WO2019162695A1 - Cellule - Google Patents

Cellule Download PDF

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Publication number
WO2019162695A1
WO2019162695A1 PCT/GB2019/050504 GB2019050504W WO2019162695A1 WO 2019162695 A1 WO2019162695 A1 WO 2019162695A1 GB 2019050504 W GB2019050504 W GB 2019050504W WO 2019162695 A1 WO2019162695 A1 WO 2019162695A1
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rtk
chimeric
cell
nucleic acid
domain
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PCT/GB2019/050504
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English (en)
Inventor
Martin PULÉ
Matteo Righi
Evangelia KOKALAKI
Shaun CORDOBA
Daniela ACHKOVA
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Autolus Limited
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Publication of WO2019162695A1 publication Critical patent/WO2019162695A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present invention relates to an engineered cell which expresses a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TOR); and in particular to approaches to control the proliferation and survival of such cells.
  • CAR chimeric antigen receptor
  • TOR transgenic T-cell receptor
  • 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
  • CAR cells may be inconsistent between different binders and structures, and the characteristics bestowing maximum proliferative incentive are elusive.
  • CAR cells are exposed to the hostile tumour microenvironment, which may not be favourable to CAR cell engraftment, survival and proliferation.
  • the present invention provides a cell which comprises (i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) a receptor tyrosine kinase (RTK) which is capable of signalling in the absence of cognate ligand.
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • RTK receptor tyrosine kinase
  • RTK which is capable of signaling in the absence of cognate ligand as provided by the present invention improves the effectiveness of engineered cells, for example CAR T cells, to survive and proliferate.
  • the present invention may improve the ability of engineered cells to survive and proliferate in a microenvironment in which the appropriate immunological cues may not be provided. Accordingly, the present invention may compensate for the lack of a complete physiological immune response in a tumour microenvironment.
  • the RTK may be encoded by an exogenous polynucleotide.
  • the exogenous polynucleotide may encode a wild-type RTK.
  • the RTK may be over-expressed such that it is capable of signaling in the absence of cognate ligand.
  • the RTK may comprise a mutation which enables the RTK to signal in the absence of cognate ligand.
  • the mutation may selected from a substitution, a deletion, an insertion, and a duplication.
  • the RTK may be a chimeric RTK.
  • the chimeric RTK may comprise an ectodomain which mediates dimerization or oligomerization of the chimeric RTK.
  • the chimeric RTK may comprise an endodomain which mediates dimerization or oligomerization of the chimeric RTK.
  • the chimeric RTK may be an intracellular molecule.
  • the domain which mediates dimerization or oligomerization of the chimeric RTK may comprise a disulphide bond.
  • the domain which mediates dimerization or oligomerization of the chimeric RTK may comprise a hinge domain.
  • the domain which mediates dimerization or oligomerization of the chimeric RTK may comprise a chemically operable dimerization or oligomerization domain.
  • the dimerization or oligomerization of the chimeric RTK may inducible by an agent.
  • the cell may comprise a first and a second chimeric RTK, wherein the first chimeric RTK comprises a first chemical inducer of dimerization/oligomerization (CID) binding domain (CBD1) and the second chimeric RTK comprises a second CID binding domain (CBD2), wherein CBD1 and CBD2 are capable of simultaneously binding to a CID.
  • CBD1 and CBD2 are capable of simultaneously binding to a CID.
  • the CBD1 and CBD2 may comprise different CID binding domains and the CID may comprise two different binding moieties.
  • One CBD may comprise the rapamycin binding domain of FK-binding protein 12 (FKBP12), and the other CBD may comprise the FKBP12-Rapamycin Binding (FRB) domain of mTOR; and the CID may be rapamycin or a derivative thereof which is capable of causing the the chimeric RTK to homodimerize.
  • the CID may be rapamycin, temsirolimus, everolimus, ridaforolimus or an analogue thereof.
  • CBD1 and CBD2 may be similar and the CID may comprise two identical binding moieties.
  • CBD1 and CBD2 may comprise a FK506-binding protein (FKBP). Dimerization or oligomerization of the chimeric RTK may be inhibited by an agent.
  • FKBP FK506-binding protein
  • the cell may comprise a first and a second chimeric RTK, wherein the first chimeric RTK comprises a first chemical disruptor of dimerization/oligomerization (CDD) binding domain (CDD1) and the second chimeric RTK comprises a second CDD binding domain (CDD2), wherein dimerization between CDD1 and CDD2 is disrupted in the presence of a CDD.
  • CDD dimerization/oligomerization
  • the chimeric RTK may comprise (i) an intracellular adaptor molecule which comprises at least two CDD1s and (ii) at least two intracellular molecules which each comprise a RTK signaling domain and a CDD2, wherein dimerization between CDD1 and CDD2 is disrupted in the presence of a CDD.
  • One CDD may comprise the Tet repressor (TetR), the other dimerization domain may comprise TetR interacting protein (TiP) and the agent may be tetracycline, doxycycline, minocycline or an analogue thereof.
  • TetR Tet repressor
  • TiP TetR interacting protein
  • the cell may be a cytolytic immune cell.
  • the cell may be an alpha-beta T cell, a gamma- delta T cell, a NK cell or a cytokine induced killer cell.
  • the present invention provides a chimeric receptor tyrosine kinase (RTK) receptor capable of signalling in the absence of cognate ligand comprising: (i) a domain which mediates dimerization or oligomerization; and (ii) an intracellular signalling component comprising a RTK signalling domain.
  • RTK receptor tyrosine kinase
  • the chimeric RTK may be a chimeric RTK as defined in the first aspect of the invention.
  • the present invention provides a polynucleotide which encodes a chimeric RTK as provided by the present invention.
  • 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 T-cell receptor (TCR); and (ii) a second nucleic acid sequence which encodes a receptor tyrosine kinase (RTK).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • RTK receptor tyrosine kinase
  • the second nucleic acid sequence encodes a RTK as defined in the first aspect of the invention.
  • the present invention provides a nucleic acid construct which comprises a first and a second nucleic acid sequence encoding a first and second chimeric RTK according to the present invention.
  • 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 T-cell receptor (TCR); (ii) nucleic acid sequences encoding a first and second chimeric RTK as provided by the present invention.
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • the nucleic acid sequences of the nucleic acid constructs may be separated co-expression sites.
  • the present invention provides a kit of nucleic acid sequences comprising: (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic (TCR); and (ii) a second nucleic acid sequence which encodes a receptor tyrosine kinase (RTK) as provided by the present invention.
  • a kit of nucleic acid sequences comprising: (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic (TCR); and (ii) a second nucleic acid sequence which encodes a receptor tyrosine kinase (RTK) as provided by the present invention.
  • CAR chimeric antigen receptor
  • TCR transgenic
  • RTK receptor tyrosine kinase
  • the first and second nucleic acid sequences may encode a first and second chimeric RTK as provided by the present invention.
  • the present invention provides a kit of nucleic acid sequences comprising (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) nucleic acid sequences encoding a first and second chimeric RTK as provided by the present invention.
  • a kit of nucleic acid sequences comprising (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) nucleic acid sequences encoding a first and second chimeric RTK as provided by the present invention.
  • the present invention provides a kit of vectors which comprises (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic T- cell receptor (TCR); and (ii) nucleic acid sequences encoding a first and second chimeric RTK as provided by the present invention.
  • a kit of vectors which comprises (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic T- cell receptor (TCR); and (ii) nucleic acid sequences encoding a first and second chimeric RTK as provided by the present invention.
