CA2322659A1 - Compositions and methods for gene-based vaccines to provoke t cell responses - Google Patents

Abstract

This invention provides a polynucleotide encoding an antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC) and an antigen that is processed and presented with an MHC Class II molecule on the APC. Compositions containing these polynucleotides are further provided by this invention. Methods of increasing presentation of a peptide on the surface of an APC, and APCs produced by the methods, are further provided.
Also provided are diagnostic and immunomodulatory methods using polynucleotides, APCs, and immune effector cells of the invention.

Description

COMPOSITIONS AND METHODS FOR GENE-BASED
VACCINES TO PROVOKE T CELL RESPONSES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. ~ 119(e) to U.S.
Provisional Patent Application 60/078,725, filed March 20, 1998, the contents of which are hereby incorporated by reference into the present disclosure.
TECHNICAL FIELD
This invention is in the field of molecular immunology and medicine. In particular, the present invention provides compositions and methods for inducing CD4 and CD8 T cell responses in a subject.
BACKGROUND OF THE INVENTION
The mammalian immune system is capable of generating responses to foreign antigens, to self antigens present on cancerous cells, and to self antigens present on normal tissues. The immune system comprises two types of antigen-specific cells: B cells and T cells. B cells synthesize both membrane-bound and secreted antibody. T cells can be characterized phenotypically by the manner in which they recognize antigen, by their cell surface markers, and by their secreted products. T cells express distinctive membrane molecules. Included among these are the T cell antigen receptor (TCR), which appears on the cell surface in association with CD3; and accessory molecules such as CDS, CD28 and CD45R.
Subpopulations of T cells can be distinguished by the presence of additional membrane molecules. Thus, for example, T cells that express CD4 recognize antigen associated with class II MHC molecules and generally function as helper cells whose roles include enhancement of antibody production by B cells, while T

cells that express CD8 recognize antigen associated with class I MHC molecules and generally function as cytotoxic cells.
Immune cells recognize discrete sites, known as epitopes or antigenic determinants, on the antigen. Epitopes are regions of an immunogen or antigen that bind to antigen-specific membrane-bound receptors on immune cells or to their soluble counterparts, such as antibodies. Both membrane-bound antibody on the surface of a B lymphocyte and secreted antibody recognize soluble antigen.
Unlike B cells, which recognize soluble antigen, T cells recognize antigen only when the antigen is associated with self major histocompatibility complex (MHC) gene products on the surface of an antigen presenting cell. This antigen can be displayed together with MHC molecules on the surface of antigen-presenting cells or on virus-infected cells, cancer cells, and grafts.
Disease states can result from invasion by a pathogenic organisms, including bacterial, viral, and protozoan pathogens, and subsequent inefficient or ineffective immune response to the invader. Disease states can also result from the activation of self reactive T lymphocytes, from the activation of T
lymphocytes that provoke allergic reactions, or from the activation of autoreactive T lymphocytes following certain bacterial and parasitic infections, which can produce antigens that mimic human protein, rendering these protein "autoantigens". These diseases include, but are not limited to, the autoimmune diseases. autoimmune disorders that occur as a secondary event to infection with certain bacteria or parasites, T cell mediated allergies, and certain skin diseases such as psoriasis and vasculitis. Furthermore, undesired rejection of a foreign antigen can result in graft rejection or even infertility, and such rejection may be due to activation of specific T lymphocyte populations.
Foreign antigens include macromolecules associated with pathogens such as bacteria, viruses. and protozoans; allergens; and allografts.
Self antigens, under normal physiological conditions, are usually non-immunogenic. However, self antigens can also be immunogenic, as is the case with autoimmune diseases. Autoimmune diseases affect approximately 5% of adults in Europe and North America, often causing chronic debilitating illnesses.

2 Steinman (1993) ScientifrcAmerican 269:107-114. Autoimmunity is characterized by activation of auto-reactive clones of T or B cells, generating humoral or cell-mediated responses against self antigens.
Tumor cell antigens are also self antigens, and frequently do not elicit an S immune response that results in elimination of the cancerous cells effective to control or eliminate the disease.
In addition, there are other, specific situations in which induction of an immune response to a seif antigen is desirable. These include the induction of an immune response to certain antigens as a means of contraception.
The introduction into an animal of an antigen has been widely used for the purposes of modulating the immune response, or lack thereof, to the antigen for a variety of purposes. These include vaccination against pathogens, induction of an immune response to a cancerous cell, reduction of an allergic response, reduction of an immune response to a self antigen that occurs as a result of an autoimmune disorder, reduction of allograft rejection, and induction of an immune response to a self antigen for the purpose of contraception.
A critical target of vaccines is the specialized professional antigen-presenting cell ("APC"), the most immunologically powerful of which is the bone marrow-derived dendritic cell ("DC"). Presentation of antigenic peptides on the surface of APCs in the context of MHC class I molecules leads to a cellular response while presentation of antigenic peptides by MHC class II molecules provokes a humoral response. Arca et al. (1997) J. Immunol. 20(2):138-45, examined the interactions of CD4+ and CD8+ T cells involved in the immune response to a poorly immunogenic tumor transduced to secrete GM-CSF. The authors report CD4+ and CD8+ cells are independently sensitized during tumor growth and both have functional capacity as effector cells in adoptive transfer.
Both CD4+ and CD8+ T cells were induced concurrently against a poorly immunogenic tumor.
In general, all nucleated cells have the capacity to present endogenously produced antigen (including, for example, self antigens as well as viral antigens) on MHC Class I molecules. Antigen-presenting cells, however, have the capacity to present endogenously produced antigen on MHC Class I molecules, and soluble exogenous (i.e., extracellular) antigens on both MHC CIass I and MHC Class II
molecules, but they generally present soluble exogenous antigens on Class I
molecules relatively inefficiently. Much has been learned in recent years about S antigen presenting pathways for both endogenously produced antigen and soluble exogenous antigen. Extracellular antigens are internalized by antigen-presenting cells and are processed in endocytic vesicles, where they encounter and bind to MHC Class II molecules. In all nucleated cells, viral and cellular proteins synthesized within the cell are hydrolyzed by proteasomes in the cytoplasm into peptides, some of which are transported by transporters associated with antigen processing (TAPs) into the endoplasmic reticulum, where they encounter and bind MHC Class I molecules. Raychaudhuri and Rock (1998) Nature 16:1025-1031;
and Lindauer et al. (1998) 3. Mol. Med. 76:32-47.
It would be beneficial to utilize both pathways, i.e, MHC Class I and Class II presenting pathways, in the same antigen-presenting cell, to modulate a humoral and a cellular immune response in a subject against a given antigen.
This invention provides this and related benefits which have, until this invention, remained unrealized.
DISCLOSURE OF THE INVENTION
This invention provides a polynucleotide encoding first antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC), and a second coding sequence encoding a second antigen that is processed and presented with an MHC Class II molecule on the APC. The first and second antigens may or may not have the same amino acid sequence. In some embodiments, a polynucleotide of the invention may comprise: (a) a first coding sequence which comprises a nucleotide sequence encoding an antigen; and (b) a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER).

In other embodiments, a polynucieotide of the invention may comprise: (a) a first coding sequence which comprises a nucleotide sequence encoding an antigen; (b) a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER);
and (c) a third coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that directs (or promotes retention of, or targets) the encoded antigen into a non-endosomal MHC Class II pathway.
In some of these embodiments, the encoded antigen is a naturally occurring antigen or fragment of a naturally occurring antigen. In other embodiments, the antigen is a synthetic antigen. An encoded antigen can be secreted or a cell-surface protein.
The invention further provides methods of increasing antigen presentation on the surface of an APC, comprising introducing a polynucleotide of the invention into the APC under conditions which favor expression of the polynucleotide. The invention also provides APCs produced by these methods.
These APCs have enhanced presentation of antigen encoded by a polynucleotide of the invention in both Class I and Class II MHC molecules.
Polynucleotides of the invention are useful in a variety of methods of modulating an immune response to the antigen thereby encoded. In some ... . embodiments, the polynucleotides of the invention encode tumor antigens, or synthetic antigens corresponding to tumor antigens, and are thus useful as vaccines against tumor cells expressing cell surface tumor antigen. In other embodiments, the polynucleotides of the invention encode self antigens on normal (i.e., non-cancerous) tissues. In other embodiments, the polynucleotides of the invention encode foreign (non-self] antigens, such as those associated with organisms such as pathogenic bacteria, viruses and protozoans. In still other embodiments, the polynucleotides of the invention encode antigens present on allografts which mediate their rejection.

The invention further provides polypeptides encoded by polynucleotides of the invention, and compositions comprising the polypeptides. The polypeptides are useful in immunomodulatory methods of the invention.
Compositions containing the polynucleotides are further provided by this invention. The polynucleotides can be contained in gene delivery vehicles such as viral vectors and liposomes, and in host cells. These polynucleotides are useful diagnostically and therapeutically, as well as in methods of expressing the polynucleotide for reproduction, expression and purification of the antigenic products.
The polynucleotides can be used to modulate an immune response to an antigen in a subject. Accordingly, the invention further provides methods for modulating a cellular and/or a humoral immune response to the antigen in a subject by administering to the subject an effective amount of the polynucleotide.
The polynucleotide can be delivered as naked DNA or in a gene delivery vehicle.
In one aspect, a host cell containing the polynucleotide is administered to the subject.
BRIEF DESCRIPTION OF THE FIGURE
The figure is a schematic of one embodiment of the polynucleotide of this invention. The figure shows a single transcription cassette that encodes intracellular and secreted forms of the same antigen. This polynucleotide can be . . .. . . .. . incorporated into a replication defective adenoviral vector deleted in the El, E3 and/E4 regions. A suitable helper cell line is utilized to produce infectious virions. Alternatively, a helper plasmid or helper virus can be co-transfected with the viral vector so that infectious, replication defective virions capable of expressing the polynucleotide are produced.
MODES FOR CARRYING OUT THE INVENTION
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present 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 bioiogy, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A
LABORATORY MANUAL, 2"d edition (1989}; CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (1987) and updates); the series METHODS IN ENZYMOLOGY (Academic Press. Inc.): PCR 2: A PRACTICAL
APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. ( 1995)}, Harlow and Lane eds. (1989) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL
CULTURE (R.I. Freshney ed. (1987)).
As used herein, certain terms may have the following defined meanings.
As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.
For example, the term "a cell" includes a plurality of cells, including mixtures thereof.
The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their .. . . analogs. Nucleotides may have any three-dimensional structure, and may perform .
any function, known or unknown. The term "polynucleotide" includes, for example, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
A
nucleic acid molecule may also comprise modified nucleic acid molecules.
Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.

The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6 X SSC to about 10 X SSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 6 X SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C
to about 50°C; buffer concentrations of about 9 X SSC to about 2 X SSC;
formamide concentrations of about 30% to about 50%; and wash solutions of about 5 X SSC
to about 2 X SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about 1 X
SSC to about 0.1 X SSC; formamide concentrations of about 55% to about 75%;
and wash solutions of about 1 X SSC, 0.1 X SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCI and 15 mM citrate buffer. It is understood that equivalents of SSC
. . . , using other buffer systems can be employed. _ , , . . .. .
A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none;
strand =
both; cutoff= 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE: Databases = non-redundant. GenBank +
EMBL + DDBJ + pDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address:
http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.
As used herein, the term "classical MHC Class I processing pathway"
intends that a protein is processed within a proteasome, transported to the ER, where it associates with an MHC Class I molecule.
As used herein, the term "an endoplasmic reticulum retention sequence"
intends an amino acid sequence which, when in the same translation unit with an amino acid sequence of a peptide antigen, increases the probability that the antigen will be located in the endoplasmic reticulum (ER) of a eukaryotic cell, compared with the same antigen not in the same translation unit with the amino acid sequence. "Increases the probability that the antigen will be located in the ER" intends that a given antigen which is in the same translation unit with an amino acid sequence of an amino acid sequence which promotes retention in the ER will be found in the ER at levels at least about 2-fold, more preferably at least about 5-fold, even more preferably at least about 10-fold or higher that the same antigen which is in a translation unit lacking the amino acid sequence which .promotes retention in the ER. Methods of determining,whether an antigen is in ."
the endoplasmic reticulum are known in the art and include, but are not limited to, a process involving disrupting the cell, fractionating the subcellular components, and determining in which subcellular compartment the protein is located by, e.g., immunoassay.
As used herein, the term "classical MHC Class II processing pathway"
intends that a protein is taken up by an antigen-presenting cell by endocytosis or phagocytosis and the protein is processed in an endosome, before associating with MHC Class II molecules. This is also referred to herein as "processing by the endosomal pathway". In the present invention, the terms "endosome", "endocytic compartment", "endocytic vesicle", and "endosomal compartment' are used interchangeably.
As used herein, the term "an amino acid sequence that promotes processing of a protein by a non-endosomal MHC Class II pathway", is an amino acid sequence which, when in the same translation unit with an amino acid sequence of a peptide antigen, increases the probability that the antigen will be located in a subcellular compartment of a eukaryotic cell which is nat an endosome, and where processing of the protein and subsequent association with MHC CIass II molecules can occur, compared with the same antigen not in the same translation unit with the amino acid sequence. "Increases the probability"
intends that a given antigen which is in the same translation unit with an amino acid sequence of an amino acid sequence which promotes retention in a non-endosomal subcellular compartment where processing and subsequence association with MHC Class II molecules can occur, will be found in such a subcellular compartment at levels at least about 2-fold, more preferably at least about 5-fold, even more preferably at least about 10-fold or higher that the same antigen which is in a translation unit lacking the amino acid sequence which promotes retention in such a subcellular compartment. Non-endosomal subcellular compartments where processing and subsequence association with MHC Class II molecules can occur include, but are not limited to. lysosomes, Golgi, and melanosomes. Methods to determine whether a protein is in a given subcellular compartment are described above.
The term "antigen" is well understood in the art and includes substances which are immunogenic, i.e., immunogens, as well as substances which induce immunological unresponsiveness, or anergy, i.e., anergens. An antigen may comprise one or more antigenic determinants, or epitopes. In the description of the present invention, the terms "antigen", "antigenic determinant'', and "epitope"
are used interchangeably. As used herein, the term "antigen" includes naturally occurring antigens, synthetic antigens, foreign antigens, self antigens, modified self antigens, and altered antigens.

