Classifications

• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/31—Chimeric antigen receptors [CAR]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/32—T-cell receptors [TCR]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/421—Immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/421—Immunoglobulin superfamily
  • A61K40/4211—CD19 or B4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • 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
  • 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
  • C12N5/0636—T lymphocytes
  • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
• • 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
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/70—Enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells

Definitions

• Antigen-specific T-cells may be generated by selective expansion of peripheral blood T-cells natively specific for the target antigen. However, it is difficult and quite often impossible to select and expand large numbers of T-cells specific for most cancer antigens.
• Gene-therapy with integrating vectors affords a solution to this problem as transgenic expression of Chimeric Antigen Receptor (CAR) allows generation of large numbers of T-cells specific to any surface antigen by ex vivo viral vector transduction of a bulk population of peripheral blood T-cells.
• CAR Chimeric Antigen Receptor
• the solid tumour microenvironment can be hostile to T-cells.
• inhibitory receptors may be upregulated.
• the tumour microenvironment may contain diverse types of inhibitory cells such as inhibitory T-cells, myeloid or stromal cells.
• T-cells which gain access to the solid tumour may be inhibited in their activity.
• the factors noted above may also form a barrier which prevents the CAR T-cell from entering and engrafting in the solid tumour.
• lymphoid tumours may be more difficult to kill than lymphoid cancer cells.
• lymphoid tumours are often close to apoptosis and a single CAR T-cell / tumour cell interaction may be sufficient to induce killing of the lymphoid tumour cells.
• engineered cells in particular engineered immune cells expressing a CAR or a transgenic TCR in targeting solid tumours.
• the present inventors now provide an engineered cell which encodes a transgenic synthetic biology pathway that enables the engineered cell to produce a small molecule, in particular a therapeutic small molecule.
• small molecules can - for example - penetrate into cells and disrupt key intracellular pathways including signalling pathways and metabolic pathways.
• the present invention provides an engineered cell which comprises; (i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) one or more engineered polynucleotides which encode one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in the cell.
• CAR chimeric antigen receptor
• TCR transgenic T-cell receptor
• the one or more enzymes may be encoded by one or more engineered polynucleotides.
• the one or more enzymes may be encoded by one engineered polynucleotide.
• the engineered polynucleotide may be an operon.
• the one or more enzymes may be encoded in one or more open reading frames.
• the one or more enzymes may be encoded in a single open reading frame.
• each enzyme may be separated by a cleavage site.
• the cleavage site may be a self-cleavage site, such as a sequence encoding a FMD-2A like peptide.
• the present invention provides a nucleic acid construct which comprises: (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic TCR; and (ii) one or more nucleic acid sequences which encode one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in a cell.
• CAR chimeric antigen receptor
• TCR transgenic TCR
• the present invention provides a pharmaceutical composition according to the present invention for use in treating and/or preventing a disease.
• the present invention relates to a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the present invention to a subject in need thereof.
• the method may comprise the following steps:
• the cell may be autologous.
• the cell may be allogenic.
• the present invention relates to the use of a pharmaceutical composition according to present invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
• the disease may be cancer.
• the cancer may be a solid tumour cancer.
• the present invention relates to a method for making a cell according to the present invention which comprises the step of introducing: a nucleic acid construct; a first nucleic acid sequence and a second nucleic acid sequence; a vector or a first and a second vector of the present invention into the cell.
• the cell may be from a sample isolated from a subject.
• An advantage of the present invention is that it allows a very high local concentration of an otherwise toxic small molecule at the site of a solid tumour.
• the small molecule can easily diffuse from the engineered cell of the present invention and can diffuse into a tumour cell to enact a direct toxic or modulatory effect. Accordingly, production of a therapeutic small molecule by the engineered cell of the present invention can ameliorate some the difficulties associated with targeting a solid tumour whilst reducing the drawbacks of potentially toxic effects associated with systemic administration of the therapeutic small molecule.
• Figure 1 - a Schematic diagram illustrating a classical CAR.
• Figure 2 (a) Summary of the violacein biosynthetic pathway; (b) Operon for violacein converted into a eukaryotic format with all 5 enzymes coded for as a single frame separated by FMD-2A like peptides.