  • the present invention provides a kit of vectors which comprises: (i) a first vector which comprises a nucleic acid sequence which encodes (i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) a second vector which comprises a nucleic acid sequence which encodes a chimeric receptor tyrosine kinase (RTK) as defined in the first aspect of the invention.
  • a first vector which comprises a nucleic acid sequence which encodes (i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) a second vector which comprises a nucleic acid sequence which encodes a chimeric receptor tyrosine kinase (RTK) as defined in the first aspect of the invention.
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • RTK chimeric receptor tyrosine kinase
  • the present invention provides a kit of vectors which comprises first and second vectors which comprise nucleic acid sequences encoding a first or second chimeric RTKs as provided by the present invention.
  • the present invention provides a pharmaceutical composition which comprises a plurality of cells, a chimeric RTK, a nucleic acid construct, a vector or a first and second vector according to the present invention.
  • the present invention provides a pharmaceutical composition according to the 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 invention to a subject in need thereof.
  • the method may comprise the following steps:
  • the method may further comprise monitoring toxic activity in the subject and optionally further comprise the step of administering an agent which is capable of inhibiting signaling of the RTK, such as a small molecular RTK inhibitor, to the subject in order to reduce adverse toxic effects.
  • an agent which is capable of inhibiting signaling of the RTK such as a small molecular RTK inhibitor
  • the agent may be a small molecular RTK inhibitor, such as a small molecule RTK inhibitor listed in Table 5.
  • the RTK may a chimeric RTK wherein dimerization or oligomerization of the chimeric RTK may be disrupted by an agent and the method may optionally comprise the step of administering an agent which is capable of disrupting dimerization or oligomerization of the chimeric RTK in order to reduce toxic effects.
  • the RTK is a chimeric RTK wherein dimerization or oligomerization of the chimeric RTK may be inducible by an agent and the method may comprise the step of administering an agent capable of inducing dimerization or oligomerization of the RTK to the subject to provide acceptable levels of disease progression and/or toxicity.
  • the cell may be autologous.
  • the cell may be allogenic.
  • the present invention relates to the use of an agent which is capable of inhibiting signaling of a RTK such as a small molecular RTK inhibitor, an agent which is capable of inducing dimerization or oligomerization of the RTK wherein dimerization or oligomerization of the chimeric RTK may be inducible by an agent or an agent which is capable of disrupting dimerization or oligomerization of the chimeric RTK wherein dimerization or oligomerization of the chimeric RTK may be disrupted by an agent to reduce signalling of a RTK as defined in any of claims 1 to 28.
  • a RTK such as a small molecular RTK inhibitor
  • the present invention further relates to the use of a plurality of cells, a chimeric RTK, a nucleic acid construct, a first nucleic acid sequence and a second nucleic acid sequence, a vector, a first and second vector, or a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the disease may be cancer.
  • the invention further relates to a method for making a cell, which comprises the step of introducing into a cell a nucleic acid construct, a first nucleic acid sequence and a second nucleic acid sequence, a vector or a first and second vector according to the present invention.
  • the cell may be from a sample isolated from a subject.
  • Figure 1 - a Schematic diagram illustrating a classical CAR.
  • FIG. 3 Illustrative structure of a constitutively active RTK chimera.
  • the chimeric RPTK comprises an ectodomain, which is the Fc spacer (CH2-CH3) of immunoglobulin as well as the hinge.
  • the hinge domain mediates dimerization of the RTK chimera.
  • the endodomain contains the intracellular moiety of the RTK, which associates with JAK proteins.
  • the JAK kinases Upon dimerization of the chimeric protein, the JAK kinases auto-phosphorylate each other and recruit STAT proteins thus initiating downstream pathways.
  • the depicted chimera is constitutively active due to the constitutive hinge-mediated dimerization.
  • FIG. 4 Illustrative structure of a tunable RTK chimera.
  • the RTK chimeric protein delineated in this figure constitutes of the intracellular domain of an RTK protein.
  • the RPTK endodomain associates with JAK kinases that auto-phosphorylate upon dimerization of the receptor.
  • the extracellular domain of the chimera comprises a spacer and a dimerization inducible moiety, the FKBP46V, which renders the receptor tunable.
  • Administration of a Chemical-Dimerization Inducer (CID) mediates dimerization of the FKBP46V domains, and thus of the RTK chimera initiating the auto-phosphorylation and downstream signal transmission.
  • CID Chemical-Dimerization Inducer
  • FIG. 5 Illustrative structure of a chimeric RTK adaptor system.
  • the endodomain of each construct is derived from the intracellular domain of a member of the RTK family. All constructs are expressed as dimers (dimerization occurs through the Fc-stalk) ensuring constitutive tonic RTK signalling.
  • B) Transduced T cells were normalised to 30% transduction efficiency and labelled with the dye Cell Trace Violet (CTV). Labelled T cells were cultured in the absence of cytokine support for 7 days. At the end of the 7-day period cells were analysed by flow cytometry to measure the dilution of the Cell Trace Violet which occurs as the T cells divide. NT and 35665 are negative and positive controls; respectively.
  • the present invention provides a cell which comprises: (i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) a receptor tyrosine kinase (RTK) which is capable of signalling in the absence of cognate ligand.
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • RTK receptor tyrosine kinase
  • RTKs and protein phosphatases are protein families that play crucial roles in cell signalling.
  • the main role of RTKs is the control of cell growth, proliferation, and metabolism. Their structure is conserved among the twenty subfamilies.
  • the extracellular domain ectodomain
  • ectodomain bestows ligand specificity, and typically contains diverse a globular domain such as immunoglobulin-like, fibronectin or EGF-like domain.
  • the cytoplasmic moiety bears the tyrosine kinase domain, which comprises an activation loop and the catalytic domain.
  • Wild-type RTKs are membrane-bound protein and, as such, the extracellular domain and the intracellular domain are separated by a transmembrane domain.
  • the RTK may be selected from ERBB2, R ⁇ QRBb, KLK2/FLT3, EPHA3, EPHA6, EPHA7, EPHB1 , EPHB2, EPHB4, EPHB6, DDR1 , DDR2, LTK, ALK, TRKC, RON, AXL, and MER.
  • RTKs signal through ligand-induced dimerization/oligomerisation, which leads to auto phosphorylation of tyrosine residues in the kinase domain activation loop of their cytoplasmic tail.
  • Ligand-mediated oligomerisation of the RTK leads to a two-step activation, the increase in catalytic activity and the creation of docking sites for downstream signalling proteins.
  • RTKs typically homodimerize in order to signal.
  • the RTK auto-phosphorylation may occur in cis or in trans.
  • the phosphorylated tyrosine residues constitute docking sites for numerous SH2-containing signalling molecules.
  • all RTKs signal through common downstream signal proteins such as: PI3 kinase, Ras-Raf-MAPK, JNK, and PLCy.
  • the signalling is mediated by the JAK-STAT pathway.
  • the RTKs are stringently regulated by multiple layers of auto-inhibitory mechanisms. In the unstimulated state, the activation loop could obstruct the substrate tyrosine-binding site. Pathological mutation of RTKs causes oncogenic transformation, with dysregulation of cells growth, enhanced proliferation and tissue penetration.
  • Wild-type RTKs generally require the binding of ligand for productive signalling.
  • RTKs for use in the present invention are capable of signalling in the absence of cognate ligand.
