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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • 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
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
  • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
• • 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
• • 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
  • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
  • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• This invention relates to viral vectors and methods for their use, especially for example for transducing cells, e.g. malignant ceils of haematopoietic lineage, and for inducing expression of foreign genetic material in such cells.
• the invention also relates to pharmaceutical compositions based on such viral vectors, to production of cells infected with such viral vectors, to pharmaceutical preparations based on such cells, to their use for administration to humans and non-human animals in order to express foreign genetic material in vivo, and to the use of materials as described herein for the manufacture of preparations for treatment and other applications as mentioned herein. Methods according to the invention can be used for example in cancer immunotherapy.
• Recombinant viral vectors are among several known agents available for the introduction of foreign genes into cells so that they can be expressed as protein.
• a central element is the target gene itself under the control of a suitable promoter sequence that can function in the cell to be transduced.
• Known techniques include non-viral methods, such as simple addition of the target gene construct as free ONA; incubation with complexes of target DNA and specific proteins designed for uptake of the DNA into the target celt; and incubation with target DNA encapsulated for example in liposomes or other lipid-based transfection agents.
• a further option is the use of recombinant virus vectors engineered to contain the required target gene, and able to infect the target ceils and hence earn Into the cell the target gene in a form that can be expressed.
• viruses has been used for this purpose including retroviruses, adenoviruses, and adeno-associated viruses.
• Breakefield describes he ⁇ es simplex virus type 1 expression vectors capable of infecting and being propagated in a ⁇ on-mitotic cell, and for use in treatment of neurological diseases, and to produce animal and in vitro models of such diseases.
• Recombinant viruses are known in particular for use in ⁇ e.g. corrective) gene therapy applied to gene deficiency conditions.
• genes used or proposed to be used in corrective gene therapy include: the gene for human adenosine deaminase (ADA), as mentioned in for example WO 92/10564 (KW Culver et al: US Secretary far Commerce & Cellco
• tumour cells may express one or more proteins or other biological macromolecules that are distinct from normal healthy cells, and which might therefore be used to target an immune response to recognise and destroy the tumour cells.
• tumour targets may be present ubiquitously in tumours of a certain type.
• cervical cancer where the great majority of tumours express the human papiilomavirus E6 and E7 proteins.
• the tumour target is not a self protein, and hence its potential as a unique tumour- specific marker for cancer im unotherapv is clear.
• tumour target antigens There is increasing evidence that certain self proteins can also be used as tumour target antigens. This is based on the observation that they are expressed consistently in tumour cells, but not in normal healthy cells. Examples of these include the MAGE family of proteins. It is expected that more self proteins useful as tumour targets remain to be identified.
• Tumour associated antigens and their role in the immunobiology of certain cancers are discussed for example by P van der Bruggen et al, in Current Opinion in Immunology, 4(5) (1992) 608-612.
• Other such antigens, of the MAGE series are identified in T. Boon, Adv Cancer Res 58 (1992) pp 177-210, and MZ2-E and other related tumour antigens are identified in P. van der Bruggen et al, Science 254 (1991 ) 1643-1647; tumour-associated mucins are mentioned in PO Livingston, in current Opinion in Immunology 4 (5) (1992) pp 624-629; e.g. MUC1 as mentioned in J Burchell et al, Int J Cancer 44 (1989) pp 691 -696.
• tumour-specific markers Although some potentially useful tumour-specific markers have thus been identified and characterised, the search for new and perhaps more specific markers is laborious and time-consuming.
• WO 86/07610 discloses expression of human IL-2 in mammalian ceils by means of a recombinant poxvirus comprising all or part of a DNA sequence coding for a human IL-2 protein.
• Specification EP 0 259 212 discloses viral vectors of the pox, adeno or h ⁇ s types, for controlling tumours, containing a heterologous DNA sequence coding for at least the essential regions of a tumour-specific protein.
• Specification WO 88/00971 discloses recombinant vaccine comprising a pox.
• herpes or adeno virus vaccine vector especially vaccinia, including a nucleotide sequence expressing at least part of an antigenic polypeptide and a second sequence expressing at least part of a lymphokine (interleukin 1 , 2, 3 or 4, or gamma interferon) which increases immune response to the antigenic polypeptide; and specification WO 94/16716 (E Paoletti et al: Virog ⁇ netics Corp.) describes attenuated recombinant vaccinia viruses containing DNA coding for a cytokine or a tumour antigen, e.g. for use in cancer therapy.
• a lymphokine interleukin 1 , 2, 3 or 4, or gamma interferon
• Herpesvirus saimiri a virus of non-human primates, have been described as leading to gene expression in human lymphoid cells (B ⁇ ckenstein & R Grassmann, Gene 102(2) (1991), pp 265-9). However, it has been considered undesirable to use such vectors in a clinical setting.
• tumour celts of haematopoietic lineage such as leukaemias
• to do this efficiently e.g. for pu ⁇ oses of corrective gene therapy or cancer immunotherapy.
• HSCs haematopoietic stem cells
• primary cells from human haematopoietic malignancies as targets for gene transfer and expression.
• culture of target cells e.g. HSCs.
• haematopoietic growth factors such as cytokines
• haematopoietic growth factors such as cytokines
• cytokines has been tried, with a view to induce the HSCs into cycle and increase the efficiency of retrovirus-mediated gene transfer to these target cells, but unfortunately the cytokines in the culture media appear to have induced differentatio ⁇ with loss of the desired self-renewal capacity of the cells.
• adeno-associated viral vectors have been proposed for use instead of retroviral vectors, but it has appeared that the efficiency of integration of such vectors is low.
• adenoviral vectors have limitations. Thus, while they can infect approximately 50% of haematopoietic cells under certain conditions, nevertheless gene expression is often delayed for several days. It has also been found in certain tests that transduction of a heterologous gene into acute leukaemia cells by a recombinant adenovirus vector or a retrovirus vector led to either negligible or at best about 3% transduction yield, and that thus there can be a problem of efficiency of transduction yield with such vectors.
• the present inventors consider that the prior art leaves it still desirable to provide further viral vectors and processes for their use in transforming human and animal ceils.
• target cells for transduction by herpesviral vectors can be chosen for example from among cells of haematopoietic lineages; from lymphoid or myeloid cells, from stem cells or
• CD34+ cells e.g. cell preparations containing such cells, as for example obtained or prepared in connection with bone-marrow transplantation; or cells of neuro ⁇ ctodermal origin, especially malignant such cells, and transduced with viral vectors as described herein.
• cell preparations containing such cells as for example obtained or prepared in connection with bone-marrow transplantation
• cells of neuro ⁇ ctodermal origin especially malignant such cells, and transduced with viral vectors as described herein.
• the present invention aims to provide materials and methods to facilitate the use of tumour cells as immunogens and vaccines.
• the invention alms to facilitate the transduction of cells of haematopoietic lineage and provide useful compositions and procedures based thereon.
• the present invention al ⁇ o aims to provide means for creating immunogens and therapeutic vaccines that can be used to induce immune responses against tumour-specific antigens, e.g. in patients with pre-existing tumours.
• the invention is particularly applicable for example for gene transfer into haematopoietic cells such as lymphoid cells, that are nonpermissive for expression of late lytic genes of herpesvirus such as herpes simplex virus.
• a process of treating a human or non-human animal cell to introduce heterologous genetic material e.g. material comprising a heterologous gene, into said cell, e.g. to express said genetic material in said cell, comprising the steps of (a) providing a recombinant herpesviral vector which is an attenuated or replication-defective and non-transforming mutant herpesvirus, and which carries heterologous genetic material, e.g.
• a gene encoding a heterologous protein and (b) transducing human or non-human animal cells selected from: haematopoietic cells, malignant cells related to blood cells, and malignant or non-malignant CD34 + cells; by contacting said cells with said virus vector to transduce said cells.
• said genetic material is then expressed in said cell. Transduction takes place by infection of the live target cell by the viral vector in per-se known manner.
• Such a process can for example comprise treating a human or non-human animal cell to introduce heterologous genetic material into said cell to render said cell more highly immunogenic, comprising the steps of (a) providing a recombinant herpesviral vector which is an attenuated or replication-defective and non-transforming mutant herpesvirus. and which carries e.g.