A ''native" or "natural" antigen is a polypeptide, protein or a fragment which contains an epitope, which has been isolated from a natural biological source, and which can specifically bind to an antigen receptor, in particular a T
cell antigen receptor (TCR), in a subject.
A synthetic antigen is said to "correspond" to a native epitope if the peptide binds to the same TCR as the natural epitope.
The term "peptide", used interchangeably herein with "polypeptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester. ether. etc. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L
optical isomers. and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
The term "sequence motif' refers to a pattern present in a group of molecules (e.g., amino acids or nucleotides). A typical pattern may be identified by characteristic amino acid residues, such as hydrophobic, hydrophilic, basic, acidic. and the like.
A "signal sequence" is a short amino acid sequence that directs newly synthesized secretory or membrane proteins to and through cellular membranes, including, but not limited to, the endoplasmic reticulum, and an endosomal compartment (or endosome). Signal sequences can be in the amino-terminal (N-terminal), and/or the carboxy-terminal (C-terminal) portion of a polypeptide and are generally cleaved after the polypeptide has crossed the membrane.
A ''gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides;
artificial viral envelopes; metal particles; and bacteria, viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
The term "antigen-presenting matrix", as used herein, intends a molecule or molecules which can present antigen in such a way that the antigen can be bound by a T-cell antigen receptor on the surface of a T cell. An antigen-presenting matrix can be on the surface of an antigen-presenting cell (APC), on a vesicle preparation of an APC, or can be in the form of a synthetic matrix on a solid support such as a bead or a plate. An example of a synthetic antigen-presenting matrix is purified MHC class I molecules complexed to /32-microglobulin, or purified MHC Class II molecules, or functional portions thereof, attached to a solid support.
The term "antigen presenting cell", as used herein, intends any cell which presents on its surface an antigen in association with a major histocompatibility complex molecule, or portion thereof, or, alternatively, one or more non-classical MHC molecules, or a portion thereof. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen presenting cells. Methods of making hybrid APCs have been described. see, for example, International Patent Application No. WO 98/46785; and WO 95/16775.
Dendritic cells (DCs) are potent antigen-presenting cells. It has been shown that DCs provide all the signals required for T cell activation and proliferation. These signals can be categorized into two types. The first type, which gives specificity to the immune response, is mediated through interaction between the T-cell receptor/CD3 ("TCR/CD3") complex and an antigenic peptide presented by a major histocompatibility complex ("MHC") class I or II protein on the surface of APCs. This interaction is necessary, but not sufficient, for T
cell activation to occur. In fact, without the second type of signals, the first type of signals can result in T cell anergy. The second type of signals, called co-stimulatory signals, is neither antigen-specific nor MHC-restricted, and can lead to a full proliferation response of T cells and induction of T cell effector functions in the presence of the first type of signals. As used herein, "dendritic cell"
is to include. but not be limited to a pulsed dendritic cell, a foster cell or a dendritic cell hybrid.
A "foster antigen presenting cell" is a genetically modified dendritic cell that, from the restricted association of endogeneous peptides, will take up and present exogeneous antigen on the cell surface.
The terms "major histocompatibility complex" or "MHC" refers to a complex of genes encoding cell-surface molecules that are required for antigen presentation to T cells and for rapid graft rejection. In humans, the MHC
complex is also known as the HLA complex. The proteins encoded by the MHC complex are known as "MHC molecules" and are classified into class I and class II MHC
molecules. Class I MHC molecules include membrane heterodimeric proteins made up of an a chain encoded in the MHC associated noncovalently with ~i2-microglobulin. Class I MHC molecules are expressed by nearly 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 include membrane heterodimeric proteins consisting of noncovalently associated I and ~ chains. Class II MHC are known to be present on CD4+ T cells and, in humans, include HLA-DP, -DQ, and DR. The term "MHC restriction" refers to a characteristic of T cells that permits them to recognize antigen only after it is processed and the resulting antigenic peptides are displayed in association with either a class I or class lI MHC molecule.
Methods of identifying and comparing MHC are well known in the art and are described in Allen et al. (1994) Human Imm. 40:25-32; Santamaria et al. (1993) Human Imm.
37:39-50 and HurIey et al. (1997) Tissue Antigens 50:401-415.
"Co-stimulatory molecules" are involved in the interaction between receptor-ligand pairs expressed on the surface of antigen presenting cells and T
cells. Research accumulated over the past several years has demonstrated convincingly that resting T cells require at least two signals for induction of cytokine gene expression and proliferation (Schwartz, R.H. ( 1990) Science 248:1349-1356; Jenkins, M.K. (1992) Immunol. Today 13:69-73). One signal, the one that confers specificity, can be produced by interaction of the TCR/CD3 complex with an appropriate MHC/peptide complex. The second signal is not antigen specific and is termed the "co-stimulatory" signal. This signal was originally defined as an activity provided by bone-marrow-derived accessory cells such as macrophages and dendritic cells, the so called "professional" APCs.
Several molecules have been shown to enhance co-stimulatory activity. These are heat stable antigen (HSA) (Liu, Y., et al. (1992) J. Exp. Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas, M.F., et al.
(1993) Cel174:257-268), intracellular adhesion molecule 1 (ICAM-1) (Van Seventer, G.A. ( 1990) J. Immunol. 144:4579-4586), B7-l, and B7-2/B70 (Schwartz, R.H. ( 1992) Cell 71:1065-1068). These molecules each appear to assist co-stimulation by interacting with their cognate ligands on the T
cells. Co-stimulatory molecules mediate co-stimulatory signals) which are necessary, under normal physiological conditions, to achieve full activation of naive T
cells.
One exemplary receptor-ligand pair is the B7 co-stimulatory molecule on the surface of APCs and its counter-receptor CD28 or CTLA-4 on T cells (Freeman et al. ( 1993 ) Science 262: 909-911; Young et al . ( 1992) J. CI in. Invest. 90:
229;
Nabavi et al. (1992) Nature 360:266-268). Other important co-stimulatory molecules are CD40, CD54, CD80, CD86. The term "co-stimulatory molecule"
encompasses any single molecule or combination of molecules which, when acting together with a peptide/MHC complex bound by a TCR on the surface of a T cell, provides a co-stimulatory effect which achieves activation of the T
cell that binds the peptide. The term thus encompasses B7, or other co-stimulatory molecules) on an antigen-presenting matrix such as an APC, fragments thereof (alone. complexed with another molecule(s), or as part of a fusion protein) which, together with peptide/MHC complex, binds to a cognate ligand and results in activation of the T cell when the TCR on the surface of the T cell specifically binds the peptide. Co-stimulatory molecules are commercially available from a variety of sources, including, for example, Beckman Coulter. It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified co-stimulatory molecules (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.
As used herein, "solid phase support'' or "solid support", used interchangeably, is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art.
Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels. As used herein, "solid support"
also includes synthetic antigen-presenting matrices, cells, and liposomes. A
suitable solid phase support may be selected on the basis of desired end use and suitability for various protocols. For example, for peptide synthesis, solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE~ resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene 1 S resin grafted with polyethylene glycol (TentaGel~, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California).
The term "genetically modified" means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous nucleotides.
As used herein, the term "cytokine" refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation. Non-limiting examples of cytokines which may be 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-I alpha (IL-II ), interleukin-11 (IL-I I), MIP-lI , leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The present invention also includes culture conditions in which one or more cytokine is specifically excluded from the medium. Cytokines are commerciaily available from several vendors such as, for example, Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D
Systems and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purif ed cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.
"Vector" means a self replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and/or between host cells. The term is intended to include vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication vectors that function primarily for the replication of nucleic acid and expression vectors that function for transcription and/or translation of the DNA or RNA. Also intended are vectors that provide more than one of the above functions.
"PCR primers" refer to primers used in "polymerase chain reaction" or "PCR," a method for amplifying a DNA base sequence using a heat-stable polymerase such as Taq polymerase, and two oligonucleotide primers, one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (- )-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce exponential and highly specific amplification of the desired sequence. (See, e.g., PCR 2: A PRACTICAL APPROACH, supra). PCR also can be used to detect the existence of the defined sequence in a DNA sample.
"Host cell" is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous nucleic acid molecules, polynucleotides and/or proteins. It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
The cells may be procaryotic or eucaryotic, and include but are not limited to bacterial cells, yeast cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.

An "antibody" is an immunoglobulin molecule capable of binding an antigen. As used herein, the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity.
An "antibody complex" is the combination of antibody (as defined above) and its binding partner or ligand.
The term "immunomodulatory agent", as used herein, is a molecule, a macromolecular complex, or a cell that modulates an immune response and encompasses an antigenic peptide of the invention alone or in any of a variety of formulations described herein; a polypeptide comprising an antigenic peptide of the invention; a polynucleotide encoding a peptide or polypeptide of the invention; an antigenic peptide of the invention bound to a Class I or a Class II
MHC molecule on an antigen-presenting matrix, including an APC and a synthetic antigen-presenting matrix (in the presence or absence of co-stimulatory molecule(s)); an antigenic peptide of the invention covalently or non-covalently complexed to another molecules) or macromolecular structure; and an educated, antigen-specific immune effector cell which is specific for a peptide of the invention.
The term ''modulate an immune response'' includes inducing (increasing, eliciting) an immune response; and reducing (suppressing) an immune response.
An immunomodulatory method (or protocol) is one that modulates an immune response in a subject.
As used herein, the term "inducing an immune response in a subject" is a term well understood in the art and intends that an increase of at least about fold, more preferably at least about 5-fold, more preferably at least about 10-fold, more preferably at least about 100-fold, even more preferably at least about fold, even more preferably at least about 1000-fold or more in an immune response to an antigen (or epitope) can be detected (measured), after introducing the antigen (or epitope) into the subject, relative to the immune response (if any) before introduction of the antigen (or epitope) into the subject. An immune response to an antigen (or epitope), includes, but is not limited to, production of an antigen-specific (or epitope-specific) antibody, and production of an immune cell expressing on its surface a molecule which specifically binds to an antigen (or epitope). Methods of determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art. For example, antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, wherein, for example, binding of an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig antibody). Immune effector cells specific for the antigen can be detected any of a variety of assays known to those skilled in the art, including, but not limited to, FACS, or. in the case of CTLs, '~Cr-release assays, or 3H-thymidine uptake assays.
The term "immune effector cells" refers to cells capable of binding an antigen or which mediate an immune response. These cells include, but are not limited to. T cells, B cells. monocytes, macrophages, NK cells and cytotoxic T
lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates. Certain diseased tissue expresses specific antigens and CTLs specific for these antigens have been identified. For example, approximately 80% of melanomas express the antigen known as gp100.
The term "immune effector molecule", as used herein, refers to molecules capable of antigen-specific binding, and includes antibodies, T cell antigen receptors, and MHC Class I and Class II molecules.
A "naive" immune effector cell is an immune effector cell that has never been exposed to an antigen.
As used herein, the term ''educated, antigen-specific immune effector cell", is an immune effector cell as defined above, which has encountered antigen and which is specific for that antigen. An educated, antigen-specific immune effector cell may be activated upon binding antigen. "Activated" implies that the cell is no longer in Go phase, and begins to produce cytokines characteristic of the cell type. For example, activated CD4+ T cells secrete IL-2 and have a higher WO 99/47641 PG"TNS99/06030 number of high affinity IL-2 receptors on their cell surfaces relative to resting CD4+ T cells.
A peptide or polypeptide of the invention may be preferentially recognized by antigen-specific immune effector cells, such as B cells and T cells. In the S context of T cells, the term "recognized" intends that a peptide or polypeptide of the invention, comprising one or more antigenic epitopes, is recognized, i.e., is presented on the surface of an APC together with (i.e., bound to) an MHC
molecule in such a way that a T cell antigen receptor (TCR) on the surface of an antigen-specific T cell binds to the epitope wherein such binding results in activation or anergy of the T cell. The term "preferentially recognized"
intends that a polypeptide of the invention is substantially not recognized, as defined above. by a T cell specific for an unrelated antigen. Assays for determining whether an epitope is recognized by an antigen-specific T cell are known in the art and are described herein.
The term "autogeneic", or "autologous", as used herein, indicates the origin of a cell. Thus, a cell being administered to an individual (the "recipient") is autogeneic if the cell was derived from that individual (the "donor") or a genetically identical individual. An autogeneic cell can also be a progeny of an autogeneic cell. The term also indicates that cells of different cell types are derived from the same donor or genetically identical donors. Thus, an effector cell and an antigen presenting cell are said to be autogeneic if they were derived from the same donor or from an individual genetically identical to the donor, or if they are progeny of cells derived from the same donor or from an individual genetically identical to the donor.
Similarly, the term "allogeneic", as used herein, indicates the origin of a cell. Thus, a cell being administered to individual (the "recipient") is allogeneic if the cell was derived from an individual not genetically identical to the recipient;
in particular, the term relates to non-identity in expressed MHC molecules. An allogeneic cell can also be a progeny of an allogeneic cell. The term also indicates that cells of different cell types are derived from genetically non-identical donors, or if they are progeny of cells derived from genetically non-identical donors. For example, an APC is said to be allogeneic to an effector cell if they are derived from genetically non-identical donors.
As used herein, the term "a disease or condition related to a population of CD4+ or CD8+ T cells" is one which can be related to a population of CD4+ or S CD8+ T cells, such that these cells are primarily responsible for the pathogenesis of the disease; it is also one in which the presence of CD4+ or CD8+ T cells is an indicia of a disease state; it is also one in which the presence of a population CD4+
or CD8+ T cells is not the primary cause of the disease, but which plays a key role in the pathogenesis of the disease; it is also one in which a population of CD4+ or CD8+ T cells mediates an undesired rejection of a foreign antigen. Examples of a condition related to a population of CD4+ or CD8+T cells include. but are not limited to, autoimmune disorders, graft rejection, immunoregulatory disorders, and anaphylactic disorders.
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor cells", "cancer" and "cancer cells", (used interchangeably) refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division). Neoplastic cells can be malignant or benign.
"Suppressing" tumor growth indicates a growth state that is curtailed when compared to growth without contact with educated. antigen-specific immune effector cells described herein. Tumor cell growth can be assessed by any means known in the art, .including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3H-thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor cell growth means any or all of the following states: slowing, delaying, and stopping tumor growth, as well as tumor shrinkage.
The term "culturing'' refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (either morphologically, genetically, or phenotypically) to the parent cell. By "expanded" is meant any proliferation or division of cells.

A "subject" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, marines, simians. humans, farm animals, sport animals, and pets.
As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al. (1989) supra ). Similarly, an eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors can be obtained commercially or assembled by the sequences described in methods well known in the art, for example, the methods described below for constructing vectors in general.
An "isolated" or "purified" population of cells, nucleic acids, peptides or proteins that is individually substantially free of cells and materials with which it is associated in nature. By substantially free or substantially purified is meant at . list SD% of the composition contains the population.of interest, preferably.at least 70%, more preferably at least 80%, and even more preferably at least 90%
free of molecules which associate with the molecule of interest in nature.
A "composition" is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
A "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivv.

