JP2002522067A - Engineered antigen-presenting cells expressing a group of antigens and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for presenting a plurality of disease-related antigens on antigen-presenting cells. This method can be used to generate a prophylactic or therapeutic immune response against a disease associated with the antigen. This method uses differential screening of nucleic acid sequences expressed by target and non-target cells. By identifying nucleic acid sequences that are preferentially expressed in a target cell population and expressing the identified sequences in antigen presenting cells, one of skill in the art is restricted to a single antigen previously identified. Without stimulating an immune response directed at the target cell population.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION [0002] The present invention relates to T lymphocytes by antigen-presenting cells that have been genetically modified to express and present on a surface a group of antigens that are differentially expressed by a target population. And immunotherapy comprising the activation of. Antigen presenting cells can be genetically modified by transducing with a nucleic acid sequence identified by differentially screening nucleic acid sequences expressed in a target population as compared to a non-target population.

BACKGROUND OF THE INVENTION [0003] Dendritic cells specialized as antigen presenting cells process antigens from diseased tissues,
And can be used to present them to the immune system. Dendritic cells are leukocytes from bone marrow and are significantly more potent than other antigen presenting cells with respect to the presentation and activation of cytotoxic T lymphocytes (CTLs). . Publications on dendritic cells include: Young et al., 1996, J. Am. Exp. Me
d. 183: 7-11, Alavaikoko et al., 1994, Am. J. Clin
. Pathol. 101: 761-767; Shunici et al., 1995, C.
ancer 75: 1478-1483; Hsu et al., 1996, Nature.
Medicine 2: 52-58; Mayordomo et al., 1995, Nat.
ure Medicine 1: 1297-1302; Paglia et al., 199
6, J. Exp. Med. 183: 317-322; and Boczkowsk
i et al., 1996, J. Am. Exp. Med. 184: 465-472.

[0004] There is a need for vaccines capable of boosting multiple CTL clones to enhance the immune response and to avoid escaping tumors from immune selection.

[0005] Thus, there is provided an antigen presenting cell with (i) a broad spectrum of potent anti-tumor antigens, and (ii) presenting the antigen in a form in which it can be efficiently processed and present. There is a need for a system for:

SUMMARY OF THE INVENTION The present invention provides a system for priming antigen presenting cells having a repertoire of antigens of a particular cell type (eg, tumor cells or virus-infected cells).
Furthermore, this approach need not be limited to stimulating an immune response against diseased tissue. This can be used to mount an immune response against any target tissue and to excise any target cells that express a particular set of antigens.

[0007] The present invention provides a method of making at least one vector encoding a group of antigens for expression in antigen presenting cells. In one embodiment, the method includes comparing a first nucleic acid sequence expressed by a target cell population to a second nucleic acid sequence expressed by a non-target cell population. The method further comprises selecting a nucleic acid sequence that is preferentially expressed by the target cell population as compared to the non-target cell population, and expressing the selected nucleic acid sequence in an antigen presenting cell. Into at least one vector capable of directing E. coli. In one embodiment, the antigen presenting cells are dendritic cells, macrophages, B
Cells, monocytes or fibrous cells. In another embodiment, the vector further comprises:
Includes dendritic cell targeting element.

[0008] In one embodiment, the first nucleic acid sequence and the second nucleic acid sequence are of the same tissue origin. The selected nucleic acid sequence can consist of one or more numbers. For example, the selected nucleic acid sequence can include at least three different nucleic acid sequences, at least five different nucleic acid sequences, at least seven different nucleic acid sequences, or at least nine different nucleic acid sequences. The vector may further include a nucleic acid sequence encoding an immunomodulatory cofactor. Immunomodulatory cofactors include, for example, Il-2, IL-3, IL-8, OKT3,
It can be α-interferon, γ-interferon, or MIP-1α. The vector may further encode at least one selectable marker. Examples of selectable markers include, but are not limited to, PLAP, GFP, and neomycin resistance. In one embodiment, the target cells are cancer cells. In another embodiment, the target cells are pathogenic agents (eg, viruses, bacteria, or parasites).

[0009] The present invention further provides a composition comprising at least one vector produced by the above method. In one embodiment, the vector further comprises an antigen presenting cell targeting element. The composition may further include antigen presenting cells.

[0010] The present invention also provides a method of generating an antigen presenting cell that presents a group of antigens. The method includes comparing a first nucleic acid sequence expressed by a target cell population to a second nucleic acid sequence expressed by a non-target cell population. The method further comprises selecting a nucleic acid sequence that is preferentially expressed by the target cell population as compared to the non-target cell population, and genetically modifying the antigen-presenting cells to express the selected nucleic acid sequence Performing the steps of: Also provided is an antigen presenting cell produced by the above method, or a genetically modified antigen presenting cell with a vector produced by the method of the present invention.

[0011] The present invention provides a method of activating immune cells. This method uses immune cells
Contacting with a genetically modified antigen presenting cell according to the present invention.
In one embodiment, the immune cells are T cells. Examples of T cells activated by this method include, but are not limited to, cytotoxic T lymphocytes (CTL) and helper T cells. Also provided are methods of inducing a tolerogenic response. The method comprises the step of contacting an immune cell with a genetically modified antigen presenting cell according to the invention. In one embodiment, the immune cells are helper T cells (eg, _TH2 cells). In one embodiment of the method of activating T cells or inducing a tolerogenic response, the step of contacting occurs in vivo. Alternatively, the step of contacting can occur ex vivo.
The present invention also provides immune cells (eg, CTL and _TH2 cells) activated by the methods of the present invention. The activated immune cells can be provided in the form of a composition.

[0012] The present invention provides a method of activating T cells in vivo, comprising the step of administering to a subject a composition comprising a vector or antigen presenting cell of the present invention. Also provided is a method of killing a target cell in vivo, the method comprising administering to the subject a composition, vector, or antigen presenting cell of the invention.
The present invention also provides methods of preventing infection, treating cancer, and treating viral infection, the method comprising administering a composition, vector, or antigen presenting cell of the invention to a subject. Process.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a strategy for the presentation of multiple antigens on antigen presenting cells, wherein the cells are prophylactically or against one or more target cell populations to which the antigen binds. Can be used to generate a therapeutic immune response. This strategy makes use of comparing and selecting nucleic acid sequences expressed by target and non-target cells. By identifying nucleic acid sequences that are preferentially expressed in a target cell population and expressing the identified sequences in antigen presenting cells, the target cell population is not limited to previously identified antigens Can stimulate an immune response directed at the The use of a panel of antigens may elicit a more effective immune response by directing lymphocytes to various antigen targets. This multiple antigen strategy targets the immune response to more antigens associated with a given disease, and immunizes against a disease for which the effective antigen for a particular patient or unknown disease is unknown. It is advantageous for both targeting the response.

[0014] The vectors and antigen-presenting cells of the present invention can be prepared without prior purification of individual disease-associated antigens. This is particularly useful if a given tissue or cell expresses more than one disease-associated antigen, or if the target antigen is unknown. By using differential screening to identify preferentially expressed nucleic acid sequences, one can avoid problems associated with lack of specificity. This is because nucleic acid sequences encoding non-target antigens can be excluded.

[0015] The present invention provides complex preparations of antigen-encoding vectors for expression in antigen-presenting cells. The cells can then be used to activate T cells in the subject, in vivo, ex vivo, or in vitro, without specific prior knowledge of the relevant disease-associated antigen in the subject. Complex preparations can be used to activate various T cells and can be directed against one or more antigens and against one or more disease or target cell populations.

Definitions All scientific and technical terms used in this application have the meaning commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the specified meanings.

As used herein, “target cell population” refers to one or more cells that share at least one general characteristic, for which removal or protection of that characteristic is desired. . Examples of target cells include infectious agents (eg, viruses, bacteria, or parasites), cells susceptible to an autoimmune attack, and disease cells (eg, cancer cells (eg, highly metastatic cancer cells and low Including metastatic cancer cells)). When used in the context of a target cell, "infectious agent" includes both the infectious agent itself and cells infected by the agent. For example, if the target cell is a virus, the target cell can be the virus alone, a substantially infected cell, or both.

