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WO2005006961A2 - Peptides d'antigenes dd96 - Google Patents

Peptides d'antigenes dd96 Download PDF

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Publication number
WO2005006961A2
WO2005006961A2 PCT/US2004/022854 US2004022854W WO2005006961A2 WO 2005006961 A2 WO2005006961 A2 WO 2005006961A2 US 2004022854 W US2004022854 W US 2004022854W WO 2005006961 A2 WO2005006961 A2 WO 2005006961A2
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cells
cell
gene
peptide
antigen
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PCT/US2004/022854
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English (en)
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WO2005006961A3 (fr
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Charles A Nicolette
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Genzyme Corporation
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Publication of WO2005006961A3 publication Critical patent/WO2005006961A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages

Definitions

  • the invention relates to compositions and methods useful in therapeutic, diagnostic and screening methods for human cancers and related malignancies.
  • Antigen-specific T cell activity has provided a venue for developing novel strategies for anti-cancer vaccines based on the observation that antigenic epitopes presented by molecules of the Major Histocompatibility Complex (MHC) play a central role in the establishment, maintenance and execution of mammalian immune response.
  • MHC Major Histocompatibility Complex
  • Antigen-specific cytotoxic T lymphocytes (CTLs) that recognize certain cancer antigens have been used to attack cells expressing these antigens to reject established tumors.
  • CTLs cytotoxic T lymphocytes
  • Anti-tumor T cells are localized within various cancer patients' tissues, including in the blood (where they can be found in the peripheral blood mononuclear cell fraction), in primary and secondary lymphoid tissue, e.g., the spleen, in ascites fluid in ovarian cancer patients (tumor associated lymphocytes or TALs) or within the tumor itself (tumor infiltrating lymphocytes or TILs).
  • TILs have been the most useful in the identification of tumor antigens and tumor antigen-derived peptides recognized by T cells.
  • In vitro studies have shown that the ability of a particular peptide to function as a T cell epitope requires that it bind effectively to the antigen presenting domain of an MHC molecule and also that it display an appropriate set of amino acids that can be specifically recognized by a T cell receptor molecule.
  • the development of peptide-based vaccines and therapeutics has relied either on the direct characterization of peptides associated with Class I molecules or, alternatively, on the identification of putative epitopes by inspection of the primary structures of proteins for motif sequences. (See e.g., Deroo and Muller, (2001) Comb. Chem.
  • TILs manipulated to specifically recognize, bind and lyse cancer cells have been used as a means of adoptive T-cell therapy.
  • Conventional methods to generate TELs involve mincing tumor biopsy tissue and culturing the cell suspension in vitro in the presence of the T cell growth factor interleukin-2 (IL-2). Over a period of several days, the combination of the tumor cells and IL-2 can stimulate the proliferation of tumor specific T cells at the expense of tumor cells. In this way, the T cell population is expanded.
  • IL-2 T cell growth factor interleukin-2
  • the T cells derived from the first expansion are subsequently mixed with either mitomycin C-treated or irradiated tumor cells and cultured in vitro with IL-2 to promote further proliferation and enrichment of tumor reactive T cells.
  • IL-2 IL-2-induced tumor reactive T cells.
  • a potent anti-tumor T cell population can be recovered and used to identify tumor antigens via conventional but tedious expression cloning methodology.
  • these immunotherapeutic strategies while initially promising, have not provided a cancer vaccine.
  • tolerance a state of immunologic unresponsiveness to antigens, whether self or foreign.
  • T and B cells (1) clonal deletion, in which antigenic recognition leads to the activation-induced death of specific lymphocytes; (2) clonal anergy, in which lymphocytes are not killed but are rendered unresponsive to the recognized antigen; and (3) T cell-mediated suppression, in which regulatory T cells actively inhibit an immune response to an antigen.
  • Scientific American Medicine infra. Several factors help determine which of those responses will occur. Immature lymphocytes are more susceptible to induction of tolerance than are mature lymphocytes.
  • T cells thymus
  • B cells bone marrow
  • T cells recognize antigens that have been processed into peptides and presented in a complex with MHC molecules (self-MHC/peptide complexes). Consequently, immature T cells must be screened for their ability to recognize self-MHC. This screening takes place in the thymus gland. T cells bearing receptors that recognize self-MHC are subjected to the processes of positive and negative selection.
  • T cells bearing receptors with a moderate affinity for self-MHC/peptide complexes receive survival and maturation signals after receptor ligation. Once these cells mature, they are exported to the periphery.
  • Negative selection occurs when T cells bearing receptors with a high affinity for self-MHC/peptide complexes undergo activation-induced death.
  • the thymus gland is capable of presenting many self-antigens that are normally expressed outside of the thymus or during restricted developmental stages. This allows the elimination of most T cells bearing high-affinity receptors for self-MHC/peptide complexes and plays a major role in preventing autoimmunity in peripheral organs and thus explains the failure of recent therapeutic approaches. (See, Ochoa-Garay et al. (1997) Mol.
  • T cells bearing receptors capable of low-affinity interactions with self-MHC/peptide complexes potentially self-reactive T cells are normally found in peripheral lymphoid organs.
  • Peripheral tolerance prevents these cells from inducing autoimmune disease.
  • Peripheral tolerance is achieved in one of three ways. Perhaps the most common mechanism is the failure of T cells bearing low-affinity receptors to recognize self-antigen in the periphery. In this situation, the potentially self-reactive T cell is not activated and remains functionally na ⁇ ve. These cells are functional, however, as is shown by the fact that they can be activated by immunization with self-antigen delivered in the presence of immune adjuvants.
  • a second mechanism of peripheral tolerance involves the elimination of self-reactive T cells by apoptosis. This process is analogous to clonal deletion in the thymus. Whether peripheral deletion plays an important role in tolerance to self-antigens is not known.
  • a third mechanism of peripheral tolerance involves the acquisition of anergy after ligation of the T cell receptor complex. This antigen-nonresponsive state can be induced in several distinct ways. The most extensively characterized mechanism of anergy induction occurs when the T cell receptor is ligated in the absence of costimulation.
  • T cells After a T cell has bound with an antigen, the cell requires a so-called second signal delivered by one or more costimulatory molecules to be primed for an immune response.
  • T cells express several surface molecules that can transmit this second signal. These costimulatory receptors are engaged by ligands expressed on the surface of APCs.
  • T cells that are activated in the absence of costimulation acquire defects in the transcriptional control pathways that allow the production of IL-2, an important T cell autocrine growth factor. In vitro anergy can often be overcome by supplying exogenous IL-2 to anergic T cells.
  • Costimulatory signals can be delivered to T cells by soluble factors or cell-surface molecules expressed on APCs.
  • the most potent costimulatory signals are delivered when CD28, CD154 or both are ligated on the surface of T cells.
  • the ligand for CD 154 is CD40, a protein expressed on the surface of activated B cells, DCs and macrophages.
  • the ligands for CD28 (B7-1 (CD80), B7-2 (CD86) and related proteins) are expressed on the surface of APCs, such as DCs, monocytes and B cells. Their expression is induced when APCs are activated in the course of microbial infection. This property heightens the immune response in the setting of perceived danger.
  • B7-1 and B7-2 have overlapping immunostimulatory roles: mice lacking either protein are only partially deficient in generating an immune response to foreign antigen.
  • CTLA-4 a structurally related protein that turns off activated T cells.
  • the activated T cell initiates a program that will ensure its elimination at the conclusion of the immune response.
