JP2002506633A - Compositions and uses of gene-based vaccines for eliciting a T cell response - Google Patents

Abstract

  (57) [Summary] The present invention provides antigens that are engineered and presented with MHC class I molecules on antigen presenting cells (APCs), and polynucleotides that encode engineered antigens that are presented with MHC class II molecules on APCs. Compositions comprising these polynucleotides are further provided by the present invention. Also provided are methods of increasing the presentation of peptides on the surface of APCs and APCs produced by the methods. Also provided are diagnostic and immunomodulatory methods using the polynucleotides, APCs and immune effect cells of the invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is, 35U. S. C. 119 (e) is claimed in U.S. Provisional Patent Application No. 60 / 078,725, filed March 20, 1998, the contents of which are incorporated herein by reference. You.

TECHNICAL FIELD The present invention belongs to the fields of molecular immunology and medicine. In particular, the present invention provides compositions and methods for eliciting CD4 and CD8 T cell responses in a patient.

BACKGROUND OF THE INVENTION [0003] The mammalian immune system is capable of producing a response to foreign antigens, self-antigens present on cancer cells, and self-antigens present on normal tissues. The immune system consists of B cells, T
Cells include two types of antigen-specific cells. B cells synthesize both membrane-bound and secreted antibodies. T cells can be phenotypically characterized by antigen cell surface labeling and methods of recognizing antigen by antigen secretions. T cells express unique cell membrane molecules. Included among these are T3s that appear on the cell surface with CD3.
Cellular antigen receptors (TCRs) and modifying molecules such as CD5, CD28, and CD45R. Subpopulations of T cells can be identified by the presence of additional membrane molecules. Thus, for example, expressing a CD4 recognition antigen associated with a class II MHC molecule,
And T cells that generally function as helper cells, including the role of promoting antibody production by B cells, and, on the other hand, express CD8-recognition antigens associated with class I MHC molecules and generally function as cytotoxic cells T cells.

[0004] Immune cells recognize specific sites, known as epitopes or antigenic determinants on antigens. An epitope is a region of an immunogen or antigen that binds to an antigen-specific membrane-bound receptor on immune cells, or a soluble counterpart, such as an antibody. Both membrane-bound and secreted antibodies on the surface of B lymphocytes recognize soluble antigens. Unlike B cells that recognize soluble antigens, T cells
Recognize an antigen only when it is associated with its major histocompatibility complex (MHC) gene product on the surface of antigen presenting cells. This antigen can be presented with MHC molecules on the surface of antigen presenting cells or on virus infected cells, cancer cells and grafts.

[0005] Diseases are infested by pathogenic organisms, including bacterial, viral and protozoan pathogens.
And subsequent poor or ineffective immune response to invaders. The disease may also activate self-reactive T lymphocytes, trigger an allergic response to T
Activation of lymphocytes or T-cells associated with certain bacterial and parasitic infections that can produce antigens that mimic human proteins, proteins that are described as "self-antigens"
It may be due to activation of lymphocyte self-response. These diseases include, but are not limited to, autoimmune diseases, which are autoimmune disorders that occur as a secondary event of the infection of certain bacteria or parasites. T cells mediate allergies and certain skin diseases such as psoriasis and vasculitis. In addition, unexpected foreign antigen rejection can lead to graft rejection or even infertility, and such rejection can be due to activation of certain T lymphocyte populations.

[0006] Foreign antigens include allergens and implants, which are macromolecules associated with pathogens such as bacteria, viruses, and protists. Autoantigens are usually non-immunogenic under normal physiological conditions. However, autoantigens can also be immunogens, as in autoimmune diseases. Autoimmune diseases are diseases that affect approximately 5% of adults in Europe and North America and are often chronically debilitating. Steinman (1993) Scienti
fic American 269: 107-114. Autoimmunity is characterized by autoreactive clones of T or B cells that produce a humoral or cell-mediated response to self antigens.

[0007] Tumor cell antigens are also autoantigens and often do not elicit an immune response that is effective in controlling or eliminating disease, which leads to cancer cell elimination. Further, there are other specific situations where it is desired to elicit an immune response to a self-antigen.
These include eliciting an immune response to an antigen as a method of contraception.

[0008] The introduction of an antigen into an animal has been widely used for the purpose of modulating an immune response or lack of an immune response to an antigen for various purposes, or a lack of an immune response therefrom to that antigen. Was. These include vaccination against pathogens, induction of immune responses against cancer cells, reduced allergic reactions, reduced immune response to autoantigens resulting from autoimmune disorders, reduced allograft rejection, and contraception Eliciting an immune response to self-antigens for the purpose of

[0009] The critical target of the vaccine is specialized specialized antigen presenting cells ("APC"), the most immunologically powerful of which are bone marrow-derived dendritic cells ("DC"). Presentation of antigenic peptides by MHC class II molecules elicits a humoral response, while presentation of antigenic peptides on the surface of APCs in the presence of MHC class I molecules provokes a cellular response. Arca et al. (1997) J. Am. Immunol. 20 (
2): 138-45 analyzed the interaction of CD4 ⁺ and CD8 ⁺ T cells involved in the immune response to vulnerable tumors transformed to secrete GM-CSF. The authors report that CD4 ⁺ and CD8 ⁺ cells are independently sensitized during tumor growth, and that both are capable of functioning as effector cells in adaptive transfer. . Both CD4 ⁺ and CD8 ⁺ cells were simultaneously induced on tumors with a weak immune response.

In general, all nucleated cells are capable of presenting endogenously produced antigens (including, for example, viral antigens as well as self antigens) on MHC class I molecules. However, antigen presenting cells have the ability to present endogenously produced antigens on MHC class I molecules and the ability to present soluble exogenous (ie extracellular) antigens on MHC class I and MHC class II molecules. However, presenting soluble foreign antigens on MHC class I molecules is generally relatively inefficient. In recent years, the antigen presentation pathway of exogenously produced antigens and soluble endogenous antigens has been extensively investigated. Extracellular antigens are internalized by antigen presenting cells and processed in endocytic cells where they encounter and bind MHC class II molecules. In all nucleated cells, viruses and cellular proteins synthesized in the cell are hydrolyzed to peptides by proteosomes in the cytoplasm, and some of the peptides are transported to the endoplasmic reticulum by carriers (TAPs) involved in antigen processing. Transported, where it encounters and binds to MHC class I molecules. Raychaudhuri and Rock
(1998) Nature 16: 1025-1031; and Linda.
uer et al. (1998) J. Am. Mol. Med. 76: 32-47. Utilizing both the MHC class I and MHC class II presentation pathways in the same antigen presenting cell would be effective in modulating humoral and cellular immune responses to a given antigen in a patient. The present invention provides the above and related advantages that were not recognized before the present invention.

DISCLOSURE OF THE INVENTION The present invention relates to polynucleotides encoding a first antigen that are processed and presented on an antigen presenting cell (APC) with MHC class I molecules, as well as processed, MHC
A second coding sequence is provided which encodes a second antigen presented on the APC with the class II molecule. The first and second antigens may or may not have the same amino acid sequence. In some embodiments, the polynucleotide of the invention comprises (a) a first coding sequence comprising a nucleotide sequence encoding the antigen, and (b) a nucleotide sequence encoding the antigen, and in the endoplasmic reticulum (ER) of the encoded antigen. A second coding sequence comprising a nucleotide sequence encoding an amino acid sequence that promotes retention of

In another aspect, the polynucleotide of the invention comprises (a) a first coding sequence comprising a nucleotide sequence encoding the antigen, (b) a nucleotide sequence encoding the antigen, and an endoplasmic reticulum (ER) of the encoded antigen. A) a nucleotide sequence that encodes an amino acid sequence that facilitates retention in, and (c) a nucleotide sequence that encodes the antigen, and a non-endosomal MHC class I
A third coding sequence comprising a nucleotide sequence encoding an amino acid sequence directed (or promoted or targeted for retention) to the I pathway.

[0013] In some of these embodiments, the encoded antigen is a naturally occurring antigen or a fragment of a naturally occurring antigen. In another aspect, the antigen is a synthetic antigen. The encoded antigen can be a secreted or cell surface protein.

[0014] The invention further provides a method of increasing antigen presentation on the surface of an APC, comprising introducing the polynucleotide of the invention into the APC under conditions suitable for expression of the polynucleotide. The present invention also provides APCs produced by these methods. These APCs promoted the presentation of the antigens encoded by the polynucleotides of the present invention on both class I and class II MHC molecules.

[0015] The polynucleotides of the present invention are useful in a variety of ways to modulate the immune response to the encoded antigen. In one aspect, the polynucleotide of the invention comprises:
It is useful as a vaccine against tumor cells that encode a tumor antigen or a synthetic antigen corresponding to the tumor antigen and therefore express the tumor antigen on the cell surface. In other aspects,
A polynucleotide of the invention encodes a self-antigen on normal (ie, non-cancerous) tissue. In other aspects, the polynucleotides of the invention encode foreign (non-self) antigens associated with organisms such as pathogenic bacteria, viruses and protists. In yet another aspect, a polynucleotide of the invention encodes an antigen present on an allograft that mediates rejection.

The present invention further provides polypeptides encoded by the polynucleotides of the present invention, and compositions comprising the polypeptides. Polypeptides are useful in the immunomodulation methods of the invention.

[0017] Compositions comprising the polynucleotides are further provided by the present invention. Polynucleotides can be included in gene delivery vehicles such as viral vectors and liposomes, and host cells. These polynucleotides are useful in diagnostic and therapeutic methods, as well as in polynucleotide expression methods for the production, expression and purification of antigen products.

[0018] Polynucleotides may be used to modulate the immune response to an antigen in a patient. Accordingly, the present invention further provides a method of modulating a cellular and / or humoral immune response to an antigen in a subject by administering an effective amount of the polynucleotide to the subject. Polynucleotides can be delivered as exposed DNA or on a gene delivery vehicle. In one aspect, a host cell comprising the polynucleotide is administered in an embodiment.

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

Definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology and recombination included in the art. For example, S
ambrook, Fritsch and Maniatis, MOLECULA
R CLONING: A LABORATORY MANUAL, 2nd e
edition (1989); CURRENT PROTOCOLS INM
OLECULAR BIOLOGY (FM Ausubel et al.
eds. The series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
PCR 2: A PRACTICAL APPROACH (MJ MacPh
erson. B. D. Hames and G.S. R. Taylor eds.
(1995)), Harlow and Lane eds. (1989) A
NTIBODIES, A LABORATORY MANUAL, and
ANIMAL CELL CULTURE (RI Freshney ed.
(1987)). As used herein, certain words may have the following defined meanings.

As used in the specification and claims, the singular forms “a”, “a”
Unless otherwise specified, n "and" the "include plural forms, for example, the word" cell "includes a plurality of cell groups and includes a mixture of cells.

The terms “polynucleotide” and “nucleic acid molecule” interchangeably refer to multimeric forms of nucleotides of any length. A polynucleotide can include deoxyribonucleotides, ribonucleotides, and / or types thereof. Nucleotides contain a three-dimensional structure and can perform any function, known or unknown. The term “polynucleotide” is used, for example, for single-stranded, double-stranded and three-stranded
Heavy helix molecule, gene or gene fragment, exon, intron, mRNA, t
Includes RNA, rRNA, ribozymes, cDNA recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid molecules can also include modified molecules.

Hybridization refers to the reaction of one or more polynucleotides to form a stabilized complex via hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hogstein binding, or other sequence-specific manner. The complex can be double-stranded to form a dimeric structure, three or more strands to form a heavy chain, self-hybridizing single-stranded, or any combination thereof. Hybridization reactions may constitute steps of a wider range of methods, such as initiating a PCR reaction, or enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include incubation temperatures of about 25 ° C. to about 37 ° C., about 6 × SSC to about 10 × SS
Contains hybridization buffer at a concentration of C, formamide at a concentration of approximately 0% to approximately 25%, and a wash solution of approximately 6 × SSC. Moderate (mod
erate) Examples of hybridization conditions are from about 40 ° C. to about 50 ° C.
Incubation temperature of ° C., hybridization buffer at a concentration of about 9 × SSC to about 2 × SSC, formamide at a concentration of about 30% to about 55%, and a wash solution of about 5 × SSC to about 2 × SSC. Examples of highly stringent conditions include incubation temperatures of about 55 ° C. to about 68 ° C., hybridization buffer at a concentration of about 1 × SSC to about 0.1 × SSC, and concentrations of about 55% to about 75%. Contains a wash solution of formamide, and approximately 1 × SSC, 0.1 × SSC or deionized water. Generally, the incubation time for hybridization is from 5 minutes to 24 hours, with one, two or more washing steps and a wash incubation time of 1, 2, or 15 minutes. SSC contains 0.15 M NaCl and 15
mM citrate buffer. It is understood that other buffer systems may be used as well as SSC.

A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (eg, 80%, 85%, 90% or 95%) of “sequence identity” to another sequence, When aligned, it means that the proportion of bases (or amino acids) is the same when comparing the two sequences. The juxtaposition and homology percentages or sequence identities can be determined, for example, using the Current Protocol
in Molecular Biology (FM Ausbel et.
al. , Eds. , 1987; Supplement 30, section.
7.7.18, Table 7.7.1) and can be determined using software programs known in the art. Preferably, default parameters are used in parallel. A preferred parallel program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP using the following default parameters. Genetic code = standard, selection = none, strand = both, separation = 60, expected value = 10, matrix = BLOSUM62, description = 50 sequences, distribution method = high score, database =
Non-overlapping, GenBank + EMBL + DDBJ + PDB + GenBank CD
S translations + SwissProtein + SPUpdate
+ PIR. Details of these programs can be found at the following Internet addresses: http: // www. ncbi. nlm. nih. gov / cgi-
bin / BLAST As used herein, the term "classical MHC class I processing pathway" refers to the process in which proteins are processed in the proteosome and transported to the ER where they associate with MHC class I molecules. Is intended to be.

As used herein, the term “ER retention sequence” refers to the same translation unit as the amino acid sequence of a peptide antigen if the same antigen is not in the same translation unit as the amino acid sequence of the peptide antigen. An amino acid sequence is contemplated that increases the likelihood that an antigen may be located in the endoplasmic reticulum (ER) of a eukaryotic cell, as compared to when. "Increasing the likelihood that an antigen can be localized to the ER" means at least about 2 times, more preferably at least about 5 times, the same antigen present in the translation unit lacking an amino acid sequence that promotes retention in the ER.
Even more preferably, a given antigen is found in the same translation unit as the amino acid sequence of an amino acid sequence that promotes retention in the ER, which may be found in the ER at least about 10-fold or higher. Methods for determining that an antigen is in the endoplasmic reticulum are known in the art, and destroy cells, fractionate subcellular components, and localize the protein to any subcellular component. Including, but not limited to, determining whether to do so by, for example, an immunoassay.

As used herein, the term “classical MHC class II processing pathway” refers to a protein in which the protein is taken up by antigen presenting cells by endocytosis or phagocytosis and the MHC class II molecule It is intended to be processed in the endosome before binding to the. This is also referred to herein as "processing by the endosomal pathway". In the present invention, “endosome (endosome)
some), “endocytic compar”
tment) "," endocytic vesicles "
le) "and" endosomal compartments "
) "Are used interchangeably.

As used herein, the term “amino acid sequence that facilitates processing of a protein by the non-endosomal MHC class II pathway” refers to the same amino acid sequence when present together with the amino acid sequence of a peptide antigen in the same translation unit. A subcellular eukaryotic cell in which the antigen is not endosomes and where protein fragmentation and subsequent binding to MHC class II molecules can occur, as compared to when the antigen is not in the same translation unit as the amino acid sequence. Amino acid sequences that increase the likelihood of being localized in the sexual compartment. "Increasing potential" is given in the same translational unit as the amino acid sequence of the amino acid sequence, which promotes retention in non-endosomal subcellular compartments where fragmentation and subsequent binding to MHC class II molecules can occur. Antigen promotes retention in such subcellular compartments to at least about 2-fold, more preferably at least about 5-fold, even more preferably at least about 10-fold or higher levels in such subcellular compartments It is contemplated that the same antigen present on the translation unit lacking the amino acid sequence may be found in such subcellular compartments. Non-endosomal subcellular compartments where processing and subsequent binding to MHC class II molecules can occur include, but are not limited to, lysosomes, Golgi, and melanosomes. Methods for determining whether a protein is present in a subcellular compartment are described above.

The term “antigen” is well known in the art and includes immunogenic substances, ie, immunogens, as well as substances that cause immunological non-responsiveness or allergy, ie, allergens. An antigen contains one or many antigenic determinants, ie, epitopes. In the description of the present invention, "antigen", "antigenic determinant" and "antigenic determinant"
The term "epitope" is used interchangeably. As used herein, the term “antigen” refers to naturally occurring antigens, synthetic antigens, foreign antigens, self antigens,
Includes modified self and mutant antigens.

A “native or natural” antigen is a polypeptide, protein or fragment having an epitope, which is isolated from a natural biological source and which is an antigen receptor, especially a T cell antigen receptor in a patient The polypeptide, protein or fragment capable of specifically binding to the body (TCR).

