JP2002506832A - Methods for inducing an anti-cancer immune response - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for inducing an antigen-specific immune response. In one embodiment, an effective amount of an antigenic peptide binding protein (APBP) and a cytotoxic agent are administered to a subject. In another embodiment, an effective amount of a first polynucleotide encoding an antigenic peptide binding protein and a second polynucleotide encoding a cytotoxic agent are administered to a subject. In other aspects, the methods described herein also include administration of an effective amount of an antigen presenting cell (APC) recruiting factor, cytokine or costimulatory molecule. In still other embodiments, the cytotoxic agent and the antigen presenting cell recruiting factor are encoded by one polynucleotide. In yet another aspect, the invention provides for adoptive immunotherapy by administering to a subject a population of educated antigen-specific immune effector cells. In this case, an effective amount of the cytokine and / or co-stimulatory molecule is administered together or not.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is hereby incorporated by reference . S. C. US Provisional Patent Application No. 60/07 under §119 (e)
No. 8,931 (filed on Mar. 20, 1998), the contents of which are incorporated herein by reference.

TECHNICAL FIELD The present invention is in the fields of molecular immunology and medicine. In particular, methods for inducing an antigen-specific immune response are provided.

[0003] Despite the many advances of background medical research, cancer remains the second leading cause of death in the United States. In developed countries, almost one in five people die from cancer. Conventional clinical medicine, such as surgical resection, radiation therapy and chemotherapy, has a significant failure rate, especially for solid tumors. Reasons for failure may be refractory initial tumors or recurrence due to regrowth at the original site, and / or metastasis.

[0004] Cellular immunotherapy is emerging as a technically and intelligently implemented anti-cancer therapy.
The formation of an immune response to tumors has been demonstrated in several animal models and suggested by reports of spontaneous tumor regression in humans (Stoter and Lotze (
1990) Cancer Cells, 2: 44-55). Cytotoxic T lymphocyte (CTL) responses can be directed to antigens specifically presented by tumor cells, in vivo or in vitro, without having to know in advance the molecular mechanism by which the tumor has developed. In animal models, established tumors can be cured by adaptive transmission of educational T cells that specifically recognize malignant cells (Beun et al. (1994)).
Immunol. Today, 15: 11-15).

[0005] In order to form specific T cells, naive T cells must be exposed to antigen presenting cells expressing a tumor specific antigen. APCs, such as antigens expressed on dendritic cells and macrophages, induce T cells to be antigen-specific.

[0006] Heat shock proteins (HSPs) appear to be involved in antigen presentation by binding to antigenic peptides. HSPs are synthesized in response to stressful conditions (eg, heat) and are found in most eukaryotic cell compartments. For example, HSP7
0 is found in the cytosol, while BiP is found in the endoplasmic reticulum. HSPs assist in folding and membrane translocation of nascent proteins, degradation of misfolded proteins, and other regulatory processes. They exhibit controlled ATP binding and hydrophobic domain release, preventing nascent chain aggregation on polysomes induced by the hydrophobic effect. In addition, HSPs display misfolded proteins by ubiquitination and proteasome-based degradation.

[0007] It has recently been shown that heat shock proteins have the ability to make tumor cells more immunogenic (Lukacs et al. (1997) Gene Therapy).
, 4: 346). Suto et al. (1995) Science, 269
1585-1588 report that presenting heat shock protein: peptide complexes purified from tumor cells to macrophages in vitro can present tumor antigens.

[0008] Vaccines made with peptides conjugated to carrier proteins (eg heat shock proteins) have been proposed as having potential for cancer therapy (Sriv)
astava et al. (1994) Current Opinion i
n Immun. 6: 728-732; Srivastava et al. (
1993) Advances in Cancer Research, 62:
153-177; and Tamura et al. (1997) Sciencec
e, 278: 117-120). Theoretically, the carrier: peptide complex is purified from the patient's tumor biopsy, and then the complex is taken up by the patient's own antigen presenting cells (APC), and the antigen peptide is released to release the patient's own A
The same patient can be injected again with the hope that it will be presented by MHC molecules on the surface of the PC, thereby inducing an anti-tumor cell immune response. However, this method is time and labor intensive since a tailor-made tumor specific vaccine must be created for each individual patient's tumor. The present invention overcomes the deficiencies of prior art therapies and improves methods of inducing an immune response.

DISCLOSURE OF THE INVENTION The present invention provides a method of inducing an antigen-specific immune response, particularly an immune response against a tumor antigen.

[0010] In one aspect, the present invention provides a method for inducing an antigen-specific immune response, comprising:
In embodiments, this results in an effective amount of an antigenic peptide binding protein (APBP)
) And administering a cytotoxic substance to the subject to suppress the growth of neoplastic or tumor cells in the subject. Preferably, when the cytotoxic substance is administered to the subject, the cytotoxic substance is further activated with an activating agent.
The antigenic peptide binding protein may be a heat shock protein (HSP), a soluble major histocompatibility complex (MHC) class I molecule, and an antibody that has been genetically engineered to bind an antigenic peptide. In one embodiment, the cytotoxic agent is herpes simplex virus thymidine kinase (HSV-tk) and the activating compound is ganciclovir.

[0011] In another aspect, the invention provides administering to a subject an effective amount of a first polynucleotide encoding an antigenic peptide binding protein and a second polynucleotide encoding a cytotoxic agent in the subject. Methods for inducing a sexual immune response are provided. When the cytotoxic substance is administered to the subject, the cytotoxic substance may be further activated with an activating agent. The polynucleotide may be naked DNA or one or more gene delivery vehicles, such as retroviral vectors, adenoviral vectors, adeno-associated viral vectors and / or
Alternatively, it can be administered in liposomes. For this aspect of the invention, the antigenic peptide binding protein is engineered to bind heat shock protein (HSP), soluble major histocompatibility complex (MHC) class I molecules, and antigenic peptide. Antibodies. In one embodiment, the cytotoxic agent is herpes simplex virus thymidine kinase (HSV-tk),
The activating compound is ganciclovir, wherein the first and second polynucleotides are naked DNA.

[0012] In another aspect, the methods described herein comprise an effective amount of an antigen presenting cell (APC).
And / or) administering a recruitment factor. APC supplementation factors include interleukin 4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), Sepragel and / or
Alternatively, it may be macrophage inflammatory protein 3 alpha (MIP3α). In one embodiment, the APC supplement is administered in a gene delivery vehicle. The antigenic peptide binding protein may be a heat shock protein (HSP), a soluble major histocompatibility complex (MHC) class I molecule, and an antibody that has been genetically engineered to bind an antigenic peptide. In one embodiment, the cytotoxic agent is herpes simplex virus thymidine kinase (HSV-tk),
The activator is ganciclovir.

In another embodiment, the antigenic peptide binding protein and the cytotoxic agent are encoded by one polynucleotide. In other embodiments, the antigenic peptide binding protein, cytotoxic agent and antigen presenting cell recruiting factor are encoded by one polynucleotide. In other embodiments, the antigen peptide binding protein,
And the cytotoxic agent or antigen presenting cell recruiting factor is encoded by one polynucleotide. In still other embodiments, the cytotoxic agent and the antigen presenting cell recruitment factor are encoded by one polynucleotide.

In yet another aspect, the invention provides for adoptive immunotherapy by administering to a subject a population of educated antigen-specific immune effector cells. These cells are na 免疫 vely immunized with antigen presenting cells previously exposed to an antigen peptide binding protein: peptide complex derived from a lysate of tumor cells genetically modified to express the antigen peptide binding protein. It is prepared by culturing effector cells (with or without a cytotoxic gene product). After modification and education, the educated antigen-specific immune effector cells are re-administered to the subject. The cytotoxic agent is further characterized in that it is activated by the activating agent when the tumor cells are cultured with the activating agent.

As will become apparent, one preferred feature and feature of the invention is applicable to any other aspect of the invention. Embodiments of the Invention Various publications, patents and published patent specifications are referenced herein by reference. In order to more fully describe the state of the art in the fields relevant to this specification, these publications,
Patent and published patent specifications are hereby incorporated by reference.

The practice of the present invention will employ, unless otherwise indicated, routine methods of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology, which can be readily accomplished by those skilled in the art. adopt. Such techniques are explained fully, for example, in the following documents: "
Molecular Cloning: A Laboratory Manua
1 ", 2nd edition (Sambrook et al., 1989);" Oligon
nucleotide Synthesis "(edited by MJ Gait, 1984)
"Animal Cell Culture" (edited by RI Freshney, 1987); series "Methods in Enzymology" (Academic Press); "Handbook of Experimenta
l Immunology "(DM Weir & CC Blackwee)
11); "Gene Transfer Vectors for Mamm
allian Cells "(edited by JM Miller & MP Calos, 1987);" Current Protocols in Molecul.
ar Biology "(edited by FM Ausubel et al., 1987 and periodic updates);" PCR: The Polymerase Chain Rea
ction "(Mullis et al. eds., 1994);" Current
Protocols in Immunology "(JE Colliga)
ed., et al., 1991).

Definitions Certain terms used herein have the meanings defined below: The singular forms used in the specification and claims, unless defined otherwise, have the plural meaning. Is included. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

The term “immune effector cells” refers to cells that specifically recognize an antigen present on, for example, neoplastic or tumor cells. For the purposes of the present invention, immune effector cells include B cells, monocytes, macrophages, NK cells, and T cells, such as cytotoxic T lymphocytes (CTLs), such as CTL lines, CTL clones, and tumor, inflammation or other Includes CTL from infiltrates. "T lymphocytes"
Means that the phenotype is CD3 ⁺ and is generally detected using an anti-CD3 monoclonal antibody in combination with an appropriate labeling method. The T lymphocytes of the present invention are generally positive for CD4, CD8 or both. The term "naive" immune effector cells refers to immune effector cells that have never encountered an antigen and is intended to be unprimed and synonymous with virginity. "
"Educated" refers to immune effector cells that have differentiated into antigen-specific cells by interacting with the antigen.

