WO1996031241A1 - Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface - Google Patents

Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface Download PDF

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WO1996031241A1
WO1996031241A1 PCT/US1996/004648 US9604648W WO9631241A1 WO 1996031241 A1 WO1996031241 A1 WO 1996031241A1 US 9604648 W US9604648 W US 9604648W WO 9631241 A1 WO9631241 A1 WO 9631241A1
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cells
seq
cell
donor
virus
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PCT/US1996/004648
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Gillis R. Otten
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Cell Genesys, Inc.
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Priority to CA002217297A priority Critical patent/CA2217297A1/fr
Priority to AU55338/96A priority patent/AU712415B2/en
Priority to EP96912564A priority patent/EP0820311A4/fr
Priority to JP8530474A priority patent/JPH11503024A/ja
Publication of WO1996031241A1 publication Critical patent/WO1996031241A1/fr
Priority to NO974596A priority patent/NO974596L/no

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    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the field of the subject invention is the transplantation of cells and tissue that are genetically modified to express low levels of class I MHC molecules for therapy.
  • MHC major histocompatibility complex
  • the MHC proteins serve as an identifier of "self.
  • Foreign antigens are generally recognized as "self + X", where the combination of self-MHC bound to a foreign peptide is recognized.
  • self + X the combination of self-MHC bound to a foreign peptide is recognized.
  • a transplantation is made from an allogeneic host, the transplant is recognized as foreign, and destroyed by the immune system, unless the transplant is MHC matched with the host, or the host is immunocompromised.
  • a transplant includes immunocompetent lymphocytes or monocytes, a graft may attack the host as foreign, resulting in graft-versus-host disease.
  • lymphocytes such as T-cells
  • T-cells tumor necrosis factor-producing cells
  • certain cells are lacking, such as islets of Langerhans in the case of diabetes; cells that secrete dopamine in the case of Parkinson's disease; bone marrow cells in various hematopoietic diseases; muscle cells in muscle wasting disease, or retinal epithelial cells in visual disorders, it would be desirable to be able to provide cells that can perform the desired function by expressing and secreting a gene product.
  • herpes simplex virus ICP47 protein in inhibiting antigen presentation to CD8 + T lymphocytes is discussed in York et al. (1994) Cell 77:525-535.
  • the human cytomegalovirus proteins involved in MHC protein down-regulation are described in Gilbert et al. (1993) J. Virol. 67:3461- 3469 and Beersma et al. (1993) J. Immunol. 151:4455-4464.
  • Virus induced loss of class I MHC molecules from the surface of cells infected with myxoma virus is described in Boshnov et al. (1992) J. Immunol. 148:881-887.
  • Suitable cells for transplantation are taken from a donor, and transfected with an expression vector containing genes encoding one or more virus-derived MHC down- regulatory proteins.
  • the cells are expanded ex vivo, if appropriate, and transplanted into a recipient host.
  • the decreased levels of class I MHC protein allow the transplanted cells to survive under conditions where the genetically unmodified cells would otherwise be subject to attack by the recipient host's immune system.
  • the cells may be additionally genetically modified to comprise constructs for altered effector function or for targeting diseased or virally infected cells in the host.
  • Figure 1 is a fluorescence activated cell sorter (FACS) profile of 143B osteosarcoma cells transduced with retrovirus encoding US11 , and stained with FITC-conjugated antibody specific for HLA Class I as described in Example 4, infra.
  • FACS fluorescence activated cell sorter
  • Figure 2 shows the lysis of C1 R-A2.1 + targets by HLA-A2-specific CTLs as described in Example 5, infra.
  • the Y axis is percent specific lysis and the X axis is the effector to target (E:T) ratio.
  • Figure 3 is a FACS profile of C1 R-A2.1 cells transduced with retrovirus encoding US11and CD4-zeta (top) or CD4-zeta (bottom) and labeled with PE-conjugated anti-CD4 antibody (Y axis) and FITC-conjugated anti-HLA antibody (X axis) as described in Example 5, infra.
  • Figure 4 is a FACS profile of C1 R-A2.1 clones transduced with retrovirus encoding US11 and stained with FITC-conugated anti-HLA Class I antibody as described in Example 5, infra.
  • Figure 5 (A) shows the resistance of C1 R-A2.1 clones transduced with retrovirus encoding US11 to lysis by HLA-A2-specific CTLs as described in Example 5, infra.
  • Figure 5 (B) shows the susceptibility of C1 R-A2.1 cells transduced with retrovirus encoding CD4-zeta to lysis by HLA-A2-specific CTLs as described in Example 5, infra.
  • the Y axis is percent specific lysis and the X axis is the effector to target (E:T) ratio.
  • Cells are genetically modified to have decreased class I MHC proteins on their surface by the introduction of an expression construct encoding one or more virus- derived MHC down-regulatory proteins.
  • the modified cells are used in cell or tissue transplantation, because they are resistant to being killed by the transplant recipient's immune system.
  • the subject methods are particularly useful for modifying cells, such as hematopoietic stem cells and T lymphocytes, that have been found to have low levels of transduction or transformation.
  • a significant down-regulation of Class I MHC molecules is achieved in such cells through the use of one or more techniques that enhance the effect of the subject methods.
  • Techniques of interest include the use of combinations of down-regulatory genes, particularly in conjunction with vector elements that permit multiple ribosomal entry in a single mRNA molecule (IRES elements); enhancement of transformation or transduction in vitro through the use of adhesion molecules or antibodies to adhesion molecules during in vitro culture; and selection for subpopulations of cells that express either low levels of MHC class I molecules, or high levels of a marker indicative of transformed or transduced cells.
  • the utility of modified T cells may also be enhanced by in vitro culture techniques that reduce the susceptibility of the cells to lysis by natural killer cells.
  • transplantation antigens expressed by the donor cells are mediated by transplantation antigens expressed by the donor cells.
  • the principal transplantation antigens are the class I and class II MHC proteins.
  • Cells that are deficient in class I MHC molecules are generally not recognized by cytolytic T cells, and therefore can be transplanted and maintained in a variety of individuals.
  • Down regulation of class I MHC proteins in the subject invention is accomplished by introducing into host cells an expression vector comprising virus-derived genes, whose products inhibit the cell surface expression of class I MHC proteins in the host cells.
  • the mammalian MHC class I molecule is a noncovalent, trimolecular complex that consists of an alpha chain encoded by genes within the MHC locus, a ⁇ 2 -microglobulin light chain and a small peptide derived from the intracellular proteolytic degradation of an endogenous cellular protein or foreign protein.
  • the complete class I molecule is assembled from its components intracellularly, within the endoplasmic reticulum. Only upon assembly of the complete complex is the complete class I MHC molecule transported to the plasma membrane.
  • post- translational inhibition of class I MHC expression is achieved by the introduction of virus-derived MHC down-regulatory proteins.
  • MHC down-regulatory proteins of interest include human adenovirus type 2 E3/19 kd gene (SEQ ID NO: 1 ) and human adenovirus type 5 E3/19 kd gene (SEQ ID NO:2); herpes simplex virus ICP47 gene (SEQ ID NO:3); human cytomegalovirus gene US11 (SEQ ID NO:4); human cytomegalovirus gene US5 (SEQ ID NO:5), and the like.
  • Other viruses known to down-regulate class I MHC expression include myxoma virus and rabbit fibroma virus, from which down-regulatory genes useful in the subject invention can be used.
  • the cells for modification may be any normal, i.e., non-transformed, mammalian cells of interest that find use in cell therapy, research, interaction with other cells in vitro or the like.
  • Suitable cells include epidermal cells such as keratinocytes; retinal epithelial cells; myoblasts; hematopoietic cells such as hematopoietic stem cells and T lymphocytes; endothelial cells, including venule and arterial endothelial cells; myoblasts; ⁇ -cells from the islets of Langerhans; and other cells that are readily manipulated in vitro.
  • the cells can be maintained and expanded, if desired, in culture and may be introduced into a host where the cells will remain viable and functional for long periods of time.
  • Hematopoietic cells of interest include naive or mature lymphocytes such as T-cells isolated from lymph nodes, peripheral blood, etc.