As used herein, the term "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers. such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of Garners, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. Scl., 15th Ed. (Mack Publ. Co., Easton (1975)).
As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others.
"Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination.
Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of" shall mean excluding more than trace I 5 elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.
This invention provides improved vaccines and, methods of using the vaccines to modulate cellular and humoral immune responses to an antigen or antigens in a subject. An antigen, for the purposes of the present invention, is a protein antigen. The term "protein antigen'' is used in its broadest sense and includes minimal epitopes, chimeric molecules, synthetic antigens, in addition to isolated full length proteins. The epitopes can also be derived altered antigens.
While the embodiments described below are directed to tumor antigens. it should be understood, although not explicitely stated, that any protein or polypeptide which induces an immune response is intended to be within the scope of this invention. Such antigens include, but are not limited to tumor antigens, viral antigens, bacterial antigens, and self antigens. The antigen of this vaccine may be autologous or heterologous (i.e., allogeneic) or a homolog from a isolated species, e.g., a murine antigen administered to a human patient.
Polynucleotides of the invention The poiynucleotides of this invention comprise at least two coding sequences. The first sequence encodes a first antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC).
The second coding sequence codes for second antigen that is processed and presented with an MHC Class II molecule on the APC. In some embodiments, the first and second antigens have the same amino acid sequence. In an alternative embodiment, the polynucleotides encode antigens comprising epitopes that are recognized by the same T cell receptor. In a further embodiment. encoded antigens comprise different epitopes from the same native protein. In a further embodiment, the first and second antigens share at least about SO% amino acid sequence identity with one another, as determined using alignment programs using default parameters, or are encoded by polynucleotides that hybridize to each other under stringent conditions.
A polynucleotide of the invention may comprise:
(a) a first coding sequence which comprises a nucleotide sequence encoding an antigen; and (b) a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER).
The polynucleotide can be constructed such that the amino acid sequence that promotes retention of the encoded antigen in the ER is appended to the N-terminus or the C-terminus of the encoded antigen. Alternatively, the sequence that promotes retention of the encoded antigen in the ER can occur internally, i.e., between the N-terminus and C-terminus of the encoded antigen.

A number of amino acid sequences that promote retention in the ER are known in the art. For example, the amino acid sequence Lys-Asp-Glu-Leu (KDEL), when appended to the carboxy-terminus of a protein. promotes retention of that protein in the ER. Pelham ( 1990) Trends in Biochemical Sciences 15:483-486. Others have reported that the carboxy-terminal peptide His-Asp-Glu-Phe (HDEF), promotes retention (targets) the appended protein to the ER. In addition to the "KDEL" and "HDEL" sequences, other sequences such as "DDEL", "ADEL", "SDEL", "RDEL", "KEEL", "QEDL", "HIEL", "HTEL" and "KQDL"
are known as signals for staying in endoplasmic reticulum, and polypeptides having these sequences are generally retained in the ER. Pelham (1990) TIBS, 15:483. Accordingly, in some embodiments, the amino acid sequence that promotes retention of the encoded antigen in the ER is selected from the group consisting of KDEL, HDEL, DDEL, ADEL, SDEL, RDEL, KEEL, QEDL, HIEL, HTEL, and KQDL
In some embodiments, the amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER) is KDEL. In these embodiments, when the antigen encoded by the coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding KDEL is transcribed and translated, the translation unit comprises the antigen having KDEL as the carboxy-terminal amino acids.
In other embodiments, the amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER) is HDEF. In these embodiments, when the antigen encoded by the coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding HDEF is transcribed and translated, the translation unit comprises the antigen having HDEF as the carboxy-terminal amino acids.
In other embodiments, a polynucleotide of the invention comprises:
(a) a first coding sequence which comprises a nucleotide sequence encoding an antigen;
(b) a second coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER);
and (c) a third coding sequence which comprises a nucleotide sequence encoding an antigen and a nucleotide sequence encoding an amino acid sequence that directs (or promotes retention of, or targets) the encoded antigen into a non-endosomal MHC Class II pathway.
The polynucleotide can be constructed such that the amino acid sequence that directs the encoded antigen into a non-endosomal MHC Class II pathway is appended to the N-terminus or the C-terminus of the encoded antigen.
Alternatively, the sequence that directs the encoded antigen into a non-endosomal MHC Class II pathway can occur internally, i.e., between the N-terminus and C-terminus of the encoded antigen.
A non-endosomal MHC Class II pathway includes, but is not limited to, a lysosome, the Golgi, and a melanosome. Amino acid sequence that direct proteins into a non-endosomal MHC Class II pathway are known in the art.
Amino acid sequences that direct (or promote retention of, or target) an antigen to a melanosome (Xu et al. (1998) J. Invest. Dermatol. 110:324-331; and Jimbow et al. ( 1997) Pigment Cell Res. 10:206-213 ); a lysosome (Kornfeld et al. ( 1987) FASEB J. 1:462-468; and Hasilik et al. (1992) Experientia 48:130-151); and to the Golgi (Nilsson and Warren ( 1994) Curr. Opinion Cell Biol. 6:517-521 ) have been reported in the literature and can be used in the present invention.
In some embodiments, the encoded antigen is a naturally occurring antigen. -- -or fragment of a naturally occurring antigen. In other embodiments. the antigen is a synthetic antigen. The antigen can be "self' or foreign, and can be derived from any organism. The antigen may be autologous or heterologous (i.e., allogeneic or a homolog from a isolated species, e.g., a murine antigen administered to a human patient). Each coding sequence is operatively linked to operational and regulatory sequences which control transcription and translation of the coding sequences.
These operational and regulatory sequences are well known to those skilled in the art.

The polynucleotides of this invention comprise coding regions which encode, in one embodiment, previously characterized tumor-associated antigens such as gp100 (Kawakami et al. (1997) Intern. Rev. Immunol. 14:173-192), MUC-I (Henderson et al. ( 1996) Cancer Res. 56:3763-3770), MART-1 S (Kawakami et al. (1994) Proc. Natl. Acad. Sci. 91:3515-3519; Kawakami et al.
(1997) Intern. Rev. Immunol. 14:173-192; Ribas et al. (1997) Cancer Res.
57:2865-2869), HER-2/neu (U.S. Patent No. 5,550,214), MAGE
(PCT/US92/04354) HPV16, 18E6 and E7 (Ressing et al. (1996) Cancer Res.
56(1):582-588; Restifo (1996) Current Opinion in Immunol. 8:658-663; Stern (1996) Adv. Cancer Res. 69:175-211; Tindle et al. (1995) Clin. Exp. Immunol.
101:265-271; van Driel et al. (1996) Annals of Medicine 28:471-477} CEA (U.S.
Patent 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 Research 53:227-30), tyrosinase (U.S. Patent Nos.
5,530,096 and 4,898,814; Brichard et al. (1993) J. Exp. Med. 178:489-49);
tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2), NY-ESO-1 (Chen et al.
(1997) Proc. Natl. Acad. Sci. U.S.A. 94:1914-18), or the GA733 antigen (U.S.
Patent No. 5,185,254).
In some embodiments, an antigen encoded by a polynucleotide of the invention is a secreted protein, or an antigen that has been recombinantly altered to be secreted from the cell.
The secreted form of the antigen can.be synthesized by.splicing a signal sequence onto the sequence encoding the wild-type antigen or by removing the sequences coding for the cytoplasmic and transmembrane portions of the antigen and/or by removing the sequences that 1 ) direct the encoded protein to an intracellular compartment such as the cytoplasm, nucleus or mitochondia, or 2}
cause the encoded protein to be inserted into cellular membranes (i.e., a transmembrane region), or 3) cause the encoded protein to be retained within cellular compartments such as a hydrophobic anchoring sequence or endoplasmic reticulum retention signal.

More specifically, a secreted antigen can be generated by fusing a secretory signal sequence to the wild-type antigen using standard recombinant DNA methodology familiar to one of skill in the art. The secretory signal sequence would typically be positioned at the N-terminus of the desired protein but can be placed at any position suitable to allow secretion of the antigen.
By way of illustration, the figure shows a transmembrane cassette comprising DNA
sequences encoding a wild-type antigen fused in frame at the 5' end with a DNA
sequence encoding a secretory signal sequence such as an endoplasmic reticulum targeting sequence (ERTS). An internal ribosome entry sequence (IRES) is inserted between the wild type gene and the modified gene to ensure that both genes are translated from the bi-cistronic mRNA produced by transcription of the cassette as directed by the CMV promoter. Suitable secretory signal sequences include signal sequences or derivatives of signal sequences of known secretory proteins. A variety of secretory proteins have been identified. They include, but I 5 are not limited to, certain growth factors such as f broblast growth factors 4-6, epidermal growth factor, and lymphokines such as interleukins 2-6.
Alternatively, a secreted antigen can be generated by appending a secretory signal sequence onto the antigen and optionally removing sequences that 1 ) direct the encoded protein to an intracellular compartment such as the cytoplasm, nucleus or mitochondia, or 2) cause the encoded protein to be inserted into cellular membranes (i.e., a transmembrane region), or 3) cause the encoded pr4tein to_be, retained within cellular compartments such as a hydrophobic anchoring sequence or endoplasmic reticulum retention signal. The modified antigen lacks the sequences necessary for membrane anchorage, but retains the sequences required for entry into the rough endoplasmic reticulum/golgi complex and eventual secretion from the cell.
In other embodiments, an antigen encoded by a polynucleotide of the invention is a cell-surface protein. In some of these embodiments, the polynucleotide encoding an antigen further comprises a sequence which encodes an amino acid sequence which causes the encoded protein to be inserted into cellular membranes (i.e., a transmembrane region).