  • CTLA-4 has a higher affinity for B7-1 and B7-2.
  • CD28-B7 interactions may play a role in promoting tolerance to self-antigens.
  • CD28-B7 interactions are required for the maturation of a distinct class of regulatory T cells that help maintain peripheral tolerance.
  • These regulatory T cells (T reg ) are included in a subpopulation (5%-15%) of peripheral blood CD4 + T cells that express CD25, a subunit of the IL-2 receptor.
  • T reg regulatory T cells
  • the selective removal of CD4 + and CD25 + T cells from BALB/c mice results in the development of T cell-mediated autoimmune thyroiditis, gastritis and diabetes.
  • the CD4 + and CD25 + T reg cells that mature in the thymus gland bear receptors that have an intermediate affinity for self-MHC/peptide complexes.
  • CD4 + and CD25 + T cells In the periphery, antigen exposure confers the ability to suppress the activation of CD4 + and CD25 + T cells in an antigen-independent, cell contact-dependent manner. Although these cells secrete IL-10, a potent anti-inflammatory cytokine, their suppressive activity is cytokine independent.
  • CD4 and CD25 + T cells can suppress graft versus host disease in allotransplants and they can prevent autoimmune disease in several different animal models. Consequently, these cells probably play an essential role in maintaining peripheral tolerance to self-antigens.
  • compositions and methods for aiding in the diagnoses of the condition of a cell for identifying and/or distinguishing normal and neoplastic cells and for identifying potential therapeutic agents to reverse neoplasia and/or ameliorate the symptoms associated with the presence of neoplastic cells in a subject.
  • compositions and methods to reverse neoplasia and/or ameliorate the symptoms associated with neoplastic cells in vivo comprise a 9 amino acid DD96 polypeptide, having the sequences set forth in Table 1, infra.
  • the composition comprise polynucleotides encoding these polypeptides.
  • embodiments of the invention are directed to methods of diagnosing the condition of a cell by screening for the presence of a differentially expressed DD96 gene isolated from a sample containing or suspected of containing a cell that differentially expresses DD96, in which the differential expression of DD96 is indicative of the neoplastic state of the cell.
  • These cells include but are not limited to cells derived from epithelial origin, e.g., ovarian, prostate or lung.
  • Detection can be by any appropriate method, including for example, detecting the quantity of mRNA transcribed from the DD96 gene or the quantity of cDNA produced from the reverse transcription of the mRNA transcribed from the gene or the quantity of the polypeptide or protein encoded by the gene. These methods can be performed on a sample by sample basis or modified for high throughput analysis.
  • databases containing quantitative full or partial transcripts or protein sequences isolated from a cell sample can be searched and analyzed for the presence and amount of transcript or expressed gene product.
  • the methods are particularly useful for aiding in the diagnosis of cancers of epithelial origin, e.g., lung, prostate or ovarian.
  • Another aspect of the invention is a screen to identify therapeutic agents that reverse or treat neoplasia and tumors, wherein the cell and/or tumor is characterized by the differential expression of at least polypeptide DD96.
  • the method comprises contacting the cell previously identified as possessing this genotype with an effective amount of a potential agent and assaying for reversal of the neoplastic condition.
  • peptides and polynucleotides encoding the peptides that are useful as immunogens to treat or protect from neoplasia.
  • the peptides are presented in the context of an MHC complex.
  • the peptides identified in Table 1A are, in one aspect, provided in conjunction with an HLA-A1 molecule.
  • the peptides identified in Table IB are provided in conjunction with an HLA-
  • the peptides identified in Table 1C are provided in conjunction with an HLA-A3 molecule.
  • polynucleotides encoding the proteins, fragment(s) thereof or polypeptides shown in Table 1 (also referred to herein as gene expression product), gene delivery vehicles comprising these polynucleotides and host cells comprising these polynucleotides.
  • the proteins, polypeptides or fragment(s) thereof are also useful to generate antibodies that specifically recognize and bind to these molecules.
  • the antibodies can be polyclonal or monoclonal. These antibodies can be used to isolate protein or polypeptides expressed from the genes encoding the polypeptides. These antibodies are further useful for passive immunotherapy when administered to a subject.
  • the invention also provides isolated host cells and recombinant host cells that contain a polynucleotide encoding the peptides identified in Table 1 and/or fragment(s) thereof.
  • the cells can be prokaryotic or eukaryotic and by way of example only, can be any one or more of bacterial, yeast, animal, mammalian, human and particular subtypes thereof, e.g., stem cells, antigen presenting cells (APCs) such as dendritic cells (DCs) or T cells.
  • APCs antigen presenting cells
  • DCs dendritic cells
  • kits comprises at least one agent (e.g., probe, primer or antibody) that detects expression of DD96 and instructions for use.
  • agent e.g., probe, primer or antibody
  • the invention provides methods for active immunotherapy, such as, inducing an immune response in a subject by delivering the proteins, polypeptides and fragment(s) thereof, as described herein, to the subject.
  • the proteins and/or polypeptides can be delivered in the context of an MHC molecule.
  • the invention also provides immune effector cells raised in vivo or in vitro in the presence and at the expense of an antigen presenting cell that presents a polypeptide identified in Table 1 , supra, in the context of an MHC molecule.
  • the invention also provides a method of adoptive immunotherapy comprising administering an effective amount of these immune effector cells to a subject.
  • Yet another embodiment of the present invention is a method of reversing the neoplastic condition of a lung cell, wherein the cell is characterized by differential expression of DD96, by contacting the cell with a therapeutic agent.
  • FIGS. 1A through 1C show the results of the CTL assay.
  • the targets tested with each effector cell population were autologous DCs infected with either an empty vector adenoviral construct (lacking a transgene) or the construct containing the gene of interest (indicated to the right of each graph).
  • Figure 2 shows that DD96-educated T cells lyse tumor.
  • Ad/DD96-infected DCs were used to generate DD96-specific T cells as described herein.
  • the ability of these CTL to lyse a 51 Cr-labeled HLA-A2 " was assessed in breast tumor cell line, 21PT.
  • 21PT is was previously determined to express high levels of DD96 endogenously and is HLA-A2 " .
  • the cell line was assayed either infected with Ad/EV (-•-) or Ad/HLA-A2 (-
  • This experiment confirms that Ad/DD96-educated CTL can lyse tumor endogenously expressing the antigen in an HLA-A2-restricted fashion. Therefore, it confirms that at least one HLA-A2-restricted epitope derived from DD96 is naturally processed and presented by this tumor cell line.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polymer.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for guanine when the polynucleotide is RNA.
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • a “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.
  • a “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
  • polypeptide is used interchangeably with the term “protein” and in its broadest sense refers 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.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short.
  • peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. "Operatively linked” refers to a juxtaposition wherein the elements are in an arrangement allowing them to function.
  • 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.
  • compositions 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 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.
  • isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, are normally associated with in nature.
  • an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated with in its native or natural environment, e.g., on the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
  • a "concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated” or less than “separated” than that of its naturally occurring counterpart.
  • a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide.
  • a protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.
  • Gene delivery is terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction.
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • vector-mediated gene transfer by, e.g., viral infection/transfection or various other protein-based or lipid-based gene delivery complexes
  • techniques facilitating the delivery of "naked" polynucleotides such as electroporation, "gene gun” delivery and various other techniques used for the introduction of polynucleotides.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are known in the art to be capable of mediating transfer of genes to mammalian cells.
  • a "gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; recombinant yeast cells, metal particles; and bacteria or 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.