• a gene encoding a heterologous immunomodulatory protein selected from cytokines and immunological co-stimulatory molecules and chemo-attractants and (b) transducing malignant or non-malignant human or non-human animal cells, which can be selected for example from: malignant celts related to blood ceils, haematopoietic cells, malignant or non-malignant CD34-+ cells, by contacting said cells with said virus vector to transduce said cells and render said cells more highly immunogenic.
• compositions provided and used according to certain embodiments of the invention for use in transducing human or non-human animal ceils selected from: haematopoietic cells; malignant cells related to blood ceils; and malignant or non-malignant CD34+ cells; can comprise a recombinant herpesviral vector which is an attenuated or replication-defective and non- transforming mutant herpesvirus, and which carries heterologous genetic material, e.g. a gene encoding a heterologous protein.
• compositions provided and used according to certain embodiments of the invention can comprise human or non-human animal cells selected from: haematopoietic cells; malignant cells related to blood cells; and malignant or non-malignant CD34+ cells; said cells having been infected with a recombinant herpesviral vector which is an attenuated or replication-defective and non-transforming mutant herpesvirus, and which carries e.g. a gene encoding a heterologous protein.
• Also within the invention is a proces of treating a subject hich is a human subject or a non-human animal subject in order to achieve expression of a foreign gene in vivo, comprising administering to said subject a pharmaceutical composition of the kinds mentioned above and described herein; and a process of treating a subject which Is a human subject or a non-human animal subject in order to elicit an immune response, which comprises administering to said subject a pharmaceutical composition of the kinds mentioned above and described herein.
• An aspect of the invention concerns provision and use of a recombinant herpesvirus vector, e.g. based on a non-transforming herpesvirus, carrying a gene encoding a protein, e.g. an immunom ⁇ dulator ⁇ protein, or a protein useful for expression in connection with gene therapy: also provided by the invention is its use in transducing cells to render them more highly immunogenic: among the cells that can usefully be treated in this way are for example malignant cells of human and non-human animals, especially for example malignant cells related to blood cells, e.g. leukaemic ceils, or haematopoietic cells, including CD34 + cells. ⁇ whether malignant or non-malignant.
• suitable cells for treatment include for example haematopoietic progenitor cells such as healthy CD34+ cells, which when transduced with herpesvirus vectors carrying a heterologous gene that it is desired to express in the treated cell, can carry a high copy number of the heterologous gene, enabling homologous recombination with the genome of the treated cell without the need for an integrase.
• haematopoietic progenitor cells such as healthy CD34+ cells, which when transduced with herpesvirus vectors carrying a heterologous gene that it is desired to express in the treated cell, can carry a high copy number of the heterologous gene, enabling homologous recombination with the genome of the treated cell without the need for an integrase.
• GM-CSF GM-CSF
• interleukin 2 production by tumour cells bypasses T helper function in the generation of an antitumour response (ER Fearon et al, Cell 60 (1990) pp 397 et seq), and it has been reported in the case of murine GM-CSF (G Dranoff et al. Proc Nat Acad Sci USA
• a recombinant herpesvirus for example a recombinant HSV
• a recombinant HSV can be used as a vector for transduction of (for example) leukaemia cells so as to produce expression of inserted genetic material, e.g. a gene encoding an immunomodulatory protein or other protein relevant to cancer immunotherapy or gene therapy, in such cells.
• a recombinant h ⁇ es simplex virus whether HSV1 or HSV2, engineered to contain a heterologous gene as part of its genome, can be used to deliver the gene with good efficiency to leukaemia cells, to evoke effective expression of the heterologous gene within the tumour cells, and the transduced cells can then be used for example as a cellular immunogen such as a vaccine for cancer immunotherapy, and thereby, among other effects, mediate immune effects on tumour cells other than cells infected with the virus vector.
• the invention also provides useful methods for gene transduction of leukaemia calls among others.
• methods of using a recombinant herpesvirus such as HSV, e.g.
• a replication- defective h ⁇ svirus such as replication-defective HSV, whether HSV1 or HSV2.
• a replication- defective h ⁇ svirus such as replication-defective HSV, whether HSV1 or HSV2.
• HSV replication-defective HSV
• HSV1 or HSV2 for transduction of various cell types based on cells of haematopoietic lineage, and other cell types, e.g. ⁇ euroblastomas, e.g. to introduce immunomodulatory genes, or other genes for the purpose of gene therapy or cancer immunotherapy, into such cells.
• tumour cells which can be cells that (prior to transduction) either have not been incubated at all under cell culture conditions, or else have not been incubated for more than a few hours (e.g. not more than about 2 or up to 4 hours, or not incubated as long as ovemight), e.g. freshly- sampled tumour cells, can be exposed to a recombinant herpesvirus vector as mentioned herein carrying suitable genetic material.
• This can be genetic material that is not being expressed, or is not being substantially expressed, by the tumour cells, e.g.
• the genetic material encoding an immunomodulatory protein such as for example GM-CSF, thereby to infect the cells with the recombinant h ⁇ svirus vector; and the resulting infected cells can be used for example either for reinfusio ⁇ into the subject from whom the parent cells were obtained, or for reaction with leukocytes in vitro.
• an immunomodulatory protein such as for example GM-CSF
• freshly sampled human leukaemia ceils can be exposed to a virus vector carrying a gene encoding human GM-CSF or inter alia one of the other immunomodulatory proteins mentioned herein, and reinfused into the patient as an immunogenic cell preparation, e.g. using some or ail of the procedural steps mentioned below, with a useful extent of transduction of the cells.
• a virus vector carrying a gene encoding human GM-CSF or inter alia one of the other immunomodulatory proteins mentioned herein reinfused into the patient as an immunogenic cell preparation, e.g. using some or ail of the procedural steps mentioned below, with a useful extent of transduction of the cells.
• a corresponding retrovirus vector it has proved necessary to culture the tumour cells in vitro for some days before they could be transduced usefully; e.g. in order to drive cells into cell division and render them susceptible to retroviral transduction.
• Cytotoxic T-cells can be activated and/or expanded, e.g. in vitro, e.g. for purposes of cancer immunotherapy, by the use of viral ly-tran ⁇ duced presenting or target cells, e.g. especially target cells of haematopoietic lineage, CD34 + cells, where the virus used for transduction is a vector as described herein carrying a gene encoding an antigen relevant to the desired therapy, e.g. an antigen encoded by EBV or HPV, and in addition, if desired, encoding an immunomodulatory protein as mentioned herein.
• an antigen relevant to the desired therapy e.g. an antigen encoded by EBV or HPV
• donor T-lymphocytes can be activated and expanded in relation to target cells, e.g. of types as mentioned above, expressing EBV or HPV antigens as a result of transduction by viral vectors as described herein carrying corresponding heterologous genes, e.g. HPV E6 or E7 genes.
• Recombinant herpesvirus as mentioned herein can also transduce other tumour cell types, such as neuroblastoma cells, with good efficiency.
• the recombinant herpesvirus used as a vector according to this invention can contain a gene encoding an immunomodulatory protein, or other protein relevant to cancer immunotherapy or gene therapy.
• Genes encoding any of several immunomodulatory proteins can be used in this way to render tumour cells immunogenic, in humans and non-human animals.
• the resulting immune responses can be used in prevention and treatment of tumour growth.
• Immunomodulators are molecules that can enhance or repress immunological responses. They include cytokines (soluble glycoproteins which initiate or enhance activation, growth and differentiation of immune system cells), co-stimulatory molecules (structures present on the surface of cells within the body that interact with immune cells to help stimulate immune responses) and (immunological) chemo-attracta ⁇ t molecules which serve to attract immune cells to sites of immune or inflammatory activity, e.g. at which antigens can be presented.
• cytokines soluble glycoproteins which initiate or enhance activation, growth and differentiation of immune system cells
• co-stimulatory molecules structures present on the surface of cells within the body that interact with immune cells to help stimulate immune responses
• chemo-attracta ⁇ t molecules which serve to attract immune cells to sites of immune or inflammatory activity, e.g. at which antigens can be presented.
