WO2008027456A2 - Procédés, compositions et ensembles concernant des cellules tumorales présentant des antigènes - Google Patents
Procédés, compositions et ensembles concernant des cellules tumorales présentant des antigènes Download PDFInfo
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- WO2008027456A2 WO2008027456A2 PCT/US2007/019048 US2007019048W WO2008027456A2 WO 2008027456 A2 WO2008027456 A2 WO 2008027456A2 US 2007019048 W US2007019048 W US 2007019048W WO 2008027456 A2 WO2008027456 A2 WO 2008027456A2
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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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5152—Tumor cells
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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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5156—Animal cells expressing foreign proteins
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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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- 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
- C12N2510/00—Genetically modified cells
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- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
Definitions
- Epithelial ovarian cancer is the second most common gynecologic cancer, though it accounts for the majority of gynecologic cancer deaths and will be responsible for 90% of the projected 16,090 deaths due to ovarian cancer in 2004 (Jemal et al., 2004, CA Cancer J. Clin. 54:8-29). Seventy percent of patients present with advanced disease at diagnosis. While first-line chemotherapy yields initial response rates greater than 65%, most patients eventually relapse and die of chemotherapy-resistant disease (Kristensen and Trope, 1997, Lancet 349:113-117). Therefore, there is a need for additional strategies, such as immunotherapy, to complement extant therapies (Hwu and Freedman, 2002, J. Immunother. 25:189-201).
- antigen-presenting cells the most potent of which are dendritic cells, are currently a central focus of tumor immunotherapy, due to their ability to activate T cells and induce anti-tumor immune responses (Schuler et al., 2000, Curr. Opin. Immunol. 15:138-147; Banchereau et al., 2000, Annu. Rev. Immunol. 18:767-811 ; Fong et al., 2000, Annu. Rev. Immunol. 18:245-273).
- Ovarian cancer like many other human tumors, presents a paradox: the coexistence of tumor cells and tumor-specific T cells, indicating the initiation but not completion of a tumor specific immune response (Monsurro et al., 2003, Semin. Cancer Biol. 13:473-480; Marincola et al., 2003, Trends Immunol. 24:335-342).
- the anti-tumor response in ovarian cancer is not wholly ineffective, as the presence of intratumoral T cells is correlated with increased progression-free or overall survival (Zhang et al., 2003, N. Eng. J. Med. 348:203-213).
- Tumor cells in the form of whole cell vaccines, have a long history in tumor immunotherapy (Ward et al., 2002, Cancer Immunol. Immunother. 51:351-357). In recent years, it has become evident that T cell subsets have complex requirements for survival, proliferation, and function. These complexities were not taken into account in many of the earlier attempts to modify tumor cells with costirnulatory molecules. There are numerous examples of attempts to enhance the immunogenicity of tumor cells, to in effect convert them into antigen-presenting cells, by introduction of costimulatory molecules and/or cytokines (for example, see Qian, et al., 2002, Int. J. Gynecol.
- FCS fetal calf serum
- tumor cells also have numerous features that would seem to preclude their use as antigen-presenting cells.
- tumor cells are antigenic, they are poorly immunogenic. Since most tumors do not express costimulatory molecules, they can induce anergy or tolerance based on MHC I-restricted antigen presentation in the absence of costimulation (Abken et al., 2002, Trends Immunol. 23:240-245).
- the present invention includes an isolated antigen presenting tumor cell (APTC), said APTC comprising a human tumor cell transduced with a first and a second vector, wherein the first vector comprises a nucleic acid encoding a human Fc receptor and the second vector comprises a nucleic acid encoding a co-stimulatory ligand.
- APTC isolated antigen presenting tumor cell
- the Fc receptor is CD32 or CD64.
- the co-stimulatory ligand is at least one of the group consisting of CD86, CD80, CCL 21, and 4- IBBL.
- the humor cell is transuced with a third vector, wherein the third vector comprises a nucleic acid encoding an HLA molecule, preferably HLA-2A.
- the vector is a lentiviral vector.
- the human tumor cell is an ovarian cancer cell.
- the human tumor cell is an ovarian cancer cell line.
- the present invention includes a method for inducing proliferation of a T cell, said method comprising contacting said T cell with an APTC, wherein contacting said T cell induces proliferation of said T cell.
- the present invention includes a method of treating a tumor in a mammal, said method comprising contacting a population of T cells from said mammal with an APTC, expanding said population of T cells, and administering said population of T cells to said mammal, thereby treating a tumor in a mammal.
- the present invention includes a method for inducing a T cell response in a mammal, said method comprising administering an APTC to said mammal, wherein said APTC induces proliferation of a T cell in said mammal, thereby inducing a T cell response in said mammal.
- the present invention includes a method of treating a tumor in a mammal, said method comprising administering an APTC to said mammal, wherein said APTC induces proliferation of a T cell in said mammal, thereby treating a tumor in said mammal.
- the present invention includes a kit for inducing proliferation of a T cell, said kit comprising an effective amount of an APTC wherein said APTC comprises an ovarian cancer cell transduced with a first and a second vector, wherein the first vector comprises a nucleic acid encoding a human Fc receptor and the second vector comprises a nucleic acid encoding a co-stimulatory ligand, said kit further comprising an applicator and an instructional material for the use of said kit.
- the present invention includes a kit for inducing a T cell response in a mammal, said kit comprising an effective amount of an APTC wherein said APTC comprises an ovarian cancer cell transduced with a first and a second vector, wherein the first vector comprises a nucleic acid encoding a human Fc receptor and the second vector comprises a nucleic acid encoding a co-stimulatory ligand, said kit further comprising an applicator and an instructional material for the use of said kit.
- the present invention includes a kit for treating a tumor in a mammal, said kit comprising an effective amount of an APTC wherein said APTC comprises an ovarian cancer cell transduced with a first and a second vector, wherein the first vector comprises a nucleic acid encoding a human Fc receptor and the second vector comprises a nucleic acid encoding a co-stimulatory ligand, said kit further comprising an applicator and an instructional material for the use of said kit.
- Figure 1 is a series of images depicting the morphology of ovarian cancer cell lines.
- Figure IA depicts ovarian cancer cell line ACC-OV7;
- Figure IB depicts ovarian cancer cell line ACC-OV68A;
- Figure 1C depicts ovarian cancer cell line ACC-OV551;
- Figure ID depicts ovarian cancer cell line ACC-OV684;
- Figure IE depicts ovarian cancer cell line ACC-OV552.
- the bar in Figure IA depicts 100 ⁇ m.
- Figure 2 is a series of images depicting the expression of lenti viral-vector encoded molecules by transduced ACC-O V7 Cells.
- Figure 2A and Figure 2B depict flow cytometry analysis of CD86 and CD32 expression on three-month cultures of transduced ACC-OV7 cells.
- Figure 2A illustrates untransduced cells while Figure 2B illustrates transduced cells.
- the numbers next to the boxed section of the flow cytometry analysis represent the percentage of CD32/CD86 double positive cells.
- Figure 2C depicts flow cytometry analysis of 4- IBBL expression in untransduced ACC-OV7 cells and Figure 2D depict flow cytometry analysis of 4- IBBL expression on the transduced ACC-0V7 cells depicted in Figure 2B.
- Figures 2E and 2F depict the intracellular expression of 4- IBBL.
- Figure 2E depicts cells that were fixed and permeabilized
- Figure 2F depicts cells that were fixed but not permeabilized prior to intracellular staining for 4- IBBL.
- Figure 3 is a series of images depicting stimulation of allogeneic CD8+ T cell proliferation by ACC-0V7 cells expressing costimulatory molecules.
- Figure 3 A and 3B depict the stimulation of short- term CD8+ T cell proliferation by ACC-O V7/32/86/4- IBBL cells.
- Figure 3 A depicts the proliferation profile of CFSE-labeled CD8+ T cells stimulated with ACC-OV7/32/86/4- IBBL cells in the presence (open histogram) or the absence (filled histogram) of ⁇ CD3.
- Figure 3B depicts the proliferation profile of CFSE labeled CD8+ T cells stimulated with K32 cells in the presence of ⁇ CD3 and ⁇ CD28.
- Figure 3C depicts sustained CD8+ T cell proliferation induced by ACC-OV7/CD32/86/4- IBBL cells.
- CD8+ T cells were stimulated with either ACC-OV7 (open circles) or ACC-O V7/32/86/4- IBBL (triangles) in the presence of ⁇ CD3, or irradiated K32/4-1BBL cells (filled circles) in the presence of ⁇ CD3 and ⁇ CD28.
- the arrow indicates the timing of restimulation.
- Figure 4 is a table summarizing the ovarian cancer cells, their source, days in culture, number of passages, medium supplement and extracellular matrix.
- Figure 5 is a table summarizing the antigen profile and growth characteristics of ovarian cancer cells.
- Figure 6 is a table summarizing the array Comparative Genomic
- FIG. 7 is an image of SEQ ID NO: 1.
- Figure 8 is an image of SEQ ID NO:2
- Figure 9 is an image of SEQ ID NO: 3
- Figure 10 comprising Figures 1 OA through 1 OF, is a series of images depicting the expression of lentiviral-vector encoded molecules by transduced ACC- OV87 cells.
- Figure 1OA and Figure 1OB depict flow cytometry analysis of CD64 expression of transduced ACC-OV87TA2.64 cells.
- Figures 1OC through 1OF depict flow cytometry analysis of A2 FITC, CD64 FITC, 4 IBBL PE, and CD86 APC expression, respectively in ACC-OV87TA2.64.86.41BBL cells.
- Figure 11 is a series of images depicting the expression of lentiviral-vector encoded molecules by transduced ACC- OV79 cells.
- Figures 1 IA through 1 ID depict flow cytometry analysis of A2 FITC 5 4 IBBL PE 5 CD64 FITC 3 and CD 86 APC expression, respectively in ACC- OV87TA2.64.86.41BBL cells.
- the present invention encompasses methods and compositions for the treatment of cancer and for developing an immune response to a tumor cell.
- the present invention encompasses ovarian cancer cell lines engineered to function as antigen presenting tumor cells, thus stimulating a response to a tumor antigen and/or a tumor cell.
- ovarian cancer (OvCa) cells and cell lines are transduced with a lentiviral vector comprising a nucleic acid that expresses a polypeptide that initiates and maintains T cell proliferation.
- the data disclosed herein demonstrate, for the first time, methods for determining which OvCa cell lines are the best candidates to use as antigen presenting tumor cells (APTCs) using CD8 T cells from donors.
- the data disclosed herein further demonstrate methods to measure the ability of APTCs to induce anti-tumor immune responses.
- the present invention discloses engineered APTCs that can induce antigen (tumor)-specific proliferation of CD8 T cells.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the present invention further includes expanded cells assayed for their ability to both recognize and kill tumors.