With regard to nucleic acid sequences of the present invention, "isolated"
means: an RNA or DNA polymer, portion of genomic nucleic acid, cDNA, or synthetic nucleic acid which, by virtue of its origin or manipulation: (i) is not associated with all of a nucleic acid with which it is associated in nature (e.g. is present in a host cell as a portion of an expression vector); or (ii) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature. By "isolated" it is further meant a nucleic acid sequence: (i) amplified in vitro by, for example, polymerise chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.
The nucleic acid sequences of the present invention may be characterized, isolated, synthesized and purified using no more than ordinary skill. See Sambrook et al., ( 1989) supra.
The polynucleotides of the present invention also can serve as primers for the detection of genes or gene transcripts that are expressed in APC, for example, to confirm transduction of the polynucleotides into host cells. In this context, amplification means any method employing a primer-dependent polymerise capable of replicating a target sequence with reasonable fidelity.
Amplification may be carried out by natural or recombinant DNA-polymerises such as T7 DNA
polymerise, Klenow fragment of E.coli DNA polymerise, and reverse transcriptase. A preferred length of the primer is the same as that identified for probes, above.
The invention further provides the isolated polynucleotide operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA. As used herein, the term "operatively linked" means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule. Examples of such promoters are SP6, T4 and T7. In certain embodiments, cell-specific promoters are used for cell-specific expression of the inserted polynucleotide.
Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well is a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are well known in the art and commercially available. For general methodology and cloning strategies, see GENE EXPRESSION TECHNOLOGY (Goeddel ed., Academic Press, Inc. ( 1991 )) and references cited therein and VECTORS: ESSENTIAL DATA SERIES
(Gacesa and Ramji, eds., John Wiley & Sons, N.Y. ( 1994)), which contains maps, functional properties, commercial suppliers and a reference to GenEMBL
accession numbers for various suitable vectors. Preferably, these vectors are capable of transcribing RNA in vitro or in vivo.
Methods of increasing presentation of peptide on the surface of an APC
The present invention provides methods to enhance, or increase, presentation of a peptide on the surface of an antigen-presenting cell.
comprising introducing into the cell a polynucleotide of the invention. Since polynucleotides of the invention encoding an antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC) and an antigen that is processed and presented with an MHC Class II molecule on the APC, one observes increased presentation of peptide antigens to CD4+ and to CD8+ T
cells.
The invention further provides an APC produced by the method of the present invention. These APCs exhibit enhanced presentation of a peptide antigen by both Class I and Class II MHC molecules. These levels of antigen loading on the APC surface was not achieved by prior art methods, which enhance either Class I or Class II presentation, but not both. Accordingly, these methods, and the APCs produced thereby, are navel and non-obvious over the prior art.
Whether presentation of peptide on the surface of an APC is enhanced or increased can be determined by comparing the CD4+ and CD8+ T cell response to the encoded peptide presented on the surface of an APC into which has been introduced a polynucleotide of the invention under conditions which favor expression (i.e., transcription and translation), to the CD4+ and CD8+ T cell response to the peptide when the peptide expressed on the surface of a control APC, i.e, an APC presenting the peptide encoded by a polynucleotide lacking either the nucleotide sequences encoding an amino acid sequence that promotes retention in the ER, the nucleotide sequences encoding an amino acid sequence that directs the encoded antigen into a non-endosomal MHC Class II pathway, or both. The methods of the present invention Various methods are known to evaluate T cell activation. see, for example. Gong et al. (1997) Gene Therapy 4:1023-1028. CD8+ T cell activation can be detected by any known method, including but not limited to, tritiated thymidine incorporation (indicative of DNA synthesis), and examination of the population for growth or proliferation, e.g., by identification of colonies.
Alternatively, the tetrazolium salt MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5-diphenyl tetrazolium bromide) may be added. Mossman (1983) J. Immunol.
Methods 65:55-63; Niks and Otto ( 19901 J. Immunol. Methods 130:140-1 S 1.
Succinate dehydrogenase, found in mitochondria of viable cells, converts the MTT to formazan blue. Thus, concentrated blue color would indicate metabolically active cells. In yet another embodiment, incorporation of radiolabel, e.g., tritiated thymidine, may be assayed to indicate proliferation of cells. Similarly, protein synthesis may be shown by incorporation of 35S-methionine. Cytotoxicity and cell killing assays, such as the classical chromium release assay, may be employed to evaluate epitope-specific CTL activation. To detect activation of CD4+ T cells, any of a variety of methods can be used, including, but not limited to. measuring cytokine production; and proliferation, for example, by tritiated thymidine incorporation Production of polynucleotides of the invention Any known method of producing, replicating, and expressing the isolated polynucleotides of the invention can be used. Vectors and methods for in vitro and in vivo transduction are briefly described below and are well known in the art.
The incorporation and expression of the exogenous nucleic acid can be confirmed using RT-PCR, Northern and Southern blotting analysis. Sambrook et al. (1989) supra.
The polynucleotides of this invention can be replicated using PCR. PCR
technoiogy is the subject matter of United States Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 and described in PCR: THE POLYMERASE
CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston ( 1994)) and references cited therein.
Alternatively, one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA. Accordingly, this invention also provides a process for obtaining the polynucleotides of this invention by providing the linear sequence of the polynucleotide. appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (procaryotic or eucaryotic) for replication and amplification. The DNA so amplified can be isolated from the cell by methods well known to those of skill in the art. A process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained.
RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be inserted by any appropriate method, e.g., by the use of an appropriate gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; the RNA can then be isolated using methods well known to those of skill in the art, for example, as set forth in Sambrook et al. (1989) Supra. For instance, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. ( 1989) Supra or extracted by nucleic-acid-binding resins following the accompanying instructions provided by manufactures.
Gene delivery vehicles comprising polynucleotides of the invention The present invention also provides delivery vehicles suitable for delivery of a polynucleotide of the invention into cells (whether in vivo, ex vivo, or in vitro). A polynucleotide of the invention can be contained within a cloning or expression vector. These vectors (especially expression vectors) can in turn be manipulated to assume any of a number of forms which may, for example, facilitate delivery to and/or entry into a cell.
Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, etc. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and efficient transformation of cells both in vitro and in vivo. When a nucleic acid is inserted into a suitable host cell, e.g., a procaryotic or a eucaryotic cell and the host cell replicates, the protein can be recombinantly produced. Suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using well known methods. See Sambrook, et al. (1989) Supra. In addition to the use of viral vector for insertion of exogenous nucleic acid into cells, the nucleic acid can be inserted into the host cell by methods well known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran; electroporation; or microinjection. See Sambrook et al. ( 1989) Supra for this methodology. Thus, this invention also provides a host cell, e.g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a procaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.
When the vectors are used for gene therapy in vivo or ex vivo, a pharmaceutically acceptable vector is preferred, such as a replication-incompetent retroviral or adenoviral vector. Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression of the inserted polynucleotide. As used herein, the term "pharmaceutically acceptable vector" includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells. An example of such a vector is a "replication-incompetent" vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell. An example of a replication-incompetent retroviral vector is LNL6. Miller et al. ( 1989) BioTechniques 7:980-990. The methodology of using replication-incompetent retroviruses for retroviral-mediated gene transfer of gene markers is well established. Correll et al. ( 1989) PNAS USA 86:8912; Bordignon ( 1989) PNAS USA 86:8912-52; Culver ( 1991 ) PNAS USA 88:3155; and Rill ( 1991 ) Blood 79( 10):2694-700.
In general, genetic modifications of cells employed in the present invention are accomplished by introducing a vector containing a poIynucleotide comprising sequences encoding a synthetic antigenic peptide of the invention.
A
variety of different gene transfer vectors, including viral as well as non-viral systems can be used.
A wide variety of non-viral vehicles for delivery of a polynucleotide of the invention are known in the art and are encompassed in the present invention. A
polynucleotide of the invention can be delivered to a cell as naked DNA. WO
97/40163. Alternatively, a polynucleotide of the invention can be delivered to a cell associated in a variety of ways with a variety of substances (forms of delivery) including, but not limited to cationic lipids; biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides;
polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles;
and bacteria. A delivery vehicle may take the form of a microparticle.
Mixtures or conjugates of these various substances can also be used as delivery vehicles. A
polynucleotide of the invention can be associated with these various forms of delivery non-covalently or covalently.
Included in the non-viral vector category are prokaryotic plasmids and eukaryotic plasmids. Non-viral vectors (i.e., cloning and expression vectors) having cloned therein a polynucleotide(s) of the invention can be used for expression of recombinant polypeptides as well as a source of polynucleotide of the invention. Cloning vectors can be used to obtain replicate copies of the polynucleotides they contain, or as a means of storing the polynucleotides in a depository 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 where it is desirable to express polypeptides, encoded by an operably linked polynucleotide, in an individual, such as for eliciting an immune response via the polypeptide(s) encoded in the expression vector(s). Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and need not be described in detail herein. For example, see Gacesa and Ramji, Vectors, John Wiley & Sons (1994).
Cloning and expression vectors typically contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode proteins) that (a) confer resistance to antibiotics or other toxins substances, e.g., ampicillin, neomycyin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art. Cloning and expression vectors also typically contain a replication system recognized by the host.
Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUCl8, pUCl9, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.
Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide encoding a polypeptide of interest. The polynucleotide encoding the polypeptide of interest is operably linked to suitable transcriptional controlling elements, such as promoters, enhancers arid terminators. For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons. A polynucleotide sequence encoding a signal peptide can also be included to allow a polypeptide, encoded by an operably linked polynucleotide, to cross and/or lodge in cell membranes or be secreted from the cell. A number of expression vectors suitable for expression in eukaryotic cells including yeast, avian, and mammalian cells are known in the art. Examples of mammalian expression vectors contain both prokaryotic sequence to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Examples of mammalian expression vectors suitable for transfection of eukaryotic cells include the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pRSVneo, and pHyg derived vectors. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1 ), or Epstein-Barr virus (pHEB, pREP derived vectors) can be used for expression in mammalian cells. Examples of expression vectors for yeast systems, include YEP24, YIPS, YEP51, YEP52, YES2 and YRP17, which are cloning and expression vehicles useful for introduction of constructs into S.
cerevisiae. Broach et al. (1983) Experimental Manipulation of Gene Expression, ed. M. Inouye, Academic Press. p. 83. Baculovirus expression vectors for expression in insect cells include pVL-derived vectors (such as pVL 1392, pVL1393 and pVL941), pAcUW-derived vectors and pBlueBac-derived vectors.
Viral vectors include, but are not limited to, DNA viral vectors such as those based on adenoviruses, herpes simplex virus, poxviruses such as vaccinia virus, and parvoviruses, including adeno-associated virus; and RNA viral vectors, including, but not limited to, the retroviral vectors. Retroviral vectors include marine leukemia virus, and lentiviruses such as human immunodeficiency virus.
Naldini et al. ( 1996) Science 272:263-267.
Replication-defective retroviral vectors harboring a polynucleotide of the invention as part of the retroviral genome can be used. Such vectors have been described in detail. (Miller et al. ( 1990) Mol. Cell Biol. 10:4239; Kolberg, R.
( 1992) J. NIH Res. 4:43; Cornetta et al. ( 1991 ) Hum. Gene Ther 2:21 S).
Adenovirus and adeno-associated virus vectors useful in the genetic modifications of this invention may be produced according to methods already taught in the art. (See, e.g., Karlsson et al. (1986) EMBO 5:2377; Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzcyzka ( 1992) Current Top.
Microbiol. Immunol. 158:97-129; GENE TARGETING: A PRACTICAL APPROACH
( I 992) ed. A. L. Joyner, Oxford University Press, NY). Several different approaches are feasible.
Additional references describing viral vectors which could be used in the methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.) viROLOGY, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F. et al., pp. 109-128 in METHODS IN MOLECULAR BIOLOGY, Vol. 7: GENE TRANSFER AND EXPRESSION
PROTOCOLS, Murray, E. (ed.), Humana Press, Clifton, N.J. (1991); Miller et al.
(1995) FASEBJournal 9:190-199, Schreier (1994) PharmaceuticaActa Helvetiae 68:145-159; Schneider and French (1993) Circulation 88:1937-1942; Curiel et al.
(1992) Human Gene Therapy 3:147-154; Graham et al., WO 95/00655 (5 January 1995); Falck-Pedersen WO 95/16772 (22 June 1995); Denefle et al. WO 95/23867 (8 September 1995); Haddada et al. WO 94/26914 (24 November 1994);
Perricaudet et al. WO 95/02697 (26 January 1995); Zhang et al. WO 95/25071 (12 October 1995).
The efficiency of transduction of DCs or other APCs can be assessed by immunofluorescence using fluorescent antibodies specific for the tumor antigen being expressed (Kim et al. ( 1997) J. Immunother. 20:276-286). Alternatively, the antibodies can be conjugated to an enryme (e.g. HRP) giving rise to a colored product upon reaction with the substrate. The actual amount of antigenic polypeptides being expressed by the APCs can be evaluated by ELISA.
In vivo transduction of DCs, or other APCs, can be accomplished by administration of a viral vectors comprising a polynucleotide of the invention via different routes including intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery. One method which can be used is cutaneous delivery of Ad vector at multiple sites using a total dose of approximately 1 x 10 ~ °-1 x 10 ~ 2 i.u.
Levels of in vivo transduction can be roughly assessed by co-staining with antibodies directed against APC markers) and the peptide epitope being expressed. The staining procedure can be carried out on biopsy samples from the site of administration or on cells from draining lymph nodes or other organs where APCs (in particular DCs) may have migrated. Condon et al. ( 1996) Nature Med. 2:1122-1128; Wan et al. (1997) Human Gene Therapy 8:1355-1363. The amount of antigen being expressed at the site of injection or in other organs where transduced APCs may have migrated can be evaluated by ELISA on tissue homogenates.
APCs can also be transduced in vitro%x vivo by non-viral gene delivery methods such as electroporation, calcium phosphate precipitation or cationic lipid/plasmid DNA complexes. Arthur et al. ( 1997) Cancer Gene Therapy 4:17-25. Transduced APCs can subsequently be administered to the host via an intravenous. subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
In vivo transduction of DCs, or other APCs, can potentially be accomplished by administration of cationic lipid/plasmid DNA complexes delivered via the intravenous, intramuscular, intranasal, intraperitoneal or cutaneous route of administration. Gene gun delivery or injection of naked plasmid DNA into the skin also leads to transduction of DCs. Condon et al.
( 1996) Nature Med. 2:1122-1128; Raz et al. ( 1994) PNAS 91:9519-9523.
Intramuscular delivery of plasmid DNA may also be used for immunization.
Rosato et al. (1997) Human Gene Therapy 8:1451-1458.

The transduction efficiency and levels of transgene expression can be assessed as described above for viral vectors.
Host cells comprising polynucleotides of the invention The present invention further provides host cells comprising polynucleotides of the invention. Host cells containing the polynucleotides of this invention are useful for the recombinant replication of the polynucleotides and for the recombinant production of peptides of the invention. In addition, host cells comprising a polynucleotide of the invention can be used to induce an immune response in a subject in the methods described herein.
Host cells which are suitable for recombinant replication of the polynucleotides of the invention, and for the recombinant production of peptides of the invention can be prokaryotic or eukaryotic. Host systems are known in the art and need not be described in detail herein. Prokaryotic hosts include bacterial I S cells, for example E. coli, B. subtilis, and mycobacteria. Among eukaryotic hosts are yeast, insect, avian, plant, C. elegans (or nematode) and mammalian cells.
These cells are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
When the host cells are antigen presenting cells, they can be used to expand a population of immune effector cells such as tumor infiltrating lymphocytes which in turn are useful in adoptive immunotherapies. Antigen presenting cells are described in more detail below.
In some of these embodiments, isolated host cells are APCs. APCs include, but are not limited to, dendritic cells (DCs), monocytes/macrophages, B
lymphocytes or other cell types) expressing the necessary MHC/co-stimulatory molecules.
In some embodiments, the immune effector cells and/or the APCs are genetically modified. Using standard gene transfer, genes coding for co-stimulatory molecules and/or stimulatory cytokines can be inserted prior to, concurrent to or subsequent to expansion of the immune effector cells.

In one embodiment. the host cell containing the genes) coding for the cytokine and/or co-stimulatory molecule is a professional antigen-presenting cell such as a dendritic cell which includes, but is not limited to, a pulsed dendritic cell, a dendritic cell hybrid or an antigen-presenting foster cell.
Antigen presenting cells APCs suitable for use in the present invention are capable of presenting exogenous peptide or protein or endogenous antigen to T cells in association with an antigen-presenting molecule, such as an MHC molecule. APCs include, but are not limited to, macrophages, dendritic cells, CD40-activated B cells, antigen-specific B cells, tumor cells, virus-infected cells, and genetically modified cells.
APCs can obtained from a variety of sources, including but not limited to, peripheral blood mononuclear cells (PBMC), whole blood or fractions thereof containing mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, lymph nodes, e.g., lymph nodes draining from a tumor. Suitable donors include an immunized donor, a non-immunized (naive) donor, treated or untreated donors. A "treated" donor is one that has been exposed to one or more biological modifiers. An "untreated"
donor has not been exposed to one or more biological modifiers. APC's can also be treated in vitro with one or more biological modifiers.
The APCs are generally alive but can also be irradiated. mitomycin C
treated, attenuated, or chemically fixed. Further, the APCs need not be whole cells. Instead, vesicle preparations of APCs can be used.
APCs can be genetically modified, i.e., transfected with a recombinant polynucleotide construct such that they express a polypeptide or an RNA
molecule which they would not normally express or would normally express at lower Levels. Examples of polynucleotides include, but are not limited to, those which encode an MHC molecule; a co-stimulatory molecule such as B7; and a peptide or poIypeptide of the invention.
Cells which do not normally function in vivo in mammals as APCs can be modified in such a way that they function as APCs. A wide variety of cells can function as APCs when appropriately modified. Examples of such cells are insect cells, for example Drosophila or Spodoptera; and foster cells, such as the human cell line T2. For example, expression vectors which direct the synthesis of one or more antigen-presenting polypeptides, such as MHC molecules, optionally also accessory molecules such as B7, can be introduced into these cells to effect the expression on the surface of these cells antigen presentation molecules and, optionally, accessory molecules or functional portions thereof. Alternatively, antigen-presenting polypeptides and accessory molecules which can insert themselves into the cell membrane can be used. For example, glycosyl-phosphotidylinositol (GPI)-modified polypeptides can insert themselves into the membranes of cells. Hirose et al. (1995) Methods Enzymol. 250:582-614; and Huang et al. ( 1994) Immunity I :607-613. Accessory molecules include. but are not limited to, co-stimulatory antibodies such as antibodies specific for CD28, CD80, or CD86; costimulatory molecules, including, but not limited to, B7.1 and B7.2; adhesion molecules such as ICAM-1 and LFA-3; and survival molecules such as Fas ligand and CD70. See, for example, PCT Publication No. WO
97/46256.
Foster antigen presenting cells are particularly useful as APCs. Foster APCs are derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules. Zweerink et al.
( 1993) J. Immunol. 150:1763-1771. This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1, TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC class 1-restricted CD8+ CTLs. In effect, only "empty" MHC class I molecules are presented on the surface of these cells. Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif. These T2 cells are referred to herein as "foster" APCs. They can be used in conjunction with this invention to present antigen(s).
Transduction of T2 cells with specific recombinant MHC alleles allows for redirection of the MHC restriction profile. Libraries tailored to the recombinant allele will be preferentially presented by them because the anchor residues will prevent efficient binding to the endogenous allele.
High level expression of MHC molecules makes the APC more visible to the CTLs. Expressing the MHC allele of interest in T2 cells using a powerful transcriptional promoter (e.g., the CMV promoter) results in a more reactive APC
(most likely due to a higher concentration of reactive MHC-peptide complexes on the cell surface}.
Methods for determining whether an antigen-presenting cell is capable of presenting antigen to an immune effector cell in such a manner as to effect activation of the immune effector cell, are known in the art and include, for example. 3H-thymidine uptake by effector cells, cytokine production by effector cells. and cytolytic 5' Cr-release assays.
In some embodiments, an antigenic peptide of the invention is presented on an antigen-presenting cell in a Class I or Class II MHC molecule such that the peptide is bound by a TCR on a CD4+ or CD8+ T cell, but the antigen-presenting cell lacks one or more co-stimulatory molecules required for activation of the T
cell. These antigen-presenting cells induce T cell anergy (unresponsiveness), and are useful in methods described herein for reducing or suppressing an immune response. Methods for determining whether an antigen-presenting cell is capable of presenting antigen to an immune effector cell, in such a manner as to effect T
cell anergy, are known in the art.
The following is a brief description of two fundamental approaches for the isolation of APC. These approaches involve ( 1 ) isolating bone marrow precursor cells (CD34+) from blood and stimulating them to differentiate into APC; or (2) collecting the precommitted APCs from peripheral blood. In the first approach, the patient must be treated with cytokines such as GM-CSF to boost the number of circulating CD34+ stem cells in the peripheral blood.
The second approach for isolating APCs is to collect the relatively large numbers of precommitted APCs already circulating in the blood. Previous techniques for isolating committed APCs from human peripheral blood have involved combinations of physical procedures such as metrizamide gradients and adherence/nonadherence steps (Freudenthal et al. ( 1990) PNAS 87:7698-7702);
Percoll gradient separations (Mehta-Damani et al. ( 1994) J. Immunol. 153:996-1003); and fluorescence activated cell sorting techniques (Thomas et al.
(1993) J.
Immunol. 151:6840-52).
One technique for separating large numbers of cells from one another is known as countercurrent centrifugal elutriation (CCE). In this technique, cells are subject to simultaneous centrifugation and a washout stream of buffer which is constantly increasing in flow rate. The constantly increasing countercurrent flow of buffer leads to fractional cell separations that are largely based on cell size.
In one aspect of the invention, the APC are precommitted or mature dendritic cells which can be isolated from the white blood cell fraction of a mammal, such as a marine, simian or a human (See, e.g., WO 96/23060). The white blood cell fraction can be from the peripheral blood of the mammal. This method includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukopheresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation, (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF
and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c), and (e) collecting the enriched fraction of step (d), preferably at about 4°C.
One way to identify the dendritic cell-enriched fraction is by fluorescence-activated cell sorting. The white blood cell fraction can be treated with calcium ionophore in the presence of other cytokines, such as recombinant (rh) rhIL-12, rhGM-CSF, or rhIL-4. The cells of the white blood cell fraction can be washed in buffer and suspended in Ca++/Mg~ free media prior to the separating step. The white blood cell fraction can be obtained by leukopheresis. The dendritic cells can be identified by the presence of at least one of the following markers: HLA-DR, HLA-DQ, or B7. 2, and the simultaneous absence of the following markers: CD3, CDI4, CD16, 56, 57, and CD 19, 20. Monoclonal antibodies specific to these cell surface markers are commercially available.