A synthetic antigen is said to “correspond” to a natural antigen if the peptide binds to the same TCR as the natural epitope. The term "peptide" as used interchangeably with "polypeptide" herein is used in a broad sense to refer to two or more amino acid subunits, amino acid analogs or peptidomimetic compounds. Used. The subunits may be linked by peptide bonds. In other embodiments, the subunits may be linked by other bonds, for example, esters, ethers, etc. As used herein, the term "amino acid" refers to either natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics Intended. Peptides of three or more amino acids are commonly referred to as oligopeptides if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or protein.

The term “sequence motif” refers to the manner in which a population of molecules (eg, amino acids or nucleotides) exists. Typical modes are hydrophilic, hydrophobic, basic,
It can be identified by characteristic amino acid residues such as acidic, and the like.

A “signal sequence” is used to direct newly biosynthesized secreted or membrane proteins towards and through the cellular membrane, including, but not limited to, the endoplasmic reticulum and subcellular compartments (or endosomes). It is a short amino acid sequence to be directed. The signal sequence may be at the amino-terminal (N-terminal) and / or carboxy-terminal (C-terminal) portion of the polypeptide, and is generally cleaved after the polypeptide has passed through the membrane.

“Gene delivery vehicle” is defined as a molecule that can carry an inserted polynucleotide into a host cell. Examples of gene delivery vehicles include liposomes, biocompatible polymers including natural and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial virus envelopes, metal particles and bacteria, baculoviruses, adenoviruses and retroviruses. Viruses such as viruses, bacteriophages, cosmids, plasmids, fungal vectors, and simple protein expression and genes typically used in the art described for expression in a variety of prokaryotic and eukaryotic hosts Other recombinant delivery vehicles that can be used for therapy.

As used herein, the term “antigen presentation matrix” refers to a molecule or group of molecules that can present an antigen in such a way that the antigen can bind to the surface of a T cell via a T cell antigen receptor. Intended. Antigen presenting matrix is antigen presenting cell (APC)
On a vesicle preparation of APC, or in the form of a synthetic matrix on a solid support such as beads, plates. Examples of synthetic antigen-presenting matrices are purified MHC class I molecules, or complex MHC class II molecules, that complex with β2-microglobulin, or their functional sites attached to a solid support.

As used herein, an “antigen-presenting cell” is an example of a major histocompatibility complex molecule or a portion thereof, or one or more non-classical MHC molecules or portions thereof. A cell that presents its antigen on the surface is intended. Appropriate AP
Examples of Cs are described in detail below and include, but are not limited to, macrophages, dendritic cells, B cells, APCs hybrids, and whole cells such as antigen presenting mother cells. Methods for making APCs hybrids have been described previously. See, for example, International Patent Applications WO 98/46785 and WO 95/16775.

[0037] Dendritic cells (DCs) are powerful antigen presenting cells. DCs have been shown to supply all signals necessary 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 through the T cell receptor / CD3 ("TCR / CD3") complex and major histocompatibility complex ("MHC") class I or II proteins on the surface of APCs. It is mediated by interaction with the presented antigenic peptide. This interaction is necessary but not sufficient for T cell activation to occur. Indeed, without the second type of signal, the first type of signal may result in T cell immune anergy. A second type of signal, called a costimulatory signal, is neither antigen-specific nor MHC-restricted, and, in the presence of the first signal, can trigger a full proliferative response of T cells and T cell effector function. As used herein, "dendritic cells" include, but are not limited to, pulsed dendritic cells, mother cells, or dendritic cell hybrids.

[0038] "Foster antigen present cells"
"ing cell" is a dendritic cell that has been genetically modified to take up an exogenous antigen from the limited binding of endogenous peptides and present it on the cell surface.

The term “major histocompatibility complex” or “MHC” refers to a gene complex encoding a cell surface molecule that is required for antigen presentation to T cells and acute debris rejection. In humans, the MHC complex is also known as the HLA complex. M
The protein encoded by the HC complex is known as "MHC molecule"
And class I and class II MHC molecules. Class I MHC molecules include a membrane heterodimeric protein consisting of an MHC-encoded α chain non-covalently associated with β2 microglobulin. Class I MHC molecules are expressed by almost all nucleated cells and have been shown to function in antigen presentation to CD8 ⁺ T cells. Class I molecules include HLA-A, -B, and -C in humans. Class II MHC molecules also have non-covalently attached I and 9
Includes membrane heterodimeric proteins consisting of chains. Class II MHC is also available on CD
It is present on + T cells and is known to contain HLA-DP, -DQ and -DR in humans. The term "MHC restriction" refers to the characteristic of a T cell that can be antigen-recognized only after it has been processed and the resulting antigenic peptide bound and presented with a class I or class II MHC molecule. Methods for identifying and comparing MHC are well known in the art and are described in Allen et al (1994) Human Imm. 40: 2
5-32; Santamaria et al. (1993) Human Im
m. 37: 39-50 and Hurley et al. (1997) Tiss
ue Antigens 50: 401-415.

“Co-stimulatory molecules” are involved in the interaction between a T-cell and a receptor-ligant pair expressed on the surface of an antigen presenting cell. Active research over the past several years has convincingly shown that resting T cells require at least two signals to induce cytokine gene expression and proliferation (Schwartz. RH (199).
0) Science 248: 1349-1356; Jenkins. M. K. (
1992) Immunol Today 13: 69-73). One signal that confers specificity can be produced by the interaction of the appropriate MHC / peptide complex with the TCR / CD3 complex. The second signal is not antigen-specific and "
It is called the "co-stimulation" signal. This signal is originally defined as the activity supplied by macrophages, called "professional" APCs, and accessory cells derived from bone marrow, such as dendritic cells. Several molecules have been shown to promote costimulatory activity. These are thermostable antigens (HSA) (Liu, Y., et al.
1992). Exp. Med. 175: 437-445), chondrotin sulfate modified MHC invariant chain (Ii-CS) (Naujokas, MF, et al.).
. (1993) Cell 74: 257-268), Intracellular adhesion molecule 1 (ICA)
M-1) (Van Eventer. GA (1990) J. Immunol)
. 144: 4579-4586), B7-1 and B7-2 / B70 (Schw
arz, R .; H. (1992) Cell 71: 1065-1068).
These molecules appear to assist costimulation by interacting with cognate ligands on T cells. The costimulatory molecule mediates the costimulatory signal (s) required under normal physiological conditions for the acquisition of full activation of sensitive T cells. One exemplary receptor-ligand pair is the B7 costimulatory molecule on the surface of APCs and its complementary receptor CD28 or CTLA-4 on T cells (Freeman et a
l. (1993) Science 262: 909-911; Young et.
al. (1992) J.I. Clin Invest. 90: 229; Nabav
i et al. (1992) Nature 360: 266-268). Other important costimulatory molecules are CD40, CD53, CD80, CD86.

The term “co-stimulatory molecule” refers to a co-stimulatory molecule that, when functioning with the peptide / MHC complex bound to the TCR on the surface of the T cell, achieves activation of the T cell binding peptide. Includes any single molecule or combination of molecules that provides a stimulating effect. Thus, the term is used in conjunction with B7 or the peptide / MHC complex
If the cell surface TCR specifically binds to the peptide, APC, a fragment thereof (alone, complexed with other molecule (s), or Other co-stimulatory molecule (s) on the antigen presentation matrix, such as a portion of the fusion protein). Co-stimulatory molecules include, for example, Beckm
Available from a variety of sources, including an Coulter. Molecules with biological activity similar to wild-type or purified costimulatory molecules (eg, recombinantly produced or variants thereof) are not always explicitly stated,
It is intended to be used within the spirit and scope of the present invention.

As used herein, the terms “solid support” or “solid”, which are used interchangeably,
"Solid support" is not limited to a particular type of support. Rather, a large number of supports are available and known to those skilled in the art. Solid supports include silica gel, resins, derived plastic films, glass beads, cotton, plastic beads, alumina gel. As used herein, "solid support" also includes synthetic antigen-presenting substrate cells and liposomes. Suitable solid supports may be selected based on the desired end use and suitability for various protocols. For example, for peptide synthesis, the solid support may be a polystyrene (eg, Bachem Inc ,.
PAM-res obtained from Peninsula Laboratories
in, etc.), POLYHIPE registered trademark (obtained from Aminotech, Canada), polyamine resin (Peninsula Laboratorie)
s), and a polystyrene resin (T
entaGel registered trademark, Rapp Polymere, Tubingen,
Germany) or polydimethylacrylamide resin (Milligen /
(Obtained from Biosearch, California).

The term “genetically modified” means containing and / or expressing a foreign gene or nucleic acid sequence and modifying the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion, or destruction of a cell's endogenous nucleotides.

As used herein, the term “cytokine” refers to any one of a number of factors that have various effects on a cell, such as growth or proliferation. Non-limiting examples of cytokines used alone or in combination in the practice of the present invention include interleukin-2 (IL-2), stem cell factor (SCF)
), Interleukin-3 (IL-3), interleukin-6 (IL-6),
Interleukin-12 (IL-12), G-CSF, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1I
), Interleukin-11 (IL-11), MIP-11, leukemia inhibitory factor (
LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The invention also includes culturing conditions in which one or more cytokines are specifically excluded from the medium. Cytokines are available from several trading companies, such as Gen
zyme (Framingham. MA), Genentech (South)
San Francisco, CA), Amgen (Thousand Oa)
ks, CA), R & D Systems and Immunex (Seattle
, WA) are commercially available. Although not always explicitly seen, molecules having similar biological activities, such as wild-type or purified cytokines (eg, recombinantly produced or muteins therefrom), are contemplated by the spirit and scope of the invention. It is intended to be used in

“Vector” means a self-replicating nucleic acid molecule that introduces an inserted nucleic acid molecule into and / or between host cells. This word refers to a vector having a function of mainly introducing a nucleic acid molecule insert into a cell, a replication vector having a function of mainly replicating a nucleic acid, and an expression vector having a function of transcription and / or translation of DNA or RNA. It is intended to include. Some vectors also provide more than one of the functions described above.

A “PCR primer” is a DNA sequence which is complementary to a thermostable synthase such as Taq synthase, one is complementary to the (+) strand at one end of the sequence to be amplified, and the other is the opposite. "Polymerase chain reaction" or "PCR", a method of amplifying using two oligonucleotide primers that are complementary to the terminal (-) strand
Reference will be made to the primers used in. The newly synthesized DNA strand can subsequently be used as an additional template for the same primer sequence, so that successive repetitions of primer annealing, chain extension, and dissociation will result in an exponential and highly specific sequence of the desired sequence. (Eg, PCR2: APR as described above).
ACTICAL APPROACH). PCR can also be used to detect the presence of a defined sequence in a DNA sample.

A “host cell” is a vector or exogenous nucleic acid molecule, polynucleotide and / or
Or, it is intended to include individual cells or cell cultures that can or have become recipients of a mixture of proteins. This is also intended to include the progeny of a single cell, wherein the progeny are completely identical (morphologically or genetically, or wholly) to the original parent cell by natural, accidental, or deliberate mutations. DNA complementation). The cells can be prokaryotic or eukaryotic, including but not limited to bacterial cells, yeast cells, animal cells, and mammalian cells such as mice, rats, monkeys or humans.

“Antibodies” are immunoglobulin molecules that can bind to an antigen. As used herein, this word refers to intact immunoglobulin molecules as well as anti-idiotype antibodies, variants, fragments, fusion proteins, humanized proteins, and antigen recognition sequences of the desired specificity. And modified immunoglobulin molecules containing

An “antibody conjugate” is a combination of an antibody (as defined above) and its binding partner or ligand. The term "immunomodulatory agent" as used herein is a molecule, macromolecular complex, or cell that modulates an immune response and is described in the antigenic peptide alone, or at the outset of the invention. Including any of a variety of formulations. Various formulations include; a polypeptide comprising an antigenic peptide of the invention; a polynucleotide encoding a peptide or polypeptide of the invention; an antigen comprising an APC and a synthetic antigen-presenting matrix (in the presence or absence of a costimulatory molecule). Class I or Class II MHC on presentation matrix
An antigenic peptide of the invention that binds to a molecule; an antigenic peptide of the invention that is covalently or non-covalently complexed with another molecule or macromolecular structure; and an educated antigen-specific immunity specific for the peptide of the invention. Effect cells.

The term “modulating an immune response” includes the induction (increase, elicitation) of an immune response; and the reduction (suppression) of an immune response. An immunomodulation method (or protocol) is a method of modulating an immune response in a subject.

As used herein, the term “induces an immune response in a subject” refers to the term
A word that is well understood in the art and is at least about twice as
Preferably at least about 5 times, more preferably at least about 10 times.
More preferably at least about 100-fold, more preferably at least about 5
00 fold, more preferably at least 1000 fold or more,
(Or epitope) before the immune response (if any)
The antigen (or epitope) after presenting the antigen (or epitope) to the subject
) Are intended to be detectable (measurable). Antigen (or epitome)
Response to antigen-specific (or epitope-specific) antibody production
Expressing on the surface a molecule that specifically binds to an antigen (or epitope)
Including but not limited to cell production. Given antigen (or epitome)
Methods for determining whether an immune response to
Well known. For example, antigen-specific antibodies are known in the art.
Using one of a variety of immunoassays, including but not limited to ELISA
It can be detected if used. In ELISA, for example, the immobilization of antibodies in a sample
Binding to an antigen (or epitope) is detected by a detectably labeled secondary antibody (eg,
(Enzyme-labeled mouse anti-human Ig antibody). Antigen-specific immunity
Effector cells are FACS or, in the case of CTL,⁵¹Cr-dissociation analysis or ^Three It can be detected by one of a variety of analytical methods known to those of skill in the art, including but not limited to H-thymidine incorporation analysis.

The term “immune effector cell” refers to a cell capable of binding an antigen or mediating an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells, and cytotoxic T cells (CTL), such as CTL lineages, CTL clones, and
Includes CTL from tumors, inflammation, and other invasions. A CTL that expresses an antigen specific to a diseased tissue and is specific to the antigen has been isolated. For example, about 80% of melanomas express an antigen known as gp100.

The term “immune effect molecule”, as used herein, refers to a molecule capable of binding antigen-specifically, including antibodies, T cell antigen receptors, and class 1 and class 2 MHC molecules. Including.

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 cell" is an immune effector cell as defined above that has encountered an antigen and has become specific for that antigen. The educated, antigen-specific immune effector cells can be activated by antigen binding. "Activated" cells without anymore ₀ phase G, meaning that begin to produce the characteristic cytokines cell types. For example, activated CD4 + T cells secrete IL-2 and have a higher amount of high affinity IL-2 receptor on their surface compared to quiescent CD4 + T cells.

The peptide or polypeptide of the present invention may be preferentially recognized by antigen-specific immune effector cells such as B cells and T cells. In the context of T cells,
The word "recognized" means that a peptide or polypeptide of the invention comprising one or more antigenic epitopes is recognized, ie, a T cell antigen receptor (T cell antigen receptor present on the surface of antigen-specific T cells) (TCR) binds to an epitope and is presented on the surface of the APC with the MHC molecule in a manner such that the binding causes activation or unresponsiveness of the T cell. The word "preferentially recognized" intends that the polypeptide of the invention will not subsequently be recognized by unrelated antigen-specific T cells as defined above. Assays for determining whether an epitope is recognized by antigen-specific T cells are known in the art and are described herein.

“Autogenic” or “autologous”
The word "" as used herein indicates a source of cells. That is, if the cells administered to an individual ("recipient") are from the individual ("donor") or a genetically identical individual, the cells are autologous. An autologous cell may also be a progeny of an autologous cell. The word also indicates when cells of different cell types are from the same donor or genetically identical donor. That is, if they are from the same donor or an individual genetically identical to the donor, or are descendants of cells from the same donor or an individual genetically identical to the donor, the effector and antigen presenting cells will be autologous. Called.

Similarly, the word “allogenic”, as used herein, indicates the source of the cell. That is, if the cells administered to an individual ("recipient") are from an individual who is not genetically identical to the recipient, the cells are allogeneic; Related to gender. Allogeneic cells may also be progeny of allogeneic cells. The word also indicates when cells of different cell types are derived from genetically non-identical donors or are progeny of cells from genetically non-identical donors. For example, if the APC is derived from a genetically non-identical donor, the APC may be allogeneic with the effector cell.

As used herein, the term “disease or condition associated with a population of CD4 + or CD8 + T cells” is one that can be associated with a population of CD4 + or CD8 + T cells, ie, these cells Which is first responsive to the pathogenicity of the disease; this also indicates that the presence of CD4 + or CD8 + T cells is indicative of a disease state; and that the presence of a population of CD4 + or CD8 + None, but in playing a key role in the pathogenesis of the disease; it is also in that the population of CD4 + or CD8 + T cells mediates unwanted rejection of foreign antigens. CD4 + or CD8 + T
Examples of conditions associated with populations of cells include autoimmune diseases, graft rejection, immune dysregulation,
And anaphylactic disorders.