• Immunomodulating or “immunomodulatory” protein includes one or more proteins which can enhance a host's immune response, e.g. to a mutant vims, or to an antigen such as an immunogen from a pathogen or source exogenous to the virus, or to a tumour associated antigen, which can for example be produced by the mutant virus.
• the immunomodulating proteins are not those presently used as immunogens in themselves.
• the immunodulating proteins for which encoding nucleotide sequences are expressibly carried by viruses as described herein can for example usefully have sequences native to the species which is to receive vaccination by the recombinant viruses, or which is otherwise to receive cells transduced with the recombinant viruses, e.g. it is recommended to use an immunomodulating protein of substantially human sequence for transducing a cell preparation to be used as a human immunogen or vaccine, or to be used otherwise in connection with humans.
• the proteins can be selected to enhance the effect of the mutant virus as an immunogen or vaccine in the context in which it is employed.
• immunomodulating proteins examples include cytokines for example interleukins 1 to 15 (IL1 to IL15), i ⁇ terferons alpha, beta or gamma, tumour necrosis factor (TNF), granulocyte-macrophage colony stimulating factor
• cytokines for example interleukins 1 to 15 (IL1 to IL15), i ⁇ terferons alpha, beta or gamma, tumour necrosis factor (TNF), granulocyte-macrophage colony stimulating factor
• GM-CSF macrophage colony stimulating factor
• M-CSF macrophage colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1 a and MIP-1 b, complement components and their receptors, accessory molecules such as one of the B7 famiiy of T cell co-stimulators such as B7.1 or B7.2, ICAM-1 , 2 or 3, 0X40 ligand and cytokine receptors.
• nucleotide sequences encoding more than one immunomodulating protein may comprise more than one cytokine or may be a combination of cytokine(s) and accessory m ⁇ lecule(s).
• cytokine a combination of cytokine(s) and accessory m ⁇ lecule(s).
• accessory m ⁇ lecule(s) a combination of cytokine(s) and accessory m ⁇ lecule(s).
• Many further kinds of immunomodulatory proteins and genes can be useful in this invention.
• immunomodulatory proteins examples include GMCSF; IL-2; IL4; IL7; IL12; B7.1 ; TNF-alpha; interferon gamma; CD40L and lymphotactin.
• the genetic material encoding an immunomodulatory protein can be carried in the mutant viral genome as an expressible open reading frame encoding a hybrid or fusion protein which comprises a polypeptide region having homology to and functionality of an immunomodulatory protein, linked to a polypeptide region having another homology and optionally another functionality.
• the immunomodulatory protein can be, comprise, or correspond in functionality to the gp34 protein identified as a binding partner to human OX-40 (see W Godfrey et al, J Exp Med 180(2) 1994 pp 757-762, and references cited therein, including 5 Miura et al, Mol Cell Biol 1 1 (3) 1991 , pp 1313-1325).
• the version of this protein functionality that can be encoded in the mutant viral genome can correspond to the natural gp34 sequence itself, or to a fragment thereof, or to a hybrid expression product e.g. based on the (C-terminal) extracellular (binding) domain of gp34 fused to another protein, e.g. to the constant region of an immunoglobulin heavy chain such as human IgGI , e.g. with the extracellular domain of gp34 (a type 2 membrane protein) fused at its N- terminal to the C-terminal of the immunoglobulin constant domain.
• immunomodulatory proteins can also be carried and expressed in corresponding or other derivative and hybrid forms. It is also understood that mutations of the aminoacid sequences of such immunomodulatory proteins can be inco ⁇ orated. Included here are proteins having mutated sequences such that they remain homologous, e.g. in sequence, function, and antigenic character, with a protein having the corresponding parent sequence. Such mutations can preferably for example be mutations involving conservative aminoacid changes, e.g. changes between aminoacids of broadly simitar molecular properties. For example, interchanges within the aliphatic group alanine, valine, leucine and isoleucine can be considered as conservative. Sometimes substitution of glycine for one of these can also be considered conservative.
• Interchanges within the aliphatic group aspartate and glutamate can also be considered as conservative. Interchanges within the amide group asparagine and glutamine can also be considered as conservative. Interchanges within the hydroxy group serine and threonine can also be considered as conservative. Interchanges within the aromatic group phenylalanine, tyrosine and tryptophan can also be considered as conservative. Interchanges within the basic group lysine, arginine and histidine can also be considered conservative. Interchanges within the sulphur-containing group methionine and cysteine can also be considered conservative. Sometimes substitution within the group methionine and leucine can also be considered conservative.
• Preferred conservative substitution groups are aspartate-glutamat ⁇ ; asparagine-glutamine; vatine-leucine-isoleucine; alanine-valine; phenylalanine-tyrosine; and lysine- arginine.
• mutated sequences can comprise insertion and/or deletions.
• derivative and hybrid forms include proteins with sequences having deleted therefrom any of: a transmembrane segment, an intracellular sequence portion, an N-terminal or C-terminal sequence, e.g. a sequence of from 1-5 ami ⁇ oacids upwards; and/or sequences having added thereto any of e.g. an N-terminal or C-terminal sequence, e.g. a sequence of from 1 -5 aminoacids upwards, or a further functional sequence e.g. as described above.
• the immunomodulating protein can comprise a cytokine, e.g. granulocyte macrophage colony stimulating factor (GM-CSF).
• GM-CSF granulocyte macrophage colony stimulating factor
• Murine GM-CSF gene encodes a polypeptide of 141 amino acids, the mature secreted glycoprotein having a molecular weight of between 14k-30k daltons depending on the degree ⁇ f glycosylation.
• GM-CSF is a member of the haematopoietic growth factor family and was first defined and identified by its ability to stimulate in vitro colony formation in haematopoietic progenitors.
• GM ⁇ CSF is a potent activator of neutrophils, eosinophils and macrophage-monocyte function, enhancing migration, phagocytosis, major histocompatibility complex (MHC) expression, ar... initiating a cascade of bioactive molecules which further stimulate the immune system.
• Human GM-CSF is currently being evaluated in the clinic for the treatment of neutropenia following chemotherapy and as an adjuvant in cancer therapy.
• the heterologous nucleotide sequence employed may comprise a heterologous gene, gene fragment or combination of genes.
• the invention is also applicable to corrective gene therapy, to improve the target cell's usefulness or viability.
• normal CD34+ cells can be transduced with viral vector as described herein encoding a DNA repair enzyme such as 06-methylguanine DNA methyltransferase (MGMT). for protection of target cells e.g. during chemotherapy e.g. with nitrosourea. see T Moritz et al.
• MGMT 06-methylguanine DNA methyltransferase
• Heterologous DNA e.g. further DNA
• an integras ⁇ such as one that is known to be able to act to integrate viral-vector DNA into the host genome so that the vector DNA becomes propagated when host cell mitosis occurs; and for other pu ⁇ oses.
• materials and methods to insert corrective or lethal genetic material to destroy or modulate malignant blast cells can be done for example by the expression in the target cell, by means ⁇ f the herpesviral vector methods described herein, of antisense RNA or ribozyme sequences corresponding to genetic material encoded by the vector: for example as indicated in D Marcola et al, 'Antisense Approaches to Cancer Gene Therapy', Cancer Gene Ther 2 (1995) pp 47 et seq.
• nucleotide sequences e.g. of at least about 12 nucleotides complementary in sequence to the sequence of a chosen target; by choosing from among known promoters suitable to the cellular environment in which they are to be effective, and other measures well known per se.
• RNA molecules for use of antisense RNA to disrupt expression of a target gene are indicated (in connection with a sialidase gene) in specification WO 94/26908 (Genentech: TG Warner et al).
• Techniques for using antisense oligonucleotides capable of binding specifically to mRNA molecules are also indicated in specification WO 94/29342 (La Jolla Cancer Research Foundation and the Regents of the University of Michigan: R Sawada et al) (in particular connection with mRNA encoding human lamp-derived polypeptides).