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof and a therapeutic gene.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • Retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Retro viruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
  • a DNA viral vector such as an adenovirus (Ad) or adeno-associated virus (AAV)
  • Ad adeno-associated virus
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof and a transgene.
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See e.g., WO 95/27071. Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild- type virus, have also been constructed. See e.g., WO 95/00655 and WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cell's genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466- 6470 and Lebkowski et al. (1988) Mol.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wl). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression. Gene delivery vehicles also include several non- viral vectors, including
  • DNA liposome complexes DNA liposome complexes, recombinant yeast cells and targeted viral protein-DNA complexes.
  • Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragment(s) thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
  • a "probe" when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
  • a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction.
  • Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes and proteins, including enzymes.
  • a "primer” is a short polynucleotide, generally with a free 3' -OH group that binds to a target or "template” potentially present in a sample of interest by hybridizing with the target and, thereafter, promoting polymerization of a polynucleotide complementary to the target.
  • PCR polymerase chain reaction
  • PCR is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers” or a “set of primers” consisting of an "upstream” and a “downstream” primer and a catalyst of polymerization, such as a DNA polymerase and, typically, a thermally-stable polymerase enzyme.
  • Methods for PCR are well- known in the art, and taught, for example in “PCR: A PRACTICAL APPROACH” (M. MacPherson et al, IRL Press at Oxford University Press (1991)).
  • a primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook et ah, supra.
  • An expression “database” denotes a set of stored data that represent a collection of sequences, which in turn represent a collection of biological reference materials.
  • cDNAs refers to complementary DNA that is mRNA molecules present in a cell or organism made into cDNA with an enzyme such as reverse transcriptase.
  • a “cDNA library” is a collection of all of the mRNA molecules present in a cell or organism, all turned into cDNA molecules with the enzyme reverse transcriptase, then inserted into “vectors” (other DNA molecules that can continue to replicate after addition of foreign DNA).
  • vectors for libraries include bacteriophage (also known as “phage"), viruses that infect bacteria, for example, lambda phage.
  • the library can then be probed for the specific cDNA (and thus mRNA) of interest.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides or proteins.
  • expression may include splicing of the mRNA in a eukaryotic cell.
  • "Differentially expressed” as applied to a gene refers to the differential production of the mRNA transcribed and/or translated from the gene or the protein product encoded by the gene.
  • a differentially expressed gene may be overexpressed or underexpressed as compared to the expression level of a normal or control cell (See also, Table 2 for expression levels of DD96 in various cancers).
  • 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, microarrays and chips.
  • solid support also includes synthetic antigen-presenting matrices, cells and liposomes.
  • a suitable solid phase support maybe selected on the basis of desired end use and suitability for various protocols.
  • 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 resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California).
  • polystyrene e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.
  • POLYHIPE® resin obtained from Aminotech, Canada
  • polyamide resin obtained from Peninsula Laboratories
  • a polynucleotide also can be attached to a solid support for use in high throughput screening assays.
  • PCT WO 97/10365 discloses the construction of high density oligonucleotide chips. See also, U.S. Patent
  • probes are synthesized on a derivatized glass surface also known as chip arrays.
  • Photoprotected nucleoside phosphoramidites are coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask and reacted with a second protected nucleoside phosphoramidite. The coupling/deprotection process is repeated until the desired probe is complete.
  • “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.
  • Hybridization reactions can be performed under conditions of different
  • stringency In general, a low stringency hybridization reaction is carried out at about 40 °C in lOx SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in l SSC. When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called “annealing" and those polynucleotides are described as “complementary”.
  • a double-stranded polynucleotide can be "complementary” or “homologous” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • “Complementarity” or “homology” is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.
  • a polynucleotide or polynucleotide 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.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • Metaplasia is a form of controlled cell growth in which one type of fully differentiated cell substitutes for another type of differentiated cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. 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. A metastatic cell or tissue means that the cell can invade and destroy neighboring body structures.
  • “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 3 H-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 "variable” when used to describe an antibody region refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the constant domains of antibodies are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • Two identical antigen binding fragments, called “Fab” fragments, each with a single antigen binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily can be produced by papain digestion.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'- SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • Immunoglobulins originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.
  • the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda (1), based on the amino acid sequences of their constant domains. Immunoglobulins are also assigned to different "classes" depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes).
  • 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.
  • 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 vivo.
  • 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.
  • stabilizers and adjuvants see, Martin, REMINGTON'S PHARM. SCI, 15th Ed. (Mack Publ. Co., Easton (1975)).
  • An "effective amount” is an amount sufficient to effect beneficial or desired results such as prevention or treatment. An effective amount can be administered in one or more administrations, applications or dosages.
  • a "subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals and pets.
  • a “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive” or “negative”.
  • the purpose of the experiment is to determine a correlation of an altered expression level of a gene with a particular type of cancer
  • a positive control a subject or a sample from a subject, ca ⁇ ying such alteration and exhibiting syndromes characteristic of that disease
  • a negative control a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease
  • DD96 is a 17-Kd membrane-associated, antibody-accessible protein of 114 amino acids. DD96 was shown to interact with PDZkl, which is overexpressed in selected tumors of epithelial origin. DD96 polynucleotides, polypeptides and fragment(s) thereof, can be obtained by using the sequence information provided in Table 1 (and the sequence listing infi-a) and chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or 431 A,' Foster City, CA, USA.
  • the synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Accordingly, this invention also provides a process for chemically synthesizing the proteins of this invention by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.
  • the proteins and polypeptides can be obtained by well-known recombinant methods as described herein using the host cell and vector systems as described herein.
  • the host cell can be prokaryotic or eukaryotic. Host cell systems are described supra.
  • this invention provides various methods for aiding in the diagnosis of the state of a cell that is characterized by abnormal cell growth in the form of, e.g., malignancy, hyperplasia or metaplasia.
  • the methods are particularly useful for aiding in the diagnosis of cancers of epithelial origin, e.g., lung, ovarian and prostate.
  • the neoplastic state of a cell can be determined by noting whether the growth of the cell is not governed by the usual limitation of normal growth.
  • the term also is to include genotypic changes that occur prior to detection of this growth in the form of a tumor and are causative of these phenotypic changes.
  • the phenotypic changes associated with the neoplastic state of a cell include a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens and the like. (See, Luria et al. (1978) GENERAL VIROLOGY, 3 d edition, 436-446 (John Wiley & Sons, New York)).
  • one embodiment is a method of diagnosing the condition of a cell by screening for the presence of a differentially expressed DD96 polynucleotide or polypeptide isolated from a sample containing or suspected of containing having cells that express said gene, in which the differential expression of DD96 is indicative of the neoplastic state of the cell.
  • DD96 is expressed more in a cancer or tumor cell, wherein said cell is one or more of lung, ovarian or prostate, as compared to a counterpart normal or healthy cell or tissue.
  • Detection can be by any appropriate method, including for example, detecting the quantity of mRNA transcribed from the gene or the quantity of cDNA produced from the reverse transcription of the mRNA transcribed from the gene or the quantity of the polypeptide or protein encoded by the gene. Probes for each of these methods are provided by reverse translating the peptides identified in Table 1 and using the polynucleotides encoding the peptides. These methods can be performed on a sample by sample basis or modified for high throughput analysis. Additionally, databases containing quantitative full or partial transcripts or protein sequences isolated from a cell sample can be searched and analyzed for the presence and amount of transcript or expressed gene product.