More specifically, the method requires collecting an enriched collection of white cells and platelets from leukopheresis that is then further fractionated by countercurrent centrifugal elutriation (CCE). Abrahamsen et al. ( 1991 ) J.
Clin.
Apheresis. 6:48-53. Cell samples are placed in a special elutriation rotor.
The rotor is then spun at a constant speed of, for example, 3000 rpm. Once the rotor has reached the desired speed, pressurized air is used to control the flow rate of cells. Cells in the elutriator are subjected to simultaneous centrifugation and a washout stream of buffer which is constantly increasing in flow rate. This results in fractional cell separations based largely but not exclusively on differences in cell size.
Quality control of APC and more specifically DC collection and confirmation of their successful activation in culture is dependent upon a simultaneous mufti-color FACS analysis technique which monitors both monocytes and the dendritic cell subpopulation as well as possible contaminant T
lymphocytes. It is based upon the fact that DCs do not express the following markers: CD3 (T cell); CD14 (monocyte); CD16, 56, 57 (NK/LAK cells); CD19, (B cells). At the same time, DCs do express large quantities of HLA-DR, significant HLA-DQ and B7.2 (but little or no B7.1 ) at the time they are circulating in the blood (in addition they express Leu M7 and M9, myeloid 20 markers which are also expressed by monocytes and neutrophils).
Once collected, the DC rich/monocyte APC fractions (usually 150 through 190) can be pooled and cryopreserved for future use, or immediately placed in short term culture.
Alternatively, others have reported that a method for upregulating (activating) dendritic cells and converting monocytes to an activated dendritic cell phenotype. This method involves the addition of calcium ionophore to the culture media convert monocytes into activated dendritic cells. Adding the calcium ionophore A23187, for example, at the beginning of a 24-48 hour culture period resulted in uniform activation and dendritic cell phenotypic conversion of the pooled "monocyte plus DC" fractions: characteristically, the activated population becomes uniformly CD14 (Leu M3} negative, and upregulates HLA-DR, HLA-DQ, ICAM-1. B7.1, and B7.2.
Specific combinations) of cytokines have been used successfully to amplify (or partially substitute) for the activation/conversion achieved with calcium ionophore: these cytokines include but are not limited to purified or recombinant human ("rh") rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when given alone is inadequate for optimal upregulation.
Foster Antigen Presenting Cells A foster antigen presenting cell is a genetically modified dendritic cell that, from the restricted association of endogeneous peptides, will take up and present exogeneous antigen on the cell surface. In one embodiment, the human cell line 174xCEM.T2, referred to as T2, contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763-1771 ) and therefore can be used for the manufacture of a foster antigen presenting cell. (The cell line is available from the ATCC.) This is due to a large homozygous deletion in the MHC class II region encompassing the genes TAP1, TAP2, LMP1, and LMP2, which are required for antigen presentation to MHC
class 1-restricted CD8+ CTLs. In effect, only "empty" MHC class I molecules are presented on the surface of these cells. Exogenous peptide added to the culture medium binds to these MHC molecules provided that the peptide contains the allele-specific binding motif. These T2 cells are referred to herein as "foster"
APCs. They can be used in conjunction with this invention to present the antigens.
Transduction of T2 cells with specific recombinant MHC alleles allows for redirection of the MHC restriction profile. Libraries tailored to the recombinant allele will be preferentially presented by them because the anchor residues will prevent efficient binding to the endogenous allele.
Transduction of non-professional APCs with allogeneic MHC alleles aids greatly in the immunogenicity of the recombinant cell line (Leong et al.
(1994) Inter. J. Cancer 59:212-216; Ostrand-Rosenberg et al. ( 1991 ) Inter. J.
Cancer Suppl. 6:61-68). Immunogenicity was reported to be proportional to the level of expression of the MHC proteins. T2 cells are ideal APCs.
High level expression of MHC molecules makes the APC more visible to the CTLs. Expressing the MHC allele of interest in T2 cells using a powerful transcriptional promoter (e.g., the CMV promoter) results in a more reactive APC
(most likely due to a higher concentration of reactive MHC-peptide complexes on the cell surface). Note that since only one type of MHC allele will be able to interact with a given library, the presence of or expression level of the endogenous allele will not compromise specificity if the library is designed to bind to the newly transduced cells.
Hybrid cells also will present antigens) and therefore, are useful in the methods of this invention.
Preparation of Hybrid Cells Utilizing Dendritic Cells Hybrid cells typically retain the phenotypic characteristics of the APCs.
Thus. hybrids made with dendritic cells will express the same MHC class II
proteins and other cell surface markers. Moreover, the hybrids will express those antigens expressed on the cells from which they are derived. The procedure for making these hybrids is described in WO 96/30030 and Gong et al. (1997) Nature Medicine 3(5):558-561.
A population of APCs are collected and isolated. Preferably, the ratio of APCs:antigen-expressing cells is between about 1:100 and about 1000:1. For example, in one aspect, the fraction enriched for antigen-expressing cells is then fused to APCs, preferably dendritic cells. Fusion between the APCs and antigen-expressing cells can be carried out with any suitable method, for example using polyethylene glycol (PEG) or Sendia virus. In a preferred embodiment. the hybrid cells are created using the procedure described by Gong et al. ( 1997) Nat.
Med.

3(5):558-561.
Typically, unfused cells will die off after a few days in culture, therefore, the fused cells can be separated from the parent cells simply by allowing the culture to grow for several days. In this embodiment, the hybrid cells both survive more and, additionally, are only lightly adherent to tissue culture surfaces.
The parent cells are strongly adherent to the containers. Therefore, after about 5 to 10 days in culture. the hybrid cells can be gently dislodged and transferred to new containers, while the unfused cells remained attached.
Alternatively, it has been shown that fused cells lack functional hypoxanthine-guaninephosphoribosyl transferase ("HGPRT") enzyme and are, therefore, resistant to treatment with the compound HAT. Accordingly, to select these cells HAT can be added to the culture media. However, unlike conventional HAT selection, hybrid cell cultures should not be exposed to the compound for more than I2 days. The APCs as described above can be assayed for antigens) expression as described below.
In addition to their use as vaccines, they are useful to expand immune effector cell population that can be used in adoptive immunotherapy.
Presentation of Antigen to the APC
The following briefly describes various methods for expression or presentation of antigen on APCs.
Paglia et al. (1996) J. Exp. Med. 183:317-322 has shown that APC
incubated with whole protein in vitro were recognized by MHC class I-restricted CTLs, and that immunization of animals with these APCs led to the development of antigen-specific CTLs in vivo. Hsu et al. (1996) Nature Medicine 2(1):52-58;
Celluzzi et al. (1996) J. Exp. Med. 183:283-287; Mayordomo et al. (1995) Nature Medicine 1(I2):1297-1302; Bakker et al. (1995) Cancer Research 55:5330-5334;
and Mayordomo et al. (1997) Stem Cells 15:94-103, reported on the successful use of peptide-pulsed dendritic cells in cancer immunotherapy.
In addition, several different techniques have been described which lead to the expression of antigen in the cytosol of APCs, such as DCs. These include ( 1 ) the introduction into the APCs of RNA isolated from tumor cells, (2) infection of APCs with recombinant vectors to induce endogenous expression of antigen, and (3) introduction of tumor antigen into the DC cytosol using liposomes. (See Boczkowski et al. (1996) J. Exp. Med. 184:465-472; Rouse et al. (1994) J.
Virol.
68:5685-5689; and Nair et al. ( 1992) J. Exp. Med. 175:609-612).
Genetically modified dendritic cells and their use in immunotherapy have been described. Brossart et al. ( 1997) J. Immunol. 3270-3276 and Wan et al.
(1997) Human Gene Therapy 8:1355-1363; Gong et al. (1997) Gene Therapy