• ribozymes to disrupt gene expression
• techniques for making and administering ribozymes (or antisense oligonucleotides) in order to cleave a target mRNA or otherwise disrupt the expression of a target gene are indicated in specification WO 94/13793 (Apollon: CJ Pachuk et al) (as applied to ribozymes that target certain mRNAs relevant to leukemias).
• a review of ribozyme techniques is given in M Kashani- Sabet and KJ Scanlon, ch. 8 pp 91-101 in RE Sobol and KJ Scanlon (eds.)
• a lethal gene can also be inserted into the vector to destroy the transduced cell: for example a gene that is lethal in connection with an administered pharmaceutical, as described in e.g. specification WO 95/14100 (Wellcome Foundation: C Richards et al), exemplifying a gene encoding cytosine deaminase (CDA) under control of a CEA promoter, which when introduced into a cell is lethal in connection with administration of 5-fluorocytosine, transformed by the CDA into toxic 5-f luorouracil.
• CDA cytosine deaminase
• the recombinant h ⁇ esvirus used to carry a gene encoding an immunomodulatory protein or other genetic material as discussed herein, is preferably an attenuated and/or replication-defective herpesvirus.
• the mutant h ⁇ svirus can usefully be a mutant of any suitable he ⁇ esvirus; e.g. a non-transforming mutant of a mammalian herpesvirus; e.g. a mutant of a non-transforming human h ⁇ svirus, especially for example a coated or enveloped mutant herpesvirus.
• suitable he ⁇ esvirus e.g. a non-transforming mutant of a mammalian herpesvirus
• a mutant of a non-transforming human h ⁇ svirus especially for example a coated or enveloped mutant herpesvirus.
• examples of he ⁇ esviruses of which mutants are provided and can be used as vectors according to embodiments of the invention include herpes simplex virus of type 1 (HSV-1 ) or type 2 (HSV-2), a human or animal cytomegalovirus (CMV), e.g.
• EBV human cytomegalovirus
• VZV varicella zoster virus
• EBV is less desirable, except in the form of a non-transforming mutant, because of its normally transforming properties.
• Animal viruses of which mutants are provided according to embodiments of the invention include pse ⁇ dorabies virus (PRV), equine and bovine herpesvirus including EHV and BHV types such as IBRV, and Marek's disease virus (MDV) and related viruses.
• PRV pse ⁇ dorabies virus
• EHV and BHV types such as IBRV
• MDV Marek's disease virus
• Suitable he ⁇ esviruse ⁇ to be used as a basis for recombination to produce a vector suitable for use according to the present invention include defective herpesviruses conforming with the general or specific directions in specification WO 92/05263 (Inglis et ai: Immunology Limited) (the disclosure of which is incorporated herein by reference), which describes for example the use as an immunogen or vaccine of a mutant virus whose genome is defective in respect of a gene essential for the production of infectious virus, such that the virus can infect normal host cells and undergo replication and expression of viral antigen genes in such cells but cannotm produce infectious virus.
• defective herpesviruses conforming with the general or specific directions in specification WO 92/05263 (Inglis et ai: Immunology Limited) (the disclosure of which is incorporated herein by reference), which describes for example the use as an immunogen or vaccine of a mutant virus whose genome is defective in respect of a gene essential for the production of infectious virus, such that the virus can infect
• WO 92/05263 particularly describes an HSV virus which is disabled by the deletion of a gene encoding the essential glycoprotein H (gH) which is required for virus infectivity (A Forrester et al, J Virol 66 (1992) 341-348). In the absence of gH protein expression non-infectious virus particles providing almost the complete repertoire of viral proteins are produced. These progeny particles, however, are not able to infect host cells and spread of the virus within the host is prevented. Such a virus has been shown to be an effective immunogen and vaccine in animal model systems (Farrell et al, J Virol 68 (1994) 927-932; McLean ⁇ t al, J Infect Dis, 170
• Such mutant viruses can be cultured in a cell line which expresses the gene product in respect of which the mutant virus is defective.
• ceil Iines expressing proteins of herpes simplex virus the gB glycoprotein (Cai et al, in J Virol 61 (1987) 7 4-721 ), the gD glycoprotein (Ligas and Johnson, in J Virol 62 (1988) 14B6) and the Immediate Early protein ICP4 (Deluca et al, in J Virol 56 (1985) 558). These too have been shown capable of supporting replication of viruses inactivated in respect ⁇ f the corresponding genes.
• gH glycoprotein is known to have homologues in CMV and VZV (Desai et al, in J Gen Virol 69 (1988) 1 147).
• Suitable examples of such genes are genes for essential viral glycoproteins, e.g. (late) essential viral glycoproteins such as gH, gL, gD, and/or gB, and other essential genes.
• Essential and other genes of human herpesviruses are identifiable from DJ McGeoch, 'The Genomes of the Human Herpesviruses'.
• virus vectors in the present invention are for example the mutants such as HSV-1 mutant (in1814) unable to trans-induce immediate early gene expression, and essentially avirulent when injected into mice, described by Cl Ace et al. J Virol 63(5) 9B9 pp 2260-2269.
• Specification WO 91/02788 (CM Preston & Cl Ace: University of Glasgow) describes useful HSV1 mutants including in1814 capable of establishing latent infection in a neuronal host cell and of causing expression of an inserted therapeutic gene.
• virus vectors useful in the invention are based on a mutation in a herpesvirus immediate early gene, e.g. a gene corresponding to ICPO, ICP4, ICP22 and ICP27. Mutations can be used in combination, e.g. as disclosed in WO 96/04395
• virus vectors for use in the present invention are such neuroattenuated HS 1 mutants as mutant 1716 (BP Randazzo et al. Virology 21 1 (1995) pp 94-101 ).
• herpesviruses reference is further made to data published for example in respect of human cytomegalovirus CMV (Weston and Barr ⁇ ll in J Mol Biol 192 (1986) 177-208), and varicella zoster virus VZV (AJ Davison et al, in J Gen Virol 67 (1986) 759-816).
• a genetically inactivated virus immunogen such as a vaccine provides an useful carrier for genes encoding immunomodulatory proteins.
• the virus vaccine can infect cells of the vaccinated host leading to intracellular synthesis of the immunomodulatory proteins. If the genetically inactivated vaccine is also acting as a vector for delivery of foreign antigens, then the immune response against the foreign antigen may be enhanced or altered.
• the heterologous nucleotide sequence is inserted into the genome of the mutant virus at the locus of the deleted essential gene, and most preferably, the heterologous nucleotide sequence completely replaces the gene which is deleted in its entirety.
• the heterologous nucleotide sequence completely replaces the gene which is deleted in its entirety.
• Materials and methods according to the invention can be used to evoke immunological effector mechanisms activated by cellular immunogens such as therapeutic vaccines, in particular to evoke specific cytotoxic T lymphocytes (CTLs) directed against target antigens.
• CTLs cytotoxic T lymphocytes
• Such CTLs can exert a beneficial effect by tending to recognise and destroy tumour ceils, and can also be used ex-vivo in a variety of diagnostic and/or therapeutic methods.
• tumour cells where there are antigenic differences between tumour cells and normal ceils, they can be recognised by the immune system, provided that the tumour-specific antigens are available in the correct form to stimulate an immune response. This avoids the need to identify tumour specific markers.
• CTLs destroy cells on the basis of antigen recognition in conjunction with host major histocompatibility complex (MHC) antigens; peptides generated from the antigenic target within the cytoplasm of the host cell form a complex with host MHC molecules and are transported to the cell surface, where they can be recognised by receptors on the surface of CTLs.
• MHC major histocompatibility complex
• One method of using the vectors, provided by this invention is therefore to prepare a cellular immunogen such as a vaccine from tumour material derived from one or more individuals and to administer this as an immunogen or vaccine for treatment of other subjects, e.g. patients. If a CTL response against the tumour cells is desired, however, for the reasons outlined above, the target antigens should be presented in the context of the correct MHC molecules.
• An immunogen or vaccine prepared from a tumour of one individual may not always therefore be appropriate for another individual with a different MHC type. Since MHC molecules vary from individual to individual, it is generally necessary, in order to activate CTL responses against the target antigens, to present the relevant target antigen to the immune system in the correct MHC context.