  • the database contains at least one of the sequences shown in Table 1 and/or the polynucleotide encoding it.
  • gene expression is determined by noting the amount (if any, e.g., altered) expression of the gene in the test system at the level of an mRNA transcribed from DD96.
  • augmentation of the level of the polypeptide or protein encoded by DD96 is indicative of the presence of the neoplastic condition of the cell.
  • the method can be used for aiding in the diagnosis of lung, ovarian or prostate cancer. Thus, by detecting this genotype prior to tumor growth, one can predict a predisposition to cancer and/or provide early diagnosis and treatment.
  • Cell or tissue samples used for this invention encompass body fluid, solid tissue samples, tissue cultures or cells derived there from and the progeny thereof and sections or smears prepared from any of these sources or any other samples that may contain a cell having a gene described herein.
  • the sample comprises cells prepared from a subject's tissue, e.g., prostate, ovarian or lung.
  • nucleic acid contained in the aforementioned samples is first extracted according to standard methods in the art. 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.
  • the mRNA of a DD96 contained in the extracted nucleic acid sample is then detected by hybridization (e.g., Northern blot analysis) and/or amplification procedures according to methods widely known in the art or based on the methods exemplified herein.
  • Nucleic acid molecules having at least 10 nucleotides and exhibiting sequence complementarity or homology to at least one polynucleotide encoding a peptide identified in Table 1 find utility as hybridization probes. It is known in the art that a "perfectly matched" probe is not needed for a specific hybridization. Minor changes in probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the hybridization specificity.
  • a probe useful for detecting mRNA is at least about 80% identical to the homologous region of comparable size contained in the genes or polynucleotides encoding the peptides identified in Table 1.
  • the probe is 85% identical to the corresponding polynucleotide sequence after alignment of the homologous region or, alternatively, it exhibits 90% identity.
  • radioassays e.g., Southern and Northern blot analysis
  • the total size of fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the particular nucleic acid segment. Smaller fragments derived from the known sequences will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between about 10 and about 100 nucleotides or even full-length according to the complementary sequences one wishes to detect. In one aspect, nucleotide probes having complementary sequences over stretches greater than about 10 nucleotides in length are used, so as to increase stability and selectivity of the hybrid and, thereby, improving the specificity of particular hybrid molecules obtained.
  • nucleic acid molecules having gene-complementary stretches of more than about 25 or alternatively more than about 50 nucleotides in length or even longer where desired.
  • Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology with two priming ohgonucleotides as described in U.S. Patent No. 4,603,102 or by introducing selected sequences into recombinant vectors for recombinant production.
  • a probe is about 50 to about 75, nucleotides or, alternatively, about 50 to about 100 nucleotides in length.
  • probes can be designed from the sequence of full length DD96 (see, SEQ ID NO: 80).
  • an appropriate means such as a label
  • a wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • colorimetric indicator substrates which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • Hybridization reactions can be performed under conditions of different "stringency". Relevant conditions include temperature, ionic strength, time of incubation, the presence of additional solutes in the reaction mixture such as formamide and the washing procedure. Higher stringency conditions are those conditions, such as higher temperature and lower sodium ion concentration, which require higher minimum complementarity between hybridizing elements for a stable hybridization complex to form. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art. See, for example, Sambrook et al. (1989) supra.
  • nucleotide probes of the present invention can also be used as primers and detection of genes or gene transcripts that are differentially expressed in certain body tissues.
  • a primer useful for detecting the aforementioned differentially expressed mRNA is at least about 80% identical to the homologous region of comparable size contained in the previously identified sequences encoding the peptides identified in Table 1.
  • "amplification” means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase and reverse transcriptase.
  • PCR A known amplification method is PCR, MacPherson et al., PCR: A PRACTICAL APPROACH, (1-RL Press at Oxford University Press (1991)).
  • PCR conditions used for each application reaction are empirically determined. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg 2+ ATP concentration, pH and the relative concentration of primers, templates and deoxyribonucleotides.
  • the resulting DNA fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
  • a specific amplification of differentially expressed genes of interest can be verified by demonstrating that the amplified DNA fragment has the predicted size, exhibits the predicated restriction digestion pattern and/or hybridizes to the correct cloned DNA sequence.
  • the probes also can be attached to a solid support for use in high throughput screening assays using methods known in the art.
  • the probes of this invention are synthesized on a derivatized glass surface.
  • Photoprotected nucleoside phosphoramidites are coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask and reacted with a second protected nucleoside phosphoramidite. The coupling/deprotection process is repeated until the desired probe is complete.
  • the expression level of a gene can also be determined through exposure of a nucleic acid sample to a probe-modified chip. Extracted nucleic acid is labeled, for example, with a fluorescent tag, preferably during an amplification step.
  • Hybridization of the labeled sample is performed at an appropriate stringency level.
  • the degree of probe-nucleic acid hybridization is quantitatively measured using a detection device, such as a confocal microscope. See, U.S. Patent Nos. 5,578,832 and 5,631,734. The obtained measurement is directly correlated with gene expression level.
  • the probes and high density oligonucleotide probe arrays also provide an effective means of monitoring expression of DD96. They are also useful to screen for compositions that upregulate or downregulate the expression of DD96.
  • the methods of this invention are used to monitor expression of DD96 which specifically hybridize to the probes of this invention in response to defined stimuli, such as an exposure of a cell or subject to a drug.
  • the hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids.
  • the labels may be incorporated by any of a number of means known to those of skill in the art. However, in one aspect, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acid.
  • PCR polymerase chain reaction
  • transcription amplification as described above, using a labeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.
  • a label may be added directly to the original nucleic acid sample (e.g., mRNA, polyA, mRNA, cDNA, etc.) or to the amplification product after the amplification is completed.
  • Means of attaching labels to nucleic acids include, for example nick translation or end-labeling (e.g., with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein and the like), radiolabels (e.g., H, I, S, C or P) enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA) and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • Patents teaching the use of such labels include U.S. Patents Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Means of detecting such labels are known to those of skill in the art.
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate and colorimetric labels are detected by simply visualizing the colored label.
  • the label may be added to the target (sample) nucleic acid(s) prior to or after the hybridization.
  • detectable labels that are directly attached to or inco ⁇ orated into the target (sample) nucleic acid prior to hybridization.
  • indirect labels are joined to the hybrid duplex after hybridization.
  • the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization.
  • the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected.
  • nucleic acid sample also may be modified prior to hybridization to the high density probe array in order to reduce sample complexity thereby decreasing background signal and improving sensitivity of the measurement using methods known in the art, e.g., the methods disclosed in WO 97/10365.
  • Results from the chip assay are typically analyzed using a computer software program. See, for example, EP 0717 113 A2 and WO 95/20681.
  • the hybridization data is read into the program, which calculates the expression level of the targeted gene(s) i.e., the genes identified in Table 1. This figure is compared against existing data sets of gene expression levels for diseased and healthy individuals. A correlation between the obtained data and that of a set of diseased individuals indicates the onset of a disease in the subject patient. Also within the scope of this application is a database useful for the detection of neoplastic lung tissue comprising one or more of the sequences, polynucleotides encoding the peptides, or parts thereof, of the peptides listed Table 1. These polynucleotide sequences are stored in a digital storage medium such that a data processing system for standardized representation of the genes that identify a lung cancer cell is compiled.
