "tree" Only the "dendritic cell" fraction can elicit an immune response in a naＢve B + T cell population, The "dendritic cell" fraction contains antigen presenting cells capable of initiating an immune response in vitro. And proved. Dendritic cells are the only cells capable of initiating an immune response in vitro. As previously identified as a population, this “dendritic cell” population Should contain dendritic cells.   The above data indicates that dendritic cells and macrophages transduced in vivo Demonstrated that it can induce a primary immune response in vivo and in vitro. The ability of ex vivo transduced cells to stimulate an immune response was also studied. Low density The cell fraction was separated by percoll gradient separation of the naive spleen, Stimulated with a cocktail of Itokine to promote cell division. Proliferating cells Recombination with an expression vector encoding HIV gp160 / 120 using swell culture And transduced with a retrovirus. Transduced dendritic cells and macrophage cells Aliquots were transferred to a naive host, followed by restimulation in vitro, and And performed CTL analysis. As expected, recipient mice elicit superior CTL responses And confirmed the role of these cells in inducing immunity.   The relative potency of inducing the immune response of transduced dendritic cells compared to other cell types For evaluation by the ex vivo method, transduced dendritic cells were Transduced fibroblasts expressing comparable levels of the same antigen injected by Col And compared. Transduction of dendritic cells based on retrovirus-mediated luciferase expression The efficiency of entry is approximately equal to the same protocol, followed by standardization based on protein content. 0.1%. Based on this transduction efficiency, Mouse is 5 × 10^FiveAverage about 5 × 10 in injected cells^TwoReceived transduced dendritic cells It was calculated. Immune response induced by these ex vivo transduced dendritic cells The level is 3 x 10 expressing the same antigen^Four~ 1 × 10^FiveSame for injecting fibroblasts Comparable to the level of immune response generated using the same injection protocol. Based on this calculation Thus, a single transduced dendritic cell was isolated from 60-200 transduced fibroblasts. Induces an immune response equivalent to the epidemic response.   The above data shows that the protein expressing cells are relatively rare. Derived anti To assess the frequency of antigen presenting cells between cells expressing the original or protein, These rare antigen-presenting cells are isolated at the injection site and at various sites using the following procedure. Identified in the organ organ.   C57BL / 6 mice were injected with ovalbumin-encoding recombinant retrovirus. Pooled spleen was fractionated into B, T, matalophage, and "dendritic cells" . These potential antigen presenting cells express LacZ protein upon antigen stimulation Incubated overnight with B3.Z cells, a CTL hybridoma. These cultures Articles were stained with X-gal to assess the level of B3.Z activation. As a control , B3.Z cells were incubated with the following reagents: plate-bound anti-T cell receptor A known number of E7.G cells transfected with the ovalbumin gene; Immune splenocytes; or unfractionated immune splenocytes. The standard curve is analyzed using a fixed number of E7.G cells. Generated from the frequency of activated blue B3.Z cells incubated with and each spleen fraction From which the frequency of antigen presenting cells was assessed. On average about 100 anti-spleens per spleen Original presentation cells, total 10⁷Detected in mice injected with recombinant retrovirus of cfu did. Splenocytes pooled from these mice were obtained one week after in vitro stimulation. Generated 40% lysis in CTL assays performed at 100: 1 effector: target ratio I did   In another series of experiments, 10 C57BL / 6 mice were used as described above. Una 6 × 10⁷The same gene delivery vehicle of cfu was injected. Place cells at the injection site and Isolated from the rat organs. Comparison to a standard curve generated from E7.G cell recognition 1 × 10^FiveAnd 4 × 10^FourAntigen-presenting cells were isolated from inguinal lymph nodes and popliteal phosphorus, respectively. It was shown that it was recovered from Pak node. 2 × 10^FourRecover antigen presenting cells from the injection site Was. This data indicates that antigen presenting cells are quite rare. Therefore, gene therapy Treatment-mediated immunity induction is achieved by increasing the transduction of selected cell types as described below. Can be improved.   As shown above, the cells affected by gene delivery vehicle-mediated transduction There is a hierarchy. Cells containing the expression vector can be transferred to various sources (whole organs). , Purified cell populations, or tissue sections in situ) Can be detected by such techniques. Among cells containing an expression vector (Group I), In some cases, only some express proteins (Group II), but Group I cells The relative size of the population and the group II cell population can be compared in many instances. You. Group II cells are examined by various techniques, including histochemistry and immunohistochemistry. I can put it out. When examining the duration of heterologous protein expression, loss of expression is Although not occurring during the course of the study (10 days), the duration of expression It appears to vary depending on the offspring delivery vehicle.   