WO1996032140A1 - Procedes d'elaboration de cellules veto artificielles - Google Patents

Procedes d'elaboration de cellules veto artificielles Download PDF

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WO1996032140A1
WO1996032140A1 PCT/US1996/005187 US9605187W WO9632140A1 WO 1996032140 A1 WO1996032140 A1 WO 1996032140A1 US 9605187 W US9605187 W US 9605187W WO 9632140 A1 WO9632140 A1 WO 9632140A1
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cell
antigen
polypeptide
antibody
class
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David R. Kaplan
Mark L. Tykocinski
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Tkb Associates Limited Partnership
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4612—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464436—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4648—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464839—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2510/00—Genetically modified cells

Definitions

  • This invention relates to methods and reagents for engi ⁇ neering artificial veto cells ("AVCs") for purposes of imrnunotherapy.
  • Antigen-specific (hereinafter referred to as "specific") immunotolerization is a therapeutic endpoint in subjects in need of the selective suppression of untoward immune responses to defined antigens.
  • specific T lymphocytes Given the centrality of pathogenic T lymphocytes in autoimmune, alloimrrvune, and other acute and chronic inflammatory diseases, an objective in the field of imrnunotherapy is the development of strategies for inhibiting specific T- cells.
  • Antigen-presenting cells such as dendrit ⁇ ic cells, B lymphocytes and macrophages provide one efficient means for accessing antigen-specific T-cells (T lymphocytes) .
  • APCs are responsible for initiating most immune responses through their pivotal role in antigen presentation to T-cells.
  • endogenously processed nominal antigen peptides associate intracellularly with either class I major histo- compatibility complex ("MHC") heterodimers or with class II MHC heterodimers, and the resulting heterotrimeric complexes are then translocated to APC surfaces (reviewed in Germain and Margulies, 11 Annu. Rev. Immunol. 403, 1993; Germain, 76 Cell 287, 1994) .
  • MHC major histo- compatibility complex
  • MHC-bearing cells For purposes of antigen presentation, it is common to employ one of two categories of cells, that is, cells that naturally express MHC heterodimers and cells transfected with an MHC gene expression cassette.
  • another category of an MHC-bearing cell is one in which the MHC molecule has been exogenously attached to the cell surface.
  • a method for delivering an MHC molecule to an APC surface has been described which entails the use of an HLA-A2.1-streptavidin chemical conjugate which can be added to a pre-biotinylated cell (Elliott and Eisen, 87 Proc. Natl. Acad. Sci. USA 5213, 1990) .
  • the chemical moiety was shown to be alloantigenic, but no data was presented that it can bind and present a nominal antigen peptide.
  • U.S. Patent No. 5,242,687 disclose a method for effecting conversion of a T-cell activator into a T-cell inhibitor by expressing a CD8 polypeptide on the surface of an APC.
  • the CD8 molecule functions as a "coinhibitor” , triggering an inhibition program within the T-cell.
  • the present invention provides different methods and compositions for molecularly engineering APCs to tailor their T-cell modulatory properties, for both in vivo and ex vivo applications.
  • Applicant has determined a broad set of methods for converting APCs from T-cell activators into T-cell inhibitors.
  • An APC functioning in an T-cell inhibitory mode can be referred to as either a "deletional APC” (since it is deleting a specific T-cell through either the induction of anergy or apoptosis) or a "veto cell” (since it is vetoing a specific T-cell in a manner that mimics the natural veto function of an ill- defined mononuclear cell population in the periphery) .
  • CD8 coating was the only known method for engineering AVCs.
  • the present invention offers a series of additional methods for generating non-naturally occur- ring veto cells, establishing the concept that "artificial veto cells (AVCs) " constitute a broader class of cells that can be purposefully engineered through diverse methods.
  • AVCs artificial veto cells
  • a key aspect of the present invention is that the APC is being used as a delivery vehicle for an inhibitory reagent.
  • the APC provides an efficient means for specific T-cell targeting, thereby focusing the reagent upon specific T-cell responders that are engaged by the anti- genic peptides presented at the APC surface.
  • the antigen presentation function of the APC is being used to advantage.
  • the inhibitory molecular reagent can be delivered locally, bypassing the need to administer it systemically and thus avoiding potential toxicities associated with systemic administration.
  • the T- cell modulator is acting locally, either through cell-to- cell contact or local transit.
  • AVCs can thus be pulsed with extracts from target tissues of a given disease, and the AVC (via its APC function) can present the diverse repertoire of potential pathogenic peptides to the T-cell population of the patient.
  • Applicant has determined those molecules that can be newly expressed on or in APCs in order to convert them into AVCs.
  • Reagents useful for AVC engineering include both cell surface-associated and soluble molecules. Applicant believes that in each instance an inhibitory reagent provided by the APC binds to a receptor for the reagent on the T-cell, and this binding event serves to trigger an inhibitory program within the T-cell.
  • any one of a number of molecules with known T-cell inhibitory activity can be used as a reagent for AVC engineering.
  • Agents known to induce apoptosis in T-cells or other types of cells constitute candidates.
  • a preferred polypeptide for AVC generation is Fas ligand ("Fas-L”) (also known as APO- 1 ligand) which is known to be capable of inducing apopto ⁇ sis in both resting and activated T-cells via signaling through Fas (the Fas-L receptor, Fas antigen) molecules on the T-cell surfaces.
  • Fas ligand Fas ligand
  • Fas-L receptor Fas-L receptor
  • Fas antigen Fas antigen
  • soluble antibodies with specificity for either the class I MHC heavy chain or the class I MHC ⁇ 2 microglobulin light chain can inhibit the proliferation and triggering of cytotoxicity of T- cells, and it is further believed that in at least some contexts such antibodies evoke T-cell apoptosis.
  • another preferred reagent is an anti-class I MHC antibody or an anti-3 2 microglobulin antibody expressed by or on the AVC.
  • soluble functional Fvs directed against other target molecules and these provide clearcut guidelines for the design and production of functional Fvs. Both cell surface-associated and soluble forms of the antibodies or Fvs can be employed.
  • T-cell inhibitory potential can be employed for AVC engineering. Such molecules may either act directly on the T-cell or may modulate other molecular systems of the AVC itself, conferring or enhancing the T- cell inhibitory action of the AVC.
  • Preferred reagents in this category include known immunosuppressive cytokines, for example, interleukin-10 ("IL-10") , transforming growth factor ⁇ , heterodimeric placental protein 14, homodimeric/monomeric placental protein 14, and viral proteins.