• an immunogen such as a therapeutic vaccine, in general it is considered that the selected target antigen is best Introduced into the treated subject's or patient's own ceils in order to generate an appropriate CTL response.
• tumour immunogen or vaccine on a patient's own tumour cells, a procedure known as autologous vaccination.
• a further major advantage of this way of use is that it can take advantage of antigenic targets that may be unique to a particular tumour; it is considered that the deregulated cell cycle control that is the basis of tumour growth can, over a period of time, lead to the accumulation of genetic changes manifested as new antigenic determinants.
• the last-mentioned embodiments of the present invention can avoid or solve a problem with autojogous vaccination procedure, namely that autologous tumour cells are poorly immunogenic.
• Procedures according to examples of the invention can involve introduction of a target gene into tumour cells removed from a subject, by laboratory procedures after which the cells so treated are reintroduced into a subject to be treated (ex-vivo treatment).
• An alternative procedure according to certain examples of the invention is to introduce the target gene directly into tumour cells of the patient (in vivo treatment).
• the advantage of an in-vivo procedure is that no laboratory manipulation of the patient's tumour cells is required.
• a drawback can be that effective gene transduction may be more difficult to achieve in vivo, or more difficult to achieve to a desired degree.
• Other, non- tumour, cells can also usefully be transfected with the virus vectors.
• the recombinant he ⁇ esvirus is based on a disabled form of the herpes simplex virus carrying a deletion in the glycoprotein H (gH) gene, a protein present on the surface of the virus particle that is involved in entry of virus into susceptible cells.
• This virus can only be replicated
• a producer cell line that complements the essential function missing in the viral genome a useful example of a recombinant complementing cell line is one which has been engineered to express stably the same HSV gH gene as was deleted from the virus vector.
• the virus generated from the producer cell line acquires the cell-encoded gH gene product as part of its structure and is infectious. This virus preparation can infect normal cells in the same manner as wild type virus.
• the virus genome can be intracellularly replicated, and genes earned by the genome can be expressed as protein.
• the absence of a functional gH protein when the defective virus infects a normal cell results in failure to generate new infectious virus particles.
• the gH-deleted virus is considered to be safe to administer as a vaccine or a gene delivery vehicle.
• a vector such as a HSV vector for cancer immunotherapy i ⁇ fully disabled and unable to spread within the treated host.
• a useful vector can, however, be based on any HSV virus that is deemed sufficiently safe to be used in a clinical setting.
• heterologous genes incorporated into such a gH-deleted HSV genome are inserted at tha locus from which the gH gene was removed, to minimise the risk of transfer of he heterologous gene by homoloqous recombination to wild type HSV that might co-exist in the treated individual.
• the heterologous gene can however instead be inserted at any site within the virus genome.
• a further adaptation of the method within the scope of the invention is to deliver the appropriate genetic material, e.g. a gene encoding an immunomodulatory protein, in the form of herpesviral amplicons packaged within herpesviral particles.
• Amplicon DNA is DNA that contains an origin of replication of a herpesviral genome together with DNA sequences that can direct packaging of this DNA into virus particles. Where such amplicons are present in cells along with corresponding h ⁇ svirus (helper virus), expression of amplicon DNA can occur along with expression of herpesviral DNA.
• Foreign genes can be cloned into such amplicons and thus expressed in cells infected with the amplicons as well as with herpesvirus.
• Particles containing packaged amplicons can be phenotypically equivalent to the corresponding helper virus and hence able to infect the same host cell and are considered herein as among the defective mutant herpesvirus suitable as vectors for use in the practice of the invention.
• virally-packaged amplicons can also be used to deliver selected DNA to desired cells. Amplicons and processes for their preparation that can be used or readily adapted for use in examples of the performance of this invention, along with further details, are described in further detail in WO 96/29421 (Efstathiou et al: Cantab Pharmaceuticals Research Ltd and Cambridge University Technical Services Ltd).
• the HSV helper virus used for packaging the amplicons is, by itself, not harmful to the host, and so a disabled virus with an essential gene deleted, such as the gH-deleted virus described above provides an ideal helper virus as described in WO 92/05263 and other related references cited herein.
• Other useful helper viruses can, however, be based on herpesvirus sufficiently attenuated or disabled to be used in a clinical setting, not necessarily one that is entirely replication-defective.
• the invention described here can be used to deliver chosen genetic material, e.g. DNA encoding a chosen protein such as an immunomodulatory protein, to tumour cells for the purposes of therapy.
• chosen genetic material e.g. DNA encoding a chosen protein such as an immunomodulatory protein
• the range of genes that can be delivered for the purpose of stimulating an immune response includes genes for cytokines, immunostimulators, lymphotactin, CD40, 0X40, 0X40 ligand, and other genetic material mentioned herein, which can be included in the vector as single genes or multiple genes, or multiple copies of one or more genes.
• normal and malignant human haematopoietic progenitor cells can be rapidly transduced with efficiencies ranging from 60% to 100%; the levels of transduction and gene expression that have been achieved are considered to represent high efficiency, particularly for these targets.
• Embodiments of the present invention can also produce useful rapidity of expression of a transferred gene. For example under conditions as specifically described herein, positivity for expression of the transferred gene has been obtained in 80% to 100% of CD34+ ceils as well as AML and ALL blasts within 24 hours after exposure to the vector. It has also been found that embodiments of the invention can provide a preparation of tranduced cells that produce, and for example release, the product of the transferred gene for at least 7 days at a level proportional to the MOI (multiplicity of infection, usually reckoned in plaque- forming units (pfu)/cell), for example at MOI in the range 0.05-20. e.g. in the case of GM-CSF produced in human primary leukaemic cells by expression of the corresponding gene transferred by a gH-deletant h ⁇ sviral vector.
• MOI multiplicity of infection
• embodiments of the present invention enable the production of immunogens, e.g. human leukaemia immunogens, in cases where the production of corresponding immunogens has presented logistic problems up to now.
• immunogens e.g. human leukaemia immunogens
• embodiments of the present invention have been found to enable consistently achievable useful high proportions of leukaemic blasts to be transduced from all patients so far tested, thus presenting useful advantage in clinical work.
• the present invention is further described below by the help of examples of procedures and products and of parts of procedures and products given by way of example only and not of limitation.
• Figures 1 to 6 are diagrams illustrating the construction of plasmids plMMB45, plMMB56, plMME-46, plMCl4, pi MR . and plMR3, respectively. These vectors are referred to in the description below;
• Figures 7 and 8 show results of transducing cells, in accordance with particular embodiments of the invention, with genetically disabled he ⁇ esvirus constructed as described below.
• Delivery of the vectors into cells such as haematopoietic stem cells, and engraftment of cells into a patient to be treated therewith, can be carried out by ready adaptation of techniques per-se well-known in the field. For example, methods as indicated in MK Brenner et al, Cold Spring Harbor Symposia in Quantitative Biology, vol LIX (1994), pp 691-697, or in references cited therein, or in MK Brenner et al. Lancet 342 (6 Nov 993) pp 1 134-1 137, or in references cited therein, can be readily applied and adapted.
• the gH-deieted HS 1 virus and gH-deleted HSV2 virus are propagated in the complementing cell lines. These cell lines have been engineered to express the HSV-1 gH gene or the HSV-2 gH gene respectively.
• Such cell Iines can be constructed as descnbed in WO94/05207 and W094/21807 and references cited therein. The following section provides a further description of the construction of suitable cell lines, and starts with the construction of certain plasmids.
• HSV viral DNA is required, it can be made for example (in the case of HSV2) from the strain HG52 by the method of Walboomers and Ter Schegget (1976) Virology 74, 256-258, or by suitable adaptations of that method.
• An elite stock of the HG52 strain is kept at the Institute of Virology, MRC Virology Unit,
• strains G and MS for example can be obtained from the ATCC, Rockville, Maryland, USA.
• a 4.3kb SsM fragment encoding the HSV-1 (HFEM) gH gene and upstream HSV-1 gD promoter (- 392 to + 1 1 ) was excised from the plasmid pgDBrgH (Forrester et al., op. cit.), and cloned into pUC1 19 (Vieira & Messing, 1987) to produce plasmid pUCI 19gH.
