Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/19—Dendritic cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4244—Enzymes
  • A61K40/4246—Telomerase or [telomerase reverse transcriptase [TERT]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons

Definitions

• the present invention is related to the catalytic protein subunit of human telomerase.
• the invention provides methods and compositions relating to medicine, immunology, and molecular biology.
• telomerase ribonucleoprotein complex is a specialized polymerase that maintains teiomeres, the specialized structures at the ends of eukaryotic chromosomes.
• the length and integrity of teiomeres in a cell is correlated with the entry of the cell into a senescent stage (t ' .e., loss of proliferative capacity), or, alternatively, the ability of a cell to escape senescence.
• t ' senescent stage
• t ' loss of proliferative capacity
• telomerase activity is detected in immortal cell lines and a diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Patents Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; also see, Morin, 1989, Cell 59: 521; Shay and Bacchetti, 1997, Ear. J Cancer 33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992, EMB0J. 11:1921; Counter et al., l994, Proc. Natl. Acad. Sci. U.S.A.
• telomerase plays an important role in the control of cell proliferation and in tumorigenesis.
• human telomerase is an ideal target for preventing and treating human diseases relating to cellular proliferation and senescence, such as cancer.
• the present invention provides immunological methods for preventing and treating these and other diseases in humans and nonhuman animals.
• the present invention provides a method of activating a T lymphocyte by contacting the T lymphocyte with a dendritic cell that expresses a telomerase reverse transcriptase (TRT) polypeptide encoded by a recombinant nucleic acid.
• TRT polypeptide is a human TRT (hTRT) polypeptide, and may have the sequence set forth in Figure 1, or may have a subsequence thereof.
• the hTRT polypeptide is full-length.
• the dendritic cell is a human cell. The dendritic cell may contact the T lymphocyte in vivo or in vitro.
• the invention provides a recombinant dendritic cell which comprises a recombinant TRT expression cassette.
• the recombinant expression cassette is transduced into a stem cell, and the stem cell is then differentiated into the dendritic cell.
• the stem cell is differentiated in vitro.
• the invention also provides a pharmaceutical composition comprising the aforementioned dendritic cell and a pharmaceutically acceptable carrier.
• the invention provides a method of eliciting an immune response in a human patient by (a) obtaining human dendritic cells, (b) transducing a TRT expression cassette into the cells so that they are capable of expressing a hTRT polypeptide, and (c) administering the cells to the human patient.
• the dendritic cells are isolated from the human patient to which they are administered, and or are obtained from hematopoietic precursor cells.
• the invention provides a method of eliciting an immune response in a human patient by (a) obtaining human dendritic cells, (b) pulsing the cells with a hTRT antigen, and (c) administering the cells pulsed with the hTRT antigen to the human patient.
• the dendritic cells are isolated from the human patient to which they are administered, and/or are obtained from hematopoietic precursor cells.
• the cells are pulsed with one or more hTRT antigenic peptides that are less than 50 amino acid residues in length.
• the invention provides a method for identifying a cell expressing hTRT.
• a dendritic cell is transduced with a recombinant expression cassette comprising a nucleic acid encoding a hTRT polypeptide; a T lymphocyte is contacted with the transduced dendritic cell, thereby providing an activated T lymphocyte; and a target cell is contacted with the activated T lymphocyte. The effect of the activated T lymphocyte on the target cell is then monitored.
• Figure 1 shows the amino acid sequence (in the one-letter code) of a 1132-residue hTRT protein.
• Figure 2 shows a nucleic acid sequence of a cDNA encoding the hTRT protein shown in Figure 1.
• telomerase reverse transcriptase refers to the catalytic protein subunit of the telomerase ribonucleoprotein particle.
• TRT from humans has been characterized, human TRT (hTRT) genomic and cDNA sequences have been cloned and their sequences determined. See, e.g., Nakamura et al., 1997, Science 277:955 and copending U.S. patent applications serial nos 08/912,951 and 08/974,549.
• GenBank accesion No.