An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered by one or more administrations, applications or applications. The polynucleotide of the present invention can be administered or applied transdermally, orally, subcutaneously, intramuscularly, intravenously or parenterally. An effective amount of a polynucleotide for the purposes of the present invention is an amount that will induce an antigen-specific immune response in the subject.

The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably to refer to macromolecular nucleotides of any length. Polynucleotides can include deoxyribonucleotides, ribonucleotides and / or their analogs. Nucleotides can have any three-dimensional structure and can perform any function, known or unknown. The term "polynucleotide" includes single-stranded, double-stranded and triple-helical molecules.

“Oligonucleotide” refers to single or double stranded DNA of about 5 to about 100 nucleotides. Oligonucleotides, also known as oligomers or oligobodies, can be isolated from genes or synthesized chemically by methods known in the art. "Primer" refers to a normally single-stranded oligonucleotide that provides a 3'-hydroxy terminus for the initiation of enzyme-mediated nucleic acid synthesis.

The following are non-limiting examples of polynucleotides: genes or gene fragments, exons, introns, mRNA, tRNA, rR
NA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Nucleic acid molecules can also include modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art and include, but are not limited to: aziridinyl cytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5 -Carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentynylurauracil and 2,6-diaminopurine. The use of uracil instead of thymine in deoxyribonucleic acid is also considered a similar form of pyrimidine. By way of illustration and not limitation of the invention, the polynucleotide encodes a peptide, ribozyme or antisense sequence.

“Protein”, “oligopeptide”, “polypeptide” and “peptide”
The term is used interchangeably to describe amino acid polymers of any length. These polymers may be linear or branched, may contain modified amino acids, and may be interrupted with non-amino acids. These terms may be modified naturally or by intervention such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled compound. Amino acid polymers. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids and the like), and other modifications known in the art.

The term “culturing” refers to the in vitro growth of cells or organisms on or in various types of media. It is understood that the progeny of a cell grown in culture may not be completely equivalent (in morphological, genetic or phenotypic) to the parent cell. "Expanded" means any growth or division of a cell.

A “subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rats, monkeys, humans, farm animals, sport animals and pets.

An “antigen peptide binding protein” is any protein capable of binding a peptide, preferably a peptide that triggers an antigenic response. For example, heat shock proteins and major histocompatibility complex (MHC) molecules have a binding groove that allows them to bind to peptide sequences. "Major histocompatibility complex" or "M
The term "HC" refers to a complex of genes encoding cell surface molecules required for antigen presentation to T cells and rapid graft rejection. In humans, MHC is HLA
Also known as a complex. The protein encoded by the MHC complex is "MH
Known as "C molecules", they are classified into Class I and Class II MHC molecules.
Class I MHC molecules include membrane heterodimer proteins consisting of non-covalently linked α-chain encoded in MHC with β2-microglobulin. Class I MHC molecules are expressed by almost all nucleated cells and have been shown to function in presenting antigen to CD8 ⁺ T cells. Class I molecules include HLA-A, -B and -C in humans. Class II MHC molecules also include membrane heterodimer proteins consisting of non-covalently linked α and β chains. Class II MHC molecules are known to function in CD4 ⁺ T cells, including HLA-DP, -DQ and -DR in humans.

The term “heat shock protein (HSP)” is present in most animals,
An animal represents a group of proteins that are encoded by genes that are suddenly, rapidly, and coordinately transcribed when exposed to certain stresses, such as a sudden increase in temperature. Without limitation, examples of HSPs are HSP gp96, HSP90, HSP
70, HSP65, HSP28 and the like. These are commercially available from StressGen Biotechnologies (Victoria, Canada). Polynucleotide sequences encoding these proteins are known in the art.

“Cytotoxic agents” include proteins and other molecules that are toxic to cells, for example, by disrupting the normal function of the cell. The cytotoxic agent may be a molecule that is itself toxic to the cell, or it is an "activating compound"
That is, it may be "conditionally activated" that produces a cytotoxic substance when given a prodrug. An example of a conditionally cytotoxic agent is herpes simplex virus thymidine kinase (HSP-tk), which is activated by the prodrug ganciclovir.

The term “antigen presenting cell recruiting factor” or “APC recruiting factor” includes both intact whole cells and other molecules that can recruit antigen presenting cells. Proper A
PC supplementation factors include interleukin 4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), sepragel and macrophage inflammatory protein 3 alpha (MIP3α). These are available from Genzyme (Flamingham, MA), Imnex, Schering-Plow and R & D Systems (Minneapolis, MN). They are CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (FM.
Ausubel et al. (1987)). Peptides, proteins and compounds having the same biological activity as the above factors are included in the scope of the present invention.

For the purposes of the present invention, it is provided that a single protein can have more than one of the three functions mentioned above, ie the function of antigen presenting cell recruitment, cytotoxic substances and / or antigen peptide binding. The multifunctional molecule may be encoded by a natural gene or may be engineered using recombinant techniques.

“Co-stimulatory molecules” are:
It is involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T cells. Studies accumulated over the last few years have provided compelling evidence that resting T cells require at least two signals for induction of cytokine gene expression and proliferation (Schwartz, RH (1990). )
Science, 248: 1349-1356; Jenkins, M .; K. (1
992) Immunol. Today, 13: 69-73). One signal,
That is, what imparts specificity can be generated by the interaction between the TCR / CD3 complex and an appropriate MHC / peptide complex. The second signal is not antigen specific and is called a "co-stimulation" signal. This signal was originally defined as the activity conferred by so-called "professional" APCs, such as accessory cells from bone marrow, such as macrophages and dendritic cells. Several molecules have been shown to enhance costimulatory activity. These are thermostable antigens (HSA) (L
iu, Y .; et al. (1992) J.I. Exp. Med. , 175: 437-
445); Chondroitin sulfate-modified MHC non-mutant chain (Ii-CS) (Naujo
kas, M .; F. et al. (1993) Cell, 74: 257-268).
Intracellular adhesion molecule 1 (ICAM-1) (Van Seventer, G.A .;
1990) J. Immunol. 144: 4579-4586); B7-1 and B7-2 / B70 (Schwartz, RH (1992) Cell, 71).
: 1065-1068)). Each of these molecules appears to assist co-stimulation by interacting with their cognate ligand on T cells. Co-stimulatory molecules mediate the co-stimulatory signal (s) necessary to achieve full activation of naive T cells under normal physiological conditions. An example of a receptor-ligand pair is a B7 costimulatory molecule on the surface of an APC and its counter receptor C on a T cell.
D28 or CTLA-4 (Freeman et al. (1993)).
Science, 262: 909-911; Young et al. (199
2) J. Clin. Invest. 90: 229; and Nabavi et.
al. (1992) Nature, 360: 266-268). Other important costimulatory molecules are CD40, CD54, CD80, CD86. "Co-stimulatory molecules"
The term is used to describe any single signal molecule or molecule that, when combined with the peptide / MHC complex bound by the TCR on the surface of a T cell, provides a costimulatory effect that achieves activation of the T cell binding peptide. And combinations of Thus, the term refers to B7, or other costimulatory molecule (s) on an antigen presenting matrix (eg, APC), a fragment thereof (alone, other molecule (s))
When the TCR on the surface of the T cell specifically binds the peptide (eg, as a complex with or as part of a fusion protein), it binds the cognate ligand together with the peptide / MHC complex to bind the T cell Including those that activate. Co-stimulatory molecules are commercially available from various vendors, including, for example, Beckman Coulter. Although not necessarily affirmed, molecules having a biological activity similar to wild-type or purified costimulatory molecules (eg, recombinantly produced, or mutant proteins thereof) are also used within the spirit and scope of the present invention. And

As used herein, the term “cytokine” refers to any of a number of factors that exert a variety of effects on a cell, for example, the induction of growth or proliferation. Examples of cytokines that can be used alone or in combination in the practice of the present invention include, but are not limited to: interleukin-2 (IL-2),
Stem cell factor (SCF), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-12 (IL-12), G-CSF, granulocyte macrophage colony stimulating factor (GM-CSF) ), Interleukin-1 alpha (IL-1α), interleukin-11 (IL-11), MIP-1α
, Leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO
) And flt3 ligand. The present invention also includes culture conditions that specifically exclude one or more cytokines from the culture. Cytokines are sold by several vendors, such as Genzyme (Flamingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R & D Systems and Imnex (Seattle, WA). Although not necessarily affirmed, molecules having biological activity similar to wild-type or purified cytokines (eg, those produced recombinantly or mutated proteins thereof) are also intended to be used within the spirit and scope of the invention. .

The term “expression” as used herein refers to the process by which a polynucleotide is transcribed into mRNA and translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA, provided that a suitable eukaryotic host is selected. The regulatory elements required for expression include a promoter sequence that binds RNA polymerase, and a transcription initiation sequence for ribosome binding. For example, bacterial expression vectors include a promoter, such as the lac promoter, and a Shine-Dalgarno sequence for initiation of transcription, and an initiation codon, AUG (Sambrook et al.).
al. (1989) supra). Similarly, eukaryotic expression vectors contain a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal,
Includes start codon AUG, and stop codon for ribosome desorption. Such vectors are commercially available or can be assembled by methods well known in the art, for example, as described in the methods described below for the construction of vectors in general.