• FIG 11 Production of violacein in SupT1 cells by dual transduction of SupT1 T cell line
• Figure 12 Violacein produced by SupT1 cells is toxic to SKOV3 tumour cells DETAILED DESCRIPTION OF THE INVENTION
• an engineered cell which comprises (i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) one or more engineered polynucleotides which encode one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in the cell.
• an "engineered polynucleotide” refers to a polynucleotide which is not naturally present in the cell genome.
• Such engineered polynucleotides may be introduced into a cell using, for example, standard transduction or transfection methods as described herein.
• engineered polynucleotide may be transferred to a cell using retroviral vectors.
• the one or more enzymes may be referred to herein as a transgenic synthetic biology pathway.
• the one or more enzymes comprise at least two, at least three, at least four or at least five enzymes.
• the transgenic synthetic biology pathway may comprise or consist of 2, 3, 4, 5 or more enzymes.
• the cell of the present invention may encode a set of enzymes which when translated effect the stepwise conversion of a starting material in the cell to a therapeutic small molecule.
• the one or more enzymes are encoded one or more engineered polynucleotides.
• the one or more enzymes may be encoded by one, two, three, four, five or more engineered polynucleotides.
• each enzyme of the transgenic synthetic biology pathway is encoded by a separate engineered polynucleotide.
• the expression of each enzyme of the transgenic synthetic biology pathway may be controlled by a regulatory sequence such as a promoter.
• the expression of each enzyme of the transgenic synthetic biology pathway may be controlled by related regulatory sequences so that each enzyme is expressed at the same time in the cell.
• the expression of each enzyme of the transgenic synthetic biology pathway may be controlled by the same regulatory sequences so that each enzyme is expressed at the same time in the cell.
• the expression one or more enzymes of the transgenic synthetic biology pathway may be controlled by an inducible regulatory element so that production of the therapeutic small molecule can be induced in a controllable manner.
• an inducible regulatory element for example a rate-limiting enzyme in the transgenic synthetic biology pathway
• the enzymes may be encoded as a single-reading frame under the control of the same regulatory elements (e.g. the same promoter).
• the co-expression site may be a sequence encoding a cleavage site, such that the engineered polynucleotide encodes the enzymes of the transgenic synthetic biology pathway joined by a cleavage site(s).
• a co-expression site is located between adjacent polynucleotide sequences which encode separate enzymes of the transgenic synthetic biology pathway.
• the same co-expression site is used (i.e. the same co-expression site is present between each adjacent pair of nucleotide sequences encoding separate enzymes of the transgenic synthetic biology pathway.
• the co-expression site is a cleavage site.
• the cleavage site may be any sequence which enables the two polypeptides to become separated.
• the cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
• cleavage is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage.
• FMDV Foot-and-Mouth disease virus
• various models have been proposed for to account for the "cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82: 1027-1041).
• the exact mechanism of such "cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.
• the cleavage site may be a furin cleavage site.
• Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family.
• the members of this family are proprotein convertases that process latent precursor proteins into their biologically active products.
• Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites.
• Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor.
• Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X- (Arg/Lys)-Arg') and is enriched in the Golgi apparatus.
• the cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.
• TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo.
• the consensus TEV cleavage site is ENLYFQ ⁇ S (where 'V denotes the cleaved peptide bond).
• Mammalian cells such as human cells, do not express TEV protease.
• the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.
• the cleavage site may encode a self-cleaving peptide.
• a 'self-cleaving peptide' refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately "cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.
• the co-expression sequence may be an internal ribosome entry sequence (IRES).
• the co-expressing sequence may be an internal promoter.
• the engineered polynucleotide may be an operon.
• An operon is a functioning polynucleotide unit which comprises a plurality of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo trans-splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. THERAPEUTIC SMALL MOLECULE
• the therapeutic small molecule may be any small molecule which is efficacious in the treatment of cancer.
• “Therapeutic small molecule” is used herein according to its usual meaning to refer to a pharmaceutical molecule with a low molecular weight (e.g. less than 900 daltons).
• the small molecule may be an alkaloid, terpenoid, flavonoid, polyketides or non- ribosomal peptides, sugar or sugar alcohol.