• hTRT polypeptides that may be used in the present invention include, in addition to the hTRT polypeptides having amino acid sequences described in the aforecited references and deposits, other naturally occurring variants, including allelic variants and processing variants.
• hTRT polypeptide or variant characteristic of a particular subject (e.g., expressed in tumor cells from the subject) or characteristic of a particular tumor type is used.
• Such tumor or subject-specific hTRT can be obtained using standard methods, e.g., cloning using hTRT PCR primers, purification using anti-hTRT antibodies, and other techniques. Exemplary uses of hTRT polypeptides and polynucleotides, and nonhuman homologs, are described in additional detail in U.S.
• Patent Application Serial Numbers 08/974,549 (filed November 19, 1997), 08/974,584 (filed November 19, 1997), 08/915,503 (filed August 14, 1997), 08/912,951 (filed August 14, 1997), 08/911,312 (filed August 14, 1997), 08/854,050 (filed May 9, 1997), 08/851,843 (filed May 6, 1997), 08/846,017 (filed April 25, 1997), 08/844,419 (filed April 18, 1996), 08/724,643 (filed October 1, 1996), and 08/979,742 (filed November 26, 1997).
• Each of the aforementioned patent applications is explicitly incorporated herein by reference in its entirety and for all purposes.
• a “dendritic cell” is an antigen presenting cell (APC) with a characteristic morphology including lamellipodia extending from the dendritic cell body in several directions.
• APC antigen presenting cell
• Several phenotypic criteria are also typical, including high levels of MHC molecules and costimulatory molecules, a lack of markers specific for granulocytes, NK cells, B lymphocytes, and T lymphocytes, but can vary depending on the source of the dendritic cell.
• DCs are able to initiate antigen specific primary T lymphocyte responses in vitro and in vivo, and direct a strong mixed leukocyte reaction (MLR) compared to peripheral blood leukocytes, splenocytes, B cells and monocytes.
• MLR mixed leukocyte reaction
• DCs can be derived from a hematopoietic stem cell, e.g., a CD34 + precursor cell.
• Dendritic cells are described generally by Steinman, 1991, Annu Rev Immuno
• a cell is “transduced” with a selected nucleic acid when the nucleic acid is translocated into the cell.
• a cell is “stably transduced” with a selected nucleic acid when the selected nucleic acid is replicated and passed on to progeny cells.
• a cell is “transformed” with a selected nucleic acid when the selected nucleic acid is integrated into the cell's genome.
• a "TRT expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, that includes a nucleic acid encoding a TRT polypeptide or fragment, and a promoter.
• the expression cassette also includes other genetic elements, e.g., an origin of replication, and/or chromosome integration elements such as retroviral LTRs.
• the TRT expression cassette may be plasmid, virus genome, nucleic acid fragment, or the like.
• a recombinant polynucleotide is a polynucleotide synthesized or otherwise manipulated in vitro (e.g., using molecular biological techniques), and is typically linked to sequence (e.g., a heterologous promoter, vector sequence or other sequence) with which it is not normally linked in nature and/or comprises mutations (e.g., deletions) compared to naturally occurring forms;
• a "recombinant polypeptide” is a polypeptide that is produced by expression of a recombinant nucleotide;
• a “recombinant cell” is a cell comprising a recombinant polynucleotide.
• activation when used in reference to a T lymphocyte, has the ordinary meaning in the art of immunology and refers to characteristic changes (e.g., calcium ion influx, tyrosine kinase activation) that follow ligand-receptor interactions between a T lymphocyte and antigen presenting cell.
• T cell activation ordinarily results in clonal expansion of antigen-reactive T lymphocytes.
• the methods and reagents of the invention are useful for eliciting an in vivo immune response to telomerase, a telomerase protein, and/or a cell expressing telomerase or a telomerase protein.
• the methods and reagents of the invention are used to treat or prevent diseases or conditions related to cell proliferation, such as cancers.
• TRT telomerase reverse transcriptase
• most normal somatic cells in mammals have no or very low levels of TRT expression.