  • IL-10 interleukin-10
  • transforming growth factor ⁇ for example, transforming growth factor ⁇
  • heterodimeric placental protein 14 transforming growth factor ⁇
  • heterodimeric placental protein 14 homodimeric/monomeric placental protein 14
  • viral proteins examples include those originating from the human immunodeficiency virus (HIV) .
  • HIV immunoregulatory protein is tat, but HIV immunoregulatory proteins are not limited to tat.
  • AVC engineering can be accomplished through both gene and protein transfer.
  • Gene transfer is especially well-suited for expressing immunosuppressive genes in AVCs that encode for the production of secreted proteins, but is obviously also applicable to the expression of cell surface reagents.
  • vectors for example, retroviral, adenoviral, adeno-associated viral, and naked DNA vectors
  • episomal (extrachromasomally-replicating) vectors for example, Epstein-Barr virus and BK virus vectors)
  • AVC engineering can also be accomplished by protein transfer, that is, the external application of a cell surface-associating or soluble protein.
  • cell surface-associating proteins artificially lipid- modified variants of polypeptides are preferred.
  • Lipid modification can be accomplished by covalently conjugating lipids to soluble derivatives of the inhibitory protein of interest.
  • An example of a lipid modification is palmitoylation.
  • GPI- odified proteins are a class of native cell surface molecules that can be exogenously reincorporated back into cell membranes after purification (Medof et al . , 160 J. Exp. Med. 1558, 1984; Moran et al . , 149 J. Immunol . 1736, 1992; Zhang et al . , 89 Proc. Natl. Acad. Sci. USA 5231, 1992; Bulow et al . , 27 Biochemistry 2384, 1988; Hitsumoto et al . , 5 Int . Immunol.
  • Polypeptide sequences can be artificially GPI- modified by linking their coding sequences to a GPI modi ⁇ fication signal sequence (Tykocinski et al . , 85 Proc . Natl. Acad. Sci. USA 3555, 1988; Straus et al . , 82 Proc. Natl. Acad. Sci. USA 6245, 1985; Caras et al . , 238 Science 1280, 1987; Waneck et al. , 85 Proc. Natl. Acad. Sci. USA 577, 1988) .
  • GPI modi ⁇ fication signal sequence Tykocinski et al . , 85 Proc . Natl. Acad. Sci. USA 3555, 1988; Straus et al . , 82 Proc. Natl. Acad. Sci. USA 6245, 1985; Caras et al . , 238 Science 1280, 1987; Waneck et al. ,
  • the coding sequence for the reagent of interest can be linked in-frame to a GPI modification signal sequence from the carboxy terminus of a naturally GPI-modified protein such as human decay- accelerating factor.
  • a naturally GPI-modified protein such as human decay- accelerating factor.
  • Such artificial GPI-modified proteins can be produced in large scale using mammalian ( f or example , glutamine synt het a s e amplification/expression system) or yeast (for example, Pichia expression system) over-expression systems.
  • the artificial GPI-modified protein can then be purified by immunoaffinity chromatography or other standard biochemical purification methods.
  • a polyhistidine tag By incorporating a polyhistidine tag into the chimeric polypeptide, in between the two distinct coding sequences, one can simplify purification through the use of nickel-sepharose chromatography. Artificial GPI-modified inhibitors can be painted onto AVC surfaces by simply combining them with the cells, optimally for one hour at room temperature or 37°C.
  • soluble reagent protein transfer can be accomplished through the use of liposomes. Methods are well described for designing liposomes to be used as polypeptide carriers, and these can be directly applied to the delivery of immunoregulatory soluble molecule to AVCs. Alternatively, inert carriers, for example, polysaccharide beads, can be pre-coated with soluble molecules and delivered to the cytoplasm of cells. Additionally, APCs can be induced to pinocytose high concentrations of soluble molecules. In all of these cases, the immunoregulatory molecule is released by the APC slowly over time. In another aspect, the invention features soluble inhibitory reagents that have been genetically engineered to confer special properties to them.
  • One useful modification is to combine the sequences for more than one reagent into a single polypeptide. This provides for cooperative inhibitory functions without the need for using multiple reagents. In designing such "multi ⁇ functional reagents", one can draw upon published experience with chimeric hematopoietic cytokines which have been successfully used to modulate hematopoiesis.
  • Another useful modification is lipid modifying reagents such as cytokines that normally exist as soluble forms in order to allow them to be anchored into the APC surface. Certain cytokines are known to function in both soluble and cell surface-associated modes.
  • immunosuppressive cytokines such as IL-10 and TGFS are produced in a lipid-modified, for example, GPI-modified, form and then coated onto APC surfaces to generate AVCs.
  • lipid-modified for example, GPI-modified
  • Such tethered immunoregulatory cytokines retain the inhibitory function of their natural soluble counterparts.
  • the invention features cells other than conventional APCs that are engineered to function as AVCs.
  • AVCs that are engineered to function as AVCs.
  • antigen presentation is accomplished by either using endogenous MHC molecules which can be loaded (or pulsed) with exogenous antigenic peptides, or by transfecting the cells with MHC genes and then loading (or pulsing) the expressed proteins, or by coating the cells with pre-formed MHC:antigenic peptide complexes.
  • a non-conventional APC that can be converted into an AVC is a grafted cell. Cells of a graft, such as epithelial cells, can be coated with an inhibitory molecule prior to transplantation into the recipient. This represents a method for promoting engraftment, since cytotoxic T-cell effectors can be ablated.
  • AVCs can be administered to such patients, for example, intravenously, subcuta- neously, intramuscularly, or intraperitoneally to inhibit such pathogenic T-cells.
  • the patient's own APCs can be obtained, for example, by purifying them from phlebotomized blood via standard Ficoll-hypaque centrifugation and subsequent isolation methods.
  • the cells are then converted into AVCs via inhibitory expression, pulsed with a source of pathogenic antigenic peptides, and then administered back to the patient.
  • the patient can be treated in a similar fashion with allogeneic AVCs prepared from the graft donor's blood.
  • the transplanted marrow can be pre-treated with AVCs ex vivo.
  • the invention features a method for producing an artificial veto cell capable of specifically inhibiting the proliferation, cytotoxicity or cytokine release of a T-cell or inducing apoptosis or necrotic cell death in a T-cell directed to an alloantigen or processed antigen.
  • the method entails providing an antigen presenting cell having a specific alloantigen or processed antigen. Then the extracellular surface of the antigen presenting cell is externally contacted with a chimera comprising a non-CD8 polypeptide capable of reducing T-cell proliferation, cytotoxicity, or cytokine secretion or inducing T-cell apoptosis or necrotic cell death and a moiety sufficient to bind the chimera to the surface of the antigen presenting cell in a manner which presents the polypeptide on the cell's surface such that the polypeptide is able to reduce T-cell proliferation, cytoxicity or cytokine secretion or induce T-cell apoptos ⁇ is or necrotic cell death.