As used herein, “neoplastic cell”
The words "neoplasia", "tumor", "tumor cells", "cancer" and "cancer cells" (used interchangeably) indicate relatively autonomous growth, Reference is made to cells that exhibit an abnormal growth phenotype characterized by an important defect in the control of cell proliferation (ie, disinhibition of cell division). Neoplastic cells can be malignant or benign.

“Inhibition” of tumor growth refers to a shortened growth state as compared to growing without contacting the educated antigen-specific immune effect cells as described herein. Tumor cell growth is known in the art, including, but not limited to, measuring tumor size, using ³ H-thymidine incorporation analysis to determine if tumor cells are growing, or counting tumor cells. Can be assessed by the method "Inhibition" of tumor cell growth refers to one or all of the following conditions, in which the growth of the tumor is slowed, delayed and stopped, as well as the tumor shrinks.

The word “culture” refers to the in vitro growth of cells or organs on or in various types of media. It is understood that progeny of cell growth in culture may not be completely identical (morphologically, genetically, or phenotypically) to the parent cell. By "expanded" is meant the growth or division of a cell.

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

As used herein, “expression” 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 includes splicing of the mRNA if a suitable eukaryotic host is selected. Regulators required for expression include a promoter sequence that binds RNA synthase and a translation initiation sequence for ribosome binding. For example, bacterial expression vectors include a promoter, such as the lac promoter, and a Shine-Dalgarno sequence for translation initiation, and an initiation codon AUG (Sambrook et al., (1989) supra). Similarly,
Eukaryotic expression vectors contain a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, an initiation codon AUG and a stop codon for ribosome dissociation. These vectors are commercially available or
Alternatively, it is assembled by the sequences described in methods well known in the art, such as those described below for the construction of general vectors.

A population of “isolated” or “purified” cells, nucleic acids, peptides, or proteins is substantially dissociated individually from cells and naturally associated material. Substantially dissociated or substantially purified, wherein at least 50% of the composition comprises the desired population, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% % Means dissociating molecules that naturally associate with the intended molecule.

“Composition” means an active agent and a mixture of other compounds or compositions, an inactive agent (eg, a detectable agent or label), or an active agent such as an adjuvant. A "pharmaceutical composition" comprises a mixture of active agents in a carrier, whether inactivated or activated, for diagnosis or therapy in vitro, in vivo.
It is intended to make a composition suitable for in vivo or ex vivo use.

As used herein, the term “pharmaceutically acceptable carrier” refers to phosphate buffered saline, water and oil / water or water / oil emulsions or various types of wetting agents and the like. And other common pharmaceutical carriers. The composition also contains a stabilizer and a preservative. Examples of carriers, stabilizers and adjuvants include Martin, REMINGTO
N'S PHARM. SCI. , 15th Ed. (Mack Publ
. Co. , Easton (1975)).

As described herein, the word “comprising” is intended to mean that the compositions and methods include the recited elements but do not exclude others. “Consisting essentially of” when used to define compositions and methods, means excluding other elements of the basic importance of the compound. That is, a composition consisting essentially of the elements as defined herein will contain trace amounts of contamination from isolation and purification methods, as well as pharmaceutically acceptable amounts of phosphate buffered salt solutions, preservatives and the like. Does not exclude acceptable carriers. "Consisting of" means other components of the trace element, and
It is meant to exclude more than a substantial method step for administering the composition of the invention. The embodiments defined by each of these transition words are within the scope of the present invention.

An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more controlled, effective, or applied doses.

The present invention provides improved vaccines and methods of using the vaccines to modulate cellular and humoral immune responses to an antigen or an antigen in a subject.
Antigens for the purposes of the present invention are protein antigens. The word "protein antigen" is used in its broadest sense and includes isolated full-length proteins as well as minimal epitopes, chimeric molecules, synthetic antigens. The epitope may be from an altered antigen. While the embodiments described below are intended as tumor antigens, but not explicitly positioned, it is understood that any protein or polypeptide that induces an immune response is intended to be within the scope of the present invention. Such antigens are not limited to tumor antigens, but also include viral, bacterial and autoantigens. The antigens of this vaccine can be autologous or heterologous (ie, allogeneic), homologues from an isolated species, such as mouse antigens administered to human patients,
It is.

Polynucleotides of the Invention The polynucleotides of the invention contain at least two coding sequences. The first sequence encodes a first antigen that is processed and presented with MHC class I molecules on antigen presenting cells (APCs). The second sequence encodes a second antigen that is processed and presented with MHC class II molecules on the APC. In some embodiments, the first and second antigens have the same amino acid sequence. In yet another aspect,
The polynucleotide encodes an antigen that contains an epitope recognized by the same T cell receptor. In a further aspect, the encoded antigens comprise different epitopes from the same native protein. In a further embodiment, the first and second antigens share at least 50% identical amino acid sequence with each other, as determined using an alignment program using default variables, or
Encoded by polynucleotides that hybridize to each other under stringent conditions.

The polynucleotides of the present invention include: (a) a first coding sequence that includes a nucleotide sequence that encodes an antigen; and (b) a nucleotide sequence that encodes an antigen and the ER
(ER) a second coding sequence comprising a nucleotide sequence that encodes an amino acid sequence that facilitates retention.

The polynucleotide can be constructed such that an amino acid sequence that facilitates retaining the encoded antigen in the ER is appended to the N-terminus or C-terminus of the encoded antigen. Alternatively, sequences that promote retention of the encoded antigen in the ER include:
It may occur internally, that is, between the N- and C-termini of the encoded antigen.

Many amino acid sequences that facilitate retention in the ER are known in the art. For example, the amino acid sequence Lys-Asp-Glu-Leu (KDEL), when attached to the carboxy terminus of a protein, promotes retention of the protein in the ER. Pelham (1990) Trend
s in Biochemical Sciences 15: 483-486.
. Others include the carboxy terminal peptide His-Asp-Glu-Phe (HDEF
) Was reported to promote retention of the attached protein (target) in the ER. In addition to the “KDEL” and “HDEL” sequences, “DDEL”, “ADEL”
EL, SDEL, RDEL, KEEL, QEDL, HIE
Other sequences such as "L", "HTEL" and "KQDL" are known as signals for staying in the endoplasmic reticulum, and polypeptides having these sequences are usually E. coli.
It is held in R. Pelham (1990) TIBS, 15: 483. Thus, in some embodiments, the amino acid sequence that promotes retention of the encoded antigen in the ER is KDEL, HDEL, DDEL, ADEL, SDEL, R
It is selected from the group consisting of DEL, KEEL, QEDL, HIEL, HTEL, and KQDL.

[0074] In some embodiments, the amino acid sequence that facilitates retention of the encoded antigen in the endoplasmic reticulum (ER) is KDEL. In these embodiments, when the antigen encoded by the coding sequence comprising the nucleotide sequence encoding the antigen and the nucleotide sequence encoding KDEL is transcribed and translated, the translation unit comprises an antigen having KDEL as the carboxy-terminal amino acid.

In another embodiment, the amino acid sequence that facilitates retention of the encoded antigen in the endoplasmic reticulum (ER) is HDEF. In these embodiments, when the antigen encoded by the coding sequence comprising the nucleotide sequence encoding the antigen and the nucleotide sequence encoding HDEF is transcribed and translated, the translation unit comprises an antigen having HDEF as the carboxy terminal amino acid.

In other embodiments, the polynucleotides of the invention comprise: (a) a first coding sequence comprising a nucleotide sequence encoding an antigen; (b) a nucleotide sequence encoding an antigen; ER (
(ER) a second coding sequence comprising a nucleotide sequence encoding an amino acid sequence that facilitates retention; and (c) a nucleotide sequence encoding an antigen and directing the encoded antigen to a non-endosomal MHC class II pathway. (Or promote retention,
Or a target)) nucleotide sequence encoding an amino acid sequence.

The polynucleotide can be constructed such that an amino acid sequence that directs the encoded antigen to the non-endosomal MHC class II pathway is appended to the N-terminus or C-terminus of the encoded antigen. Alternatively, the sequence that directs the encoded antigen to the non-endosomal MHC class II pathway may occur internally, ie, between the N- and C-termini of the encoded antigen.

Non-endosomal MHC class II pathways include, but are not limited to, lysosomes, Golgi apparatus, and melanosomes. Amino acid sequences that direct proteins to non-endosomal MHC class II pathways are known in the art.
Antigens were purified from melanosomes (Xu et al., (1998) J. Invest. Derm.
atol. 110: 324-331; and Jimbow et al., (1997) P.
iigment Cell Res. 10: 206-213); lysosomes (K
Ornfeld et al., (1987) FASEB J. et al. 1: 462-468; and Hasilik et al. (1992) Experientia 48: 130-1.
51); and Golgi apparatus (Nilsson and Warren (1994) Cur.
r. Opinion Cell Biol. 6: 517-521) (or that promotes retention or targets) amino acid sequences have been reported in the literature and can be used in the present invention.

[0079] In some embodiments, the encoded antigen is a naturally occurring antigen or a fragment of a naturally occurring antigen. In other embodiments, the antigen is a synthetic antigen. Antigens may be "self" or foreign and may be derived from any organism. Antigens may be autologous or heterologous (ie, homologues from allogeneic or isolated species,
For example, a mouse antigen administered to a human patient). Each coding sequence is operably linked to operational and regulatory sequences that control the transcription and translation of the coding sequence. These operational and regulatory sequences are well known to those skilled in the art.

The polynucleotide of the present invention, in one embodiment, comprises gp100 (Kawak)
Ami et al., (1997) Intern. Rev .. Immunol. 14
173-192), MUC-1 (Henderson et al., (1996) Ca
ncres. 56: 3763-3770), MART-1 (Kawak)
Ami et al., (1994) Proc. Natl. Acad. Sci. 9
1: 3515-3519; Kawakami et al., (1997) Intern.
. Rev .. Immunol. 14: 173-192; Ribas et al., (
1997) Cancer Res. 57: 2865-2869), HER-
2 / neu (US Pat. No. 5,550,214), MAGE (PCT / US92 / 0).
4354) HPV 16, 18E6 and E7 (Ressing et al., (1996)
Cancer Res. 56 (1): 582-588; Restifo (1
996) Current Opinion in Immunol. 8: 6
58-663; Stern (1996) Adv. Cancer Res
. 69: 175-211; Tindle et al., (1995) Clin. E
xp. Immunol. 101: 265-271; van Driel et al., (1996) Annuals of Medicine 28: 471-4.
77) CEA (US Pat. No. 5,274,087), PSA (Lundwall,
A. (1989) Biochem. Biophys. Research
Communications 161: 1151-59), prostate membrane-specific antigen (PSMA) (Israeli et al., (1993) Cancer Rese).
arch 53: 227-30), tyrosinase (U.S. Patent Nos. 5,530,096 and 4,988,814; Brichard et al., (1993) J. Exp M.
ed. 178: 489-49); Tyrosinase-related protein 1 or 2 (T
RP-1 and TRP-2), NY-ESO-1 (Chen et al., (1997) P
rc. Natl. Acad. Sci. U. S. A. 94: 1914
-18), or a previously characterized tumor-associated antigen, such as the GA733 antigen (US Pat. No. 5,185,254).

[0081] In some embodiments, the antigen encoded by the polynucleotide of the invention is a secreted protein or an antigen that has been recombinantly modified to be secreted from the cell.

The secreted form of the antigen may be spliced with a signal sequence to the sequence encoding the wild-type antigen, or remove sequences encoding the cytoplasmic and transmembrane regions of the antigen, and / or 1) encode the Directed to the intracellular fraction, such as the cytoplasm, nucleus or mitochondria, or 2) inserts the encoded protein into the cell membrane (ie, a transmembrane region), or 3) inserts the encoded protein into a hydrophobic anchor sequence or It can be synthesized by removing a sequence retained in an intracellular fraction such as an endoplasmic reticulum maintenance signal.

More specifically, the secreted antigen is a standard recombinant D-protein known to those of skill in the art.
Expression can be achieved by fusing a secretory signal sequence to the wild-type antigen using NA methodology. The secretory signal sequence is typically located at the N-terminus of the required protein, but may be located at any suitable position to allow secretion of the antigen. As an illustration, the figure shows that the DNA sequence encoding the wild-type antigen has a DNA encoding a secretory signal sequence such as the endoplasmic reticulum target sequence (ERTS).
Shown is a transmembrane cassette containing the sequence fused to the 5 'end in reading frame. An internal ribosome entry sequence (IR) between the wild-type gene and the gene to be modified
ES) is inserted, ensuring that both genes are translated from the bicistronic mRNA produced by transcription of the cassette as governed by the CMV promoter. Suitable secretory signal sequences include known secretory protein signal sequences or signal sequence derivatives. A variety of secretory proteins have been identified. They include, but are not limited to, certain growth factors such as fibroblast growth factor 4-6, epidermal growth factor, and lymphokines such as interleukin 2-6.

Alternatively, the secreted antigen can be obtained by adding a secretory signal sequence to the antigen, and optionally 1) directing the encoded protein to intracellular compartments such as the cytoplasm, nucleus and mitochondria, or 2) encoding Causing the protein to be inserted into the membrane of the cell (ie, a transmembrane region), or 3) retaining the encoded protein in a compartment of the cell, such as a hydrophobic anchor sequence or an endoplasmic reticulum retention signal;
It can also be expressed by removing the sequence. The modified antigen retains the sequences necessary for entry into the rough endoplasmic reticulum / Golgi mixture and eventual secretion from cells, but lacks the sequences required for membrane anchoring.

[0085] In another embodiment, the antigen encoded by the polynucleotide of the present invention is a cell surface protein. In some of these embodiments, the polynucleotide encoding the antigen further comprises a sequence encoding an amino acid sequence that allows the encoded protein to insert into the membrane of a cell (ie, a transmembrane region).

“Isolated” with respect to the nucleic acid sequences of the present invention, by their origin or operation;
i) does not associate with all nucleic acids that are associated in nature (eg, is present in the host cell as part of an expression vector); or (ii) associates with nucleic acids or other chemical components other than those associated with nature. Or (iii) a non-naturally occurring RNA or DNA polymer, a portion of a genomic nucleic acid, a cDNA, or a synthetic nucleic acid. "Isolated" further refers to (i) amplified in vitro, for example, by the polymerase chain reaction (PCR); (ii) synthesized, for example, by chemical synthesis; (iii) recombinantly produced by cloning Or (iv) further purified nucleic acid sequences, for example by cleavage and gel separation.

The nucleic acid sequences of the present invention can also be characterized, isolated, synthesized and purified using slightly conventional techniques. Sam mentioned above
Brook et al. , (1989).

A polynucleotide of the present invention can also serve as a primer for the detection of a gene or gene transcript expressed in an APC, eg, to confirm transduction of the polynucleotide into a host cell. It is. In this context, amplification refers to any method that uses a primer-dependent polymerase that is capable of replicating the target sequence with appropriate fidelity. Amplification is T7
DNA polymerase, E. It can also be performed by a Klenow fragment of E. coli DNA polymerase and a natural or recombinant DNA polymerase such as reverse transcriptase. The preferred length of the primer is the same as that identified for the probe described above.

The present invention relates to an isolated RNA operably linked promoter for RNA transcription and other regulatory sequences for DNA and RNA replication and / or transient and constant expression. A polynucleotide is further provided. As used herein, "operably linked"
) "Means that the promoter is arranged in a manner that directs RNA transcription from the DNA molecule. Examples of such promoters are SP6, T
4 and T7. In some embodiments, a cell-specific promoter is used for cell-specific expression of the inserted polynucleotide. A promoter or promoter / enhancer, and a cloning site that has a stop codon and a selectable marker sequence, and may also be capable of operably linking the inserted DNA fragment to the promoter. Vectors are well known in the art and are commercially available. For general methodology and cloning strategies, see GENE EXPRESSION TECHN
OLOGY (Goeddel ed., Academic Press, Inc.
. (1991)) Reconsidered references cited in the above references, as well as VECTORS
: ESSENTIAL DATA SERIES (Gacesa and Ra
mji, eds. , John Wiley & Sons, N.W. Y. (1994
See)). They include queries for maps, functional properties, commercial suppliers and GenEMBL accession numbers for a variety of suitable vectors. Preferably, these vectors are capable of transcribing RNA in vitro or in vivo.

Methods for Increasing the Presentation of Peptides on the Surface of APCs The present invention relates to methods for enhancing or increasing the presentation of peptides on the surface of antigen presenting cells, including transducing the polynucleotides of the invention into cells. To provide a way. The polypeptides of the present invention can be engineered and presented together with MHC class I molecules on antigen presenting cells (APCs), as well as the engineered MHC class I molecules on APCs.
Increased presentation of peptide antigens to CD4 + and CD8 + T cells is observed because they encode antigens presented with the I molecule.

The present invention further provides an APC produced by the method of the present invention. These APCs show enhanced presentation of peptide antigens by both MHC class I and class II molecules. The amount of these antigens loaded on the APC surface was not achieved with prior art methods. That is, in the prior art, only either class I or class II presentation is enhanced, but not both. Thus, these methods, and the APCs produced thereby, are novel and non-obvious to the prior art.