• Alkaloids are nitrogen-containing compounds of low molecular weight produced by a large variety of organisms, including bacteria, fungi, plants, and animals. Most alkaloids are derived through decarboxylation of amino acids such as tryptophan, tyrosine, ornithine, histidine, and lysine, and possess important pharmacological activities. For example, sanguinarine has shown potential as an anticancer therapeutic, bisbenzyliso-quinoline alkaloid tetrandrine has immunomodulatory effects, and a number of indolocarbazole alkaloids have entered clinical trials for inhibiting neovascularization and as cancer treatments.
• Alkaloids can be classified into a number of groups such as morphinane-, protoberberine-, ergot-, pyrrolidine-, quinolizidine- and furanoquinoline-alkaloids according to the amino acids from which they originate.
• Benzylisoquinoline alkaloids such as sanguinarine
• Benzylisoquinoline alkaloids are synthesized from tyrosine via reticuline in Magnoliaceae, Ranunculaceae, Berberidaceae, Papaveraceae, and many other species.
• the early pathway from tyrosine to reticuline is common among many plant species, whereas there is more diversity in late pathways.
• the therapeutic small molecule may be selected from a cytotoxic molecule; a cytostatic molecule; an agent which is capable of inducing differentiation of the tumour; and a proinflammatory molecule.
• a cytotoxic molecule refers to a molecule which is directly toxic to a cell and is capable of inducing cell death.
• a cytotoxic molecule may disrupt DNA synthesis, protein synthesis and/or metabolic processes within the cell.
• Illustrative cytotoxic molecules include, but are not limited to, violacein, mycophenolic acid, terpenes/isoprenoids (e.g. geraniol, sesterterpenes such as ophiobolin derivatives; Taxol), triterpenoids (e.g.
• ginsenosides oleanolic acid, ursolic acid, betulinic acid or protopanaxadiol
• cyclosporin Tacrolimus
• Methotrexate sanguinarine and fluorouracil.
• the cytotoxic molecule may be selected from one of the following types: alkylators, such as cyclophosphamide; anthracyclines, such as daunorubicin; antimetabolites, such as cytarabine; vinca alkaloids, such as vincristine; and topoisomerase inhibitors, such as etoposide.
• alkylators such as cyclophosphamide
• anthracyclines such as daunorubicin
• antimetabolites such as cytarabine
• vinca alkaloids such as vincristine
• topoisomerase inhibitors such as etoposide.
• a cytostatic molecule refers to molecules which are capable of modulating cell cycle and cell growth, in particular molecules which are capable of inducing cell growth arrest.
• ATRA trans retinoic acid
• ATRA can induce differentiation of certain types of acute myeloid leukaemia.
• the therapeutic small molecule may be violacein
• Violacein is an indole derivative, isolated mainly from bacteria of the genus Chromobacterium. Violacein exhibits important anti-tumour properties - for example violacein has activity against MOLT-4 leukaemia, NCI-H460 non-small-cell lung cancer and KM12 colon-cancer cell lines.
• Violacein is formed by enzymatic condensation of two tryptophan molecules, requiring the action of five proteins (see Figure 2).
• the genes required for its production may be referred to as vioABCDE (see August et al. Journal of Molecular Microbiology and Biotechnology, vol. 2, no. 4, pp. 513-519, 2000 - herein incorporated by reference) and have been cloned and expressed within other bacterial hosts, such as E. coli.
• the vioABCDE genes encode the enzymes VioA, VioB, VioC, VioD and VioE.
• the one or more engineered polynucleotides may encode VioA, VioB, VioC, VioD and VioE such that the engineered cell of the present invention is capable of synthesising violacein from tryptophan.
• SEQ ID NO: 6 An illustrative violacein single operon reading frame comprising the VioA, VioB, VioC, VioD and VioE polypeptides in frame with each other and separated by foot-and-mouth like 2A sequences is shown as SEQ ID NO: 6. In this sequence, the 2A peptide sequences are shown in bold and italic. A nucleic acid sequence which encodes the violacein ORF is shown as SEQ ID No. 7.
• the one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in the cell may comprise one or more of the sequences shown as SEQ ID NO: 1 to 6, or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant VioA, VioB, VioC, VioD and/or VioE polypeptides retain the capacity to provide the required to form violacein from tryptophan in a cell.