• Reagents such as the dendritic cells described infra, that induce differentiation and proliferation of T lymphocytes that specifically target telomerase expressing cells may be used for the prevention or treatment of cancer.
• Induction of an anti-TRT immune response in a human or nonhuman subject will inhibit growth of a tumor in the subject, and/or result in regression of the tumor.
• the methods and reagents (e.g., cells) of the invention may also be used prophylactically, to elicit an immune response that decreases a subject's risk of developing a cancer.
• an immune response in a subject may be elicited by administration of TRT proteins and fragments and derivatives thereof, polynucleotides encoding such TRT proteins, fragments, and derivatives, and antigen presenting cells (e.g., dendritic cells) comprising the aforementioned polypeptide and polynucleotide reagents, e.g., dendritic cells displaying TRT antigens in the context of peptide/MHC complexes.
• the immune response that is evoked may be primarily humoral (i.e., antibody meditated), primarily cell-mediated (e.g., cytotoxic T-lymphocyte- mediated), or have both humoral and cellular components.
• T lymphocytes may be activated ex vivo and administered to a subject.
• the immune response to TRT or a TRT-expressing cell is elicited by methods including one or more of the steps of (1) administering to a subject an antigen presenting cell (APC) that presents a TRT antigenic peptide; (2) administering to a subject T-lymphocytes that recognize a TRT peptide/MHC complex; (3) administering an immunogenic amount of a polynucleotide encoding a TRT protein, polypeptide, or fragment; (4) administering an immunogenic amount of a TRT protein, polypeptide, or fragment.
• APC antigen presenting cell
• the polynucleotide of (3) or polypeptide of (4) are usually administered in an amount capable of inducing a Class I MHC-restricted cytotoxic T-lymphocyte response against cells expressing a TRT protein or, alternatively, in an amount capable of inducing the production of antibodies by the subject.
• antigen presenting cells are used to activate T lymphocytes in vivo or ex vivo, to elicit an immune response against TRT-expressing cells.
• APCs are highly specialized cells, including macrophages, monocytes, and dendritic cells (DCs), that can process antigens and display their peptide fragments on the cell surface together with molecules required for lymphocyte activation.
• dendritic cells are superior to other antigen presenting cells for inducing a T lymphocyte mediated response (e.g., a primary immune response).
• DCs may be classified into subgroups, including, e.g., follicular dendritic cells, Langerhans dendritic cells, and epidermal dendritic cells.
• DCs have been shown to be potent simulators of both T helper (Th) and cytotoxic T lymphocyte (CTL) responses. See Schuler et al., 1997, Int. Arch. Allergy Immunol. 112:317-22.
• Th T helper
• CTL cytotoxic T lymphocyte
• MHC class I and MHC class II proteins The loading of MHC class I molecules usually occurs when cytoplasmic proteins (including proteins such as TRT that are ultimately transported to the nucleus) are processed and transported into the secretory compartments containing the MHC class I molecules.
• MHC Class II proteins are normally loaded in vivo following sampling (e.g., by endocytosis) by APCsof the extracellular milieu.
• DCs migrate to lymphoid organs where they induce proliferation and differentiation of antigen-specific T lymphocytes, i.e., Th cells that recognize the peptide/MHC Class II complex and CTLs that recognize the peptide/MHC Class I complex.
• T lymphocytes and cell mediated immunity An introduction to T lymphocytes and cell mediated immunity is found in Paul, 1993, FUNDAMENTAL IMMUNOLOGY, THIRD EDITION Raven Press, New York, NY and the references cited therein.
• DCs or DC precursor cells
• antigenic peptide fragments ex vivo referred to as "antigen pulsing"
• genetically modified ex vivo to express a desired antigen
• the pulsed or genetically modified DCs can be cultured ex vivo with T lymphocytes (e.g., HLA-matched T lymphocytes) to activate those T cells that specific for the selected antigen.