Antigen processing and presentation involves many steps and parameters: Including: processing of antigenic proteins into peptides; transport of peptides to the endoplasmic reticulum Binding of peptides to appropriate MHC class I molecules; and peptide-MHC class I complexes A microenvironment in which the body can interact with cytotoxic T cells. Thus, protein expressing cells Only a sub-population appears to present antigenic peptides to the immune system (group III). The above results confirm this hypothesis, and antigen presenting cells are quite rare Find out for sure.   Finally, it does not contain an expression vector and therefore cannot express the antigen, Nevertheless, there is a different group of cells presenting antigen (Group IV). these As an example of a cell, a primary transduced cell or a fragment thereof is taken up and To reprocess and present the antigenic peptide for its own MHC molecule ( That is, cross-priming) and phagocytes are mentioned.   The above results indicate that antigen presentation after direct administration of the gene delivery vehicle And that it is mediated by macrophage cells. Tied to a particular theory Without transfection, the final transduced cells at the injection site are caused by needle damage. It appears that it can be replenished by this inflammatory response. After transduction, the cells It is excreted into the spleen via a tube and appears to present antigen. Therefore, Vivo transduced monocytes / macrophages and dendritic cells transfer the immune response An excellent source for Therefore, self- or haplotype-matched, Dendritic cells or monocytes / macrophages can be used for immunotherapy to treat or prevent disease. Can be used in therapeutic approaches. However, ex-vivo gene therapy Due to the stickiness and other complications, gene delivery vehicles can be delivered directly to patients. Is preferred. In a particularly preferred approach, in vivo transduction of dendritic cells Is performed. In order to enhance such transduction, patients can be selectively treated with leukocytes. Gene delivery vehicles encoding cytokines such as GM-CSF that cause proliferation Can be treated by pre-injection or co-injection. Alternatively, such immunomodulation A recombinant version of the cofactor may be administered. In a particularly preferred approach, The gene delivery vehicle is targeted to dendritic cells in vivo. Such targeted genes An example of a representative production of a delivery vehicle is described in Example 2 below.                                 Example 2 Retrograde to induce prophylactic and therapeutic immune responses against tumor-specific antigens Mouse dendritic cells transduced with the ils vector 1.Tumor treatment   Human papillomavirus (HPV) is used in several human cancers, especially in the cervical region and anal Involved in the development of squamous cell tumors of the hilar region. Some papillomavirus types E7 protein is derived from cervical epithelium by binding to the tumor suppressor gene Rb product. Mediates oncogenic transformation of vesicles. The E7 gene is immortalized, and Cells (Kanda, T et al., J. Virol, 62: 610-613, 1988) and primary human fibroblasts (Wa tanabe, S et al., J. Virol, 63, 965-969, 1989). Protein Mutational analysis of E7, intended to elucidate the relationship between quality structure and function, A single point mutation at acid 24 eliminates transforming ability, while transactivating activity (Edmonds, C et al., J. Virology 63, 2650-2656, 1989) Therefore, this E7 variant was called p24. Theoretically, present in most cervical cancers E7 proteins are attractive targets for in vivo stimulated T cell responses.   Since the mouse cell line is not permissive for HPV infection, two surrogate HPV regions were added to DA / p24 Produced by transducing a retroviral vector from a producer strain did. The DA / p24 producer strain was transformed with the p24 gene (Edmonds, C et al., J. Virology 63, 26). 50-2656, 1989) was inserted into the retroviral backbone instead of the ras gene. JC (ATCC No. C), a mammalian carcinoma cell line constructed as described in Example 2 above RL-2116) and CT26, a colorectal carcinoma cell line (Michael Brittain at Baylor Co.) llege of Medicine) was transduced with DA / p24, and JC.p24 and CT26.p24, respectively. Was prepared.   The transduced dendritic cell fraction was prepared as follows. Slaughter native mice The spleen dendritic cell (SP-DC) fraction was prepared as described in Example 1. Bone marrow dendritic cells (BM-DC) was prepared by washing long bones followed by complement-mediated removal of B cells. Made. They were converted to RPMI 1640 medium containing GM-CSF (500 U / ml) and IL-4 (1000 U / ml). Put in. DA / p24, without leaking producer cells across the membrane, 0.45 Transwell with micron pore size (Corning Costar, Cambridge, MA ). The dendritic cell fraction was transferred to low attachment tissue culture wells (Corning Costar, Cam bridge, MA) and place the transwell containing DA / p24 on the dendritic cell fraction. placed. After 7 days of co-cultivation, the recovered cells are transferred to a natural recipient for prevention. 