- the present invention discloses the use of engineered APTCs as a tumor vaccine in a mammalian model, and the ability of the APTCs to promote a sustained, effective anti-tumor immune response in vivo.
- an element means one element or more than one element.
- Allogeneic refers to a cell or biological compound derived from a different animal of the same species.
- autologous is meant to refer to any material derived from the same mammal to which it is later to be re-introduced into the mammal.
- to "alleviate” a disease means reducing the severity of one or more symptoms of the disease.
- apper any device including, but not limited to, a hypodermic syringe, a pipette, and the like, for administering the compounds and compositions of the invention.
- a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated, then the animal's health continues to deteriorate.
- a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
- an effective amount is meant an amount that when administered to a mammal, causes a detectable level of T cell response compared to the T cell response detected in the absence of the compound. T cell response can be readily assessed by a plethora of art-recognized methods.
- the amount of the compound or composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.
- instructional material includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and/or compound of the invention in the kit for effecting alleviating or treating the various diseases or disorders recited herein.
- the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue or a mammal, including as disclosed elsewhere herein.
- the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition.
- the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.
- pharmaceutically acceptable carrier means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
- physiologically acceptable ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
- a "coding region" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
- a "coding region" of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon.
- the coding region may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
- Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from.
- a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
- Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
- “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
- An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
- Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
- a first region of an oligonucleotide "flanks" a second region of the oligonucleotide if the two regions are adjacent one another or if the two regions are separated by no more than about 1000 nucleotide residues, and preferably no more than about 100 nucleotide residues.
- fragment as applied to a nucleic acid, may ordinarily be at least about 18 nucleotides in length, preferably, at least about 24 nucleotides, more typically, from about 24 to about 50 nucleotides, preferably, at least about 50 to about 100 nucleotides, even more preferably, at least about 100 nucleotides to about 200 nucleotides, yet even more preferably, at least about 200 to about 300, even more preferably, at least about 300 nucleotides to about 400 nucleotides, yet even more preferably, at least about 400 to about 500, and most preferably, the nucleic acid fragment will be greater than about 500 nucleotides in length.
- a "fragment" of a stimulatory or costimulatory ligand protein or an antigen is about 6 amino acids in length. More preferably, the fragment of a protein is about 8 amino acids, even more preferably, at least about 10, yet more preferably, at least about 15, even more preferably, at least about 20, yet more preferably, at least about 30, even more preferably, about 40, and more preferably, at least about 50, more preferably, at least about 60, yet more preferably, at least about 70, even more preferably, at least about 80, and more preferably, at least about 100 amino acids in length amino acids in length.
- a “genomic DNA” is a DNA strand which has a nucleotide sequence homologous with a gene as it exists in the natural host.
- a fragment of a chromosome is a genomic DNA.
- "Homologous” as used herein refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then ⁇ they are completely or 100% homologous at that position.
- the percent homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% identical, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
- the DNA sequences 5 ⁇ TTGCC3' and 5 'TATGGC3' share 50% homology.
- isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
- nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
- the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
- the following abbreviations for the commonly occurring nucleic acid bases are used.
- A refers to adenosine
- C refers to cytidine
- G refers to guanosine
- T refers to thymidine
- U refers to uridine.
- two polynucleotides as "operably linked” is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other.
- a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region.
- the nucleic acid encoding the desired protein further comprises a promoter/regulatory sequence
- the promoter/regulatory is positioned at the 5' end of the desired protein coding sequence such that it drives expression of the desired protein in a cell.
- the nucleic acid encoding the desired protein and its promoter/regulatory sequence comprise a "transgene.”
- promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
- this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
- the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
- a “constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell under most or all physiological conditions of the cell.
- An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer which corresponds to the promoter is present in the cell.
- tissue-specific promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
- a “polyadenylation sequence” is a polynucleotide sequence which directs the addition of a poly A tail onto a transcribed messenger RNA sequence.
- a “polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
- nucleic acid typically refers to large polynucleotides.
- oligonucleotide typically refers to short polynucleotides, generally, no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G 3 C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U” replaces "T.” Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.
- a "portion" of a polynucleotide means at least at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.
- Recombinant polynucleotide refers to a polynucleotide having sequences that are not naturally joined together.
- An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell.
- a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
- a non-coding function e.g., promoter, origin of replication, ribosome-binding site, etc.
- a "recombinant polypeptide” is one which is produced upon expression of a recombinant polynucleotide.
- Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
- protein typically refers to large polypeptides.
- peptide typically refers to short polypeptides.
- polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl -terminus.
- transgene means an exogenous nucleic acid sequence which exogenous nucleic acid is encoded by a transgenic cell or mammal.
- a “recombinant cell” is a cell that comprises a transgene. Such a cell may be a eukaryotic cell or a prokaryotic cell. Also, the transgenic cell encompasses, but is not limited to, an APTC 5 an ovarian tumor cell comprising the transgene and a prokaryotic cell comprising the transgene.
- exogenous nucleic acid is meant that the nucleic acid has been introduced into a cell or an animal using technology which has been developed for the purpose of facilitating the introduction of a nucleic acid into a cell or an animal.
- tag polypeptide is meant any protein which, when linked by a peptide bond to a protein of interest, may be used to localize the protein, to purify it from a cell extract, to immobilize it for use in binding assays, or to otherwise study its biological properties and/or function.
- to "treat” means reducing the frequency with which symptoms of a disease (i.e., viral infection, tumor growth and/or metastasis, or other effect mediated by decreased numbers and/or decreased activity of T cells, and the like) are experienced by a patient.
- symptoms of a disease i.e., viral infection, tumor growth and/or metastasis, or other effect mediated by decreased numbers and/or decreased activity of T cells, and the like
- vector any plasmid or virus encoding an exogenous nucleic acid.
- the term should also be construed to include non- plasmid and non- viral compounds which facilitate transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like.
- the vector may be a viral vector which is suitable as a delivery vehicle for delivery of a nucleic acid that encodes a protein and/or antibody of the invention, to the patient, or to the aAPC, or the vector may be a non-viral vector which is suitable for the same purpose.
- viral and non-viral vectors for delivery of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94: 12744-12746).
- viral vectors include, but are not limited to, a lentiviral vector, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian pox virus, and the like (Cranage et al., 1986, EMBO J. 5:3057-3063; International Patent Application No. WO 94/17810, published August 18, 1994; International Patent Application No.
- non- viral vectors include, but are not limited to, liposomes, polyamine derivatives of DNA, and the like.
- a "therapeutic" treatment is a treatment administered to a patient who exhibits signs of pathology for the purpose of diminishing or eliminating those signs and/or decreasing or diminishing the frequency, duration and intensity of the signs.
- stimulation is meant a primary response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction.
- Stimulation can mediate altered expression of certain molecules, such as downregulation of T cell suppressors, and/or reorganization of cytoskeletal structures, and the like.
- Activation refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions.
- the term “activated T cells” refers to, among other things, T cells that are undergoing cell division.
- specifically binds is meant an antibody, or a ligand, which recognizes and binds with a cognate binding partner (e.g., a stimulatory and/or costimulatory molecule present on a T cell) protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.
- a “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an APTC) can specifically bind with a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
- an antigen presenting cell e.g., an APTC
- a cognate binding partner referred to herein as a "stimulatory molecule”
- a “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell (e.g., an APTC of the invention, among others).
- an antigen presenting cell e.g., an APTC of the invention, among others.
- “Loaded” with a peptide, as used herein, refers to presentation of an antigen in the context of an MHC molecule.
- Co-stimulatory ligand includes a molecule on an antigen presenting cell (e.g., an APTC, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, an antibody to CD3 and/or an antibody to CD28, mediates a T cell response, including, but not limited to, proliferation, differentiation, and the like.
- an antigen presenting cell e.g., an APTC, and the like
- a co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-Ll, PD-L2, 4-1 BBL, OX40L, inducible COStimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA- G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, and a ligand that specifically binds with B7-H3.
- a co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4- IBB, OX40, CD30, CD40, PD-I, ICOS, lymphocyte function-associated antigen-1 (LFA-I) 5 CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
- an antibody that specifically binds with a co-stimulatory molecule present on a T cell such as, but not limited to, CD27, CD28, 4- IBB, OX40, CD30, CD40, PD-I, ICOS, lymphocyte function-associated antigen-1 (LFA-I) 5 CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
- LFA-I lymphocyte function-associated antigen-1
- a "co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co- stimulatory response by the T cell, such as, but not limited to, proliferation.
- treat means to reduce the frequency of the disease or disorder reducing the frequency with which a symptom of the one or more symptoms disease or disorder is experienced by an animal.
- vaccine is meant a composition, a protein or a nucleic acid encoding a protein, or an APTC of the invention, which serves to protect an animal against a disease and/or to treat an animal already afflicted with a disease by inducing an immune response, compared with an otherwise identical animal to which the vaccine is not administered or compared with the animal prior to the administration of the vaccine.
- a tumor antigen means a protein, a polypeptide, or a peptide, which constitutes part of the tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes.
- a tumor antigen peptide can be a peptide that is generated as a result of degradation of the tumor antigen in a tumor cell and can induce or activate tumor-specific cytotoxic T lymphocytes upon being expressed on the cell surface by binding to an HLA molecule (e.g., HLA-A*02).
- an HLA molecule e.g., HLA-A*02
- the site of the amino acid sequence which is capable of inducing tumor-specific cytotoxic T lymphocytes that is present in a tumor antigen is referred to a tumor antigen epitope (tumor antigen determinant).
- the invention relates to the discovery that human tumor cells, more specifically, human ovarian cancer cells, can be modified to express costimulatory molecules and other immune modulators to induce sustained proliferation of allogeneic tumor-specific cells and maintain anti-tumor effector functions.
- the present invention includes tumor cells that can be readily transduced using lentivirus vectors to express numerous molecules, including, but not limited to, stimulatory ligands, costimulatory ligands, soluble immune modulators, and the like, in order to induce the proliferation of tumor-specific CD8 cells and prevent and/or inhibit tumor growth or kill tumors.
- the present invention is based, in part, on the use of a lentiviral vector to introduce an array of costimulatory molecules into tumor cells, thereby generating a tumor antigen-presenting cell (APTC) that stimulates proliferation of cytotoxic CD8 cells, thus providing broader anti-tumor immunity.
- APTC tumor antigen-presenting cell
- the data disclosed herein is applicable to the use in an allogeneic setting, and therefore, an APTC of the present invention can be used to initiate proliferation of cytotoxic cells in a variety of patients.
- the APTC comprises, among other things, a costimulatory ligand that specifically binds a cognate costimulatory molecule expressed on the T cell surface- stimulates the T cell and induces T cell proliferation such that large numbers of specific T cells can be readily produced.