  • the data processing system is useful to analyze gene expression between two cells by first selecting a cell suspected of being of a neoplastic phenotype or genotype and then isolating polynucleotides from the cell. The isolated polynucleotides are then sequenced. The sequences from the sample are compared with the sequence(s) present in the database using homology search techniques described above, hi one aspect, greater than 90% is selected or, alternatively, greater than 95% is selected or, alternatively, greater than or equal to
  • 97% sequence identity is selected, between the test sequence and at least one sequence, or polynucleotide encoding it, identified in Table 1 or its complement, is a positive indication that the polynucleotide has been isolated from a lung, prostate or ovarian cancer cell as defined above. Alternatively, one can compare a sample against a database. Briefly, multiple sequence, or polynucleotide encoding it, identified in Table 1 or its complement, is a positive indication that the polynucleotide has been isolated from a lung, prostate or ovarian cancer cell as defined above. Alternatively, one can compare a sample against a database. Briefly, multiple
  • RNAs are isolated from cell or tissue samples using methods known in the art and described for example, in Sambrook et al. (1989) supra.
  • the gene transcripts can be converted to cDNA.
  • a sampling of the gene transcripts are subjected to sequence-specific analysis and quantified. These gene transcript sequence abundances are compared against reference database sequence abundances including normal data sets for diseased and healthy patients.
  • the patient has the disease(s) with which the patient's data set most closely correlates which includes the overexpression of the transcripts identified herein.
  • Differential expression of DD96 can also be determined by examining the protein product. A variety of techniques are available in the art for protein analysis.
  • radioimmunoassays include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), "sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.
  • One means to determine protein level involves (a) providing a biological sample containing polypeptides; and (b) measuring the amount of any immunospecific binding that occurs between an antibody reactive to the expression product of a gene of interest and a component in the sample, in which the amount of immunospecific binding indicates the level of the expressed proteins.
  • Antibodies that specifically recognize and bind to the protein products of these genes are required for these immunoassays. These may be purchased from commercial vendors or generated and screened using methods well known in the art. See, Harlow and Lane (1988) supra and Sambrook et al. (1989) supra. Alternatively, polyclonal or monoclonal antibodies that specifically recognize and bind the protein product of a gene of interest can be made and isolated using known methods. In diagnosing malignancy, hype ⁇ lasia or metaplasia characterized by a differential expression of genes, one typically conducts a comparative analysis of the subject and appropriate controls.
  • a diagnostic test includes a control sample derived from a subject (hereinafter “positive control”), that exhibits the predicted change in expression of a gene of interest and clinical characteristics of the malignancy or metaplasia of interest.
  • a diagnosis also includes a control sample derived from a subject (hereinafter “negative control”), that lacks the clinical characteristics of the neoplastic state and whose expression level of the gene at question is within a normal range.
  • a positive correlation between the subject and the positive control with respect to the identified alterations indicates the presence of or a predisposition to said disease.
  • a lack of correlation between the subject and the negative control confirms the diagnosis.
  • the method is used for diagnosing cancers of epithelial origin, e.g., lung, ovarian or prostate, on the basis of a differential expression of DD96.
  • the present invention also provides a screen for identifying leads, drugs, therapeutic biologies and methods for reversing the neoplastic condition of the cells or selectively inhibiting growth or proliferation of the cells described above.
  • the screen identifies lead compounds or biological agents which are useful for the treatment of malignancy, hype ⁇ lasia or metaplasia characterized by differential expression of DD96.
  • suitable cell cultures or tissue cultures are first provided.
  • the cell can be a cultured cell or a genetically modified cell which differentially expresses DD96 associated with a neoplastic cell.
  • the cells can be from a tissue biopsy.
  • the cells are cultured under conditions (temperature, growth or culture medium and gas (CO 2 )) and for an appropriate amount of time to attain exponential proliferation without density dependent constraints. It also is desirable to maintain an additional separate cell culture; one which does not receive the agent being tested as a control.
  • the method can be modified for high throughput analysis and suitable cells may be cultured in microtiter plates and several agents may be assayed at the same time by noting genotypic changes, phenotypic changes and/or cell death.
  • the agent is a composition other than a DNA or RNA nucleic acid molecule
  • the suitable conditions comprise directly added to the cell culture or added to culture medium for addition.
  • the screen involves contacting the agent with a test cell characterized by differential expression of DD96 and then assaying the cell for the level of DD96 expression. In some aspects, it may be necessary to determine the level of DD96 expression prior to the assay.
  • This provides a base line to compare expression after administration of the agent to the cell culture, hi another embodiment, the test cell is a cultured cell from an established cell line that differentially expresses a gene of interest.
  • an agent is a possible therapeutic agent if gene expression is returned (reduced or increased) to a level that is present in a cell in a normal or non-neoplastic state, or the cell selectively dies, or exhibits reduced rate of growth.
  • the test cell or tissue sample is isolated from the subject to be treated and one or more potential agents are screened to determine the optimal therapeutic and/or course of treatment for that individual patient.
  • an "agent" is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide.
  • oligomers such as oligopeptides and ohgonucleotides and synthetic organic compounds based on various core structures; these compounds are also included in the term "agent”.
  • various natural sources can provide compounds for screening, such as plant or animal extracts and the like. It should be understood, although not always explicitly stated that the agent is used alone or in combination with another agent, having the same or different biological activity as the agents identified by the inventive screen. The agents and methods also are intended to be combined with other therapies.
  • the term "reversing the neoplastic state of the cell” is intended to include apoptosis, necrosis or any other means of preventing cell division, reduced tumorigenicity, loss of pharmaceutical resistance, maturation, differentiation or reversion of the neoplastic phenotypes as described herein.
  • lung cells having differential expression of a gene of interest that results in the neoplastic state are suitably treated by this method. These cells can be identified by any method known in the art that allows for the identification of differential expression of the gene.
  • the agent is a nucleic acid, it can be added to the cell cultures by methods known in the art, which includes, but is not limited to calcium phosphate precipitation, microinjection or electroporation.
  • the nucleic acid can be inco ⁇ orated into an expression or insertion vector for inco ⁇ oration into the cells.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art and briefly described infra.
  • Polynucleotides are inserted into vector genomes using methods well known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of restricted polynucleotide.
  • oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability transcription termination and RNA processing signals
  • kits containing the agents and instructions necessary to perform the screen and in vitro method as described herein also are claimed.
  • the method provides a convenient animal model system which can be used prior to clinical testing of the therapeutic agent or alternatively, for lead optimization, hi this system, a candidate agent is a potential drug if gene expression is returned to a normal level or if symptoms associated or correlated to the presence of cells containing differential expression of a gene of interest are ameliorated, each as compared to untreated, animal having the pathological cells. It also can be useful to have a separate negative control group of cells or animals which are healthy and not treated, which provides a basis for comparison.
  • Therapeutic agents provided by this invention include, but are not limited to small molecules, polynucleotides, peptides, antibodies, antigen presenting cells and include immune effector cells that specifically recognize and lyse cells expressing DD96.
  • the therapeutic agent is administered in an amount effective to treat cancer of epithelial origin, e.g., lung, ovarian and prostate.
  • Therapeutics of the invention can also be used to prevent progression from a pre- neoplastic or non-malignant state into a neoplastic or a malignant state.
  • Various delivery systems are known and can be used to administer a therapeutic agent of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (See e.g. , Wu and Wu ( 1987) J. Biol. Chem. 262 :4429-4432), construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc.
  • Methods of delivery include but are not limited to intra-arterial, intra-muscular, intravenous, intranasal and oral routes.
  • the agents identified herein as effective for their intended piupose can be administered to subjects or individuals susceptible to or at risk of developing a disease correlated to the differential expression of DD96.