As used herein, "preferentially expressed" refers to a nucleic acid sequence that is substantially more abundant in a target sample when compared to a non-target sample. Substantially greater can be at least about 20% greater. In one embodiment, substantially greater is about 50% greater. In another embodiment, substantially greater is about twice the amount. In a preferred embodiment, substantially greater is at least about 10 times the amount. In more preferred embodiments, substantially more is at least about 20, 50, or 10
The amount is 0 times. The relationship between target and non-target samples is chosen to optimize the identification of antigen-encoding sequences that are relatively specific for the target cells. For example, the target cells can be colon cancer cells, and the corresponding non-target cells are non-cancerous colon cells. In another example, target cells can be highly pathogenic viruses and non-target cells can be less pathogenic viruses. Such differential screening between target and non-target populations can result in nucleic acid sequences encoding antigens associated with a particular disease or pathogen without requiring knowledge of the relevant antigens.

As used herein, “vector” refers to a construct capable of delivering, and preferably expressing, one or more genes or sequences of interest into a host cell. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, DNA or RNA expression vectors with cationic aggregating agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (eg, Producer cells).

As used herein, “expression control sequence” refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter (eg, a constitutive or an inducible promoter, or an enhancer). The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

The term “nucleic acid” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, in a manner similar to naturally occurring nucleotides And known analogs of natural nucleotides that hybridize to nucleic acids. Unless otherwise indicated, a particular nucleic acid sequence optionally includes complementary sequences.

As used herein, “antigen presenting cell” or “APC” means a cell that can handle an antigen and present it to lymphocytes. Examples of APCs include macrophages, Langerhans-dendritic cells, follicular dendritic cells,
Including but not limited to B cells, monocytes, fibroblasts and fibrocytes. Dendritic cells are a preferred type of antigen presenting cell. Dendritic cells are found in many non-lymphoid tissues, but can migrate through the afferent lymphatic or blood stream to T-dependent areas of lymphoid organs. In non-lymphoid organs, dendritic cells include Langerhans cells and intestinal dendritic cells. In lymph and blood, they are
Including afferent lymph veil cells and blood dendritic cells. In lymphoid organs,
They are lymphoid dendritic cells and interdigital cells
ng cell). As used herein, each of these cell types and each of their progenitors is referred to as a "dendritic cell" unless otherwise indicated.

As used herein, “antigen presenting cell targeting element” refers to a molecule that can specifically bind antigen presenting cells (eg, dendritic cells). If a biological effect is seen in the cell type after binding of the targeting element and its complement, or between binding of the targeting element bound to dendritic cells and non-target cells, a 10-fold increase Difference greater than, and preferably 25, 50 or 100
If there is more than a two-fold difference, the targeting element specifically binds to dendritic cells. Generally, the targeting element is less than 10 ^-5 M, preferably less than 10 ^-6 M,
More preferably less than 10 ^-7, and most preferably less than 10 ^-8 M K _D (Scatchard analysis (Scatchard, 1949, Ann.N.Y.Acad.
Sci. 51: 660-672)). Suitable targeting elements are preferably non-immunogenic, not degraded by proteolysis, and are not removed by the immune system. Certain preferred targeting elements are found in animals in the context of (cleaning a
gent) (in the absence of gent). Examples of dendritic cell surface markers that antibodies (or antigen-binding domains derived therefrom) can be made to give rise to dendritic cell targeting elements include those in FIG. 1 (eg, CD1, CD11
a, CD11c, CD23, CD25, CD32, CD40, CD45, CD5
4, CD58, MHC class I, MHC class II, Mac-1, Mac-2,
Mac-3), but not limited thereto.

As used herein, an “immunomodulatory cofactor” is an agent that, when expressed on an APC, has enhanced quality or strength from an immune response elicited in the absence of a cofactor. And cofactors that elicit an immune response to the antigen presented by the APC. The quality or intensity of the response can be measured by various assays known in the art, including, for example, in vitro assays to measure cell proliferation (
For example, ³ H-thymidine incorporation), and in vitro cytotoxicity assays (eg, measuring ⁵¹ Cr release; Warner et al., 1991, AIDS Res. And Human Retroviruses 4: 645-655). In another embodiment, the immunomodulatory cofactor is not encoded in the expression vector but is added exogenously before, simultaneously with, or after administration of the vector. Examples of immunomodulatory cofactors include cytokines and chemokines (eg,
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, I
L-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-
14, IL-15, OKT3, α-interferon, β-interferon,
γ-interferon, MIP-1α (LD-78), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
Tumor necrosis factor (TNF), CD3, CD8, ICAM-1, ICAM-2, LF
A-1, LFA-3, and other proteins (eg, HLA class I molecules, H
LA class II molecules, B7, B7-2, β-microglobulin, chaperone, and MHC binding transporter protein or analog thereof)). The choice of an immunomodulatory cofactor is based on the therapeutic effect of this factor. Preferred immunomodulatory cofactors include α-interferon, γ-interferon and IL-2.

The present invention relates to nucleic acid sequences that encode a group of antigens that are preferentially expressed in a population of target cells and that can be used to genetically modify antigen presenting cells (APCs) I will provide a. The invention further provides a method for preparing these nucleic acid sequences.
These nucleic acid sequences can be prepared by comparing a first nucleic acid sequence expressed by a population of target cells to a second nucleic acid sequence expressed by a population of non-target cells. A nucleic acid sequence that is preferentially expressed by the target cell population relative to the non-target cell population is then selected. This method provides nucleic acid sequences that can be used to express a panel of antigens in an APC without prior knowledge of the antigen and without compromising the specificity of the population of target cells. Nucleic acid sequences include DNA, RNA, and their synthetic derivatives.

A method for comparing sets of nucleic acid sequences and selecting for differentially expressed sequences comprises:
It is known in the art. The comparison can be performed using a nucleic acid sequence isolated from the sample (eg, from one or more cell types), or it can be performed, for example, using a public database (eg, GenBank, EMBL, or DNA).
It can be performed by comparing sequence data obtained from Database of Japan (DDBJ). For example, when two nucleic acid samples are compared (eg, a sequence from a population of target cells and a sequence from a population of non-target cells), each sample is an isolated pool of nucleic acid sequences, database sequence information. , From any other source of nucleic acid sequence information, or a combination of two or more sources. Conventional techniques for differential display of mRNA and differential screening of cDNA libraries are described in F.S. M. Ausubl et al., 1
996, Current Protocols in Molecular B
iology, John Wiley & Sons, Inc. Unit 5.8
And Sections 5.8.6 and 5.8.14.

One example of detecting differential expression of a gene is described in US Pat. No. 5,677,125 for a method of detecting and diagnosing pre-invasive breast cancer. In this example, epithelial cells are isolated from a sample of abnormal breast tissue exhibiting histological and cytological characteristics of pre-invasive breast cancer, and mRNA is isolated from this sample. A cDNA library of one or more abnormal breast tissues is prepared from the isolated mRNA. One or more cDNA libraries are then prepared by similar means from samples of normal breast tissue. Abnormal and normal library cD
NA is compared to detect the expression of at least one gene in an abnormal sample that is different from the gene expressed in a normal sample. A similar approach can be applied for introduction into APCs to obtain a panel of nucleic acid sequences that are preferentially expressed by a target cell population over a non-target cell population.