  • the T cells may be separated as to specific subsets or specificities after harvesting.
  • antigen specific T cells Large quantities of T cells with a particular antigenic specificity may be produced by antigen stimulation and in vitro culture, as known in the art (see Lamers et al. (1992) Int. J. Cancer 51 :973-979).
  • allogeneic T cells genetically modified so as to have an altered effector function or to be reactive, for example cytotoxic, with a specific target.
  • T cells may be genetically modified to express a chimeric receptor as described in U.S. Patent number 5,359,046, issued October 25, 1994, to target diseased or infected cells, or cancer cells, in addition to expressing MHC down-regulatory genes.
  • Epidermal cells may be harvested from skin sections by disaggregation of the tissue sample, and separation of subsets of interest by conventional methods as known in the art.
  • the cells to be modified will be selected to achieve a particular function when introduced into a mammalian host or used for research or other purpose. Also of interest will be the stem cells which act as the progenitors for any of the above cells, which may be the original progenitor or a progenitor cell that is already dedicated to a particular lineage.
  • the cells for modification are harvested from a suitable donor.
  • the donor will be non-autologous, e.g. allogeneic, with respect to the recipient. However, in some cases, particularly those related to autoimmune disease, the donor will be autologous.
  • the cells may be obtained from any mammalian host, including murine and other rodents, lagomorphs, porcine, feline, bovine, canine, primate, etc., particularly human.
  • the method of harvesting will depend on the nature of the cells.
  • Hematopoietic cells may be fetal, neonate or adult, and are collected from fetal liver, bone marrow, lymphoid tissue or blood, particularly peripheral blood that has been treated with progenitor cell mobilizing agents, e.g. G-CSF, GM-CSF, etc. as known in the art. Separation of hematopoietic cells into subsets of interest may be performed by a number of techniques known in the art, including elutriation, density separation, leukophoresis, flow cytometry, high or low gradient magnetic separation, and the like.
  • keratinocyte stem cells from the stratum basalis. Normal myoblasts are obtained from tissue samples, which may include fetal, neonatal or adult tissue. Diverse muscles such as limb, trunk and extra-ocular may be used. The cells may be dissociated prior to genetic modification.
  • DNA or RNA constructs providing for expression of virus-derived MHC down-regulatory genes are introduced into the cells using standard methods for introduction of nucleic acids.
  • Such recombinant constructs will comprise at least one down-regulatory gene, as previously described.
  • Combinations of interest include SEQ ID NO:1 or SEQ ID NO:2 combined with SEQ ID NO:3; SEQ ID NO:1 or SEQ ID NO:2 combined with SEQ ID NO:4; SEQ ID NO:1 or SEQ ID NO:2 combined with SEQ ID NO:5; SEQ ID NO:3 combined with SEQ ID NO:4; SEQ ID NO:3 combined with SEQ ID NO:5; and SEQ ID NO:4 combined with SEQ ID NO:5, and the like.
  • the combined sequences may be present on a single vector, or may be introduced on separate vectors introduced sequentially or together into the cell.
  • the expression of the down-regulatory gene or combination of genes will preferably decrease the level of surface MHC class I molecules by at least about 70% as compared to the unmodified cell, more usually at least about 80%, and preferably by at least about 90%.
  • the level of expression may be affected by the activation state of the cell, where quiescent cells may have reduced MHC molecules when compared to activated cells. Detection and quantitation of surface expression of MHC molecules may be performed by standard immunoassay, antibody staining and flow cytometry, etc., as known in the art. In some cases it may be possible to achieve greater reduction in HLA class I with a combination of two or more class I inhibiting genes than with a single gene.
  • allogeneic cells may be even more resistant to class l-reactive CTL and may survive longer upon transplantation into an allogeneic recipient, being more resistant to immune rejection.
  • the various class I inhibiting genes use distinct mechanisms to reduce HLA class I expression.
  • US11 from cytomegalovirus (CMV) causes dislocation of HLA class I heavy chains from the endoplasmic reticulum to the cytosol where the heavy chains are rapidly degraded (Wiertz (1996) Cell 84:769; Jones (1995) J. Virol. 69:4830).
  • HSV Herpes Simplex Virus
  • the E3/19 proteins of Adenovirus type 2 and type 5 reside in the endoplasmic reticulum and bind to the lumenal domains of class I HLA heavy chains, thereby preventing further transport (Gooding (1990) Crit. Rev. Immunol. 10:53: PSSbo (1989) Adv. Cancer Res. 52:151 : Wold (1989) Mol. Biol. Med. 6:433: Wold (1991 ) Life Sci. Adv. 10:89: Wold (1991 ) Vj[pl 184:1 ).
  • simultaneous expression of two genes may have multiplicative effect on class I reduction.
  • the constructs that will be employed will normally include a marker that allows for selection of cells into which the DNA carrying the Class I inhbiting genes has been integrated, as against cells which have not integrated the recombinant construct.
  • a marker that allows for selection of cells into which the DNA carrying the Class I inhbiting genes has been integrated, as against cells which have not integrated the recombinant construct.
  • markers exist, particularly antibiotic resistance markers, such as resistance to G418, hygromycin, and the like.
  • negative selection may be used, where the marker is the HSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir.
  • the constructs can be prepared in a variety of conventional ways.
  • the recombinant constructs may be further modified to include functional entities other than the down-regulatory sequences, which may find use in the preparation of the construct, amplification, transformation of the host cell, etc.
  • Constructs useful for insertion of down-regulatory genes include retroviral vectors, adenoviral vectors and adeno-associated viral vectors.
  • retroviral vectors combinations of retroviruses and an appropriate packaging line where the capsid proteins are functional for infecting human cells may be suitable in the present invention.
  • Various amphotropic virus-producing cell lines are known, such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902): and CRIP (Danos et al. (1988) PNAS 85:6460-6464).
  • PA12 iller et al. (1985) Mol. Cell. Biol. 5:431-437
  • PA317 iller et al. (1986) Mol. Cell. Biol. 6:2895-2902
  • CRIP Disanos et al. (1988) PNAS 85:6460-6464.
  • Retroviral vectors containing more than one down-regulatory gene or also containing another gene, such as a selectable marker may be constructed by preparing retroviruses expressing a multicistronic messenger RNA.
  • IRES elements internal ribosome entry sites
  • ECMV encephalomyocarditis virus
  • Retroviruses containing two or more down-regulatory genes are of particular use to decrease the expression of Class I molecules in cells that are refractory to transduction, such as hematopoietic stem cells and other primary lymphoid cells.
  • IRES elements may also be used to construct retroviruses expressing additional proteins such as novel homing receptors, cytokines, cytokine receptors, cell survival genes, and suicide genes.
  • adenoviral vectors methods well known in the art, such as those described in Wang et al. (1995) Gene Therapy 2:775-783, may be used.
  • More than one down-regulating gene may also be introduced into target cells by sequential transduction of target cells with retroviruses encoding a single down-regulating gene by techniques well known in the art.
  • two or more down-regulating genes may be introduced into target cells by transducing the target cells simultaneously with recombinant retroviral vectors that are packaged into viruses comprising envelope proteins that interact with different receptors on the surface of the target cells.
  • a construct encoding US11 may be packaged into a virus comprising amphotrophic envelope proteins and a construct encoding Ad2 E3/E19 may be packaged into a virus containing a Gibbon Ape Leukemia Virus envelope protein (U.S. Patent No. 5,470,726).
  • Transduction of the cells may be accomplished by the incubation of cells and virus for at least 24 hours in the culture medium. The cells are then allowed to grow in the culture medium for at least about two weeks, and may be allowed to grow for at least about five weeks or more, before transplantation.
  • those cells that show the desired phenotype may then be further analyzed by restriction analysis, electrophoresis, Southern blot analysis, polymerase chain reaction, staining with antibodies specific for the donor MHC class I ⁇ chains or for ⁇ 2-microglobulin, or the like.
  • the resulting cells will then be screened to ensure that substantially no Class I MHC antigens are expressed on the surface. If necessary, selection methods such as flow cytometry, high gradient magnetic separation, etc. may be used to enrich for cells that have low MHC class I expression.