3 washes with PBS prior to injection into tomato or tumor-bearing mice for treatment experiments did. The parental immortalized dendritic cell line, D2SC / 1 (WO 94/28113), Were transduced. The resulting two immortalized dendritic cell lines D2SC / 1.β-gal and And D2SC / 1.p24 were used as controls.   For treatment experiments, either JC.p24 or CT26.p24 tumor-bearing BALB / c mice were used. And syngeneic BM-DC transduced by co-culture with the DA / p24 producer cell line. Or treated with SP-DC dendritic cells. Tumors were inoculated on day 1 followed by p24 traits Treated weekly with introduced BM-DC or SP-DC. Native BALB / c mice for immunization experiments Were immunized twice on days 1 and 7 with p24-transduced BM-DC or SP-DC, followed by 1 On day 4, she was attacked with JC.p24 or CT26.p24. All mice were treated twice a week with tumor cells The product was measured. For all experiments, the mean tumor volume was Plotted. A.CT26.p24 model   2 × 10^Five CT26.p24 cells were transferred to 50 BALB / c mice (5 groups of 10 mice each) ) And dendrites from p24-transduced BM or spleen on days 7, 14, and 21 The cells were treated. D2SC / 1. β-gal and DS2C / 1.p24 at 5 × 10⁶Cell / Mau Injections / treatment. BM-DC and SP-DC were 2-3 × 10⁶Cells / mouse / Treatment or 4-10 × 10^FiveInjected cells / mouse / treatment. BM-DC and SP-DC efficiency Is estimated to be between 0.1 and 1%, thus 2 × 10^Three~ 3 × 10^Four(BM-DC) or 4 × 10^Two ~ 1 × 10^Four(SP-DC) transduced dendritic cells / mouse / treatment seemed to be injected .   The results on the effect of dendritic cell therapy using CT26.p24 cells are shown in FIG. p24 Mice immunized with transduced SP-DC or BM-DC were compared to control mice. Showed less than a 30% reduction in tumor volume. B.JC.p24 model   3 × 10^Five JC. P24 cells were transferred to 50 BALB / c mice (5 groups of 10 mice each) And treated on days 7, 14, and 21 with transduced dendritic cells. D2SC / 1.β-gal and D2SC / 1.p24 at 5 × 10⁶Injected cells / mouse / treatment. BM-D C and SP-DC were treated with 2-3 × e6 cells / mouse / treatment or 4-10 × 10 6, respectively.^FiveFine Injected in vesicles / mouse / treatment. Transwell co-culture for BM-DC and SP-DC The transduction efficiency was estimated to be between 0.1 and 1% and therefore 2 × 10^Three~ 3 × 10^Four(BM-DC) Or 4 × 10^Two~ 1 × 10^Four(SP-DC) transduced dendritic cells / mouse / immune injected .   The results on the effect of dendritic cell therapy using JC.p24 are shown in FIG. SP-DC immunity Mice show about a 60% reduction in tumor volume when compared to control animals. Indicated. Does the fact that BM-DC vaccinated animals were injected only once with BM-DC? Nevertheless, some protection was still observed. Some animals have reduced tumor burden showed that. D2SC / 1.β-gal immunized mice were unimmunized control and D2SC / 1.p24 immunized It showed an intermediate level of protection between the animals. 2.Tumor prevention   A.JC.p24 tumor   Fifty BALB / c mice were immunized twice on day 1 and 7.3 x 10^Five JC.p24 cells, 5 0 BALB / c mice (5 groups of 10 mice each) were injected and 7,14, And on day 21 they were treated with transduced dendritic cells. D2SC / 1.β-gal and D2SC / 1.p24 is 5 × 10⁶Injected cells / mouse / treatment. BM-DC and SP-DC, respectively , 2-3 × 10⁶Cells / mouse / treatment or 4-10 × 10^FiveInjected cells / mouse / therapy . The transduction efficiency of BM-DC and SP-DC by transwell co-culture is 0.1 to 1 % And therefore 2 × 10^Three~ 3 × 10^Four(BM-DC) or 4 × 10^Two~ 1 × 10^Four( Transduced dendritic cells / mouse / immunization of (SP-DC) were injected.   The results on the effect of dendritic cell immunization using JC.p24 are shown in FIG. BM-DC Waku Despite the fact that chin-inoculated animals were injected only once with BM-DC, some Was still observed (FIG. 6). Some animals showed reduced tumor burden . D2SC / 1.β-gal immunized mice (FIG. 7) were treated with non-immune control and D2SC / 1.p24 It showed an intermediate level of protection between immunized animals. B.CT26.p24 model   Dendritic cell-based prophylaxis and treatment is more effective if the tumor is immunogenic It has been suggested in the literature (Zitvogel et al., Exp J. Med. 183: 87, 1996). I have. Dendritic cell prevention or treatment achieves complete protection or regression of immunogenic tumors But weakly immunogenic tumors can only achieve partial protection or regression . JC.p24 is weakly immunogenic and, therefore, probably less sensitive to immunotherapy. A similar prophylactic experiment with immunogenic CT26.p24 would give better protection since it was not (See FIG. 8). 