• a Not 1 site was introduced into plasmid pUC1 19gH by site-directed mutagenesis. 87 bp downstream of the gH stop codon.
• the resulting plasmid, plMC03 was used to generate a Not 1 -Sst 1 fragment which was repaired and ligated into the eucaryotic expression vector pRc/CMV (Invitrogen Co ⁇ oration), pre-digested with Not 1 and Nru 1 to remove the CMV IE promoter.
• the resulting plasmid, plMC05 contains the HSV-1 gH gene under the transc ⁇ ptio ⁇ al control of the virus inducible gD promoter and BGH
• Bovine Growth Hormone poly A contains the neomycin resistance gene for selection of G418 resistant stable cell Iines.
• ⁇ H-deleted HSV-1 complementing cell line The plasmid pIMCO ⁇ was transfected into Vero (ATCC no. 88020401 ) cells using the calcium phosphate technique (Sambrook, Fritsch & Maniatis, A Laboratory Manual, Cold Spring Harbor Laboratory Press 1989). Cells were selected by dilution cloning in the presence of G418 and a clonal cell line was isolated. Following expansion and freezing, cells were seeded into 24 well plates and tested for their ability to suppo ⁇ the growth of gH-negative virus, by infection with SCI 6 (del)gH (Forrester et al, op. cit) at 0.1pfu/cell. Vims plaques were observed 3 days post infection confirming expression of the gH gene.
• TK- thymidine kinase negative BHK cells
• Plasmid plMMB24 containing the HSV- 2 gH gene is constructed from two adjacent BamHI fragments of HSV-2 strain 25766.
• the plasmids are designated pTW49, containing the approximately 3484 base pair BamHI R fragment, and pTW54, containing the approximately 331 1 base pair BamHI S fragment, both cloned into the BamHI site of pBR322.
• Equivalent plasmids can be cloned easily from many available strains or clinical isolates ⁇ f HSV-2.
• the 5' end of the HSV- 2 gene is excised from pTW54 using BamHI and Kpnl. to produce a 2620 base pair fragment which is gel-purified.
• HSV-2 gH gene is excised from pTW49 using BamHI and Sail, to produce a 870 base pair fragment which is also gel-purified.
• the two fragments are cloned into pUC1 19 which had been digested with SalHI and Kpnl. This plasmid now contains the entire HSV- 2 gH gene.
• Plasmid p!MC08 containing the HSV-2 (strain 25766) gH gene was constructed as follows. Plasmid plMMB24 was digested with Ncol and BstXI and the fragment containing the central portion of the gH gene was purified from an agarose gel. The 5' end of the gene was reconstructed from two oligonucleotides CE39 and CE40 which form a linking sequence bounded by Hindlll end Ncol sites.
• the 3' end of the gene was reconstructed from two oligonucleotides CE37 and CE38 which form a linking sequence bounded by BstXI and Not! sites.
• the two oligonucleotide linkers and the purified Ncol-BstXI gH fragment were cloned in a triple ligation into Hindlll-Notl digested plMC05, thus replacing the HSV-l gH gene by the HSV-2 gH gene.
• the resultant plasmid was designated plMC08.
• the plasmid plMC08 contains the HSV-2 gH gene under the transcriptional control of the virus inducible gD promoter and BGH (Bovine
• the plasmid piMC08 was transfected into Vero (ATCC no. 88020401 ) cells using the calcium phosphate technique (Sambrook, Fritsch & Maniatis, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). Cells were selected by dilution cloning in the presence of G418 and a clonal call line was isolated. Following expansion and freezing, these cells, designated CR2 ceils, were seeded into 24 well plates, and infected with the gH deleted HSV-1 (SCI 6 (del)gH) at O.lpfu/ceil. Virus plaques were observed 3 days post infection confirming expression of the gH gene.
• plMMB56 + plMMB56 + is a vector with a lacZ cassette flanked by HSV-2 sequences from either side of the gH gene. It is made as follows: the two PCR fragments made by oligos MB97-MB96 and by oligos MB57-MB58 are digested with the restriction enzymes appropriate to the sites that have been included in the PCR oligonucleotides. The MB97-MB96 fragment is digested with Hindlll and Hpal. The MB57-MB58 fragment is digested with Hpal and EcoRI. These fragments are then ligated into the vector pUCl 19 which has been digested with Hindlll and EcoRI. The resultant plasmid is called plMMB45 (Fig 1).
• Hindlll MB97 5' TCGAAgCTTCAGGGAGTGGCGCAGC 3' Hpal MB96: 5 * TCAGJTA_&CGGACAGCATGGCCAGGTCAAG 3'
• MB58 5' TCAGAATTCGAGCAGCTCCTCATGTTCGAG 3'
• the E.coli beta- galactosidase gene under the control of an SV40 promoter, is inserted into plMMB45.
• the SV40 promoter plus beta-galactosidase gene is excised from the plasmid pCHl 10 (Pharmacia) using BamHI and Tth III 1. The ends are filled in using the Klenow fragment of DNA polymerase. The fragment is gel-purified.
• the plasmid plMMB45 is digested with Hpal.
• plMMB46 plMMB46 contains sequences flanking the HSV-2 gH gene, with a central unique
• any gene cloned into this site can be inserted by recombination into the HSV- 2 genome at the gH locus. If the virus is a TK-negative gH-negative virus, (for example made using the plMMB56 + plasmid described above) then the plasmid will replace the 3' end of the TK gene, thus restoring TK activity and allowing selection for TK-positive virus.
• TK-negative gH-negative virus for example made using the plMMB56 + plasmid described above
• T e two PCR fragments made by oligos MB94-MB109 and by oligos MB57-MB108 are digested with the restriction enzymes appropriate to the sites that have been included in the PCR oligonucleotides.
• the MB94-MB109 fragment is digested with Hindlll and Hpal.
• the MB57-MB108 fragment is digested with Hpal and EcoRI. These fragments are then ligated into the vector pUCl 19 which has been digested with Hindlll and EcoRI.
• the resultant plasmid i ⁇ called plMMB46 see Fig 3).
• the oligonucleotides used are as follows:
• Hpal MB57 5' TCAGTTAACGCCTCTGTTCCTTTCCCTTC 3'
• EcoRI MB108 5' TCAGAATTCGTTCCGGGAGCAGGCGTGGA 3'
• Hindlll MB94 5' TCAAAGCJTATGGCTTCTCACGCCGGCCAA 3' Hpal MB109: 5' TCAGTTAACTGCACTAGTTTTAATTAATACGTATG 3'
• the plasmid pRc/CMV (Invitrogen Co ⁇ oration) was digested with the restriction enzymes Nrul, Pvull and Bsml and a 1066 base pair Nrul-Pvull fragment was isolated from an agarose gel. The fragment was cloned into Hpal digested plMMB4 ⁇ (see Fig 4). The resultant is named plMC14.
• the pRc/CMV fragment contains the cytomegalovirus major immediate early promoter (CMV-IE promoter) and the bovine growth hormone (BGH) poly A addition site.
• CMV-IE promoter cytomegalovirus major immediate early promoter
• BGH bovine growth hormone
• the plasmid pi MRI is a recombination vector for the insertion of the murine GM-CSF gene, under the control of the CMV-IE promoter, into a DISC
• plMC1 is digested with Xbal, phosphatased with CIAP, gel purified and the overhanging ends made flush with Klenow polymerase.
• the murine GM-CSF gene is excised from the plasmid pGM 3.2FF (referred to as pGM3.2 in Gough et al. EMBO Journal 4, 645-653. 1985) (or from the equivalent plasmid constructed as described below), by a two stage procedure. Firstly pGM
• plMR1 The resulting plasmid is designated plMR1 (see Fig 5).
• An alternative plasmid equivalent to pGM3.2. can be constructed as follows.
• a library of cDNA clones is constructed from a cloned T-lymphocyte line (from a BALB/c strain of mouse), such as LB3 (Kelso et al, J Immunol. 132, 2932, 1984) in which the synthesis of GM-CSF is inducible by concanavalin A.