  • the method entails inserting a genetic sequence encoding a non-CD8 cell surface polypeptide or a chimera into the antigen presenting cell.
  • Another embodiment of the method features inserting into an antigen presenting cell a genetic sequence encoding a secreted molecule that is capable of reducing T-cell proliferation, cytotoxicity or cytokine secretion or inducing apoptosis or necrotic cell death in a T-cell.
  • a secreted molecule is delivered to the antigen presenting cell in a form that allows for subsequent localized release.
  • an artificial veto cell is meant a non- naturally occurring cell that is capable of inhibiting the proliferation of, cytotoxicity of, or cytokine release by a T-cell or of inducing apoptosis or necrotic cell death in a T-cell.
  • the cell bears an antigenic peptide at its cell surface that can bind to the T-cell receptor of a T- cell that is being inhibited or induced to apoptose or undergo necrotic cell death.
  • This antigenic peptide permits targeting of the inhibitory effect to antigen- specific T-cells.
  • the cell is a molecularly modified conventional antigen-presenting cell, such as a dendritic cell or a B-cell.
  • a non-antigen processing cell can also be converted to an artificial veto cell by providing an antigenic peptide to its cell surface.
  • proliferation is meant cell division which increases the number of cells present .
  • Cell division is associated with DNA synthesis and can be monitored ex vivo by measuring 3 H-thymidine incorporation.
  • cytotoxicity is meant the capacity of one cell to kill another cell. This cell function can be monitored ex vivo by measuring 51 Cr release from radio- labeled target cells.
  • cytokine secretion is meant the release from the cell of a protein that mediates an immune response.
  • apoptosis programmed cell death in response to a variety of different triggers.
  • necrosis cell death mediated by an environment made so hostile to the cell, by means of poisons or inappropriate pH or lack of oxygen, that the cell cannot maintain a state of homeostasis.
  • alloantigen is meant either class I or class II MHC molecules with or without associated antigenic peptides from a different individual of the same species.
  • processed antigen is meant fragments of a foreign substance, usually a protein, that bind to class I or class II MHC molecules.
  • An antigen presenting cell can be isolated in a number of ways. They may be obtained from peripheral blood. The blood is fractionated by density gradients to obtain either the mononuclear cells or the polynuclear cells. This procedure is well known to persons skilled in the art. Various techniques can be utilized to isolate APCs (B lymphocytes, dendritic cells, or monocytes) such as adherence, adherence and release, fluorescence activated cell sorting with lineage specific antibodies, magnetic cell sorting with lineage specific antibodies, complement mediated killing with lineage specific antibodies. Polymorphonuclear blood cells can also be used as APCs. Various tissues also contain cells that can act as APCs. These tissues can be dissociated by physical and enzymatic means to release APCs.
  • APCs B lymphocytes, dendritic cells, or monocytes
  • endothelial cells can be obtained from vessels and yoblasts from muscle.
  • the ability of some of these cells to act as APCs can be enhanced by treatment of the cells with cytokines such as interferon gamma.
  • cytokines such as interferon gamma.
  • isolation of the APC from a particular source will determine the type of alloantigen presented by the APC.
  • a specific processed antigen can be expressed on an isolated APC by either feeding unprocessed antigenic peptide to the APC and allowing the APC to process the antigenic peptide or exposing the APC to processed antigen.
  • Those in the art are familiar with both these techniques. In this manner, an antigen presenting cell having an alloantigen or processed antigen can be provided.
  • contacting the extracellular surface is meant contacting the cell surface from the exterior as opposed to insertion into a cell membrane via an intracellular route.
  • chimera is meant a polypeptide that possesses at least one domain from one protein and at least another domain from a different protein.
  • non-CD8 polypeptide a protein other than CD8, that is involved in immunoregulation by inhibition of cytokine release, proliferation or cytotoxicity or by inducing apoptosis or necrotic cell death.
  • molecules include: secreted molecules and cell surface associated molecules.
  • moiety sufficient to bind said chimera to said surface is meant a molecule or domain of a molecule that allows for attachment to or incorporation into the cell membrane.
  • a molecule may be a lipid.
  • inserting into said antigen presenting cell a genetic sequence is meant any procedure that allows for introduction of nucleic acids into a cell, e.g., transfection, electroporation, liposo e transfer.
  • non-CD8 cell surface polypeptide By “genetic sequence encoding a non-CD8 cell surface polypeptide” is meant the sequence of nucleic acids specific for the non-CD8 cell surface polypeptide in the context of other regulatory sequences that enable the polypeptide sequence to be transcribed into mRNA and then translated into an active protein.
  • secreted molecule is meant a molecule that can be released by an artificial veto cell so that it is free from the antigen presenting cell.
  • genetic sequence encoding a secreted molecule is meant the sequence of nucleic acids specific for the secreted molecule in the context of other regulatory sequences that enable the molecules sequence to be transcribed into mRNA and then translated into an active protein.
  • delivering a secreted molecule is meant providing a secreted molecule in a form that allows for its uptake by an APC and subsequent local release.
  • the non-CD8 polypeptide is selected from the group consisting of polypeptides that comprise amino acid sequences from Fas ligand, an anti- class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti- ⁇ 2 microglobulin antibody, an anti- / S 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas-antigen Fv chimeric polypeptide, interleukin-10, transforming growth factor ⁇ , heterodi eric placental protein 14, ho odimeric placental protein 14, and an immunoregulatory viral protein.
  • Fas ligand is a membrane bound polypeptide that is known to bind to a T-cell surface molecule Fas antigen and via this binding event induce apoptosis in said T- cell.
  • the polypeptide to be used for AVC engineering comprises either the entire Fas ligand (amino acid residues 1-281) or the extracellular portion of Fas ligand (amino acid residues 103-281) .
  • Fas ligand is a membrane bound polypeptide that is known to bind to a T-cell surface molecule Fas antigen and via this binding event induce apoptosis in said T- cell.
  • Fas antigen is defined as CD95, a cell surface molecule that binds Fas ligand.
  • anti-class I MHC heavy chain Ab is meant an antibody molecule with specificity for the heavy polypeptide chain of a class I MHC heterodimer.
  • anti-class I MHC antibodies that are capable of inhibiting a T-cell are 25.99, W6/32, CR1-S63, CR10-215, CR11-115, CR11-351, 5H7, Q6/64, Ql/28, 6/31, CR1, 01.65, and Bl-23-2 (Tanabe, et al . , 148 J. Immunol. 3202, 1992; De Felice, et al . , 122 Cell.