• the percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST, which is freely available at http://blast.ncbi.nlm.nih.gov. Suitably, the percentage identity is determined across the entirety of the reference and/or the query sequence. Synthesis of Geranyl diphosphate derived terpenoids
• the therapeutic small molecule may be a terpenoid.
• IDP and DMADP are combined by a variety of enzymes to produce a number of intermediates of differing five carbon combinations such as geranyl diphosphate (C10), geranygeranyl diphosphate (C20) and squalene (C30) (see Figure 4).
• Simple isoprenoids may be synthesized from mevalonate pathway precursors using a single enzymatic step.
• geraniol a monoterpenoid synthesized by many plant species, is a major component of rose oil and has been shown to possess anti-cancer functions.
• Geraniol can be synthesized in yeast cells from geranyl diphosphate by expression of a single geraniol synthase gene from Valeriana officinalis (Zhao, J. et al.; (2016); App. Microbiol, and Biotech. 100, 4561-4571 - incorporated herein by reference).
• the one or more enzymes for use in the present invention may comprise a geraniol synthase enzyme.
• An illustrative geraniol synthase from Valeriana officinalis is shown as SEQ ID NO: 8 (corresponding to UniProt Accession Number - KF951406).
• the one or more enzymes for use in the present invention may comprise a ophiobolin F synthase enzyme.
• An illustrative ophiobolin F synthase from Aspergillus clavatus is shown as SEQ ID NO: 9 (corresponding to UniProt Accession Number - A18C3).
• the ophiobolin F synthase may comprise the sequence shown as SEQ ID NO: 9 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retains the capacity to produce an ophiobolin from dimethylallyl diphosphate (DMAPP), Geranyl diphosphate, farnesyl diphosphate or geranylgeranyl diphosphate.
• DMAPP dimethylallyl diphosphate
• Geranyl diphosphate farnesyl diphosphate or geranylgeranyl diphosphate.
• Geraniol and ophiobolins are a relatively simple isoprenoid, but their synthesis demonstrates the feasibility of synthesizing more complex isoprenoids using multiple enzymes.
• a further example of a terpene derivative is Taxol, a complex tricyclic diterpene, requiring up to 19 enzymes to synthesize from I DP and DMADP precursors required for geraniol synthesis. This synthetic pathway and the enzymes involved are reviewed in Croteau et al (2006) Taxol biosynthesis and molecular genetics Phytochem Rev. 5:75-97. Synthesis of triterpenoids from squalene
• the therapeutic small molecule may be a triterpenoid.
• Cholesterol is a cellular product derived from the mevalonate pathway requiring similar precursors to prenylation precursors, but enzymes directing the synthesis of squalene divert from the pathway to produce cholesterol (Figure 3).
• Squalene is a triterpene and is a precursor for the synthesis of a wide variety of triterpene derived compounds ( Figure 5) many of which have anticancer activity.
• ginsenosides By expression of four plant derived enzymes it has been possible to produce complex ginsenosides in yeast (Wang, P. et al.; (2015); Metabolic Engineering. 29, 97-105 - incorporated herein by reference). In addition to ginsenosides having anti-cancer activity, precursor compounds such as oleanolic acid or protopanaxadiol have anticancer properties.
• the one or more enzymes for use in the present invention may comprise a group of enzymes capable of producing ginsenosides.
• An illustrative group of four enzymes capable of producing ginsenosides are shown as SEQ ID NO: 10-13.
• SEQ ID NO: 10 Protein sequence of Dammarenediol 12-hydroxylase from Panax ginseng (Uniprot H2DH16) MAAAMVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVKYRK EKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVEKMFPRSHGESNADNFSKVRGKMMFLLKVD GMKKYVGLMDRVMKQFLETDWNRQQQINVHNTVKKYTVTMSCRVFMS IDDEEQVTRLGSS IQNIEAGLLAVPINI PGTAMNRAIKTVKLLTREVEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYT TLNGTITFVLNYLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRI IPPGVGTF
• transgenic synthetic biology pathway may be expressed in an inducible manner
• mechanisms by which the transgenic synthetic biology pathway may be expressed in an inducible manner include, but are not limited to, (a) expression triggered by a factor in the tumour microenvironment (e.g. binding of cognate antigen to the CAR or transgenic TCR); and (b) expression trigger by a small molecule pharmaceutical.