• the library is searched by colony hybridisation with a sequence specific to the murine GM-CSF gene (see Gough et al, EMBO J, 4. 645, 1985 for sequence).
• a example of an oligonucleotide usable in this case is 5' TGGATGACAT GCCTGTCACA TTGAATGAAG AGGTAGAAGT 3'.
• the open reading frame for the GM-CSF gene is preceded by a short open reading frame (ORF) of 15 base pairs. Because it is possible that this might interfere with the expression of GM-CSF, the plasmid pIMRI was altered so that this small reading frame was removed.
• pIMRI was digested with NotI and PpuMI.
• the digested vector was phosphatased with calf intestinal alkaline phosphatase (CIAP) and gel-purified. The sequences between the two restriction enzyme sites were replaced by a short piece of double- stranded DNA generated by the annealing of two oligonucleotides CE55 and
• the oligonucleotides are constructed so as to have overhanging ends compatible with the NotI and PpuMI ends generated by the digestion of pIMRI .
• the two oligonucleotides are annealed, phosphorylated, and ligated to the Notl- PpuMI-digest ⁇ d pIMRI .
• the resultant vector was designated plMR3. The sequences in the relevant region are shown below:
• plMMB34 This is a recombination vector containing sequences flanking the HSV-1 gH gene. The left side flanking sequences inactivate TK gene which lies adjacent to the gH gene.
• the two PCR fragments made by oligos MB97-MB100 and by oligos MB61-MB58 are digested with the restriction enzymes appropriate to the sites that have been included in the PCR oligonucleotides.
• the MB97-MB1 Q0 fragment is digested with Hindlll and Hpal.
• the MB61-MB58 fragment is digested with Hpal and EcoRI. These fragments are then ligated into the vector pUC119 which has been digested with Hindlll and EcoRI.
• the resultant plasmid is called plMMB34.
• the oligonucleotides used are as follows:
• Hpal MB61 5' TCASJTAA ⁇ AGCCCCTCTTTGCTTTCCCTC 3'
• the E.coli beta- galactosidase gene under the control of an SV40 promoter is inserted into plMMB34.
• the SV40 promoter plus beta-galactosidase gene is excised from the plasmid pCH110 (Pharmacia) using BamHI and Tth III 1. The ends are filled in using the Klenow fragment of DNA polymerase. The fragment is gel-purified.
• the plasmid plMMB34 is digested with Hpal, phosphatased with Calf Intestinal Alkaline Phosphatase (CIAP) to abolish self ligation, anc* gel-purified.
• the gel- purified fragments are then ligated together to produce the plasmid plMMB55 + .
• plMMB63 is made from HSV-1 strain KOS (m) DNA. plMMB63 contains sequences flanking the HSV-1 gH gene, with a central unique Hpal site. Any gene cloned into this site can be inserted by recombination into the HSV-1 genome at the gH locus. If the virus is a TK-negative virus (for example made using the plMMB55 + plasmid described above) then the plasmid will replace the
• the two PCR fragments made by oligos MB98-MB63 and by oligos MB61 - MB58 are digested with the restriction enzymes appropriate to the site ⁇ that have been included in the PCR oligonucleotides.
• the MB98-MB63 fragment is digested with Hindlll and Hpal.
• the MB61-MB58 fragment is digested with Hpal and EcoRI. These fragments are then ligated into the vector pUC119 which has been digested with Hindlll and EcoRI.
• the resultant plasmid is called plMMB63.
• the oligonucleotides used are as follows:
• Hindlll MB98 5' TCAAAGCTTATGGCTTCGTACCCCTGCCAT 3'
• Hpal MB63 5' TCASTTAAJCGGACCCCGTCCCTAACCCACG 3'
• Hpal MB61 5' TC AGTTA ACAG CCCCTCTTTG CTTTCCCTC 3'
• This plasmid is a general recombination plasmid with unique sites for the insertion of foreign genes which can then be recombined into an HSV-1 gH- deleted DISC vector.
• the plasmid pRc/CMV was digested with Nrul and Pvull and a 1066 bp fragment, which contains CMV IE promoter and a polyA signal, was blunt ended with Klenow polymerase and inserted into the unique Hpal site of plasmid plMMB63.
• This plasmid is named pIMXI .O.
• the multiple cloning site contained between the CMV IE promoter and the polyA signal is ideal for cloning other genes into the plasmid and their subsequent introduction into DISC HSV-1 .
• the plasmid plMX3.0 is a recombination vector for the insertion of murine GM-CSF, under the control of CMV IE promoter, into the deleted gH region of type 1 DISC HSV.
• This plasmid was constructed by inserting the murine GM-CSF which was excised out from plasmid pGM3.2FF (op. cit.) with Smal and Dral, into the unique BsaBI site of pIMXI.O.
• This plasmid, plMX3.0. is the HSV-1 equivalent of plMR3.
• Recombinant virus expressing GM-CSF was made in two stages. In the first stage the gH gene, and part of the TK gene are replaced by a "IacZ cassette", consisting of the SV40 promoter driving the E.coli IacZ gene. This virus has a TK minus phenotype and also gives blue plaques when grown under an overlay containing the colourige ⁇ ic substrate X-gal. This recombinant virus can now be conveniently used for the insertion of foreign genes at the gH locus.
• Recombinant virus was constructed by transfection of viral DNA with the plasmid plMMB56 + (for HSV-2) or plMMB55 + (for HSV-1 ).
• Viral DNA is purified on a sodium iodide gradient as described in Walboomers & Ter Schegget (1976) Virology 74, 256-258.
• a transfection mix is prepared by mixing 5 ⁇ g of viral DNA, 0.5//g of linearised plasmid DNA (linearised by digestion with the restriction enzyme Seal) in 1 ml of HEBS buffer (137mM NaCl. 5mM KCI, 0.7mM Na2HP0 4 , 5.5mM glucose, 20m M Hepes, pH 7.05). 70 l of 2M CaCl z is added dropwise, and mixed gently. The medium is removed from a sub-confluent 5cm dish of CR1 or
• CR2 cells (gH-expressing Vero cells) and 500 /I of the transfection mix is added to each of two dishes. The cells are incubated Bt 37°C for 40 minutes, when
• FCS foetal calf serum
• the plaques are allowed to grow to full CPE and harvested by scraping into the medium. Multiple rounds of plaque-purification are carried out until a pure stock of virus is obtained.
• the structure of the first stage recombinant is confirmed as follows.
• Sodium iodide purified viral DNA is prepared as before, and digested with BamHI.
• This digest is separated on an agarose gel and transferred to a nylon membrane.
• TK-positive recombinant viruses are selected by growth on BHK gH-positive TK-negative cells, in the presence of 0.6//M methotrexate, 15/iM Thymidine, 9.5/ M Glycine, 4.75j/M Adenosine and 4.75//M
• virus infected cells are harvested as before and screened on gH-deleted HSV1 complementary cells. Overlays are added as before and white plaques are selected in the presence of Xgal. Plaques are picked as before and plaque-purified three times on said gH-deleted HSV1 complementary cells.
• the structure of the viral DNA is analysed as before.
• Cos 1 ceils (ECACC No. 88031701 ) are transfected with plasmid DNA using DEAE dextran as described in Gene Transfer and Expression, A laboratory Manual, Michael Kriegler. Supernatants from transfected Cos 1 cells or infected CR2 cells are screened for GM-CSF activity by bioassay. An IL-3/GM-CSF responsive murine haematopoietic cell line designated C2GM was obtained from
• the cell line C2GM is maintained in Fischers media with 20% horse serum, 1 % glutamine and 10% conditioned cell media.
• the conditioned cell media is obtained from exponentially growing cultures of Wehi 3b cells (ECACC No. 86013003) which secrete murine IL-3 into the media. W ⁇ hi 3b cells are maintained in RPMI 1640 media, 10% FCS and 1 % glutamine.
• HSV- 1 and HSV- 2 mutants which are gH-negative and which express GM-CSF, etc.