- the APTC expands the T cell "specifically" in that only the T cells expressing the particular costimulatory molecule are expanded by the APTC. Further, because an APTC is derived from a tumor cell, preferably an ovarian tumor cell, the T cell is specific for that tumor cell, thus developing an immune response to that tumor cell.
- the immune response induced by contacting a T-cell with an APTC can comprise a cytotoxic immune response, such as those elicited in CD8 T cells, or a helper T cell response, such as those elicited in CD4 T cells,
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the present invention is also based, in part, on an understanding of the complex costimulatory signaling pathways required to generate an effective anti-tumor CTL response, often lacking in previous attempts to convert tumor cells into antigen presenting cells.
- the present invention encompasses the introduction of the appropriate costimulatory molecules into a tumor cell in order to convert tumor cells into professional antigen-presenting cells, capable of efficiently activating and arming tumor- specific T cells.
- the converted tumors cells or otherwise referred to as APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the costimulatory molecules and cytokines expressed by the APTCs of the present invention include, but are not limited to: CD32, CD86, 4-1 BBL, CD80, CD83,
- CD32 is the FC ⁇ RII receptor. This receptor is expressed on APTCs and binds exogenous stimulatory antibodies and presents them to T cells (Maus et al., 2002, Nat. Biotechnol. 20: 143-148; Thomas et al., 2002, Clin. Immunol. 105 : 259-272). Expression of CD32 on OvCa cells allows the APTCs of the present invention to be loaded with, for example, an anti-CD3 antibody, as well as other IgG antibodies that bind various antigens described herein and known in the art, thus providing a antigen-independent "signal one" T cell growth stimulus.
- CD64 An alternative to using CD32 is CD64.
- CD86 (B7-2), along with CD80 (B7-1) is the prototypic costimulatory ligand, and both CD80 and CD86 share CD28 and CTLA-4 as receptors (June et al., 1994, Immunol. Today 15: 321-331). While CD80 and CD86 are often viewed as interchangeable costimulatory molecules, CD80 may interact more productively with the negative T cell regulator CTLA-4, while CD86 may be a more potent CD28 ligand (Sansom et al., 2003, Trends Immunol. 24: 314-319).
- CD 86 has been reported to generate more immunogenic tumors than transfection of CD80 (LaBeIIe et al., 2002, Blood 99: 2146-2153; Martin-Fontecha et al., 2000, J. Immunol. 164: 698-704; Martin-Fontecha et al., 1996, Eur. J. Immunol. 26: 1851-1859).
- 4- IBBL is a type II transmembrane protein that is expressed by a broad range of professional APCs.
- 4-1BB (CD137) is a member of the TNF receptor family that promotes survival of CD8 T cells (Hurtado et al., 1997, J.
- 4-1BB stimulation preferentially activates CD8 T cells in vitro and amplifies generation of CTL responses in vivo and improves survival of activated CD8 T cells (Shuford et al., 1997, J. Exp. Med. 186: 47-55). Further, it has been demonstrated that a 4-1 BBL mediated signaling is necessary to induce long-term expansion of CD8 T cells (Maus et al., 2002, Nat. Biotechnol. 20: 143-148).
- the APTCs of the present invention can further comprise CD83, ICOS-L, HLA- A2, IL-7, IL- 15 and CCL 21, among other co-stimulatory molecules described elsewhere herein and known in the art.
- CD83 is a marker of mature DCs whose role in T cell activation has recently been investigated (Lechmann et al., 2002, Trends Immunol. 23: 273-275; Scholler et al., 2001, J. Immunol. 166: 3865-3872).
- Co-immobilization of anti-CD3 and CD83-Ig fusion protein enhanced the ratio of CD8 to CD4 T cells, suggesting that CD83 ligation preferentially activates CD8 T cells (Scholler et al., 2002, J. Immunol.
- CD83-expressing tumor cells were rejected, and they primed the immune system to reject parental CD83 deficient tumors (Yang et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101: 4990-4995).
- ICOS-L binds the CD28-related molecule ICOS 5 delivering a potent costimulatory signal to T cells that enhances production of effector cytokines (IFN- ⁇ , IL- 4, and IL- 13) but is unable to produce high levels of IL-2 (Hutloff et al., 1999, Nature 397: 263-266) or induce the survival factor Bcl-xL (Parry et al., 2003, J. Immunol. 171 : 166-174).
- ICOS-L stimulation promotes effector functions in both CD4 and CD8 T cells (Mittrucker et al., 2002, J. Immunol. 169: 5813-5817; Wallin et al., 2001, J.
- the APTCs disclosed herein can express HLA-A2 (e.g., HLA-A*02), and the expression of HLA-A2, or other HLA antigens is augmented to enhance endogenous tumor cell antigen presentation.
- the APTCs of the invention can be engineered to exhibit HLA- A2 (or other HLA antigens) to augment the presentation of tumor-specific antigens thereby enhancing the expansion of CD8+ T cells.
- the APTC engineered to exhibit HLA on its surface is able to expand a population of T cells that recognize a combination of tumor antigens.
- the APTC engineered to exhibit HLA on its surface is able to expand a population of T cells that recognizes a combination of tumor antigens associated with the autologous APTC.
- an ovarian tumor cell can be isolated from a primary tumor sample and engineered to exhibit on its surface HLA (e.g., HLA-A*02) and at least one immune co-stimulatory molecule.
- HLA e.g., HLA-A*02
- the engineered ovarian tumor cell can expand a population of T cells to recognize a combination of ovarian tumor antigens.
- the engineered ovarian tumor cell can expand a population of T cells to recognize all ovarian tumor antigens associated with the ovarian tumor cell.
- IL-7 is produced only by stromal cells (Sudo et al., 1989, J. Exp. Med 170: 333-338) and is essential for naive T cell homeostatic expansion (Tan et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98: 8732-8737). IL-7 is also a critical CD8 memory cell survival factor (Tan et al. 3 2002, J. Exp. Med. 195: 1523-1532; Schluns et al. ⁇ 2000, Nat. Immunol. 1 : 426-432).
- IL-15 is a key CD8 T cell survival factor and is produced by activated macrophages and DCs (Waldmann et al., 2001, Immunity 14: 105-110). IL-15 biological activity does not appear to correlate with mRNA expression levels (Dubois et al., 2002, Immunity 17: 537-547; Musso et al., 1999, Blood 93: 3531-3539). Recent studies have demonstrated that IL-15 and the IL-15R ⁇ chain are both required for the basal proliferation of memory CD8 T cells, and that IL-15 must be presented in trans by bone marrow derived APCs (Schluns et al., 2004, Blood 103: 988-994).
- IL-15 has enhanced anti-tumor CD8 T cell activity (Klebanoff et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101: 1969-1974).
- CCL21 also known as secondary lymphoid organ chemokine (SLC)
- SLC secondary lymphoid organ chemokine
- APTCs producing CCL21 enhance T cell activation by recruiting T cells to the APTC. Further, while CCL 21 may not have a measurable effect during the ex vivo expansion of T cells, this chemokine significantly effects the generation of tumor specific responses in in vivo models. As disclosed elsewhere herein, in ovarian cancer patients, tumor cells and tumor-specific T cells coexist, a clear manifestation of an ultimately ineffective immune response.
- This inadequate anti-tumor immune response could be due to: (1) inadequate numbers and/or: (2) inadequate effector functions, of tumor-specific T cells.
- the present invention provides methods, compositions and kits to strengthen the anti-tumor immune response, quantitatively and qualitatively, by providing greater numbers of tumor- specific T cells with improved anti-tumor effector functions.
- the present invention demonstrates that the anti-tumor immune response can be augmented by modifying the tumor cells themselves to function as APCs. That is, tumor cells that are modified to express the appropriate costimulatory molecules can induce sustained proliferation of autologous tumor-specific CD8 cells, while maintaining anti-tumor effector functions.
- APTCs antigen-presenting tumor cells
- the APTCs can stimulate ex vivo the proliferation and anti-tumor activity of autologous CD8 cells isolated from peripheral blood or malignant peritoneal effusions. Furthermore, the APTCs can stimulate CD8 cells in vivo, in both adoptive therapy and active vaccination settings.
- a cell line established from a tumor e.g., an ovarian tumor according to the methods disclosed herein is a valuable reagent because such a cell line comprises a renewable source of tumor-associated antigens.
- the cell serves as a source of tumor antigens.
- the cell line established from a tumor can be used as a source of antigens that are unique to the autologous tumor.
- the cell isolated from a tumor is a non- haematopoietic cell.
- the cell is not a dendritic cell.
- the cell is preferrably derived from a cancer patient.
- the cell can be isolated from an ovarian tumor specimen.
- the cell isolated from the cancer patient can be manipulated according to the methods disclosed herein to generate an antigen presenting tumor cell (APTC).
- APTC antigen presenting tumor cell
- the invention includes a cell line engineered to exhibit on its surface at least one immune co-stimulatory molecule. Accordingly, the present invention encompasses an APTC. Further, because an APTC is derived from a tumor cell, preferably an ovarian tumor cell, the APTC is specific for a tumor antigen or a combination of tumor antigens, thus capable of developing an immune response to that tumor cell.
- the immune response induced by contacting a T-cell with an APTC can comprise a cytotoxic immune response, such as those elicited in CD8 T cells, or a helper T cell response, such as those elicited in CD4 T cells.
- the APTC of the invention comprises an ovarian cancer cell, preferably human in origin, transduced using a lentiviral vector (LV).
- LV lentiviral vector
- any cancer cell is encompassed in the present invention.
- the LV encodes at least one immune co-stimulatory molecule. While the data disclosed herein demonstrate that multiple different molecules transduced into an ovarian cancer cell (OvCa cell) were stably and highly expressed in long-term culture, there is nothing to suggest that this is a limit in the number or kinds of molecules that can be introduced into these cells. Instead, any molecule, whether stimulatory, co-stimulatory, cytokine, antigen, and the like, can be introduced into these cells to produce an APTC of the invention.
- the APTC of the invention encompasses a tumor cell isolated from a primary cancer specimen modified by any means (e.g., genetically, physically, or chemically manipulated) to have on its surface at least one molecule capable of binding to a T-lymphocyte and inducing a primary activation event and/or a proliferative response or capable of binding a molecule having such an affect thereby acting as a scaffold.
- any means e.g., genetically, physically, or chemically manipulated
- the tumor cell is a non-haematopoietic cell.
- the tumor cell is not a dendritic cell.
- the APTC is engineered to express a molecule that binds to the Fc portion of an antibody.
- an APTC comprises a cell line engineered to stably express a molecule capable of binding to the Fc portion of an antibody. The APTC can then be loaded, or have attached thereto, any variety of antibodies that recognize cell surface molecules present on the surface of T lymphocytes, e.g. CD3, or a component of the TCT/CD3 complex, CD28, 4-1BB, TCR 3 etc.