[0028] The non-target cells may be normal cells or diseased cells, but have a different spectrum of antigens than the target cells. For example, it may be preferable to obtain an antigen specific for a particular stage in the disease. Thus, the non-target cells may be cells at different stages of the target disease. An example of a non-target cell population is one or more selected from stage 1 and stage 2 cells in cervical carcinoma. Antigen presenting cells that present an antigen specific for the desired stage (eg, stage 3 cervical cancer) can be provided. In another example, the target cell population is a high metastatic colon cancer cell line and the non-target cell population is a low metastatic colon cancer cell line. Differential screening of nucleic acid sequences expressed by two cell lines can be used to select sequences encoding antigens specific for highly metastatic colon cancer cells. If the non-target cells are normal cells, differential screening will remove or reduce nucleic acid sequences common to normal cells, thereby avoiding an immune response to antigens present on normal cells. If the non-target cells are normal cells, differential screening will remove or reduce nucleic acid sequences common to normal cells, thereby avoiding an immune response to antigens present on normal cells. Examples of combinations of target non-target cells suitable for differential screening include, but are not limited to: KM12L4A cells (high metastatic colon cancer line; Morikawa et al., 1988, Cancer Res. 48: 1943).
/ KM12C (low metastatic colon cancer line; Morikawa et al., 1988, Cance.
r Res. 48: 6863); MDA-MB-231 cells (highly metastatic breast cancer strain;
Brinkley et al., 1980, Cancer Res. 40: 3118) / M
CF-7 cells (non-metastatic breast cancer line; Yang et al., 1998, Anticancer)
Res. 18 (1A): 53-59); and MV-522 cells (highly metastatic lung cancer line; Varki et al., 1987, Int. J. Cancer 40:46) / U.
CP-3 cells (low metastatic lung cancer cell line; Varki et al., 1987, supra).

Differential screening has also been tailored for various purposes of antigen presenting cells (ta
Illustrated) combinations can be provided. One combination of the APC of the present invention is:
Includes antigens for related symptoms. For example, HIV infected patients often have CMV
Infected by Thus, antigen presenting cells can be prepared. It expresses genes that are preferentially associated with HIV infected cells. Then a second preparation can be prepared. Here, the antigen presenting cells express an antigen that is preferentially expressed by cells infected with CMV. These two preparations can be combined and administered to a subject. This provides simultaneous treatment for both conditions. Examples of other relevant conditions in which preparations of antigen presenting cells may be combined include HIV and Epstein-
Burr virus (EBV) positive lymphoma; and HIV and hepatitis C virus (HCV).

Another example where separate preparations can be combined provides APCs for targeting tumors of uncertain origin. In this case, antigen-presenting cells that have been genetically modified to express nucleic acid sequences from the spectrum of the tumor type can be used to elicit a broad anti-tumor immune response.

A third example where separate preparations can be combined provides for targeting a disease associated with a different spectrum of antigens in different patients. Preparations from various patients containing most or all repertoires of the antigens associated with the disease in most or all patients can be performed without having to first determine which antigen is expressed in the disease of the individual subject. Can be combined and administered to a patient.

The combination can be prepared at the nucleic acid level. In this case, the preferentially expressed nucleic acids are combined and then introduced into antigen-presenting cells in vitro. Alternatively, separate antigen presenting cells can be prepared using nucleic acids that are preferentially expressed. This can then be combined to provide a complex antigen preparation.

In one embodiment of the invention, the mR preferentially expressed by the target cell population
Using NA, a nucleic acid, such as a cDNA, encoding a full-length sequence is obtained. The nucleic acid sequence so obtained can be used to identify preferentially expressed antigens associated with the target cell population. The antigens so obtained can then be tested to determine which are most effective as immunogens.

In one example of testing the immunogenicity of an antigen, peripheral blood cells are removed from a subject. The dendritic cells are then isolated and pulsed with the antigen to be tested or genetically modified to express the test antigen. The T cells of the subject can then be stimulated in vitro with the pulsed or modified dendritic cells. The ability of the modified dendritic cells to stimulate T cells indicates the immunogenicity of the test antigen.

(Vector) The present invention provides a vector containing the nucleic acid sequence selected by the differential screening as described above. The selected sequence can be introduced into at least one vector. Preferably, the vector is capable of directing the expression of a selected nucleic acid sequence in antigen presenting cells (eg, dendritic cells). A vector may encode a single antigen or multiple antigens. Where multiple antigens are encoded by a single expression vector, these antigens may be from the same or different cell types. Alternatively, compositions comprising more than one vector, each encoding one or more antigens associated with the same or a different cell type as the cell type encoded by the other vector, can also be prepared.

In one embodiment, the invention provides a method for producing at least one vector encoding a group of antigens for expression in antigen presenting cells. In one embodiment, the method comprises: a first nucleic acid sequence expressed by the target cell population;
Comparing to a second nucleic acid sequence expressed by the non-target cell population. The method comprises the steps of selecting a nucleic acid sequence that is preferentially expressed by the target cell population as compared to a non-target cell population, and selecting the selected nucleic acid sequence for expression of the selected nucleic acid sequence in antigen presenting cells. The method further comprises the step of introducing into at least one vector that can be directed. In one embodiment, the antigen presenting cells are dendritic cells, macrophages, B cells, monocytes or fibrocytes. In another embodiment, the vector further comprises a dendritic cell targeting element.

[0037] In one embodiment of the method, the first nucleic acid sequence and the second nucleic acid sequence are the same tissue of origin. The selected nucleic acid sequence can be one or more. For example, the selected nucleic acid sequence can include at least three different nucleic acid sequences, at least five different nucleic acid sequences, at least seven different nucleic acid sequences, or at least nine different nucleic acid sequences. The vector may further include a nucleic acid sequence encoding an immunomodulatory cofactor.
This immunomodulatory cofactor is, for example, IL-2, IL-3, IL-8, OKT3, α-
It can be interferon, γ-interferon or MIP-1α. The vector may further encode at least one selectable marker. Examples of selectable markers include PLAP (US Ser. No. 09 / 006,298, 1998).
(Filed January 13, 2012), GFP and neomycin resistance, but are not limited thereto. In one embodiment, the target cells are cancer cells. In another embodiment, the target cells are infectious agents (eg, viruses, bacteria or parasites).

The vectors of the invention can be used to genetically modify APCs, such as dendritic cells, either in vivo or in vitro. Several methods for genetically modifying APCs are known, including transduction with viral vectors, either directly or via retroviral producer cells, calcium phosphate precipitation, Of recipient cells with bacterial protoplasts, including DN
Treatment of recipient cells with liposomes containing A, DEAE dextran, receptor-mediated endocytosis, electroporation, microinjection, and many other techniques known to those skilled in the art. For example, Meth
ods in Enzymology, 185, Academic Press
, Inc. , San Diego, CA (ed. By DV Goeddel) 1990.
Or M. Krieger, Gene Transfer and Expr
ession-A Laboratory Manual, Strokto
n Press, New York, NY, 1990, and references cited therein, as well as Berger and Kimmel, Guide to Mo.
rectangular Cloning Technologies, Methods i
n Enzymology 152 Academic Press, Inc.
, San Diego, CA (Berger); Sambrook et al., Mole.
cultural Cloning-A Laboratory Manual (2nd
Edition) 1-3 1989; and Current Protocols in
Molecular Biology, F.M. M. Ausubel et al., Green
ne Publishing Associates, Inc. , And Joh
n Wiley & Sons, Inc. , (Supplement 1994), WO93 / 2
4640; Mannino and Gould-Fogerite 1988, B
iotechniques 6 (7): 682-691; U.S. Pat. No. 5,279.
WO91 / 06309; and Felgner et al., Proc. N
atl. Acad. Sci. ScL USA 84: 7413-7414 1987.

[0039] Examples of viral vectors include, for example, retroviral vectors based on HSV, HIV, mouse retrovirus, gibbon leukemia virus and other viruses such as adeno-associated virus (AAV) and adenovirus. However, it is not limited to these. (Miller et al., 1990, Mol
Cell Biol. 10: 4239; Kolberg 1992, NIH
Res. 4:43, and Cornetta et al., 1991, Hum. Gene
Ther. 2: 215). Widely used retroviral vectors include mouse leukemia virus (MuLV), gibbon monkey leukemia virus (GaL
V), autotrophic retroviruses, human immunodeficiency virus (HIV) and combination based retroviral vectors. For example, Buchscher
Et al., 1992; Virol. 66 (5): 2731-2739; Johan
n et al., 1992, J. Am. Virol. 66 (5) 1635-1640; Somem
rfeld et al., 1990, Virol. 176: 58-59; Wilson et al.
1989, J.A. Virol. 63: 2374-2378; Miller et al., 19
91; Virol. 65: 2220-2224, and Rosember
g and Fauci 1993, Fundamental Immunolog
y, 3rd edition, W.C. E. FIG. Paul (eds.) Raven Press, Ltd. , Ne
w York and references therein; Miller et al., 1990, Mol. C
ell. Biol. 10: 4239; Kolberg 1992, J. Mol. NI
H Res. 4:43; and Cornetta et al., 1991, Hum. Ge
ne Ther. 2: 215.