  • markers for separation a wide variety of fluorescent or magnetically labeled molecules can be employed, which may be conjugated as labels to antibodies specific for cellular markers that identify MHC antigens.
  • Fluorescent markers that are available include fluorescein, Texas Red, phycobiliproteins, allophycocyanin, cyanine derivatives, rhodamine, and tandem conjugates for surface markers.
  • the cells may then be grown in an appropriate nutrient medium for expansion, and used in a variety of ways. The cells may be used for transplantation, to become part of an existing tissue, or may be grown to form tissue for transplantation into a non-syngeneic host.
  • the cells may be used for replacement of skin in the case of burns, where keratinocytes may be grown to form a continuous layer prior to application.
  • the keratinocytes may be used in the case of plastic surgery to replace skin removed from the host for use at another site.
  • Other uses for the keratinocytes include transplantation in decubitus ulcers.
  • islets of Langerhans they may be grown and introduced into capsules or otherwise for insertion into a host for the production of insulin.
  • retinal epithelial cells they may be injected into the subretinal space of the eye to treat visual disorders, such as macular degeneration.
  • immune cells In the case of immune cells, they may be injected into the bloodstream or elsewhere to treat immune deficiency. In the case of myoblasts, they may be injected at various sites to treat muscle wasting diseases, such as Duchenne muscular dystrophy. For organ transplants, transplants of non-syngeneic tissue such as xenogeneic grafts of heart or liver may be performed between related species.
  • adhesion molecules and antibodies to adhesion molecules as a coating for tissue culture plates during transduction serves to increase the efficiency of retroviral transduction (described in co-pending U.S. patent application no. 08/517,488).
  • the adhesion molecules may be fibronectin or the CS-1 domain of fibronectin.
  • Antibodies to VLA-4, VLA-5, CD29, CD11a, CD11b and CD44 may also be used.
  • ICAM-1 or LFA-3 adhesion molecules, or antibody to CD2 or LFA-1 may be used.
  • B cells gp39 molecules or antibody to CD40 may be used.
  • the retroviral, adenoviral or AAV vector may also serve for introduction of therapeutic proteins in the transplanted cells, where the proteins may be retained intracellularly or be secreted.
  • Production of proteins may include growth factors, e.g. G-, M-, and GM-CSF, epidermal growth factor, platelet derived growth factor, transforming growth factor, etc.; lymphokines, such as the interleukins; hormones, such as ACTH, somatomedin, insulin, angiotensin, etc., coagulation factors, such as Factor VIIIC; normal versions of the proteins associated with genetic diseases such as adenosine deaminase or the protein associated with cystic fibrosis; protective agents, such as ⁇ 1 -antitrypsin; regulatory proteins or enzymes associated with the production of amino acid free products, such as the expression of tyrosine hydroxylase for the production of L-dopamine, and the like.
  • genes may be under the transcriptional control of a constitutive promoter or inducible promoter (including enhancer sequence). In the latter situation, regulation may result by induction by a naturally occurring signal or as a result of introduction into the host of an exogenous signal.
  • genes encoding proteins for redirecting the donor cells to target diseased or dysfunctional cells, or to enhance effector function may be included on the vector or on separate vectors for introduction into the donor cells.
  • the cells may be employed as layers, introduced in containers for maintenance at a particular site, or as solid masses impregnated in inert matrices or independent of a matrix.
  • the number of cells administered will vary widely, depending upon the particular application and the manner in which the cells are administered. Administration may be by injection, topical application, or incision and placement, in the appropriate location.
  • the level of immunosuppressive regimen which is employed with the modified cells will be substantially less rigorous than would normally be used in a comparable treatment with unmodified cells.
  • the amount of immunosuppression required for maintenance of the modified cells will vary depending upon the nature of the match between the donor and recipient cells at minor histocompatibility loci, the level of activity of the host's immune system, the manner in which the cells are introduced, the particular site at which the foreign cells are introduced, and the type and number of transplanted cells.
  • the regimens which can be employed may be based on existing regimens associated with the transplantation of allogeneic tissue. Therefore the dosage level, frequency of administration, manner of administration and formulations for different situations and patients will have been established.
  • the subject invention provides for a reduction in the adverse effects of immunosuppression, where the reduction may be as a result of lower dosages, reduced frequency of administration, delaying the initiation of administration of the drug, or combinations thereof.
  • the immunosuppressive therapy can be maintained to prevent initiation of rejection by monitoring the transplant and/or immune system and regulating the administration of the immunosuppressive agent to maintain the graft at or preferably below initiation of rejection.
  • the immunosuppressive regimen may take many forms and may be combinations of forms. Immunosuppressive regimens include irradiation, chemotherapy, specific immunosuppressive agents, and the like.
  • immunosuppressive agents such as cyclosporin A, cyclosporin G and FK-506; azathioprine, corticosteroids, e.g. prednisolone and methylprednisolone, monoclonal antibodies against various surface membrane proteins of the lymphoid and/or myeloid lineage, etc.
  • the isolated virus DNA was amplified by the polymerase chain reaction (PCR) with Taq polymerase, according to the manufacturers directions. Oligonucleotide primers were designed and made to specifically amplify the US5 (SEQ ID NO:5) and US11 (SEQ ID NO:4) reading frames.
  • the primers for US5 were as follows: (SEQ ID NO:6) 5' GCC ACC ATG CAT ACA CAA CGG GCC 3'; (SEQ ID NO:7) 5' TCC TAG CCA CCG GTT GTT A 3'.
  • the primers for USH were as follows: (SEQ ID NO:8) 5" GCC ACC ATG AAC CTT GTA ATG C 3'; (SEQ ID NO:9) 5' TCA GTC TAT ATA TCA CCA CTG G 3'.
  • PCR amplified DNA products were electrophoresed on preparative agarose gels (0.8% agarose, 1 X TAE buffer). Bands at 648 and 381 bp were excised, extracted 2X with phenol chloroform, ethanol precipitated, and resuspended in 10 mM Tris, 1 mM EDTA. The isolated fragments were ligated to the PCR II cloning vector (Invitrogen, according to the manufacturers instructions).
  • Competent E. coli One ShotTM (Invitrogen, San Diego, CA) were transformed with the ligated vector. Transformants were selected by growth in the presence of ampicillin. Transformants containing the appropriate inserts were identified by PCR amplification with the appropriate primer set, followed by gel electrophoresis of the amplification product and detection of the appropriately sized band. Plasmids containing the correct DNA sequence were then purified.
  • HSV-1 Herpes Simplex Virus
  • Vero cells were infected with HSV-1 at an MOI of 0.01. Four days later virus was collected from the culture by high speed centrifugation and then frozen at -70 * C. Viral DNA was isolated from thawed virus by standard methods.
  • the ICP47 gene was amplified by PCR using primers corresponding to (SEQ ID NO:3, 1-20) and (SEQ ID NO:3, 247-267). The amplification product was ligated to the pCR II vector as described above.
  • Adenovirus Type 2 Ad2 E3/19 KD Gene.
  • Ad2 Adenovirus Type 2
  • pCRII.Ad2.E3/19 which encodes the E3/19 gene from Adenovirus 2.
  • the EcoRV C fragment comprising the E3 transcription unit of Ad2 was cloned as described in Korner ef al. (1992) P.N.A.S. 89:11857-11861.
  • the E3 gene was amplified from the plasmid by PCR, using oligonucleotides that flank the complete E3/19 kd gene.
  • the amplification product was ligated to the pCR II vector as described above.
  • Adenovirus Type 5 (Ad5) E3/19 KD gene This example describes the construction of pCR3.Ad5 E3/19, which encodes the E3/19 gene from Adenovirus 5. The gene is isolated as described in Wold ef al. (1985) J. Biol. Chem.
  • Retrovirus vectors and packaging cell lines similar to those described in Finer ef al. (1994) Blood 83:43. are used.
  • DNA fragments encoding SEQ ID NO:1 ; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; and SEQ ID NO.5 are excised from the plasmids by digestion with EcoRI restriction endonuclease, and purified by preparative gel electrophoresis. The purified DNA fragments are ligated to the retrovirus vector r/ af2 (Finer ef al., supra).