- an APTC can be generated by directly engineering a cell line to stably express the ligands for cell surface molecules present on the surface of T lymphocytes, e.g. CD3, or a component of the TCR/CD3 complex, CD28, 4-1 BB, TCR, etc.
- the APTC can be further engineered to stably express one or more co-stimulatory molecules, for example CD86 or 4-1BB ligand.
- an APTC is engineered to express the human low-affinity Fc ⁇ receptor, CD32 (or any other Fc ⁇ receptor such as CD64) and the CD86 molecule.
- an APTC is engineered to express CD64 and the 4- IBB ligand.
- the APTC is engineered to exhibit on its surface CD64, CD86, and 4- IBBL.
- the APTC of the present invention can be generated that express membrane bound ScFv or a fragment thereof, that recognize any cell surface molecule of interest, such as CD3, CD28, 41BB and the like, or that recognize other antibodies, such as through binding to the Fc portion.
- the APTC can be armed with secondary antibodies that bind through recognition of the Fc portion.
- an APTC may be engineered to express a variety of molecules useful for the stimulation and activation of T lymphocytes and/or be loaded or otherwise coated with or otherwise have attached thereto a variety molecules useful for the stimulation and activation of T lymphocytes.
- numerous immunoregulatory molecules can be used to produce an almost limitless variety of APTCs once armed with the teachings provided herein.
- T cell mediated immune responses and the factors mediating them, are well-known in the art. More specifically, a primary signal, usually mediated via an antibody to CD3, initiates the T cell activation process. Additionally, numerous co-stimulatory molecules present on the surface of a T cell are involved in regulating the transition from resting T cell to cell proliferation.
- co-stimulatory molecules also referred to as "co-stimulators", which specifically bind with their respective ligands.
- co-stimulators include, but are not limited to, CD32, CD86, CD80, 4- IBBL, CD83, ICOS-L, HLA-A2, IL-7, IL-15 and CCL 21.
- the primary stimulatory signal mediates T cell stimulation, but the co-stimulatory signal is then required for T cell activation, as demonstrated by proliferation.
- the APTC of the invention encompasses a cell comprising a stimulatory ligand that specifically binds with CD3 such that a primary signal is transduced.
- the APTC further comprises at least one co-stimulatory ligand that specifically binds with at least one co-stimulatory molecule present on a T cell, which co-stimulatory molecule includes, but is not limited to, CD32, CD86, 4-1BBL, CD80, CD83, ICOS-L, HLA-A2, IL-7, IL- 15 and CCL 21.
- co-stimulatory molecule includes, but is not limited to, CD32, CD86, 4-1BBL, CD80, CD83, ICOS-L, HLA-A2, IL-7, IL- 15 and CCL 21.
- co-stimulatory ligands are encompassed in the invention, as would be understood by one skilled in the art armed with the teachings provided herein.
- Such ligands include, but are not limited to, mutants, variants, fragments and homologs of the natural ligands described previously.
- mutant or variant of the ligand is encompassed in the invention and can be transduced into a cell using a LV to produce the APTC of the invention and such mutants and variants are discussed more fully elsewhere herein. That is, the invention includes using a mutant or variant of a ligand of interest and methods of producing such mutants and variants are well-known in the art.
- the APTC of the invention comprises at least one stimulatory ligand and at least one co-stimulatory ligand, such that the APTC can stimulate and expand a T cell, preferably a CD8 T cell, comprising a cognate binding partner stimulatory molecule that specifically binds with the stimulatory ligand on the APTC and a cognate binding partner co-stimulatory molecule that specifically binds with the co-stimulatory ligand on the APTC such that interaction between the ligands on the APTC and the corresponding molecules on the T cell mediate, among other things, T cell proliferation, expansion and an immune response as desired.
- the immune response is a tumor specific, cytotoxic CD8 mediated immune response.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- an APTC of the invention can be readily produced to expand that T cell.
- a panel of APTCs of the invention can be used to determine which molecules, and combinations thereof, can expand that T cell.
- the present invention provides tools for expansion of desirable T cells, as well as tools for elucidating the molecules on particular T cells that mediate T cell activation and proliferation, particularly activation and proliferation that is detrimental to a tumor cell.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the nucleic acids of the invention, expressed by an LV vector encompass an RNA or a DNA sequence encoding a protein of the invention, and any modified forms thereof, including chemical modifications of the DNA or RNA which render the nucleotide sequence more stable when it is cell free or when it is associated with a cell. Chemical modifications of nucleotides may also be used to enhance the efficiency with which a nucleotide sequence is taken up by a cell or the efficiency with which it is expressed in a cell. Any and all combinations of modifications of the nucleotide sequences are contemplated in the present invention.
- any number of procedures may be used for the generation of mutant, derivative or variant forms of a protein of the invention using recombinant DNA methodology well known in the art such as, for example, that described in Sambrook and Russell (2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY), and Ausubel et al. (2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY). Procedures for the introduction of amino acid changes in a protein or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in these, and other, treatises.
- the APTC represents a source of cells to stimulate T cells in an antigen specific manner.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the APTC of the present invention can be used in vivo or ex vivo to stimulate tumor-specific T cells, autoantigen- specific T cells, and or viral-specific T cells.
- the APTC can be generated such that tolerance to such tumor, auto, or viral antigens is broken, either in an in vivo or in vitro setting.
- the APTC can be administered locally to a tumor site, a site of viral infection or site of autoimmune disease, or alternatively can be administered systemically.
- T cells that have been stimulated using the APTC as described herein can then be infused into a mammal in need thereof.
- the APTC of the invention is useful for stimulating and expanding a population of antigen specific T cells.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the APTCs are useful in therapeutic situations where it is desirable to regulate an immune response (e.g. , induce a response or enhance an existing response in an antigen specific manner).
- the APTC can be used to activate T cells in vitro, whereby the activated T cells ' can be administered to the mammal in need thereof.
- the APTC can be used in vivo to enhance a T cell response against tumor-associated antigens.
- Tumor cells from a mammal typically express tumor-associated antigens but may be unable to stimulate a co-stimulatory signal in T cells (e.g., because they lack expression of co-stimulatory molecules).
- tumor cells modified to express at least one co-stimulatory molecule can be contacted with T cells from the mammal (e.g., in vitro or in vivo) to generate tumor-antigen-specific T cells.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the tumor-antigen-specific T cells can further be expanded according to the method of the invention and the specific T cells can be returned to the mammal.
- T cells may be desirable to contact T cells with the APTC of the invention in vivo, e.g., via vaccination.
- the specific T cells may be isolated and contacted in vitro and expanded with the APTC according to the method of the invention.
- the vaccination using the APTC of the invention is sufficient to elicit an immune response in the mammal.
- the invention includes a nucleic acid encoding a costimulatory molecule, a costimulatory ligand or antigen, wherein a nucleic acid encoding a tag polypeptide is covalently linked thereto. That is, the invention encompasses a chimeric nucleic acid wherein the nucleic acid sequences encoding a tag polypeptide is covalently linked to the nucleic acid encoding at least one protein of the invention, or biologically active fragment thereof.
- tag polypeptides are well known in the art and include, for instance, green fluorescent protein (GFP), an influenza virus hemagglutinin tag polypeptide, a herpesvirus tag polypeptide, myc, myc-pyruvate kinase (myc-PK), His6, maltose binding protein (MBP), a FLAG tag polypeptide, and a glutathione-S-transferase (GST) tag polypeptide.
- GFP green fluorescent protein
- influenza virus hemagglutinin tag polypeptide hemagglutinin tag polypeptide
- myc-PK myc-pyruvate kinase
- MBP myc-pyruvate kinase
- MBP myc-pyruvate kinase
- MBP myc-pyruvate kinase
- MBP myc-pyruvate kinase
- MBP myc-pyruvate kinase
- MBP myc-
- the nucleic acid comprising a nucleic acid encoding a tag polypeptide can be used to localize a protein of the invention, or a biologically active fragment thereof, within a cell, a tissue, and/or a whole organism (e.g., a human, and the like), and to study the role(s) of the protein in a cell.
- a tag polypeptide facilitates isolation and purification of the "tagged" protein such that the proteins of the invention can be produced and purified readily.
- expression of a costimulatory ligand comprising a tag allows the detection of expression of the ligand, and further permits isolation of cells expressing the ligand using many methods, including, but not limited to, cell sorting.
- the present invention also provides for analogs of proteins or peptides which comprise a costimulatory ligand as disclosed herein.
- Analogs may differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.
- Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutarnine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.
- Modifications include in vivo, or in vitro, chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
- the present invention also includes polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
- Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring synthetic amino acids.
- the peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.
- the present invention should also be construed to encompass "mutants,"
- mutants, derivatives and variants are costimulatory ligands, cytokines, antigens (e.g., tumor cell, viral, and other antigens), which are altered in one or more amino acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or more base pairs) such that the resulting peptide (or DNA) is not identical to the sequences recited herein, but has the same biological property as the peptides disclosed herein, in that the peptide has biological/biochemical properties of a costimulatory ligand, cytokine, antigen, and the like, of the present invention (e.g., expression by an APTC where contacting the APTC expressing the protein with a T cell, mediates proliferation of, or otherwise affects, the T cell).
- costimulatory ligands e.g., tumor cell, viral, and other antigens
- a costimulatory ligand which when transduced into a OvCa cell is expressed and, when the cell is contacted with a T cell expressing a cognate costimulatory molecule on its surface, it mediates activation and stimulation of the T cell, with induced proliferation.
- the present invention provides a powerful novel screening assay for the identification of mutants, variants, fragments, and homologs of costimulatory ligands in that a potential novel form of a costimulatory ligand can be transduced and expressed in the APTC of the invention.
- the ability of the APTC to stimulate and/or activate a T cell, preferably a CD8 T cell can be assessed and compared with the ability of an APTC comprising the wild type or "natural" costimulatory ligand to stimulate and/or activate an otherwise identical T cell.
- functional variants demonstrating the ability to activate/stimulate the T cell to a greater, lesser or equal extent as the control wild type ligand, can be readily identified, isolated and characterized.
- Such novel variants of costimulatory ligands are potential research tools for elucidating T cell processes, and also provide important potential therapeutics based on inhibiting or inducing T cell activation/stimulation, such as, but not limited to, administration of a variant with inhibitory activity which can compete with the natural ligand to inhibit unwanted T cell responses such as, but not limited to, transplant rejection.
- a variant demonstrating greater costimulatory ligand activity can be used to increase a desired T cell response, such as, but not limited to, a cytotoxic tumor specific immune response.
- an exemplary variant ligand can be engineered to be more effective than the natural ligand or to favor the binding of a positive costimulatory molecule (CD28) at the expense of a negative regulator (CTLA-4).