  • engineered cells which express a RTK which is capable of signalling in the absence of cognate ligand transmit survival and/or proliferative signals when present in a hostile tumour microenvironment. Accordingly, such cells will have improved engraftment and expansion compared to corresponding engineered cells which do not comprise a RTK which is capable of signalling in the absence of cognate ligand.
  • RTK signalling may be determined using methods which are known in the art.
  • RTK activation and signalling may be analysed by quantifying autophosphorylation of the RTK signalling domain and/or signalling proteins downstream of the RTK.
  • Downstream signalling proteins which may be analysed include STAT molecules, Erk and IKK, for example.
  • Methods for determining phosphorylation of suitable signalling molecules include, but are not limited to, western blot using phosphorylation-specific antibodies, mass spectrometry, colorimetric, radioactive, or fluorometric detection, ELISA, cell-based ELISA, and intracellular flow cytometry.
  • Productive RTK signalling may also be determined by assessing a functional effect in a cell which expresses the RTK capable of signalling in the absence of cognate ligand, for example, by determining cell proliferation using a cell trace violet dye dilution via FACS.
  • a RTK which is capable of signalling in the absence of cognate ligand may be capable of inducing phosphorylating of the RTK intracellular signalling domain in the absence of cognate ligand.
  • a RTK which is capable of signalling in the absence of cognate ligand may be capable of inducing the phosphorylation of downstream signalling molecules such as STATs, Erk and/or IKK, in the absence of cognate ligand.
  • a RTK which is capable of signalling in the absence of cognate ligand may increase the proliferation of cells in which RTK expression has been modified compared to equivalent, control cells in which RTK expression has not been modified.
  • the assessment of cell proliferation should be determined in the absence of exogenous RTK ligand and keeping all conditions, other than RTK expression, consistent between the cells with modified RTK expression and the control cells.
  • the cells with modified RTK expression may have at least 1.5-, at least 2-, at least 5-, at least 10-, at least 25-, at least 50-, or at least 100-fold greater proliferation compared to unmodified control cells.
  • a RTK which is capable of signalling in the absence of cognate ligand may increase the proliferation and/or survival of cells in which RTK expression has been modified compared to equivalent control cells in which RTK expression has not been modified.
  • the assessment of cell proliferation and/or survival should be determined in the absence of exogenous RTK ligand and keeping all conditions, other than RTK expression, consistent between cells with modified RTK expression and the control cells.
  • cell proliferation and survival may be determined using a cell trace violet dye dilution via FACS in combination with a determination of cell number under starvation culture conditions. Suitable starvation culture conditions are known in the art and may refer - for example - to cell culture performed in the absence of exogenous serum (e.g.
  • the cells with modified RTK expression may have at least 1.5-, at least 2-, at least 5-, at least 10-, at least 25-, at least 50-, or at least 100-fold greater proliferation compared to unmodified control cells.
  • the cells with modified RTK expression may have at least 1.5-, at least 2-, at least 5-, at least 10-, at least 25-, at least 50-, or at least 100-fold greater cell number compared to unmodified control cells.
  • inhibitors include, but are not limited to, imatinib mesylate (STI571 ; Gleevec), gefitinib (Iressa), erlotinib (OSI-1774; Tarceva), lapatinib (GW- 572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU 11248), and leflunomide (SU101) (Arora and Scholar, 2005).
  • STI571 imatinib mesylate
  • Iressa gefitinib
  • OSI-1774 erlotinib
  • lapatinib GW- 572016
  • canertinib CI-1033
  • semaxinib SU5416
  • vatalanib PTK787/ZK222584
  • sorafenib BAY 43-9006
  • Table 5 A table of RTK inhibitors and their targets
  • the RTK for use in the present invention may be encoded by an exogenous polynucleotide.
  • exogenous polynucleotide means that the polynucleotide which expresses the RTK is not part of the endogenous genome of the cell.
  • the exogenous polynucleotide may be an engineered nucleic acid construct or a vector.
  • the exogenous nucleotide may encode a wild-type RTK.
  • a“wild- type” RTK refers to a RTK comprising a ligand-binding domain which is capable of binding a known RTK ligand and a RTK signalling domain.
  • the wild-type RTK may be a RTK as listed in Table 2, for example.
  • the wild-type RTK may be MET/c-MET or FGFR-1.
  • the wild-type RTK may comprise an amino acid sequence shown as SEQ ID NO: 1 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 signal via a tyrosine kinase domain.
  • the wild-type RTK may comprise an amino acid sequence shown as SEQ ID NO: 2 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 signal via a tyrosine kinase domain.
  • the RTK is over-expressed compared to an equivalent, control cell which does not comprise the exogenous polynucleotide.
  • the comparison between the cell over-expressing the RTK and the control cell should be performed under conditions which are equivalent for each cell (apart from the level of RTK expression). Expression levels may be determined by methods which are known in the art, for example, real-time quantitative PCR, western blot and/or flow cytometry.
  • the cell of the present invention may express at least 1.5-, at least 2-, at least 5-, at least 5-, at least 10-, at least 20-, at least 50-, or at least 100-fold greater levels of the RTK compared to a corresponding, unmodified cell.
  • the RTK expressed by the exogenous polynucleotide may not be detectable in a corresponding, unmodified cell.
  • the over-expression of a RTK leads to high membrane density of the RTK.
  • This high membrane density causes trans auto-phosphorylation of the cytoplasmic tail/signalling domain of the RTK and enables the RTK to signal in the absence of cognate ligand.
  • Over expression of a wild-type RTK which enables signalling in the absence of cognate ligand may be determined using known methods for assaying productive RTK signalling, as described herein - for example.
  • the RTK which is over-expressed may be a wild-type RTK.
  • wild-type RTK is intended to cover variants of the wild-type RTK polypeptide which provide substantially the same function as the corresponding wild-type RTK.
  • the RTK may comprise a variant having at least 80, 85, 90, 95, 98 or 99% sequence identity to the corresponding wild-type RTK (for example, the wild-type RTK polypeptides referred to in Table 2).
  • RTK overexpression may lead to productive RTK signalling in the absence of cognate ligand.
  • the overexpression of EGFR or HER-2/neu overexpression in breast cancer is increased the proliferation rates.
  • the RTK which is over-expressed may be Met/c-Met, FGFR1 , FLT3, EGFR, ErbB2/HER2, Kit/SCFR, Flt4/VEGFR3 orFRGF2/K-SAM.
  • the RTK which is over-expressed may be Met/c-Met.
  • the RTK which is over-expressed may be FGFR1
  • the RTK which is over-expressed may be FLT3.
  • the RTK which is over-expressed may be EGFR.
  • the RTK which is over-expressed may be ErbB2/HER2.
  • the RTK which is over-expressed may be Kit/SCFR.
  • the RTK which is over-expressed may be Flt4/VEGFR3.
  • the RTK which is over-expressed may be FRGF2/K-SAM.
  • the RTK may comprise a mutation compared to the corresponding, wild-type RTK amino acid sequence, or the polynucleotide sequence encoding the RTK polypeptide, which mutation enables the RTK to signal in the absence of cognate ligand.
  • the mutation may be a substitution, a deletion, an insertion or a duplication, for example.
  • BCR-ABL The best characterised fusion protein is BCR-ABL. This translocation happens between the long arms of chromosome 9 and 22 and is found in over 90% of chronic myelogenous leukaemia patients.
  • the BCR-ABL fusion protein consists of the SH1/SH2 (tyrosine kinase) of ABL fused in frame to the coiled-coil oligomerisation domain of BCR. This juxtaposition results in constitutive and ligand-independent dimerization and activation.