• T lymphocytes e.g., HLA-matched T lymphocytes
• antigen-laden DC may be used to boost host defense against tumors (see, e.g., Hsu, et al., 1996, Nature Med.2:52-58; Young et al., 1996, J Exp Med. 183:7-11; McArthur et al., 1998, J Immunother. 21 :41-47; Tuting et al., 1997, Eur. J. Immunol. 27:2702-2707; Nair et al., 1997, Int. J. Cancer 70:706-715).
• target antigen e.g., target "tumor” antigen
• TRT cytoplasmic proteins and nuclear proteins
• the present invention relates to the use of polypeptides and polynucleotides encoding TRT (especially human hTRT), and antigen presenting cells (especially dendritic cells), to elicit an immune response against TRT-expressing cells, such as cancer cells, in a subject.
• TRT especially human hTRT
• antigen presenting cells especially dendritic cells
• this involves (1) isolating a hematopoietic stem cells, (2) genetically modifying the cells to express a TRT polypeptide, (3)
• the process involves (1) isolating DCs (or isolation and differentiation of DC precursor cells) (2) pulsing the cells with TRT peptides, and (3) administering the DCs to the subject.
• the TRT pulsed or expressing DCs of the invention are used to activate T lymphocytes ex vivo.
• DC stem cells are isolated for transduction with a TRT- encoding polynucleotide, and induced to differentiate into dendritic cells.
• the genetically modified DCs express the TRT polypeptide, and display peptide fragments on the cell surface. Isolation of DC Precursor Cells
• Human hematopoietic progenitor and stem cells are characterized by the presence of a CD34 surface membrane antigen, which may be used in purification.
• human hematopoietic stem cells are obtained by bone marrow aspiration, and the bone marrow mononuclear cells are separated from the other components by means of Ficol density gradient centrifugation and adherence to plastic.
• the light density, non-adherent cells are obtained and further selected using an anti-CD34 antibody (preferably monoclonal) by standard methods (e.g., incubation of cells with the anti-CD34 antibody, subsequent binding to an immobilized secondary antibody, and removal of nonbound components; see, e.g., Harlow and Lane, 1988, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York)
• an anti-CD34 antibody preferably monoclonal
• standard methods e.g., incubation of cells with the anti-CD34 antibody, subsequent binding to an immobilized secondary antibody, and removal of nonbound components; see, e.g., Harlow and Lane, 1988, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York
• cells can be obtained by leukapheresis of peripheral blood and anti-CD34 chromatography (see, e.g., Reeves et al, 1996, Cancer Res. 56:5672
• the DC or DC precursor cell is genetically modified to express a TRT polypeptide (e.g., transduced ex vivo with a polynucleotide encoding TRT).
• a TRT polypeptide e.g., transduced ex vivo with a polynucleotide encoding TRT.
• Exogenous TRT-encoding polynucleotides may be incorporated into DC as TRT expression cassettes using methods such as those described infra.
• the DC is transformed with an expression cassette comprising a region encoding a TRT polypeptide (or one or more fragments thereof).
• the TRT polypeptide is processed into antigenic peptides expressed on the surface of the DC as complex with MHC class I and II surface molecules.
• the TRT expression cassette includes an operably linked promoter (to drive expression of the TRT coding sequences).
• a strong promoter such as a t-RNA pol III promoter, or a pol II promoter with strong constitutive expression is used.
• Suitable promoters include the constitutive adeno virus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter- enhancer combinations known in the art.
• the TRT coding sequence is introduced into the DC precursor without a linked promoter.
• the TRT transcription is directed by an endogenous promoter (e.g., following integration of the TRT coding sequence into the cell chromosome) or a separately introduced promoter (e.g., that becomes linked by recombination).
• the TRT expression cassette is contained in an expression vector such as a plasmid or viral vector, which may also include other elements, e.g., an origin of replication, chromosome integration elements such as retroviral LTRs, and/or selection (e.g., drag resistance) sequences.
• all of most (e.g., at least about 60%, at least about 75% or at least about 90%) of the TRT protein is expressed (i.e., coded for) in the TRT expression cassette. In some cases, however, a shorter fragment may be expressed.
• TRT coding sequence will encode at least about 8, more often 12, still more often at least 30 or at least 50 contiguous TRT amino acid residues.