• tumour associated antigens are known, as shown in the following Table 1.
• the antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.
• the spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk.
• the spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk.
• a human lgG1 spacer may be altered to remove Fc binding motifs.
• CD3-zeta endodomain which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound.
• CD3-zeta may not provide a fully competent activation signal and additional co- stimulatory signalling may be needed.
• chimeric CD28 and OX40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together (illustrated in Figure 1 B).
• the percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST, which is freely available at http://blast.ncbi.nlm.nih.gov. Suitably, the percentage identity is determined across the entirety of the reference and/or the query sequence.
• the T lymphocyte When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction.
• heterologous TCR molecules it is possible to engineer cells to express heterologous (i.e. non-native) TCR molecules by artificially introducing the TRA and TRB genes; or TRG and TRD genes into the cell using vector.
• the genes for engineered TCRs may be reintroduced into autologous T cells and transferred back into patients for T cell adoptive therapies.
• Such 'heterologous' TCRs may also be referred to herein as 'transgenic TCRs'.
• the T cell may be an alpha-beta T cell or a gamma-delta T cell.
• the cell may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells.
• an immortalized T-cell line which retains its lytic function may be used.
• the cell may be a haematopoietic stem cell (HSC).
• HSCs can be obtained for transplant from the bone marrow of a suitably matched donor, by leukopheresis of peripheral blood after mobilization by administration of pharmacological doses of cytokines such as G-CSF [peripheral blood stem cells (PBSCs)], or from the umbilical cord blood (UCB) collected from the placenta after delivery.
• PBSCs peripheral blood stem cells
• URB umbilical cord blood
• the marrow, PBSCs, or UCB may be transplanted without processing, or the HSCs may be enriched by immune selection with a monoclonal antibody to the CD34 surface antigen.
• the cell of the present invention is an engineered cell. Accordingly, the first nucleic sequence encoding a CAR or transgenic TCR and one or more nucleic acid sequences which encodes one or more enzymes capable of synthesising a therapeutic small molecule are not naturally expressed by the alpha-beta T cell, a NK cell, a gamma-delta T cell or a cytokine-induced killer cell.
• the one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in a cell are encoded on a single nucleic acid sequence.
• polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other. It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code.
• nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides.
• a co-expression site is used herein to refer to a nucleic acid sequence enabling co- expression of both (i) a CAR or a TCR; and (ii) one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in a cell as defined herein.
• the present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) or nucleic acid construct(s) of the invention.
• a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it expresses a CAR or transgenic TCR and one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in the cell.
• the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.
• the vector may be capable of transfecting or transducing a cell.
• the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
• the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
• Such a formulation may, for example, be in a form suitable for intravenous infusion.
• a method for treating a disease relates to the therapeutic use of the cells of the present invention.
• the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
• the method for preventing a disease relates to the prophylactic use of the cells of the present invention.
• the cells may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease.
• the subject may have a predisposition for, or be thought to be at risk of developing, the disease.
• the present invention provides a cell, a nucleic acid construct, a first nucleic acid sequence and a second nucleic acid sequence, a vector, or a first and a second vector of the present invention for use in treating and/or preventing a disease.
• the present invention provides a cell of the present invention for use in treating and/or preventing a disease
• the disease to be treated and/or prevented by the method of the present invention may be immune rejection of the cell which comprises (i) a chimeric antigen receptor (CAR) or a transgenic TCR; and (ii) one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in a cell as defined herein.
• CAR chimeric antigen receptor
• TCR transgenic TCR
• the cell, in particular the CAR cell, of the present invention may be capable of killing target cells, such as cancer cells.
• the target cell may be recognisable by expression of a TAA, for example the expression of a TAA provided above in Table 1.
• CAR or transgenic TCR- expressing cells of the present invention may be generated by introducing DNA or RNA coding for the CAR or TCR and one or more enzymes which are capable of synthesising a therapeutic small molecule when expressed in combination in the cell by one of many means including transduction with a viral vector, transfection with DNA or RNA.