A “host cell” is any individual cell or cell culture that can be, or has been, the recipient of a vector or can take up exogenous nucleic acid molecules, polynucleotides and / or proteins. It shall include things. It shall also include the progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental or deliberate mutation. The cells can be prokaryotic or eukaryotic, and include bacterial cells, yeast cells, animal cells, and mammals such as mice,
Includes, but is not limited to, rat, monkey or human cells.

“Gene delivery vehicle” is defined as any molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vehicles include liposomes,
Viruses such as baculoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors, and other recombinant vehicles commonly used in the art for expression in a variety of eukaryotic and prokaryotic hosts. Which can be used for gene therapy and simple protein expression.

“Viral vector” is defined as a recombinantly produced virus or viral particle containing a polynucleotide that is delivered into a host cell in vivo, ex vivo or in vitro. Examples of viral vectors include:
Retrovirus vectors, adenovirus vectors, adeno-associated virus vectors and the like are included. In embodiments where gene transfer is mediated by a retroviral vector, the vector construct represents a polynucleotide comprising the retroviral genome or a portion thereof and a therapeutic gene. Non-viral gene delivery vehicles include DN
A / liposome complex, and target virus protein DNA complex. To enhance delivery to cells, the nucleic acids or proteins of the invention can be conjugated to a cell surface antigen, such as an antibody that binds TCR, CD3 or CD4, or a binding fragment thereof. Liposomes that also contain a targeting antibody or fragment thereof can be used in the methods of the invention. The present invention also provides targeting conjugates for use in the methods disclosed herein.

As used herein, the term “immune effector molecule” refers to a molecule capable of antigen-specific binding, including antibodies, T cell antigen receptors, and MHC class I and class II molecules. .

A “naive” immune effector cell is an immune effector cell that has not been exposed to an antigen. As used herein, the term "educated antigen-specific immune effector cells" is an immune effector cell as defined above that has encountered an antigen and is specific for that antigen. The educated antigen-specific immune effector cells can be activated upon binding the antigen. "Activation" indicates that the cells are no longer _zero phase G, begin producing characteristic cytokine to the cell type.
For example, activated CD4 + T cells secrete IL-2 and have a higher number of high affinity IL-2 receptors on their cell surface compared to resting CD4 + T cells.

The term “preferentially recognized” indicates that the polypeptide of the invention is not substantially recognized by T cells specific for an unrelated antigen as defined above. Assays for determining whether an epitope is recognized by an antigen-specific T cell are well known in the art and are described herein.

As used herein, “neoplastic cells”, “neoplasms”, “tumors”, “tumor cells”, “
The terms “cancer” and “cancer cell” (used interchangeably) indicate relatively autonomous growth and, therefore, an abnormally proliferative expression characterized by a significant loss of control of cell growth (ie, deregulation of cell division). Represents cells that show a type, neoplastic cells can be malignant or benign.

“Inhibitory” tumor growth indicates a reduced growth rate compared to growth without contact with the educated antigen-specific immune effector cells described herein. Tumor growth rate can be assessed by any means known in the art, including measuring tumor size, measuring the presence or absence of tumor cell growth using a ³ H-thymidine incorporation assay, or counting tumor cells. "Inhibitory" tumor growth refers to any or all of the following conditions: slowing, delaying and stopping tumor growth, and shrinking tumors.

The term “culture” refers to the in vitro growth of cells or organisms on or in various types of media. It is understood that the progeny of a cell grown in culture may not be completely equivalent (in morphological, genetic or phenotypic) to the parent cell. "Expanded" means any growth or division of a cell.

“Composition” is intended to mean a combination of an active substance with another compound or composition that is inert (eg, a detectable substance or label) or active (eg, an adjuvant).

“Pharmaceutical composition” is intended to include the combination of the active substance with an inert or active carrier to render the composition suitable for in vitro, in vivo or ex vivo diagnosis or treatment.

As used herein, the term “pharmaceutically acceptable carrier” refers to any standard pharmaceutical carrier, such as phosphate buffered saline, water, and emulsions (eg, oil / water or water / oil) Emulsions), and various types of wetting agents. The composition may contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHA
RM. SCI. 15th edition (Mac Publishing, Easton (1975))
reference.

As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, means excluding other elements that have significance to the combination. For example, a composition consisting essentially of the elements defined herein can contain trace amounts of contaminants from isolation and purification methods, and a pharmaceutically acceptable carrier, such as phosphate buffered saline,
Do not exclude preservatives. “Consisting of” means excluding more than trace amounts of other component elements or substantial method steps for administering the compositions of the present invention. The embodiments defined by each of these transition terms are within the scope of the present invention. Materials and Methods In one aspect, the invention provides an effective amount of an antigenic peptide binding protein (APB).
A method for inducing an antigen-specific immune response in a subject by administering to the subject P) and a cytotoxic agent. APBP and cytotoxic substances are described in further detail below. The APBP and cytotoxic agent can be administered to the subject as molecules, eg, proteins, or as polynucleotides encoding these compounds, and the polynucleotides can be introduced into the subject by a gene delivery vehicle. In other embodiments, at least one APBP or cytotoxic agent is administered as a whole molecule and the other is administered by a polynucleotide inserted into a gene delivery vehicle.

In another embodiment, an antigen-specific immune response is induced in a subject by administering an effective amount of an APBP, a cytotoxic agent, and an effective amount of an antigen presenting cell (APC) recruiting factor. In this embodiment, the APBP, cytotoxic agent and APC
The recruiting factors as whole molecules; by polynucleotides encoding these molecules;
Alternatively, it can be administered as a combination of the whole molecule and the polynucleotide. Suitable APC supplementation factors are described herein.

The methods described herein can be performed in vivo, ex vivo or in vitro. In particular, the APBP, cytotoxic agent, and optionally the APC supplement can be administered to a subject either directly as a molecule or as encoded by a polynucleotide in a gene delivery vehicle. Alternatively, ex vivo methods involve treating cells obtained from a subject in the manner described herein and re-administering the cells to a patient. Finally, the invention can be used in in vitro cell culture for the formation of antigen-specific immune effector cells. These cells can then be used as therapeutics.

Thus, the present invention provides a method of inducing an immune response that destroys distant metastases by locally administering a molecule of the present invention to an accessible mass, such as a skin area or a nodule. The APBP: peptide complex is released from the cancer cells as the antigen peptides in the cancer cells are loaded on the APBPs which are rich and the cells are destroyed by the action of cytotoxic substances. The released APBP: peptide conjugate is taken up by APC recruited to the treatment site (eg, by an APC recruiting factor), resulting in the presentation of the antigenic peptide. When the peptide is presented on the cell surface, an anti-tumor cell immune response is mounted, and these educated antigen-specific immune effector cells bind local and distant cells expressing the tumor-specific antigen. Destroy. These and other advantages of the present invention are discussed herein.

Antigen Peptide Binding Protein For the purposes of the present invention, antigen peptide binding protein (APBP) includes any molecule capable of binding an antigen peptide, preferably a peptide. In a preferred embodiment, the APBP is of human origin. Probably the best known natural A
PBP may be a membrane-bound glycoprotein encoded by a gene known as major histocompatibility complex (MHC). These proteins are involved in the presentation of various antigens on the cell surface. In the protein region farthest away from the cell membrane, a crack (
Grooves) join. Through a complex series of process steps, the antigenic peptide bound to the groove in the MHC is then presented on the cell surface where the peptide can be recognized by the immune system.

In a preferred embodiment, the APBP is a heat shock protein (HSP), such as HSP65. Like MHC proteins, heat shock proteins also have peptide-binding grooves and can bind short peptide sequences generated by proteosome complex-mediated proteolysis of intracellular proteins. Heat shock proteins are also involved in loading these bound peptide fragments onto MHC molecules and then presenting them to the cell surface. Thus HSP:
Peptide conjugates provide a rich source of antigenic peptides, and conjugates purified from transformed cells are capable of eliciting an immune response specific to the transformed cells. Anti-HSP antibody assays can be used as a means of monitoring therapy using the methods of StressGen Biotechnologies (Victoria, Canada) and commercially available materials. Alternatively, as known in the art, Harlow and
Anti-HSP monoclonal antibodies can be produced using the technique described in Lane (1989), supra, and used to monitor therapy.

Thus, presentation of antigens, such as tumor-associated antigens, by APCs triggers a strong immune response, resulting in the destruction of tumor cells by antigen-specific immune effector cells, eg, cytotoxic T lymphocytes (CTL). You. Induction of a CTL response is one way to assay for a positive response to therapy and a means to confirm the biological activity of the novel factors useful in the methods of the present invention. The presence of a large number of T cells in the tumor has sometimes correlated with favorable prognosis (Whitesides).
and Parmiani (1994) Cancer Immunol. Im
munother. 39: 15-21). Woolley et al. (19
95) Immunology, 84: 55-63, showed that implanting a polyurethane sponge containing irradiated tumor cells can efficiently capture anti-tumor CTL (four times more than lymph and 50 times more than spleen or peripheral blood). ). After being activated by T cell cytokines in the presence of a properly presented recognition antigen, TIL
Proliferate in culture and acquire effective anti-tumor cytolytic propeptides. Weid
mann et al. (1994) Cancer Immunol. Immu
other. 39: 1-14.