• the TRT polypeptide expressed has a sequence of a naturally occurring TRT. It will be recognized, however, that the invention is not limited
• TRT polypeptide may comprise mutations such as deletions, insertions, or amino acid substitutions when compared to a naturally occurring TRT polypeptide, so long as at least one TRT peptide epitope can be processed by the DC and presented on a MHC class I or II surface molecule. It will be appreciated that it may sometimes be desirable to use TRT sequences other than "wild type,” in order to, for example, increase antigenicity of the TRT peptide or to increase TRT peptide expression levels.
• the introduced TRT sequences encode TRT variants such as polymorphic variants (e.g., a variant expressed by a particular human patient) or variants characteristic of a particular cancer (e.g., a cancer in a particular patient).
• polymorphic variants e.g., a variant expressed by a particular human patient
• variants characteristic of a particular cancer e.g., a cancer in a particular patient.
• the TRT expression sequence may be introduced (transduced) into DCs or stem cells in any of a variety of standard methods, including transfection, recombinant vaccinia viruses, adeno-associated viruses (AAVs), and retroviruses (see, e.g., Reeves et al., 1996, Cancer Res. 56:5672; Brossart et al., 1997, J Immunol. 158:3270; Ribas et al., 1997, Cane. Res. 57:2865; Carter etal, 1993, WO 93/24641; Kotin, 1994, Human Gene Therapy 5:793-801; Specht et al., 1997, J. Exp. Med.
• TRT- encoding polynucleotide can be packaged into viral particles using packaging cell lines, which are incubated with the DC stem cells.
• TRT-peptide expressing hematopoietic progenitor cells described supra are induced to differentiate into DCs by conventional methods, e.g., by exposure to cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), flt-3 ligand, tumor necrosis factor alpha c-kit ligand (also called steel factor or mast cell factor).
• cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), flt-3 ligand, tumor necrosis factor alpha c-kit ligand (also called steel factor or mast cell factor).
• interleukin-4 interleukin-4
• TNF-alpha when mixed with undifferentiated stem cells, increases the likelihood that the stem cells will develop as dendritic cells.
• calcium ionophore is used to stimulate the maturation of isolated monocytes into dendritic cells (U.S. Patent 5,643,786).
• DCs are obtained from CD34+ hematopoietic progenitor cells from the blood (e.g., of cancer patients) according to the method described by Bernhard et al., 1995, Cancer Res.
• a DC maturation factor may be used to cause "immature DCs" to stably express dendritic cell characteristics (e.g., dendritic cell markers p55 and CD83 ; see WO 97/29182).
• immature DCs may be used to activate T cells (Koch et al., 1995, J. Immunol.155:93-100).
• the transformed DCs of the invention are introduced into the subject (e.g., human patient) where they induce a immune response.
• the immune response includes a CTL response against target cells bearing TRT antigenic peptides (e.g., in a MHC class I/peptide complex). These target cells are typically cancer cells.
• the DCs of the invention are to be administered to a patient, they are preferably isolated from, or derived from precursor cells from, that patient (i.e., the DCs are administered to an autologous patient).
• the cells may be infused into HLA- matched allogeneic, or HLA-mismatched allogenic patients. In the latter case, immunosuppressive drugs may be administered to the recipient.
• the cells are administered in any suitable manner, preferably with a pharmaceutically acceptable carrier (e.g., saline).
• a pharmaceutically acceptable carrier e.g., saline
• administration will be intravenous, but intra-articular, intramuscular, intradermal, intraperitoneal, and subcutaneous routes are also acceptable.
• Administration i.e., immunization
• Infusions of DC may be combined with administration of
• cytokines that act to maintain DC number and activity (e.g., GM-CSF, IL-12)
• the dose administered to a patient should be sufficient to induce an immune response as detected by assays which measure T cell proliferation, T lymphocyte cytotoxicity, and/or effect a beneficial therapeutic response in the patient over time, e.g., to inhibit growth of cancer cells or result in reduction in the number of cancer cells or the size of a tumor.