  • Recombinant plasmids containing the down-regulatory gene inserted into the retroviral vector are identified by PCR amplification using the appropriate primer set, followed by gel electrophoresis of the amplification product, and identification of a band of the appropriate size.
  • Recombinant plasmids containing the down-regulatory gene are prepared as described in Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook, E. F. Fritsch, T. Maniatis, CSHL, Cold Spring Harbor, NY, 1989.
  • T cells are isolated from a sample of peripheral blood from a suitable immunocompetent human donor.
  • the mononuclear cells are isolated by Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation (600g for 20 min. at 20 * C). After centrifugation, interphase cells are collected, resuspended in buffer and sedimented at 300 x g and then once again resuspended in buffer and centrifuged at 200 x g to remove platelets.
  • Polyclonal mouse anti-CD8 or anti-CD4 is added to the cell suspension and incubated for 15 minutes on ice.
  • the cells are washed, and FITC conjugated anti-mouse antibodies added to the cells.
  • the cells are incubated and washed, and sorted by flow cytometry for CD8 positive, and CD4 positive cells.
  • Purified T cells are cultured in vitro with interleukin-2, and antibody reactive with the T cell receptor/CD3 complex, e.g. anti-CD3 as described in Lamers et al. (1992) Int. J. Cancer 51:973-979. and Riddell and Greenberg (1990) J. Immunological Methods 128:189-201.
  • Retrovirus-mediated Gene Transfer After about 5 days of culture, retrovirus is added to the cells together with 2-8 ⁇ g/ml polybrene at an MO I of 1-10. One to three aliquots of virus are added at 1 day intervals during a period of 2-3 days. Cells are subsequently washed, and then recultured in the presence of IL-2. Within several days of transduction, T cells are analyzed for expression of the transduced gene, and expressing cells are separated from nonexpressing cells. Cells are stained for expression of cell surface class I MHC molecules using pan-specific, or donor specific anti-class I MHC monoclonal antibodies. The gene-expressing, class I deficient transduced cells are separated from the non-expressing transduced cells by fluorescence activated cell sorting. Confirmation of the presence of the transduced gene is performed by PCR amplification using primers specific for the virus-derived gene.
  • T cells prepared above are then transduced with a second down-regulatory gene, using the same method of virus infection.
  • This example provides a protocol for the transplantation into a human recipient of allogeneic hematopoietic stem cells that have been genetically modified to down-regulate the class I MHC molecules on the cell surface.
  • the donor is given GM-CSF subcutaneously at a dose of 5 ⁇ g/kg/day.
  • peripheral blood is collected.
  • Leukophoresis is performed using a 9 liter, 3 hour treatment set to collect peripheral blood mononuclear cells (see L. Campos, ef al. (1993) Leukemia 7:1409-15; A. Grigg, et al. (1993) Bone Marrow Transplant 11. Suppl. 2:23-9).
  • CD34 + cells are isolated using a CellPro LC34 affinity column (CellPro, Bothell, WA).
  • Recovered cells are plated out in Myelocult H5100 media (Stem Cell Technologies Inc., Vancouver, B.C.) containing 100 ng/ml huSCF, 50 ng/ml hulL-3, 10 ng/ml hulL-6, and 10 _6 M hydrocortisone for a period of 48 hours for "pre-stimulation".
  • the transduction method for introducing the down-regulatory into stem cells is essentially the same as described in Example 1. After transduction the cells are expanded and differentiated in vitro in Myelocut medium with addition of 100 ng/ml hu SCF, 50 ng/ml hu IL-3, 10 ng/ml hu IL-6, and 10 ⁇ M gancyclovir to inhibit 293 cell proliferation. These 293 cells will not survive under gancyclovir selection, due to their being transfected with the thymidine kinase gene. At approximately 10 days after transfection, the cells are analyzed for
  • MHC class I expression by staining with commercially available FITC conjugated polyclonal anti-human ⁇ 2-microglobulin. If necessary, the cells are sorted to select for cells having low levels of surface class I molecules.
  • the recipient is administered with the genetically modified cells infused i.v. at a rate of 10 cc/min. of 10 6 cells/ml.
  • the recipient is given a total of 10 8 cells in equal aliquots over a period of three days.
  • donor cells can be provided that are resistant to immune destruction by host cytotoxic T lymphocytes or HLA Class I specific antibodies.
  • the method of genetically modifying cells find use in transplantation for therapy, where transplanted allogeneic cells would otherwise be rejected by the recipient.
  • the methods are also useful in modifying autologous cells from a patient suffering from an autoimmune disease, where the cells would otherwise be subject to attack by the host immune system. In this way, a wide range of diseases resulting from the loss of number and/or function of cells may be treated, where the introduced cells will survive, multiply and function.
  • Animal cells may be genetically modified by the subject methods and may then be used as a source of tissues and cells for transplantation, as a model for transplantation therapies, and experimentally to test for drug efficacy.
  • This example describes the construction of retroviral vectors containing down-regulatory genes and the production of recombinant retroviruses to be used in the transduction of host cells.
  • Table 1 lists the retroviral vectors produced from the plasmids described in Example 1. These retroviruses produced encode either the down-regulatory gene alone (monocistronic retrovirus vector), or the down-regulatory gene in combination with another gene, the CD4/zeta gene, as a marker for transduction of the host cells (bicistronic retrovirus vector).
  • A. Monocistronic retroviral vectors Monocistronic vectors. Monocistronic vectors were constructed by insertion of cloned virus gene into the vector pRT43.267.
  • This retroviral plasmid contains, from 5' to 3', a cytomegalovirus (CMV) immediate early enhancer/promoter region, a moloney murine sarcoma virus (MMSV) R/U5 region, a splice donor site, part of the murine moloney leukemia virus (MMLV) gag gene, an MMLV splice acceptor, the polylinker described below, and an MMLV 3' LTR. Sequences containing the US11 and ICP47 genes from the plasmids described in Example 1 were inserted into the polylinker as described in example C and G below.
  • the retrovirus vector pRT43.267 was derived from pRT43.2F3
  • pRT43.2F3 was digested with EcoRI and Apal, and the vector fragment was isolated.
  • An oligonucleotide containing a BamHI site, a Notl site and a Sail site was ligated to the vector fragment between the EcoRI and Apal sites on the vector fragment.
  • B. Bicistronic retroviral vectors Bicistronic vectors. Bicistronic vectors were constructed by insertion of cloned virus genes into pRT43.267TNFgsig.ic. This retroviral plasmid contains, from 5' to 3', a cytomegalovirus (CMV) immediate early enhancer/promoter region, an MMSV R/U5 region, a splice donor site, part of the MMLV gag gene, an MMLV splice acceptor, the polylinker described below, an encephalomyocarditis virus (ECMV) IRES element, a CD4-zeta gene and an MMLV 3' LTR.
  • CMV cytomegalovirus
  • MMSV R/U5 region a splice donor site
  • a splice donor site part of the MMLV gag gene
  • an MMLV splice acceptor the polylinker described below
  • the retroviral vector pRT43.267gsig.ic was derived from pRT43.2F3 with the following changes: the IRES element from EMCV (residues 286-871 from Genbank locus EVCGAA, Accession no. X74312) was inserted immediately 5" to the ATG initiation codon of the CD4-zeta gene in pRT43.2F3. 102 bp of the 5' untranslated portion of the CD4-zeta gene upstream of the ATG initiation codon was deleted. A polylinker sequence containing the recognition sites for Bam HI, Not I, and Sail was inserted 5' of the IRES element. These changes in ⁇ RT43.2F3 were accomplished using techniques well known in the art.
  • retroviral vectors were co-transfected, along with the plK6.1 MCVampac plasmid (which encodes gag-pol-amphotropic env as described in WO 94/29438) into TSA201 293 cells (Heinzel et al. (1988) J Virol. 62( 10): 3738-3746). Retrovirus containing cell culture supernatant was collected after 48 hours, filtered, aliquoted and frozen. Titer was determined on NIH 3T3 cells and was approximately 1 x 10 6 -1 x 10 7 /ml.
  • Example 4 inhibits expression of multiple class I alleles in several different human cell types This example describes the transduction of a variety of different human cell types with retroviral vectors containing the HCMV US11 down-regulatory gene and the reduction in HLA Class I expression in these cells upon expression of the US11 gene.
  • Bicistronic retroviruses encoding US11 inhibit HLA expression Cells were transduced with the bicistronic (IRES containing) retrovirus described in Example 3 encoding HCMV US11 and CD4/zeta. The following cells were transduced: 143B (human osteosarcoma cells (ATCC CRL-8303);
  • two additional fresh aliquots of virus were added at one day intervals.
  • cells were transduced with retrovirus encoding CD4/zeta only.