Whether the presentation of a peptide on the surface of an APC is enhanced or increased depends on the transduction of the polypeptide of the invention into it under conditions suitable for expression (ie, transcription and translation). Response of CD4 + and CD8 + T cells to the encoded peptide presented on the surface of the
Can also be determined by comparing the response of CD4 + and CD8 + T cells to the peptide when expressed on the surface of A control APC is either a nucleotide sequence that encodes an amino acid sequence that promotes retention in the endoplasmic reticulum, a nucleotide sequence that encodes an amino acid sequence that directs the encoded antigen into a non-endosomal MHC class II pathway, or both. Is an APC in which a peptide encoded by a polynucleotide lacking is present.

[0093] The method of the invention. Various methods for examining T cell activation are known. For example, Gong
et al. (1997) Gene Therapy 4: 1023-1028.
See, Activation of CD8 + T cells can be, but is not limited to, by incorporation of tritiated thymidine (an indicator of DNA synthesis) and, for example, by colony identification.
It can be detected by any known method, including examining a cell population for growth or proliferation. Alternatively, tetrazolium salt MTT (3- (4,5-
Dimethyl-tazol-2-yl) -2,5-diphenyltetrazolium bromide) may be added. Mossman (1983) J. Mol. Immunol. Method
s 65: 55-63; Niks and Otto (1990) J. Am. Immu
nol. Methods 130: 140-151. Succinate dehydrogenase, found in live mitochondria, converts MTT to formzan blue. Thus, the enriched blue color would suggest metabolically active cells. In yet another embodiment, a radiolabel, such as tritiated thymidine,
Can be analyzed as an indicator of cell proliferation. Similarly, protein synthesis can be indicated by incorporation of ³⁵ S-methionine.
Epitope-specific CTL activation may be assessed using cytotoxicity and cell killing assays, such as the classic chromium release assay. Any of a variety of methods can be used to detect CD4 ⁺ T cell activation, including, but not limited to, measuring cytokine production and proliferation, eg, by incorporation of tritiated thymidine.

Production of the Polynucleotides of the Invention Any known method for producing, replicating and expressing the isolated polynucleotides of the present invention can be used. Vectors and methods for transduction in vitro and in vivo are described briefly below and are very well known in the art. Incorporation and expression of exogenous nucleic acids is performed at RT
-PCR, Northern and Southern blotting analysis, Sambrook as described above.
et al. (1989).

[0095] The polynucleotides of the present invention can be replicated using PCR.
PCR technology is disclosed in U.S. Pat. Nos. 4,683,195, 4,800,159,
754,065, and 4,683,202, and PCR:
The POLYMERASE CHAIN REACTION (Mullis
et al. eds, Birkhauser Press, Boston (1
994)) and references cited in the literature.

Alternatively, one skilled in the art can use a sequence provided herein and a commercial DNA synthesizer to replicate DNA to replicate DNA. Thus, the present invention also provides linear sequences of polynucleotides, appropriate primer molecules, chemicals such as enzymes, and instructions for replication, and nucleotides in the proper orientation to obtain the polynucleotide. Also provided are methods for obtaining the polynucleotides of the present invention by chemically replicating or linking In a separate embodiment, these polynucleotides are further isolated. Furthermore, one of skill in the art can insert the polynucleotide into a suitable replicating vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA thus amplified can be isolated from cells by methods well known to those skilled in the art. A method for obtaining a polynucleotide according to this method, and the polynucleotide so obtained, are further provided herein.

[0097] RNA can also be obtained by first inserting a DNA polynucleotide into a suitable host cell. DNA can be inserted by any suitable method, for example, by using a suitable gene delivery vehicle (eg, liposomes, plasmids or vectors) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; the RNA is then, for example, described in Sambrook et al. (1989), it can also be isolated by using methods well known to those skilled in the art. For example, mRNA can be prepared using various lytic enzymes or chemical solutions, as described above in Sambr.
OK et al. (1989), or can be extracted using a nucleic acid binding resin according to the instructions provided by the manufacturer.

Vehicles for Gene Carriers Comprising the Polynucleotides of the Invention The present invention provides (in vivo, ex vivo or in vitro)
A delivery vehicle suitable for delivery of a polynucleotide of the invention into a cell is also provided. A polynucleotide of the invention can be included in a cloning or expression vector. Then, these vectors (especially expression vectors)
Can be manipulated to take any of a number of forms that can facilitate, for example, transport into and / or entry into cells.

[0099] Expression vectors containing these nucleic acids are useful for obtaining host vector systems for producing proteins and polypeptides. These expression vectors mean that they must be capable of replicating in the host organism either as episomes or as a complete piece of chromosomal DNA. Suitable expression vectors include plasmids, adenoviruses, adeno-associated viruses, viral vectors including retroviruses, cosmids, and the like. Adenovirus vectors are used for high-level expression and efficient cell transformation both in vitro and in vivo.
It is particularly useful for transducing a gene into a tissue in vivo. When the nucleic acid is inserted into a suitable host cell, such as a prokaryotic or eukaryotic cell, and the host cell replicates, the protein can be produced recombinantly. Suitable host cells depend on the vector and may include mammalian, animal, human, monkey, insect, yeast, and bacterial cells constructed using well-known methods. Sa described above
mbrook et al. (1989). In addition to using viral vectors for the insertion of foreign nucleic acids into cells, nucleic acids may be used to transform bacterial cells; calcium phosphate precipitation of mammalian cells; or DEAE-dextran; electroporation; or microinjection. It can also be inserted into host cells by methods well known in the art, such as transduction. See Sambrook et al., Supra, for this methodology. (1989). Thus, the invention also provides a host cell, eg, a prokaryotic cell, such as a mammalian cell, an animal cell (rat or mouse), a human cell, or a bacterial cell, comprising a polynucleotide encoding a protein or polypeptide or an antibody.

When the vector is used for gene therapy in vivo or ex vivo,
Pharmaceutically acceptable vectors, such as non-replicable retroviruses or adenoviruses, are preferred. Pharmaceutically acceptable vectors containing the nucleic acids of the invention can be further modified for transient or constant expression of the inserted polynucleotide. As used herein, the term "pharmaceutically acceptable vector" is capable of, but not limited to, selectively targeting and having the ability to introduce nucleic acids into dividing cells. Includes vector or transport vehicle.
An example of such a vector is a "non-replicable" vector, defined by its lack of ability to produce viral proteins that prevents the spread of the vector in infected host cells. An example of a non-replicable retroviral vector is LNL6.
Miller et al. (1989) BioTechniques 7: 9.
80-990. The methodology of using non-replicable retroviruses for retrovirus-related gene transfer of genetic markers is very well established. Correl
et al. (1989) PNAS USA 86: 8912; Bordign.
on (1989) PNAS USA 86: 8912-52; Culver (1
991) PNAS USA 88: 3155; and Rill (1991) Bl.
wood 79 (10): 2694-700.

In general, the genetic modification of the cells used in the present invention is achieved by an introducing vector comprising a polynucleotide comprising a sequence encoding a synthetic antigenic peptide of the present invention. A variety of different gene transfer vectors can be used, including viral and non-viral systems.

A wide variety of non-viral vehicles for delivery of a polynucleotide of the invention include:
It is known in the art and is included in the present invention. Or,
The polynucleotide of the present invention can be delivered to cells as bare DNA. WO 97/40163. Alternatively, the polynucleotides of the present invention include, but are not limited to, cationic lipids; biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; It can be delivered to relevant cells in a variety of ways using a variety of substrates (forms of delivery), including metal particles; and bacteria. The delivery vehicle may take the form of particulates. Mixtures or conjugates of these various substrates can also be used as delivery vehicles. The polynucleotides of the present invention may be non-covalently or covalently associated with these various modes of delivery.

[0103] Included in the category of non-viral vectors are prokaryotic and eukaryotic plasmids. Non-viral vectors in which the polynucleotides of the present invention have been cloned (ie, cloning and expression vectors) can be used for expression of recombinant polypeptides, as well as sources of the polynucleotides of the present invention. Cloning vectors can also be used to obtain duplicate copies of the polynucleotides they contain, or as a means of storing the polynucleotides in a repository for future recovery. Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain. They may also be used when it is preferable to express the polypeptide encoded by the operably linked polynucleotide in an individual, for example, to elicit an immune response with the polypeptide encoded in the expression vector. Can be done. Suitable cloning and expression vectors include any known in the art, for example, vectors used in bacterial, mammalian, yeast, and insect expression systems. Specific vectors and suitable host cells are known in the art, and thus need not be described in detail herein. For example, Gacesa
and Ramji, Vectors, John Wiley & Sons (19
94).

[0104] Cloning and expression vectors typically include a selectable marker, such as a gene encoding a protein necessary for the survival or growth of the host cell into which the vector is transformed, but not the most Such a marker gene may be performed in a separate polynucleotide sequence, which is introduced together into the host cell. Only those host cells into which the selection gene has been transduced will survive and / or grow under the selection conditions. Typical selection genes provide (a) resistance to antibiotics or other toxic substances such as ampicillin, neomycin, methotrexate, etc .; (b) complement auxotrophy; or (c) use from mixed media. Encoding protein, providing vital nutrients that cannot. Selection of the appropriate marker gene will depend on the host cell. And suitable genes for different hosts are known in the art. Cloning and expression vectors also typically include a replication system that is recognized by the host.

[0105] Suitable cloning vectors can be constructed according to standard techniques, and may be selected from the majority of cloning vectors available in the art.
While the cloning vector chosen will vary according to the host cell to be used, useful cloning vectors will generally have the capacity for self-renewal and will have one of the unique restriction enzymes It may possess the target and / or may carry a marker gene that can be used in the clone of choice containing the vector. Suitable examples are plasmids and bacterial viruses such as pU
C18, pUC19, Bluescript (eg, pBS SK +) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pC
R1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are Bi
Available from trading companies such as oRad, Strategene, and Invitrogen.

[0106] Expression vectors generally include a replicable polynucleotide construct that includes a polynucleotide that encodes the desired polypeptide. A polynucleotide encoding a polypeptide of interest is operably linked to a suitable transcriptional regulator, such as a promoter, enhancer, and terminator. Expression (ie,
For translation), one or more translational regulators, such as a ribosome binding site, a translation initiation site, and a stop codon, are also usually required. A polynucleotide encoding a signal peptide to allow the polypeptide encoded by the operably linked polynucleotide to cross and / or remain at the cell membrane or to be secreted from the cell An array may also be included. Numerous expression vectors suitable for expression in eukaryotic cells, including yeast, bird, and mammalian cells, are known in the art. Examples of mammalian expression vectors include both prokaryotic sequences for promoting the growth of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Examples of suitable mammalian expression vectors for transfection of eukaryotic cells include pcDNAI / amp, pcDNAI / neo, pRc / CMV, p
Includes vectors derived from SV2gpt, pSV2neo, pRSVneo, and pHyg. Alternatively, bovine papillomavirus (BPV-1) or Epstei
Viral derivatives such as the n-Barr virus (pHEB, a vector derived from pREP) can be used for expression in mammalian cells. Examples of expression vectors for the yeast system are YEP24, YIP5, Y. Useful cloning and expression vectors for transducing the construct into S. cerevisiae.
EP51, YEP52, YES2 and YRP17. Broach
t al. (1983) Experimental Manipulation
of Gene Expression, ed. M. Inouye, Acad
emic Press, p. 83. Baculovirus expression vectors for expression in insect cells include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors and pBlueBac-derived vectors.

[0107] Viral vectors include, but are not limited to, DNA virus vectors, such as adenoviruses, including herpes simplex virus, poxviruses such as vaccinia virus, and viruses based on parvovirus, including adeno-associated viruses; , RNA virus vectors, including retrovirus vectors. Retroviral vectors include mouse leukemia virus and lentivirus such as human immunodeficiency virus. Naldini et al. (1996) Science 272: 263-267.

It is also possible to use a replication defective retroviral vector harboring a polynucleotide of the invention as part of the retroviral genome. Such vectors have been described in detail. (Miller et al. (1990) Mol.
Cell Biol. 10: 4239; Kolberg, R .; (1992) J.I.
NIH Res. 4:43; Cornetta et al. (1991) Hu
m. Gene Ther 2: 215).

Adenovirus and adeno-associated virus vectors useful in the genetic modification of the present invention can also be produced according to methods already taught in the art. (See, eg, Karlsson et al. (1986) EMB.
O 5: 2377; Carter (1992) Current Opinion.
in Biotechnology 3: 533-539; Muzcyzka
(1992) Current Top. Microbiol. Immunol.
158; 97-129; GENE TARGETING: A PRATICAL
APPROACH (1992) ed. A. L. Joiner, Oxford
See University Press, NY). Several different methods are possible.

Further references describing viral vectors that can be used in the methods of the invention include: Horwitz, M .; S. , Adenoviridae
and Their Replication, in Fields, B.A. ,
et al. (Eds.) VIROLOGY, Vol. 2, Raven Pre
ss New York, pp. 1679-1721, 1990); Graha
rn, F.R. et al. Pp. 109-128 in METHODS I
NM MOLECULAR BIOLOGY, Vol. 7: GENE TRANS
FER AND EXPRESSION PROTOCOLS, Murray,
E. FIG. (Ed.), Humana Press, Clifton, N.M. J. (19
91); Miller et al. (1995) FASEB Journal
9: 190-199, Schreier (1994) Pharmaceuti.
ca Acta Helvetiae 68: 145-159; Schneid
er and French (1993) Circulation 88:19.
37-1942; Curiel et al. (1992) Human Gen
e Therapy 3: 147-154; Graham et al. , WO 95/00655 (5 January 1995); Falck-Pede
rsen WO 95/16722 (22 June 1995); Denef
le et al. WO 95/23867 (8 September 199
5); Haddada et al. WO94 / 26914 (24 Novem
ber 1994); Perricaudet et al. WO 95/02
697 (26 January 1995); Zhang et al. WO
95/25071 (12 October 1995).

The transduction efficiency of DC or other APCs may be evaluated by immunofluorescence using fluorescent antibodies specific for the tumor antigen being expressed (Kim et al.
1997) J. Amer. Immunother. 20: 276-286). Alternatively, the antibody can be conjugated to an enzyme (eg, HRP) that produces a colored product upon reaction with a substrate. The actual amount of antigenic polypeptide expressed by APC may be assessed by ELISA.

In vivo transduction of DCs, or other APCs, involves polynucleotides of the invention via different pathways, including intravenous, intramuscular, intranasal, intraperitoneal, cutaneous delivery. This can be achieved by administering a viral vector. One method that could be used is about 1 × 10 ¹⁰ -1 × 10 ¹² i.
u. Multi-site Ad vector skin delivery using the total amount of (immune units). The level of transduction in vivo can be roughly assessed by co-staining with antibodies to the expressed APC marker and peptide epitope. Staining may be performed on biopsy samples from the site of administration, or on cells from draining lymph nodes or other organs into which APCs (especially in DCs) may have metastasized. Condon et al. (1996) Nature M
ed. 2: 1122-1128; Wan et al. (1997) Human
Gene Therapy 8: 1355-1363. The amount of antigen expressed at the site of administration or other organs into which transduced APCs may have metastasized may be assessed by ELISA on tissue homogenates.

APCs may also be transduced in vitro / ex vivo by non-viral gene delivery methods such as electroporation, calcium phosphate precipitation or cationic lipid / plasmid DNA mixtures. Arthur et al. (1997) C
ancer Gene Therapy 4: 17-25. Transduced AP
C may then be administered to host cells by delivery via the intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route.

In vivo transduction of DC, or other APCs, is performed by intravenous, intramuscular, intranasal, intraperitoneal, transdermal administration of cationic lipid / plasmid DNA.
It can also potentially be achieved by administration of the mixture. Gene gun delivery or injection of bare plasmid DNA into the skin also leads to DC transduction. C
ondon et al. (1996) Nature Med. 2: 1122-
1128; Raz et al. (1994) PNAS 91: 9519-95.
23. Intramuscular delivery of plasmid DNA may also be used for immunization. Ros
ato et al. (1997) Human Gene Trapy 8:
1451-1458.

The transduction efficiency and level of transgene expression can be evaluated in a manner similar to the viral vector method described above. Host cells containing the polynucleotide of the present invention The present invention further provides host cells containing the polynucleotide of the present invention. Host cells containing a polynucleotide of the present invention are useful for recombinant replication of the polynucleotide and for recombinant production of a peptide of the present invention. In addition, host cells containing the polynucleotides of the invention can be used to induce an immune response in a subject in the methods described herein.

[0116] Suitable host cells for the recombinant replication of the polynucleotides of the invention and for the recombinant production of the peptides of the invention may be prokaryotic or eukaryotic. Host systems are known in the art and need not be described in detail herein. Host prokaryotes are bacterial cells, such as E. coli. coli, B.E. subtilis, and mycobacteria. Among host eukaryotes are yeast, insects, birds, plants, C. elg
ans (or nematodes) and mammalian cells. These cells are modified as appropriate for inducing a promoter, selecting a transformant, or amplifying a gene encoding a required sequence, and cultured in a conventional nutrient medium.