Monitoring tumor regression in vivo, or using assays well known in the art to measure T cell responses, determines whether the objectives of the methods described herein have been achieved. Available for For example, any method of comparing T cell counts before and after therapy can be employed. Furthermore, induction of costimulatory molecules by these methods may also stimulate anergic or low affinity autoreactive CTL clones. C
Methods for assaying TL clones include: Kawakami
et al. (1988) J. Am. Exp. Med. 168: 2183-91 Standard a ⁵¹ Cr release assay described in; To summarize, the addition of cytotoxic T cells targeted cells loaded with previously ⁵¹ Cr, measuring the release of of ⁵¹ Cr from the target cells lysed. Kawakami et al. (1994) PNAS, 91: 351.
5-19. Cytokine release assay described in 5-19; Briefly, cytotoxic T cells are added to target cells, and the amount of IFNγ released is measured by ELISA. To determine the relative percentage of immune effector cells that recognize a particular target in a mixed population, see Czerkinsky et al. (1988) J. Am. Im
munol. Meths. The enzyme-linked immunospot (ELISPOT) assay described at 110: 29-36 is used. Briefly, a 96-well nitrocellulose bottom plate is coated with an anti-cytokine antibody, generally anti-interferon-γ. Target cells and immune effector cells, such as cytotoxic T cells (CTL), are added to the wells. Cytokines released from CTL are captured with anti-interferon-γ antibody and quantified in a standard ELISA format.

As described above, the amount of the APBP: antigen complex can also be measured, for example, by formatting an antibody against APBP and using an immunological technique, such as an ELISA assay. For example, when HSP65 is used as APBP, the antibody is HSP
65 would be specific.

The present invention uses a cytotoxic agent to destroy cancer cells, thereby reducing APB
It also includes releasing the P: peptide complex. A substance that is toxic to cells includes any substance that disrupts cell function, preferably so as to lyse the target cell. In some embodiments, a cytotoxic compound is toxic to both the cell in which it is taken up and also to neighboring cells, such that it is bystander.
der) effect.

[0056] In one embodiment, the cytotoxic agent is cytotoxic without further modification. Examples include cytotoxic agents that do not require a prodrug, including: toxins (eg, ricin toxin), specific tumor suppressor gene products (eg, p53), triggering cell death. Signal-generating molecules that can be pulled (eg, Fas ligand and TNFα), pro-apoptotic factors (eg, bcl-x
s; Clarke et al. (1995) PNAS, 92: 1104-11.
029), ribozymes, proteases, DNAses and RNAses specific for antisense RNA or mRNA encoding proteins necessary for cell survival.

In a preferred embodiment, the cytotoxic agent is activated with an activator. That is,
An activating compound must be administered to produce a cytotoxic agent. For example, herpes simplex virus thymidine kinase (HSV-tk) can be administered to a subject as a peptide or by a gene delivery vehicle carrying a polynucleotide encoding the peptide. However, HSV-tk does not become cytotoxic until the addition of an activating compound such as ganciclovir or acyclovir (available from Hoffman-La Roche; Natley, NJ).
Examples of other cytotoxic agents that require a prodrug include E. coli.
Includes cytosine deaminase, E. coli purine nucleoside phosphorylase, E. coli nitroreductase, mammalian cytochrome p450 isozymes, carboxypeptidase G2, and mammalian thymidine phosphorylase.

The purpose of the cytotoxic agent is to release these complexes from the tumor cells when the antigen-specific APBP: peptide conjugate is at the highest concentration, thus controlling when the cytotoxic agent destroys the cells It is desirable to do. In one embodiment, this may be by controlling the transient expression of the cytotoxic agent gene and the peptide binding protein gene, or at some point after APBP delivery, to deliver the cytotoxic compound (or the gene encoding the compound) to cancer cells. Achieved by delivering. Alternatively, a conditionally active cytotoxic gene encoding a protein that itself becomes cytotoxic when supplemented with a cofactor or activating component or a protein that can form a cytotoxic compound when supplied with a cofactor or prodrug. And may be activated at an appropriate time.

In one embodiment, a polynucleotide encoding a cytotoxic agent and APB
The polynucleotide encoding P is delivered in a separate gene delivery vehicle. In this case, it is unlikely that all cancer cells expressing one polynucleotide will express the other. Thus, in order to maximize the destruction of cells containing high intracellular concentrations of APBP: peptide conjugate, the cytotoxic agent had a significant bystander effect, ie the gene delivery vehicle was successfully introduced and transcribed. It is desirable to also lyse cells adjacent to the cells. The need for a cytotoxic agent to have a significant bystander effect is significantly less in embodiments where the cytotoxic agent and the polynucleotide encoding APBP are delivered in the same gene delivery vehicle.

Antigen presenting cell (APC) recruiting factor An antigen presenting cell (APC) is a cell capable of inducing the presentation of one or more antigens, preferably those containing class I MHC molecules. APCs are intact whole cells and other molecules, such as macrophages, dendritic cells, B cells, purified MHC class I molecules complexed to β2-microglobulin, hybrid APCs, and Foster antigen presenting cells. May be.

[0061] Dendritic cells (DCs) are considered to be the most effective antigen presenting cells (APCs).
DCs were shown to have all the signals required for T cell activation and proliferation. These signals can be classified into two types. The first type that confers specificity on the immune response is the T cell receptor / CD3 ("TCR / CD3") complex,
Major histocompatibility complex ("MHC") class I or II on PC surface
It is mediated by the interaction of the protein with the presented antigenic peptide. This interaction is necessary, but not sufficient, for T cell activation to occur. In fact, in the absence of a second type of signal, a first type of signal can result in T cell anergy. A second type of signal, called a costimulatory signal, is not antigen-specific and not MHC-restricted, but can result in a complete proliferation response of T cells and induction of T cell effector function in the presence of the first type of signal .

In one aspect of the invention, one or more factors that recruit antigen presenting cells are also administered. The APC recruiting factor ensures that high localized concentrations of antigen presenting cells are obtained near the destroyed cancer cells, and that uptake of free APBP: peptide complexes is achieved. APCs recruited to the site have the ability to process the resulting APBP: peptide complex for MHC molecules, thereby inducing an anti-cancer immune response through education of immune effector cells. In other embodiments, an effective amount of a cytokine and / or costimulatory molecule is administered in the form of a protein or a polynucleotide encoding the protein to enhance an immune response.

Gene Delivery Vehicle In one embodiment, the gene delivery vehicle can be used to deliver a polynucleotide encoding one or more APBPs, cytotoxic agents, or APC recruiting factors to a subject. The method of the present invention is intended to include any method for delivering a gene to a subject. Examples of delivery mechanisms include, but are not limited to, virus-mediated gene transfer, liposome-mediated transfer, transformation, transfection and transduction, such as adenovirus, adeno-associated virus and herpes virus. Virus-mediated gene transfer using DNA virus-based and retrovirus-based vectors.

Vectors Useful for Gene Modification In general, in vitro, ex vivo and in vivo genetic modification of cells for use in the present invention is accomplished by introduction of a vector containing a heterologous or altered antigen-encoding polypeptide or transgene. You. A variety of gene transfer vectors can be used, including viral and non-viral systems. Viral vectors useful for the genetic modification of the present invention include adenovirus, adeno-associated virus vectors, retroviral vectors and adeno-retroviral chimeric vectors.

Construction of Recombinant Adenovirus or Adeno-Associated Virus Vectors Adenovirus or adeno-associated virus vectors useful for the genetic modification of the present invention can be prepared by methods already taught in the art (eg, See: Karlsson et al. (1986) EMBO, 5: 2.
377; Carter (1992) Current Opinion in B
iotechnology, 3: 533-539; and Muzcyzka (1
992) Current Top. Microbiol. Immunol. Fifteen
8: 97-129; GENE TARGETING: A PRACTICAL
APPROACH (1992) Editor A. L. Joyner, Oxford
University Press, New York). Several different approaches can be taken. A helper-independent replication-deficient human adenovirus system is preferred.

A recombinant adenovirus vector based on human adenovirus 5 (Virolo
gy, 163: 614-617, 1988) lacks the essential early genes from the adenovirus genome (usually EIA / EIB), and thus in a permissive cell line that supplies the missing gene product in trans. It cannot be replicated without growing. Instead of this missing adenovirus genomic sequence, the transgene can be cloned and expressed in cells infected with a replication-defective adenovirus. Adenovirus-based gene transfer does not integrate the transgene into the host genome (less than 0.1% of the transgene is incorporated into host DNA by adenovirus-mediated transfection) and is therefore not stable, Viral vectors grow at high titers and can transfect non-replicating cells. Human 293 cells, or human fetal cells transformed with the adenovirus EIA / EIB gene, are a representative example of a useful permissive cell line and are commercially available from ATCC (Manassas, Va.). However, other cell lines capable of replicating replication-defective adenovirus vectors in cells, such as HeLa cells, can also be used.