  • cDNAs can then be subcloned into expression vectors, and the resulting expression vectors can be used to express the antibodies in cells being converted to artificial veto cells.
  • a membrane anchoring moiety can be appended to the carboxy-terminus.
  • This can consist of a linker peptide, a hydrophobic transmembrane peptide and cytoplasmic extension (for example, an amino acid sequence corresponding to the hydrophobic transmembrane peptide and cytoplasmic extension of membrane IgG) or of a GPI modification signal sequence from a polypeptide that is naturally GPI-anchored to the membrane (for example, human decay-accelerating factor, spanning an amino acid sequence comprising Pro311 to Thr343 of this protein) .
  • Gene chimerization is readily carried out using splice-by-overlap-extension polymerase chain reaction ("SOE-PCR") technology. (Horton et al . , 8 Biotechniques 528, 1990; Horton et al. , 217 Methods Enz mol . 270, 1993) .
  • anti-class I MHC heavy chain Fv chimeric polypeptide a recombinant fusion polypeptide comprising a leader peptide, the variable domain of the immunoglobulin heavy chain of an anti-class I MHC heavy chain Ab, a linker peptide (for example, [ (Gly) ,Ser] 3 ) , and the variable domain of the immunoglobulin light chain of an anti-class I MHC heavy chain Ab.
  • a membrane anchoring moiety is appended to the carboxy-terminus .
  • This can consist of a linker peptide, a hydrophobic transmembrane peptide and cytoplasmic extension (for example, an amino acid sequence corresponding to the hydrophobic transmembrane peptide and cytoplasmic extension of membrane IgG) or of a GPI modification signal sequence from a polypeptide that is naturally GPI-anchored to the membrane (for example, human decay-accelerating factor, spanning an amino acid sequence comprising Pro311 to Thr348 of this protein) .
  • a stop codon is appended immediately following the light chain variable domain.
  • anti-j ⁇ 2 microglobulin antibody an antibody molecule with specificity for the ⁇ 2 microglobulin chain of a class I MHC heterodimer.
  • An example of an anti-,S 2 microglobulin antibody that is capable of inhibiting a T-cell is described in Bach et al. , 182 Science 1350, 1973. The strategy for cloning and expressing such an antibody is as described above for an anti-class I MHC heavy chain antibody.
  • anti-3 2 microglobulin Fv chimeric polypeptide is meant a recombinant fusion polypeptide comprising a leader peptide, the variable domain of the immunoglob ⁇ lin heavy chain of an anti-? 2 microglobulin Ab, a linker peptide (for example, [ (Gly) 3 Ser] 3 ) , and the variable domain of the immunoglobulin light chain of an anti- / S 2 microglobulin Ab.
  • a membrane anchoring moiety is appended to the carboxy- terminus.
  • This can consist of a linker peptide, a hydrophobic transmembrane peptide ' and cytoplasmic extension (for example, an amino acid sequence corresponding to the hydrophobic transmembrane peptide and cytoplasmic extension of membrane IgG) or of a GPI modification signal sequence from a polypeptide that is naturally GPI-anchored to the membrane (for example, human decay-accelerating factor, spanning an amino acid sequence comprising Pro311 to Thr348 of this protein) .
  • a stop codon is appended immediately following the light chain variable domain.
  • interleukin-10 is meant a soluble cytokine known by that designation with well-described T-cell inhibitory function.
  • the complete amino acid sequence of this cytokine, or functional derivatives thereof, are used according to the present invention for AVC engineering. (Vieira et al. , 88 Proc. Natl. Acad. Sci. , USA 1172, 1991) , incorporated herein by reference.
  • transforming growth factor ⁇ is meant a soluble cytokine with described T-cell inhibitory function.
  • the complete amino acid sequence of this cytokine, or functional derivatives thereof, are used according to the present invention for AVC engineering. (Derynck et al . , 316 Nature 701, 1985, incorporated herein by reference; Wahl et al. , 140 J. Immunol. 3026, 1988) .
  • heterodimeric placental protein 14 is meant a soluble cytokine with described T-cell inhibitory function.
  • cDNAs corresponding to the PP14.1 and PP14.2 chains of the heterodimer have previously been cloned from cells of the platelet lineage (Morrow, et al . , 145 Amer. J. Pathol. 1485, 1994, incorporated herein by reference) .
  • the complete amino acid sequence of this cytokine, or functional derivatives thereof, are used according to the present invention for AVC engineering.
  • homodimeric placental protein 14 is meant a soluble cytokine with described T-cell inhibitory function.
  • cDNA corresponding to this protein has previously been cloned from pregnant endometrium.
  • the complete amino acid sequence of this cytokine, or functional derivatives thereof, are used according to the present invention for AVC engineering.
  • Soluble molecules such as interleukin-10, transforming growth factor ⁇ , heterodimeric placental protein 14 and homodimeric placental protein 14, and the extracellular domain of the Fas ligand can modified into membrane associated variants by the above described methods.
  • an immunoregulatory viral protein is meant a viral-encoded polypeptide with immunoregulatory characteristics.
  • a viral protein shown to regulate or modify the immune response of T cells includes the native complete sequence or any partial sequence that maintains the immunoregulatory function.
  • proteins with immunoregulatory characteristics are those of the immunodeficiency virus, HIV.
  • Membrane associated variants can be constructed by the above described methods.
  • the secreted molecule is interleukin-10, transforming growth factor ⁇ , an anti-class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti-? 2 microglobulin antibody, and anti-S 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas-antigen Fv chimeric polypeptide, a chimeric bacterial toxin molecule, HIV-tat, CTLA4Ig, a viral immunoregulatory protein, heterodimeric placental protein 14, homodimeric placental protein 14, or an antibody against a T-cell activation antigen.
  • HIV tat is meant a soluble protein known by that designation and encoded by human immunodeficiency virus with well-described T-cell inhibitory function.
  • An active domain of HIV tat consists of amino acids 1-72. (Viscidi, et al. , 246 Science 1606, 1989, incorporated herein by reference) .
  • the complete amino acid sequence of this protein, or functional derivatives thereof, are used according to the present invention for AVC engineering.
  • any activation antigen of a T-cell is meant cell surface molecules that are not expressed on T cells not stimulated by antigens but are expressed on T cells stimulated by antigens. Such molecules include: CD25, CD38, CD30, CD49a, CD49b, CD49c, CD49d, CD26, CD69, CD70, CD71, CD96, CD98, CD109, and class II MHC molecules.
  • Antibodies to an activation antigen of a T-cell can be whole molecules or Fv.