When the host cell is an antigen presenting cell, the host cell can be used to expand a population of immune effector cells, such as tumor infiltrating lymphocytes, and the immune effector cells are useful in adoptive immunotherapy. is there. Antigen presenting cells are described in more detail below.

[0118] In some of these embodiments, the isolated host cell is an APC. AP
C includes, but is not limited to, dendritic cells (DC), monocytes / macrophages, B lymphocytes or other cell types that express the required MHC / costimulatory molecules.

[0119] In some embodiments, the immune effector cells and / or APC are genetically modified. Using standard gene transfer, genes encoding costimulatory molecules and / or stimulatory cytokines can be simultaneously, prior to expansion of the immune effector cells,
Alternatively, it can be inserted later.

In certain embodiments, the host cell comprising a gene encoding a cytokine and / or costimulatory molecule includes, but is not limited to, a pulsed dendritic cell, a dendritic cell hybrid or a tree comprising an antigen presenting Foster cell. Specialized antigen presenting cells such as dendritic cells.

Antigen Presenting Cells Suitable APCs for use in the present invention are capable of presenting a foreign peptide or protein, or endogenous antigen to T cells in association with an antigen presenting molecule such as an MHC molecule. . APC includes, but is not limited to, macrophages, dendritic cells, CD
Includes 40 activated B cells, antigen-specific B cells, tumor cells, virus infected cells, and genetically modified cells.

APC can be obtained by, but not limited to, peripheral blood mononuclear cells (PBMC), whole blood or a fraction thereof including mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, leukocyte export. Cells, lymph nodes, e.g., lymph nodes drained from a tumor,
Can be obtained from a variety of sources. Suitable donors include immunized donors, non-immunized (natural) donors, treated or untreated donors. A "treated" donor is one that has been exposed to one or more biological modifiers. A "raw" donor has not been exposed to one or more biological denaturants. APC may also have been treated in vitro with one or more biological denaturants.

APCs are generally viable, but may be irradiated, treated with mitomycin C, attenuated, or chemically fixed. In addition, AP
C need not be the whole cell. Alternatively, a vesicle preparation of APC may be used.

The APC is genetically modified, ie, a recombinant polynucleotide construct that expresses a polypeptide or RNA molecule that is not normally expressed or is usually only expressed in small amounts. May be transfected. Examples of polynucleotides include, but are not limited to, an MHC molecule; a costimulatory molecule such as B7;
And polynucleotides encoding the peptides or polypeptides of the invention.

Cells that do not normally function as APCs in vivo in mammals may be modified in such a way as to function as APCs. A wide variety of cells, when appropriately modified, can also function as APCs. Examples of such cells are insect cells, such as Drosophila or Spodoptera; and Foster cells, such as the human cell line T2. For example, expression vectors that direct the synthesis of one or more antigen presenting polypeptides, such as MHC molecules, and optionally accessory molecules such as B7, are introduced into these cells, and these cellular antigen presenting molecules, and If desired, the expression of the accessory molecule or a functional part thereof on the surface may be affected. Alternatively, antigen presenting polypeptides and accessory molecules that can insert themselves into the cell membrane may be used. For example, a glycosylphosphatidylinositol (GPI) modified polypeptide can insert itself into the membrane of a cell. Hirose et al. (1995) Met
hods Enzymol. 250: 582-614; and Huang et.
al. (1994) Immunity 1: 607-613. Accessory molecules include, but are not limited to, co-stimulatory antibodies such as CD28, CD80, or CD86-specific antibodies; co-stimulatory molecules, including but not limited to B7.1 and B7.2; ICAM-
1 and adhesion molecules such as LFA-3; and surviving molecules such as Fas ligand and CD70. For example, PCT Publication No. WO97 /
See 46256.

Foster antigen presenting cells are particularly useful as APCs. Foster APC is a human cell line 174 × CEM. It is derived from T2. T2 contains a mutation in its antigen processing pathway that limits the binding of endogenous peptides to MHC class I molecules on the cell surface. Zweerlink et al. (1993) J
. immunol. 150: 1763-1771. This means that TAP1, TA
P2, LMP1, and LMP2 genes (these are MHC class I restricted CD8 +
(Necessary for antigen presentation to CTLs) resulting in a large homozygous deletion in the MHC class II region. In fact, only "empty" MHC class I molecules are displayed on the surface of these cells. Exogenous peptides added to the culture medium will bind to these MHC molecules as long as the peptide contains a binding motif allele-specifically. These T2 cells are referred to herein as "foster" APCs. They can be used to present antigen in relation to books.

Transduction of T2 cells with specific recombinant MHC alleles (alleles)
Enables re-control of HC-limited properties. Libraries prepared for recombinant alleles will not prevent efficient binding to endogenous alleles,
They will be presented preferentially.

High-level expression of MHC molecules makes APCs more visible to CTLs. By expressing the desired MHC allele in T2 cells using a strong transcription promoter (eg, the CMV promoter) (possibly with reactive MH on the cell surface)
Stronger reactive APC results (due to the higher concentration of C-peptide complex).

In such rules that affect the activation of immune effector cells, methods for determining whether antigen presenting cells are capable of presenting antigen to immune effector cells are described in the art. It is known in the art and includes, for example, ³ H-thymidine incorporation by effector cells, production of cytokines by effector cells, and a cytolytic ^51- Cr release assay.

In some embodiments, the antigenic peptides of the invention are presented to antigen presenting cells in MHC class I or class II molecules such that the peptides are bound by the TCR on CD4 + or CD8 + T cells, Presenting cells lack one or more costimulatory molecules required for T cell activation. These antigen presenting cells induce T cell anergy (reactivity) and are useful in the methods described herein for reducing or suppressing an immune response. In such a law that affects T cell anergy, a method for determining whether an antigen presenting cell is capable of presenting an antigen to an immune effector cell comprises:
It is known in the art.

The following is a brief description of two basic techniques for the isolation of APC. These approaches (1) isolate bone marrow progenitor cells (CD34 +) from blood and
Stimulating them to differentiate into C; or (2) collecting pre-committed APCs from peripheral blood. In the first approach, the method must treat patients with cytokines such as GM-CSF to boost large numbers of circulating CD34 + hepatocytes in peripheral blood.

A second approach to isolating APCs is to collect a relatively large number of precommitted APCs that already circulate in the blood. Previous techniques for isolating committed APCs from human peripheral blood include metrizamide gradients and the sticky / non-sticky step (Freudenthal et al.
(1990) PNAS 87: 7698-7702); Percoll gradient separation (Mehta-Danini et al. (1994) J Immunol.
153: 996-1003); and a fluorescence activated cell sorting technique (Thomas).
et al. (1993) J. Amer. Immunol. 151: 6840-52).

One technique for separating large numbers of cells from one another is known as countercurrent centrifugal elution (CCE). In this technique, cells are simultaneously centrifuged and exposed to being washed away by a constantly increasing flow of buffer. The constantly increasing countercurrent flow of the buffer leads to fractional cell separation based primarily on cell size.

In one aspect of the present invention, the APC is a precursor or mature dendritic cell that can be separated from a leukocyte cell fraction of a mammal such as a mouse, monkey or human (see WO 96/23060). . The white blood cells can be from peripheral blood of a mammal. The method includes the following steps. (A) supplying leukocyte cells obtained from a mammalian source by a method known in the art such as a leukocyte export method, and (b) subjecting the leukocyte cell fraction of step (a) to 4 or 4 by countercurrent centrifugal elution. Separated into further sub-fractions, (c) calcium ionophore, GM-CSF and I
Stimulating the conversion of monocyte cells to dendritic cells in one or more fractions from step (b) by contacting the cells with L-13 or GM-CSF and IL-4; Identifying the dendritic cell-enriched fraction from step (c); and (e)
The enriched fraction of step (d) is collected, preferably at about 4 ° C. One method of identifying fractions enriched in dendritic cells is by fluorescence activated cell sorting. The leukocyte cell fraction can be treated with a calcium ionophore in the presence of other cytokines such as recombinant (rh) rhIL-12, rhGM-SCF, or rhIL-4. The cells of the leukocyte fraction are washed with buffer prior to the separation step and Ca +
+ / Mg ++ may be suspended in a non-existent solvent. The leukocyte cell fraction is obtained by a leukocyte export method. Dendritic cells were labeled with the following labels (HLA-DR, HLA-DQ, or BLA).
7.2) is present and at least one of the following labels (CD3, CD1
4, CD16, 56, 57 and CD19, 20) can be identified by their non-existence. Monoclonal antibodies specific for these cell surface labels are commercially available.

More specifically, the method is derived from the leukapheresis method and is countercurrent centrifugal elution (
It requires collecting a richer collection of white blood cells and platelets further fractionated by CCE). Abrahamsen et al. (1991
J.). Clin. Apheresis. 6: 48-53. The cell sample is placed in a specific elution rotor. The rotor is then rotated at a constant speed of, for example, 3000 rpm. Once the rotor has reached the desired speed, pressurized air is used to regulate the flow rate of the cells. The cells in the elutriator are simultaneously centrifuged and exposed to being washed away by a constantly increasing flow of buffer. This results in a predominantly, but not exclusively, fractional cell separation based on differences in cell size.

The control of the properties of APCs and, more specifically, the DC collection and their effective activation in the culture medium, both monocyte and dendritic cell subpopulations, are a possible contaminant, T It relies on a simultaneous multicolor FACS analysis technique that also measures cells. It is possible that the DCs are labeled as follows: CD3 (T cells), CD14 (monocytes), CD16
, 56, 57 (NK / LAK cells) and CD19, 20 (B cells). At the same time, as DC circulate in the blood, they express large amounts of HLA-DR, significant HLA-DQ and B7.2 (but little or no B7.1) (additionally. , They express Leu M7 and M9, bone marrow markers that are also expressed by monocytes and neutrophils).

Once collected, the enriched DC / monocyte APC fraction (usually 150-190
) May be pooled and cryopreserved for later use, or immediately placed in short-term cultures.

Alternatively, others have reported methods of positively controlling (activating) dendritic cells and converting monocytes to the activated dendritic cell phenotype. The method involves that the addition of a calcium ionophore to the culture medium converts monocytes into activated dendritic cells. For example, calcium ionophore A23187 at the beginning of a culture time of 24 to 48 hours
Results in uniform activation of the pooled "monocyte + DC" fraction and phenotypic conversion to dendritic cells. Characteristically, the activated population is uniformly CD14 (Le
u M3) negative and HLA-DR, HLA-DQ, ICAM-1, B
Control 7.1 and B7.2 positively.

[0139] Specific combinations of cytokines have been effectively used to amplify (or partially replace) the activation / conversion made with calcium ionophores. These cytokines are purified or recombinant human ("rh") rhGM-CSF
, RhIL-2 and rhIL-4. Both cytokines are inappropriate for optimal positive control when given alone.

Antigen Presenting Foster Cells Antigen presenting Foster cells are dendritic cells that have been genetically modified to absorb and present foreign antigens on the cell surface from restricted binding of endogenous peptides. In some embodiments, the human cell line 174xCEM. T2 (referred to as T2) contains a mutation in the antigen processing pathway that limits the binding of endogenous peptides to cell surface type I MHC molecules (Zweerink et al. (1993) J. Immunol. 1).
50: 1763-1177), and therefore can be used in the production of facilitating antigen presenting cells. (The cell line is available from the ATCC.) This is a large isomorphism in the type II MHC region containing the TAP1, TAP2, LMP1 and LMP2 genes, which are required for antigen presentation of type I MHC restricted CD8 + CTL. This is due to a junction type defect. In fact, only "empty" type I MHC molecules are displayed on these cell surfaces. Foreign peptides added to the culture medium will bind to these MHC molecules as long as they contain a motif that binds allele-specifically. These T2 cells are referred to herein as "Foster" APCs. They can be used to present antigens in the context of the present invention.

Transduction of T2 cells with specific recombinant MHC alleles allows redirection of MHC restriction properties. Libraries prepared for recombinant alleles will be predominantly presented by them as they will prevent anchor residues from effectively binding to endogenous alleles.

Transduction of allogeneic MHC alleles of non-specialized APCs greatly assists in the development of immunity in recombinant cell lines. (Leong et al.
1994) Inter. J. Cancer 59: 212-216; Ostra
nd-Rosenberg et al. (1991) Inter. J. Can
cer Suppl 6: 61-68). It has been reported that immune development is proportional to the expression level of MHC protein. T2 cells are the ideal APC.

High expression of MHC molecules makes APCs recognizable by CTL. Desired MH in T2 cells using a strong transcription promoter (eg, CMV promoter)
Expression of the C allele results in more reactive APCs (possibly due to high concentrations of cell surface reactive MHC-peptide complexes). Since only certain types of MHC alleles are believed to be able to interact with a given library, presentation of the endogenous allele or if the library is designed to bind to newly transduced cells. It should be noted that the expression level does not seem to compromise specificity.

[0144] Hybrid cells can also present antigen, and thus are useful in the methods of the invention. Preparation of Hybrid Cells Using Dendritic Cells Hybrid cells (hybrid cells) typically retain the phenotypic characteristics of APCs.
Thus, hybrids made with dendritic cells would express similar MHC class II proteins and other cell surface markers. Further, it is believed that the hybrids express antigens that are expressed on the cells from which they were derived. Methods for making hybrids are described in WO 96/3030 and Gong et al. (199
7) Nature Medicine 3 (5): 558-561.

The population of APC is collected and separated. Preferably, the ratio of APC: antigen expressing cells is between about 1: 100 and about 1000: 1. For example, in one aspect, the fraction enriched in antigen presenting cells is then fused to APCs, preferably dendritic cells. Fusion between APC and antigen presenting cells can be performed by any suitable method, for example using polyethylene glycol (PEG) or Sendai virus. In a preferred embodiment, the hybrid cells are Gong et.
al. (1997) Nat. Med. 3 (5): 558-561.

Typically, unfused cells are expected to die after a few days in culture, so fused cells may be separated from parent cells simply by growing the culture for several days. It is possible. In this embodiment, the hybrid cells are both more viable and even only lightly attached to the tissue culture surface. The parent cell is strongly attached to the container. Thus, after about 5 to 10 days of culture, the hybrid cells can be gently removed and transferred to a new container, while the unfused cells remain attached.

Alternatively, the fused cells have been shown to lack functional hypoxanthine-guanine phosphoribosyltransferase ("HGPRT") and thus be resistant to treatment with the compound HAT. Thus, HAT can be added to the culture medium to select for these cells. However, the conventional HAT
Unlike selection, the hybrid cell medium should not be exposed to the compound for more than 12 days. The APCs described above can be analyzed for antigen presentation as described below.

[0148] In addition to their use as vaccines, they are subject to adoptive immunotherapy (adopt
It is useful to expand the immune effector cell population that can be used for live immunotherapy.

Presenting Antigens to APCs The following briefly describes various methods for expressing or presenting antigens to APCs. Paglia et al. (1996) J. Amer. Exp. Med. 183: 31
7-322 show that APCs cultured with whole proteins in vitro with shaking are recognized by MHC class I restricted CTLs, and that immunization of animals with these APCs leads to the generation of antigen-specific CTLs in vivo. Is shown. Hsu et al. (1996) Nature Medicine 2 (1
): 52-58; Celluzzi et al. (1996) J. Amer. Exp. M
ed. 183: 283-287; Mayordomo et al. (1995
) Nature Medicine 1 (12): 1297-1302; Bak
ker et al. (1995) Cancer Research 55: 5
330-5334; and Mayordomo et al. (1997) St
em Cells 15: 94-103 reported the effective use of peptide applied dendritic cells in cancer immunotherapy.

In addition, several different techniques have been described which lead to the presentation of antigen in the cytosol of APCs such as DC. These include (1) introduction of RNA isolated from tumor cells into APC, and (2) A of recombinant vector to induce endogenous expression of antigen.
PC infection, and (3) introduction of tumor antigens into DC cytosol using liposomes. (Boczkowski et al. (1996) J. Exp.
Med. 184: 465-472; Rose et al. (1994) J. Am.
Virol. 68: 5685-5689; and Nair et al. (19
92) J. Exp. Med. 175: 609-612).

[0151] Genetically modified dendritic cells and their use in immunotherapy have been described. Brossart et al. (1997) J. Am. Immunol. 3
270-3276 and Wan et al. (1997) Human Gen
e Therapy 8: 1355-1363; Gong et al. (19
97) Gene Therapy 4: 1023-1028; Kim et a
l. (1997) J. Am. Immunotherapy 20 (4): 276-28
6; and Song et al. (1997) J. Am. Exp. Med. 186 (
8): 1247-1256 reported that adenoviruses containing antigenic peptides could be used to transduce DCs, and that animals could then be administered DCs to induce an immune response. Cordon et al. (1996)
Nature Medicine 2 (10): 1122-1128 is a naked (
Genetic immunization of skin with naked DNA results in the induction of potent antigen-specific, cytotoxic T lymphocyte-mediated protective cotton. Art
hur et al. (1997) Cancer Gene Therapy
4 (1): 17-25 compare and analyze methods of gene transfer in human dendritic cells.