4:1023-1028; Kim et al.(1997) J. Immunotherapy 20(4):276-286; and Song et al.
( 1997) J. Exp. Med. 186(8):1247-1256, reported that adenovirus containing antigenic peptides can be used was used to transduce DCs which are then administered to animals to induce an immune response. Condon et al. (1996) Nature Medicine 2(10):1122-1128 showed that cutaneous genetic immunization with naked DNA resulted in a potent, antigen-specific, cytotoxic T-lymphocyte-mediated protective immunity. Arthur et al. ( 1997) Cancer Gene Therapy 4(1):17-25 compare and analyze the gene transfer methods in human dendritic cells.
However, none of the above report on the use of APCs to present cell-surface and secreted forms of the antigen to induce an immune response as claimed herein.
Insertion of the genes) into the APCs requires the making of appropriate gene delivery vehicles and methods for efficient transduction. The following are useful for in vitro and in vivo transduction with the genes) of interest.
Immune Effector Cells The present invention makes use of the above-described APCs to stimulate production of an enriched population of antigen-specific immune effector cells.
Accordingly, the present invention provides a population of cells enriched in educated. antigen-specific immune effector cells, specific for an antigen encoded by a polynucleotide of the invention. In some embodiments, the antigen corresponds to an antigen on the surface of tumor cells and the educated, antigen-specific immune effector cells of the invention suppress growth of the tumor cells.
When APCs are used, the antigen-specific immune effector cells are expanded at the expense of the APCs, which die in the culture. The process by which nave immune effector cells become educated by other cells is described essentially in Coulie (1997) Molec. Med. Today 3:261-268.
The APCs prepared as described above are mixed with naive immune effector cells. Preferably, the cells may be cultured in the presence of a cytokine, for example IL2. Because dendritic cells secrete potent immunostimulatory cytokines, such as IL-12, it may not be necessary to add supplemental cytokines during the first and successive rounds of expansion. In any event, the culture conditions are such that the antigen-specific immune effector cells expand (i.
e.
proliferate) at a much higher rate than the APCs. Multiple infusions of APCs and optional cytokines can be performed to further expand the population of antigen-specific cells.
In one embodiment, the immune effector cells are T cells. In a separate embodiment, the immune effector cells can be genetically modified by transduction with a transgene coding for example, IL-2, IL-11 or IL-13.
Methods I 5 for introducing transgenes in vitro, ex vivo and in vivo are well known in the art.
See Sambrook, et al. (1989) Supra.
An effector cell population suitable for use in the methods of the present invention can be autogeneic or allogeneic, preferably autogeneic. When effector cells are allogeneic, preferably the cells are depleted of alloreactive cells before use. This can be accomplished by any known means, including, for example, by mixing the allogeneic effector cells and a recipient cell population and incubating them for a suitable time, then depleting CD69+ cells, or inactivating alloreactive cells, or inducing anergy in the alloreactive cell population.
Hybrid immune effector cells can also be used. Immune effector cell hybrids are known in the art and have been described in various publications.
See, for example, International Patent Application Nos. WO 98/46785; and WO
95/16775.
The effector cell population can comprise unseparated cells, i.e., a mixed population. for example, a PBMC population, whole blood, and the like. The effector cell population can be manipulated by positive selection based on expression of cell surface markers, negative selection based on expression of cell surface markers, stimulation with one or more antigens in vitro or in vivo, treatment with one or more biological modifiers in vitro or in vivo, subtractive stimulation with one or more antigens or biological modif ers, or a combination of any or all of these.
Effector cells can obtained from a variety of sources, including but not limited to, PBMC, whole blood or fractions thereof containing mixed populations, spleen cells, bone marrow cells, tumor infiltrating lymphocytes, cells obtained by leukapheresis, biopsy tissue, lymph nodes, e.g., lymph nodes draining from a tumor. Suitable donors include an immunized donor, a non-immunized (naive) donor, treated or untreated donors. A "treated" donor is one that has been exposed to one or more biological modifiers. An ''untreated" donor has not been exposed to one or more biological modifiers.
Methods of extracting and culturing effector cells are well known. For example, effector cells can be obtained by leukapheresis, mechanical apheresis using a continuous flow cell separator. For example, lymphocytes and monocytes can be isolated from the huffy coat by any known method, including, but not limited to, separation over Ficoll-HypaqueTM gradient, separation over a Percoll gradient. or elutriation. The concentration of Ficoll-HypaqueTM can be adjusted to obtain the desired population, for example, a population enriched in T cells.
Other methods based on affinity are known and can be used. These include, for example. fluorescence-activated cell sorting (FACS), cell adhesion, magnetic bead separation, and the like. Affinity-based methods may utilize antibodies, or portions thereof, which are specific for cell-surface markers and which are available from a variety of commercial sources, including, the American Type Culture Collection (Manassas, MD). Affinity-based methods can alternatively utilize ligands or ligand analogs, of cell surface receptors.
The effector cell population can be subjected to one or more separation protocols based on the expression of cell surface markers. For example, the cells can be subjected to positive selection on the basis of expression of one or more cell surface polypeptides, including, but not limited to, ''cluster of differentiation"
cell surface markers such as CD2, CD3, CD4, CDB, TCR, CD45, CD45R0, CD45RA, CD 11 b, CD26, CD27, CD28, CD29, CD30, CD31, CD40L; other markers associated with lymphocyte activation, such as the lymphocyte activation gene 3 product (LAG3), signaling lymphocyte activation molecule (SLAM), T1/ST2; chemokine receptors such as CCR3, CCR4, CXCR3, CCRS; homing receptors such as CD62L, CD44, CLA, CD146, a4~37, aE~37; activation markers such as CD25, CD69 and OX40; and lipoglycans presented by CD1. The effector cell population can be subjected to negative selection for depletion of non-T
cells and/or particular T cell subsets. Negative selection can be performed on the basis of cell surface expression of a variety of molecules, including, but not limited to, B cell markers such as CD19, and CD20; monocyte marker CD14; the NK cell marker CD56.
An effector cell population can be manipulated by exposure, in vivo or in vitro, to one or more biological modifiers. Suitable biological modifiers include, but are not limited to, cytokines such as IL-2, IL-4, IL-10, TNF-a, IL-12, IFN-y;
non-specific modifiers such as phytohemagglutinin (PHA), phorbol esters such as phorbol myristate acetate (PMA), concanavalin-A, and ionomycin; antibodies specific for cell surface markers, such as anti-CD2, anti-CD3, anti-IL2 receptor, anti-CD28; chemokines, including, for example, lymphotactin. The biological modifiers can be native factors obtained from natural sources, factors produced by recombinant DNA technology, chemically synthesized polypeptides or other molecules, or any derivative having the functional activity of the native factor. If ~~ ~ - more than~one biological modifier is used; the exposure can-be simultaneous or sequential.
The present invention provides compositions comprising immune effector cells, which may be T cells, enriched in antigen-specific cells, specific for an antigen encoded by a polynucleotide of the invention. By "enriched" is meant that a 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 enriched from an original naive cell population. The proportion of the enriched cell population which comprises antigen-specific cells can vary substantially, from less than 10%
up to 100% antigen-specific cells. If the cell population comprises at least 50%, preferably at least 70%. more preferably at least 80%, and even more preferably at least 90%, antigen-specific immune effector cells, specific for a peptide of the invention, then the population is said to be "substantially pure". The percentage which are antigen-specific can readily be determined, for example, by a 3H-thymidine uptake assay in which the effector cell population (for example, a T-cell population) is challenged by an antigen-presenting cell presenting an antigen encoded by a polynucleotide of the invention.
Expansion of Immune Effector Cells The present invention makes use of the above-described APCs to stimulate production of an enriched population of antigen-specific immune effector cells.
The antigen-specific immune effector cells are expanded at the expense of the APCs, which die in the culture. The process by which naive immune effector cells become educated by other cells is described essentially in Coulie ( 1997) Molec. Med. Today 3:261-268; Hwu et al. (1993) J. Immunol. 150:4104-4115;
and Rosenburg,et al. (I985) N. Eng. J. Med. 313(23):1485-1492.
In one embodiment, the antigen-specific immune effector cell population comprises both CD4+ and CD8+ T cells.
In one aspect, the cytotoxic T cells (i.e., CD8+ T cells) are polyclonal T
cells isolated from a site of cytotoxic T cell infiltration from a subject.
Alternatively, such cells may be isolated from a site of cytotoxic T cell infiltration ,. . .: from .two or more subjects or human patients;. in which the subjects share an MHC
halotype. In another embodiment, the CTLs may be two or more cytotoxic T cell lines. In yet another embodiment, the CTLs 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 tumors from which cells or cell lines are obtained can be the same type of tumor in different individuals with a shared MHC halotype or different types of tumors from different subjects who share an MHC haplotype.

Alternatively, CTL infiltrates can be from sites of viral infection.
autoimmune inflammation, transplantation rejection, an like sites of inflammation or lymphocyte/leukocyte infiltration.
The APCs prepared as described above are mixed with naive immune effector cells. Preferably, the cells may be cultured in the presence of a cytokine, for example IL2. Because dendritic cells secrete potent immunostimulatory cytokines. such as IL 12, it may not be necessary to add supplemental cytokines during the first and successive rounds of expansion. In any event, the culture conditions are such that the antigen-specific immune effector cells expand (i.e.
proliferate) at a much higher rate than the APCs. Multiple infusions of hybrid cells and optional cytokines can be performed to further expand the population of antigen-specific cells.
In one embodiment, the immune effector cells are T cells and are specific for tumor-specific antigens which are presented by the APCs.
Polypeptide Antigens of the Invention The invention further provides peptide antigens encoded by the polynucleotides of the invention. Peptide antigens encoded by polynucleotides of the invention can be produced by any known method. Isolated peptides of the present invention can be synthesized using an appropriate solid state synthetic procedure. Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San ... .. Francisco..Calif..(19,68)..A preferred method is the Merrifield process. Merrifield, Recent Progress in Hormone Res., 23:451 ( 1967). The antigenic activity of these peptides may conveniently be tested using, for example, the assays as described herein.
Once an isolated peptide of the invention is obtained, it may be purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. For immunoaffmity chromatography, an epitope may be isolated by binding it to an affinity column comprising antibodies that were raised against that peptide. or a related peptide of the invention, and were affixed to a stationary support.
Alternatively, affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Methods Enzymol. 194:508-509), and glutathione-S-transferase can be attached to the peptides of the invention to allow easy purification by passage over an appropriate affinity column. A DNA aff nity column using DNA containing a sequence encoding the peptides of the invention could be used in purification.
Isolated peptides can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
Also included within the scope of the invention are antigenic peptides that are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, crosslinking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson,et al., ( 1988) Ann.
Rev.
Biochem.57:285-320).
Assaying Antigen Specificity The immune effector cells described herein are selected for their ability to modulate an immune response. In one aspect, the immune effector cells are selected both for their ability to actively lyse the cells expressing the specific antigen and for their ability to increase a humoral response to the antigen.
.... . .Cytolytic activity of.the.cells can be measured in various ways, including, butpot. . .
limited to, tritiated thymidine incorporation (indicative of DNA synthesis), and examination of the population for growth or proliferation, e.g., by identification of colonies. (See, e.g., WO 94/21287). In another embodiment, the tetrazolium salt MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5-diphenyl tetrazolium bromide) may be added (Mossman (1983) J. Immunol. Methods 65:55-63: Niks and Otto (1990) J.
Immunol. Methods 130:140-151). Succinate dehydrogenase, found in mitochondria of viable cells, converts the MTT to fonmazan blue. Thus, concentrated blue: color would indicate metabolically active cells. Similarly, protein synthesis may be shown by incorporation of 3'S-methionine. In still another embodiment, cytotoxicity and cell killing assays, such as the classical chromium release assay, may be employed to evaluate epitope-specific CTL
activation. Other suitable assays will be known to those of skill in the art.
Stimulation of a humoral response to an antigen can be measured by specific antibody production, using any known method, including ELISA and RIA.
Compositions of the invention This invention also provides compositions containing any of the above-mentioned peptides, polypeptides, polynucleotides, host cells, antigen-presenting cells, immune effector cells, vectors, antibodies and fragments thereof, and an acceptable solid or liquid carrier. When the compositions are used pharmaceutically, they are combined with a "pharmaceutically acceptable carrier"
for diagnostic and therapeutic use. These compositions also can be used for the preparation of medicaments for the diagnostic and immunomodulatory methods of the invention.
Methods using the polynucleotides, antigen presenting cells, and immune effector cells of the invention The present invention provides diagnostic and immunomodulatory methods using polynucleotides, and host cells (including APCs and educated immune effector cells), i.e., immunomodulatory agents, of the invention.
,. . . ,. ... , ~ . ...
Diagnostic methods The present invention provides diagnostic methods using the polynucleotides and APCs of the invention. The methods can be used to detect the presence of an antigen-specific CD4+ and/or CD8+ T cell which binds an antigen encoded by a polynucleotide of the invention.
The diagnostic methods of the invention include: ( 1 ) assays to predict the efficacy of an antigen encoded by a polynucleotide of the invention; (2) assays to determine the precursor frequency (i.e., the presence and number of) of immune effector cells specific for an antigen encoded by a polynucleotide of the invention and/or its natural counterpart: and (3) assays to determine the efficacy of an antigen encoded by a polynucleotide of the invention once it has been used in an immunomodulatory method of the invention.
Diagnostic methods of the invention are generally carned out under suitable conditions and for a sufficient time to allow specific binding to occur between an antigen encoded by a polynucleotide of the invention, usually presented by an APC of the invention, and an immune effector molecule, such as a TCR, on the surface of an immune effector cell, such as a CD4+ or CD8+ T
cell.
"Suitable conditions" and "sufficient time" are generally conditions and times suitable 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 within a pH range of between S and 9. A variety of buffered solutions are known in the art, can be used in the diagnostic methods of this invention, and include, but are not limited to, phosphate-buffered saline. Sufficient time for binding and response will generally be between about 1 second and about 24 hours after exposure of the sample to the antigen encoded by a polynucleotide of the invention.
In some embodiments, the invention provides diagnostic ssays to predict the efficacy of an antigen encoded by a polynucleotide of the invention, presented by an APC of the invention. In some of these embodiments, defined T cell epitopes are used to clinically characterize tumors and viral pathogens in order to .. . _ ,. determine, in advance, the predicted efficacy of an in vivo vaccine trial. This can be achieved by a simple proliferation assay of a patient's peripheral blood mononuclear cells using defined T cell epitopes as stimulators. Peptides which elicit a response are viable vaccine candidates for that patient.
In other embodiments, assays are provided to determine the precursor frequency (i.e., the presence and number of) of resting (naive) immune effector cells specific for an antigen encoded by a polynucleotide of the invention and which therefore have the potential to become activated. In these embodiments, an antigen-presenting cell bearing on its surface an antigen encoded by a polynucleotide of the invention is used to detect the presence of immune effector cells in a biological sample which bind specifically to the natural epitope. A
functional assay is used to determine (and quantitate) the antigen-specific immune effector cells. As an illustrative example, PBMCs are isolated from a subject with a tumor. A sample of these PBMCs is cultured together for a suitable time with the tumor cells from the same subject. A second sample of these PBMCs is cultured together for a suitable time with surrogate APCs pulsed with an antigen encoded by a polynucleotide of the invention which corresponds to a natural epitope(s) expressed on the surface of the tumor. Both tumor cells and surrogate APCs are loaded with''Cr. By comparing the amount of 5'Cr release from the tumor cell and the antigen-pulsed surrogate APC, one can determine the precursor frequency of immune effector cells which are specific for tumor and the precursor frequency of immune effector cells which are specific for an antigen encoded by a polynucleotide. Functional assays include, but are not limited to, immune effector cell proliferation, cytokine production, specific lysis of an APC.
In other embodiments, the efficacy of an immunomodulatory method, including immunomodulatory methods of the invention, in modulating an immune response to an antigen encoded by a polynucleotide of the invention and/or its natural counterpart, can be tested using diagnostic assays of the invention.
These diagostic assays are also useful to assess or monitor the efficacy of an immunotherapeutic agent. In some of these embodiments, the method allows detection of immune effector cells, which may be activated CD4+ or CD8+ T
cells, . . which. have become. activated or anergized as a result of exposure to an.
antigen encoded by a polynucleotide of the invention. A sample containing cells from a subject can be tested for the presence of CD4+ or CD8+ T cells which have become activated or anergized as a result of binding to an antigen encoded by a polynucleotide of the invention. In some embodiments, the method comprises the steps of: (a) contacting an immobilized APC presenting an antigen encoded by a polynucleotide of the invention on its surface bound to Class I and Class II
MHC
molecules with a biological sample under suitable conditions and for a time sufficient to allow binding of an immune effector cell which bears on its surface an antigen receptor specific for the peptide, thereby immobilizing the antigen-specific immune effector cell; and (b) contacting the immobilized immune effector cell with a detestably labeled molecule, such as an antibody, which specifically binds the immune effector cell. In other embodiments, the method comprises the steps of (a) contacting an immobilized antigen-presenting matrix which presents an antigen encoded by a polynucleotide of the invention on its surface bound to a Class I or Class II MHC molecule with a biological sample under suitable conditions and for a time sufficient to allow binding of an immune effector cell which bears on its surface an antigen receptor specific for the peptide, thereby immobilizing the antigen-specific immune effector cell; and (b) performing a functional assay on the immobilized immune effector cell. Once the immune effector cell is bound to the immobilized APC presenting on its surface an antigen encoded by a polynucleotide of the invention, it can be labeled on the basis of characteristic cell surface molecules, including, but not limited to, CD4, CDB, and cell surface markers specific for activated T cells. A variety of cell surface markers specific to populations of immune effector cells are known to those skilled in the art and have been described in numerous publications.
see, for example, The Leukocyte Antigen Facts Book, Barclay et al., eds., 1995, Academic Press. Antibodies to these markers are commercially available from. inter alia, Beckman Coulter. The immobilized immune effector cell can also be characterized by presence of mRNA and/or proteins in the cytosol which are characteristic of a given T cell type in a given activated or anergic state. A
,. ~ .. ,. characteristic mRNA can be detected by any known.~means, including, but not limited to, a polymerase chain reaction. A detestably labeled antibody to a cell surface marker can be contacted with the immobilized immune effector cell under suitable conditions and for a time sufficient to allow specific binding. If necessary or desired, the labeled cells can be physically removed from unbound label or excess unbound label can be inactivated. The requirements of an antibody specific for a cell surface marker on an immune effector cell are that the antibody bind specifically and that the antibody not interfere with binding between a TCR and the immobilized synthetic antigenic peptide epitope.