  • the agent When the agent is admimstered 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.
  • a tumor sample is removed from the patient and the cells are assayed for the differential expression of DD96.
  • 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 agent.
  • the method is useful to further confirm efficacy of the agent.
  • groups of nude mice (Balb/c NCR nu/nu female, Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 10 5 to about 10 9 hype ⁇ roliferative, cancer or target cells as defined herein.
  • the agent is administered, for example, by subcutaneous injection around the tumor.
  • Tumor measurements to determine reduction of tumor size are made in two dimensions using venier calipers twice a week. Other animal models may also be employed as appropriate.
  • 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 pu ⁇ ose 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.
  • 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.
  • the pharmaceutical compositions can be admimstered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form.
  • the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compositions of the invention.
  • an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parental (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 being treated.
  • the agent should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the agent, optionally in saline or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient. Desirable blood levels of the agent maybe maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
  • operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component antiviral agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects.
  • compositions comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic agents.
  • Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients, hi general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. , povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • compositions for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol or oil.
  • a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.
  • the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances abso ⁇ tion or penetration of the agent through the skin or other affected areas.
  • Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at lease one emulsifier with a fat or an oil or with both a fat and an oil.
  • a hydrophihc emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the agent.
  • a suitable carrier especially an aqueous solvent for the agent.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the agent, such carriers as are known in the art to be appropriate.
  • Formulations suitable for nasal administration wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops or by aerosol administration by nebulizer include aqueous or oily solutions of the agent.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, thickening agents and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above-recited, or an appropriate fraction thereof, of an agent.
  • DD96 can be used to generate transgenic animal models.
  • geneticists have succeeded in creating transgenic animals, for example mice, by manipulating the genes of developing embryos and introducing foreign genes into these embryos. Once these genes have integrated into the genome of the recipient embryo, the resulting embryos or adult animals can be analyzed to determine the function of the gene. The mutant animals are produced to understand the function of known genes in vivo and to create animal models of human diseases. (See e.g.,
  • ANTIBODIES Also provided by this invention is an antibody capable of specifically forming a complex with the expression product of a DD96 polypeptide.
  • the term "antibody” includes polyclonal antibodies and monoclonal antibodies and variants of these antibodies presently known to those of skill in the art.
  • the antibodies include, but are not limited to mouse, rat and rabbit or human antibodies.
  • the antibodies are useful to identify and purify gene expression products as well as APCs expressing the polypeptides.
  • Laboratory methods for producing polyclonal antibodies and monoclonal antibodies, as well as deducing their corresponding nucleic acid sequences, are known in the art; see, Harlow and Lane (1988) supra and Sambrook et al. (1989) supra.
  • the monoclonal antibodies of this invention can be biologically produced by introducing protein or a fragment thereof into an animal, e.g., a mouse or a rabbit.
  • the antibody producing cells in the animal are isolated and fused with myeloma cells or hetero- myeloma cells to produce hybrid cells or hybridomas.
  • the hybridoma cells producing the monoclonal antibodies of this invention also are provided.
  • one of skill in the art can produce and screen the hybridoma cells and antibodies of this invention for antibodies having the ability to bind the proteins or polypeptides.
  • a monoclonal antibody being tested binds with the protein or polypeptide, then the antibody being tested and the antibodies provided by the hybridomas of this invention are equivalent. It also is possible to determine without undue experimentation, whether an antibody or its variant has the same specificity as the monoclonal antibody of this invention by determining whether the antibody being tested prevents a monoclonal antibody of this invention from binding the protein or polypeptide with which the monoclonal antibody is normally reactive. If the antibody being tested competes with the monoclonal antibody of the invention as shown by a decrease in binding by the monoclonal antibody of this invention, then it is likely that the two antibodies bind to the same or a closely related epitope.
  • the monoclonal antibody of this invention can pre-incubate the monoclonal antibody of this invention with a protein with which it is normally reactive, and determine if the monoclonal antibody being tested is inhibited in its ability to bind the antigen. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same or a closely related, epitopic specificity as the monoclonal antibody of this invention.
  • antibody also is intended to include antibodies of all isotypes.
  • Particular isotypes of a monoclonal antibody can be prepared either directly by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the "sib selection" technique to isolate class switch variants using the procedure described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J. Immunol.
  • Antibody variants also include biological active fragments of the polyclonal and monoclonal antibodies described above. These "antibody fragments" retain some ability to selectively bind with its antigen or immunogen. Such antibody fragments can include, but are not limited to:
  • F(ab') 2 (4) Fv, and (5) SCA
  • a specific example of "a biologically active antibody fragment” is a CDR region of the antibody. Methods of making these fragments are known in the art, e.g., Harlow and Lane (1988) supra. Single-chain antibody fragments are also known to be useful to import substances into cells. U.S. Patent No. 6,635,248 and PCT/FR98/01740. Techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used. See, e.g., U.S. Patent Nos. 6,054,297; 6,407,213; 6,639,055; 6,719,971; .
  • Fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody.
  • Single chain Fv can also be used as is convenient. They can be made from vaccinated transgenic mice, if desired. Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. See, e.g., U.S. Patent Nos.: 6,750,041 and 5,648,237 and methods disclosed in U.S. Patent Application No.: US 2002/0019517.
  • the antibodies of this invention also can be modified to create chimeric antibodies.
  • Chimeric antibodies are those in which the various domains of the antibodies' heavy and light chains are coded for by DNA from more than one species. See, e.g., U.S. Patent No.: 4,816,567.
  • the term "antibody variant” also includes "diabodies" which are small antibody fragments with two antigen-binding sites, wherein fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (VH V L ).
  • antibody variant further includes ""linear antibodies".
  • the procedure for making the is known in the art and described in Zapata et al. (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-VH -C H I) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. The isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibodies of the invention can also be accomplished by one of ordinary skill in the art by producing anti-idiotypic antibodies (Herlyn et al. (1986) Science 232:100).
  • An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest. Idiotypic identity between monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using antibodies to the epitopic determinants on a monoclonal antibody it is possible to identify other hybridomas expressing monoclonal antibodies of the same epitopic specificity. It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the mirror image of the epitope bound by the first monoclonal antibody.
  • the anti-idiotypic monoclonal antibody could be used for immunization for production of these antibodies.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the antibodies of this invention can be linked to a detectable agent or label.
  • haptens such as biotin, which reacts avidin or dinitrophenol, pyridoxal and fluorescein, which can react with specific anti-hapten antibodies. See, Harlow and Lane (1988) supra.
  • the monoclonal antibodies of the invention also can be bound to many different carriers.
  • this invention also provides compositions containing the antibodies and another substance, active or inert.
  • compositions containing the antibodies, fragment(s) thereof or cell lines which produce the antibodies are encompassed by this invention. When these compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.
  • the present invention provides a method of inducing an immune response comprising delivering the biologies and compositions of the invention (e.g., peptides identified in Table 1) in the context of an MHC molecule.
  • the polypeptides of this invention can be pulsed into antigen presenting cells using the methods described herein.
  • Antigen-presenting cells include, but are not limited to dendritic cells (DCs), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules.
  • DCs dendritic cells
  • monocytes/macrophages a cell type(s) expressing the necessary MHC/co-stimulatory molecules.
  • Isolated host cells which present the polypeptides of this invention are further useful to expand and isolate a population of educated, antigen-specific immune effector cells.