However, none of the above report the use of APCs to present cell surfaces and the secreted antigens for inducing an immune response as claimed herein.

Insertion of the gene (s) into the APC requires a method for producing an appropriate gene carrier vehicle and efficient gene transfer. The following are useful for in vitro and in vivo transduction of the desired gene.

Immune Effector Cells The present invention stimulates the production of an enriched population of antigen-specific immune effector cells by use of the APCs described above. Thus, the present invention provides a cell population enriched in the educated antigen-specific immune effector cells, which are specific for the antigen encoded by the polynucleotide of the present invention. In some embodiments, the antigen corresponds to an antigen on the surface of a tumor cell, and the educated antigen-specific immune effector cells of the invention inhibit tumor cell growth. When APC is used, antigen-specific immune effector cells are expanded by consuming APC, and APCs die in the medium. A method by which untreated immune effector cells become educated by other cells is described in Coulie (199).
7) Molec. Med. Today 3: 261-268.

APC prepared as described above is mixed with untreated immune effector cells. Preferably, the cells may be cultured in the presence of a cytokine, for example, IL-2. Because dendritic cells secrete potent immunostimulatory cytokines, such as IL-12, it may not be necessary to add supplemental cytokines during the initial and continuous circulation of expansion. In any case, the culture conditions are such that antigen-specific immune effect cells expand (ie, proliferate) with much higher efficiency than APCs. Multiple fusions of APC and desired cytokines may be performed to further expand the population of antigen-specific cells.

In one aspect, the immune effector cells are T cells. In another embodiment, the immune effector cells may be genetically modified, for example, by transducing a transgene encoding IL-2, IL-11 or IL-13. Methods for introducing transgenes in vitro, ex vivo and in vivo are well known in the art. Sambrook et al. (1989) See Supra.

Effector cell populations suitable for use in the methods of the invention may be autologous or allogeneic, but are preferably autologous. Effector cells are allogeneic
In this case, the cells are preferably depleted of atypically reactive cells before use. This may include, for example, mixing the allogeneic effector cells and the recipient cell population and shaking them for a suitable period of time and then depleting CD69 + cells or inactivating atypically responsive cells, or Can be performed in any known manner, including by inducing anergy in the cell population.

[0158] Hybrid immune effect cells can also be used. Hybrids of immune effectors are known in the art and have been described in various publications. See, for example, International Patent Applications WO 98/46785; and WO 95/16775.

The effector cell population is an unseparated cell, ie, a mixed population, such as a PBMC population, whole blood, etc.
May be included. Effector cell populations can be positively selected based on expression of cell surface labels, negative selection based on expression of cell surface labels, stimulated with one or more antigens in vitro or in vivo, one or more in vitro or in vivo. , Negatively stimulated with one or more antigens or biological modifiers, or manipulated by any or all combinations thereof.

The effector cells can be obtained from various sources. Sources include mixed populations, spleen cells, bone marrow cells, lymphocytic infiltrating tumor cells, cells obtained by leukapheresis, biopsy samples, lymph nodes, eg, PBMCs, including lymph nodes drained from tumor cells, whole blood, Alternatively, it includes, but is not limited to, its fraction. Suitable donors include immunized donors, unimmunized (untreated) donors, treated or untreated donors. A "treated" donor has been exposed to one or more biological modifiers. An “untreated” donor is one that has not been exposed to one or more biological modifiers.

Methods for extracting and culturing effector cells are well known. For example, effector cells can be obtained by leukapheresis, mechanical ablation therapy using a continuous efflux cell separator. For example, lymphocytes and monocytes can be separated from buffy coats by any known method, including but not limited to separation by Ficoll-Hypaque ^™ gradient, Percoll gradient separation or elution. Ficoll-
The concentration of Hypaque ^™ can be adjusted to obtain a desired population, eg, a population enriched in T cells. Other methods based on affinity are known and can be used. These include, for example, fluorescence activated cell separation (FACS), cell adhesion, magnetic bead separation, and the like. Affinity-based methods are specific for cell surface labeling and are available from a variety of commercial sources (American Type Culture Collection).
antibodies (including Manassas, MD) or a portion thereof. Affinity-based methods can alternatively utilize ligands or ligand analogs of cell surface receptors.

The effector cell population can be exposed to one or more separation protocols based on the expression of cell surface labels. For example, cells can be subjected to positive selection based on the expression of one or more cell surface polypeptides. The cell surface polypeptide is CD2, CD3
, CD4, CD8, TCR, CD45, CD45RO, CD45RA, CD11
b, CD26, CD27, CD28, CD29, CD30, CD31, CD40
L, etc. “Differentiated cluster” cell surface labeling, lymphocyte activating gene 3 product (LAG
3), Signal lymphocyte activating molecule (SLAM), other markers related to lymphocyte activation such as T1 / ST2, chemokine receptors such as CCR3, CCR4, CXCR3, CCR5, CD62L, CD44, CLA, CD146 , Α4β7, αE
Including but not limited to homing receptors such as β7, activation labels such as CD25, CD69 and OX40, and lipoglycans presented by CD1. The effector cell population can be exposed to negative selection to delete non-T cells and / or specific T cell subpopulations. Negative selection is based on various molecules (CD19
And labeled B cells such as CD20, labeled CD14 on monocytes, labeled CD56 on NK cells
Including, but not limited to. ) Can be performed based on the expression on the cell surface.

The effector cell population can be engineered by exposing one or more biologically modified genes in vivo or in vitro. Suitable biological modifiers are IL-2, IL-
4, cytokines such as IL-10, TNF-α, IL-12 and IFN-γ, phytohemagglutinin (PHA), phorbol esters such as phorbol myristate acetic acid (PMA), concanavalin-A and ionomycin Non-specific modifiers, such as, but not limited to, antibodies specific for cell surface labeling, such as anti-CD2, anti-CD3, anti-IL-2 receptor, anti-CD28, and the like, including lymphokactin. Not. Biological modifiers have the functional activity of natural factors obtained from natural sources, factors produced by recombinant DNA technology, chemically synthesized polypeptides or other molecules, or natural factors. Any derivative may be used. If one or more biological modifiers are used, the exposure can be simultaneous or sequential.

The present invention provides a composition comprising an immune effector cell, wherein the immune effector cell comprises a T cell enriched with an antigen-specific cell specific for an antigen encoded by a polynucleotide of the invention. May be. "Enriched" means that the cell population is at least about 50-fold, more preferably at least about 500-fold, and even more preferably at least about 5000-fold or more, from the original untreated cell population. That means. The proportion of the enriched cell population that encompasses the antigen-specific cells can vary substantially from less than 10% to 100% antigen-specific cells. If the cell population encompasses at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the antigen-specific immune effector cells specific for the peptide of the invention, The group is "
It is referred to as "substantially pure." The percentage that is antigen-specific is, for example, the ³ H-thymidine incorporation such that the effector cell population (eg, the T cell population) is attacked by antigen-specific cells presenting the antigen encoded by the polynucleotide of the invention. It can be easily determined by the assay.

Expanding Immune Effector Cells In the present invention, the use of the APCs described above stimulates the product of an enriched population of antigen-specific immune effector cells. Antigen-specific immune effect cells die in culture A
Increased by PC consumption. The manner in which untreated immune effector cells become educated by other cells is essentially as described in Coulie (1997) Molec. Me
d. Today 3: 261-268; Hwu et al. (1993) J,
Immunol. 150: 4104-4115; and Rosenburg
t al. (1985) N.P. Eng. J. Med. (23): 1485-149
2.

[0166] In one embodiment, the antigen-specific immune effector cell population is CD4 + and CD8
+ Wraps both T cells. In one aspect, the cytotoxic T cells (ie, CD8 + T cells) are polyclonal T cells isolated from the site of cytotoxic T cell infiltration of a subject. Or,
Such cells may be isolated from the site of cytotoxic T cell infiltration in two or more subjects or human patients, where the subjects share an MHC haplotype. In other embodiments, the CTL may be more than one cytotoxic T cell line. In yet another aspect, the CTL may be any combination of the foregoing.

In a further aspect of the invention, the site of cytotoxic T cell infiltration is a tumor.
The tumor from which the cell or cell line is obtained can be a homologous tumor of a different individual sharing the MHC haplotype or an atypical tumor from a different subject sharing the MHC haplotype.

Alternatively, CTL infiltration can be from viral infections, autoimmune inflammation, sites of transplant rejection, inflammation or similar sites of lymphocyte / leukocyte infiltration. APC prepared as described above is mixed with untreated immune effector cells. Preferably, the cells may 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 add supplemental cytokines during the initial and continuous circulation of expansion. In any case, the culture conditions are such that the antigen-specific immune effect cells expand (ie, proliferate) with much higher efficiency than APCs. Multiple fusions of hybrid and desired cytokines can be made to further expand the population of antigen-specific cells.

In one embodiment, the immune effector cells are T cells and are specific for a tumor-specific antigen presented by APC. The polypeptide antigen of the present invention The present invention further provides a peptide antibody encoded by the polynucleotide of the present invention. Peptide antigens encoded by the polynucleotides of the present invention can be produced by any known method. An isolated peptide of the invention may be synthesized using a suitable solid phase synthesis procedure. Steward and Young,
Solid Phase Peptide Synthesis, Freema
ntle, San Francisco, Calif. (1968). The preferred method is the Merrifield method. Merrifield, Recent Pr
ogreess in Horne Res, 23: 451 (1967). The antigenic activity of these peptides can be conveniently tested, for example, using an assay as described herein.

Once the isolated peptides of the present invention are obtained, standard techniques including chromatography (eg, ion exchange, affinity, and size column chromatography), centrifugation, differential solubility, or protein purification It can be purified by any other standard technique. For immunoaffinity chromatography, the antigenic determinant is separated by binding it to an affinity column containing the antibody created for the peptide or related peptide of the invention and attached to a constant support. Can be done.

Alternatively, hexa-His (Invitrogen), maltose binding domain (New England Biolabs), influenza coat sequence (
Kolodziej et al. (1991) Methods Enzymo
l. 194: 508-509) and glutathione-S-transferase may be added to the peptides of the invention so that they can be easily purified by passing through an appropriate affinity column. A DNA affinity column using DNA containing a sequence encoding the peptide of the present invention can be used for purification.

[0172] The isolated peptide can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography. Also included in the scope of the present invention are, for example, phosphorylation, glycosylation, cross-linking,
An antigenic peptide that is differentially modified during or after translation by acylation, proteolytic cleavage, binding to an antibody molecule, membrane molecule or other ligand. . (Ferg
uson et al. (1998) Ann. Rev .. Biochem. 57:
285-320) Antigen Specificity Assay The immune effector cells described herein are selected for their ability to alter the immune response. In one aspect, the immune effector cells have both an ability to lyse cells expressing a specific antigen and an ability to increase a humoral response to the antigen,
Selected for. The cell lysing ability of a cell is measured by a variety of methods, including but not limited to tritiated thymidine incorporation (indicating DNA synthesis) and investigation of populations of growth or proliferation, eg, by colony identification. (See WO 94/21287). In another embodiment, the tetrazolium salt MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide) can be added (Mossm).
an (1983) J. Am. Immunol. Methods 65: 55-63; Ni
ks and Otto (1990) J. Am. Immunol. Methods13
0: 140-151). Succinate dehydrogenase, found in mitochondria of living cells, converts MTT to formazan blue. Therefore, it is considered that the concentrated blue color indicates metabolically active cells. Similarly, protein synthesis can be indicated by incorporation of ³⁵ S-methionine. In still other embodiments,
Cytotoxicity and cell killing assays, such as the classic chromium release assay, can be employed to assess epitopic-specific CCTL activity. Other suitable assays will be known in the art. Stimulation of a humoral response to an antigen can be measured by an antigen-specific product using any known method, including ELISA and LIA.

Compositions of the Invention The present invention relates to peptides, polypeptides, polynucleotides, host cells, antigen presenting cells, immune effectors, vectors, antibodies and fragments thereof as described above, acceptable solid or liquid carriers. A composition comprising any of the following is provided. When the compositions are used pharmaceutically, they are combined with a "pharmaceutically acceptable carrier" for diagnostic and therapeutic uses. These compositions can also be used in the preparation of a medicament for the diagnostic and immunomodulatory methods of the present invention.

Methods of Using the Polynucleotides, Antigen Presenting Cells and Immune Cells of the Invention The present invention uses the polynucleotides of the invention and host cells (including APCs and educated immune cells), ie, immunomodulators. Diagnostic and immunomodulatory methods are provided.

Diagnostic Methods The present invention provides diagnostic methods using the polynucleotides and APCs of the present invention. The method can be used to detect the presence of antigen-specific CD4 + and / or CD8 + that binds to an antigen encoded by a polynucleotide of the invention.

The diagnostic methods of the present invention include: (1) an assay that predicts the efficacy of the antigen encoded by the polynucleotide of the present invention; (2) a precursor of an immune effector cell specific to the antigen encoded by the polynucleotide of the present invention and / or its natural counterpart. Assay to determine frequency (ie presence and number), and (3)
An assay for determining the potency of an antigen encoded by a polynucleotide of the invention, once used in an immunomodulatory method of the invention.

The diagnostic methods of the invention generally involve, under appropriate conditions, and antigens encoded by the polynucleotides of the invention (usually presented by the APCs of the invention) and CD4 + or CD8 + T cells, etc. The effect is carried out for a time sufficient to allow specific binding to take place with the immune effector molecule such as TCR on the surface of the cell. "Suitable conditions" and "sufficient time" are generally those conditions and times appropriate for specific binding. Suitable conditions occur between about 4 ° C and about 40 ° C, preferably between about 4 ° C and about 37 ° C, in a buffered solution, and in a pH range between 5 and 9. Various buffered solutions are known in the art and can be used in the diagnostic methods of the invention, and include, but are not limited to, phosphate buffered saline. Not limited. Sufficient time for binding and reaction will generally be between about 1 second and about 24 hours after exposing the sample to the antigen encoded by the polynucleotide of the present invention.

In one aspect, the invention provides a diagnostic assay for predicting the efficacy of an antigen encoded by a polynucleotide of the invention, presented by an APC of the invention. In any of these embodiments, the defined T cell antigenic determinants can be used to clinically characterize tumors and viral pathogens and determine the predicted efficacy of in vivo vaccine trials in advance. This is the defined T
It can be accomplished by a simple proliferation assay of peripheral blood mononuclear cells of patients using cell determinants as stimulants. The peptide that elicits the response is a potential vaccine candidate for the patient.

In another embodiment, the frequency of precursors of resting (untreated) immune effector cells that have the potential to be specific and activated for the antigen encoded by the polynucleotide of the present invention Such assays for determining (ie, presence and number) are provided. In these embodiments, the presence of an immune effector cell that specifically binds to a natural antigenic determinant in a biological sample is determined using antigen-presenting cells having an antigen encoded by the polynucleotide of the present invention on the surface thereof. Used to detect. Functional assays are used to determine (and quantify) antigen-specific immune effector cells. Illustratively, PBMCs are separated from tumor-bearing objects. These PBMC samples are co-cultured with tumor cells from the same subject for an appropriate time. A second sample of these PBMCs is cultured for an appropriate period of time with a surrogate APC pulsed with an antigen encoded by a polynucleotide of the invention corresponding to a natural antigenic determinant expressed on the surface of the tumor. Both tumor cells and surrogate APC are loaded with 51Cr. By comparing the amount of 51Cr released from tumor cells and surrogate APCs pulsed with the antigen, the frequency of tumor-specific immune effector cell precursors and the immune effector cells specific for the antigen encoded by the polynucleotide It is possible to determine the frequency of the precursor. Functional assays include, but are not limited to, immune effect cell proliferation, cytokine production, specific lysis of APCs.