• the skilled person can readily adapt the present teaching to the preparation of other mutant viruses which are defective in respect of a first gene essential for the production of infectious virus, such that the vims can infect normal cells and undergo replication and expression of viral antigen in these cells but cannot produce named infectious virus and which also express a heterologous nucleotide sequence which encodes an immunomodulating protein or other genetic material as mentioned herein.
• mutant viruses can be made on the basis of deletion or other inactivation (for example) of the following essential genes in the following viruses and vims types :- in herpes simplex viruses, essential genes such as gB. gD. gL. ICP4, ICP8 and/or ICP27 can be deleted or otherwise inactivated as well as or instead of the gH gene used in the above examples.
• known essential genes such as any known essential homologues tD the gB, gD, gL, gH, ICP4, ICP8 and/or ICP27 genes of HSV, can be selected for deletion or other inactivation.
• Cytomegalovirus can e.g. be genetically disabled by deleting or otherwise activating genes responsible for temperature-sensitive mutations, for example as identifiable from Dion et al, Virology 158 (1987) 228-230.
• cell Iines can be derived from cancer patients by biopsy or otherwise and can be used directly or following culture in vitro.
• Cells from patients with solid tumours can be obtained following surgical removal of the tumour or of metastases, or from biopsy material from the tumour or metastases.
• Tumour biopsies or re-sected material can be used to prepare single cell suspensions either by mechanical or enzymic disaggregation or by other well known methods.
• Infection/transduction of tumour ceils or cell Iines with the recombinant defective HSV vector carrying a gene of choice can be carried out in vitro by dispensing aliquots of a single-cell suspension into suitable tissue culture vessels such as 24-well plates or flasks.
• a suitable cell concentration can be 0.5 to 2.0x10 * 6 cells/well in 1 or 2 ml of medium.
• Vims can then be added at a multiplicity of infection far example in the range 0.01-20, for example 0.05 to 0.1 pfu/cell, or up to 1 or up to about 5 pfu/cell, and the culture is incubated for 2 h to allow the virus to enter the cell.
• the cells can be used for immunotherapeutic and other pu ⁇ os ⁇ s as mentioned herein either directly or after culture in fresh medium for varying lengths of time, e.g. for up to 1 to 7 days. For test purposes, as in the test experiments described below, culture was carried out for 1 to 7 days.
• Samples of the cells infected by the virus vector can be examined for expression of the heterologous gene carried within the virus vector.
• cells infected with a recombinant defective HSV vector containing the lac Z gene can be tested for the presence of ⁇ -galactosidase activity either by using an antibody or antiserum preparation directed against ⁇ -galactosidase. or by using a galactosidase substrate (e.g. Fluoreporter (TM)) which upon cleavage by ⁇ - galactosidase gives a fluorescent product.
• the fluorescent product or antibody can then be detected by fluorescence microscopy or by flow cytometry. The proportion ⁇ %) of cells showing fluorescence, indicates the proportion expressing the gene product and can be calculated from the results of the detection step.
• a suitable test system to test and illustrate the effectiveness of transduction in accordance with the present invention, using a recombinant virus vector is as follows, and can be adapted to other examples of herpesvirus vector.
• the vector used in the test described here contains a Iacz reporter gene: generally a different vector having a gene encoding an immunomodulatory protein or other protein, or other genetic material as mentioned herein. In place of the reporter gene (or in addition to it) is used in the practice of the invention.
• a Iacz gH-deleted HSV mutant was constructed as described herein above, with reference to the 'first stage' mutant virus.
• This first stage in the production of the vector containing the gene for the immunomodulatory protein is a suitable test vector used in the test procedure described below.
• such mutants can also be constructed as described in specification WO 94/21807, corresponding with the 'first stage' recombinant mentioned in WO 94/21807, constmction ⁇ f which is described on page 28 line 28 to page 29 line 26 with associated description (hereby incorporated by reference).
• the IacZ gene is used here as a test and marker gene. Using the techniques described herein and in the mentioned specifications, other useful genes can readily be i ⁇ co ⁇ orated in the place of the Iacz or as well as the Iacz gene.
• A Two independent cell lines derived from acute lymphoblastic leukaemias (ALL); (AD and RS human pre-B-leukaemic cell lines established at St Jude Children's Research Hospital, Memphis, TN from clinical samples and cultured in RPMI 1640 (Biowhittaker) supplemented with 10% FCS (Biowhittaker. Walkersville, MD), 1001 U/ml penicillin and 100mu-g/ml streptomycin (Biowhittaker), and 2 mmol/l L-glutamine)); B: Three independent cell lines derived from neuroblastoma (NB);
• C Primary cells freshly isolated from four patients with ALL; D: Primary cells derived from three patients with acute myeloid leukaemia (AMD; and E: Primary cells derived from two patients with NB.
• Leukaemic blast cells were isolated from patients with > 80% blast cells by Ficoll sedimentation of peripheral blood or bone marrow mononuclear cells. M ⁇ eloblasts can be maintained in liquid culture in RPMI supplemented as above (Biowhittaker). Lymphoblasts can be maintained in liquid culture or where necessary on allogeneic skin fibroblasts as stromal support.
• the recombinant defective HSV vector has also shown a surprisingly high capacity for transduction of fresh NB cells and NB cell lines.
• the transduction efficiency for two of the three NB cell Iines wa ⁇ > 70%, and for the fresh isolates it was 65% and 100% respectively.
• Bone marrow was obtained from two normal donors. The mononuclear fraction (by Ficoll sedimentation) was passed down an anti-CD34 column (Cellpro. Seattle, WA) to enrich the CD34+ progenitor ceil population. These cells were then exposed to the lacz-encoding disabled herpesvirus described above at a number of different multiplicities of infection (MOI), ranging from 0.05-20 (pfu/cell). After 2 hours exposure, the cells were divided into two portions and could be maintained either in stromal support cultures or in culture with cytokines as mentioned below.
• MOI multiplicities of infection
• the stromal support cultures with 8x10 A 5/sq.cm of surface area were established in Fisher's medium (Life Technologies, Grand Island, NY), with 15% horse serum and 5% fetal calf serum (FCS: Summit Biotechnology. Ft Collins, CO), 1 x10 * -6 mol/l hydrocortison ⁇ (Abbott, Chicago, III.). 10 * -4 mol/l mercaptoethanol (Sigma, St Louis, MO) and 400 mu-l/ml transferrin (Life Technologies).
• Stromal cells were then employed as feeder layers and reseeded with transduced CD34 + cells obtained as described above.
• An alternative culture method for a portion of the transduced cells is to grow them in liquid media supplemented with foetal bovine serum, IL3 and stem cell factor. The other of the portions was mixed in methylcellulose and grown in tissue culture dishes at a density of 10 A 5/ml.
• Iac2 transduction and expression of Iac2 are readily adaptable to the expression of other desired proteins and genetic material by the use of alternative virus vectors carrying corresponding other genetic material in place of Iacz a ⁇ described above.
• GM-CSF cytokine
• MLL murine lymphoblastic leukaemia
• FIG. 7 shows bar charts expressing results of tests for GM-CSF secretion by different transduced cell lines at different MOI (multiplicity of infection: ratio of viral pfu to cell count).
• the contiguous bars in each set of three refer to the production at 1 , 3 and 7 days respectively under a given indicated set of conditions (cell type, MOI).
• the vertical axis indicates scale of GM-CSF production per 5x10 * 5 cells per 24 hours.
• Figure 8 is a FACS plot showing the result of a successful transduction of CD34 + primary bone marrow ceils (haematopoietic progenitor cells) from a normal adult human souurce.
• Bone marrow cells were transformed using the disabled h ⁇ esviral vector carrying a gene encoding a cytokine (GM-CSR (gH-deletant HSV vector encoding GM-CSF).
• GM-CSR gH-deletant HSV vector encoding GM-CSF.
• the cells were purified in standard manner by CD34 selection and stained in standard manner for CD34 antigen.
• other CD34 + cells e.g. those showing malignant properties, can be transduced and thereafter used, e.g. r ⁇ infused as immunogenic therapeutic vaccine into the patient from whom the parental cells were derived, or used in-vrtr ⁇ /ex-vivo to prime or stimulate lymphocytes.

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