  • cells were stained by two color immunofluorescence for HLA class I and CD4/zeta and analyzed by flow cytometry.
  • the antibody used to detect HLA class I reacts with monomorphic determinants on the HLA-A, B and C class I molecules (J. Immunol. (1982) 128:129-135; Qs ⁇ (1979) 14:9-20).
  • the percentage of transduced cells was determined by the detection of CD4 expression of the surface of the cells and their HLA class I surface staining intensity was compared to that of the untransduced cells (CD4/zeta negat ive subpopulation) to determine fold reduction of HLA Class I expression.
  • the results are expressed in Table 2 below.
  • the reduction in HLA class I expression was highest in the 143B osteoscarcoma cells and the Raji B cells and lowest in the Jurkat T cells and the SJ27 normal T cells (40 and 14 fold reduction, respectively versus 3.5 and 1.8 fold, respectively).
  • CD4/zeta expression was also strongest in those cell lines (143B and Raji) with the greatest reduction in HLA class I expression, and weakest in the cells with the lowest reduction in HLA class I expression (Jurkat and SJ27 T cells). Since US11 and CD4/zeta are expressed from a bicistronic message, the level of CD4/zeta expression was used as an indirect measure of the level of US11 expression. Therefore, the reduction in HLA I expression directly correlates with the estimated level of expression of US11. The reason for weak expression in the T cell line is not known.
  • 143B osteosarcoma cells were transduced with a retrovirus vector that encodes US11 only. After about 4 or more days of culture, cells were harvested and class I cell surface levels were determined by immunofluorescence and flow cytometry using the W6/32 class l-specific mAb that reacts with a non-polymorphic HLA class I determinant described above. As demonstrated in Fig. 1 , 17% of the cells expressed HLA class I at a level that was 30 fold less than that expressed by non-transduced 143B cells, or by 143B cells that were transduced with a single gene retrovirus vector that encodes CD4/zeta only.
  • This example demonstrates that the reduction of HLA expression of the surface of cell lines transduced with the down-regulatory US11 gene results in the resistance of these cells to the cytolytic effect of T cells specific for HLA.
  • C1 R cells are an HLA-A ne 9 ative human B lymphoblastoid cell line
  • H LA-A2.1 P° sitive C1 R transfectants were transduced with the bicistronic (IRES containing) retrovirus vector encoding both HCMV US11 and CD4/zeta.
  • Cells were stained for MHC class I and CD4 molecules, and analyzed by two-color FACS. As shown in Fig. 3, the majority of transduced cells (CD4 + ) were found to have very low MHC class I compared to untransduced (CD4-) cells.
  • the CD4 + cells were cloned, and three clones were selected for further analysis. All of the clones had very low levels of HLA class I, as measured by flow cytometry (Fig. 4).
  • clone 8 was found to have no detectable cell surface HLA-A2.1.
  • the clones were labeled with 51 Cr and used as targets in a four hour cytolytic assay using A2.1 -specific human T cell effectors.
  • lysis of the US11 -transduced C 1 R-A2.1 + clones was equal to the background level of lysis of the untransfected, parental (HLA-A2 ne 9 ative ) C1 R cells.
  • HLA-A3-specific human CTL line is derived by repeated stimulation of HLA-A3 ne 9ative peripheral blood mononuclear Academic (PBMC) with irradiated HLA-A3P° sitive allogeneic cells.
  • PBMC peripheral blood mononuclear Cell
  • the following cells are used as allogeneic stimulator cells: either PBMC (irradiated at 2000 R) (Jelachich, J. Immunol. 141 : 1108, 1988), HLA-A3P° sitive C1R transfectants (10,000 R), Jurkat (10,000R) or Raji cells (10.000R).
  • This CTL line is first tested for its ability to lyse HLA-A3P° sitive C1 R transfectants, but not parental (HLA-A3 negative) Q1 R cells.
  • HLA-A3P 0Sitive C1 R cells are then transduced with the US11/CD4/zeta bicistronic retrovirus and CD4/zeta + , HLA-A3 ,0W cells are purified and used as targets for the HLA-A3 reactive CTL.
  • 0W C1 R cells are lysed minimally or not at all by the HLA-A3 specific CTLs. This example, in combination with Example 5, demonstrates that the US11 gene can down-regulate different HLA Class I alleles.
  • CTLp nonlytic CTL precursors
  • MLC mixed lymphocyte culture
  • MLTC mixed lymphocyte- tumor culture
  • HLA-A2.1 native or HLA-A3negative human PBMC that contain CTLp are cultured either at a 1 :1 ratio with irradiated (2000R) allogeneic HLA-A2 + or HLA-A3 + lymphocytes that have been transduced with the US11 gene or at a 10:1 ratio with irradiated (10.000R) stimulator cell lines that have been transduced with the US11 gene.
  • These stimulator cells may be derived from the HLA-A2.1 + C1 R cell line, the HLA-A2 + JY cell line or from the HLA-A3 + C1 R cell line (Takahashi et al, Proc. Natl. Acad. Sci 88: 10277, 1991 ).
  • HLA-A2.1 + targets eg. HLA-A2.1 + C1 R or JY
  • HLA-A3 + targets eg. HLA-A3 + C1 R or Jurkat
  • Non-HLA specific lytic CD56 + cells may be removed from the culture prior to the measurement of HLA-A2.1 or HLA-A3-specific CTL activity, as described in Kos ef al. J. Immunol. 155:578 (1995).
  • CTL activity is measured to demonstrate that little or no HLA class l-specific CTL activity is seen in PBMC cultures that are stimulated with US1 1 -transduced, HLA dim or negative ce
  • Ad2 E3/19 and Ad5 E3/19 Block Expression of Multiple Class I Alleles
  • adenovirus down-regulatory genes Ad2 E3/19 and Ad5 E3/19, are also able to prevent the expression of HLA class I molecules on host cells when the host cells are transduced with retroviruses containing those genes.
  • 143B is a human osteosarcoma cell line that expresses high levels of HLA class I on the cell surface.
  • Cells were transduced with bicistronic (Ad E3/19; CD4/zeta) retrovirus vectors, or with a control monocistronic vector encoding CD4/zeta molecules. After 4 or more days in culture, samples were taken and the level of cell surface HLA class I determined by immunofluorescence and flow cytometry. The percentage of cells that were transduced was measured by the expression of the CD4/zeta molecules on their cell surfaces. CD4P° sitive cells were then analyzed for HLA class I expression. As shown in Table 3, the Ad5 E3/19 gene reduced HLA expresssion by 6.6 fold and the Ad2 E3/19 gene reduced HLA expression 7.5 fold in the transduced (CD4 + ) population.
  • Example 9 Blockade of HLA Expression bv Combinations of Down-re ⁇ ulatorv Genes This example illustrates the use of more than one down-regulatory gene to inhibit the expression of HLA Class I in target cells.
  • the following combinations are used: 1 ) HCMV US 11 and Ad2 E3/19 or Ad5 E3/19; 2) US11 and ICP47; and 3) ICP47 and Ad2 E3/19 or Ad5 E1/9.
  • Combinations of the recombinant retroviruses are used to transduce mammalian cells sequentially according to methods well known in the art. Alternatively, the cells are transduced simultaneously with the different retroviruses.
  • the retroviral vectors are packaged in cells expressing different viral envelope proteins that interact with different receptors.
  • the plK6.1 MCVampac packaging vector which encodes amphotrophic env (as described in W094/29438), is used to package one of the recombinant viruses and pMOV-GaLV, which encodes the Gibbon Ape Leukemia SEATO virus envelope protein (as described in U.S. Patent no. 5,470,726) is used to package the other recombinant virus.
  • Example 1 that encode the combinations of down-regulatory genes wherein the second down-regulatory gene is substituted for the CD4-zeta gene.
  • bicistronic vectors are used to transduce the target cells as described in the preceding examples.
  • Example 10 Production of Class l-deficient Human Hematopoietic Stem Cells and T cells This example illustrates the production of human hematopoietic stem cells and T cells that have been transduced with vectors containing one or more down regulatory genes. To ensure efficient transduction of these cells and the loss of HLA Class I expression, the cells are plated on adhesion molecules or on antibodies to adhesion molecules during the transduction process.
  • the ability to maintain both self-renewing and differentiating populations of cells derived from stem cells depends upon cell - cell contact of stem cells and stromal cells in the bone marrow (Gordon and Greaves, Bone Marrow Transplantation. 4:335-338 (1989)).
  • the contact of stromal cells and hematopoietic stem cells involves many molecules, including growth factors, exemplified by the kit ligand on stromal cells and c-kit receptor found on stem cells (Zsebo el a Cell. £_:213-224 (1990)) and adhesion molecules such as fibronectin on stromal cells and VLA-4 on hematopoietic stem cells (Williams et al..
  • These contact molecules are either transmembrane or, if located extracellularly, they are proteins which contact transmembrane proteins and enable signals for either self-renewal or differentiation to be transmitted between the stromal cells and the stem cells.
  • these contact molecules are either transmembrane or, if located extracellularly, they are proteins which contact transmembrane proteins and enable signals for either self-renewal or differentiation to be transmitted between the stromal cells and the stem cells.