In another aspect, the efficacy of an immunomodulatory method, including an immunomodulatory method of the invention, modulates an immune response to an antigen encoded by a polynucleotide of the invention and / or its natural counterpart. In some cases, it can be tested using the diagnostic assays of the present invention. These diagnostic assays are also useful for assessing or measuring the efficacy of an immunotherapeutic agent. In any of these embodiments, the method can be an activated CD4 + or CD8 + T cell, which is activated or anergized as a result of exposure to an antigen encoded by a polynucleotide of the invention. Allows the detection of certain immune effector cells. A sample containing cells from the subject can be tested for the presence of CD4 + or CD8 + T cells that have been activated or anergized as a result of binding to an antigen encoded by a polynucleotide of the invention. In one embodiment, the method includes the following steps. (A) Immobilized APC presenting the antigen encoded by the polypeptide of the present invention on its surface by binding to type I and type II MHC molecules
Contacting the biological sample under suitable conditions and for a time sufficient to allow the binding of immune effect cells having an antigen receptor specific for the peptide on its surface, thereby allowing antigen-specific immunization Fixing the effector cells and (b) contacting the immobilized immune effector cells with a detectably labeled molecule such as an antibody that specifically binds to the immune effector cells. In another embodiment, the method comprises the following steps.
(A) an immobilized antigen-specific matrix presenting the antigen encoded by the polypeptide of the invention bound to its surface with type I and type II MHC molecules, under suitable conditions and peptide-specific Contacting the biological sample with a biological sample for a time sufficient to allow binding of immune effector cells having a unique antigen receptor on its surface, thereby immobilizing antigen-specific immune effector cells; and (b) Performing a functional assay on fixed immune effector cells. Once the immune effector cells present on their surface the antigen encoded by the polypeptide of the invention, the immobilized A
Once bound to PC, it can be labeled based on characteristic cell surface molecules, including but not limited to CD4, CD8, and cell surface labels specific for activated T cells It is. Various cell surface labels specific for immune effector cell populations are known in the art and have been described in numerous publications.
For example, The Leukocyte Antigen Facts Book
Barclay et al. eds. , 1995, Academic Pr
See ess. Antibodies with these labels are commercially available, inter alia, from Beckman Coulter. Fixed immune effector cells can also be characterized by the presence of mRNA and / or protein in the cytosol, which is characteristic of a given T cell in a given state of activation or anergy. Characteristic mRNAs include (but are not limited to) the polymerase chain reaction
It can be detected by any known method. The antibody detectably labeled on the cell surface label can be contacted with the immobilized immune effector cells under appropriate conditions and for a time sufficient to allow specific binding. If necessary or desirable, the labeled cells can be physically removed from the unbound label, and excess unbound label can be inactivated. The requirement for an antibody specific for cell surface labeling on immune effect cells is that the antibody binds specifically and that the antibody interferes with the binding between the TCR and the immobilized synthetic antigenic peptide antigenic determinant. It is not.

Labels that can be used are known to those skilled in the art and include, but are not limited to, conventional labeling substances such as phosphors, radioisotopes, chromophores, and magnetic particles. The enzyme label may be luciferase, eg, Aequorea victo
green fluorescent protein (GFP), such as GFP from ria or any of the many GFPs known in the art, β-galactosidase, chloramphenicol acetyltransferase. For example, Current Protocols in Molecular Biolo
gy (FM Ausubel et al., eds., 1987, and periodic revisions). Any test that detects the label, directly or indirectly, is suitable for use in the present invention. Tests include colorimetric, fluorimetric or luminescent, radioimmunoassays or other immunological tests.

Immunomodulatory Methods The present invention provides methods for modulating an immune response in an individual to an antigen encoded by a polypeptide of the present invention, ie, the corresponding natural epitope. The immunomodulation methods of the present invention include methods of inducing or increasing an immune response in a subject, and methods of suppressing or decreasing the immune response, wherein the subject is provided with an effective amount of a polynucleotide or APC of the present invention or an immune effector cell. And administration under conditions or conditions that produce the desired effect on the immune response (or lack thereof) to the peptide antigen. The immunomodulation methods of the invention include vaccine methods, adoptive immunotherapy, and methods of inducing T cell unresponsiveness or anergy.

Various methods for assessing T cell activation are known. CTL activation may be performed by any known method, including but not limited to tritiated thymidine incorporation (an indicator of DNA synthesis), and number testing for population growth or growth, such as, for example, colony identification. Can be detected by Alternatively, tetrazolium salt MTT (3- (4,5-dimethyl-2-tazolyl) -2,5-diphenyl
Tetrazolium bromide) can be added. Mossman (1983)
J. Immunol. Methods 65: 55-63; Niks and
Otto (1990) Immunol. Methods 130: 1
40-151. Succinate dehydrogenase, found in the mitochondria of living cells, converts MTT to formazan blue. Thus, a concentrated blue color may indicate metabolically active cells. In yet another embodiment, incorporation of a radiolabel, such as, for example, tritiated thymidine, can be tested to indicate cell proliferation. Similarly, protein synthesis may be indicated by incorporation of ³⁵ S-methionine. In yet another embodiment, cytotoxicity and cell killing tests, such as the classic chromium release test, can be used to assess epitope-specific CTL activation.
Any of a variety of methods can be used to detect activation of CD4 + T cells, including, but not limited to, measuring cytokine production and proliferation, for example, by incorporation of tritiated thymidine.

The release of ⁵¹ Cr from labeled target cells is a standard test that can be used to estimate the number of peptide-specific CTL in a biological sample. Tumor cells or APCs of the invention were radiolabeled with approximately 200 μCi of Na ₂ ⁵¹ CrO 4 at 37 ° C. for 60 minutes, followed by washing, as were the targets. T cells and target cells (
１1 × 10 4 / well) were combined in a 96-well U-bottom plate with various effectors, target ratios. Plates were centrifuged at 100 × g for 5 minutes, cell contact was initiated and left for 4-16 hours at 37 ° C. with 5% CO ₂ . The release of ⁵¹ Cr in the supernatant was determined and the absence of T cells (negative control) or 0.1% TRI
Compared to targets left with TON ^™ X-100 (positive control). See, eg, Mishell and Shigi, eds. Selected Metho
ds in Cellular Immunology (1980) H.
See Freeman and Co.

The formulation of the APC of the present invention may vary depending on the desired result. In general, peptides presented on APCs by class I and class II MHC molecules with appropriate costimulatory molecules can result in the induction of an immune response to the peptide. The anergic (or non-reactive) state in T lymphocytes can be induced by the presentation of antigen by the APCs of the present invention containing the appropriate MHC molecules on their surface but without the appropriate costimulatory molecules.

A polynucleotide of the present invention can be administered in a gene transfer vehicle or inserted into a host cell, and the recombinantly encoded polypeptide can be transcribed, translated, and processed. Isolated cells comprising a polynucleotide of the present invention in a pharmaceutically acceptable carrier can be combined with an appropriate and effective amount of an adjuvant, cytokine, or costimulatory molecule for effective vaccination. . In some embodiments, the host cell can be an APC, such as a dendritic cell. Host cells can be further modified by inserting a polynucleotide that encodes an appropriate amount of either or both of the cytokine and the costimulatory molecule.

The methods of the present invention can be further modified by co-administering an effective amount of a cytokine or costimulatory molecule to the subject. An agent provided herein as effective for its intended purpose may be a subject having a disease to be treated by an immunomodulatory vesicle of the invention or a subject at risk of developing or at risk of developing such a disease. It can be administered to an individual. When the reagent is administered to a subject, such as a mouse, rat, or human patient, the reagent can be added to a pharmaceutically acceptable carrier and administered systemically or locally to the subject. Clinical dosages are empirically determined and may vary with the pathology or condition being treated, the subject being treated, and the efficiency and harm of the treatment.

The amount of the polynucleotide, or APC, or immune effector cell of the present invention will depend, in part, on the intended effect, and may ultimately vary at the discretion of the medical or veterinary practitioner. Factors to consider are the condition being treated, the route of administration,
And the nature of the composition, body weight, body surface, age, and general condition of the mammal and the particular peptide being administered. Once the cells have been administered, an observation of a clinical response such as disease symptoms or a decrease in tumor mass is made. Administration can be repeated, for example, monthly or as appropriate. Those of skill in the art can agree that appropriate dosing treatment will be at the discretion of the physician or veterinary practice.

In vivo administration can be effected at certain doses, either continuously or intermittently during treatment. The most effective methods of administration and determination of dosage are well known to those skilled in the art,
And the subject to be treated. Single or multiple administrations can be carried out depending upon the dose and the method selected by the treating physician. Appropriate dosage formulations and methods of administering the drug may be set forth below.

The medicaments and compositions of the present invention may be used in the manufacture of a medicament, for the treatment of humans and other animals, by administration with conventional means, such as the active ingredient in a pharmaceutical composition. Can be used.

In particular, the agents of the present invention, also referred to herein as active ingredients, may be administered nasally, topically (dermal, aerosol, buccal, and sublingual).
)), Parenterally (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary, by any suitable route. The desired route may vary depending on the condition and age of the recipient, and the disease or condition being treated. Vaccines for Cancer Treatment and Prevention In one embodiment, the immunomodulatory methods of the invention include a vaccine for cancer treatment.
Cancer cells contain many new antigens that can potentially be recognized by the immune system. Given the rate at which epitopes were identified, TILs were isolated from patients with solid tumors, their MHC restriction was determined, and their CTLs against the appropriate library for reactive epitopes were examined and affected. Individual anti-cancer vaccines can be made for the given individuals. These vaccines can be both a treatment for affected individuals and a prophylactic treatment for recurrence (or the occurrence of disease in patients with a familial genetic risk for the disease). In many cases, the immunogenicity of high affinity peptides suggests that holes in the functional T cell repertoire are relatively rare, if any, and thus elicit a tumor antigen-specific CTL response. If not, vaccination of individuals who have never had cancer is expected to be successful as a preventive treatment (Sette et al. (1994) J.
. Immunol. , 153: 5586-5592). In mice, vaccination with the appropriate epitope not only removes established tumors, but also protects against tumor recurrence after otherwise lethal amounts of tumor cells are given. (Bystryn et al. (1993) Supra. Vaccine Against Tumors Caused by Pathogenic Organisms) The polynucleotides and APCs of the invention are also useful for inducing (or increasing or enhancing) an immune response against pathogenic organisms. Include pathogenic viruses, bacteria, and protozoa.

[0192] Viral infection is an ideal candidate for immunotherapy. Immunological responses to viral pathogens may be ineffective, as in the case of lentiviruses such as HIV, which cause AIDS. The high frequency of spontaneous mutations allows these viruses to escape the immune system. However, the saturating nature of CTL epitopes displayed on infected cells has identified antigens conserved between different polymorphisms in essential genes that are very intolerant to mutations, making a more efficient vaccine Enable design.

Adoptive Immunotherapy The expanded population of antigen-specific immune effect cells and the APCs of the invention find use in adoptive immunotherapy and as a vaccine.

Applied immunotherapies, in one aspect, comprise the production of educated, antigen-specific immune effector cells, produced by culturing untreated immune effector cells with the APCs described above, in a substantially pureer manner. Administering a population to a subject. In some embodiments, A
PC is a dendritic cell.

In one embodiment, the adoptive immunotherapy described herein is autologous. In this case, an APC is created using parental cells isolated from a single subject. The expanded population also uses T cells isolated from the subject. Finally, the expanded population of antigen-specific cells is administered to the same patient.

In a further aspect, the APC or immune effector cells are administered with an effective amount of a stimulatory cytokine or costimulatory molecule such as IL-2. Methods for Inducing T Cell Anergy The synthetic antigenic peptide epitopes of the invention are useful in methods for inducing T cell inactivity or anergy. Disorders that can be treated using these methods include autoimmune disorders, allergies, and allogeneic transplant rejection.

Autoimmune damage is a disease in which the body's immune system responds to its own tissues. These include arthritis, ulcerative colitis, and multiple sclerosis. Polynucleotides of the invention that encode antigens corresponding to endogenous factors that are recognized as foreign can be used in the development of therapy using gene therapy or other techniques.

For example, synthetic CTL epitopes that can serve as “suicide substrates” for CTLs that mediate autoimmunity can be designed as described above. In a way, peptides with high affinity for MHC alleles, but which cannot activate the TCR, can effectively mask the cellular immune response against cells presenting the antigen in question. As an aid to this approach, the long latency period of the HIV virus is due to the antiviral immune response, and the mechanism by which the virus eventually escapes the immune system is occupied by MHC molecules.
It is believed to induce T cell anergy by creating epitopes that do not stimulate the CR lytic response. Klennerman et al.
(1995) Eur, J .; Immunol. , 25: 1627-19.
31.

In vivo stimulation of T cells in the absence of other signals via the T cell-antigen receptor complex and CD3 alone induces T cell anergy or paralysis. Activation of T cells, as measured by interleukin-2 production and proliferation in vitro, is responsible for the generation of antigenic and costimulatory signals caused by cell-cell interactions between antigen-specific T cells and antigen-timed cells. You need both. Interaction of CD58 (LFA-3) protein of these CD2 proteins and antigen presenting cells on the surface of T cells, C of CD11a / CD18 (LFA-1) protein
Interaction with D54 (ICAM-1) protein and C5 of CD5 protein
Various interactions, such as interactions with the D72 protein, are such in vivo
tro may be provided. Cytokines from antigen presenting cells (eg, interleukin-1 and interleukin-6) can also provide a costimulatory signal that results in T cell activation in vitro. Transport of both antigenic and costimulatory signals results in stable transcription of the interleukin-2 gene and other central T cell activation genes. The aforementioned costimulatory signals are dependent on protein kinase C and calcium. Potential antigen presenting cells express CD80 (B7 and BB1) and other related surface proteins, and many T cells express B7 binding proteins such as CD28 and CDLA-4. CD28 and CDLA
Binding of CD80 by -4 stimulates a costimulatory pathway that is independent of protein kinase C and calcium, leading to active T cell proliferation. B cell stimulation also depends on the interaction between specific antigens and cell surface immunoglobulins. T cell-derived cytokines (eg, interleukins 1 and 4), physical contact of T cells with B cells through specific pairs of specific receptors and co-receptors, or therapies, Provides the signal or signals required for stimulation.

Conventional routes of administration are used. An immunotherapeutic antigen according to the invention in an amount that stimulates T cells or produces anergy (or a therapeutically effective amount as described above)-
The superantigen multimer is contacted with a target cell. "T cell anergy producing amount" intends an amount effective to produce a statistically significant inhibition of TCR mediated cellular activity. This can be analyzed in vitro using a T cell activation test. Typically, T cell anergy or activation is tested by incorporation of tritiated thymidine in response to a specific antigen.

One way in which T cell anergy can be induced is to present APCs that present antigen in MHC class I or class II molecules but lack the costimulatory molecules necessary to activate T cells. It is to present to. For example, cells that have been transformed by an MHC antigen for which the T cell clone has been defined, and that are otherwise normal antigen presenting cells (APCs), may be used. Appropriate peptides recognized by resting T cells in the context of MHC transformed into cellular hosts other than APCs are provided to resting T cells. Α and β of class II or MHC class I molecules
MHC is expressed as a result of the introduction of a mammalian cell other than an antigen presenting cell that has a gene that constantly expresses the chain together with the invariant chain. Importantly, these cells
MHC and peptides do not provide other proteins, either cell surface proteins or secreted proteins associated with antigen presenting cells, that together provide a costimulatory signal.

To determine that anergy is being induced, the T cells tested are
In MHC class I and class II with costimulatory molecules required for cell activation,
The cells may be cultured with APCs presenting the antigen encoded by the polynucleotide of the present invention. Cultures were incubated for 48 hours, pulsed with tritiated thymidine, and uptake was measured after about 18 hours. Antigen-presenting cells that do not stimulate T cells, because they use MHC that is not restricted or that use peptides that are insensitive, are better than controls when presented to T cells. No uptake indicates no activation. IL-2
, -3, or -4, eg, cell surface proteins associated with activation such as CD71, or other conventional tests to determine the degree of activation, such as other convenient techniques. Can be used. Another method is to determine the expression of proteins that are expressed on quiescent T cells but not on anergic T cells. U.S. Patent No. 5,747,299.

Adoptive Immunotherapy The cells and compositions of the present invention are useful as cancer vaccines and in adoptive immunotherapy. The ability of dendritic cells pulsed with autologous antigen to induce a clinically relevant immune response has been previously reported by Hsu et al. (1996)
Nature Med. 2 (1): 52-58. Using this clinical study as a guide, it is possible to administer to a subject an effective amount of APC to induce an anti-tumor immune response, as described herein. Following isolation and purification of dendritic cells (day 0), purified and pulsed dendritic cells were administered by subcutaneous injection on days 2, 28 and 56 and 5 to 6 months later. On day 16, the patient received a subcutaneous injection of keyhole limpet hemocyanin or idiotype protein in saline at a site separate from the site of intravenous injection of pulsed dendritic cells.

[0204] The expanded population of antigen-specific immune effect cells of the invention can be used in applied immunotherapy.
And find use as a vaccine. Adoptive immunotherapy, in one aspect, comprises a substantially pure population of educated antigen-specific immune effectors created by culturing untreated immune effectors with APCs as described above, Administering to a subject. Preferably, the APC is a dendritic cell.

[0205] In one embodiment, the adoptive immunotherapy described herein is autologous. In this case, APCs are made using parental cells isolated from a single subject. The expanded population also uses T cells isolated from the subject. Finally, the expanded population of antigen-specific cells is administered to the same patient.

[0206] In another aspect, the adoptive therapy is performed in an allogeneic manner. Here, cells from two or more patients are used to generate APCs to stimulate the production of immune effector cells. For example, if T cells and / or dendritic cells from a subject providing in-house biotechnology are not available, cells from other healthy or diseased subjects may be used to generate antigen-specific cells. Can be The expanded population can be administered to any of the subjects from which the cells were isolated, or to a completely different subject.

In a further embodiment, the APC or immune effector cell is an effective amount of, eg, I
Administered with a stimulatory cytokine or costimulatory molecule such as L-2. An agent identified herein as effective for its intended purpose can be administered to a subject having a tumor or to an individual who is or is at risk of developing a tumor.
When the drug is administered to a subject, such as a mouse, rat, or human patient, the drug can be added to a pharmaceutically acceptable carrier and administered to the subject, either systemically or locally. Tumor regression can be examined to determine if beneficial treatment can be provided to the patient. The amount for treatment is empirically determined and may vary depending on the pathology being treated, the subject being treated and the usefulness and toxicity of the treatment. When given to animals, this method is useful for further confirming the efficacy of the drug.