- CD28 positive costimulatory molecule
- CTL-4 negative regulator
- a costimulatory ligand also encompasses a molecule that can bind an antibody that specifically binds with a molecule present on a T cell. That is, the invention encompasses an APTC comprising not only a costimulatory ligand (e.g., CD80 and CD86, among others) that bind a costimulatory molecule on a T cell (e.g., CD28), but also encompasses a molecule, such as CD32 (an Fc ⁇ RII receptor), that specifically binds an antibody, and the antibody is specific for a T cell molecule, such as, for example, CD3. Another example of an Fc ⁇ receptor is CD64. Numerous antibodies to the T-cell molecules are presently available, or they can be produced following procedures that are well-known in the art.
- an APTC comprising an antibody, or a molecule that specifically binds an antibody
- an APTC comprising an antibody, or a molecule that specifically binds an antibody
- an APTC can be produced, as exemplified elsewhere herein, by introducing a nucleic acid encoding CD32, the human Fc ⁇ receptor (or any other Fc ⁇ receptor such as CD64), into the APTC.
- the CD32 expressed on an APTC surface can then be "loaded” with any desired antibody that binds with CD32, including, but not limited to, antibody that specifically binds CD3 and antibody that specifically binds with CD28.
- the invention encompasses an APTC wherein a nucleic acid encoding the antibody ligand of interest, perhaps linked to an IRES sequence, is transduced and expressed on the surface of the APTC thereby eliminating the need for expression of CD32 and loading thereof.
- the present invention includes an APTC transduced with a nucleic acid encoding at least one antibody that specifically binds with CD3, CD28, among others, as well as an APTC transduced with CD32 and loaded with at least one antibody that specifically binds with the aforementioned molecules.
- the invention encompasses an APTC wherein the co-stimulatory ligand is a cognate binding partner that specifically binds with a co-stimulatory molecule, as well as where the ligand is an antibody that specifically binds with a costimulatory molecule, and any combination thereof, such that a single APTC can comprise both nucleic acids encoding costimulatory ligands and/or antibodies specific for costimulatory molecules present on the T cell, and any combination thereof.
- the invention can also encompass an APTC comprising a nucleic acid encoding an antigen of interest.
- a tumor cell such as an OvCa cell
- expresses a wide range of tumor antigens endogenous to a tumor cell such as Her2/Neu, CK7, CK 18, Epithelial Antigen, Mucl, p53 and CAl 25, and presents these antigens to an immune cell, such as a CD8 cell, in order to develop an immune response to the tumor.
- the LV presently disclosed can express a wide variety of costimulatory molecules, soluble immune modulators, and the like, and therefore, the APTC of the present invention can express a variety of antigens.
- a wide plethora of antigens are included, such as, but not limited to, tumor antigens, e.g., telomerase, melanoma antigen recognized by T cells (MART-I), melanoma antigen- encoding genes, 1, 2, and 3 (MAGE-I, -2, -3), melanoma GPlOO, carcinoembryonic antigen (CEA), breast cancer antigen HER-2/Neu, serum prostate specific antigen (PSA), Wilm's Tumor 1 (WT-I), mucin antigens (MUC-I 3 -2, -3, -4), and B cell lymphoma idiotypes.
- a viral, or the antigen from any other pathogen can also be transduced and expressed by the telomerase, mel
- the invention encompasses an APTC transduced with a nucleic acid encoding a cytokine, a chemokine, or both.
- Cytokines encompassed by the APTC of the present invention include, but are not limited to, IL-7, -15, -18, and -21).
- Other cytokines that can be expressed by an APTC include, but are not limited to, interferon- ⁇ (IFN ⁇ ), tumor necrosis factor- ⁇ (TNF ⁇ ), SLC 5 IL-2, IL-4, IL-23, IL-27 and the like.
- the invention further includes, but is not limited to, chemokine RANTES, MIP- Ia, MIP-Ib, SDF-I, eotaxin, and the like.
- the invention can encompass a cytokine, including a full-length, fragment, homologue, variant or mutant of the cytokine.
- a cytokine includes a protein that is capable of affecting the biological function of another cell.
- a biological function affected by a cytokine can include, but is not limited to, cell growth, cell differentiation or cell death.
- a cytokine of the present invention is capable of binding to a specific receptor on the surface of a cell, thereby affecting the biological function of a cell, even more preferably, affecting the immunomodulatory function of a cell.
- a preferred cytokine includes, among others, a hematopoietic growth factor, an interleukin, an interferon, an immunoglobulin superfamily molecule, a tumor necrosis factor family molecule and/or a chemokine.
- a more preferred cytokine of the invention includes a granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF ⁇ ), tumor necrosis factor beta (TNF ⁇ ), macrophage colony stimulating factor (M-CSF), interleukin- 1 (IL-I), interleukin-2 (IL-2), interleukin-4 (IL- 4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin- 10 (IL-10), interleukin- 12 (IL- 12), interleukin- 15 (IL-15), interleukin-21 (IL-21), interferon alpha (IFN ⁇ ), interferon beta (IFN ⁇ ), interferon gamma (IFN ⁇ ), and IGIF, among many others.
- GM-CSF granulocyte macrophage colony stimulating factor
- TNF ⁇ tumor necrosis factor alpha
- TNF ⁇ tumor necrosis factor beta
- M-CSF macrophage colony stimulating factor
- IL- 1
- a chemokine encompasses an alpha-chemokine or a beta-chemokine, including, but not limited to, a C5a, interleukin-8 (IL-8), monocyte chemotactic protein 1 alpha (MIP l ⁇ ), monocyte chemotactic protein 1 beta (MIP l ⁇ ), monocyte chemoattractant protein 1 (MCP-I), monocyte chemoattractant protein 3 (MCP-3), platelet activating factor (PAFR), N- formyl-methionyl-leucyl-[3H]phenylalanine (FMLPR), leukotriene B4 (LTB4R), gastrin releasing peptide (GRP), RANTES, eotaxin, lymphotactin, IPlO, 1-309, ENA78, GCP-2, NAP-2 and/or MGSA/gro.
- IL-8 interleukin-8
- MIP l ⁇ monocyte chemotactic protein 1 alpha
- an APTC of the invention is not limited in any way to any particular antigen, cytokine, costimulatory ligand, antibody that specifically binds a costimulatory molecule, and the like. Rather, the invention encompasses an APTC comprising numerous molecules, either all expressed under the control of a single promoter/regulatory sequence or under the control of more than one such sequence. Moreover, the invention encompasses administration of one or more APTC of the invention where the various APTCs encode different molecules.
- the various molecules e.g., costimulatory ligands, antigens, cytokines, soluble immune modulators, and the like
- an APTC of the invention provides an almost limitless number of variations and the invention is not limited in any way to any particular combination or approach.
- the skilled artisan armed with the teachings provided herein and the knowledge available in the art, can readily determine the desired approach for each particular T cell.
- the skilled artisan can determine which approach(es) can be applied to the particular T cells to be expanded or stimulated.
- the invention encompasses the preparation and use of pharmaceutical compositions comprising an APTC of the invention as an active ingredient.
- a pharmaceutical composition may consist of the active ingredient alone, as a combination of at least one active ingredient (e.g., an effective dose of an APTC) in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional (active and/or inactive) ingredients, or some combination of these.
- the active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
- the term "pharmaceutically acceptable carrier” means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
- the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
- the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
- compositions suitable for administration to humans are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.
- compositions of the invention include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs, birds including commercially relevant birds such as chickens, ducks, geese, and turkeys, fish including farm-raised fish and aquarium fish, and crustaceans such as farm-raised shellfish.
- Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, intra-lesional, pulmonary, or another route of administration.
- Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
- a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
- a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
- the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
- compositions of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
- the composition may comprise between 0.1% and 100% (w/w) active ingredient.
- the composition can comprise a specific number of cells, suspended in or absent a pharmaceutically acceptable carrier.
- a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
- additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers and chemotherapeutic agents, interferons, cytokines, and the like. Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
- Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
- Aqueous vehicles include, for example, water and isotonic saline.
- Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
- a pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil in water emulsion or a water-in-oil emulsion.
- the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
- Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
- parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
- Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
- parenteral administration is contemplated to include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
- Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
- compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
- This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
- Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1 ,3 butane diol, for example.
- Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di-glycerides.
- compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
- An APTC of the invention and/or T cells expanded using an APTC can be administered to an animal, preferably a human. When the T cells expanded using an APTC of the invention are administered, the amount of cells administered can range from about 100,000 cells to about 300 billion.
- APTC is administered alone, or with a pharmaceutically acceptable carrier or other compound, either with or without T cells expanded thereby, they can be administered in an amount ranging from about 100,000 to about one billion cells wherein the cells are infused into the animal, preferably, a human patient in need thereof. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.
- the APTC may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.
- the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
- An APTC (or cells expanded thereby) may be co-administered with the various other compounds (cytokines, chemotherapeutic and/or antiviral drugs, among many others).
- the compound(s) may be administered an hour, a day, a week, a month, or even more, in advance of an APTC (or cells expanded thereby), or any permutation thereof.
- the compound(s) may be administered an hour, a day, a week, or even more, after administration of an APTC (or cells expanded thereby), or any permutation thereof.
- the frequency and administration regimen will be readily apparent to the skilled artisan and will depend upon any number of factors such as, but not limited to, the type and severity of the disease being treated, the age and health status of the animal, the identity of the compound or compounds being administered, the route of administration of the various compounds and an APTC (or cells expanded thereby), and the like.
- the invention encompasses a method for specifically inducing proliferation of a T cell.
- the method comprises contacting a T cell that is to be expanded with an APTC comprising, among other things, a lentivirus vector encoding a ligand that specifically binds with a co-stimulatory molecule expressed on a T cell.
- an APTC comprising, among other things, a lentivirus vector encoding a ligand that specifically binds with a co-stimulatory molecule expressed on a T cell.
- contacting a T cell with an OvCa-based APTC comprising, among other things, a costimulatory ligand that specifically binds a cognate costimulatory molecule expressed on the T cell surface, stimulates the T cell and induced T cell proliferation such that large numbers of specific T cells can be readily produced.
- the APTC expands the T cell "specifically" in that only the T cells expressing the particular costimulatory molecule are expanded by the APTC.
- the T cell to be expanded is present in a mixture of cells, some or most of which do not express the costimulatory molecule, only the T cell of interest will be induced to proliferate and expand in cell number.
- the T cell can be further purified using a wide variety of cell separation and purification techniques, such as those known in the art and/or described elsewhere herein.
- an APTC is derived from a tumor cell, preferably an OvCa tumor cell
- the T cell is specific for that tumor cell, thus developing an immune response to that tumor cell.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the immune response induced by contacting a T-cell with an APTC can comprise a cytotoxic immune response, such as those elicited in CD8 T cells, or a helper T cell response, such as those elicited in CD4 T cells.
- the T cell of interest need not be identified or isolated prior to expansion using the APTC. This is because the APTC is selective and will expand the T cell(s) expressing the cognate costimulatory molecule.