  • the immune effector cells e.g., cytotoxic T lymphocytes
  • the population can be purified using methods known in the art, e.g., FACS analysis or ficoll gradient.
  • the methods to generate and culture the immune effector cells as well as the populations produced thereby also are the inventor's contribution and invention.
  • Pharmaceutical compositions comprising the cells and pharmaceutically acceptable carriers are useful in adoptive immunotherapy.
  • the immune effector cells Prior to administration in vivo, the immune effector cells are screened in vitro for their ability to lyse tumor cells
  • the immune effector cells and/or the APCs are genetically modified.
  • 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.
  • This invention also provides methods of inducing an immune response in a subject, comprising administering to the subject an effective amount of a polypeptide described above (as shown in Table 1) under the conditions that induce an immune response to the polypeptide.
  • the polypeptide can be administered in a formulation or as a polynucleotide encoding the polypeptide.
  • the polynucleotide can be admimstered 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 the polynucleotides of this invention in a pharmaceutically acceptable carrier can therefore be combined with appropriate and effective amount of an adjuvant, cytokine or co-stimulatory molecule for an effective vaccine regimen, hi one embodiment, the host cell is an APC such as a dendritic cell.
  • the host cell can be further modified by inserting of a polynucleotide coding for an effective amount of either or both a cytokine and/or 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.
  • IMMUNOGENICITY ASSAYS The immunogenicity of therapeutic agents of this invention can be determined by known methodologies including, but not limited to those exemplified below. In one embodiment, such methodology may be employed to compare an equivalent polypeptide of the invention with nature or wild-type DD96 (see SEQ ID NO: 80; also shown under GenBank Ace. No. XM_001626). For example, an altered peptide may be considered "more active” if it compares favorably with the activity of the wild-type peptide in any one of the following assays.
  • a peptide e.g., from those shown in Table 1 which displays more activity than another immunogenic peptide, i.e., for treatment and/or diagnostic pu ⁇ oses.
  • an immunogenic peptide which is comparable with the native DD96 peptide will be suitable.
  • 51 Cr-release lysis assay Lysis of peptide-pulsed 51 Cr-labeled targets by antigen-specific T cells can be compared for target cells pulsed with either the native or altered peptides.
  • the peptides of the invention can be evaluated for the ability to elicit peptide-reactive T cell populations from normal donor or patient-derived PBMC. In this system, elicited T cells can be tested for lytic activity, cytokine-release, polyclonality and cross-reactivity. (Parkhurst, M.R. et al. (1996) J. Immunol. 157:2539). 4. Transgenic animal models.
  • Immunogenicity can be assessed in vivo by vaccinating HLA transgenic mice with either the peptides of the invention and determining the nature and magnitude of the induced immune response.
  • the hu-PBL-SCID mouse model allows reconstitution of a human immune system in a mouse by adoptive transfer of human PBL. These animals may be vaccinated with the peptides and analyzed for immune response as previously mentioned. (Shirai, M. et al. (1995) J. Immunol. 154:2733; Mosier, D.E. etal. (1993) Proc. Natl. Acad. Sci. USA 90:2443). 5. Proliferation. T cells will proliferate in response to reactive peptides.
  • Proliferation can be monitored quantitatively by measuring, for example, 3 H- thymidine uptake. (Caruso, A. et al. (1997) Cytometry 27:71). 6. Tetramer staining. MHC tetramers can be loaded with individual peptides and tested for their relative abilities to bind to appropriate effector T cell populations. (Airman, J.D. et al. (1996) Science 274(5284):94-96). 7. MHC Stabilization. Exposure of certain cell lines such as T2 cells to
  • HLA-binding ligands results in the stabilization of MHC complexes on the cell surface. Quantitation of MHC complexes on the cell surface has been correlated with the affinity of the peptide for the HLA allele that is stabilized. Thus, this technique can determine the relative HLA affinity of peptide epitopes. (Stuber, G. et al. (1995, ) Int. Immunol. 7:653). 8. MHC competition. The ability of a peptide to interfere with the functional activity of a reference peptide and its cognate T cell effectors is a measure of how well a second or altered peptide can compete for MHC binding. Measuring the relative levels of inhibition is an indicator of MHC affinity.
  • the expanded populations of antigen-specific 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 a subject a substantially pure population of educated, antigen-specific immune effector cells made by culturing na ⁇ ve immune effector cells with APCs as described above.
  • the APCs are dendritic cells.
  • the adoptive immunotherapy methods described herein are autologous.
  • the APCs are made using parental cells isolated from a single subject.
  • the expanded population also employs T cells isolated from that subject.
  • the expanded population of antigen-specific cells is administered to the same patient.
  • an effective amount, APCs or immune effector cells are administered with an effective amount of a stimulatory cytokine, such as IL-2 or a co-stimulatory molecule.
  • a stimulatory cytokine such as IL-2 or a co-stimulatory molecule.
  • the agents identified herein as effective for their intended pu ⁇ ose can be administered to subjects in need of such therapy. Method for administration of therapeutic agents are known in the art and described briefly, infra. EXPANSION OF IMMUNE EFFECTOR CELLS The present invention makes use of these APCs presenting peptides of the invention 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.
  • na ⁇ ve immune effector cells become educated by other cells is described in the art, e.g., in Coulie (1997) Molec. Med. Today 3:261-268.
  • the APCs prepared as described above are mixed with na ⁇ ve immune effector cells.
  • the cells may be cultured in the presence of a cytokine, for example IL-2. Because dendritic cells secrete potent immunostimulatory cytokines, such as IL-12, it may not be necessary to add supplemental cytokines during the first and successive rounds of expansion, hi 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.
  • the immune effector cells are T cells.
  • the immune effector cells can be genetically modified by transduction with a transgene coding for example, IL-2, IL-11 or IL-13.
  • Methods for introducing transgenes in vitro, ex vivo and in vivo are known in the art. See, Sambrook et al. (19S9) supra.
  • APCs can be transduced with viral vectors encoding a relevant polypeptides. Viral vectors are described supra.
  • In vivo transduction of DCs or other APCs can be accomplished by administration of Ad (or other viral vectors) via different routes including intravenous, intramuscular, intranasal, intraperitoneal or cutaneous delivery.
  • Transduced APCs can subsequently be administered to the host via an intravenous, subcutaneous, intranasal, intramuscular or intraperitoneal route of delivery.
  • the method is cutaneous delivery of Ad vector at multiple sites using a total dose of approximately lxl0 10 -lx 10 12 i.u.
  • Levels of in vivo transduction can be roughly assessed by co-staining with antibodies directed against
  • APC marker(s) and the TAA 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 and Wan et al. (1997) Hum. Gene Ther. 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 ELIS A on tissue homogenates.
  • DCs can also be transduced in vitro/ex 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 Ther. 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.
  • PBMC Normal or patient donor PBMC are exposed to the invention peptides either individually or as a cocktail under the appropriate reaction conditions to generate a mixed population of reactive cytotoxic T cells. These cells are tested for their ability to react with tumor cells lines known to express the antigen. This ensures that the peptide-educated CTL recognize the epitope in its native state.
  • These tumor-reactive T cells can be used as a bulk population or individual tumor-reactive clones can be derived by standard limiting dilution analysis, in order to determine whether a particular patient's tumor expresses the unknown antigen.