  • translocations involve the Platelets Derived Growth Factors Receptor Beta (PDGFR-B) and TEL.
  • PDGFR-B Platelets Derived Growth Factors Receptor Beta
  • TEL TEL
  • the translocation t(5;12)(q33;p13) results in the fusion of the SH2 domain of the PDGFR-B and the coiled coil domain of TEL.
  • this fusion protein acquires ligand-independent activation.
  • the RTK mutation may be a mutation as described herein.
  • Cognate ligand independent signalling caused by a mutation relative to a wild-type RTK sequence may be determined by assays as described herein.
  • the RTK for use in the present invention may be a chimeric RTK.
  • chimeric RTK refers to a RTK which comprises (a) a domain which mediates dimerization or oligomerization of the RTK and (b) an RTK intracellular signalling domain; wherein the domain which mediates dimerization or oligomerization of the RTK does not bind a wild-type RTK ligand.
  • the domain which mediates dimerization or oligomerization of the chimeric RTK is not a naturally occurring RTK ligand binding domain.
  • the present chimeric RTK is capable of dimerization and/or oligomerization and productive signalling in the absence of the cognate ligand for the RTK.
  • the present chimeric RTK is capable of dimerization and/or oligomerization and productive signalling in the absence of the cognate ligand for the RTK which corresponds to the RTK from which the present signalling domain in derived.
  • the present chimeric RTK does not comprise a ligand binding domain which is capable of binding to a wild-type RTK ligand.
  • the chimeric RTK may be a transmembrane protein which comprises a transmembrane domain and an RTK intracellular signalling domain.
  • a chimeric RTK which comprises a transmembrane domain further comprises an ectodomain.
  • the domain which mediates dimerization or oligomerization of the chimeric RTK may be present in the ectodomain or the endodomain of the chimeric RTK.
  • the present chimeric RTKs do not require the binding of cognate ligand to induce dimerization/oligomerization and subsequent signalling. Accordingly, the present chimeric RTKs may be intracellular, soluble molecules. In other words, the present chimeric RTKs may not comprise a transmembrane domain. In these embodiments, the chimeric RTK may comprise a dimerization/oligomerization domain and a RTK signalling domain as described herein.
  • the chimeric RTK may comprise a CAR antigen-binding domain, a RTK intracellular signalling domain and a domain which is capable of inducing dimerization/oligomerization of the chimeric RTK; as defined herein.
  • the domain which mediates dimerization or oligomerization of the chimeric RTK may be capable of mediating direct dimerization and/or oligomerization.
  • the domain may mediate dimerization and/or oligomerization through a direct interaction with a corresponding domain in a second chimeric RTK.
  • the domain which mediates direct dimerization or oligomerization may be any domain which is capable of inducing dimerization and/or oligomerization of a two or more proteins.
  • Suitable domains are well-known in the art and include, for example, hinge domains, hinge domains in combination with Fc stalk spacers, Fab antibody domains, CD28 stalks, CD8 stalks, coiled coil alpha helical zippers, and leucine zippers.
  • the domain which mediates direct dimerization or oligomerization may form at least one disulphide bond with a corresponding domain.
  • hinge domains, hinge domains in combination with Fc stalk spacers, Fab antibody domains, CD28 stalks, and CD8 stalks are each capable of forming at least one disulphide bond with a second corresponding domain.
  • the formation of disulphide bonds causes the chimeric RTK to dimerize and enables it to signal in the absence of cognate ligand.
  • the hinge domain may be a hinge domain from any suitable immunoglobulin.
  • the hinge domain may be an IgGi, lgG 2 , lgG 3 or lgG 4 hinge domain.
  • Illustrative hinge domains are shown as SEQ ID NO: 3-6.
  • SEQ ID NO: 3 (human lgG1 hinge): EPKSCDKTHTCP
  • SEQ ID NO: 4 (human lgG2 hinge): ERKCCVECPPCP
  • SEQ ID NO: 5 (human lgG3 hinge):
  • SEQ ID NO: 6 (human lgG4 hinge): ESKYGPPCPSCP
  • the hinge domain may comprise the sequence shown as SEQ ID NO: 3 to 6 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 dimerize.
  • 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 dimerization/oligomerization domain may comprise a hinge domain as described herein and further comprise an Fc stalk spacer.
  • the Fc stalk spacer may, for example comprise a CH2CH3 domain from an IgG molecule.
  • the hinge-Fc stalk spacer domain may comprise the sequence shown as SEQ ID NO: 7 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 dimerize.
  • the dimerization/oligomerization domain may be a dimerization domain from a Fab antibody fragment of any suitable immunoglobulin.
  • An illustrative Fab dimerization domain is shown as SEQ ID NO: 8 or 9.
  • SEQ ID NO: 8 Human CH1 sequence: STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
  • SEQ ID NO: 9 Human Light Kappa sequence
  • the Fab dimerization domain may comprise the sequence shown as SEQ ID NO: 8 or 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 dimerize.
  • the dimerization/oligomerization domain comprise a CD8 stalk domain.
  • An illustrative CD8 stalk domain is shown as SEQ ID NO: 10.
  • SEQ ID NO: 10 human CD8 stalk
  • the dimerization domain may comprise the sequence shown as SEQ ID NO: 10 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 dimerize.
  • the dimerization/oligomerization domain comprise a CD28 stalk domain.
  • An illustrative CD28 stalk domain is shown as SEQ ID NO: 11.
  • SEQ ID NO: 11 human CD28 stalk
  • the dimerization domain may comprise the sequence shown as SEQ ID NO: 11 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 dimerize.
  • the domain which mediates direct dimerization or oligomerization may comprise a coiled coil alpha helical zipper or a leucine zipper.
  • the coiled coil zipper or leucine zipper enable dimerization or oligomerization of the chimeric RTK, respectively, in the absence of cognate ligand. As such, the inclusion of either of these domains enables the chimeric RTK to signal in the absence of cognate ligand.
  • a coiled coil is a structural motif in which two to seven alpha-helices are wrapped together like the strands of a rope. Many endogenous proteins incorporate coiled coil domains.
  • the coiled coil domain may be involved in protein folding (e.g. it interacts with several alpha helical motifs within the same protein chain) or responsible for protein-protein interaction. In the latter case, the coiled coil can initiate homo or hetero oligomer structures.
  • multimer and ‘multimerization’ are synonymous and interchangeable with‘oligomer’ and‘oligomerization’.
  • Coiled coils usually contain a repeated pattern, hxxhcxc (SEQ ID NO: 12), of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeat.
  • the positions in the heptad repeat are usually labelled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, leucine, or valine. Folding a sequence with this repeating pattern into an alpha-helical secondary structure causes the hydrophobic residues to be presented as a 'stripe' that coils gently around the helix in left-handed fashion, forming an amphipathic structure.
  • the a-helices may be parallel or anti-parallel, and usually adopt a left-handed super-coil. Although disfavoured, a few right-handed coiled coils have also been observed in nature and in designed proteins.
  • the coiled coil domain may be any coiled coil domain which is capable of forming a coiled coil multimer such that a complex of chimeric RTKs comprising the coiled coil domain is formed.
  • coiled coil domain may be a synthetically generated coiled coil domain.