- expansion of certain T cells is achieved by using several APTC expressing a different molecule(s) on each APTC, or a single APTC expressing various molecules, including, but not limited to, an antigen, a cytokine, a costimulatory ligand, an antibody ligand that specifically binds with the costimulatory molecule present on the T cell.
- the APTC can comprise a nucleic acid encoding CD32 (or any other Fc ⁇ receptor such as CD64) such that the CD32 expressed on the APTC surface can be "loaded” with any antibody desired so long as they bind CD32, which is an Fc ⁇ receptor. This makes the "off the shelf APTC easily tailored to stimulate any desired T cell.
- the invention encompasses a method for specifically inducing proliferation of a T cell expressing a known co-stimulatory molecule.
- the method comprises contacting a population of T cells comprising at least one T cell expressing the known co-stimulatory molecule with an APTC comprising a LV encoding a ligand of the co- stimulatory molecule.
- an APTC expresses at least one co-stimulatory ligand that specifically binds with a co-stimulatory molecule on a T cell
- binding of the co-stimulatory molecule with its cognate co-stimulatory ligand induces proliferation of the T cell.
- the T cell of interest is induced to proliferate without having to first purify the cell from the population of cells.
- this method provides a rapid assay for determining whether any cells in the population are expressing a particular costimulatory molecule of interest, since contacting the cells with an APTC will induce proliferation and detection of the growing cells thereby identifying that a T cell expressing a costimulatory molecule of interest was present in the sample.
- any T cell of interest where at least one costimulatory molecule on the surface of the cell is known, can be expanded and isolated.
- the invention also includes a method for specifically expanding a T cell population subset.
- the method comprises contacting a population of T cells comprising at least one T cell of a subset of interest with an APTC capable of expanding that T cell, or at least an APTC expressing at least one costimulatory ligand that specifically binds with a cognate costimulatory molecule on the surface of the T cell.
- an APTC capable of expanding that T cell
- an APTC expressing at least one costimulatory ligand that specifically binds with a cognate costimulatory molecule on the surface of the T cell.
- T cell subsets include T helper (THl and TH2) CD4 expressing, cytotoxic T lymphocyte (CTL) (TcI or Tc2) T regulatory (Treg), TC/S, naive, memory, central memory, effector memory, and ⁇ T cells. Therefore, cell populations enriched for a particular T cell subset can be readily produced using the method of the invention.
- the invention also includes a method for identifying a co-stimulatory ligand, or combination thereof, which specifically induces activation of a T cell subset.
- the method comprises contacting a population of T cells with an APTC comprising an LV encoding at least one co-stimulatory ligand, and comparing the level of proliferation of the T cell subset contacted with an APTC with the level of proliferation of an otherwise identical T cell subset not contacted with an APTC.
- a greater level of proliferation of the T cell subset contacted with an APTC compared with the level of proliferation of the otherwise identical T cell subset which was not contacted with an APTC is an indication that at the co-stimulatory ligand specifically induces activation of the T cell subset to which that T cell belongs.
- the present method permits the identification of a costimulatory ligand that specifically expands a T cell subset where it is not previously known which factor(s) expand that T cell subset.
- the skilled artisan would appreciate that in order to minimize the number of screenings, it is preferable to transduce as many nucleic acids encoding costimulatory ligands such that the number of assays can be reduced.
- the method allows, by combining the various proteins (e.g., stimulatory ligand, costimulatory ligand, cytokine, and the like), to assess which combination(s) of factors will make the most effective APTC, or combination of APTCs, to expand the T cell subset. In this way, the various requirements for growth and activation for each T cell subset can be examined.
- the method comprises contacting various APTCs with the T cell subset without first characterizing the costimulatory molecules on the surface of the T cell subset.
- the invention encompasses a method where the costimulatory molecule(s) present on the surface of the T cell subset are examined prior to contacting the APTCs with the cell.
- the present invention provides a novel assay for determining the growth requirements for various T cell subsets.
- the invention encompasses a method for inducing a T cell response to an antigen. The T cell response can be induced ex vivo, and then the cells can be delivered to a mammal via infusion or by other methods disclosed herein.
- the present invention encompasses inducing a T cell response in a mammal.
- the method comprises administering an APTC that specifically induces proliferation of a T cell specific for the antigen, for example an antigen expressed on a tumor cell, such as an OvCa cell.
- the method comprises administering an APTC the induces proliferation of a T cell population that is polyclonal with respect to antigen reactivity.
- the invention encompasses a method for inducing a T cell response to an antigen in a mammal in need thereof, the method comprising obtaining a population of cells from the mammal wherein the population comprises T cells, contacting the T cells with an APTC presenting the antigen in the context of an MHC complex, wherein contacting the T cells with the APTC induces proliferation of T cells specific for the antigen.
- the antigen-specific T cells are administered to the mammal, thereby inducing a T cell response to the antigen in the mammal in need thereof.
- the data disclosed elsewhere amply demonstrate that antigen-specific CTLs can be readily produced by contacting a T cell with an APTC.
- the APTCs can be used to generate a T cell population that is polyclonal with respect to antigen reactivity.
- the present invention further comprises a method of inducing an immune response to a tumor cell in a mammal, preferably a human, by adoptive transfer of T cells stimulated by an APTC.
- the present method comprises inducing the proliferation and activation of T cells, preferably CD8 T cells with an APTC of the invention.
- T cells preferably CD8 T cells
- the expanded, activated T cells are then infused, or otherwise administered to a mammal, preferably a human, having a tumor, and therefore in need of such treatment.
- CD8 cells are tumor-specific, and are thus able to control and/or kill a tumor.
- the present invention further comprises a method of inducing an immune response to a tumor cell in a mammal, preferably a human, by the active immunization of a mammal in need thereof with an APTC. That is, a mammal is administered an APTC of the present invention.
- the APTC acts as an antigen presenting cell, activating and arming endogenous T cells, preferably CD8 T cells, to attack a tumor in a tumor-specific manner.
- the APTCs are administered to a mammal essentially as described elsewhere herein.
- the present invention encompasses using an APTC as a vaccine to enhance reactivity of the antigen and enhance in vivo effect.
- the APTC may be delivered to a patient in combination with a vaccine, one or more cytokines, one or more therapeutic antibodies, or in combination with the APTCs as described herein. Virtually any therapy that would benefit by a more robust T cell population is within the context of the methods of use described herein.
- the APTC and/or activated T cells of the invention can be used in conjunction with other cancer therapies, including but not limited to chemotherapy, radiation, or treatment with agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK5O6, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
- agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK5O6, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
- immunoablative agents such as CAMP
- An advantage of using APTCs of the invention in conjunction with cancer therapies is that by using the methods of the present invention to induce an immune response to a tumor cell in a mammal, the amount and/or frequency of other cancer therapies can be reduced and therefore reduce certain side effects associated with current cancer therapies. Also a benefit of the present invention is the synergistic effects of using the APTCs of the invention with current cancer therapies.
- the APTCs of the present invention may be administered to a recipient as a "one-time" therapy for the treatment of cancer. However, if desired, multiple administrations of APTCs may also be employed.
- the invention described herein also encompasses a method of personalized therapy.
- a tumor cell can be isolated from a patient exhibiting symptoms of cancer.
- the isolated tumor cell can be engineered to exhibit on its surface at least one immune co- stimulator molecule.
- This modified tumor cell is considered an APTC and is useful in inducing a tumor specific immune response in the patient.
- the APTC acts as an antigen presenting cell, activating and arming endogenous T cells, preferably CD8 T cells, to attack a tumor in a tumor-specific manner.
- a "therapeutic effective amount" of APTC is an increased amount of activate tumor specific T cells, when compared with the number of activated tumor specific T cells in the absence of the administration of the APTC. It is believed that the increased number of tumor specific T cells following administration of APTCs into the patient helps to target the immune response against the tumor cell.
- An effective amount of APTCs can be determined by comparing the number of activated tumor specific T cells in a recipient of APTCs thereto, with the number of activated tumor specific T cells present in the recipient prior to administration of APTCs thereto. An increase in the number of activated tumor specific T cells in the recipient of the APTCs indicates that the number of APTCs administered is an effective amount of APTCs.
- the APTCs of the invention can be transplanted into a patient using techniques known in the art such as Ie., those described in U.S. Pat. Nos. 5,082,670 and 5,618,531 5 each incorporated herein by reference, or any suitable site in the body.
- Transplantation of the APTCs can be accomplished using techniques well known in the art as well as those described herein or as developed in the future.
- the present invention comprises a method for introducing the APTCs into a mammal, preferably, a human. Also, methods that relate to bone transplants are well known in the art and are described for example, in U.S. Patent No. 4,678,470, and U.S. Patent No. 5,571,083, teaches methods for transplanting cells to any anatomical location in the body.
- the cells may also be encapsulated and used to deliver biologically active molecules, according to known encapsulation technologies, including microencapsulation (see, e.g., U.S. Pat Nos.
- cell number in the devices can be varied; preferably, each device contains between 10 3 - 10 9 cells, most preferably, about 10 5 to 10 7 cells.
- macroencapsulation devices may be implanted in the patient. Methods for the macroencapsulation and implantation of cells are well known in the art and are described in, for example, U.S. Patent 6,498,018.
- the dosage of the APTCs and/or activated T cells varies within wide limits and may be adjusted to the individual requirements in each particular case.
- the number of cells used depends on the weight and condition of the recipient, the number and/or frequency of administration, and other variables known to those of skill in the art.
- the mode of administration of APTCs and/or activated T cells to the patient may vary depending on several factors including the type of disease being treated, the age of the patient.
- the APTCs and/or activated T cells may be introduced to the desired site by direct injection, or by any other means used in the art for the introduction of compounds administered to a patient suffering from a particular disease or disorder.
- the APTCs and/or activated T cells can be administered into a host in a wide variety of ways.
- Preferred modes of administration are intravascular, intracerebral, parenteral, intraperitoneal, intravenous, epidural, intraspinal, intrastemal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, or intramuscular.
- the APTCs and/or activated T cells may also be applied with additives to enhance, control, or otherwise direct the intended therapeutic effect.
- the cells may be further purified by use of antibody-mediated positive and/or negative cell selection to enrich the cell population to increase efficacy, reduce morbidity, or to facilitate ease of the procedure.
- cells may be applied with a biocompatible matrix which facilitates in vivo tissue transplantation by supporting and/or directing the fate of the implanted cells.
- the invention includes various kits which comprise an APTC of the • invention, a nucleic acid encoding various proteins, an antibody that specifically binds to a costimulatory molecule on the surface of a T cell, and/or a nucleic acid encoding the antibody of the invention, an antigen, or a cytokine, an applicator, and instructional materials which describe use of the kit to perform the methods of the invention.