Other references describing adenovirus vectors and other viral vectors that can be used in the methods of the invention include: Horwitz M. S
. , Adenoviridae and Their Replication
, Fields, B .; et al. (Ed.) VIROLOGY, Vol. 2, Raven Press, New York, pp. 1679-1721 (1990); Grah
am, F.S. et al. Pp. 109-128, METHODS IN MO
LECULAR BIOLOGY, Vol. 7: GENE TRANSFER
AND EXPRESSION PROTOCOLS, Murray, E.A. (Ed.), Humana Press, Clifton, NJ (1991); Mil.
ler, N .; et al. (1995) FASEB Journal, 9:19
0-199; Schreier, H .; (1994) Pharmaceutical
Acta Helvetiae, 68: 145-159; Schneider
and French (1993) Circulation, 88: 1937.
-1942; Curiel, D .; T. et al. (1992) Human G
ene Therapy, 3: 147-154; Graham, F .; L. Et al., International Patent Application Publication WO 95/00655; Falck-Pedersen, E. et al. S
. , WO 95/16772; Denefle, P .; et al. , WO95 / 2
3867; Haddada, H .; et al. , WO 94/26914; Per
ricaudet, M .; et al. , WO 95/02697; and Zhan
g, W.S. et al. , WO 95/25071. A variety of adenovirus plasmids are also available from commercial suppliers: for example, Microvics Biosystems, Toronto, Ontario (see, eg, Microvics Product Information Sheet: Plasmids for Construction of Adenovirus Vectors, 1996). A report describing the construction and use of an adeno-retrovirus chimeric vector that can be used for genetic modification, Vi
le et al. (1997) Nature Biotechnology,
15: 840-841, and Feng et al. (1997) Natur
See also eBiotechnology, 15: 866-870.

Other references describing AAV vectors that can be used in the methods of the present invention include: Carter, B .; , HANDBOOK OF PARVO
VIRUSES, Vol. I, PP. 169-228, 1990; Berns,
VIROLOGY, pp. 1743-1764 (Raven Press, 1990);
Carter, B .; (1992) Curr. Opin. Biotechnol.
, 3: 533-539; Muzyczka (1992) Current Top.
ics in Micro. Immunol. 158: 97-129; Flot
te, T .; R. et al. (1992) Am. J. Respir. Cell
Mol. Biol. 7: 349-356; Chatterjee et al.
(1995) Ann. NY Acad. Sci. 770: 79-90; Flot
te, T .; R. Et al., International Patent Application Publication WO 95/13365; Trempe, J
. R. Et al., WO 95/13392; Kotin, R .; (1994) Human
Gene Therapy, 5: 793-801, 1994; Flotte, T
. R. et al. (1995) Gene Therapy, 2: 357-36.
2; Allen, J .; M. WO 96/17947; and Du, et al. (
1996) Gene Therapy, 3: 254-261.

Construction of Retroviral Vectors Retroviral vectors useful in the methods of the present invention are prepared recombinantly by methods already taught in the art. For example, International Patent Application Publication WO94 / 2
9438 describes retroviral packaging plasmids and packaging cell lines. As will be appreciated by those skilled in the art, retroviral vectors useful in the methods of the present invention are those capable of infecting the cells described herein. Methods used to construct vectors, transfix and infect cells are widely practiced in the art. Examples of retroviral vectors are from murine, avian or primate retroviruses. Retroviral vectors based on Moloney murine leukemia virus (MoMLV) are most commonly used because of the availability of retroviral variants that efficiently infect human cells. Other suitable vectors include gibbon ape leukemia virus (
Gibbon Ape Leukemia Virus, GALV) or HI
V-based.

In preparing retroviral vector constructs derived from Moloney murine leukemia virus (MoMLV), in most cases, the gag, pol and env sequences of the virus are removed from the virus, leaving room for insertion of foreign DNA sequences. make. Outpatient DN
The gene encoded by A is normally expressed under the control of a strong viral promoter in the LTR. If the gag, pol and env functions are provided by the packaging cell line in trans, such constructs can be efficiently packaged into viral particles. Thus, when a vector construct is introduced into this packaging cell, the gag, pol, and env proteins produced by the cell will bind to the vector RNA to produce infectious viral particles, which are secreted into the medium. The virus so produced can infect target cells and integrate into its DNA, but does not produce infectious virions due to the lack of essential packaging sequences. Most packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the required coding sequences, so that multiple sets of replication competent viruses must be prepared before replication competent virus can be prepared. A change event is required. Alternatively, the packaging cell line carries the integrated provirus. Proviruses produce all the proteins necessary for the assembly of infectious viruses, but are defective in that their RNA cannot be packaged into viruses. Instead, the RNA produced from the recombinant virus is packaged. Thus, the virus stock released from the packaging cells contains only the recombinant virus.

[0071] The range of host cells that can be infected with a retrovirus or retroviral vector depends on the viral envelope proteins. Recombinant viruses can be used to infect virtually all other cell types, recognized by the packaging cell's env protein, integrate the viral genome in transduced cells, and stabilize foreign gene products. Produce. In general, the murine ecotropic env of MoMLV can infect rodent cells, while the amphotropic (amphotr)
opic) env can infect rodents, birds and certain primate (including human) cells. Amphoteric packaging cells for use in the MoMLV system are known in the art and are commercially available. This includes, but is not limited to: PA12 and PA317, Miller, et al. (1985)
Mol. Cell. Biol. 5: 431-437; Miller, et al.
. (1986) Mol. Cell. Biol. 6: 2895-2902; and Danos, et al. (1988) Proc. Natl. Acad. Sci
. USA, 85: 6460-6464. Some xenotropic vector systems can infect human cells.

The host range of the retroviral vector was altered by replacing the env protein of the base virus with that of the second virus. The resulting "pseudotyped" virus has the host range of the virus donating the envelope protein,
Expressed by the packaging cell line. Recently, vesicular stomatitis virus (V
SV-G) was used in place of the MoMLV env protein. Burns
, Et al. (1993) Proc. Natl. Acad. Sci. USA,
90: 8033-8037; and International Patent Application Publication WO 92/14829. VSV-G pseudotyped vectors have a broad host range because infection is not dependent on specific receptors.

Normally, a vector will contain at least two heterologous genes or gene sequences: (i) a therapeutic gene to be transfected; and (ii) a marker gene that allows one to trace infected cells. As used herein, a "therapeutic gene" is
The gene may be an entire gene derived from a defective endogenous gene, or may be a gene fragment having a functional activity capable of compensating for a patient deficiency. Therapeutic genes also include antisense oligonucleotides or genes useful for antisense suppression, and ribozymes for ribozyme-mediated therapy. For example, in the present invention, the therapeutic gene may be one that neutralizes an immunosuppressive factor or that opposes its action.

Therapeutic genes encoding dominant negative oligonucleotides and peptides, as well as genes encoding regulatory proteins and oligonucleotides are also encompassed by the present invention. In general, gene therapy will involve the transmission of one therapeutic gene, even if more than one gene is required for the treatment of a particular disease. In one embodiment, the therapeutic gene is a dominant negative mutant of the wild-type immunosuppressant. Alternatively, the therapeutic gene may be a wild-type, copy, or functional analog of the defective gene.

[0075] More than one gene can be administered per vector, or more than one gene can be delivered using several compatible vectors. Depending on the genetic defect, the therapeutic gene can include regulatory and untranslated sequences. For gene therapy of human patients, the therapeutic gene is generally of human origin, but is highly homologous and biologically identical or homologous in humans, provided that the gene product does not induce an adverse immune response in the recipient. Genes from other closely related species with equivalent functions can also be used. Therapeutic genes suitable for use in treatment will vary depending on the disease.

The nucleotide sequence of a therapeutic gene is generally known in the art, or can be obtained from various sequence databases, such as Genebank. The therapy gene itself is generally available or can be isolated and cloned using polymerase chain reaction PCR (Perkin Elmer) and other standard recombinant techniques. It will be apparent to one of skill in the art that any gene can be excised as a compatible restriction fragment and inserted into a vector to properly express the therapeutic gene in hematopoietic cells.

[0077] A marker gene can be inserted into the vector to monitor the success of the transduction and to select cells that have integrated the DNA relative to cells that have not integrated the DNA construct. Various marker genes include, but are not limited to, antibiotic resistance markers, such as resistance to G418 or hygromycin. A negative choice can then be taken as a convenience. This includes (but is not limited to) those where the marker is the HSV-tk gene
This makes cells sensitive to substances such as acyclovir and ganciclovir. Alternatively, selection can be performed by using a stable cell surface marker for selecting transgene expressing cells by FACS sorting. NeoR
The (neomycin / G418 resistance) gene is conventional, but any convenient marker whose sequence is not already present in the recipient cells can be used.

Retroviral particles incorporate chimeric envelope proteins or non-viral membrane proteins to improve the stability of the viral particles and to extend host range or to allow cell type-specific targeting upon infection As such, the viral vector can be modified. Preparation of retroviral vectors with altered host range is described, for example, in International Patent Application Publication WO 92/148.
29 and WO 93/14188. Retroviral vectors that can target specific cell types in vivo are also described, for example, by Kasahar
a, et al. (1994) Science, 266: 1373-1376. Describe the construction of Moloney Leukemia Virus (MoMLV) containing a chimeric envelope protein consisting of human erythropoietin (EPO) fused to a viral envelope protein. This hybrid virus exhibits tissue tropism for human erythroid progenitors carrying receptors for EPO, and is therefore useful for sickle cell anemia and thalassemia gene therapy. Retroviral vectors that can specifically target cell infection are preferred for in vivo gene therapy.

[0079] Virus constructs can be prepared by a variety of conventional methods. The target feature, for example, L
Numerous vectors are currently available with long terminal repeats (TRs), marker genes, and restriction sites, which can be further modified by techniques known in the art. The construct may encode a signal peptide sequence to ensure that the cell-encoded or secreted protein encoded by the gene is properly post-translationally processed and, where appropriate, expressed on the cell surface. Preferably, the foreign gene (s) is under the control of a cell-specific promoter.

The expression of the infectious gene can be controlled in a variety of ways depending on the purpose of the gene transfer and the desired effect. For example, the transgene can be constitutively expressed or under the control of a promoter that causes the gene to be expressed to be expressed only under certain physiological conditions or under certain cell types.