  • proteins which contain a coiled coil domain include, but are not limited to, kinesin motor protein, hepatitis D delta antigen, archaeal box C/D sRNP core protein, cartilage-oligomeric matrix protein (COMP), mannose-binding protein A, coiled-coil serine- rich protein 1 , polypeptide release factor 2, SNAP-25, SNARE, Lac repressor or apolipoprotein E.
  • Kinesin motor protein parallel homodimer (SEQ ID NO: 13)
  • Hepatitis D delta antigen parallel homodimer (SEQ ID NO: 14)
  • Archaeal box C/D sRNP core protein anti-parallel heterodimer (SEQ ID NO: 15)
  • Mannose-binding protein A parallel homotrimer (SEQ ID NO: 16)
  • Coiled-coil serine-rich protein 1 parallel homotrimer (SEQ ID NO: 17)
  • Polypeptide release factor 2 anti-parallel heterotrimer
  • Chain B VVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFR (SEQ ID NO: 19)
  • Chain B ALSEIETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIEYNVEHAVDYVERA VSDTKKAVKY (SEQ ID NO: 21)
  • Lac repressor parallel homotetramer
  • the coiled coil domain is capable of oligomerization.
  • the coiled coil domain may be capable of forming a trimer, a tetramer, a pentamer, a hexamer or a heptamer.
  • the coiled coil domain may comprise the sequence shown as any of SEQ ID NO: 13-25 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 enable oligomerization of the chimeric RTK.
  • the coiled coil domain may be the COMP coiled coil domain.
  • COMP is one of the most stable protein complexes in nature (stable from 0°C-100°C and a wide range of pH) and can only be denatured with 4-6M guanidine hydrochloride.
  • the COMP coiled coil domain is capable of forming a pentamer.
  • COMP is also an endogenously expressed protein that is naturally expressed in the extracellular space. This reduces the risk of immunogenicity compared to synthetic spacers.
  • the crystal structure of the COMP coiled coil motif has been solved which gives an accurate estimation on the spacer length.
  • the COMP structure is ⁇ 5.6nm in length (compared to the hinge and CH2CH3 domains from human IgG which is ⁇ 8.1 nm).
  • the coiled coil domain may consist of or comprise the sequence shown as SEQ ID NO: 26 or a fragment thereof.
  • the coiled coil domain may comprise a variant of one of the coiled coil domains described above, providing that the variant sequence retains the capacity to form a coiled coil oligomer.
  • the coiled coil domain may comprise a variant of the sequence shown as SEQ ID NO: 13-26 having at least 80, 85, 90, 95, 98 or 99% sequence identity, providing that the variant sequence retains the capacity to form a coiled coil oligomer.
  • a coiled-coil domain is different from a leucine zipper.
  • Leucine zippers are super-secondary structures that function as dimerization domains. Their presence generates adhesion forces in parallel alpha helices.
  • a single leucine zipper consists of multiple leucine residues at approximately 7-residue intervals, which forms an amphipathic alpha helix with a hydrophobic region running along one side. This hydrophobic region provides an area for dimerization, allowing the motifs to "zip" together.
  • Leucine zippers are typically 20 to 40 amino acids in length, for example approximately 30 amino acids.
  • Leucine zippers are typically formed by two different sequences, for example an acidic leucine zipper heterodimerizes with a basic leucine zipper.
  • An example of a leucine zipper is the docking domain (DDD1) and anchoring domain (AD1) which are described in more detail below.
  • Leucine zippers form dimers, whereas the coiled-coiled spacers of the present invention for multimers (trimers and above). Leucine zippers heterodimerise in the dimerization potion of the sequence, whereas coiled-coil domains homodimerise.
  • the dimerization and/or oligomerization of the present chimeric RTK may be controllable by the presence or absence of an agent.
  • the agent which induces or disrupts dimerization/oligomerization of the chimeric RTK may be a small molecule.
  • the dimerization and/or oligomerization of the chimeric RTK may only occur in the presence of an agent i.e. a separate molecule acting as an “inducer” of dimerization/oligomerization.
  • RTK signalling will occur in the absence of cognate ligand only in the presence of said agent.
  • CID chemical inducer of dimerization/oligomerization
  • the CID binding domains of chimeric RTKs of the invention and the corresponding CID may be any combination of molecules/peptides/domains which enables the selective co localisation and dimerization of the chimeric RTKs in the presence of the CID.
  • the CID agent is a molecule which is able to simultaneously bind to a first and second chimeric RTK in order to induce dimerization and/or oligomerization as defined herein.
  • the CID agent therefore comprises at least two binding moieties.
  • the CID may be any pharmaceutically acceptable molecule which can simultaneously be bound by at least two binding domains.
  • the CID may be capable of being delivered to the cytoplasm of a target cell and being available for intracellular binding.
  • the binding moieties of the CID may interact with identical binding domains present on the first and second chimeric RTKs of the present invention. That is, the CID may comprise two identical binding moieties such that it can simultaneously interact with a binding domain on a first chimeric RTK and an identical binding domain on a second RTK.
  • the CID and CID binding domains may be the FK506 binding protein (FKBP) ligand dimerization system described by Clackson et at. (PNAS; 1998; 95; 10437-10442, incorporated herein by reference).
  • FKBP FK506 binding protein
  • This dimerization system comprises two FKBP-like binding domains with a F36V mutation in the FKBP binding domain and a dimerization agent (AP1903) with complementary amino acid substitutions.
  • Exposing cells engineered to express FKBP-like binding domain fusion proteins to AP103 results in the dimerization of the proteins comprising the FKBP-like binding domains but no interactions involving endogenous FKBP.
  • the dimerization system described by Farrar et at. which utilises bacterial DNA gyrase B (GyrB) binding domains and the antibiotic coumermycin as CID may also be used in the signalling system of the present invention (Methods Enzymol; 2000; 327; 421-419 and Nature; 1996; 383; 178-181 , incorporated herein by reference).
  • the binding moieties of the CID may interact with different binding domains on the first and second RTKs. That is, the CID may comprise two different binding moieties which can simultaneously interact with a binding domain on the first RTK and a different binding domain on the second RTK.
  • the CID and CID binding domains may comprise the dimerization system described by Belshaw et at. (Nature; 1996; 93; 4604-4607, incorporated herein by reference), which utilises a FK506 (Tacrolimus)/cyclosporin fusion molecule as the CID agent with FK-binding protein 12 (FKBP12) and cylcophilin A as the binding domains.
  • the CID / CID binding domain may also be the rapamycin and FKBP12/FKBP12-Rapamycin Binding (FRB) domain of mTOR system described by Rivera et at. (Nature Med; 1996; 2; 1028-1032, incorporated herein by reference) or the non-immunosupressive rapamycin analogs (rapalogs) and FKBP12/FRB system described by Bayle et at. (Chem Bio; 2006; 13; 99-107).
  • the CID may be C-20-methyllyrlrapamycin (MaRap) or C16(S)- Butylsulfonamidorapamycin (C16-BS-Rap), as described by Bayle et at.
  • the CID may be C16-(S)- 3-methylindolerapamycin (C16-iRap) or C16-(S)-7-methylindolerapamycin (AP21976/C16- AiRap) as described by Bayle et al., in combination with the respective complementary binding domains for each.
  • dimerization systems suitable for use in the present invention include an estrone/biotin CID in combination with an oestrogen-binding domain (EBD) and a streptavidin binding domain (Muddana & Peterson; Org. Lett; 2004; 6; 1409-1412; Hussey et al.] J. Am. Chem. Soc.; 125; 3692-3693); a dexamethasone/methotrexate CID in combination with a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain (Lin et al.] J. Am. Chem.