3.Comparison of Dendritic Cell Loading to Other Methods   The following experiments demonstrate that retroviral transduction is not Equivalent to vesicle protein or peptide loading. β-gal Was substituted in CT26.β-gal, a CT26 tumor transduced with the DA / β-gal vector. Used as tumor antigen. The immune response was transduced with β-gal or Induction using bone marrow dendritic cells, either peptide or protein loaded Led. The following groups were compared for immunity induction, and the results were It was shown that CTL responses derived from all of the loops could be comparable. The result is Dendritic cell loading of peptides or proteins is most important for inducing an immune response. Was also considered important because it was considered a good method. Similar potency, but optimized Was achieved by the use of a transduction method.   1. No treatment   2. BM-DC. β-gal (1 × 10⁶) Transduction (DA / β-gal co-culture, 7 days)   3. Overnight protein incubation (100 μg / ml)   4.2 hours peptide loading (20 ng / ml) + hb2m (10 μg / ml) (human β-2 Microglobulin is a standard protocol for cellular loading of antigenic peptides. Is col)   FIG. 9 shows a comparison between methods of introducing antigen into dendritic cells. As shown in this figure Equivalent level of protection against tumor attack, retroviral vector transduction Immunization using dendritic cells and dendritic cells loaded with antigenic peptides or proteins Observed from quarantined animals. Β-gal pepti used to load dendritic cells The molar ratios of the proteins or proteins were so large (> 1 million times) Retroviral vector transduction efficiency was estimated to be only 1%), retrovirus Ils transduction achieves protection equivalent to peptide or protein loading Highly encouraged.   The table below shows the levels of protein expression required for induction of immunity by dendritic cells. Indicates that there is only 1% of the level of fibroblasts. Specifically, fibroblast immunity Equivalent net CTL lysis in animals with transduced dendritic cells with 1/100 protein expression Observed in immunized mice. This result indicates that antigen presentation by dendritic cells is excellent. Prove efficiency.  Although the invention has been described above both in general and with respect to preferred embodiments, It is understood that changes and modifications will occur to those skilled in the art in light of the foregoing description. You. Accordingly, the appended claims are intended to cover all such claims as falling within the scope of the invention as claimed. It is intended to cover such changes.   In addition, publications, patents, patent applications, and the present invention may be , Particularly with respect to its implementation as described in the detailed description and examples. Other materials cited to provide details are incorporated herein by reference in their entirety. Invite to

────────────────────────────────────────────────── ─── Continuation of front page    (31) Priority claim number 08 / 772,010 (32) Priority date December 19, 1996 (December 19, 1996) (33) Priority country United States (US) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), CA, JP (72) Inventor Lee, Virginia             United States California 92007,             Cardiff, Evergreen Drive             1374 (72) Inventors Gory and Douglas J.             United States California 92924,             Leucadia, Hillcrest Drive               277

Claims (1)

[Claims] 1. Directs expression of dendritic cell targeting elements and at least one disease-associated antigen A gene delivery vehicle comprising an expression vector. 2. 2. The method of claim 1, wherein the expression vector is carried by a recombinant virus. Gene delivery vehicle. 3. 3. The genetic of claim 2, wherein said recombinant virus is a recombinant retrovirus. Child delivery vehicle. 4. The gene transfer according to claim 2, wherein the recombinant virus is an alphavirus. Vehicle. 5. The expression vector is used for cancer, heart disease, bacterial infection, parasite infection, and virus. Directing the expression of an antigen associated with a disease selected from the group consisting of 2. The gene delivery vehicle of claim 1. 6. A gene delivery vehicle and a pharmaceutically acceptable carrier according to claim 1. A pharmaceutical composition comprising: 7. An in vivo method for producing a genetically modified dendritic cell, the method comprising: Administering to the animal a gene delivery vehicle targeting dendritic cells. Wherein the gene delivery vehicle comprises a dendritic cell targeting element and at least one A method comprising an expression vector that directs the expression of a disease-associated antigen. 8. 8. The method of claim 7, wherein said gene delivery vehicle is a recombinant virus. 9. A method of stimulating a prophylactic immune response in an animal, said method comprising: Vector directing expression of a genetic element and at least one disease-associated antigen Administering to the animal a prophylactically effective amount of a gene delivery vehicle comprising Law. 10. 10. The method of claim 9, wherein said gene delivery vehicle is a recombinant virus. .