The retroviral LTRs are active in most hematopoietic cells in vivo and generally provide a basis for transcription of the inserted sequences and their constitutive expression (Ohash
i, et al. (1992) Proc. Natl. Acad. Sci. 89:
11332; and Correll, et al. (1992) Blood, 8
0: 331). Other suitable promoters include human cytomegalovirus (CM)
V) immediate early promoter and Moloney murine sarcoma virus (MMSV), rous sarcoma virus (RSV) or spleen focus forming virus (Splen Foc)
us Forming Virus, SFFV).

Examples of promoters that can be used to express the introduced sequence in a particular cell type include granzyme A for expression in T cells and NK cells.
Includes the CD34 promoter for expression in stem and progenitor cells, the CD8 promoter for expression in cytotoxic T cells, and the CD11b promoter for expression in bone marrow cells.

For gene expression under certain physiological conditions, an inducible promoter can be used. For example, an electrophilic response element can be used to induce expression of a chemical resistance gene in response to an electrophilic molecule. Therapeutic effect can be further enhanced by targeting the gene product to an appropriate cellular region, such as the nucleus, by adding an appropriate localization sequence.

The vector construct is introduced into a packaging cell line that produces infectious viral particles. Packaging cell lines capable of producing high titers of a replication defective recombinant virus are known in the art. For example, International Patent Application Publication WO 94/2943
See FIG. Viral particles are harvested and purified from the cell supernatant for in vivo infection by methods known in the art, for example, by filtering the supernatant 48 hours after transfection. Virus titer can be measured by infecting a fixed number of appropriate cells (depending on the retrovirus) by instillation of virus supernatant. 4
After 8 hours, transduction efficiency can be assayed by a variety of methods, including Southern blotting.

After viral transduction, the presence of the viral vector in the transduced cells or their progeny can be demonstrated by PCR or the like. PCR can be performed to detect marker genes or other viral transduction sequences. Generally, blood is collected periodically, and for example, when the NeoR gene is used as a marker, PCR can be easily performed using a NeoR probe. The presence of the viral transduction sequence in bone marrow cells or mature hematopoietic cells is evidence of successful reconstitution by the transduced cells. PCR methods and reagents are well known in the art:
R PROTOCOLS, A GUIDE TO MEMETHODS AND A
PPLICATIONS, Innis, Gelfand, Sinsky &
See White (Academic Press, San Diego, 1990); these are commercially available (Perkin Elmer).

[0086] Non-viral vectors, eg, plasmid vectors, useful for the genetic modification of the present invention can be prepared according to methods taught in the art. References describing the construction of non-viral vectors include: Ledley, F.
. D. (1995) Human Gene Therapy, 6: 1129-1.
144; Miller, N .; et al. (1995) FASEB Journal
al, 9: 190-199; Chonn, A .; et al. (1995) Cur
. Opin. in Biotech. 6: 698-708; Schofield
J. P. et al. (1995) British Med. Bull. 51
: 56-71; Brightham, K .; L. et al. (1993) J. Amer. Lip
osome Res. 3: 31-49; Brightham, K. et al. L. , International Patent Application Publication WO 91/06309; Felgner, P .; L. Et al., WO 91/174
24; Solodin, et al. (1995) Biochemistry,
34: 13537-13544; WO93 / 19768; Debs, et al., WO9.
3/25673; Felgner, P .; L. Et al., US Pat. No. 5,264,618.
No .; M. Et al., US Pat. No. 5,283,185; Gebeye.
hu, et al., US Patent No. 5,334,761; Felgner, P .; L. Et al., U.S. Patent No. 5,459,127; W. Et al., WO 95/28
494; Jessee, WO 95/02698; Haces and Ciccar.
one, WO 95/17373; and Lin et al., WO 96/01840.

[0087] More than one gene can be administered per vector, or more than one gene can be delivered using several compatible vectors. Depending on the genetic defect, the therapeutic gene can include regulatory and untranslated sequences. For gene therapy of human patients, the polynucleotides encoding APBP and / or APC supplementation factor are generally of human origin, but should be used in humans if the gene product does not induce an adverse immune response in the recipient. Genes from other closely related species that exhibit high homology and biologically identical or equivalent functions can also be used. Therapeutic genes suitable for use in therapy will vary depending on the disease.

The nucleotide sequence of a polynucleotide is generally known in the art, or can be obtained from various sequence databases, such as a gene bank. The polynucleotides themselves are also commonly available or can be isolated and cloned using polymerase chain reaction PCR (Perkin Elmer) and other standard recombinant techniques. It will be apparent to one of skill in the art that any gene encoding the molecule of interest can be excised as a compatible restriction fragment and inserted into a vector for proper expression in a subject.

It is known to those skilled in the art that minor modifications to the nucleotide sequence do not affect the function of the molecule encoded thereby. Accordingly, polynucleotides that are biologically equivalent to the published sequence are also useful in the methods described herein.
These polynucleotides can be identified by hybridizing under stringent conditions to sequences set forth in the publication or sequences known in the art.
Alternatively, using a sequence alignment program and default parameters, at least 80%, more preferably at least 90%, of the disclosed sequences.
%, And most preferably at least 95% identical polynucleotides.

“Hybridization” refers to the reaction of one or more polynucleotides to form a complex stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding may be performed in Watson-Crick base pairing, Hoogsteen bonding, or in any other sequence-specific manner. The complex may comprise two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, one self-hybridizing strand, or any combination thereof. it can. Hybridization reactions can constitute a step in a broader process, such as initiating a PCR reaction or enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubation temperature, about 25 to about 37 ° C .; hybridization buffer concentration, about 6 × SSC to about 10 × SSC; formamide concentration, about 0約 about 25%; and wash solution, about 6 × SSC. Examples of moderate hybridization conditions include: incubation temperature, about 40 to about 50 ° C .;
About 9 × SSC to about 2 × SSC; formamide concentration, about 30 to about 50%; and wash solution, about 5 × SSC to about 2 × SSC. Examples of high stringency conditions include: incubation temperature, about 55 to about 68 ° C; buffer concentration,
About 1 × SSC to about 0.1 × SSC; formamide concentration, about 55 to about 75%; and a wash solution, about 1 × SSC, 0.1 × SSC, or deionized water. Generally, the hybridization incubation time is 5 minutes to 24 hours, the number of washing steps is 1,
For two or more, the wash incubation time is about 1, 2 or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalent SSCs with other buffer systems can be used.

A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) may have a certain percentage (eg, 80%, 85%, 90%) relative to other sequences.
Or 95%) means that, when aligned for comparison of two sequences, that% of bases (or amino acids) are identical. This alignment and% homology or sequence identity can be determined by software programs known in the art, such as CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY (FM Ausubel, et.
al. (1987) Supplement, 30, 7.7.18, Table 7.
The determination can be made using those described in 7.1). Preferably, default parameters are used for the alignment. A preferred alignment program is BLAST using the default parameters. In particular, the preferred programs are BLASTN and BLASTP using the following parameters: Gene Code = Standard; Filter = None; Strand = Both; Cutoff = 60; Expected = 10; Matrix = BLOSUM62; According to the score (HIGH SCORE); database = non-levandant (non-
reboundant), Genebank + EMBL + DDBJ + PDB + Genebank CDS translation + SwissProtein + Spandate + PIR. Details of these programs can be found at the following internet address: http: // www. ncbi. nlm
. nih. gov / cgi-bin / BLAST.

In Vivo, Ex Vivo, or In Vitro Introduction of a Gene Delivery Vehicle A gene delivery vehicle carrying a polynucleotide described herein can be introduced into host cells in vivo, ex vivo, or in vitro. In one embodiment, the gene delivery vehicle is introduced into a subject in vivo. Vehicles can be introduced transdermally, orally, subcutaneously, intramuscularly, intravenously or parenterally (Wan, et al.).
al. (1997) Human Gene Therapy, 8: 1355-6.
3). For example, if the tumor to be treated includes sites such as skin lesions or nodules (eg, melanoma), the gene delivery vehicle (s) may be applied topically to the skin,
Alternatively, it can be injected into a subcutaneous nodule or lesion. For example, gene delivery vehicles by liposomes can penetrate the epithelium upon topical application.

[0094] In other embodiments, ex vivo gene therapy can be employed. In this case, the molecule of the gene delivery vehicle carrying the gene encoding the molecule is introduced into the cell outside of the subject, and the transduced cell is re-introduced into the subject. Preferably, cells are obtained from a biopsy sample taken from the subject. These cells are cultured and a gene delivery vehicle carrying a polynucleotide encoding APBP is introduced into the cultured cells. If desired, transduction with a cytotoxic agent can also take place during the culture. The cells are then re-introduced into the subject and the cytotoxic agent is activated in vivo, releasing the APBP: peptide complex in the subject.

In yet another aspect, the methods described herein can be performed in vitro. For example, a tumor biopsy sample is isolated as follows. Then, during the culture, APBP,
The cytotoxic agent, and optionally the molecules of the APC recruiting factor or the polynucleotides encoding them, can be introduced by one or more gene delivery vehicles. In addition, APC and naive immune effector cells must be added--APC presents tumor-specific antigens and educates naive immune cells. These antigen-specific immune effector cells can then be re-introduced into the subject and specifically recognize and destroy the tumor cells.

[0096] Tumor cell isolation Tumor cells can be isolated by any method known in the art. In one embodiment, a biopsy sample is minced to prepare a cell suspension. Preferably, tumor cells are replaced by other cells (eg, immune effector cells, eg, T cells) in a manner well known in the art.
Cells).