  • RSL1 is a synthetic non-steroidal analogue of 20-hydroxyecdysone. It is a member of a class of insecticides known as diacylhydrazines and can function to act as a non-steroidal ecdysone agonist. These molecules induce premature moulting and larvae death but are well tolerated in vertebrates.
  • RSL1 has been used in artificial transcription switches in which a two-protein transcription switch is used consisting of a fusion between the ecdysone receptor (EcR) and GAL4, and the retinoid X receptor (RXR) and VP16.
  • EcR is modified to interact specifically with RSL13
  • RXR is chimeric comprising of helices 1-8 replaced with helices 1-8 of human RXRb, and helices 9-12 from Locusta migratoria RXR.
  • the present invention may use EcR and RXR domains for dimerisation in the presence of RSL1 or a derivative thereof.
  • rapamycin and FK506 act by inducing the heterodimerization of cellular proteins. Each drug binds with a high affinity to the FKBP12 protein, creating a drug-protein complex that subsequently binds and inactivates mTOR/FRAP and calcineurin, respectively.
  • the FKBP-rapamycin binding (FRB) domain of mTOR has been defined and applied as an isolated 89 amino acid protein moiety that can be fused to a protein of interest. Rapamycin can then induce the approximation of FRB fusions to FKBP12 or proteins fused with FKBP 12.
  • one of the CID binding domains may comprise FRB or a variant thereof and the other CID binding domain may comprise FKBP12 or a variant thereof.
  • the dimerization domains may be or comprise one the sequences shown as SEQ ID NO: 27 to SEQ ID NO: 31 or a variant thereof.
  • Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 27-31 , provided that the sequences provide an effective dimerization system. That is, provided that the sequences facilitate co-localisation of a first chimeric RTK and a second chimeric RTK of the present invention.
  • The“wild-type” FRB domain shown as SEQ ID NO: 28 comprises amino acids 2025-2114 of human mTOR.
  • the FRB sequence may comprise an amino acid substitution at one of more of the following positions: 2095, 2098, 2101.
  • the variant FRB may comprise one of the following amino acids at positions 2095, 2098 and 2101 :
  • FRB variants W or F Bayle et al (Chem Bio; 2006; 13; 99-107, incorporated herein by reference) describe the following FRB variants, annotated according to the amino acids at positions 2095, 2098 and 2101 (see Table 1 of Bayle et at) ⁇ KTW, PLF, KLW, PLW, TLW, ALW, PTF, ATF, TTF, KLF, PLF, TLF, ALF, KTF, KHF, KFF, KLF. These variants are capable of binding rapamycin and rapalogs to varying extents, as shown in Table 1 and Figure 5A of Bayle et al.
  • the chimeric RTK of the present invention may comprise one of these FRB variants.
  • the surface of rapamycin which contacts FRB may be modified.
  • Compensatory mutation of the FRB domain to form a burface that accommodates the“bumped” rapamycin restores dimerizing interactions only with the FRB mutant and not to the endogenous mTOR protein.
  • Bayle et al. describe various rapamycin analogs, or “rapalogs” and their corresponding modified FRB binding domains.
  • rapamycin analogs or “rapalogs” and their corresponding modified FRB binding domains.
  • C-20-methyllyrlrapamycin (MaRap) C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap) and C16-(S)-7- methylindolerapamycin (AP21976/C16-AiRap)
  • Other rapamycins/rapalogs include sirolimus and tacrolimus.
  • the dimerization and/or oligomerization of the chimeric RTK may spontaneously occur, but be disrupted in the presence of an agent i.e. a separate molecule acting as a“disruptor” of dimerization/oligomerization.
  • RTK signalling will occur in the absence of cognate ligand, but only in the absence of said agent.
  • the agent capable of acting as a“disruptor” of dimerization/oligomerization may be referred to herein as a first chemical disruptor of dimerization/oligomerization (CDD).
  • CDD first chemical disruptor of dimerization/oligomerization
  • the CDD may be capable of being delivered to the cytoplasm of a target cell and being available for intracellular binding.
  • the agent may be a molecule, for example a small molecule, which is capable of specifically binding to the first CDD binding domain or the second CDD binding domain at a higher affinity than the binding between the first CDD binding domain and the second CDD binding domain.
  • the binding system may be based on a peptide:peptide binding domain system.
  • the first or second CDD domain may comprise the peptide binding domain and the other binding domain may comprise a peptide mimic which binds the peptide binding domain with lower affinity than the peptide.
  • the use of peptide as agent disrupts the binding of the peptide mimic to the peptide binding domain through competitive binding.
  • the peptide mimic may have a similar amino acid sequence to the“wild-type” peptide, but with one of more amino acid changes to reduce binding affinity for the peptide binding domain.
  • the agent may bind the first binding domain or the second binding domain with at least 10, 20, 50, 100, 1000 or 10000-fold greater affinity than the affinity between the first binding domain and the second binding domain.
  • Small molecules agents which disrupt protein-protein interactions have long been developed for pharmaceutical purpose (reviewed by Vassilev et a/; Small-Molecule Inhibitors of Protein- Protein Interactions ISBN: 978-3-642-17082-9, incorporated herein by reference).
  • the proteins or peptides whose interaction is disrupted can be used as the first and/or second CDD and the small molecule may be used as the agent.
  • TetR Tet repressor
  • TetR Tet repressor
  • TiP TetR interacting protein
  • tetracycline system Tet operon
  • the Tet operon is a well-known biological operon which has been adapted for use in mammalian cells.
  • the TetR binds tetracycline as a homodimer and undergoes a conformational change which then modulates the DNA binding of the TetR molecules.
  • Klotzsche et at. (Nucleic Acids Res. 2009 Apr;37(6): 1778-88, incorporated herein by reference), described a phage-display derived peptide which activates the TetR.
  • This protein (TetR interacting protein/TiP) has a binding site in TetR which overlaps, but is not identical to, the tetracycline binding site.
  • TiP and tetracycline compete for binding of TetR.
  • the first CDD binding domain may be TetR or TiP
  • the second CDD binding domain may be the corresponding, complementary binding partner or variants thereof.
  • the agent may be tetracycline, doxycycline, minocycline or an analogue thereof.
  • An analogue refers to a variant of tetracycline, doxycycline or minocycline which retains the ability to specifically bind to TetR.
  • Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 32-33, provided that the sequences provide an effective dimerization system which is disrupted in the presence of tetracycline, doxycycline, minocycline or an analogue thereof. That is, provided that the sequences facilitate co-localisation of a first chimeric RTK and a second chimeric RTK of the present invention which may be disrupted in the presence of tetracycline, doxycycline, minocycline or an analogue thereof.
  • the chimeric RTK may comprise (i) an intracellular adaptor molecule which comprises at least two CDD binding domains and (ii) at least two intracellular molecules which each comprise a RTK signalling domain and a second CDD binding domain wherein dimerization between the first and second CDD binding domains is disrupted in the presence of a CDD.
  • the CDD binding domains of the adaptor molecule may be connected via a linker peptide.
  • the linker peptide may be any peptide which is able to connect the CDD binding domains such that each CDD binding domain can independent dimererize with a second CDD binding domain.
  • Suitable linker peptides are well known in the art and include, for example, the linker shown as SEQ ID NO: 34.
  • the linker may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as the sequence shown as SEQ ID NO: 35.
  • the linker may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 34 or 35.
  • the present chimeric RTK may comprise a signalling domain from any RTK.