  • reconstruction of the cell-cell contacts as described in co-pending U.S. application no. 08/517,488, Finer ef al., may be used.
  • Fibronectin, the CS-1 domain of fbronectin or antibody to VLA-4 is added to the culture dishes as described below.
  • C D34 + cells are isolated from the peripheral blood of patients undergoing cyclophosphamide and G-CSF treatment.
  • Mononuclear cells are isolated from leukophoresed blood by fractionation using a standard Ficoll gradient (Pharmacia, Piscataway, NJ).
  • the CD34 + cells are isolated using positive selection on a CellPro CEPRATE LC affinity column (CellPro, Bothell, WA).
  • This population of cells is then cultured for a period of 48-72 hours at a density of 0.5 - 1 X 10 6 cells/ml in "prestimulation medium” which contains Myeloid Long Term Culture Medium supplied as a complete medium from Terry Fox Labs, (Vancouver, Canada) with the addition of 100 ng/ml human Stem Cell Factor (SCF), 50 ng/ml human IL-3, and 10 ng/ml human IL-6 (Genzyme, Cambridge, MA).
  • SCF Stem Cell Factor
  • IL-6 Genzyme, Cambridge, MA
  • 293 cells are transfected by first plating at a density of 1.4 X10 6 cells/10cm dish 24 hours prior to transfection, followed by co-transfection with the retroviral vectors described in the previous examples and 7.5 ug of the packaging plasmid plK6.1MCVampac. Eighteen hours later, transfection media is removed and replaced with 10 ml IMDM (JRH Biosciences, Woodland CA) + 10% FBS. Viral supernatant is then collected 24-36 hours later and 100ng/ml human SCF, 50 ng/ml human IL-3, 10 ng/ml human IL-6, and 8 ug/ml polybrene are added.
  • Fibronectin and CS-1 coated plates are made by adding 30ug/ml PBS of fibronectin, derived from human plasma, or CS-1 (Sigma, St Louis, MO) to tissue culture plates. The plates are then incubated at 37 * overnight and washed with PBS ("24 hour method").
  • the plate is placed under UV light for 1 hour with the lid off and then an additional hour with the lid on, the PBS is removed, one ml of 2% BSA is added for 20 minutes, and the plates are washed with DPBS/0.2% HEPES ("2 hour method")(Williams et al. suora).
  • Cell-cell contact plays an important role for the activation and growth of many cells of the hemapoietic lineage. For example, many cell-cell contacts have been identified that are essential for T cell activation (Bolhuis ef al., Cancer Immunol. Immunother. 34:1-8 (1991 )) including the interactions of receptor/co-receptor pairs on T lymphocytes and antigen presenting cells such as LFA-1 and ICAM-1 , and CD-2 and LFA-3. In B lymphocytes, the CD40 / gp39 interaction takes place between B lymphocytes and T lymphocytes and is necessary for B lymphocyte activation (Armitage ef al., Sem. Immunol.. 6:267-278 (1994)).
  • CD2 Antibodies to CD2 (Springer ef al.. Nature 323:262 (1987)) or CD40 can substitute for the ligands and mediate cell-cell interaction and activation.
  • the transduction of T and B lymphocytes by supernatant infection has been reported to be of low efficiency (Hwu ef al., J. Immunol. 9:4104-4115 (1993); Baker ef al.. Nucleic Acids Res. 20:5234 (1992)).
  • antibodies to the receptor present on the target cells i.e., anti-CD2 or LFA1 antibody for T lymphocytes and anti-CD40 antibody for B lymphocytes
  • anti-CD2 or LFA1 antibody for T lymphocytes and anti-CD40 antibody for B lymphocytes which have been shown to activate their respective cell types, can also be used to enhance the supernatant transduction efficiency of these cells.
  • PBMCs Peripheral blood mononucleocytes
  • D-PBSE/CMF PBS containing 1 mM EDTA, Ca and Mg free
  • resuspended at 5x10 7 cells in 4 ml of D-PBSE/CMF containing 0.5% of human gamma globulins and incubated at room temperature for at least 15 minutes.
  • CD8 + T cells are purified from the PBMC cell suspension by positive panning.
  • the PBMC suspension is loaded into a pre-washed T-25 tissue culture flask coated with an antibody specific for the human CD8 protein (AIS CD8 selection flask (Applied Immune Sciences, Santa Clara, CA)) at a density of 5x10 7 cells per 4 ml per T-25 flask.
  • Cells are incubated for one hour at room temperature, and the non-adherent cells removed by gentle pipetting and washing the flask three times with the D-PBSE/CMF.
  • the CD8 + T cells are simultaneously released from the flask and activated by adding 10 ml of T cell medium containing 10 ng/ml OKT3 (Ortho Pharmaceuticals, Raritan, NJ) and 10% IL2 (Pharmacia).
  • the physical separation is accomplished using complement mediated lysis, fluorescence-activated cell sorting, magnetic beads, affinity chromatography, or the like.
  • Cells are then incubated with T cell media for 48 hours, harvested from the flask, and washed once with T cell medium, and finally resuspended in fresh T cell medium (10% FCS, Hyclone; RPMI1640, CellGro; 10mM Hepes buffer (Gibco); 1% Sodium pyruvate (Gibco); 1 % non-essential amino acids (Gibco); 2mM glutamine (Gibco); 25 ⁇ M 2-mercaptoethanol (Sigma) and 1 % streptomycin/penicillin) plus 10% IL2 at a density of 0.5-1.0x10 6 /ml in 24 well plates.
  • 293 cells are plated at 1x10 6 cells/6 well plate, and then transfected with the appropriate construct after 48 hours. 24 hours post transfection, the transfection media is removed and replaced with T cell growth media.
  • Antibody plates are prepared as described above. 0.5x10 6 purified and activated human CD8+ T cells prepared as described above (usually at day 4 or 5 post-purification/activation) are plated on the antibody coated plates and incubated with 1 ml of fresh T cell medium (plus 10% IL2 and 2 ⁇ g/ml polybrene) together with 1 ml of viral supernatant obtained from the 293 transient transfection system described above. After an 8 hour incubation period, 1.5 ml of medium is removed from each well, and replaced with 0.5 ml of fresh T cell medium together with 1.0 ml of viral supernatant (polybrene at 2 ⁇ g/ml and IL2 at 10%). After a 12 hour incubation period, the two step supernatant procedure is repeated.
  • the transduced CD8 + T cell population is subsequently maintained in T cell medium.
  • T cells are periodically re-stimulated every 7 to 10 days by the addition of OKT3 at 10ng/ml or by exposing the cells to immobilized OKT3 in a T-25 tissue culture flask at a density of 1-2x10 7 CD8+ T cells/10 ml T cell medium plus 10% IL2. Cells are incubated for 48 hours, washed 1x with T cell medium, and resuspended in fresh medium plus 10% IL2 at 0.5-1.0 x 10 6 /ml.
  • HLA class I deficient T cell and stem cell subpopulations prepared as described above are optionally further enriched by immunoaffinity techniques, e.g. HLA class I specific antibody plus complement mediated lysis, fluorescence-activated cell sorting, adherence to a plastic substratum, magnetic beads, affinity chromatography, or the like.
  • immunoaffinity techniques e.g. HLA class I specific antibody plus complement mediated lysis, fluorescence-activated cell sorting, adherence to a plastic substratum, magnetic beads, affinity chromatography, or the like.
  • Pan-specific W6/32 anti-HLA Class I antibody and/or allele-specific BB7.2 anti-HLA-A2.1 class I antibody are used (ATCC HB-82; Hum. Immunol. 3: 277-299, 1981 ).
  • HLA Class I deficient human T cells This example describes a method to allow the survival of HLA Class I deficient human T cells after transplantation.
  • a potential obstacle to the use of HLA class I deficient human T cells is that lymphoid cells that are deficient in cell surface class I HLA have been found to be susceptible to lysis by natural killer (NK) cells .
  • NK natural killer
  • human NK cells will recognize and lyse class l-disparate allogeneic lymphoblasts in vitro.
  • HLA class I deficient human or murine T cells are incubated for prolonged periods in vitro (e.g. 5 days) prior to transplantation, to reduce their susceptibility to NK recognition and lysis.
  • Normal human T cells were isolated as described above and stimulated with the polyclonal activator PHA and were kept in culture for two or five days. The resultant T lymphoblasts were then labeled with 51 Cr and tested as targets for lysis by freshly isolated allogeneic NK cells. It was observed that 5 day lymphoblasts were resistant to NK lysis, whereas 2 day lymphoblasts were susceptible to lysis.
  • HLA class l-deficient human lymphocytes As a model for HLA class l-deficient human lymphocytes, the susceptibility of class l-deficient murine T lymphoyctes (derived from ⁇ 2-microglobulin knockout mice as described in W093/16177) to NK lysis was investigated. Surprisingly, it was found that class l-deficient mouse T lymphocytes were highly resistant to NK lysis if the T lymphocytes were maintained in culture for 5 days or more. In contrast, 2 day lymphoblasts were susceptible, as expected from the prior art. On the other hand, HLA class I deficient LPS-activated B lymphoblasts remained completely susceptible to NK lysis after 5 days of culture.
  • ADDRESSEE FLEHR, HOHBACH, TEST, ALBRITTON & HERBERT
  • GCGCAGTATA CGCTGATGAT GGTGGCAGTG ATTCAAGTGT TTTGGGGGCT GTATGTGAAA 600