It is generally desirable to isolate the initial inoculated population from neoplastic cells prior to culture. Separation of various cell types from neoplastic cells can be performed by any of a number of methods, including cell sorters, magnetic beads, and packed columns. Other separation methods include, but are not limited to: physical separation, magnetic separation, use of antibody-coated magnetic beads, affinity chromatography, bound to or used with monoclonal antibodies Cytotoxic substances (
"Panning" using antibodies attached to solid matrices (eg, plates), including but not limited to complement and cytotoxins.
) ", Elutriation, or other convenient methods.

The use of physical separation methods includes those based on differences in physical properties (density gradient centrifugation and countercurrent centrifugation elutriation) and those based on differences in cell surface properties (lecithin and antibody affinity). , And those based on differences in vital staining properties (mitochondrial binding dyes r
ho123 and the DNA binding dye Hoechst 33342). These methods are well known to those skilled in the art.

[0099] Monoclonal antibodies are other useful substances that can be used to identify markers that are associated with a particular cell lineage and / or stage of differentiation. Antibodies can be bound to a solid support to allow for coarse separation. The separation method used should be such that the viability of the collected fractions is maximized. A variety of methods with different efficiencies can be used to obtain "relatively crude" isolates. In such separations, the absence of the marker may remain with the cell population to be retained, up to 10% of the total cells present, usually about 5% or less, preferably 1% or less. . The particular method used will depend on the efficiency of separation, the attendant cytotoxicity, ease and speed of implementation, and the need for complex equipment and / or technical skill.

Another method for separating cell fractions is described in Kawakami et al. (1
988). Exp. Med. , 168: 2183-91, by employing culture conditions for preferentially growing a target cell population.

Evaluation of Gene Transfer Efficiency In Vitro or In Vivo The efficiency of gene transfer into a cell of interest can be monitored by any method known in the art. For example, as described above, a gene delivery vehicle can include a reporter gene or marker gene to facilitate identification of cells that have successfully taken up the vehicle (Kass-Eisler, et al. (1994) Gen.
e Therapy, 1: 395-402). Particularly for in vitro and ex vivo, marker genes will prove to be particularly useful. A screening marker or reporter gene is a gene that encodes a product that can be easily assayed. Non-limiting examples of screening markers include a gene encoding green fluorescent protein (GFP) or a gene encoding a modified fluorescent protein.

[0102] Preferably, the marker gene contained in the delivery vehicle is a selectable marker. A “positive” selectable marker gene encodes a product in which only cells bearing this gene can survive and / or grow under certain conditions. For example, plant and animal cells expressing the introduced neomycin resistance (Neo ^r ) gene are resistant to compound G418. Cells without the ^Neor gene marker are killed by G418. A negative selectable marker gene encodes a product that can selectively kill cells that express the product. For example, as described above, a conditionally activated cytotoxic agent, such as HSV-tk, is also a selectable marker.
Cells expressing this gene can be killed using ganciclovir or acyclovir.

Other methods that can be employed to measure the degree of gene transfer include the following: (1) Quantification of vector-specific DNA by PCR; (2) Transgene-specific mRNA by RT-PCR (3) quantification of serum transgene product amount by ELISA when the transgene encodes a soluble secretory factor, for example, α1-antitrypsin; (4) quantification of serum anti-transgene product amount by ELISA And (5) transfecting the target tissue by homogenizing the tissue and assaying the enzyme activity or treating the tissue with a suitable substrate that is converted by the transgene product to a compound that can be quantified colorimetrically; Quantification of biological activity of gene products; Isolation, culture and expansion of APCs including dendritic cells Two basic methods for APC isolation are briefly described below. These methods include (1) isolating bone marrow precursor cells (CD34 ⁺ ) from blood and stimulating them to differentiate into APCs; or (2) isolating APCs from peripheral blood.
Precommitted APCs that are directed to differentiate into PCs
). In the first method, the patient must be treated with a cytokine, such as G-CSF, to increase the number of circulating CD34 ⁺ stem cells in the peripheral blood.

The second method of APC isolation involves the collection of relatively rare progenitor cells that are already circulating in the blood and are committed to APC differentiation. Conventional methods for isolating progenitor cells that have been committed to APCs from human peripheral blood include a combination of a metrizamide concentration gradient and a physical method such as an attachment / non-adhesion step (Freden).
thal p. S. et al. (1990) PNAS, 87: 7698-77.
02); Percoll concentration gradient separation method (Mehta-Damani, et a
l. (1994) J. Am. Immunol. 153: 996-1003); and a method for selecting fluorescence-activated cells (Thomas R. et al. (1993) J. Am.
Immunol. 151: 6840-52).

One method for separating large numbers of cells from each other is known as countercurrent centrifugation (CCE). In this method, cells are treated simultaneously by centrifugation and washing with a stream of buffer that constantly increases in flow rate. The constantly accelerating counter-current buffer flow separates the cells, generally based on cell size.

In one aspect of the invention, the APCs are precursor cells or mature dendritic cells that have been committed to APCs, which can be isolated from a mammalian, eg, murine, monkey or human leukocyte fraction. (For example, International Patent Application Publication WO9)
6/23060). The leukocyte fraction may be from peripheral blood of a mammal. The method includes the following steps: (a) obtaining a leukocyte fraction from a mammalian source by methods known in the art, such as leukophoresis; (b) the leukocyte fraction of step (a). For 4 more times by countercurrent centrifugal elutriation.
(C) stimulating the conversion of monocytes to dendritic cells by contacting the monocytes in one or more fractions from step (b) with a calcium ionophore; (D) identifying the dendritic cell enriched fraction from step (c); and (e) step (d).
) Is collected, preferably at about 4 ° C. One method for identifying dendritic cell enriched fractions is by fluorescence activated cell sorting. The leukocyte fraction is treated with a calcium ionophore in the presence of other cytokines such as rhIL-12, rhGM-CSF or rhIL-4. Prior to the separation step, the cells of the leukocyte fraction can be washed with a buffer and suspended in a Ca ⁺⁺ / Mg ^++- free medium.
The leukocyte fraction is obtained by leukapheresis. Dendritic cells have the following markers: HLA-DR, HLA-DQ or B7.2.
Together with the presence of at least one of the following markers: CD3, CD14, C
It can be identified by the substantial absence of D16, 56, 57 and CD19, 20. Monoclonal antibodies specific for these cell surface markers are commercially available.

[0107] More particularly, the method comprises the steps of leukapheresis.
) Requires enrichment of leukocytes and platelets and then further countercurrent centrifugation (CCE) (Abrahamsen, TG et al. (1)
991) J. Clin. Apheresis, 6: 48-53). The cell sample is loaded into a special elutriation rotor. Next, the rotor is rotated at a constant speed of, for example, 3000 rpm. When the rotor reaches the target speed, the flow rate of the cells is controlled using pressurized air. The cells in the elutriator are treated simultaneously by centrifugation and washing with a buffer stream of constantly increasing flow rates. As a result, cells are generally (but not limited to)
Fractionation is based on cell size.

The quality control of APCs, and more specifically the validation of their successful activation in DC harvesting and culture, is a critical factor in simultaneously monitoring monocyte and dendritic cell subpopulations and the potential for T lymphocyte contamination. By color FACS analysis. This is based on the fact that DC does not express the following markers: CD3 (T cells); CD14 (monocytes); CD
16, 56, 57 (NK / LAK cells); CD19, 20 (B cells). At the same time D
C indicates that large amounts of HLA-DR, significant amounts of HLA-DQ as they circulate in the blood
And B7.2 (although B7.1 expresses little or no) (and they express Leu M7 and M9; these are bone marrow markers also expressed by monocytes and neutrophils).

[0109] In one embodiment, the cells expressing APC and one or more antigens are autologous. In other embodiments, the APCs and antigen-expressing cells are allogenic; that is, from different subjects.

Therapeutic Applications The methods described herein are useful for providing in vivo, ex vivo and in vitro therapy of diseases associated with an antigen-specific immune response, particularly cancer. APBP
And when cytotoxic agents are administered to tumor sites in vivo, they act on tumor cells there to release large amounts of tumor-specific peptides bound to APBP. These APBP: peptide complexes are presented by antigen presenting cells recruited to the site, preferably using a variety of recruiting factors. The APC then educates immune effector cells and acts to specifically bind and destroy tumor cells throughout the subject.

[0111] These methods involve isolating tumor cells from a subject, introducing an effective amount of APBP and a conditionally activated cytotoxic agent to the cells, and administering the cells to the subject. Ex vivo can also be applied. Activation of the cytotoxic agent (by administration of the appropriate prodrug) results in an immunospecific response.

[0112] The methods described herein can also be used in adoptive immunotherapy regimes. Tumor cells are isolated from the biopsy and cultured in vitro. The APBP, cytotoxic agent, and optionally the APC recruiting factor (or a polynucleotide encoding these molecules) are then introduced into the culture, for example, by one or more gene delivery vehicles. Similarly, when APC and naive immune effector cells are introduced into tumor cell culture, AP
C will present the tumor-specific antigen and then educate the naive immune cells to make them tumor-specific. These antigen-specific immune effector cells can then be re-introduced into the subject and specifically recognize and destroy the tumor cells. Antigen-specific immune effector cells can be used in autologous or allogeneic immunotherapy regimes.

The present invention utilizes these APC cells to stimulate the production of an enriched population of antigen-specific immune effector cells. Antigen-specific immune effector cells expand at the expense of APCs, which die in culture. The process by which naive immune effector cells are educated by other cells is essentially the process of Coulie (1997).
Molec. Med. Today, 261-268.