  • the chimeric RTK may comprise a signalling domain from a RTK listed in Table 2.
  • the chimeric RTK may comprise a signalling domain from Met/c-Met, FGFR1 , FLT3, EGFR, ErbB2/HER2, Kit/SCFR, Flt4/VEGFR3 orFRGF2/K-SAM, or a variant thereof provided that the variant sequence retains the capacity to signal via a tyrosine kinase domain.
  • the chimeric RTK may comprise a signalling domain from Met/c-Met or FGFR1 , or a variant thereof provided that the variant sequence retains the capacity to signal via a tyrosine kinase domain.
  • the chimeric RTK may comprise a signalling domain shown as any of SEQ ID NO: 36 or 37, or a variant thereof.
  • Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 36 or 37 provided that the variant sequence retains the capacity to signal via a tyrosine kinase domain.
  • SEQ ID NO: 36 Metal/c-Met signalling domain
  • Classical CARs which are shown schematically in Figure 1 , are chimeric type I trans membrane proteins which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain).
  • the binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site or on a ligand for the target antigen.
  • mAb monoclonal antibody
  • a spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation.
  • a common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen.
  • a trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.
  • CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.
  • the antigen-binding domain is the portion of a classical CAR which recognizes antigen.
  • the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a wild-typeligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • 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 transmembrane domain is the sequence of a classical CAR that spans the membrane. It may comprise a hydrophobic alpha helix.
  • the transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the CAR or RTK of the present invention may comprise a signal peptide so that when it is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • the CAR may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain.
  • a flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
  • 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.
  • the intracellular signalling domain is the signal-transmission portion of a classical CAR.
  • 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 0X40 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 intracellular signalling domain may be or comprise a T cell signalling domain.
  • the intracellular signalling domain may comprise one or more immunoreceptor tyrosine- based activation motifs (ITAMs).
  • ITAM immunoreceptor tyrosine- based activation motifs
  • An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system.
  • the motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/i. Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/!x (6.8) YxxL/i).
  • ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of important cell signalling molecules such as the CDS and z-chains of the T cell receptor complex, the CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors.
  • the tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the ceil.
  • the intracellular signalling domain component may comprise, consist essentially of, or consist of the O ⁇ 3-z endodomain, which contains three ITAMs.
  • the CDS-z endodomain transmits an activation signal to the T cell after antigen is bound.
  • the CDS-z endodomain transmits an activation signal to the T cell after the MHC/peptide complex comprising the engineered B2M binds to a TCR on a different T cell.
  • the intracellular signalling domain may comprise additional co-stimulatory signalling.
  • 4-1 BB also known as CD137
  • CD28 and 0X40 can be used with CDS-z to transmit a proliferative / survival signal.
  • intracellular signalling domain may comprise the CDS-z endodomain alone, the CDS-z endodomain in combination with one or more co-stimulatory domains selected from 4- 1 BB, CD28 or 0X40 endodomain, and/or a combination of some or all of 4-1 BB, CD28 or 0X40.
  • the endodomain may comprise one or more of the following: an ICOS endodomain, a CD2 endodomain, a CD27 endodomain, or a CD40 endodomain.
  • the endomain may comprise the sequence shown as SEQ ID NO: 38 to 41 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 transmit an activating signal to the 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.
  • the CAR may have the general format: antigen-binding domain-TCR element.
  • TOR element means a domain or portion thereof of a component of the TOR receptor complex.
  • the TOR element may comprise (e.g. have) an extracellular domain and/or a transmembrane domain and/or an intracellular domain e.g. intracellular signalling domain of a TOR element.
  • the TOR element may selected from TOR alpha chain, TOR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, CD3 epsilon chain.
  • the TOR element may comprise the extracellular domain of the TOR alpha chain, TOR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.
  • the TOR element may comprise the transmembrane domain of the TOR alpha chain, TOR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.
  • the TCR element may comprise the intracellular domain of the TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.
  • the TCR element may comprise the TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, or CD3 epsilon chain.
  • T-cell receptor is a molecule found on the surface of T cells which is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is a heterodimer composed of two different protein chains.
  • the TCR in 95% of T cells the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR consists of gamma and delta (g/d) chains (encoded by TRG and TRD, respectively).
  • the T lymphocyte When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction.
  • antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex.
  • 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 a 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 cell of the present invention may be an immune effector cell, such as a T-cell, a wild- typekiller (NK) cell or a cytokine induced killer cell.
  • an immune effector cell such as a T-cell, a wild- typekiller (NK) cell or a cytokine induced killer cell.
  • the T cell may be an alpha-beta T cell or a gamma-delta T cell.
  • the cell may be derived from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).
  • T or NK cells for example, may be activated and/or expanded prior to being transduced with nucleic acid molecule(s) encoding the polypeptides of the invention, for example by treatment with an anti-CD3 monoclonal antibody.
  • 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.
  • cytokines such as G-CSF [peripheral blood stem cells (PBSCs)]
  • 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 CAR or transgenic TCR and the RTK are not naturally expressed by a corresponding, unmodified cell - for example an unmodified alpha-beta T cell, a NK cell, a gamma-delta T cell or cytokine- induced killer cell.
  • the present invention provides a nucleic acid construct which comprises: (i) a first nucleic acid sequence which encodes i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) a second nucleic acid sequence which encodes a receptor tyrosine kinase (RTK).
  • a nucleic acid construct which comprises: (i) a first nucleic acid sequence which encodes i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) a second nucleic acid sequence which encodes a receptor tyrosine kinase (RTK).
  • the RTK is capable of signalling in the absence of cognate ligand when it is overexpressed in a cell.
  • the RTK is a chimeric RTK according to the present invention.
  • the present invention further provides a nucleic acid construct comprising a first and a second nucleic acid sequence encoding a first and a second chimeric RTK which dimerize in a chemically-operable manner, as provided by the present invention.
  • the present invention further provides a nucleic acid construct which comprises (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic T- cell receptor (TCR); (ii) nucleic acid sequences encoding a first and a second chimeric RTK which dimerize in a chemically-operable manner, as provided by the present invention.
  • a nucleic acid construct which comprises (i) a first nucleic acid sequence which encodes a chimeric antigen receptor (CAR) or a transgenic T- cell receptor (TCR); (ii) nucleic acid sequences encoding a first and a second chimeric RTK which dimerize in a chemically-operable manner, as provided by the present invention.
  • the present invention further provides a kit comprising nucleic acid sequences according to the present invention.
  • the kit may comprise (i) a first nucleic acid sequence which encodes i) a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR); and (ii) a second nucleic acid sequence which encodes a receptor tyrosine kinase (RTK) or a first and second chimeric RTK which dimerize in a chemically-operable manner according to the present invention.
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • RTK receptor tyrosine kinase
  • polynucleotide As used herein, the terms“polynucleotide”,“nucleotide”, and“nucleic acid” are intended to be synonymous with each other.
  • the nucleic acid construct may comprise a plurality of nucleic acid sequences which encode different RTKs as provided by the present invention.
  • the nucleic acid construct may comprise two, three, four or five nucleic acid sequences which different RTKs of the invention.
  • the plurality of nucleic acid sequences may be separated by co expression sites as defined herein.
  • polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
  • the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular an immune effector cell as described herein.
  • 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 number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • the terms“variant”,“homologue” or“derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • a co-expression site is used herein to refer to a nucleic acid sequence enabling co expression of separate nucleic acid sequences of the present invention.