In vivo administration can be effected at one dose, continuously or intermittently throughout the treatment. The most effective methods and methods for determining dosage are well known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the target cell being treated and the subject being treated. obtain. Single or multiple administrations can be carried out with the dose and pattern being selected by the treating physician. Appropriate dosage formulations and methods of administering the drug can be found below.

[0209] Epitopes or wild-type antigenic peptides corresponding to proteins that have not yet been identified are also within the scope of the invention. A common strategy to look for tumor antigens is to isolate tumor-specific T cells and try to identify the antigen recognized by these cells. In cancer patients, lymphocytic infiltration existing at the tumor site
Specific CTL is obtained. Weidmann, et al. , Supra
. These TILs are the only population of cells that can be traced to the site of disease if labeled with indium and transported properly. Alternatively, large libraries for virtual antigens can be created and tested. "Phage method" (Scott
and Smith (1990) Science 249: 386-39.
0: Cwirla et al. (1990) Proc. Natl. A
cad. Sci. 87: 6387-6382: Devlin et al.
. (1990) Science 249: 404-406), and very large libraries can be constructed. Another method is, for example, the Geysen method (G
eyesen et al. (1986) Mol. Immunol. 23:
709-715; Geysen et al. (1987) Immun
ol. Method 102: 259-274) and Fodor et a.
l. (1991) Uses mainly chemical methods, such as the method of Science 251: 767-773. Furka et al. (1988) 14th
Inter. Cong. Bio. Vol. 5, Abst. FR: 01
3; Furka (1991) Inter. J. Peptide Pro
tein Res. 37: 487-493), Houghton (U.S. Patent No. 4,683,211, issued December 1986) and Rutter et al.
. (US Patent No. 5,010,175, issued April 23, 1991) describes a method of making a mixture of peptides that can be tested as agonists or antagonists.

[0210] In a further aspect of the present invention, a solid phase epitope recovery method (Solid-PHa
se Epitope Recovery, “SPHERE” PCT WO97
/ 35035) can be used to identify tumor agents. Briefly, SPHERE can be used to create a library of molecules, preferably peptides, and identify antigens by connecting one type of molecule to a support via a dissociable junction. . At least a portion of the molecule bound to the respective support can be dissociated, and it can be determined whether the peptide is recognized by the antigen-specific immune effector cells.

Thus, the invention also provides a search to screen for novel wild-type antigens that can be further modified and used to induce cellular and humoral immune responses in a subject. The antigen in vitro or in vivo and its biological activity are positive controls. Using the methods described herein, the biological activity of the isolated antigen and its secreted form can be compared for its biological activity.

[0212] The present invention further relates to c-encoding such proteins by making degenerate oligonucleotide probes or primers based on the sequence of the epitope.
Methods for cloning DNA or genomic DNA are provided. By this method and the method of detecting sequences in a sample using methods such as Northern analysis, Southern analysis and PCR, nucleic acids and carriers such as pharmaceutically acceptable carriers, solid supports or detectable labels. Further provided is a composition comprising:

[0213] Therapeutic and diagnostic oligopeptide sequences determined by the foregoing methods are further provided by the present invention. This method and Western analysis, ELISA
Oligopeptide sequences and carriers, such as pharmaceutically acceptable carriers, solid supports or detection by methods of detecting oligopeptide sequences in a sample using methods such as Harlow and Lane (1989), supra. Compositions comprising possible labels are further provided.

The following examples are provided to illustrate, but not limit, the present invention.

[0215]

【Example】

Identification of tumor-associated antigens See, eg, Kawakami et al. (1994) Proc. Natl.
Acad. Sci. Any conventional method can be used to identify novel tumor-associated antigens, such as the expression cloning method described in 91: 3515-19. Briefly, in this method, after a library of cDNAs corresponding to mRNA from tumor cells is cloned into an expression vector and transformed into target cells, the target cells are incubated with cytotoxic T cells. . CT
A single cDNA sequence capable of stimulating CTL through serial dilution and re-examination of the less complex cDNA pool, identifying a cDNA pool capable of stimulating L and thus encoding a syngeneic tumor antigen Can be obtained.

SAGE (US Pat. No. 5,695,937) and SPHERE (PCT)
WO 97/35035) can also be used to identify hypothetical antigens in use in the present invention.

SAGE to identify antigens recognized by expanded immune effector cells, such as CTL, by identifying nucleotide sequences expressed on antigen presenting cells
Analysis can be used. Briefly, SAGE analysis comprises (1) antigen-expressing population and (2)
1.) Begin by providing complementary deoxyribonucleic acid (cDNA) from a population of cells that do not express the antigen. Both cDNAs can be ligated at the primer site. Then, for example, an appropriate sequence tag is created using an appropriate primer for amplifying DNA. By measuring the difference in the set of these tags between the two cell types, sequences that are abnormally expressed in the antigen-expressing cell population can be identified.

Another method that can be used to identify antigenic epitopes is PCT WO9
7/35035 (Solid PHase E)
(Pitope Recovery, SPHERE) method. Briefly, the peptide library is loaded onto beads and inserted into a 96-well plate. A plate with 1000 beads per well can contain 10 ⁶ beads, and 10 96-well plates with 100 beads per well can contain 10 ⁵ beads. In order to minimize the amount of CTL cells and manual work required per search, the eluted peptides can be further pooled to obtain wells of any desired complexity. For example, based on experiments with a soluble library, 2 × 10 6
In 96-well plates containing only CTL cells (per plate of 10,000 peptides), it is possible to search 10 ⁷ peptide. After dissociating a percentage of the peptides from the beads, gamma-irradiated APC or Foster AP
C, and incubated with the cloned CTL line, positive wells are determined by ³ H-thymidine incorporation. Alternatively, as noted above, cytokine production or cytolytic 51Cr release assays may be used (C
outic et al. (1992) Int. J. Cancer, 50 '
289-291). Beads from each positive well are separated and individually tested using an additional percentage of peptides obtained from each bead. The individual positive beads are then decoded and the reactive amino acid sequence is identified.
Analysis of all positive clones can give a partial depiction of the conserved substituted epitope stimulating the CTL clone tested. At this time, the peptide can be resynthesized and retested. To enumerate the full nature of derivatives for which a particular CTL is tolerant, a second library (with minimal complexity) expressing all conserved substitutions can also be synthesized. Simultaneous CT (with the same MHC constraint)
Screening L allows the search for cross-reacting epitopes.

Alternatively, previously described antigens, which are mutant proteins of the antigen and allogeneic and heterologous antigens, are useful in the present invention. MART-1 and gp100 are pigment cell differentiation antigens that are specifically recognized by HLA-A2 restricted tumor-infiltrating lymphocytes (TILs) from melanoma patients and appear to be involved in tumor regression ( Kawakami et al. (1994) Proc.Natl.Aca.
d. Sci. U. S. A. 91: 6458-62; Kawakami et a
l. (1994) Proc. Natl. Acad. Sci. U. S. A. 91:
91: 3515-9). In recent years, mouse homologous genes of human MART-1 have been isolated. The full length open reading frame of mouse MART-1 is 342
It encodes a 113 amino acid residue protein consisting of base pairs and having a predicted molecular weight of about 13 KDa. 68. Comparison of amino acid sequences of human and mouse MART-1.
It showed 6% identity.

In another embodiment, the described method for identifying CD8 + MHC class I restricted CTL epitopes comprises CD4 + MHC class II restricted helper T cells (Th
) It can be applied to epitope identification. In this case, an MHC class II allele-specific library is synthesized such that haplotype-specific anchor residues appear at appropriate locations. MHC class II agretope motifs have been identified for common alleles (Rammensee (1995) Curr. Opin. Imm.
unol. 7: 85-96; Altuvia et al. (1994) Mo
l. Immunol. 24: 375-379; Ray et al. (
1994) J. Immunol. 152: 3946-3957; Verre
ck et al. (1994) Eur. J. Immunol. 24:
375-379; Sinigaglia and Hammer (1994
) Curr. Opin. Immunol. 6: 52-56; Rotz
Schke and Falk (1994) Curr. Opin. Im
munol. 6: 45-51). The overall length of the peptide can be 12-20 amino acid residues, and methods described previously can be used to limit the complexity of the library. The screening process is identical to that described for MHC class I-related epitopes, except that a B lymphoblastoma cell line (B-LCL) rather than T2 cells is used for antigen presentation. In a desirable aspect, there is a defect in antigen processing (Mellins et al. (1991) J.
. Exp. Med. 174: 1607-1615) The previously described B-LCL is used, which allows for specific presentation of exogenously added antigen. The library is searched for reactivity with CD4 + MHC class II allele-specific Th cells. See Mellins, et al. supra. Reactivity can be measured by incorporation of 3H-thymidine according to the method of, or by any of the methods previously described for MHC class I-related epitope retrieval.

The in vitro confirmation of the immunogenicity of the hypothetical antigens of the invention can be confirmed using the following method of testing for CTL production. However, weakly immunogenic antigens can also be used in making the compositions and methods of the invention.

Using these sequences and the recombinant expression techniques summarized below, polypeptides and proteins can be made for display on APCs of the invention. Tumor Protection in Animal Models The transcription cassette shown in the figures is contained in an adenovirus vector such that expression of the gene results in intracellular and secretory production of the same protein. Zhai
et al. (1996) Immunol. 156: 700-1
0 for direct immunization as described in
as et al. (1997) Cancer Research 57: 2
As described in 865-9, antigen-presenting cells (preferably dendritic cells) derived from the host have been transformed with an adenovirus vector and are genetically modified A
The adenovirus vector can be used ex vivo, where the PC is subsequently injected back into the host. Adenoviral vectors encoding the same wild-type and secreted antigens are injected subcutaneously into C57BL / 6 mice at two sites (one per site).
. 5E) IU), immunization in vivo is performed. Two weeks later, the lethal dose (
Subcutaneous injections of 2 × 10 ⁴ ) mouse B16F10 melanoma tumor cells were performed on the animals. Animals are measured for survival and tumor measurements are made. For ex vivo dendritic cell therapy, bone marrow cells were collected from C57BL / 6 mice and GM-
The cells are cultured in vitro with CSF and IL4 to obtain dendritic cells. Dendritic cells were infected overnight with the adenovirus vector at an MOI of 500,
The next day (5 × 10 ⁵ ) dendritic cells are washed and injected into the recipient C57BL / 6 mice via tail vein injection. Two weeks later, animals are injected subcutaneously with a lethal dose of mouse B16F10 melanoma tumor cells as described above, and tumor size and viability are recorded as a function of time.

While the present invention has been described in connection with the above embodiments, it is understood that the foregoing description and the following examples are intended to be illustrative, but not limiting, of the scope of the present invention. Should be. For example, any of the above compositions and / or methods may also include:
Known treatments and compositions can be combined. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[Brief description of the drawings]

FIG. 1 is an outline of one embodiment of the polynucleotide of the present invention. The figure shows a single transcription cassette encoding the intracellular and secreted forms of the same antigen. This polynucleotide can be inserted into a replication defective adenovirus vector that lacks the E1, E3 and E4 regions. Suitable helper cell lines are used for the production of infectious virus. Alternatively, a helper plasmid or helper virus may be infected with the viral vector such that an infectious, replication-defective virus capable of expressing the polynucleotide is produced.

[Procedure amendment]

[Submission date] April 3, 2001 (2001.4.3)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/04 C12N 1/21 4H045 C07K 5/00 15/00 ZNAA C12N 1/21 A61K 37/02 5 / 06 C12N 5/00 EF term (reference) 4B024 AA01 BA31 CA01 DA02 DA05 DA11 EA02 EA04 GA11 GA18 GA19 4B065 AA01X AA57X AA87X AB01 BA01 CA24 CA45 4C084 AA13 BA44 CA18 CA36 DA01 MA02 NA14 ZB022 ZB212 ZB01 MA0 MA0 MA05 NA14 ZB02 ZB21 ZB26 ZC75 4C087 AA01 BB34 BB43 CA04 NA14 ZB02 ZB21 ZB26 4H045 AA10 BA13

Claims (37)

[Claims] 1. A polynucleotide encoding an antigen that is processed and presented on an antigen presenting cell (APC) together with an MHC class I molecule, as well as an antigen that is processed and presented on an APC together with an MHC class II molecule. 2. A (a) first coding sequence comprising an antigen-encoding nucleotide sequence; and (b) an antigen-encoding nucleotide sequence and an endoplasmic reticulum of the encoded antigen (
2. The polynucleotide of claim 1, comprising a second coding sequence comprising a nucleotide sequence that encodes an amino acid sequence that facilitates retention in ER). 3. The amino acid sequence that promotes maintenance of the encoded antigen in the endoplasmic reticulum (ER) is represented by K
DEL, HDEL, DDEL, ADEL, SDEL, RDEL, KEEL, QE
3. The polynucleotide of claim 2, wherein the polynucleotide is selected from the group consisting of DL, HIEL, HTEL, and KQDL. 4. The polynucleotide of claim 2, further comprising a third coding sequence comprising a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that directs the encoded antigen to a non-endosomal MHC class II pathway. . 5. The polynucleotide of claim 2, wherein the antigen encoded by the first coding sequence is a secreted antigen. 6. The polynucleotide of claim 2, wherein the antigen encoded by the first coding sequence is a cell surface antigen. 7. The polynucleotide of claim 1, wherein the antigen is a tumor-associated antigen. 8. The tumor-associated antigen may be gp100, MUC-1, MART-1, HER-2, CE.
A, PSA, prostate-specific membrane antigen (PSMA), tyrosinase, tyrosinase-related protein 1 or 2 (TRP-1 and TRP-2), NY-ESO-1 and GA733 antigens. Polynucleotide. 9. A vehicle for gene delivery comprising the polynucleotide of claim 1. 10. A host cell comprising the polynucleotide of claim 1. 11. The host cell of claim 10, wherein the cell is an antigen presenting cell (APC). 12. The host cell of claim 11, wherein the APC is a dendritic cell. 13. A composition comprising the polynucleotide of claim 1 and a carrier. 14. A polypeptide encoded by the polynucleotide of claim 1. 15. A composition comprising a polynucleotide encoded by the polynucleotide of claim 1 and a carrier. 16. A method for expressing the polynucleotide of claim 1, comprising growing a cell under conditions suitable for expression of the polynucleotide of claim 1. 17. A method for increasing the presentation of a peptide to the surface of an antigen-presenting cell, comprising introducing the polynucleotide into a cell under conditions suitable for expression of the polynucleotide of claim 1. 18. A cell produced by the method of claim 17. 19. A method of producing an educated population of antigen-specific immune effectors comprising culturing untreated immune effectors with antigen-presenting cells transduced by the polynucleotide of claim 1. 20. A population of educated antigen-specific immune effector cells produced by the method of claim 19. 21. A method of inducing an immune response to an antigen in a subject, comprising administering to the subject an effective amount of the polynucleotide of claim 1 under conditions that induce an immune response to the antigen stomach. 22. The method of claim 21, further comprising administering to the subject an effective amount of a cytokine. 23. The method of claim 22, wherein the subject is administered a gene encoding a cytokine. 24. The method of claim 21, further comprising administering to the subject an effective amount of a costimulatory molecule. 25. The method of claim 24, wherein the gene encoding a costimulatory molecule is administered to the subject. 26. The method of claim 21, wherein the antigen is a tumor-associated antigen. 27. gp100, MUC-1, MART-1, HER-2, CEA, PSA, prostate specific membrane antigen (PSMA), tyrosinase, tyrosinase-related protein 1 or 2 (TRP-1 and TRP-2), 27. The method of claim 26, wherein a tumor-associated antigen is selected from the group comprising NY-ESO-1 and GA733 antigens. 28. A method of inducing an immune response against a natural antigen in a subject, comprising administering to the subject an effective amount of the host cell of claim 11 under conditions that induce an immune response to the antigen. 29. The method of claim 28, further comprising administering to the subject an effective amount of a cytokine. 30. The method of claim 28, wherein the subject is administered a gene encoding a cytokine. 31. The method of claim 28, further comprising administering to the subject an effective amount of a costimulatory molecule. 32. The method of claim 21, wherein the subject is administered a gene encoding a costimulatory molecule. 33. The method of claim 28, wherein the unprocessed antigen is a tumor-associated antigen. 34. gp100, MUC-1, MART-1, HER-2, CEA, PSA, prostate specific membrane antigen (PSMA), tyrosinase, tyrosinase-related protein 1
Or 2 (TRP-1 and TRP-2), NY-ESO-1 and GA733
34. The method of claim 33, wherein a tumor-associated antigen is selected from a group comprising the antigen. 35. The method of claim 28, wherein the host cell further expresses the cytokine. 36. The method of claim 28, wherein the host cell further expresses a costimulatory molecule. 37. A method of adoptive immunotherapy comprising administering to an individual an effective amount of a population of the educated antigen-specific immune effect cells of claim 20.