The APC prepared as described above is mixed with naive immune effector cells. Preferably, the cells can be cultured in the presence of a cytokine, eg, IL2. Because dendritic cells secrete potent immunostimulatory cytokines, such as IL12, it may not be necessary to replenish cytokines during the initial and subsequent rounds of expansion. In any case, the culture conditions are such that antigen-specific immune effector cells expand (ie, proliferate) much faster than APCs. Multiple injections of hybrid cells and, optionally, cytokines can be used to further expand the antigen-specific cell population.

The therapy of the present invention can be applied in vivo or ex vivo. For in vivo therapy, murine B16 melanoma cells (2 × 10 ⁴ ) are injected subcutaneously into the flank of C57BL / 6 mice. About 10-14 days later, when the tumor size was 7 mm × 9 mm, antigenic peptide binding protein (e.g., heat shock protein) and APC supply factors (e.g. GM-CSF) adenoviral vectors encoding the 5 × 10 ⁹ ~1 × Inject 10 ¹⁰ intratumorally. The next day, cytotoxic factors (eg, HSV-TK)
Is injected into the tumor. One day later, animals were dosed with the prodrug ganciclovir twice a day intraperitoneally (O'Malley, e).
t al. (1995) Cancer Research, 55: 1080-5.
And then daily for 6 days thereafter. Monitor tumor size and viability as a function of time. Unmodified B in opposite flank of tumor free animal
Re-challenge with 16 tumor cells and determine if they were protected by the generation of systemic anti-tumor immunity.

For ex vivo therapy, murine B16 melanoma cells are cultured in vitro with an antigen peptide binding protein (eg, hsp) and an APC recruiting factor (eg, G
Adenovirus encoding M-CSF, as well as cytotoxic factors (eg, H
(SV-TK). The tumor cells are then injected subcutaneously into the flank of C57BL / 6 mice. One day later, the animals are dosed with the prodrug ganciclovir twice a day intraperitoneally (as above) and then daily for 6 days. As above, tumor size and viability are monitored as a function of time to determine protection from subsequent tumor cell attack. Animals that become resistant to tumor challenge as a result of this therapy are killed and splenocytes are harvested and assayed for anti-tumor T cell reactivity.

[0117] Example melanoma or the patient or head and neck cancer patients with therapy reachable melanoma lesions head and neck cancer, one or more vectors that encode antigenic peptide binding protein, a cytotoxic factor and APC supply factors Injection. If the cytotoxic factor is HSV-TK, the prodrug ganciclovir is administered systemically by intravenous infusion. Monitor tumor regression and anti-tumor cell immune response.

While the invention has been described with respect to the foregoing embodiments, it should be understood that the above description and examples do not limit the scope of the invention. For example, any of the above compositions and / or methods can be combined with known therapies or compositions. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61K 45/00 101 4H011 101 48/00 4H045 48/00 A61P 35/00 A61P 35/00 37 / 04 37/04 43/00 111 43/00 111 C07H 21/04 B C07H 21/04 A61K 35/76 C12N 15/09 C07K 16/46 // A61K 35/76 A61K 37/52 C07K 16/46 C12N 15 / 00 A F-term (reference) 4B024 AA01 BA45 BA51 BA54 CA01 HA17 4C057 BB02 BB05 DD01 MM04 4C084 AA02 AA13 AA17 BA35 DA02 DA16 DC01 DC25 DC50 MA02 NA14 ZB262 4C085 AA14 BB17 CC03 DD23 EA03 4C0A4A01A01 CA03 CA40 DA76 EA28

Claims (39)

[Claims] 1. A method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an effective amount of an antigenic peptide binding protein (APBP) and a cytotoxic agent. 2. The method according to claim 1, further comprising activating the cytotoxic substance with an activating agent.
2. The method of claim 1, further comprising administering the activator to the subject. 3. The method according to claim 1, wherein the antigen peptide binding protein is a heat shock protein (H
2. The method of claim 1, wherein the method is selected from the group consisting of SP), a soluble major histocompatibility complex (MHC) class I molecule, and an antibody that has been genetically engineered to bind an antigenic peptide. 4. The method of claim 1, wherein the conditionally activated cytotoxic agent is herpes simplex virus thymidine kinase (HSV-tk) and the compound is ganciclovir. 5. A method for inducing an antigen-specific immune response in a subject, comprising the steps of: administering an effective amount of a first polynucleotide encoding an antigenic peptide binding protein and a second polynucleotide encoding a cytotoxic agent. A method comprising administering. 6. The method according to claim 1, further comprising activating the cytotoxic substance with an activating agent.
6. The method of claim 5, further comprising administering the activator to the subject. 7. The method according to claim 7, wherein the antigen peptide binding protein is a heat shock protein (H
6. The method of claim 5, wherein the method is selected from the group consisting of SP), a soluble major histocompatibility complex (MHC) class I molecule, and an antibody that has been genetically engineered to bind an antigenic peptide. 8. The method of claim 1, wherein the cytotoxic substance is herpes simplex virus thymidine kinase (HS).
V-tk) and the activator is ganciclovir. 9. The method of claim 5, wherein said first and second polynucleotides are naked DNA. 10. The method of claim 5, wherein the first and second polynucleotides are administered in one gene delivery vehicle. 11. The method of claim 5, wherein the first and second polynucleotides are administered in the first and second gene delivery vehicles. 12. The method of claim 10, wherein the gene delivery vehicle is selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, and a liposome. 13. The method of claim 11, wherein the first and second gene delivery vehicles are selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated virus vector and a liposome. 14. The method of claim 1 or 2, further comprising administering to the subject an effective amount of an antigen presenting cell (APC) recruiting factor. 15. The APC recruiting factor is selected from the group consisting of interleukin 4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), sepragel and macrophage inflammatory protein 3alpha (MIP3α).
The method according to claim 14. 16. The method according to claim 16, wherein the antigen peptide binding protein is a heat shock protein (
15. The method of claim 14, wherein the method is selected from the group consisting of HSP), a soluble major histocompatibility complex (MHC) class I molecule, and an antibody that has been genetically engineered to bind an antigenic peptide. 17. The method of claim 15, wherein the cytotoxic substance is herpes simplex virus thymidine kinase (H).
15. The method according to claim 14, wherein the activator is SV-tk) and the activator is ganciclovir. 18. The method of claim 1 or 2, further comprising administering to the subject an effective amount of an antigen presenting cell (APC) recruiting factor. 19. The method of claim 18, wherein the APC recruiting factor is encoded by a third polynucleotide. 20. The APC recruiting factor is selected from the group consisting of interleukin 4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), sepragel and macrophage inflammatory protein 3alpha (MIP3α).
The method according to claim 18. 21. An antigen peptide-binding protein comprising a heat shock protein (
19. The method of claim 18, wherein the method is selected from the group consisting of HSP), a soluble major histocompatibility complex (MHC) class I molecule, and an antibody that has been genetically engineered to bind an antigenic peptide. 22. The method of claim 19, wherein said first, second and third polynucleotides are DNA. 23. The method of claim 19, wherein the first, second and third polynucleotides are administered in one gene delivery vehicle. 24. The method of claim 19, wherein the first, second and third polynucleotides are administered in more than one gene delivery vehicle. 25. The method of claim 23, wherein the gene delivery vehicle is selected from the group consisting of a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, and a liposome. 26. The method of claim 24, wherein the two or more gene delivery vehicles are selected from the group consisting of a retroviral vector, an adenoviral vector, an adeno-associated viral vector, and a liposome. 27. The method according to claim 5, wherein the antigen peptide binding protein and the cytotoxic substance are encoded by one polynucleotide. 28. The method of claim 18, wherein the antigenic peptide binding protein, cytotoxic agent and antigen presenting cell recruiting factor are encoded by one polynucleotide. 29. The antigen peptide binding protein and the cytotoxic agent or antigen presenting cell recruiting factor are encoded by one polynucleotide.
9. The method according to 8. 30. The method of claim 18, wherein the cytotoxic agent and the antigen presenting cell recruiting factor are encoded by one polynucleotide. 31. Cytotoxicity along with antigen presenting cells previously exposed to an antigen peptide binding protein: peptide complex derived from a lysate of tumor cells genetically modified to express the antigen peptide binding protein Administering a population of educated antigen-specific immune effector cells prepared by culturing naive effector cells, with or without the gene product, to a subject, and targeting the educated immune effector cells Adoptive immunotherapy, including reintroduction to 32. The method of claim 31, wherein the method further comprises activating the cytotoxic agent with an activating agent, and further comprising culturing the tumor cells with the activating agent. 33. The APBP is selected from the group consisting of heat shock protein (HSP), soluble major histocompatibility complex (MHC) class I molecules, and antibodies that have been genetically engineered to bind antigenic peptides. 32. The selected,
The method described in. 34. The method in which the cytotoxic substance is herpes simplex virus thymidine kinase (H
33. The method of claim 32, wherein the activator is ganciclovir. 35. The method of claim 31, wherein the APBP and the cytotoxic agent are encoded by the first and second polynucleotides. 36. The method of claim 31, wherein the antigen-specific immune effector cells administered to the subject are autologous. 37. The method of claim 31, wherein the antigen-specific immune effector cells administered to the subject are allogeneic. 38. The method of claim 1, 5 or 31, further comprising administering to the subject an effective amount of a cytokine and / or costimulatory molecule. 39. The method of claim 38, wherein the cytokine and / or costimulatory molecule is administered as a polynucleotide encoding a cytokine and / or costimulatory molecule.