- kits which comprise an APTC of the • invention, a nucleic acid encoding various proteins, an antibody that specifically binds to a costimulatory molecule on the surface of a T cell, and/or a nucleic acid encoding the antibody of the invention, an antigen, or a cytokine, an applicator, and instructional materials which describe use of the kit to perform the methods of the invention.
- exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure.
- Each of these kits is included within the invention.
- the invention
- the kit is used pursuant to the methods disclosed in the invention. Briefly, the kit may be used to administer an APTC of the invention to a T cell expressing at least one costimulatory molecule. This is because, as more fully disclosed elsewhere herein, the data disclosed herein demonstrate that contacting a T cell with an APTC comprising a costimulatory ligand that specifically binds with the cognate costimulatory molecule present on the T cell, mediates stimulation and activation of the T cell. Further, the T cells produced using this kit can be administered to an animal to achieve therapeutic results.
- the kit further comprises an applicator useful for administering the APTC to the T cells.
- an applicator useful for administering the APTC to the T cells will depend on, e.g. , the method used to administer the APTC, as well as the T cells expanded by the APTC, and such applicators are well-known in the art and may include, among other things, a pipette, a syringe, a dropper, and the like.
- the kit comprises an instructional material for the use of the kit. These instructions simply embody the disclosure provided herein.
- the kit can further include a pharmaceutically-acceptable carrier.
- the composition is provided in an appropriate amount as set forth elsewhere herein. Further, the route of administration and the frequency of administration are as previously set forth elsewhere herein.
- the kit encompasses an APTC comprising a wide plethora of molecules, such as, but not limited to, CD32, CD86, CD80, 4- IBBL, CD83, ICOS-L, HLA- A2, IL-7, IL- 15 and CCL 21, as well as others set forth elsewhere herein.
- the kit comprises a kit where each molecule to be transduced into the APTC is provided as an isolated nucleic acid encoding a molecule, a vector comprising a nucleic acid encoding a molecule, and any combination thereof, including where at least two molecules are encoded by a contiguous nucleic acid and/or are encoded by the same vector.
- the routineer would understand that the invention encompasses a wide plethora of constructs encoding the molecules of interest to be introduced into an APTC of the invention.
- the data disclosed herein encompasses methods and compositions for generating an immune response to an ovarian tumor antigen, including an ovarian tumor and/or an ovarian tumor cell.
- the data disclosed herein further demonstrates methods to increase the success when establishing a stable cell line from a primary tumor specimen.
- a primary specimen is cultured under multiple conditions, with varied extracellular matrices and serum supplements.
- the cell lines are established without using fetal calf serum (FCS) or other xenogeneic reagents, resulting in the production of 14 ovarian tumor cell lines.
- FCS fetal calf serum
- the lines produced by the methods disclosed herein are all low passage, and with one exception, they have never been exposed to FCS. Five lines were characterized in detail.
- one line was modified using lentiviral vector-mediated transduction to express lymphocyte costimulatory molecules.
- ACC-0V7 All cultures except ACC-0V7 were initiated under multiple conditions.
- Cells were plated on either tissue culture-treated flasks (Sarstedt, Newton, NC), or flasks coated with either 0.5 ⁇ g/cm 2 human plasma fibronectin (Becton Dickinson, Bedford, MA) or 1 ⁇ g/cm 2 human placental collagen IV (Southern Biotechnology Associates, Birmingham, AL).
- Specimens were cultured in OC medium, whose formulation is similar to the previously described ACL-4 medium (Gazdar, et al., 1986, Cancer Res., 46: 6011-6012).
- OC medium consists of RPMI 1640 supplemented with IX ITES (10 ⁇ g/ml insulin, 10 ⁇ g/ml transferrin, 10 ⁇ M ethanolamine, and 10 ng/ml selenium), 2 mM L- glutamine, 10 mM HEPES, 0.5 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 1 ng/ml epidermal growth factor, 18 ng/ml hydrocortisone, and 0.1 nM triiodothyronine (all from Biowhittaker) and 10 IU/ml penicillin/10 ⁇ g/ml streptomyyin (Mediatech, Herndon, VA).
- IX ITES 10 ⁇ g/ml insulin, 10 ⁇ g/ml transferrin, 10 ⁇ M ethanolamine, and 10 ng/ml selenium
- 2 mM L- glutamine 10 mM HEPES, 0.5 mM sodium pyruv
- ACC-OV7 cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 10% FCS (Hyclone, Logan, Utah), and penicillin/streptomycin (100 IU/ml and 100. ⁇ g/ml, respectively). All cultures underwent 50% medium changes twice weekly and were subcultured using 0.05% trypsin/0.53 mM EDTA (Invitrogen, Carlsbad, CA).
- Immunocytochemistry was performed using cells cultured in 4 well Permanox Lab-Tek chamber slides (Nalge Nunc, Rochester, NY) pre-coated with the appropriate extracellular matrix. Paraformaldehyde-fixed cells were incubated with anti- p53 (clone Pab240, diluted 1:50) or anti-CA125 (Clone OvI 85:1, diluted 1 :50, both from LabVision, Fremont, CA) and reactivity was visualized using the ABC-HRP kit (Vector Labs, Burlingame, CA), in accordance with the manufacturer's instructions.
- Flow cytometry was performed using conjugated antibodies specific for Her2/neu (Clone Neu 24.7), CD32 (Clone FLI8.26), 4-1 BBL (Clone C65485), CD86 (clone 2331), all purchased from Becton-Dickinson.
- Anti-cytokeratin 7 (Clone OV- TL1230), anti-Mucl (Clone E29), anti-cytokeratin 18 (Clone DClO), and anti-epithelial antigen (Clone Ber-Ep4) were purchased from Dako, (Carpinteria, CA). Intracellular antigens were detected using the Fix-and-Perm Kit (Caltag, Burlingame, CA).
- STR analysis was performed using the AmpF STR Blue kit (Applied Biosystems, Foster City, CA), in accordance with manufacturers' instructions.
- the amplified products were electrophoresed using a 377 DNA sequencer (Applied Biosystems), data was extracted using Genescan Analysis, and peak analysis was performed using Genotyper 2.i software.
- Array CGH (aCGH) Analysis was performed using previously described procedures (Greshock, et al. s 2004, Genome Res., 14: 179-187). Arrays consisting of 4,134 BAC clones spanning the human genome at ⁇ 0.9 Mb resolution were used as targets for all hybridization experiments. For each cell line, 1 ⁇ g of genomic DNA was labeled with Cy3-CTP (Amersham, Piscataway, NJ) while the pooled normal reference sample was labeled with Cy5-dCTP (Amersham). Labeled samples were hybridized to the arrays for 72 hours at 37 0 C in the presence of 100 ⁇ g human Cot- 1 DNA (Invitrogen, Carlsbad, CA).
- RNA obtained from neutrophils, activated B cells, and mature dendritic cells were prepared from RNA obtained from neutrophils, activated B cells, and mature dendritic cells, respectively.
- the cDNAs were inserted into the self-inactivating lentiviral vector pCLPSl ⁇ , generating vectors pCLPS-CD32, pCLPS-4-lBBL, and pCLPS-CD86.
- Packaged vectors were prepared by transfecting 293T cells as described (Dull, et al., 1998, J.Virol., 72: 8463- 8471). Vector-containing supernatant was harvested 24-48 hours later and concentrated by ultracentrifugation (Reiser, 2000, Gene Ther., 7: 910-913).
- ACC-O V7 cells were transduced simultaneously with pCLPS-CD32, pCLPS-4-lBBL, and pCLPS-CD86 vectors and allowed to expand in culture for three weeks. The cells were then harvested and stained with antibodies to CD32, 4- IBBL and CD86 as described above and sorted using a Mo-Flo cell sorter (Cytomation, Fort Collins, CO). Triple-positive cells (designated ACC-OV7/32/86/4) were collected and cultured.
- CD8+ T cells As a positive control, 2.56 x 10 5 CD8+ T cells were incubated with 1.28 x 10 5 irradiated K562 cells expressing CD32 (K32) or CD32 and 4- IBBL (K32-4-1BBL) in the presence of 0.5 ⁇ g/ml anti-CD3 and 0.5 ⁇ g/ml anti-CD28 (clone 9.3) as previously described (Maus, et al., 2002, Biotechnol., 20: 143-148). CD8+ T cells stimulated with ACC-OV7 or ACC-OV7/32/86/4 were re-plated after 2 days, in the absence of tumor cells, at a concentration of 5-10 x 10 5 cells/ml.
- CD8+ T cells were re- stimulated using the same conditions as the primary stimulation. Where indicated, freshly isolated CD8+ T cells were washed, suspended in PBS, and labeled with 1.25 ⁇ M CFSE (carboxyfluorescein diacetate succinimidyl ester, Molecular Probes, Eugene, OR) as described (Thomas, et aL; 2002, Clin. Immunol., 105: 259-272) prior to stimulation.
- CFSE carboxyfluorescein diacetate succinimidyl ester
- Cultures were initiated from a total of 30 primary ovarian cancer specimens. 10 specimens consisted of solid tumor only, 9 were ascites tumor only, and 11 comprised both solid and ascites tumor. The majority of specimens were simultaneously cultured under multiple (typically 9) conditions. These included unsupplemented OC medium, as well as 0.5% human serum albumin-supplemented or 10% Human AB serum-supplemented medium. Cultures were simultaneously initiated on tissue culture plastic, collagen IV-coated-, and fibronectin-coated flasks. Stable cell lines were successfully established from 10 of the 30 primary cultures. In some instances, more than one line was established from the same specimen, resulting in a total of 14 cell lines generated. Cell line information is presented in the table in Figure 4. All lines were from late-stage (IIIC or IV) ovarian cancer patients, and with the exception of ACC- OV84, all lines were established from chemotherapy-naive patients.
- ACC-OV7 formed uniform monolayers of polygonal cells with large nuclei and relatively abundant cytoplasm. Basally oriented vacuoles were prominent in sub- confluent cultures but not evident in confluent cultures. ACC-OV551 and ACC-O V552 differed morphologically despite being isolated from the same specimen. ACC-OV551 cells were large, frequently vacuolated, and contained abundant perinuclear granules.
- ACC-OV551 cells had relatively abundant cytoplasm and prominent nucleoli, usually 1-2 per cell. They grew as discrete islands, never forming continuous monolayers. In contrast, ACC-O V552 cells were round and densely packed, with scant cytoplasm. They also had prominent nucleoli, but lacked the prominent vacuoles observed in ACC-OV551.
- the cell lines derived from patient 68, ACC-OV684 and ACC-OV68A also displayed dissimilar morphologies. ACC-O V684 cells grew as densely packed monolayers, with large nuclei containing multiple prominent nucleoli. OV68A cells were irregularly shaped, with more abundant cytoplasm. They were more vacuolated and formed less dense monolayers.
- Pathol., 183: 311-317 and ovarian tumor cell lines are Mucl -positive, expression levels vary widely.