• the cell of the invention may be made by:
• the cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.
• This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
• Violacein is a tryptophan derivative synthesized by a number of bacterial species. It is made by a complex biosynthetic pathway which also generates the recognised anticancer drugs rebeccamycin and staurosporine ( Figure 2a).
• This operon was split into 2 separate retroviral expression plasmids containing the VioA and VioB genes, and the VioC, VioD and VioE genes respectively. Expression of all 5 genes are required for violacein production.
• the violacein biosynthetic genes were introduced into SupT1 cells by retroviral transduction. Due to the natural fluorescence of violacein, it was possible to measure violacein production in SupT1 T cell line using flow cytometry analysis ( Figure 11).
• Normal human T-cells are transduced with a CAR which recognizes the myeloid antigen CD33 along with the lentiviral vector described above which codes for Violacein. Control T- cells are also generated which are only transduced with the CD33 CAR.
• Non-transduced T- cells from the same donor, CD33 CAR T-cells and CD33 CAR / Violecein T-cells are co- cultured with the AML cell line HL60 at different effector to target ratios for 1 , 2, 5 and 7 days. Quantity of remaining HL60 target cells is determined by flow cytometry. An NSG mouse model of AML using HL60 cells is tested by treating with CD33 CAR cells and CD33 CAR / Violacein cells.
• Geraniol is a monoterpenoid compound synthesized by many plant species which displays an anti-proliferative/pro-apoptotic effect against cancer cells in vitro. It is produced from the precursor geranyl diphosphate by the action of the enzyme geraniol synthase. Additionally, geranyl diphosphate is a product of the mevalonate pathway in human cells which lack geraniol synthase.
• geraniol in the SupT1 T cell line was initiated by introduction through retroviral transduction of the geraniol synthase (GS) gene from Valeria officinalis co-expressed with the human farnesyl diphosphate synthase (FDPS) gene, either as a separate enzyme or fused directly to geraniol synthase, which was introduced to boost production of precursor geranyl diphosphate molecules from the host cell metabolic pathway (see table below). All constructs were co-expressed with an anti-CD19 CAR based upon the anti-CD19 antibody HD37 and possessing a 41 BB and CD3zeta endodomain. In some cases, the FDPS also contained the K266G mutation which has been reported to enhance geraniol phosphate production.
• GS geraniol synthase
• FDPS human farnesyl diphosphate synthase
• SupT1 expressing the FDPS and GS constructs listed in the above table were co-cultured with SKOV3 cells as follows: SKOV3 cells expressing a nuclear-localized red fluorescent protein (mKATE) were plated in a 96-well plate at a density of 5,000 cells per well and allowed to adhere overnight. The following day the indicated transduced SupT1 cells were added to the SKOV3 cells at density of 20,000 cells per well in a total volume of 200ul cell culture medium.
• mKATE nuclear-localized red fluorescent protein
• Etoposide which induces the apoptosis of SKOV3 cells
• Etoposide which induces the apoptosis of SKOV3 cells
• Cells were continuously monitored in a Incycute live cell imager and the number of viable SKOV3 cells enumerated every hour by counting the presence of red fluorescent nuclei.
• Co-culture of SupT1 T cells expressing these constructs with CD19-negative SKOV3 ovarian cancer cell line resulted in increased growth inhibition of SKOV3 cells when compared to the control CAR lacking the geraniol producing GS gene ( Figure 7).
• Caffeine is a purine derivative synthesized by a number of plant species and is a known antagonist of the immunomodulatory Adenosine A2AR receptor expressed on T cells.
• Caffeine methyl transferase (CAXMT1) from Coffea arabica and caffeine synthase (CCS1) from Camellia sinensis into the SupT1 T cell line resulted in the production of caffeine by these human cell lines.
• Caffeine production could be further enhanced by the addition of the pre-cursor xanthosine ( Figure 8).
• the production of caffeine was monitored by culturing 1x10 6 transduced cells in a 2ml culture medium in the presence of the indicated amounts of Xanthosine. After 72 hours supernatants were harvested, cleared of cells by centrifugation and caffeine levels were determined by ELISA.

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