A mouse vector containing the gibbon ape leukemia virus (GaLV) envelope can be used to transduce many mammalian cells. Johan et al., 1
992, see above. The same receptor is used by the simian sarcoma-associated virus (SSAV), a strain of GaLV (Somerfeld et al., 199).
0, supra). The construction of hybrid virions with a GaLV envelope protein has been demonstrated (Wilson et al., 1989, supra; Miller et al., 19).
91, supra). Any of these vectors and methods for generating retroviral clones can be applied to the present invention. GaLV retroviral packaging cell lines are used in gene transfer in humans, hamsters, cows, cats, dogs, monkeys, chimpanzees, macaques, primates, and other species whose cells have a host cell receptor for the GaLV envelope protein. Can be used to provide infectious replication-defective hybrid virions.

Further examples of vectors include adenoviral vectors, AAV vectors, poxviral vectors (B. Moss, 1992, Current T
opics in Microbiology and Immunology
158: 25-38) (including vaccinia, chicken pox and canarypox), recombinant influenza viral vectors (A. Garci).
a-Sastre and P.A. Palese 1995, Biologicals
23: 171-178), or non-viral gene delivery technology (F. Leeds).
ley 1994, Biotechnology 5: 626-636). AAV-based vectors can be used to transduce cells with a selected nucleic acid, for example, in in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures. West et al., 1987, Virology 160: 38-47; U.S. Patent No. 4,797,368; WO 93/24641; Kotin 1994,
Human Gene Therapy 5: 793-801; and Muzy
czka 1994, J. Mol. Clin. Invest. 94: 1351.

[0042] Suitable in vitro amplification procedures for amplifying sequences to be subcloned into expression vectors are known. Examples of such in vitro amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase-mediated techniques (eg, NASBA); Berger, Sambrook et al. 1989, Molecular Clo
ning-A Laboratory Manual (2nd edition) 1-3; and U.S. Pat. No. 4,683,202; PCR Protocols A Guide
Eto Methods and Applications (Edited by Innis et al.) Academic Press Inc. San Diego, CA 19
90; Arnheim & Levinson (October 1, 1990) C & E
N36-47; NIH Res. 1991, 3: 81-94; Kwoh et al.
1989, Proc. Natl. Acad. Sci. USA 86: 1173;
Guatelli et al., 1990, Proc. Natl. Acad. Sci. US
A 87: 1874; Lomell et al., 1989, J. Am. Clin. Chem. 3
5: 1826; Landegren et al., 1988, Science 241: 1.
077-1080; Van Brunt 1990, Biotechnology.
y 8: 291-294; Wu and Wallace 1989, Gene 4.
: 560; Barriner et al., 1990, Gene 89: 117; and Sooknanan and Malek 1995, Biotechnology.
13: 563-564. An improved method for cloning in vitro amplified nucleic acids is described in US Pat. No. 5,426,039.

Antigen Presenting Cells Antigen presenting cells (APCs) process antigen and present it to lymphocytes. The present invention provides APCs that present a panel of antigens. APCs are genetically modified to express a nucleic acid sequence that is preferentially expressed by a target cell population as compared to a non-target cell population. Examples of APCs include, but are not limited to, macrophages, Langerhans dendritic cells, follicular dendritic cells, B cells, monocytes, fibroblasts and fibrocytes. In a preferred embodiment, the APC is a dendritic cell. However, the choice of the optimal APC may vary depending on the antigen to be presented (Butz and Bevan 1998, J. Immunol. 160: 2).
130-2144).

[0044] Dendritic cells have abnormal dendritic morphology, are motile, efficiently cluster, and activate T cells specific for cell surface stimulation. Typically, dendritic cells in non-lymphoid organs (eg, Langerhans cells and stromal cells) are afferent lymphoids that migrate to lymphoid tissues if they can be isolated as dendritic cells or finger cells. And become veiled cells in the blood (cells that frequently elongate and contract large lamellipodia).

Dendritic cells initiate a T-dependent response from resting lymphocytes. Once sensitized, T cells interact with other antigen presenting cells. The activity of processing dendritic cell antigens is modulated. Fresh cells isolated from the skin or lymphoid organs (ie, cells cultured for less than one day) display native protein.
Thereafter, they do not process the antigen. Furthermore, dendritic cells are not actively phagocytic.

Dendritic cells are described in Tedder and Jansen, 1997, Current.
Protocols in Immunology, John Wiley & S
ons, unit 7.32, and can be isolated and purified using conventional methods. Another method for obtaining and identifying dendritic cells is described in WO98 /
15579; WO98 / 01538; WO98 / 15615; and WO98 /
14561. For example, human dendritic cells can be isolated from blood mononuclear cells by first enriching the peripheral cell population for dendritic cells by depletion of T cells and adherent cells. Low buoyant density cells are isolated using density gradient centrifugation of this preparation over metrizamide. This population is substantially free of lymphocytes and contains 20-80% dendritic cells. Dendritic cell purity can be determined by various techniques (hemocyte counting, cytocentrifi on glass slides).
immunization and immunofluorescence staining using flow cytometric analysis). However, flow cytometry is preferred for optical quantification and consistency.

Young et al. Produced pure dendritic cell colonies from normal human CD34.
+ (Positive for this hematopoietic stem cell marker) was identified as a dendritic cell colony forming unit in bone marrow progenitors (Young et al., 1995, J. Exp. M).
ed. 182: 1111-1120). Addition of c-kit-ligand to the GM-CSF- and TNF-α-supplemented suspension of CD34 + bone marrow cells increases dendritic cell colony forming units by about 100-fold by day 14. Dendritic cells from these colonies are potent stimulators of T cells.

A partially enriched population of epidermal Langerhans cells, where Langerhans cells can comprise up to about 60% of the total cell population, can be rapidly prepared. Keratinocytes can be depleted from mouse tissues using α-thy-1 (monoclonal antibody) and complement and adhesion. Enriched preparations of human Langerhans cells can be prepared by substituting anti-CD1 antibodies for α-thy-1. In culture, neither mouse nor human Langerhans cells are active antigen presenting cells until 1-3 days after culture, and after they have expanded, express MHC class II and cell adhesion molecules, and have Fc receptors, Deleting similar blood and lymph dendritic cells. Cell populations containing more than 90% dendritic cells can be obtained from human blood without enrichment if less than 0.1% of leukocytes are dendritic cells. Such enrichment is due to T cells, monocytes, and B cells.
And achieved by continuous depletion of NK cells, which can result in an initial population ranging from 30-60% dendritic cells. Then, a higher purity, in particular, against CD45R _A, monoclonal antibodies (which selectively reacts against contaminants) is obtained by panning or fluorescence-activated cell sorting using the (FACS) (P Freudenthal et al., 1990, Proc.
Sci. (USA) 87: 7698-7702). In order to enrich for dendritic cells, generally low buoyant density, non-adhesion to plastics in culture (especially
Days or more), and selection for the absence of the marker found on other cells is performed. Such methods deplete other cell types, but do not positively select dendritic cells.

Dendritic cells express distinct patterns of markers on their cell membranes. FIG.
Exemplifies this pattern by indicating the presence or absence of several distinct cell surface markers. Other markers that can be used to select positive or negative for dendritic cells include ICAM-1 (CD-54), LFA-3 (C
D58), and CD11b. Dendritic cells isolated from human or mouse blood (but not skin) express CD11a or LFA-1. In the skin, the immunostimulatory effect of dendritic cells is influenced by cytokines, especially GM-C
It can be enhanced by SF.