  • Peptide-educated clonal populations of CTLs of the present invention can be used to construct recombinant soluble T cell receptor (TCR) protein with specificity for the native epitope corresponding to the unnamed antigen. Multimerization of the
  • TCR proteins results in a reagent that will bind to the HLA-A2 molecule in the presence of the specific epitope recognized by the TCR. Therefore, this reagent is useful to test patient tumor cells derived from biopsy or tumor resection for the presence of the unnamed native epitope. A positive result indicates that the patient's tumor expresses the unnamed antigen implying potential benefit to the patient by successful vaccination with the peptides.
  • Phage display technology has been successfully employed to identify recombinant antibodies capable of binding specifically to distinct MHC/peptide complexes (See, Denkberg, G. et al, (2002) PNAS 99(14):9421-9426). These monoclonal antibodies are useful to detect the presence of particular epitopes presented in the context of MHC molecules on the surface of tumor cells. This approach is useful to generate monoclonal antibodies against invention peptide-bound HLA-A2 molecules. These antibodies can be tested for specificity with tumor cell lines known to express or not express the antigen. Patient tumor derived from biopsy or tumor resection are tested for the presence of the native epitope/MHC surface complex by measuring antibody binding. A positive result indicates that the patient's tumor expresses the unnamed antigen implying potential benefit to the patient by successful vaccination with the peptides.
  • the following are non-limiting examples of the compositions and methods of this invention.
  • EXPERIMENTAL EXAMPLES Immunogenicity Of DD96 In Vitro T Cell Education (IVE) Normal healthy HLA-A2 + donors were apheresed. Monocytes were differentiated into dendritic cells in vitro and infected with an adeno viral construct engineered to express the antigen. The genetically modified DCs were cocultured with autologous CD8 + T cells. The resulting bulk cultures that grew after 4-5 weekly restimulations were then tested for antigen specificity in a 51 Cr-release microcytotoxicity assay. DC Preparation Normal donor monocytes were harvested by leukapheresis in a volume of -175 mis.
  • the cells were diluted 1:1 with PBS (Mg +2 , Ca +2 free) and 30 mis layered onto 20 mis Ficoll-Paque (research grade, Pharmacia Biotech., 17-0840-03). Cells were collected at the interface by centrifugation (IEC, Model GP8, 1400 RPM/40 min., room temperature, no brake, swinging bucket rotor). The cells were washed 3x with PBS and resuspended in RPMI supplemented with P/S, glutamine, 5% Human AB serum (Sigma #2520) and lOmM HEPES (complete medium) and rocked overnight on a Nutator in 50 ml conical tubes at 4°C.
  • the cells were then plated at 1.5xl0 8 cells/T-150 Flask (Corning, #430823). After 1 hour, non-adherent cells were removed by washing 3x with unsupplemented RPMI. The cells were then fed with complete RPMI plus lOOng/ml GM-CSF (Genzyme, #RH-CSF-C) and 20 ng/ml IL-4 (Genzyme, #2181-01) in a volume of 20 mls/T150 flask. On day 3, 5 mis complete RPMI (plus cytokines) was added to each flask. On day 6, the cells were processed for infection as described below.
  • DC Infection DCs to be infected were plated at a density of 1 x 10 6 /ml serum-free OptiMEM (GibcoBRL, #31985-070) (+100ng/ml GM-CSF and 20 ng/ml IL-4)/well of a 24-well cluster plate.
  • Reagent grade gradient-purified Ad2/DD96 (Genzyme) to the culture at an MOI of 500. The culture was allowed to incubate overnight in complete medium and immediately processed as described for T Cell Education.
  • DC Freeze/Thaw Day 6 DCs were harvested from flasks or plates and pelleted by centrifugation. The cells were resuspended in 90% human AB serum/10% DMSO and aliquoted to 1.5 ml Nunc cryotubes (1 ml tube) at a cell density of 1-5 x 10 6 /ml. The tubes were stored at -80°C for 24 hours and transferred to liquid nitrogen for extended frozen storage. DCs were thawed by warming the tubes in a 37°C water bath and immediately transfened to 10 mis PBS in a 15 ml conical centrifuge tube. The cells were pelleted and washed 2x with 10 mis PBS before use.
  • Adenovirus-infected DCs were cocultured with 1 x 10 7 unfractionated autologous PBMCs prepared as described in (DC Preparation, above) in 1 ml Iscove's medium/10% human AB serum in 1 well of a 24-well cluster plate. The culture was incubated at 37°C/5% CO 2 for 7 days. The cells were then transferred to a 1.5 ml eppendorf tube and pelleted by centrifugation.
  • the cell pellet was resuspended in 1 ml Iscove's medium 10% human AB serum containing 5 x 10 5 previously frozen Ad2/DD96-transduced DCs, 1 x 10 6 autologous CD8 + T cells and 50 IU/ml recombinant human IL-2 (Genzyme, E.co/t-derived, reagent grade).
  • CD8 + T cells were isolated from the apheresis product immediately after the ficoll gradient using anti-CD8 paramagnetic Dynabeads according to the manufacturer's instructions.
  • CTL Assay 1 x 10 6 genetically-modified DCs were labeled overnight in 900 ⁇ l RPMI medium 10%) Fetal bovine serum supplemented with lOO ⁇ l (lOO ⁇ Ci) 51 Cr
  • the 51 Cr-labeled target cells were transferred to a 1.5 ml eppendorf tube and washed 3x 1 ml of serum-free AIM-V medium (GibcoBRL, #12055-083).
  • l e+4 cells were transferred to each well of a 96- well V-bottom plate (Costar, polystyrene, #3894) in a volume of lOO ⁇ l AEVI-V medium/10%) human AB serum per well and the plate was spun at 1200 ⁇ m for 3 minutes.
  • Effector cells were added to appropriate wells in AIM-V medium/10%) human AB serum at the indicated E:T (Effector :Target) ratios in a volume of lOO ⁇ l for a total reaction volume of 200/11.
  • Medium no effector cells
  • the plate was then spun at 1200 ⁇ m for 3 minutes and returned to the incubator for 4 hours.
  • Results of the CTL assay are shown in Figure 1.
  • the targets tested with each effector cell population were autologous DCs infected with either an empty vector adenoviral construct (lacking a transgene) or the construct containing the gene of interest (indicated to the right of each graph).
  • RNA was purified from surgically resected primary tumors and normal tissues and reverse transcribed.
  • Antigen-specific PCR primers directed at the region of the gene harboring the identified HLA-A2-restricted epitope were designed to produce an amplicon of 50-100 base pairs.
  • 18S ribosomal RNA was chosen as an internal standard for multiplex PCR since it is not differentially expressed between normal and transformed tissue. Most samples were assayed 2-3 times.
  • the prevalence of this antigen in primary tumors of various types was assessed using a quantitative TaqMan assay and found to be overexpressed to varying degrees in each tumor type. The fold-overexpression in the tumors was calculated by

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Abstract

La présente invention concerne des procédés et compositions permettant de détecter, diagnostiquer, pronostiquer et surveiller l'évolution de cancers et de tumeurs malignes d'origine épithéliale tels que dans le cas des ovaires, des poumons et de la prostate. L'invention concerne également des nécessaires concernant à cet effet. L'invention concerne aussi des procédés de recherche systématique permettant de trouver des agonistes et des antagonistes d'antigènes associés à ces cancers et tumeurs malignes.
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* Cited by examiner, † Cited by third party
Title
KOCHER O. ET AL: 'Identification of a Novel Gene, Selectively Up-Regulated in Human Carcinomas, Using the Differential Display Technique' CLINICAL CANCER RESEARCH vol. 1, no. 10, October 1995, pages 1209 - 1215, XP002995543 *

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