Labels which may be employed are known to those skilled in the art and include, but are not limited to, traditional labeling materials such as fluorophores, radioactive isotopes, chromophores, and magnetic particles. Enzyme labels include, but are not limited to, luciferase; a green fluorescent protein (GFP), for example, a GFP from Aequorea victoria, or any of a variety of GFP known in the art; (3-galactosidase, chloramphenicol acetyl transferase. See, for example, Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987, and periodic updates). Any assay which detects the label, either by directly or indirectly, is suitable for use in the present invention. Assays include colorimetric, fluorimetric, or luminescent assays, radioimmunoassays or other immunological assays.
Immunomodulatory methods The invention provides methods of modulating an immune response in an individual to an antigen encoded by a polynucleotide of the invention, and thus to the corresponding natural epitope. Immunomodulatory methods of the invention include methods that result in induction or increase, as well as methods that result in suppression or reduction, of an immune response in a subject, and comprise administering to the subject an effective amount of a polynucleotide, or an APC, or an immune effector cell, of the invention in formulations and/or under conditions that result in the desired effect on an immune response (or lack thereof) M. ~ . . ao :the peptide antigen. Immunomodulatory methods of the invention.include ..
vaccine methods, adoptive immunotherapy, and methods to induce T cell unresponsiveness, or anergy.
Various methods are known to evaluate T celi activation. CTL activation can be detected by any known method, including but not limited to, tritiated thymidine incorporation (indicative of DNA synthesis), and examination of the population for growth or proliferation, e.g., by identification of colonies.
Alternatively, the tetrazolium salt MTT (3-(4,5-dimethyl-thazol-2-yl)-2,5-diphenyl tetrazolium bromide) may be added. Mossman (1983) J. Immunol.
Methods 65:55-63; Niks and Otto (1990) J. Immunol. Methods 130:140-151.

Succinate dehydrogenase. found in mitochondria of viable cells, converts the MTT to formazan blue. Thus, concentrated blue color would indicate metabolically active cells. In yet another embodiment, incorporation of radiolabeh e.g., tritiated thymidine, may be assayed to indicate proliferation of cells. Similarly, protein synthesis may be shown by incorporation of 3'S-methionine. In still another embodiment, cytotoxicity and cell killing assays, such as the classical chromium release assay, may be employed to evaluate epitope-specific CTL activation. To detect activation of CD4+ T cells, any of a variety of methods can be used, including, but not limited to, measuring cytokine production; and proliferation, for example, by tritiated thymidine incorporation Release of 5' Cr from labeled target cells is a standard assay which can be used to assess the number of peptide-specific CTLs in a biological sample.
Tumor cells, or APCs of the invention, are radiolabeled as targets with about p,Ci of Na2 ''Cr04 for 60 minutes at 37° C, followed by washing. T
cells and target cells (~1 x 104/well) are then combined at various effector-to-target ratios in 96-well, U-bottom plates. The plates are centrifuged at 100 x g for 5 minutes to initiate cell contact, and are incubated for 4-16 hours at 37°C with

5% C02.
Release of''Cr is determined in the supernatant, and compared with targets incubated in the absence of T cells (negative control) or with 0.1% TRITONTM X-100 (positive control). See. e.g., Mishell and Shiigi, eds. Selected Methods in Cellular Immunology (1980) W.H. Freeman and Co.
. . , . . . . . , . The: formulation of an APC of the invention will vary;
depending on the desired result. In general, peptides presented on an APC by Class I and Class II
MHC molecules, together with the appropriate co-stimulatory molecules, will result in induction of an immune response to the peptide. An anergic (or unresponsive) state may be induced in T lymphocytes by presentation of an antigen by an APC of the invention which contains appropriate MHC molecules on its surface, but which lacks the appropriate co-stimulatory molecules.
Polynucleotides of the invention can be administered in a gene delivery vehicle or by inserting into a host cell which in turn recombinantly transcribes, translates and processed the encoded polypeptide. Isolated host cells containing a polynucleotide of the invention in a pharmaceutically acceptable earner can be combined with appropriate and effective amount of an adjuvant, cytokine or co-stimulatory molecule for an effective vaccine regimen. In some embodiments, the host cell is an APC, such as a dendritic cell. The host cell can be further modified S by inserting of a polynucleotide coding for an effective amount of either or both of a cytokine a co-stimulatory molecule.
The methods of this invention can be further modified by co-administering an effective amount of a cytokine or co-stimulatory molecule to the subject.
The agents provided herein as effective for their intended purpose can be administered to subjects having a disease to be treated with an immunomodulatory method of the invention or to individuals susceptible to or at risk of developing such a disease. When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. Therapeutic amounts can be empirically determined and will vary with the pathology or condition being treated, the subject being treated and the efficacy and toxicity of the therapy.
The amount of a polynucleotide or APC or immune effector cell of the invention will vary depending, in part, on its intended effect, and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of adminitstration, and . . ~ . . , ... ., nature of the Formulation, the mammal's body weight, surface area, age, and general condition and the particular peptide to be administered. Cells can be administered once, followed by monitoring of the clinical response, such as diminution of disease symptoms or tumor mass. Administration may be repeated on a monthly basis, for example, or as appropriate. Those skilled in the art will appreciate that an appropriate administrative regimen would be at the discretion of the physician or veterinary practitioner.
Administration in vivo can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated.
Single or multiple administrations can be carned out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
The agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
More particularly, an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including nasal, topical (including transdermal, aerosol, buccal and sublingual), parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease or condition being treated.
Vaccines for cancer treatment and prevention In one embodiment, immunomodulatory methods of the present invention comprise vaccines for cancer treatment. Cancer cells contain many new antigens potentially recognizable by the immune system. Given the speed with which epitopes can be identified, custom anticancer vaccines can be generated for affected individuals by isolating TILs from patients with solid tumors, . , , . determining their MHC restriction, and assaying these CTLs against the . . .
appropriate library for reactive epitopes. These vaccines will be both treatments for affected individuals as well as preventive therapy against recurrence (or establishment of the disease in patients which present with a familial genetic predisposition to it). Inoculation of individuals who have never had the cancer is expected to be quite successful as preventive therapy, even though a tumor antigen-specific CTL response has not yet been elicited, because in most cases high affinity peptides seem to be immunogenic suggesting that holes in the functional T cell repertoire. if they exist, may be relatively rare. Sette et al.
(1994) J. Immunol., 153:5586-5592. In mice, vaccination with appropriate epitopes not only eliminates established tumors but also protects against tumor re-establishment after inoculation with otherwise lethal doses of tumor cells.
Bystryn et al. (1993) Supra.
Vaccines, for diseases caused by pathogenic organisms Polynucleotides and APCs of the present invention are also useful in methods to induce (or increase, or enhance) an immune response to a pathogenic organism. These include pathogenic viruses, bacteria, and protozoans.
Viral infections are ideal candidates for immunotherapy. Immunological responses to viral pathogens are sometimes ineffective as in the case of the lentiviruses such as HIV which causes AIDS. The high rates of spontaneous mutation make these viruses elusive to the immune system. However, a saturating profile of CTL epitopes presented on infected cells will identify shared antigens among different serotypes in essential genes that are largely intolerant to mutation which would allow the design of more effective vaccines.
Adoptive Immunotherapy Methods The expanded populations of antigen-specific immune effector cells and APCs of the present invention find use in adoptive immunotherapy regimes and as vaccines.
Adoptive immunotherapy methods involve, in one aspect, administering to a subject a substantially pure population of educated, antigen-specific immune effector cells made by culturing naive immune effector cells with APCs as .. . ~ . described above. In some embodiments, the APCs are dendritic cells.
..
In one embodiment, the adoptive immunotherapy methods described herein are autologous. In this case, the APCs are made using parental cells isolated from a single subject. The expanded population also employs T cells isolated from that subject. Finally, the expanded population of antigen-specific cells is administered to the same patient.
In a further embodiment, APCs or immune effector cells are administered with an effective amount of a stimulatory cytokine, such as IL-2 or a co-stimulatory molecule.

Methods of inducing T cell anergy Synthetic antigenic peptide epitopes of the present invention are useful in methods to induce T cell unresponsiveness, or anergy. Disorders which can be treated using these methods include autoimmune disorders, allergies, and allograft rejection.
Autoimmune disorders are diseases in which the body's immune system responds against self tissues. They include most forms of arthritis, ulcerative colitis, and multiple sclerosis. Polynucleotides of the invention encoding antigens corresponding to endogenous elements that are recognized as foreign can be used in the development of treatments using gene therapy or other approaches. For example, synthetic CTL epitopes, which can act as "suicide substrates" for CTLs that mediate autoimmunity, can be designed as described above. That is to say, peptides which have a high affinity for the MHC allele but fail to activate the TCR could effectively mask the cellular immune response against cells presenting 1 S the antigen in question. In support of this approach, it is believed that the long latency period of the HIV virus is due to an antiviral immune response and a mechanism by which the virus finally evades the immune system is by generating epitopes that occupy the MHC molecules but do not stimulate a TCR lytic response, inducing specific T cell anergy. Klenerman et al. (1995) Eur. J.
Immunol.. 25:1927-I 93 I .
In vitro stimulation of T cells through the complex of T cell-antigen . ~. receptor and CD3 alone in the absence of other signals, induces T cell anergy or paralysis. T cell activation as measured by interleukin-2 production and proliferation in vitro requires both antigenic and co-stimulatory signals engendered by cell to cell interactions among antigen-specific T cells and antigen presenting cells. Various interactions of these CD2 proteins on the T-cell surface with CD58 (LFA-3) proteins and antigen-presenting cells, those of CD1 la/CD18 (LFA-I) proteins with CD54 (ICAM-1) proteins and those of CDS proteins with CD72 proteins can impart such a co-stimulatory signal in vitro. Cytokines derived from antigen-presenting cells (e.g., interleukin-1 and interleukin-6) can also provide co-stimulatory signals that result in T-cell activation in vitro.
The delivery of both antigenic and co-stimulatory signals leads to stable transcription of the interleukin-2 gene and other pivotal T cell-activation genes. The foregoing co-stimulatory signals depend on protein kinase C and calcium. Potent antigen presenting cells express CD80 (B7 and BB I ) and other related surface proteins and many T cells express B7 binding proteins, namely CD28 and CTLA-4 proteins. Binding of CD80 by CD28 and CDLA-4 stimulates a T cell co-stimulatory pathway that is independent of protein kinase C and calcium leading to vigorous T cell proliferation. The stimulation of B cells also depends on the interaction between the specific antigen and the cell-surface immunoglobulin.
T cell derived cytokines (e.g., interleukins l and 4), physical contact between T cells and B cells through specific pairs of receptors and co-receptors, or both, provide the signal or signals essential for B cell stimulation.
Conventional routes of administration are used. A T-cell stimulating or anergy producing amount (or therapeutically effective amount as described above) of an immunotherapeutic antigen-superantigen polymer according to the invention is contacted with the target cells. By "T-cell anergy effective amount" is intended an amount which is effective in producing a statistically significant inhibition of a cellular activity mediated by a TCR. This may be assessed in vitro using T-cell activation tests. Typically, T-cell anergy or activation is assayed by tritiated thymidine incorporation in response to specific antigen.
One way in which T cell anergy can be induced is to present to a T cell an APC which presents an antigens in MHC Class I anti Class II molecules, but which lack co-stimulatory molecules necessary to activate a T cell. For example, a cell other than a normal antigen presenting cell (APC), which has been transfected with MHC antigen to which a selected T cell clone is restricted, can be used.
Resting T cells are provided with an appropriate peptide recognized by the resting T cells in the context of the MHC transfected into a cellular host other than an APC. The MHC is expressed as a result of introduction into a mammalian cell other than an antigen presenting cell of genes constitutively expressing the a and ~i chains of the MHC class II, or an MHC Class I molecule together with invariant chain. Importantly, these cells do not provide other proteins, either cell surface proteins or secreted proteins, associated with antigen presenting cells, which together with the MHC and peptide result in co-stimulatory signals.
To determine whether anergy has been induced, the T cells to be tested can be cultured together with an APC which presents an antigen encoded by a polynucleotide of the invention in MHC Class I and Class II molecules together with co-stimulatory molecules necessary to activate the T cell. The cultures are incubated for about 48 hours, then pulsed with tritiated thymidine and incorporation measured about 18 hours later. The absence of incorporation above control levels, where the T-cells are presented with antigen presenting cells which do not stimulate the T cells, either due to using an MHC to which the T cells are not restricted or using a peptide to which the T cells are not sensitive, is indicative of an absence of activation. One may use other conventional assays to determine the extent of activation, such as assaying for IL-2, -3, or -4, cell surface proteins associated with activation, e.g. CD71 or other convenient techniques. Another method is to determine the expression of a protein which is 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 this invention are useful as cancer vaccines and in adoptive immunotherapy. The ability of autologous antigen-pulsed dendritic cells to induce a clinically relevant immune response has previously been reported. Hsu et al. (1996) Nature Med. 2(1):52-58. Using this clinical study as a guide, it is possible to administer an effective amount of the APCs as described herein to a subject to induce an anti-tumor immune response. After isolation and purification of DCs (day 0) purified pulsed dendritic cells were administered by subcutaneous injection on days 2, 28 and 56 and then 5 to 6 months later. At day 16, patients received subcutaneous injections with either keyhole limpet hemocyanin or idiotype protein in saline at a site separate from intraveneous injection of the pulsed DCs.

The expanded populations of antigen-specif c immune effector cells of the present invention also find use in adoptive immunotherapy regimes and as vaccines.
Adoptive immunotherapy methods involve, in one aspect, administering to S a subject a substantially pure population of educated, antigen-specific immune effector cells made by culturing naive immune effector cells with APCs as described above. Preferably, the APCs are dendritic cells.
In one embodiment, the adoptive immunotherapy methods described herein are autologous. In this case, the APCs are made using parental cells isolated from a single subject. The expanded population also employs T cells isolated from that subject. Finally, the expanded population of antigen-specific cells is administered to the same patient.
In another embodiment, the adoptive immunotherapy methods are allogeneic. Here, cells from two or more patients are used to generate the APCs, and stimulate production of the immune effector cells. For instance, cells from other healthy or diseased subjects can be used to generate antigen-specific cells in instances where it is not possible to obtain autologous T cells and/or dendritic cells from the subject providing the biopsy. The expanded population can be administered to any one of the subjects from whom cells were isolated, or to another subject entirely.
In a further embodiment, APCs or immune effector cells are administered with an effective amount of a stimulatory cytokine, such as IL-2 or a co-stimulatory molecule.
The agents identified herein as effective for their intended purpose can be administered to subjects having tumors or individuals susceptible to or at risk of a tumor. When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject. To determine patients that can be beneficially treated, a tumor regression can be assayed.
Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the therapy.