A dendritic cell, or other APC, is a population substantially comprising dendritic cells (ie, greater than about 50% dendritic cells, more preferably greater than about 75% dendritic cells, even more preferably (With more than about 90% dendritic cells, particularly preferably more than about 95% dendritic cells). Antigen presenting cells are preferably isolated from subjects in which the activated T cells are active ("self" therapy). Or,
Cells can be obtained from a donor or a cell bank, such as a blood bank.

The present invention provides a method for preparing an antigen-presenting cell that presents a group of antigens. A
PCs can be genetically modified using one or more vectors of the invention. Antigen presenting cells are transduced with a vector encoding a group of antigens. These antigens are then expressed in the cell, processed by the cell, and the relevant processed antigen fragment is transported to the cell surface where it can be displayed.

[0052] Cell cultures, including cell lines and cells (including dendritic cells) cultured from tissue or blood samples, for use in conjunction with the present invention are well known in the art. Freshney (Culture of Animal Cells,
a Manual of Basic Technology, 3rd edition, Wille
y-Liss, New York (1994)) and references cited herein provide general guidance for culturing cells.

T cells T cells can be isolated and activated in vitro by contact with antigen presenting cells. Some such techniques are well-known. The expression of the surface marker is T
Facilitates cell identification and purification. Methods for purifying and isolating T cells include:
FACS, incubation in flasks with immobilized antibodies that bind specific cell types and panning with magnetic beads.

[0054] T cells and dendritic cells are characterized by expression of certain markers on the cell surface and lack of expression of other markers. For example, dendritic cells express MHC molecules and costimulatory molecules (eg, B7-1 and B7-2), and granulocytes,
Deletes markers specific for NK cells, B cells and T cells. In mice, some, but not all, dendritic cells are 33D1 (dendritic cells from spleen and Peyer's patches but not from skin or thymic medulla), NLDC145 (T cells of skin and some lymphoid organs). Dendritic cells in dependent regions), and CD
11c (CD11c also reacts with macrophages). T cells have various markers depending on the particular subtype, most notably CD4 and C4.
D8) is positive.

Immunoassays for the detection of cells during cell isolation or cell purification include, for example,
It can be done in any of the several settings outlined below: Maggio
(Eds.) 1980, Enzyme Immunoassays, CRC Pres
s, Boca Raton, Florida; Tijan 1985, "Pra
ctice and Theory of Enzyme Immunoass
ays ", Laboratory Technologies in Bioche
Mistry and Molecular Biology, Elsevier
r Science Publishers B.R. V. , Amsterdam;
Harlow and Lane, supra; Chan (eds) 1987, Immunoa.
ssay: A Practical Guide Academic Pres
s, Orlando, FL; Price and Newman (eds.) 1991
, Principles and Practice of Immunoas
says Stockton Press, NY; and Ngo (eds) 198
8, Non-isotopic Immunoassays Plenum P
less, NY. For a review of common immunological and immunoassay procedures, see
States and Terr (eds) 1991, Basic and Cini
See cal Immunology (7th edition). For a discussion of how to generate antibodies to selected antigens, see, eg, Coligan 1991,
Current Protocols in Immunology Wheel
y / Greene, N.M. Y. And Harlow and Lane 1989;
, Antibodies: A Laboratory Manual Cold
Spring Harbor Press, N.M. Y. ; States et al. (Eds.)
See Basic and Clinical Immunology (4th edition).

[0056] Most preferably, the cells are isolated and characterized by a flow cytometry method using fluorescence activated flow cytometry (FACS). A wide variety of flow cytometry methods are known. For a general review of FACS, see, for example, Abbas et al., 1991, Cellular and Mole.
cultural Immunology, W.C. B. Saunders Compan
y (in particular, Chapter 3), as well as Kuby 1992, Immunology, W.
. H. See Freeman and Company (especially Chapter 6).

(Method) The present invention provides a method for using an antigen-presenting cell that presents a series of antigens. In one embodiment, the invention provides a method of activating immune cells in vivo. Examples of immune cells include, but are not limited to, T cells (including cytotoxic T lymphocytes and helper T cells). In one embodiment, the method comprises administering to the subject a vector encoding a series of antigens that are preferentially expressed by the target cell population as compared to the non-target population.
The vector is preferably constructed to direct the expression of an antigen that is preferentially expressed in antigen presenting cells. Preferably, the antigen presenting cells are dendritic cells. This vector can be further modified to encode an immunomodulatory cofactor, as described above. The target cell can be, for example, a cancer cell, virus, bacterium, parasite, or other disease-associated cell. Upon administration, the vector transduces the APC in the subject, thereby genetically modifying the APC in vivo. This genetically modified APC is then available to contact and activate immune cells.

In another embodiment, the method of activating immune cells comprises genetically activating the immune cells to express a range of antigens that are preferentially expressed by the target cell population as compared to the non-target population. Contacting with the modified antigen-presenting cells. Preferably, the antigen presenting cells are dendritic cells. This contacting step can be performed in vivo or ex vivo. Examples of eliciting a CTL response in vitro are described in Nair
Et al., 1993, J. Am. Virol. 68: 5685 and F.R. J. Rouse et al.
1994, J.A. Virol. 68: 5685. T in vivo
Examples of stimulating a cellular response include: Porgador et al., 1996, J. Am. Immun
ol. 156 (8): 2918-2926; C. McKinney and J
. W. Storylein, 1989J. Immunol. 143: 1560;
And H. Takahashi et al., 1993, Int. Immunol. 5: 8
49. The APC is administered to the subject for in vivo contact.
Alternatively, immune cells are isolated from the subject and contacted with the APC in vitro or ex vivo. The immune cells (eg, T cells) are activated ex vivo and then reintroduced into the subject or provided to a different subject, in which the immune cells are then Can be contacted with target cells in the subject. Techniques for adoptive immunotherapy of cancer are described in Chang, A.
. E. FIG. And S.I. Shu, 1996, Crit. Rev .. Oncol. Hema
tol. 22 (3): 213-228; and Kradin, R.M. K. , 19
93, in Therapeutic Applications of In
terleukin-2, Atkins, M .; B. And J. W. Mier,
Marcel Dekker, Inc. NY, pp. 217-232.

[0059] In some embodiments, activation of immune cells by the above methods is used to kill target cells. Accordingly, the present invention provides a method for killing target cells, the method comprising the steps of providing an immune cell with a genetically modified A
Contacting the PC. This contacting step involves genetically modified APC
, Or by administering a vector of the present invention to obtain an AP in a subject.
C can be performed in vivo by transducing C and then contacting the APC with immune cells. Alternatively, this contacting step can be performed in vitro. Immune cells activated in vitro by contact with a genetically modified APC can be administered to a subject.

Alternatively, the choice of vector or APC can be designed to obtain a tolerogenic response, for example, by contacting the APC with T _H2 cells. Accordingly, the present invention further provides a method of inducing a tolerogenic response, comprising contacting an immune cell with a genetically modified APC according to the present invention. Inducing a tolerogenic response is in the treatment of autoimmune disorders,
And for applications such as preventing rejection of foreign tissue (eg, graft tissue or autologous cells that have been genetically modified with foreign material). Similar to methods for killing target cells, methods for inducing a tolerogenic response can be performed by genetically modified APCs in vitro or in vivo.

The present invention further provides a method of preventing a disease such as an infection or a cancer, comprising administering to a subject a composition comprising a vector or APC of the present invention. Also provided is a method of treating a disease, such as cancer or an infection, comprising administering to a subject a composition comprising a vector or APC of the invention. Examples of infections include, but are not limited to, viral, bacterial, or parasitic infections. Examples of cancer include, but are not limited to, melanoma, glioma, and cancer of the colon, breast, prostate, lung and liver.