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  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Developmental Biology & Embryology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Transplantation (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Materials For Medical Uses (AREA)

Abstract

Des cellules de mammifères sont modifiées génétiquement par introduction de gènes codant des protéines à régulation négative du complexe majeur d'histocompatibilité (CMH) issues de virus. Les cellules appropriées pour une transplantation sont prélevées sur un donneur puis transfectées par un vecteur d'expression codant une ou plusieurs protéines à régulation négative du CMH issues de virus. Les cellules du donneur génétiquement modifiées sont développées ex vivo, lorsque cela se révêle nécessaire, puis transplantées sur le récepteur. Les taux plus faibles de protéines de classe I du CMH permettent aux cellules transplantées de survivre dans des conditions où des cellules non modifiées génétiquement feraient l'objet d'une offensive du système immunitaire du récepteur. Les cellules du donneur peuvent en outre être modifiées pour cibler des cellules ou pour renforcer la fonction effectrice des cellules du donneur chez le récepteur.
PCT/US1996/004648 1995-04-04 1996-04-03 Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface WO1996031241A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA002217297A CA2217297A1 (fr) 1995-04-04 1996-04-03 Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface
AU55338/96A AU712415B2 (en) 1995-04-04 1996-04-03 Transplantation of genetically modified cells having low levels of class I MHC proteins on the cell surface
EP96912564A EP0820311A4 (fr) 1995-04-04 1996-04-03 Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface
JP8530474A JPH11503024A (ja) 1995-04-04 1996-04-03 細胞表面のクラスｉ ｍｈｃタンパク質の低レベルを有する遺伝子学的に修飾された細胞の移植
NO974596A NO974596L (no) 1995-04-04 1997-10-03 Transplantasjon av genetisk modifiserte celler som har lave nivåer av klasse I MCH proteiner på celleoverflaten