• assays which measure T cell proliferation, T lymphocyte cytotoxicity, and/or effect a beneficial therapeutic response in the patient over time, e.g., to inhibit growth of cancer cells or result in reduction in the number of cancer cells or the size of a tumor.
• 10 6 to 10° or more DCs are infused, if available.
• DCs are obtained (either from a patient or by in vitro differentiation of precursor cells) and pulsed with antigenic peptides having a sequence of TRT (e.g., hTRT).
• TRT e.g., hTRT
• the pulsing results in the presentation of TRT peptides onto the surface MHC molecules of the cells.
• the TRT-peptide/MHC complexes displayed on the cell surface are capable of inducing a MHC-restricted cytotoxic T- lymphocyte response against target cells expressing TRT polypeptides (e.g., cancer cells).
• DCs can be obtained by isolating DC precursor cells and inducing them to differentiate into DCs, as described supra.
• DCs may be isolated from both lymphoid and non-lymphoid tissues; typically they are purified from peripheral blood.
• Methods for isolation of human DCs from blood include apheresis followed by procedures involving density gradient methods, positive selection (e.g., affinity chromatography with antibodies directed to DC surface markers), negative selection, or combinations thereof (see, e.g., WO97/22349; WO95/34638; WO98/01538; WO94/02156).
• DC may be isolated from a normal human or from a patient suffering from a disease.
• individuals may be treated with colony stimulating factors to increase their number of DC prior to isolation.
• GM-CSF may be infused into an individual at 250 ⁇ g/m 2 /day for several days up to three weeks intravenously prior to obtaining the peripheral blood mononuclear leukocytes (PBML) for the purification of DC.
• PBML peripheral blood mononuclear leukocytes
• Immortalized DCs may be prepared according to the methods of copending applications USSN 08/912,951 and 08/974,549.
• DCs transformed with Epstein-Barr virus see, WO 97/04802
• retroviral vectors containing an oncogene see, e.g, WO 94/28113
• U.S. Pat. 5,648,219 may be used.
• DCs are exposed ex vivo to TRT antigens, and allowed to process the antigen so that TRT epitopes are presented on the surface of the cell in the context of a MHC class I (or MHC class II) complex. This procedure is referred to as "antigen pulsing.” The “pulsed DCs” may then be used to activate T lymphocytes.
• the TRT peptide antigens used for pulsing DCs comprise at least one linear epitopes derived from the TRT protein. TRT proteins or substantial fragments thereof may be used, as they will be taken up and processed by the DCs. Alternatively, short "peptides" may be administered to the DCs.
• TRT peptides When used for pulsing, they will usually have at least about 6 or 8 amino acids and fewer than about 30 amino acids or fewer than about 50 amino acid residues in length. In one embodiment, the immunogenic TRT peptide has between about 8 and 12 amino acids.
• a mixture of hTRT protein fragments may be used; alternatively a particular peptide of defined sequence may be used.
• the TRT peptide antigens may be produced by de novo peptide synthesis, enzymatic digestion of purified or recombinant hTRT, by purification of telomerase from a natural source (e.g., a patient or tumor cells from a patient), or expression of a recombinant polynucleotide encoding a hTRT fragment.
• the various class I MHC alleles bind peptides having, from N-terminus to C-terminus, first, second and/or third conserved amino acid residues separated by a certain number of amino acids.
• the binding motifs for several human HLA-A alleles are provided in WO 94/03205.
• the TRT fragment or peptide comprises at least one linear TRT epitope having a class I MHC binding motif, and, in particular, an HLA-A 1 binding motif, an HLA-A2.1 binding
• an HLA- A3.2 binding motif an HLA-A 11 binding motif, an HLA-A24.1 binding motif or an HLA-B7 binding motif.
• a TRT such as the hTRT sequences provided in Figure 1 and Figure 2
• a mixture of TRT peptides may be used, so that a wide variety of HLA binding motifs are represented.
• TRT antigen used for pulsing DC will depend on the nature, size and purity of the peptide or polypeptide. Typically, from about 0.05 ⁇ g/ml to about 1 mg/ml, most often from about 1 to about 100 ⁇ g/ml of TRT peptide is used.