Compositions The present invention provides compositions that are useful for treating and preventing diseases such as cancer or infection. In one embodiment, the composition is a pharmaceutical composition. The composition may comprise a therapeutically or prophylactically effective amount of a vector and / or antigen presenting cell of the invention, as described above. The composition can optionally include a carrier, such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Thus, there is a wide variety of suitable pharmaceutical compositions of the present invention. Most typically, quality control (microbiology, clonogenic assays, viability tests) is performed, and the cells are re-infused into the patient after administration of diphenhydramine and hydrocortisone. For example, M. Korbling et al., 1986, Blood 67: 529-532 and Haas et al., 1990, Exp. Hematol. 18: 94-98.

Formulations and carriers suitable for parenteral administration (eg, intra-articular (joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include the formulation, Aqueous, isotonic, sterile injectable solutions and suspensions, solubilizing agents, thickening agents, stabilizers that can include antioxidants, buffers, bacteriostats, and solutes that are isotonic with the intended recipient's blood And aqueous and non-aqueous sterile suspensions, which can include preservatives. Intravenous or intraperitoneal administration is a preferred method of administration.

Methods of Administration In one embodiment of the invention, a patient infected with a virus such as HIV-1 or a patient suffering from a cancer such as melanoma is provided with a genetically modified antigen presentation. Treated by administering the cells or introducing the T cells back into the patient using antigen-presenting cells that have been genetically modified to activate the population of T cells against infection or cancer. obtain. Accordingly, the invention provides a method for producing cytotoxic T cells in vitro, ex vivo, or in vivo. In another embodiment, the patient is treated by administering at least one vector encoding a group of antigens. Here, the vector includes a dendritic cell targeting element. The patient's own dendritic cells can then be genetically modified in vivo.

In vitro activated T cells, such as CD8 + CTL, can be introduced into a subject where they are cytotoxic to target cells bearing their corresponding antigenic peptide, wherein the T cells are: Activated to recognize class I MHC molecules. These target cells are typically cancer cells or infected cells that express a unique antigenic peptide on their MHC class I surface.

Similarly, helper T cells (eg, CD4 + T cells), which recognize antigenic peptides in the context of MHC class II, are also genetically modified antigen presenting cells (eg, dendritic cells) Which can include antigenic peptides in both class I and class II MHC contexts. These helper T cells also stimulate an immune response against target cells. Like cytotoxic T cells, helper T cells are stimulated in vitro or in vivo with genetically modified antigen presenting cells. In one embodiment, the response that produces tolerance is generated by stimulation of T _H2 cells by administration of a genetically modified APC.

[0067] The group of antigens is preferably associated with a disease selected from the group consisting of cancer, hyperproliferative diseases, bacterial infections, parasitic infections, and viral infections. Diseases suitable for treatment using immunostimulatory strategies include the following: viral infections (
For example, HBV (see WO 93/15207), HCV (WO 93/1
5207), HPV (WO 92/05248, WO 90/1045).
9, EPO 133, 123), Epstein Barr virus (EPO
173,254; JP 1,128,788; and U.S. Pat. No. 4,939,08.
8 and 5,172,414), feline leukemia virus (W
O93 / 09070, EPO377,842, WO90 / 08832, and W
O93 / 09238), feline immunodeficiency virus (U.S. Pat.
37,753, WO92 / 15684, WO90 / 13573, and JP4
126,085), HTLV I and II, and HIV (WO 91/02
805); cancers such as melanoma, cervical cancer, colon cancer, kidney cancer, breast cancer, ovarian cancer, prostate cancer, leukemia, and heart disease.

Bacterial infections that can be treated include, among others, pneumonia, sepsis, tuberculosis, and Stap
hyococcus infection, but is not limited to these.

[0069] Parasitic infections that can be treated include malaria (protozoa of the genus Plasmodium (including P. falciparum, P. malariae, P. ovale, and P. vivax)), sleeping sickness (by trypanosoma) Cause ocular onchocerciasis), but are not limited to these.

Viral infections that can be treated include hepatitis A, hepatitis B, hepatitis C, non-A, non-B
Hepatitis, delta hepatitis factor, CMV, Epstein-Barr virus, HTLV I
, HTLV II, FeLV, FIV, and those caused by HIV I.

Treatment includes prophylactic and therapeutic methods. Prophylaxis or therapy may be achieved by a single direct injection at a single point or at multiple points. Administration is also
It can be almost simultaneous to multiple sites.

Patients or subjects include mammals such as humans, cows, horses, dogs, cats, pigs, and sheep animals.

In one embodiment, T cells or antigen presenting cells are administered directly to a subject to produce T cells that are active against a target cancer cell type, infected cell type, or other cell type. These administrations are by any of the routes normally used for bringing cells into ultimate contact with mammalian blood or tissue cells. In another embodiment, at least one vector encoding a group of antigens is administered.

The compositions are typically administered parenterally (eg, intravenously, subcutaneously, and intramuscularly).
Or other traditional direct routes (eg, buccal / sublingual, rectal, oral, nasal, topical (eg, transdermal and ocular), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular,
Alternatively, it is administered in vivo via the intranasal route or via specific tissues (liver, bone marrow) or, in the case of cancer treatment, directly into the tumor. Parenteral routes include W
It is discussed further in O96 / 20732.

The cells or vectors are often administered in any suitable manner, with a pharmaceutically acceptable carrier. While suitable methods of administering cells to a patient in the context of the present invention are available and more than one route may be used to administer a particular cell composition, particular routes often involve It may provide a faster and more effective reaction than another route.

In the context of the present invention, the dose of cells (eg, activated T cells or dendritic cells) administered to a patient will result in a beneficial therapeutic response in the patient over time or inhibit the growth of cancer cells. Or should be sufficient to inhibit infection. Thus, an amount sufficient to elicit an effective immune response to a particular antigen and / or to alleviate, reduce, cure, or at least partially arrest symptoms and / or complications from the disease or infection. To be administered to patients. An amount sufficient to accomplish this is defined as a "therapeutically effective dose."

This dose depends on the activity of the generated T cells or antigen presenting cells and the condition of the patient,
As well as the weight or surface area of the patient to be treated. The size of this dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular cell in a particular patient. AIDS or cancer (eg,
In determining the effective amount of cells to be administered in the treatment or prevention of diseases such as metastatic melanoma, prostate cancer, etc.
It is necessary to assess the progress of the disease and the generation of an immune response against any introduced cell type.

[0078] Generally, at least about 10 ⁴ to 10 ^6, and cells typically between 1 × 10 ⁸ and 1 × 10 ^{1 0} in the intravenous infusion or intraperitoneally for about 60-120 minutes to 70kg patient Injected. Intravenous infusion is preferred. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and 1 hour after infusion and stored for analysis. Cell re-infusion for 1 year
Repeat approximately every month for 0-12 treatments. After the initial treatment, the infusion may be performed on an outpatient basis based on the judgment of the attending physician. If reinfusion is given as an outpatient, participants will be monitored for at least 4 hours after treatment.

For administration, cells of the invention may be applied to a patient's body weight and overall health, as cell type LD-50 (or other toxicity criterion), as well as cells at various concentrations. Administration may be at a rate determined by the type of side effect. Administration can be accomplished in single or divided doses. The cells of the invention may supplement other treatments for conditions with known conventional therapies. These conventional treatments include cytotoxic agents, nucleotide analogs and biological response modifiers (modifiers).
)including. Similarly, biological response modifiers are added as needed for treatment. For example, the cells are administered with an adjuvant, or a cytokine (eg, GM-CSF, IL-12 or IL-2), as appropriate.