  • Antibodies to T-cell activation antigens can be obtained by immunizing mice with activated T lymphocytes and producing cloned hybridomas secreting antibodies by utilizing standard methods known to those skilled in the art. These monoclonal antibodies can be selected for defined specificities by comparison with the known antibodies. Alternatively, purified activation antigens or synthetic peptides produced from their determined sequences can be used to immunize mice for the production of monoclonal antibodies.
  • CTLA4Ig is meant a soluble molecule that inhibits the ability of CD28 or CTLA4 to ligate to its receptors on APCs which are positive for B7-1 or B7-2 (Lenschow et al . , 257 Science 789, 1992; Linsley et al., 257 Science 792, 1992) .
  • chimeric bacterial toxin molecule is meant cytokines (e.g.,IL-l, 11-2, IL-4, IL-6, IL-7, TGF3, tumor necrosis factor) , antibodies (whole molecules or Fv) to activation antigens, and anti-Fas antigen molecules that are chimeric with bacterial toxins especially those from Pseudomonas (Kozak et al . , 145 . Immunol . 2766, 1990; Kreitman et al., 87 Proc. Natl. Acad. Sci., USA 8291, 1990; Lorberboum-Galski et al. 265 J. Biol. Che .
  • cytokines e.g.,IL-l, 11-2, IL-4, IL-6, IL-7, TGF3, tumor necrosis factor
  • antibodies whole molecules or Fv
  • anti-Fas antigen molecules that are chimeric with bacterial toxins especially those from Pseudomon
  • a complete cytokine sequence (e.g., IL-2) can be recombined with a bacterial toxin sequence truncated so that its cellular recognition domain is not included in the chimeric protein.
  • the cytokine domain will act as a cellular recognition domain and the bacterial toxin sequence will provide cell destructive capabilities.
  • Chimeric molecules are transferred to AVCs via gene transfer and are subsequently secreted from the APC.
  • the antigen presented by t-he antigen presenting cell is bound to a class I MHC polypeptide; the antigen is bound to a class II MHC polypeptide; the moiety comprises a lipid modification; the lipid modification comprises glycosyl- phosphatidylinositol; the GPI-modified peptide is produced by gene transfer of a chimeric gene expression construct comprising a GPI modification signal sequence into a host cell and isolation of the GPI-modified polypeptide from the host cell; the soluble molecule is delivered via a liposome; the soluble molecule is delivered via an inert bead; the soluble molecule is delivered via pinocytosis.
  • class I MHC polypeptide sequence is meant an amino acid sequence corresponding to a portion of the extracellular domain of a class I major histocompatibility complex heavy chain, for example, an HLA-A, HLA-B, or HLA- C heavy chain.
  • class II MHC polypeptide sequence is meant an amino acid sequence corresponding to a portion of the extracellular domain of either a class II major histocom ⁇ patibility complex alpha chain or a class II major histo ⁇ compatibility complex beta chain, for example, an HLA-DQ, HLA-DR, or HLA-DP alpha or beta chain.
  • lipid modification is meant the attachment of a lipid moiety to a protein by chemical or enzymatic means.
  • glycosyl-phosphatidylinositol-modified polypeptide an polypeptide which has a covalently attached glycosyl-phosphatidylinositol molecule, so as to allow membrane insertion.
  • a chimeric gene expression construct comprising a glycosyl-phosphatidylinositol modification signal sequence
  • a chimeric gene expression construct comprising a glycosyl-phosphatidylinositol modification signal sequence
  • liposome is meant a lipid encapsulated vessel for delivery of molecules to a cell.
  • inert bead is meant a substance that is chemically unreactive, physically stable, and is not a substrate for an enzyme, for example a polysaccharide bead.
  • pinocytosis is meant the process of inclusion of an extracellular substance in a cellular vesicle located in the cytosol and made from an invagination of the cell surface membrane.
  • the use of pinocytosis to cause cells to uptake molecules is known to those who practice the art.
  • the chimeric bacterial toxin comprises a cytokine that binds to a receptor on a T cell, an antibody that binds to an antigen on a T cell or a Fv molecule that binds to an antigen on a T cell; the chimeric bacterial toxin comprises the toxin domain from diphtheria toxin or Pseudomonas exotoxin.
  • cytokine that binds a receptor on a T cell are cytokines such as IL-1, 11-2, IL-4, IL-6, IL-7, TGF3, or tumor necrosis factor.
  • antibody that binds to an antigen on a T cell is meant an antibody directed toward activation antigens, or anti-Fas antigen molecules.
  • Fv molecule that binds to an antigen on a T cell is meant an Fv molecule directed toward activation antigens or anti-Fas antigen molecules.
  • toxin domain is meant a portion of a toxin that possesses toxic activity.
  • diphtheria toxin is meant the soluble toxin protein that is released by the bacterium Corynebacterium diphtheria.
  • Pseudomonas exotoxin the soluble toxic protein released by Pseudomonas bacterium.
  • the invention features a method for producing a non-naturally occurring biological membrane capable of specifically inhibiting the prolifera ⁇ tion, cytotoxicity or cytokine secretion or inducing apoptosis or necrotic cell death in the T-cell directed to an alloantigen or processed antigen.
  • the method comprises isolating a biological membrane having an alloantigen or processed antigen, and contacting the extracellular portion of the biological membrane with a chimera comprising a non-CD8 polypeptide capable of reducing T- cell proliferation, cytotoxicity or cytokine secretion or inducing T-cell apoptosis or necrotic cell death and a moiety sufficient to bind the chimera to the biological membrane in a manner which presents the polypeptide on the biological membrane such that the polypeptide is able to reduce T-cell proliferation, cytoxicity, or cytokine secretion or induce T-cell apoptosis or necrotic cell death.
  • non-naturally occurring biological membrane is meant a membrane that does not naturally have the specified T cell immunoregulatory capacity, but has been provided with such capacity.
  • isolated a biological membrane having said alloantigen or processed antigen is meant a procedure for obtaining a biological surface membrane from a cell expressing an alloantigen or processed antigen. These procedures are well known to those skilled in the art.
  • a cell presenting an alloantigen or processed antigen is used to isolate such a biological membrane.
  • Biological membranes are isolated by disrupting the cell through a variety of means such as freeze-thawing, nitrogen cavation, detergent lysis, or other techniques known to those in the art. Nuclei are sedimented and discarded leaving cytosol and cell membranes. The membranes are isolated by appropriate density gradient and centrifugation techniques.
  • fractions with plasma (surface) membrane are found in specific fractions of a density gradient and they are identified with specific enzymatic activities known to characterize the surface membranes.
  • the non-CD8 polypeptide would be bound to the membrane utilizing the same procedures as described for intact cells.