  • 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 may be used.
  • 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.
  • TSV Tobacco Etch Virus
  • 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 self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
  • the primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus.
  • apthoviruses such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus
  • the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating“cleavage” at its own C-terminus (Donelly et al (2001) as above).
  • 2A-like sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al., 2001) as above.
  • the co-expression sequence may be an internal ribosome entry sequence (IRES).
  • the co expressing sequence may be an internal promoter.
  • 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 a receptor tyrosine kinase (RTK) as defined herein.
  • RTK receptor tyrosine kinase
  • the vector may comprise a plurality of nucleic acid sequences which encode different RTKs as provided by the present invention.
  • the vector may comprise two, three, four or five nucleic acid sequences which different RTKs of the invention.
  • the plurality of nucleic acid sequences may be separated by co-expression sites as defined herein.
  • 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 present invention also relates to a pharmaceutical composition containing 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.
  • the invention relates to a pharmaceutical composition containing a cell according to the present invention.
  • 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.
  • the present invention provides a method for treating and/or preventing a disease which comprises the step of administering 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 example in a pharmaceutical composition as described above) to a subject.
  • the present methods for treating and/or preventing a disease may comprise administering a cell of the invention (for example in a pharmaceutical composition as described above) to a subject.
  • 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 method may involve the steps of:
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and any toxic activity and administering an agent which is capable of inhibiting signalling of the RTK, such as a small molecular RTK inhibitor, to the subject in order to reduce or lessen any adverse toxic effects.
  • an agent which is capable of inhibiting signalling of the RTK such as a small molecular RTK inhibitor
  • the agent may be a small molecule selected from the inhibitors listed in Table 5. It will be apparent that the inhibitor should be selected such that it is capable of inhibiting the RTK expressed in the engineered cell of the invention.
  • the method may involve monitoring the progression of the disease and any toxic activity and administering an agent which is capable of disrupting dimerization or oligomerization of the chimeric RTKs to the subject in order to reduce or lessen any adverse toxic effects.
  • the method may involve monitoring the progression of the disease and any toxic activity and adjusting the dose of the agent administered to the subject to provide acceptable levels of disease progression and toxic activity.
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and monitoring any toxic activity and adjusting the dose of the agent administered to the subject to provide acceptable levels of disease progression and toxic activity.
  • Monitoring the progression of the disease means to assess the symptoms associated with the disease over time to determine if they are reducing/improving or increasing/worsening.
  • Toxic activities relate to adverse effects caused by the cells of the invention following their administration to a subject.
  • Toxic activities may include, for example, immunological toxicity, biliary toxicity and respiratory distress syndrome.
  • the survival and proliferation of cells of the present invention may be adjusted by altering the amount of a small molecule RTK inhibitor or CID/CDD agent present, or the amount of time the small molecule RTK inhibitor or CID/CDD agent is present.
  • the present invention also provides a method for treating and/or preventing a disease in a subject which subject comprises cells of the invention, which method comprises the step of administering a small molecule RTK inhibitor or CID/CDD agent to the subject.
  • this method involves administering a suitable a small molecule RTK inhibitor or CID/CDD agent to a subject which already comprises cells of the present invention.
  • the dose of the small molecule RTK inhibitor or CID/CDD agent administered to a subject, or the frequency of administration may be altered in order to provide an acceptable level of both disease progression and toxic activity.
  • the specific level of disease progression and toxic activities determined to be‘acceptable’ will vary according to the specific circumstances and should be assessed on such a basis.
  • the present invention provides a method for altering the survival and/or proliferation of the cells of the present invention in order to achieve this appropriate level.
  • the small molecule RTK inhibitor or CID/CDD agent may be administered in the form of a pharmaceutical composition.
  • 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.
  • 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 invention also relates to the use of 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 of the present invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the invention relates to the use of a cell in the manufacture of a medicament for the treatment and/or prevention of a disease
  • the disease to be treated and/or prevented by the method of the present invention may be cancer.
  • the cancer may be such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.
  • 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.
  • the cancer may be a cancer listed 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 a receptor tyrosine kinase (RTK) as defined herein by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • RTK receptor tyrosine kinase
  • the cell of the invention may be made by:
  • transduction or transfection of the cells with one or more a nucleic acid sequence(s) or nucleic acid construct as defined above in vitro or ex vivo.
  • the cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.
  • Receptor tyrosine kinases are single-pass transmembrane cell surface receptors which upon binding of their cognate ligand, dimerise, juxtaposing the intracellular tyrosine kinase domains of each receptor monomer.
  • the dimerization causes transphosphorylation of tyrosine residues within the cytoplasmic tail which in turn recruit a variety of SH2-domain containing proteins.
  • the resulting cell signalling plays an important role in the regulation of the growth, differentiation, and survival of the cell.
  • a constitutively active signalling platform was designed.
  • Primary human T-cells were transduced with a number of chimeric constructs, each consisting of anti-CD19-binding scFv (Fmc63), fused to Fc-spacer (from lgG1), which in turn is fused to a transmembrane domain that anchors the protein on the cell surface.
  • the endodomain of each construct is derived from the intracellular domain of a member of the RTK family. All constructs are expressed as dimers (dimerization occurs through the Fc-stalk) ensuring constitutive tonic RTK signalling (see Figure 6A).
  • transduced T cells were normalised to 30% transduction efficiency and labelled with the dye Cell Trace Violet (CTV), a fluorescent dye which is hydrolysed and retained within the cell.
  • CTV Cell Trace Violet
  • Labelled T cells were cultured in the absence of cytokine support for 7 days. At the end of the 7-day period cells were analysed by flow cytometry to measure the dilution of the Cell Trace Violet which occurs as the T cells divide.
  • the histogram of the CTV fluorescence of non- transduced T cells is used as a negative control for placing a proliferation gate.
  • the CTV traces that fall within the proliferation gate form the percent proliferating T-cells.
  • Figure 6B shows a number of RTK constructs that are capable of improving proliferation of T cells cultured in the absence of cytokine support for 7 days.
  • NT and 35665 are negative and positive controls; respectively.
  • test constructs shown in Figure 6B are identified in the Table below.

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Abstract

La présente invention concerne une cellule qui comprend : (i) un récepteur antigénique chimérique (CAR) ou un récepteur de lymphocyte T transgénique (TCR) ; et (ii) un récepteur de tyrosine kinase (RTK) capable de signaler en l'absence d'un ligand cognate.
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WO2021156277A1 (fr) 2020-02-04 2021-08-12 Miltenyi Biotec B.V. & Co. KG Cellule immunitaire exprimant un récepteur d'antigène chimère adaptateur pour la détection d'antigènes solubles
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EP3915578A1 (fr) 2020-05-28 2021-12-01 Miltenyi Biotec B.V. & Co. KG Récepteur d'antigène chimérique doté d'un espaceur comprenant des domaines d'ensemble c2 de type ig
WO2022096664A1 (fr) 2020-11-09 2022-05-12 Miltenyi Biotec B.V. & Co. KG Procédés et compositions pour éliminer des cellules immunitaires modifiées
WO2023057285A1 (fr) 2021-10-06 2023-04-13 Miltenyi Biotec B.V. & Co. KG Procédé d'insertion ciblée de gènes dans des cellules immunitaires
WO2024078995A1 (fr) 2022-10-15 2024-04-18 Miltenyi Biotec B.V. & Co. KG Transduction de lymphocytes t gammadelta avec des vecteurs retroviraux pseudotypés

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