Many of the routes of administration described herein or otherwise known in the art
Administration by route can be performed at a single time or at multiple
It can be conveniently achieved by direct administration using the device. Furthermore, at a given time
Gene delivery vehicle (or ex vivo transduced cells for that matter)
"Administration" includes administration to one or more areas or by one or more routes. Book
In certain embodiments of the invention, one or more dosages may be straightforward in the indicated manner.
Administered directly: 10 intravenously^Three, 10^Five, 10⁷, 10⁹, 10^TenOr 10¹¹c
dosage above fu; 10 in artery^Three, 10^Five, 10⁷, 10⁹, 10^TenOr 10 ¹¹ Dosage above cfu; 10 intramuscularly^Three, 10^Five, 10⁷, 10⁹, 10^TenOr
10¹¹a dosage of at least cfu,^TenOr 10¹¹prefer cfu dosage
Yes; 10 in the skin^Three, 10^Five, 10⁷, 10⁹, 10^TenOr 10¹¹throw more than cfu
Dose; 10 for lung^Three, 10^Five, 10⁷, 10⁹, 10^TenOr 10¹¹throw more than cfu
Dose; 10 subcutaneously^Three, 10^Five, 10⁷, 10⁹, 10^TenOr 10¹¹more than cfu
Dosage, 10⁹, 10^TenOr 10¹¹cfu dosage is preferred;
Is 10^Three, 10^Five, 10⁷, 10⁸, 10⁹, 10^TenOr 10¹¹Medication over cfu
Quantity, 10⁸, 10⁹, 10^TenOr 10¹¹Preferred dosage of cfu; spleen
10 to lymphatic organs such as tonsils or lymph nodes^Three, 10^Five, 10⁷, 10⁸, 1
0⁹, 10^TenOr 10¹¹dosage above cfu; 10 for tumor^Three, 10^Four, 10^Five,
10^Five, 10⁷, 10⁸, 10⁹, 10^TenOr 10¹¹a dosage of at least cfu,
10⁸, 10⁹, 10^TenOr 10¹¹cfu dosage is preferred; and imported phosphorus
10 for pa^Three, 10^Five, 10⁷, 10⁸, 10⁹, 10^TenOr 10¹¹more than cfu
Dosage. For convenience, "cfu" also refers to non-viral particles,
Thus, 1 cfu equals one non-viral particle.

EXAMPLES The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. This example is not intended to limit the scope of the invention in any way or otherwise.

Example 1 Preparation of Dendritic Cells Genetically Modified to Present a Group of Antigens Dendritic cells were collected from Tedder and Jansen, 1997, Current.
Protocols in Immunology, John Willley
and Sons, Basic Protocol I as described in 7.32.1. Obtain cell lines from matched pairs of target and non-target cell types. Matched pairs of cell types include, for example, cell lines from tissues of the same origin (eg, prostate) and cell lines with different targeted characteristics. An example of a target cell is a prostate cancer cell. An example of a matched pair of cell types is prostate cells that differ in their metastatic potential. Nucleic acid sequences that are preferentially expressed in prostate cancer tissue are described in U.S. Patent Application Serial No. 60 / 088,877, filed June 11, 1998, the entire contents of which are incorporated herein by reference. . Then, to the dendritic cells,
For example, transduction of preferentially expressed nucleic acid sequences is performed using conventional techniques, such as those described in WO 97/24447, the entire contents of which are incorporated herein by reference. Successful transduction of human dendritic cells with T cell sensitization in vitro (
by priming (Albert et al., 1998, Natu).
re 392: 86-89; Nair et al., 1998, Nature Biotechnology 16 (4): 364-369; Antigen.
Processing and Presentation, Current Protocols in Immunology, Wiley, New Yo
rk, 1998). Candidate immunogenic tumor-associated antigen sequences can be determined by screening transduced dendritic cells using responder T cells obtained from peripheral blood or dLN of a tumor-bearing patient. Once a pattern of reactivity is found in a particular tumor type, the relative potency of these antigens can be assessed using mouse homologs and syngeneic mouse tumor models expressing one or more of these antigens. Example 2 describes one method for assessing efficacy.

Example 2: Dendritic cell-based immunotherapy for the treatment of metastatic tumors in combination with systemic Proleukin® IL-2 The genetically modified dendritic cells of the present invention Can be used alone or in combination with other therapies to treat tumors. This example evaluates the contribution of dendritic cells genetically modified using the mouse homologue of the nucleic acid sequence identified by the method of Example 1 to systemic proleukin IL-2 immunotherapy in mouse syngeneic tumors Here's how to do it. The C57B / 6 induced B16-F10 lung metastasis model, the CT-26 mouse colon model, or the mouse 3LL lung model can be used to generate the most effective treatment regime. These tumor models are not very immunogenic and are differentially responsive to single-drug proleukin therapy, as measured by both survival and tumor burden.

The strategy of this study was to use dendritic cells and systemic proleukin IL-2 (eg, lower amounts of IL- to maintain a high objective response rate while reducing toxicity.
Develop dendritic cells + systemic IL-2 regimen to define optimal mixture of 2) and for application to lung and / or colon mouse syngeneic tumor models that are resistant to single agent IL-2 therapy To be used.

(Experimental Design) Mouse spleen dendritic cells were isolated and described in Girolomoni et al. Im
munol. 145 (9): 2820-26. First, spleens from either untreated mice or mice bearing day 7 tumors were excised, minced, and 40 mg collagenase (Sigma, St. Lois, MO) in Hank's Balanced Salt Solution (HBSS). ) For 1 hour. The cells are then filtered through a 100 μm nylon mesh and washed. Red blood cells,
0.83% ammonium chloride, 0.1% KHCO ₂ , 0.004% EDTA (A
Dissolve in CK Lysis buffer) and pellet to 1.035 Perc
all (Pharmacia biotech) at 3 × 10 ⁷ cells / ml and overlay on an equal volume of 1.075 g / ml Percoll. The suspension is centrifuged at 2200 rpm at 4 ° C. for 20 minutes. Bands were harvested at the interface, washed twice with HBSS, and 5 × 10 ⁶ cells / m 2 in complete culture medium.
Resuspend and incubate at 37 ° C. for 90 minutes. Remove non-adherent cells and discard. Fresh complete culture medium is added and the culture is continued at 37 ° C. for 18-24 hours. Remove spleen dendritic cells by gentle pipetting.

Cells harvested from spleen cell cultures were⁶/ Ml cells 15m
1. Overlay 3 ml layer of 14.5% metrizamide-CM solution in a centrifuge tube.
Therefore, it is further concentrated. Centrifuge this gradient at 2000 rpm for 15 minutes at 4 ° C.
Separate, collect the band and wash, count and 5 × 10 5 in complete medium ⁶ Resuspend in cells / ml.

An aliquot was phenotyped (phenot) using the following three-color staining protocol
("DC" refers to dendritic cells):

[0088]

[Table 1] Bone marrow-derived dendritic cells (BM-DC) were treated with 10 ng / ml GM-CSF and 1
It is isolated from erythrocyte-depleted mouse bone marrow cells cultured for 7 days in complete medium supplemented with 0 ng / ml IL-4. On day 7, BM-DCs were harvested by gentle pipetting and 14.5% (by weight) metrizamide (Sigma, St.
(Louis, MO). The low density interface containing BM-DC is collected by gentle pipette inhalation. BM-DCs are washed twice with complete medium and counted (purity> 90%, MHC class II, CD40, CD
80, CD86, and CD11c positive co-expression).

The genetically modified dendritic cells prepared as described in Examples 1 and / or 2 are then injected either before (protective) or after (therapeutic) tumor challenge. . For protection, an intravenous (iv) tumor challenge is introduced one week after the last immunization (days -15, -8, 0). For treatment, immunizations will be given on days 4, 8, and 12 after intravenous tumor challenge, with or without proleukin.

The proleukin therapy is administered as follows: For protection, 10 mg / kg, iv, is started with a second immunization for 5 days. For treatment, intravenous, 10 mg / kg / day, 5 or 10 days, starting 2 days after intravenous tumor challenge.

Experiment # 1: Single Drug Proleukin Therapy (10 mice / group): B16-F10: 50-85,000 cells, iv, day 0 Proleukin: twice iv per day for 7 days Starting on day 3, sacrificed and counting lung metastases.