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41610695A 1995-04-04 1995-04-04
US08/416,106 1995-04-04

Publications (1)

Publication Number Publication Date
WO1996031241A1 true WO1996031241A1 (fr) 1996-10-10

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PCT/US1996/004648 WO1996031241A1 (fr) 1995-04-04 1996-04-03 Transplantation de cellules genetiquement modifiees presentant de faibles taux de proteines de classe i du complexe majeur d'histocompatibilite sur leur surface

Country Status (7)

Country Link
EP (1) EP0820311A4 (fr)
JP (1) JPH11503024A (fr)
KR (1) KR19980703665A (fr)
AU (1) AU712415B2 (fr)
CA (1) CA2217297A1 (fr)
NO (1) NO974596L (fr)
WO (1) WO1996031241A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998047914A2 (fr) * 1997-04-18 1998-10-29 Roche Diagnostics Gmbh Gene us6 issu du cytomegalovirus humain
WO1999036562A1 (fr) * 1998-01-14 1999-07-22 Human Gene Therapy Research Institute Systemes d'expression nucleotidiques a immunogenicite reduite destines a la therapie genique
EP1020520A1 (fr) * 1999-01-15 2000-07-19 Introgene B.V. Prévention d'élimination immunologiques des cellules du corps mammifère, molécules PML mutantes utiles à cet effet
EP2598166A1 (fr) * 2010-07-26 2013-06-05 Searete LLC Cellules dépourvues de cmh
WO2018193394A1 (fr) * 2017-04-19 2018-10-25 Allogene Therapeutics, Inc. Compositions de lymphocytes t améliorés et procédés
WO2019167973A1 (fr) * 2018-03-01 2019-09-06 Kyoto University Inhibiteur d'évolution de cycle cellulaire
US10428118B2 (en) 2014-07-16 2019-10-01 Oregon Health & Science University Human cytomegalovirus comprising exogenous antigens
US10532099B2 (en) 2016-10-18 2020-01-14 Oregon Health & Science University Cytomegalovirus vectors eliciting T cells restricted by major histocompatibility complex E molecules
US10688164B2 (en) 2015-11-20 2020-06-23 Oregon Health & Science University CMV vectors comprising microRNA recognition elements
US10760097B2 (en) 2011-06-10 2020-09-01 Oregon Health & Science University CMV glycoproteins and recombinant vectors
US11091779B2 (en) 2015-02-10 2021-08-17 Oregon Health & Science University Methods and compositions useful in generating non canonical CD8+ T cell responses
US11266732B2 (en) 2010-05-14 2022-03-08 Oregon Health & Science University Recombinant HCMV and RHCMV vectors and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994016065A1 (fr) * 1992-12-31 1994-07-21 Exemplar Corporation Production de cellules destinees a la transplantation et reduisant le rejet du greffon par l'hote, et cellules ainsi obtenues
US5359046A (en) * 1990-12-14 1994-10-25 Cell Genesys, Inc. Chimeric chains for receptor-associated signal transduction pathways

Patent Citations (2)

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US5359046A (en) * 1990-12-14 1994-10-25 Cell Genesys, Inc. Chimeric chains for receptor-associated signal transduction pathways
WO1994016065A1 (fr) * 1992-12-31 1994-07-21 Exemplar Corporation Production de cellules destinees a la transplantation et reduisant le rejet du greffon par l'hote, et cellules ainsi obtenues

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CELL, 20 May 1994, Vol. 77, YORK et al., "A Cytosolic Herpes Simplex Virus Protein Inhibits Antigen Presentation to CD8+ T Lymphocytes", pages 525-535. *
JOURNAL OF IMMUNOLOGY, 01 April 1990, Vol. 144, No. 7, ROUTES et al., "Resistance of Human Cells to the Adenovirus E3 Effect on Class I MHC Antigen Expression", pages 2763-2770. *
See also references of EP0820311A4 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998047914A2 (fr) * 1997-04-18 1998-10-29 Roche Diagnostics Gmbh Gene us6 issu du cytomegalovirus humain
WO1998047914A3 (fr) * 1997-04-18 1999-03-11 Boehringer Mannheim Gmbh Gene us6 issu du cytomegalovirus humain
WO1999036562A1 (fr) * 1998-01-14 1999-07-22 Human Gene Therapy Research Institute Systemes d'expression nucleotidiques a immunogenicite reduite destines a la therapie genique
EP1020520A1 (fr) * 1999-01-15 2000-07-19 Introgene B.V. Prévention d'élimination immunologiques des cellules du corps mammifère, molécules PML mutantes utiles à cet effet
US11266732B2 (en) 2010-05-14 2022-03-08 Oregon Health & Science University Recombinant HCMV and RHCMV vectors and uses thereof
EP2598166A1 (fr) * 2010-07-26 2013-06-05 Searete LLC Cellules dépourvues de cmh
EP2598166A4 (fr) * 2010-07-26 2014-11-05 Searete Llc Cellules dépourvues de cmh
US10760097B2 (en) 2011-06-10 2020-09-01 Oregon Health & Science University CMV glycoproteins and recombinant vectors
US10428118B2 (en) 2014-07-16 2019-10-01 Oregon Health & Science University Human cytomegalovirus comprising exogenous antigens
US10995121B2 (en) 2014-07-16 2021-05-04 Oregon Health & Science University Human cytomegalovirus comprising exogenous antigens
US11692012B2 (en) 2014-07-16 2023-07-04 Oregon Health & Science University Human cytomegalovirus comprising exogenous antigens
US11091779B2 (en) 2015-02-10 2021-08-17 Oregon Health & Science University Methods and compositions useful in generating non canonical CD8+ T cell responses
US10688164B2 (en) 2015-11-20 2020-06-23 Oregon Health & Science University CMV vectors comprising microRNA recognition elements
US10532099B2 (en) 2016-10-18 2020-01-14 Oregon Health & Science University Cytomegalovirus vectors eliciting T cells restricted by major histocompatibility complex E molecules
US11305015B2 (en) 2016-10-18 2022-04-19 Oregon Health & Science University Cytomegalovirus vectors eliciting T cells restricted by major histocompatibility complex E molecules
WO2018193394A1 (fr) * 2017-04-19 2018-10-25 Allogene Therapeutics, Inc. Compositions de lymphocytes t améliorés et procédés
AU2018255926B2 (en) * 2017-04-19 2023-10-12 Allogene Therapeutics, Inc. Improved T cell compositions and methods
AU2018255926B9 (en) * 2017-04-19 2023-10-19 Allogene Therapeutics, Inc. Improved T cell compositions and methods
WO2019167973A1 (fr) * 2018-03-01 2019-09-06 Kyoto University Inhibiteur d'évolution de cycle cellulaire

Also Published As

Publication number Publication date
NO974596D0 (no) 1997-10-03
AU712415B2 (en) 1999-11-04
NO974596L (no) 1997-12-03
EP0820311A4 (fr) 2002-09-25
KR19980703665A (ko) 1998-12-05
AU5533896A (en) 1996-10-23
JPH11503024A (ja) 1999-03-23
EP0820311A1 (fr) 1998-01-28
CA2217297A1 (fr) 1996-10-10

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