When delivered to an animal, the method is useful to further conf rm efficacy of the agent.
Administration in vivo can be effecte3 in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated.
Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be found below.
Also within the scope of this invention is an epitope or wild-type antigenic peptide corresponding to a yet unidentified protein. A common strategy in the search for tumor antigens is to isolate tumor-specific T-cells and attempt to identify the antigens recognized by these cells. In patients with cancer, specific CTLs have been derived from lymphocytic infiltrates present~at the tumor site.
Weidmann, et al., supra. These TILs are unique cell population that can be traced back to sites of disease when they are labeled with indium and adoptively transferred. Alternatively, large libraries of putative antigens can be produced and tested. Using the "phage method" (Scott and Smith (1990) Science 249:386-390;
Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6387-6382; Devlin et al. (1990) Science 249:404-406), very large libraries can be constructed. Another approach uses primarily chemical methods, of which the Geysen method (Geysen et al.
(1986) Mol. Immunol. 23:709-715; Geysen et al. (1987) J. Immunol. Method 102:259-274) and the method of Fodor et al. ( 1991 ) Science 251:767-773, are examples. Furka et al. (1988) 14th Inter. Cong. Bio. Vol. 5, Abst. FR:013;
Furka (1991) Inter. J. Peptide Protein Res. 37:487-493), Houghton (U.S. Patent No.
4,683,211, issued December 1986) and Rutter et al. (U.S. Patent No. 5,010,175, issued April 23, 1991 ) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
In a further aspect of this invention, Solid-PHase Epitope REcovery ("SPHERE", described in PCT WO 97/35035) can be used to identify tumor antigens. Briefly, SPHERE can be used to identify antigens by creating a library of molecules, preferably peptides, and attaching one type of molecule to a solid support via a releasable linker. At least a portion of the molecules bound to each support can be released and it can be determined if the antigen-specific immune effector cells recognized the peptide.
Thus, this invention also provides a screen to identify novel wild-type antigens that can be further modified and used to induce a cellular and a humoral immune response in the subject. The antigens and their biological activity in vitro and in vivo are positive controls. Using the methods described herein, the biological activity of the isolated antigen and its secreted form can be compared to their biological activity.
Furthermore, the invention provides a method for cloning the cDNA and genomic DNA encoding such a protein by generating degenerate oligonucleotides probes or primers based on the sequence of the epitope. Compositions comprising the nucleic acid and a carrier, such as a pharmaceutically acceptable carrier, a solid support or a detectable label, are further provided by this method as well as methods for detecting the sequences in a sample using methods such as Northern analysis, Southern analysis and PCR.
Further provided by this invention are therapeutic and diagnostic oligopeptide sequences determined according to the foregoing methods.
Compositions comprising the oligopeptide sequence and a carrier, such as a pharmaceutically acceptable Garner, a solid support or a detectable label, are further provided by this method as well as methods for detecting the oligopeptide sequence in a sample using methods such as Western analysis and ELISA.
Harlow and Lane ( 1989), supra.
The following examples are provided to illustrate, but not limit, the invention.
EXAMPLES

Identiftcation of Tumor Associated Antigens Any conventional method, e.g., expression cloning methodology as described in Kawakami et al. (1994) Proc. Natl. Acad. Sci. 91:351-19, can be used to identify a novel tumor-associated antigen. Briefly, in this method, a library of cDNAs corresponding to mRNAs derived from tumor cells is cloned into an expression vector and introduced into target cells which are subsequently incubated with cytotoxic T cells. One identifies pools of cDNAs that are able to stimulate the CTL and through a process of sequential dilution and re-testing of less complex pools of cDNAs one is able to derive unique cDNA sequences that are able to stimulate the CTL and thus encode the cognate tumor antigen.
SAGE (U.S. Patent No. 5,695,937) and SPHERE (described in PCT WO
97/35035), also can be used to identify putative antigens for use in the subject invention.
SAGE analysis can be employed to identify the antigens recognized by 1 S expanded immune effector cells such as CTLs, by identifying nucleotide sequences expressed in the antigen-expressing cells. Briefly, SAGE analysis begins with providing complementary deoxyribonucleic acid (cDNA) from ( 1 ) the antigen-expressing population and (2) cells not expressing that antigen. Both cDNAs can be linked to primer sites. Sequence tags are then created, for example, using the appropriate primers to amplify the DNA. By measuring the differences in these tag sets between the two cell types, sequences which are aberrantly expressed in the antigen-expressing cell population can be identified.
Another method which may be used to identify antigenic epitopes is Solid PHase Epitope REcovery ("SPHERE") which is described in PCT WO 97/35035.
Briefly, roughly speaking, peptide libraries are loaded onto beads and inserted into 96-well plates. The plates with 1000 beads per well will accommodate 106 beads; ten 96-well plates with 100 beads per well will accommodate 105 beads.
In order to minimize both the number of CTL cells required per screen and the amount of manual manipulations, the eluted peptides can be further pooled to yield wells with any desired complexity. For example, based on experiments with soluble libraries, it should be possible to screen 10' peptides in 96-well plates (10,000 peptides per well) with as few as 2 X lOb CTL cells. After cleaving a percentage of the peptides from the beads, incubating them with gamma-irradiated APCs or foster APCs, and the cloned CTL line(s), positive wells are determined by 3H-thymidine incorporation. Alternatively, as pointed out above. cytokine production or cytolytic ' ~ Cr-release assays may be used (Coutic et al. ( I
992) Int.
J. Cancer. 50:289-291 ). Beads from each positive well are then separated and assayed individually, utilizing an additional percentage of the peptide from each bead. Positive individual beads will then be decoded, identifying the reactive-amino acid sequence. Analysis of all positives will give a partial profile of conservatively substituted epitopes which stimulate the CTL clone tested. At this point, the peptide can be resynthesized and retested. Also, a second library (of minimal complexity) can be synthesized with representations of all conservative substitutions in order to enumerate the complete spectrum of derivatives tolerated by a particular CTL. By screening multiple CTLs (of the same MHC restriction) simultaneously, the search for crossreacting epitopes is greatly facilitated.
Alternatively, muteins of the antigen as well as allogeneic and antigens from a different species, of previously characterized antigens are useful in the subject invention. MART-1 and gp100 are melanocyte differentiation antigens specifically recognized by HLA-A2 restricted tumor-infiltrating lymphocytes (TILs) derived from patients with melanoma, and appear to be involved in tumor regression (Kawakami et al. ( 1994) Proc. Natl. Acad. Sci. U.S.A. 91:6458-62;
Kawakami et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:91:3515-9). Recently, the mouse homologue of human MART-1 has been isolated. The full-length open -ending frame of the mouse MART-I consists of 342 bp, encoding a protein of 113 amino acid residues with a predicted molecular weight of ~l 3 kDa.
Alignment of human and marine MART-1 amino acid sequences showed 68.6%
identity.
In another embodiment, the described method for the identification of CD8+ MHC Class I restricted CTL epitopes can be applied to the identification of CD4+ MHC Class II restricted helper T-cell (Th) epitopes. In this case, MHC
Class II allele-specific libraries are synthesized such that haplotype-specific anchor residues are represented at the appropriate positions. MHC Class II
agretopic motifs have been identified for the common alleles (Rammensee (1995) Curr. Opin. Immunol. 7:85-96; Altuvia et al. (1994) Mol. Immunol. 24:375-379;
Reay et al. ( 1994) J. Immunol. 152:3946-3957; Verreck et al. ( 1994) Eur. J.
Immunol. 24:375-379; Sinigaglia and Hammer ( 1994) Curr. Opin. Immunol.

6:52-56: Rotzschke and Falk ( 1994) Curr. Opin. Immunol. 6:45-51 ). The overall length of the peptides will be 12-20 amino acid residues, and previously described methods may be employed to limit library complexity. The screening process is identical to that described for MHC Class I-associated epitopes except that B
lymphoblastoid cell lines (B-LCL) are used for antigen presentation rather than T2 cells. In a preferred aspect, previously characterized B-LCLs that are defective in antigen processing (Mellins et al. ( I 991 ) J. Exp. Med.
174:1607-1615); thus allowing specific presentation of exogenously added antigen, are employed. The libraries are screened for reactivity with isolated CD4+ MHC
Class II allele-specific Th cells. Reactivity may be measured by 3H-thymidine incorporation according to the method of Mellins, et al. supra., or by any of the methods previously described for MHC Class I-associated epitope screening.
In vitro confirmation of the immunogenicity of a putative antigen of this invention can be confirmed using the method described below which assays for the production of CTLs. However, weakly immunogeneic antigens also can be used for the making of compositions and in the methods of this invention.
Using these sequences and recombinant expression techniques summarized below, polypeptides and proteins can be made for presentation on the APC of this invention.
Tumor Protection in Animal Models The transcription cassette shown in the Figure is contained within an adenoviral vector such that expression of the genes leads to the production of intracellular and secreted forms of the same protein. The adenoviral vector can be employed either in vivo for direct immunization as described in Zhai et al. ( 1996) J. Immunol. 156:700-10 or ex vivo wherein antigen presenting cells (preferably dendritic cells) derived from a host are transduced with the adenoviral vector and the genetically modified APCs are subsequently infused back into the host as described in Ribas et al. (1997) Cancer Research 57:2865-9. For in vivo immunization, C57BL/6 mice are injected subcutaneously at two sites (1.SE9 IU
per site) with the adenoviral vector encoding the wild type and secreted forms of the same antigen. Two weeks later, animals are challenged with a lethal dose (2 x 10'x) of marine B 16F 10 melanoma tumor cells by subcutaenous injection.
Animals are scored for survival and tumor measurements are taken. For ex vivo dendritic cell therapy, bone marrow cells are harvested from C57BL/6 mice and cultured in vitro with GM-CSF and IL4 to derive dendritic cells. The DCs are infected at an MOI of S00 with the adenoviral vector overnight, and the next day the DCs (S x 105) are washed and infused into recipient C57BL/6 mice via tail vein injection. Two weeks later, animals are challenged with a lethal dose of marine B 16F 10 melanoma tumor cells via subcutaneous injection as above and tumor size and survival are recorded as a function of time.
It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and the following examples are intended to illustrate and not limit the scope of the invention. For example, any of the above-noted compositions and/or methods can be combined with known therapies or compositions. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Claims (37)

1. A polynucleotide encoding an antigen that is processed and presented with an MHC Class I molecule on an antigen-presenting cell (APC) and an antigen that is processed and presented with an MHC Class II molecule on the APC.

2. The polynucleotide of claim 1, comprising:
(a) a first coding sequence which comprises a nucleotide sequence encoding the antigen; and (b) a second coding sequence which comprises a nucleotide sequence encoding the antigen and a nucleotide sequence encoding an amino acid sequence that promotes retention of the encoded antigen in the endoplasmic reticulum (ER).

3. The polynucleotide of claim 2, wherein the amino acid sequence that promotes retention of the encoded antigen in the ER is selected from the group consisting of KDEL, HDEL, DDEL, ADEL, SDEL, RDEL, KEEL, QEDL, HIEL, HTEL, and KQDL.

4. The polynucleotide of claim 2, further comprising a third coding sequence which comprises a nucleotide sequence encoding the antigen and a nucleotide sequence encoding an amino acid sequence that directs the encoded antigen into 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 polynucleotide of claim 7, wherein the tumor-associated antigen is selected from the group consisting of gp100, MUC-1, MART-1, HER-2, CEA, PSA, prostate specific membrane antigen (PSMA), tyrosinase, tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2), NY-ESO-1 and GA733 antigen.

9. A gene delivery vehicle 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 theAPC is a dendritic cell.

13. A composition comprising the polynucleotide of claim 1, a carrier.

14. A polypeptide encoded by the polynucleotide of claim 1.

15. A composition comprising a polypeptide encoded by the polynucleotide of claim 1, and a carrier.

16. A method of expressing the polynucleotide of claim 1, comprising growing a cell comprising under conditions which favor expression of the polynucleotide.

17. A method of increasing presentation of a peptide on the surface of an antigen-presenting cell, comprising introducing into the cell a polynucleotide of claim 1, under conditions which favor expression of the polynucleotide.

18. A cell produced by the method of claim 17.

19. A method of producing a population of educated, antigen-specific immune effector cells, comprising culturing naive immune effector cells with an antigen-presenting cell transduced with a 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 a polynucleotide of claim 1 under conditions that induce an immune response to the antigen.

22. The method of claim 21, further comprising administering an effective amount of a cytokine to the subject.

23. The method of claim 22, wherein a gene coding for the cytokine is administered to the subject.

24. The method of claim 21, further comprising administering an effective amount of a co-stimulatory molecule to the subject.

25. The method of claim 24, wherein a gene coding for the co-stimulatory molecule is administered to the subject.

26. The method of claim 21, wherein the antigen is a tumor-associated antigen.

27. The method of claim 26, wherein the tumor-associated antigen is selected from the group consisting of gp 100, MUC-1, MART-1, HER-2, CEA, PSA, prostate specific membrane antigen (PSMA), tyrosinase, tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2), NY-ESO-1, and GA733 antigen.

28. A method of inducing an immune response to a native antigen in a subject, comprising administering to the subject an effective amount of a host cell of claim 11 under conditions that induce an immune response to the antigen.

29. The method of claim 28, further comprising administering an effective amount of a cytokine to the subject.

30. The method of claim 28, wherein a gene coding for the cytokine is administered to the subject.

31. The method of claim 28, further comprising administering an effective amount of a co-stimulatory molecule to the subject.

32. The method of claim 21, wherein a gene coding for the co-stimulatory molecule is administered to the subject.

33. The method of claim 28, wherein the native antigen is a tumor-associated antigen.

34. The method of claim 33, wherein the tumor-associated antigen is selected from the group consisting of gp100, MUC-1, MART-1, HER-2, CEA, PSA, prostate specific membrane antigen (PSMA), tyrosinase, tyrosinase related proteins 1 or 2 (TRP-1 and TRP-2), NY-ESO-1, and the GA733 antigen.

35. The method of claim 28, wherein the host cell further expresses a cytokine.

3d. The method of claim 28, wherein the host cell further expresses a co-stimulatory molecule.

37. A method of adoptive immunotherapy, comprising administering to an individual an effective amount of a population of educated, antigen-specific immune effector cells of claim 20.