[0092]

[Table 2] Experiment # 2: Selecting the best proleukin regimen (less than 2 out of 10 mice died, lung metastases reduced by more than 50%)

[0093]

[Table 3] Experiment # 3: Selection of the most active DC regimen (reduction of lung metastasis of dual single agent proleukin)

[0094]

[Table 4] The potency of the genetically modified dendritic cells (DCs) can be assessed by short-term culture immune function assays based on antigen-induced expression of CD69. Briefly, whole blood and / or spleen cells from untreated mice, tumor-bearing mice, and DC immunized mice are prepared as single cell preparations, depleted of macrophages by adhesion,
Then resuspend to 5 × 10 ⁶ cells / ml. 500 microliters of various D
A C source (X-irradiated) was used for 12 x 75 m containing 0.5 ml of monocyte depleted responder cells
Measure in a polypropylene tube. The mixed cells are incubated at 37 ° C. for 24 hours and harvested at 6 hours for analysis. Cultures contain Brefeldin during the last 4 hours of culture. The culture is then surface stained with CD3 / CD4 / CD69 or CD3 / CD8 / CD69. Duplicate cultures were permeabilized and CD3 / CD4 / IFNγ, CD3 / CD4 / IL-4, CD3 / CD8 /
Stain with IFNγ and CD3 / CD8 / IL-4. TNFα and GM-
The reagents for CSF are used to further define CD8 reactivity to genetically modified DCs.

DCs loaded with baculovirus-producing mouse gp100 (cloned from B16F10) are used as a positive control.

Sera from immunized mice can be obtained, pelleted, heat inactivated, and analyzed for future analysis for anti-tumor binding activity, cytokine / chemokine content, sIL-2R, and other inflammatory molecules. At -70 ° C for storage.

The foregoing detailed description provides exemplary information regarding the present invention. Those skilled in the art will understand that modifications may be made without departing from the spirit and purpose of the invention.

[Brief description of the drawings]

FIG. 1 shows cell surface markers that can be used to identify dendritic cells. (
-) Indicates a marker that is not present on dendritic cells that can be used in a negative selection strategy. (+), (++), and (+++) indicate increasingly useful markers present on dendritic cell surfaces.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/12 A61P 33/00 33/00 35/00 35/00 C12N 15/00 A C12N 5/10 5 / 00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA , CH CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Escobedo, Jamie United States of America California 94507, Alamo, Livorna Road 1470 (72) Inventors Collins, Amy El. United States California 94611, Piedmont, Bonita Avenue 236 (72) Inventor Fon, Timothy United States California 94556, Moraga, Sandingham South 170 F-term (Reference) 4B024 AA01 BA31 BA36 DA03 EA02 4B065 AA92X AA92Y AB01 BA02 CA24 CA44 4C084 AA13 MA01 NA14 Z262 ZB332 ZB352 ZB372 4C086 AA01 AA02 EA16 MA01 MA04 NA14 ZB26 ZB33 ZB35 ZB37 4C087 AA01 AA02 BB43 BB63 MA01 NA14 ZB26 ZB33 ZB35 ZB37

Claims (40)

[Claims] 1. A method for producing at least one vector encoding a group of antigens for expression in antigen presenting cells, comprising: (a) first nucleic acid sequence expressed by a target cell population; Comparing to a second nucleic acid sequence expressed by the non-target cell population; (b) selecting a nucleic acid sequence that is preferentially expressed by the target cell population as compared to the non-target cell population; and (c) And b.) Introducing said selected nucleic acid sequence into at least one vector capable of directing the expression of said selected nucleic acid sequence in antigen presenting cells. 2. The method according to claim 1, wherein the antigen-presenting cells are dendritic cells, macrophages, B cells, monocytes or fibrocytes. 3. The method of claim 1, wherein said vector further comprises an antigen presenting cell targeting element. 4. The method of claim 1, wherein said first nucleic acid sequence and said second nucleic acid sequence are of the same tissue origin. 5. The method of claim 1, wherein said selected nucleic acid sequence comprises at least five different nucleic acid sequences. 6. The method of claim 1, wherein said selected nucleic acid sequence comprises at least seven different nucleic acid sequences. 7. The method of claim 1, wherein said selected nucleic acid sequence comprises at least nine different nucleic acid sequences. 8. The method of claim 1, wherein said vector further comprises a nucleic acid sequence encoding an immunomodulatory cofactor. 9. The method of claim 8, wherein said immunomodulatory cofactor is IL.
-2, IL-3, IL-8, OKT3, α-interferon, γ-interferon, or MIP-1α. 10. The method of claim 1, wherein said vector further encodes at least one selectable marker. 11. The method of claim 10, wherein said selectable marker is P
The method, wherein the method is LAP, GFP or neomycin resistant. 12. The method of claim 1, wherein said target cells are cancer cells. 13. The method of claim 1, wherein said target cells are viruses, bacteria or parasites. 14. A composition comprising at least one vector produced by the method of claim 1. 15. The composition of claim 14, wherein said vector further comprises an antigen presenting cell targeting element. 16. The composition of claim 14, further comprising an antigen presenting cell. 17. A method for producing an antigen-presenting cell that presents a group of antigens, comprising: (a) replacing a first nucleic acid sequence expressed by a target cell population with a first nucleic acid sequence expressed by a non-target cell population. (B) selecting at least one nucleic acid sequence that is preferentially expressed by the target cell population as compared to the non-target cell population; and (c) the selected nucleic acid Genetically modifying the antigen presenting cell to express the sequence. 18. The method according to claim 17, wherein said antigen presenting cells are dendritic cells, macrophages, B cells, monocytes or fibrocytes. 19. The method of claim 17, wherein said first nucleic acid sequence and said second nucleic acid sequence are of the same tissue origin. 20. The method according to claim 17, wherein the selected nucleic acid sequence comprises at least five different nucleic acid sequences. 21. The method of claim 17, wherein said selected nucleic acid sequence comprises at least seven different nucleic acid sequences. 22. The method according to claim 17, wherein said selected nucleic acid sequence comprises at least nine different nucleic acid sequences. 23. The method of claim 1, wherein said selected nucleic acid sequence further encodes at least one selectable marker. 24. The method of claim 23, wherein said selectable marker is P
The method, wherein the method is LAP, GFP or neomycin resistant. 25. The method of claim 17, wherein said target cells are cancer cells. 26. The method according to claim 17, wherein the target cells are viruses, bacteria or parasites. 27. An antigen presenting cell produced by the method of any one of claims 17 to 26. 28. A method of activating a T cell, comprising contacting the T cell with the antigen-presenting cell of claim 27. 29. The method of claim 28, wherein said T cells are cytotoxic T lymphocytes. 30. A method of inducing a tolerogenic response, wherein the T cell is a T cell.
A method comprising contacting with the antigen-presenting cell according to 7. 31. The method of claim 30, wherein said T cells are T _H2 cells. 32. The method according to claim 28 or 30, wherein said contacting step occurs in vivo. 33. The method according to claim 28 or 30, wherein said contacting step occurs ex vivo. 34. The method of claim 32 or 33, wherein said activating is in the presence of an immunomodulatory cofactor. 35. The method of claim 34, wherein the immunomodulatory cofactor is IL-2, IL-3, IL-8, OKT3, α-interferon, γ-interferon, or MIP-1α. ,Method. 36. A method of activating T cells in vivo, wherein the method activates T cells in vivo.
A method comprising the step of administering to the subject the composition according to claim 1. 37. A method for killing a target cell in vivo, comprising:
28. A method comprising administering to the subject the composition of claim 27 or the antigen-presenting cell of claim 27. 38. A method for preventing infection, comprising the step of administering the composition according to claim 14 or the antigen-presenting cell according to claim 27 to a subject. 39. A method of treating cancer, comprising administering to a subject a composition according to claim 14 or an antigen presenting cell according to claim 27, wherein the target is a target. The method, wherein the cell is a cancer cell. 40. A method of treating an infection, comprising administering to a subject a composition according to claim 14 or an antigen presenting cell according to claim 27, wherein the target is a target. The method, wherein the cell is an infectious agent.