  • the method encompasses providing an antigen presenting cell having a specified alloantigen or processed antigen, inserting into the antigen presenting cell a genetic sequence encoding a chimera comprising a non-CD8 polypeptide capable of reducing T-cell proliferation, cytotoxicity or cytokine secretion or inducing T-cell apoptosis or necrotic cell death and a moiety sufficient so that the chimera is expressed on the surface of the antigen presenting cell in a manner which presents the polypeptide so as to be able to reduce T-cell proliferation, cytoxicity or cytokine secretion or induce T-cell apoptosis or necrotic cell death, and isolating the biological membrane from the antigen presenting cell.
  • the invention features a glycosyl-phosphatidylinositol-modified polypeptide comprising a polypeptide selected from the group consisting of polypeptides that comprise amino acid sequences from the extracellular domain of Fas ligand, an anti-class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti-/S 2 microglobulin antibody, and anti- ⁇ 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas antigen Fv chimeric polypeptide, interleukin-10, transforming growth factor ⁇ , heterodimeric placental protein 14, homodimeric placental protein 14, HIV tat, and an immunoregulatory viral protein, which is separate from a membrane.
  • a polypeptide selected from the group consisting of polypeptides that comprise amino acid sequences from the extracellular domain of Fas ligand, an anti-class I MHC heavy chain antibody, an anti-class I
  • the invention features an artificial veto cell having a membrane exogenously coated with a lipid-modified non-CD8 polypeptide selected from the group consisting of polypeptides that comprise amino acid sequences from the extracellular domain of the Fas ligand, an anti-class I MHC heavy chain antibody, an anti- class I MHC heavy chain Fv chimeric polypeptide, an an:i- / S 2 microglobulin antibody, and anti-3 2 microglobuli Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas antigen Fv chimeric polypeptide, interleukin-10, transforming growth factor ⁇ , heterodimeric placental protein 14, homodimeric placental protein 14, and an immunoregulatory viral protein.
  • a lipid-modified non-CD8 polypeptide selected from the group consisting of polypeptides that comprise amino acid sequences from the extracellular domain of the Fas ligand, an anti-class I MHC heavy chain antibody, an anti- class I M
  • the artificial veto cell comprises a genetic sequence that encodes for the lipid-modified non-CD8 polypeptide.
  • genetic sequence that encodes a cell surface lipid-modified non-CD8 polypeptide is meant a nucleic acid sequence encoding a polypeptide including a lipid modification sequence and other regulatory sequences that enable the encoded sequence to be transcribed into mRNA, translated into protein and modified with a lipid moiety.
  • the invention features an artificial veto cell bearing a transfected genetic sequence that encodes a secreted molecule capable of reducing T-cell proliferation, cytotoxicity, or cytokine secretion or inducing apoptosis or necrotic cell death directed to alloantigens or processed antigens.
  • bearing a transfected genetic sequence is meant a genetic sequence encoding for a secreted molecule either integrated into a chromosome or existing extrachromosomally.
  • the invention features an artificial veto cell containing an exogenously added secreted molecule capable of reducing T-cell proliferation or cytotoxicity, or cytokine secretion or inducing apoptosis or necrotic cell death directed to alloantigens or processed antigens.
  • the artificial antigen presenting cell contains the exogenously added secreted molecule by carrier-delivery, pinocytosis, or liposome delivery.
  • the secreted molecule is selected from the group consisting of interleukin-10, transforming growth factor ⁇ , an anti-class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti-3 2 microglobulin antibody, an anti-/3 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas-antigen Fv chimeric polypeptide, a chimeric bacterial toxin molecule, HIV-tat, CTLA4Ig, a viral immunoregulatory protein, heterodimeric placental protein 14, homodimeric placental protein 14, and an antibody against a T-cell activation antigen.
  • interleukin-10 transforming growth factor ⁇
  • an anti-class I MHC heavy chain antibody an anti-class I MHC heavy chain Fv chimeric polypeptide
  • an anti-3 2 microglobulin antibody an anti-/3 2 microglobulin Fv chimeric polypeptide
  • the invention features a method for inhibiting an antigen-specific T-cell.
  • the method comprises providing an artificial veto cell which presents in, or on its surface a non-CD8 polypeptide capable of reducing T-cell proliferation, cytotoxicity or cytokine secretion or inducing T-cell apoptosis or necrotic cell death and an MHC:nominal antigen peptide complex, and exposing the T-cell capable of responding to the MHC:nominal antigen peptide complex so as to inhibit the cellular immune response of the T cells to the antigen.
  • MHC:nominal antigen peptide complex is meant a class I or class II MHC molecule with a noncovalent or covalently associated antigenic peptide.
  • exposing is meant brought into proximity so as to allow molecules either bound to or released from the antigen presenting cell to contact the T cell.
  • non-CD8 polypeptide 27 In a preferred embodiment the non-CD8 polypeptide 27
  • polypeptides that comprise amino acid sequences from Fas ligand, an anti- class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti-S 2 microglobulin antibody, and anti-3 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas antigen Fv chimeric polypeptide, interleukin-10, transforming growth factor ⁇ , heterodimeric placental protein 14, homodimeric placental protein 14, HIV tat, and an immunoregulatory viral protein.
  • the invention features a method for inhibiting an antigen-specific T-cell by providing an artificial veto cell which locally releases a secreted molecule and an MHC:nominal antigen peptide complex present on its surface, and exposing the T-cells capable of responding to the MHC:nominal antigen peptide complex so that the secreted molecule is locally released and inhibits the cellular immune response of the T cells to the antigen.
  • the secreted molecule is selected from the group consisting of interleukin-10, transforming growth factor ⁇ , an anti-class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti-/3 2 microglobulin antibody, an anti-jS 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas-antigen Fv chimeric polypeptide, a chimeric bacterial toxin molecule, HIV-tat, CTLA4Ig, a viral immunoregulatory protein, heterodimeric placental protein 14, homodimeric placental protein 14, and an antibody against a T-cell activation antigen.
  • T-cells are exposed ex vivo; T-cells are exposed in vivo; the MHC polypeptide is syngeneic with the antigen-specific T-cell; the MHC polypeptide is allogeneic with the antigen-specific T- cell.
  • ex vivo is meant outside of the body of a patient to be treated.
  • in vivo in the body of a patient to be treated.
  • synthetic is meant that the MHC molecules of the antigen-specific T-cell is identical to that of the MHC molecule on the artificial veto cell that is being transferred.
  • antigen-specif- ic T-cell bears at least one MHC allelic variant that is disparate with that of the MHC molecule on the artificial veto cell that is being transferred.
  • the invention features a method for inhibiting a pathogenic T-cell in a patient.