• the cells are then allowed sufficient time to take up and process the antigen and express antigen peptides on the cell surface in association with either class I or class II MHC. Typically this occurs in about 18-30 hours, most often about 24 hours.
• enriched DC are resuspended (10 6 cells /ml) in RPMI media (Gibco) and cultured with (50 ug/ml) hTRT peptide antigens overnight under standard conditions (e.g., 37°C humidified incubator/5%
• the pulsed DC are washed in physiologic saline and administered to a subject as described supra, Section (A)(4).
• Antigen-specific T lymphocytes may be prepared ex vivo by collecting naive T lymphocytes from an individual (e.g., CD8+ T lymphocytes), contacting them with the DCs of the invention (e.g., TRT transformed or pulsed DCs) so that TRT-reactive T lymphocytes proliferate.
• the DCs are pulsed with TRT antigens at the time of co-culture with T lymphocytes.
• the expanded TRT-reactive T lymphocyte population may then be administered to the individual, or may be used for in vitro assays.
• the ex vivo activated-T lymphocytes are cytotoxic against target cells bearing TRT antigenic peptides, such as cancer cells (see, e.g., WO 94/ 02156).
• T lymphocytes are Several techniques for isolating T lymphocytes.
• Ficoll-Hypaque density gradient centrifugation is used to separate PBMC from red blood cells and neutrophils according to established procedures.
• Cells are washed with AIM-V (GIBCO) supplemented with 2 mM glutamine, 10 ⁇ g/ml gentamicin sulfate, 50 ⁇ g/ml streptomycin and 1% fetal bovine serum).
• Enrichment for T lymphocytes is performed by negative or positive selection with appropriate monoclonal antibodies (e.g., anti-CD2, CD3, CD4, CD5 and CD8) coupled to columns or magnetic beads according to standard techniques.
• monoclonal antibodies e.g., anti-CD2, CD3, CD4, CD5 and CD8
• fluorescence-activated cell sorting is used.
• IL-4 and IL-2 An aliquot of cells is analyzed for cell surface phenotype including CD4, CD8, CD3 and CD14.
• Cells are washed and resuspended at a concentration of5 X 10 5 cells perml of AIM-V supplemented as above and containing 5% FBS and 100 U/ml recombinant IL-2.
• the T lymphocytes are then cultured with pulsed or genetically modified DCs, optionally in the presence of low doses of IL-4 and IL-2.
• the activated T lymphocytes can then be administered to a subject (e.g., infused) as described for DCs, supra.
• the expanded T lymphocytes may be administered alone, or in combination with lymphokines such as IL-2 and/or IL-4.
• the present invention provides commercially valuable assays, e.g., for identifying TRT-expressing cells.
• dendritic cells are transformed with a TRT-encoding nucleic acid or pulsed with a TRT peptide.
• the DC is used to activate isolated T lymphocytes, which are then tested for cytotoxic activity against a class of cells though to express TRT.
• Cytotoxicity e.g., as indicated in a standard assays such as a " 51 Cr release assay,” infra indicates that the cells express TRT is amounts sufficient to mediate a T lymphocyte recognition of the cell. This provides investigators with an assay for TRT-expressing cells.
• the target antigen-presenting cells and effector CTL cells are mixed in culture and target cell lysis is observed.
• a cell e.g., a cell expressing TRT, such as a cancer cell
• Any suitable method for measuring cell lysis can be used by one skilled in the art.
• a radioactivity release assay can be used to measure lysis of the target cells.
• the target cells e.g., the target cells
• Radioactive reagents such as 5I Cr, which are taken up by live cells. Following labeling, the target cells are washed and mixed with specific CTLs. Supernatants are harvested after a suitable time and counted to determine the percent radioactivity release. Other methods to determine the amount of cell lysis include trypan blue exclusion, in which living cells that exclude the dye are counted and compared to a control sample of non-presenting cells treated in the same manner.

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• 1999
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