- CK7 expression is commonly used to distinguish ovarian cancer from other neoplasms (Chu, et al, 2000, Mod. Pathol., 13: 962-972; McCluggage, 2002, Histopathology, 40: 309-326). All 5 lines were CK7-positive ( Figure 5).
- ACC-OV7, ACC-OV551, and ACC-OV552 were uniformly and strongly CK7-positive, but in the lines derived from patient 68, ACC-OV684 and ACC-OV68A, only 50-70% of the cells were CK7-positive. Again, this variability recapitulates previous observations, as at least one widely used ovarian cancer cell line, A2780, is CK7 -negative, and another, SK-OV- 3, is not uniformly CK7-positive (Stimpfi, et al., 1999, Cancer Lett., 145: 133-141).
- Her2/neu is frequently over-expressed in ovarian cancers, with estimates ranging from ⁇ 25% to approximately 50% (Hellstrom, et al., 2001, Cancer Res., 61 :
- Her2/neu surface expression was measured_by flow cytometry ( Figure 5).
- MCF-7 and SK-BR3 cells which express low and high levels of Her2/neu, respectively (Szollosi, et al., 1995, Cancer Res., 55: 5400-5407), were used as standards.
- the mean fluorescence intensity of Her2/neu on MCF-7 cells was comparable to that observed for all lines in our panel, while the mean fluorescence intensity of Her2/neu expression on SK-BR3 cells was nearly 2 logs higher.
- p53 and CAl 25 expression was examined by immunocytochemistry (Figure 5).
- p53 is over-expressed in 55% of Stage III/IV ovarian carcinomas (Kmet, et al., 2003, Cancer, 97: 389-404), and slightly higher rates have been observed in cultures established from ovarian tumors (Verschraegen, et al., 2003, Clin. Cancer Res., 9: 845- 852).
- p53 was overexpressed in all the lines in our panel, and in all cases, expression was confined to the nucleus.
- CAl 25 expression is commonly used to detect and monitor ovarian cancer (Whitehouse, et al., 2003, Gynecol. Oncol., 88: S152-S157). CA125 expression varied markedly between the cell lines, ranging from strongly positive (ACC-OV552) to completely absent (ACC-OV7). This is in agreement with previously reported observations demonstrating that surface CAl 25 expression is highly variable and often undetectable in ovarian cancer cell lines (Stimpfi, et al., 1999, Cancer Lett., 145: 133- 141).
- the alleles present in the cell lines were present in the original tumor specimen.
- alleles present in the original specimen were absent from the cell line, which may be attributable to genetic instability of the cultured cells (Orth, et al., 1994, Proc. Nat'l. Acad. Sci. USA, 91 : 9495-9499; Hiorns, et al., 2004, Br. J. Cancer, 90: 476-482).
- primary tumor specimens are mixtures of malignant and non-malignant cells. Conceivably, allelic loss occurred prior to the initiation of cell culture, but this loss was masked by the predominance of normal diploid tissue in the original specimen.
- aneuploid DNA values correlate with enhancement of some aspects of in vitro growth, such as the ability to grow under anchorage-independent conditions (Verheijen, et al., 1985, Int. J. Cancer, 35: 653-657).
- the aberrant genome compositions of the cell lines were further examined by array Comparative Genomic Hybridization (aCGH) analysis (Greshock, et al., 2004, Genome Res., 14: 179-187), the results of which are presented in Figure 6. All lines had numerous copy number aberrations. Many of the alterations detected were unique to the cell lines derived from a common primary specimen.
- ACC- OV68A had a loss at 8q24qter. All five lines tested had increases on chromosome 20. ACC-OV684 and ACC-OV-551 had gains at the level of the entire chromosome, while ACC-OV7 and ACC-OV68A had increases of the q arm, and ACC-OV552 had a gain at 20gl3.
- tumor cell line The most important criterion for a tumor cell line to serve as a whole-cell tumor vaccine is that it be immunogenic. Since tumor cells do not in general express costimulatory molecules, most tumor cells and hence tumor cell lines are poorly immunogenic. Thus, they can induce ahergy or tolerance based on MHC I-restricted antigen presentation in the absence of costimulation (Abken, et al., 2002, Trends Immunol., 23: 240-245). There have been numerous attempts to modify tumor cells by introducing costimulatory molecules and/or cytokines, many of which of have indeed resulted in enhanced immunogenicity, leading to clinical trials employing both autologous and allogeneic whole tumor cell vaccines (Pardoll, 2002, Clin.
- CD86 (B7-2), along with CD80 (B7-1), is the prototypic costimulatory ligand, and CD80 and CD86 share CD28 and CTLA-4 as receptors (Riley, et al., 2005, Blood, 105: 13-21).
- 4-1 BBL is expressed by a range of professional APCs, and 4-1 BBL:4-1BB interactions preferentially activate CD8 + T cells in vitro, enhance survival of activated CD8 + T cells, and amplify in vivo CTL responses (Shuford, et al., 1997, J. Exp. Med., 186: 47-55).
- ACC-O V7 cells transduced with lentiviral vectors encoding CD32, CD86, and 4- IBBL were collected by sterile sorting and placed in culture.
- the stability of vector-encoded gene expression in transduced ACC-O V7 cells was measured after 3 months of in vitro culture (Fig. 2).
- CD32 and CD86 expression remained high after 3 months of continuous culture, with over 90% of cells expressing both molecules (Fig. 2A).
- 4-1 BBL surface expression decreased markedly, with only ⁇ 15% of cells still positive for 4-1BBL after 3 months of culture (Fig. 2B).
- the intracellular levels of 4-1 BBL were measured (Fig. 2C).
- ovarian cell lines modified to function as a tumor vaccine are as follows.
- the ACC-OV87 cell line was modified to express the Fc ⁇ receptor CD64 and the costimulatory molecules CD86 and 4-1 BBL.
- CD64 binds exogenous IgG antibodies and presents them to T cells (see Figure 10).
- the ACC-OV79 cell line was modified to express Fc ⁇ receptor CD64 and the costimulatory molecules CD86 and 4-1 BBL.
- CD64 binds exogenous IgG antibodies and presents them to T cells (see Figure 11).
- CFSE-labeled allogeneic CD8 + T cells were cocultured for six days with ACC-OV7/32/86 cells at a 2: 1 T cell:tumor cell ratio, in the presence or absence of 0.5 ⁇ g/ml ⁇ CD3.
- CFSE-labeled CD8 + T cells were incubated with CD32-expressing K562 (K32) cells (Maus, et al., 2002, Nat. Biotechnol., 20: 143-148) in the presence of 0.5 ⁇ g/ml each of ⁇ CD3 and ⁇ CD28 antibodies.
- ACC-OV7/32/86 cells induced nearly 100% of the CD8 + T cells to proliferate, as measured by CFSE dilution (Fig. 3A). Similar results were obtained when CD8 + T cells were stimulated with ⁇ CD3 and ⁇ CD28-coated K32 cells. In contrast, CD8 + T cell proliferation was not observed in CD8/ACC-OV7/32/86/4 cocultures when ⁇ CD3 mAb was omitted. This experiment demonstrated that in short- term proliferation assays, ACC-O V7/32/86/4 cells stimulated robust, ⁇ CD3-dependent CD8 + T cell proliferation.
- CD8 + T cells were mixed with either ⁇ CD3-coated ACC-OV7 cells or ⁇ CD3 coated ACC-O V7/32/86/4 cells at a 2:1 T celhtumor cell ratio as described elsewhere herein.
- CD8 + T cells were mixed with ⁇ CD3/ ⁇ CD28-coated K32-4-1 BBL cells. When the mean T cell volume declined to near-resting cell levels, fresh stimulator cells and antibodies were added.
- CD8 + T cells stimulated through CD28-mediated pathways only enter a plateau phase after ⁇ 2 weeks of growth and become unresponsive to further stimulation (Maus, et al., 2002, Nat. Biotechnol., 20: 143- 148).
- soluble (cleaved) 4- IBBL can stimulate T cell growth (Salih, et al., 2001, J. Immunol., 167: 4059-4066). Therefore, cleaved soluble 4- IBBL may still be functioning in the present system.
- the present disclosure describes a strategy of initiating cultures under multiple conditions simultaneously to establish a panel of low passage ovarian cancer cell lines that, with one exception, have never been exposed to FCS.
- the likelihood of maximizing cell line establishment is achieved by varying a few basic parameters known to those of skill in the art, such as medium supplementation and extracellular matrix composition.
- Augmenting anti-tumor CTL responses by manipulating T cell-antigen- presenting cell interactions is a fundamental theme of tumor immunotherapy. Enhancing the immunogenicity of tumor cells by introduction of costimulatory molecules is disclosed herein, and anti-tumor CD8 + T cell responses have been observed in a subset of pancreatic cancer patients who received modified allogeneic tumor preparations (Jaffee, et al., 2001, J. Clin.
- Human MHC HLA class I downregulation is a widespread phenomenon in tumor biology and is believed to contribute to tumor escape mechanisms by way of decreasing the role of HLA molecules (e.g., HLA-A* 02) in presenting immunogenic peptides to T cells.
- HLA-A* 02 natural killer
- NK natural killer
- the recognition, activation and destruction of a target by the preformed NK cells in the circulation or tissues requires only a few hours.
- Such a mechanism is believed to be a special relevance when HLA deficient tumor cells reach the circulation during metastases.
- NK surveillance tumor cells clearly spread and metastasize.
- a tumor cell can be isolated from a primary specimen and engineered to be used as a tumor vaccine.
- the engineered tumor cell can be used to generate a polyclonal and functional tumor antigen-specific T cells.
- the tumor cells engineered by way of introducing a costimulatory molecule into them can further be modified by also introducing an HLA molecule into them.
- any of the ovarian tumor cell lines disclosed herein can be transduced with lentiviral vectors encoding HLA-2A (e.g., HLA-A*02), CD32, CD86, and 4- IBBL (or any combination thereof).
- Transduction with HLA-2A serves to increase the amount of HLA-2A molecules expressed on the surface of the ovarian tumor cell. It is believed that the increased ⁇ gxpression of HLA-2A in the ovarian tumor cell augments the presentation of tumor-specific antigens and therefore increases the amount of antigens that can be recognized by T cells.
- the engineered ovarian tumor call can be used to generate a T cell population that is polyclonal with respect to antigen reactivity. It is believed that the engineered ovarian tumor cell can be used to expand a population of T cells that recognizes a combination of ovarian tumor antigens associated with the ovarian tumor cell.
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Abstract
L'invention concerne des cellules tumorales présentant des antigènes, transduites avec des vecteurs lentiviraux, des procédés de déclenchement d'une prolifération de lymphocytes T, des procédés de déclenchement d'une réponse immunitaire de lymphocytes T, et un procédé de traitement d'une tumeur chez un mammifère au moyen des cellules tumorales présentant des antigènes.
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