  • the method comprises administering to the patient an artificial veto cell which presents in, or on its surface a polypeptide selected from the group consisting of poly ⁇ peptides that comprise amino acid sequences from Fas ligand, an anti-class I MHC heavy chain antibody, an anti- class I MHC heavy chain Fv chimeric polypeptide, an anti- / S 2 microglobulin antibody, and anti- / S 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas antigen Fv chimeric polypeptide, interleukin-10 , transforming growth factor ⁇ , heterodimeric placental protein 14, homodimeric placental protein 14, and an immunoregulatory viral protein, and an MHC:nominal antigen peptide complex.
  • the method for inhibiting a pathogenic T-cell in a patient may be practiced by utilizing an artificial veto cell which locally releases a molecule selected from the group consisting of interleukin 10, transforming growth factor ⁇ , an anti- class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti-/3 2 microglobulin antibody, an anti- / S 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen antibody, an anti-Fas-antigen Fv chimeric polypeptide, a chimeric bacterial toxin molecule, HIV-tat, CTLA4Ig, a viral immunoregulatory protein, heterodimeric placental protein 14, homodimeric placental protein 14, and an antibody against a T-cell activation antigen.
  • administering to the patient is meant given internally to a patient.
  • the invention features various membrane associated antibodies. These include: an anti-class I MHC antibody comprising an immunoglobi.lin heavy chain polypeptide and an immunoglobulin light chain polypeptide, an anti- / S 2 microglobulin antibody comprising an immunoglobulin heavy chain polypeptide and an immuno ⁇ globulin light chain polypeptide, an anti-Fas antigen antibody comprising an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, an anti-class I MHC Fv chimeric polypeptide, anti-3 2 microglobulin Fv chimeric polypeptide, an anti-Fas antigen chimeric polypeptide.
  • the antibodies are lipid-modified; the lipid modification is a glycosyl- phosphatidylinositol moiety; the polypeptide comprises a poly-histidine tag.
  • poly-histidine tag two or more clustered histidines that are inserted into a polypeptide sequence in order to permit purification of the polypep ⁇ tide by nickel-sepharose chromatography.
  • an optimal site for insertion of the polyhistidine amino acid sequence is in between the sequence for the polypeptide of interest and the GPI modification signal sequence.
  • the N-terminal signal peptide and C-terminal GPI moiety preclude polyhistidine insertion into the conventional N-terminal and C-terminal sites. Insertion of the polyhistidine sequence is accomplished by inserting the coding sequence for this polyhistidine sequence into the desired site of an expression construct .
  • the invention features a method for producing an artificial veto cell utilizing a non-antigen presenting cell.
  • the methods are analogous to those described for antigen presenting cells.
  • a non-antigen presenting cell is isolated.
  • Such a cell is manipulated so that an alloantigen or processed antigen is presented on the extracellular surface of the cell.
  • non-antigen presenting cell is meant a cell that does not efficiently process antigenic proteins into antigenic peptides. Generally, such a cell lacks costimulator function as well. Non-antigen presenting cells are isolated utilizing the same procedures as utilized for the isolation of antigen presenting cells.
  • manipulated so that an alloantigen or processed antigen is presented is meant treating the cell so that it presents an antigen.
  • manipulating comprises loading the cells with antigenic peptides; manipulating comprises transfecting the cells with MHC genes and loading the cells with antigenic peptides; manipulating comprises coating the cells with pre-formed MHC: antigenic peptide complexes.
  • loading the cells with antigenic peptides is meant introduction of specific antigenic peptides into association with class I or class II MHC molecules. Such loading techniques are know to those who practice the art.
  • transfecting the cells with MHC genes is meant introducing into the cells nucleic acid sequences encoding MHC proteins in the context of other regulatory sequences that enable the encoded sequences to be transcribed into mRNA and translated into MHC proteins.
  • coating the cells with pre-formed MHC:antigenic peptide complexes is meant treating cells with MHC complexed with antigenic peptides such that the complex adheres to cells.
  • polypeptide is selected from the group consisting of polypeptides that comprise amino acid sequences from Fas ligand, an anti- class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti-3 2 microglobulin antibody, and anti-?
  • the secreted molecule is selected from the group consisting of interleukin-10, transforming growth factor ⁇ , an anti-class I MHC heavy chain antibody, an anti-class I MHC heavy chain Fv chimeric polypeptide, an anti- / S 2 microglobulin antibody, an anti-j_?
  • microglobulin Fv chimeric polypeptide an anti-Fas antigen antibody, an anti-Fas-antigen Fv chimeric polypeptide, a chimeric bacterial toxin molecule, HIV-tat, CTLA4Ig, a viral immunoregulatory protein, heterodimeric placental protein 14, homodimeric placental protein 14, or an antibody against a T-cell activation antigen.
  • Chimeric coding sequences are generally assembled using the splice-by-overlap-extension polymerase chain reaction (“SOE-PCR") method (Horton, et al . , 8 BioTechnicrues 528, 1990) . This technique has been used to generate chimeric coding sequences for artificial GPI modified proteins (Huang, et al., 31 Molec. Immunol. 1017, 1994) . This is readily adapted for generating GPI- or hydrophobic peptide-anchored variants of polypeptides to be used for AVC engineering.
  • SOE-PCR splice-by-overlap-extension polymerase chain reaction
  • this approach can be used for generating Fv chimeric polypeptides and to generate chimeric bacterial toxin molecules.
  • the SOE-PCR method is rapid and is not dependent upon having convenient restriction endonuclease cleavage sites at specific regions of the native polypeptide' s coding sequence.
  • convenient restriction endonuclease sites By incorporating convenient restriction endonuclease sites into the outer SOE-PCR primers, subsequently subcloning into expression vectors is greatly simplified.
  • the present invention encompasses a wide range of alterations of the non-CD8 polypeptides, e.g. substitutions and deletions, that keep intact the active portion of the molecule.
  • the active portion of the molecule generally comprises the extracellular domain.
  • the capacity to bind to specific receptors is essential although not always sufficient for activity.
  • Active domains or essential components of proteins can be determined by mutagenesis procedures well known in the art. For example, portions of the Fas ligand molecule can be truncated or mutated or otherwise modified and then tested to see if the altered molecule retains its ability to inhibit T lymphocytes.
  • the extracellular domain includes amino acids 103 to 281 (Takahashi et al . , 6 Inter. Immunol . 1567, 1994) .
  • an IL-2-bacterial toxin chimera the deletion of the glutamine at amino acid position 74 of the IL-2 moiety or a mutation of alanine at amino acid position 50 to tryptophan results in molecules with biological activity equivalent to the native IL-2 molecule.
  • For HIV tat amino acid residues 1 to 72 have been shown to possess immunoregulatory effects. (Viscidi et al . 246 Science 1606, 1989) .
  • the treatment of a patient with an inflammatory bowel disease includes the following steps to be performed for the inhibition or elimination of anti-bowel T cells using Fas ligand coated artificial veto cells.
  • peripheral blood is obtained from the patient via phlebotomy, a mononuclear cell fraction is isolated from the blood by Ficoll-hypaque gradient centrifugation, and the B-cell population is enriched using negative immuno- magnetic bead selection.
  • the B-cells are activated by incubating with killed Staphylococcus aureus, Cowen strain or with protein A coated sepharose beads or with a combination of anti-IgM and lipopolysaccharide according to well-established methods. Cytokines may be added to these cultures to enhance the activation process. After 3 days the activated B cells are fed colonic cell extract for 2 hours at 37°C to allow for the uptake and processing of antigen. After the cells are washed, they are incubated for an additional hour at 37°C in the absence of serum in the presence of RPMI 1640 with 25 micrograms per ml of lipid-conjugated Fas-Ligand (extracellular domain) . These artificial veto cells, either untreated or irradiated or chemically treated to irreversibly prevent further growth are then introduced into the patient . Standard clinical protocols are used to establish an optimal dosing schedule.
  • the prospective transplant recipient undergoes pretransplant transfusion with donor activated B cells coated with lipidated Fas-ligand.
  • Cell retrieval and protein transfer are performed as described above; of note, antigenic pulsing of the cells is not required in this circumstance.
  • Fas ligand cDNA in an expression vector can be transferred into the B-cells and Fas ligand expressing cells are utilized.
  • Artificial veto cells containing genetic sequences encoding for soluble molecules, e.g., cytokines, Fv or heavy chain antibodies or chimeric bacterial toxin molecules, or artificial veto cells to which soluble molecules have been delivered, can be utilized as described above, with or without antigenic pulsing.
  • Some of the other diseases that could be treated utilizing the methods and compositions of the present invention include graft versus host rejection, diabetes, pernicious anemia, autoimmune hepatitis, and allergic diseases.
  • the treatment of a bone marrow specimen in order to prevent graft versus host disease subsequent to transplantation into a recipient includes the following steps to be performed when using Fas-Ligand coated artificial veto cells.
  • peripheral blood is obtained from the prospective recipient via phlebotomy
  • a mononuclear cell fraction is isolated from the blood by Ficoll/hypaque gradient centrifugation, and the B lymphocyte population is enriched using negative immuno- magnetic bead selection.
  • the B cells are activated by incubating with killed Staphylococcus aureus, Cowen strain or with protein A coated sepharose beads or with a combination of anti-IgM and lipopolysaccharide according to well-established methods.
  • Cytokines may be added to these cultures to enhance the activation process. After 3 days the activated B cells are washed and then incubated for an hour at 37°C in the absence of serum in the presence of RPMI 1640 with 25 micrograms per ml of lipid- conjugated Fas-Ligand (extracellular domain) . These artificial veto cells, either irradiated or chemically treated to irreversibly prevent further growth or not treated, are then added to the bone marrow specimen and allowed to incubate overnight at 37°C in appropriate culture medium, so as to delete bone marrow T cells with specificity against the recipient.

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Abstract

Procédé d'obtention d'une cellule veto artificielle capable d'inhiber spécifiquement la prolifération, la cytotoxicité ou la sécrétion de cytokine, ou d'induire l'apoptose des cellules T ou la mort des cellules nécrotiques dirigées vers des aloantigènes ou des antigènes traités. Ledit procédé comporte les étapes suivantes: partir d'une cellule porteuse d'un antigène du type aloantigène ou antigène traité; établir un contact extérieur entre la surface extracellulaire de la cellule porteuse d'antigène et une chimère comportant un polypeptide non-CD8 capable de réduire la prolifération des cellules T, la cytotoxicité ou la sécrétion de cytokine, ou d'induire l'apoptose des cellules T ou la mort des cellules nécrotiques, et un fragment suffisant pour lier la chimère à la surface de la cellule porteuse d'antigène de manière à présenter le polypeptide sur la surface de la cellule pour qu'il puisse réduire la prolifération des cellules T, la cytotoxicité ou la sécrétion de cytokine, ou induire l'apoptose des cellules T et/ou la mort des cellules nécrotiques
PCT/US1996/005187 1995-04-10 1996-04-08 Procedes d'elaboration de cellules veto artificielles WO1996032140A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8871515B2 (en) 2008-09-17 2014-10-28 Isogenis, Inc. Construction of fully-deleted adenovirus-based gene delivery vectors and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5242687A (en) * 1989-03-15 1993-09-07 Tkb Associates Limited Partnership Method of reducing cellular immune response involving T-cells using CD8-bearing antigen presenting cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5242687A (en) * 1989-03-15 1993-09-07 Tkb Associates Limited Partnership Method of reducing cellular immune response involving T-cells using CD8-bearing antigen presenting cells

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 76, issued 28 January 1994, GERMAIN R.N., "MHC-Dependent Antigen Processing and Peptide Presentation: Providing Ligands for T Lymphocyte Activation", pages 287-299. *
INTERNATIONAL IMMUNOLOGY, Volume 6, No. 10, issued 1994, TAKAHASHI et al., "Human Fas Ligand: Gene Structure, Chromosomal Location and Species Specificity", pages 1567-1574. *
KIDNEY INTERNATIONAL, Volume 43, Supplement 39, issued 1993, TYKOCINSKI et al., "Prospects for Anti-Rejection Therapies Based Upon CD8-Dependent Immunoregulation", pages S-120 - S-123. *
MOLECULAR IMMUNOLOGY, Volume 31, No. 13, issued 1994, HUANG et al., "Alloantigenic Recognition of Artificial Glycosyl Phosphatidylinositol-Anchored HLA-A2.1", pages 1017-1028. *
NATURE, Volume 370, issued 25 August 1994, LOWIN et al., "Cytolytic T-Cell Cytotoxicity is Mediated Through Perforin and Fas Lytic Pathways", pages 650-652. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8871515B2 (en) 2008-09-17 2014-10-28 Isogenis, Inc. Construction of fully-deleted adenovirus-based gene delivery vectors and uses thereof
US9169493B2 (en) 2008-09-17 2015-10-27 Isogenis, Inc. Construction of fully-deleted adenovirus-based gene delivery vectors and uses thereof
US9719107B2 (en) 2008-09-17 2017-08-01 Isogenis, Inc. Construction of fully-deleted adenovirus-based gene delivery vectors and uses thereof

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