WO2023198849A1 - Lymphocytes t modifiés pour expression de l'interleukine 7 et du ligand 19 de la chimiokine à motif c-c - Google Patents

Lymphocytes t modifiés pour expression de l'interleukine 7 et du ligand 19 de la chimiokine à motif c-c Download PDF

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WO2023198849A1
WO2023198849A1 PCT/EP2023/059714 EP2023059714W WO2023198849A1 WO 2023198849 A1 WO2023198849 A1 WO 2023198849A1 EP 2023059714 W EP2023059714 W EP 2023059714W WO 2023198849 A1 WO2023198849 A1 WO 2023198849A1
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cells
cell
cancer
population
tcr
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Katherine Jane ADAMS
Louise Vera RICE
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Adaptimmune Limited
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    • 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
    • A61K39/4632—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
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4635—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4637—Other peptides or polypeptides
    • 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
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464486—MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5418—IL-7

Definitions

  • T Cells Engineered to Express Interleukin 7 and C-C motif Chemokine Ligand 19 Field The present invention relates to the modification of T cells to increase their cytotoxic activity and the use of modified T cells in immunotherapy, for example for the treatment of solid cancers.
  • Background T cells or T lymphocytes
  • T cells are found widely distributed within tissues and the tumour environment.
  • T cells are distinguished from other lymphocytes by the presence of T cell receptors (TCRs) on the cell surface.
  • TCRs T cell receptors
  • the TCR is a multi-subunit transmembrane complex that mediates the antigen-specific activation of T cells.
  • the TCR confers antigen specificity on the T cell, by recognising an antigen peptide ligand that is presented on the target cell by a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • peptides derived from altered or mutated proteins in tumour target cells can be recognised as foreign by T cells expressing specific TCRs, many antigens on tumour cells are simply upregulated or overexpressed (so called self-antigens) and do not induce a functional T cell response. Therefore, studies have focussed on identifying target tumour antigens which are expressed, or highly expressed, in the malignant but not the normal cell type.
  • CT cancer/testis
  • NY-ESO-1 cancer/testis
  • MAGE- A family of CT antigens which are expressed in a very limited number of healthy tissues.
  • Identification of such antigens has promoted the development of targeted T cell-based immunotherapy, which has the potential to provide specific and effective cancer therapy (Ho, W.Y. et al. Cancer Cell 2003; 3:1318-1328; Morris, E.C. et al. Clin.
  • IL-7 interleukin 7
  • Rosenberg, S.A. Nature 2001; 411:380-384; Boon, T. and van der Bruggen P. J. Exp. Med.1996; 183:725-729 The intravenous administration of interleukin 7 (IL-7) has been proposed to improve outcomes in T cell-based immunotherapy.
  • IL-7 is known to bolster the persistence of tumour- specific T-cells (Melchionda, F. et al. J. Clin.
  • T-cells genetically modified to either secrete IL-7 or overexpress the IL-7 receptor display enhanced antitumour efficacy in preclinical models (Vera, J.F. et al. Mol. Ther.2009;17:880-8, Markley, J.C. and Sadelain, M. Blood 2010;115:3508- 3519).
  • systemic administration of cytokines to patients with cancer has caused significant toxicity (Sportes, C. et al. Clin. Cancer Res.2010; 16:727-35, Conlon, K.C. t al. J. Clin. Oncol.2015;33:74-82, Brudno, J.N.
  • a first aspect of the invention provides a T cell that expresses a heterologous T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4 and further expresses heterologous interleukin 7 (IL-7) and heterologous C-C motif chemokine ligand 19 (CCL19).
  • TCR heterologous T cell receptor
  • IL-7 heterologous interleukin 7
  • CCL19 heterologous C-C motif chemokine ligand 19
  • a T cell of the first aspect may comprise; (i) a first heterologous nucleotide sequence encoding a T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4; (ii) a second heterologous nucleotide sequence encoding C-C motif chemokine ligand 19 (CCL19); and (iii) a third heterologous nucleotide sequence encoding interleukin 7 (IL-7).
  • TCR T cell receptor
  • CCL19 C-C motif chemokine ligand 19
  • IL-7 interleukin 7
  • a second aspect of the invention provides a nucleic acid construct comprising; (i) a first heterologous nucleotide sequence encoding a T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4; (ii) a second heterologous nucleotide sequence encoding C-C motif chemokine ligand 19 (CCL19); and (iii) a third heterologous nucleotide sequence encoding interleukin 7 (IL-7).
  • TCRs of the first and second aspect are human affinity enhanced TCRs.
  • a third aspect of the invention provides a vector, for example a lentiviral vector, comprising a nucleic acid construct of the second aspect.
  • a fourth aspect of the invention provides a population of T cells according to the first aspect.
  • a fifth aspect of the invention provides a pharmaceutical composition comprising a population of T cells according to the fourth aspect and a pharmaceutically acceptable excipient.
  • a sixth aspect of the invention provides a population of T cells according to the fourth aspect for use in a method of treatment of the human or animal body, for example a method of treatment of cancer in an individual.
  • Related aspects provide the use of a population of T cells according to the fourth aspect in the manufacture of a medicament for the treatment of cancer in an individual and a method of treating cancer comprising administering to an individual with cancer a population of T cells according to the fourth aspect.
  • a seventh aspect of the invention provides a method of producing a population of modified T cells comprising; introducing a first heterologous nucleotide sequence encoding a T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4; a second heterologous nucleotide sequence encoding C-C motif chemokine ligand 19 (CCL19); and a third heterologous nucleotide sequence encoding interleukin 7 (IL-7) into a population of T cells obtained from a donor individual to produce a population of modified T cells.
  • TCR T cell receptor
  • CCL19 C-C motif chemokine ligand 19
  • IL-7 interleukin 7
  • a method of the seventh aspect may comprise introducing a nucleic acid construct or a vector according to the second or third aspects into a population of T cells obtained from a donor individual to produce a population of modified T cells.
  • An eighth aspect of the invention provides a method of producing a population of modified T cells comprising; introducing a first heterologous nucleotide sequence encoding a T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4; a second heterologous nucleotide sequence encoding C-C motif chemokine ligand 19 (CCL19); and a third heterologous nucleotide sequence encoding interleukin 7 (IL-7) into a population of progenitor cells; and differentiating the population of progenitor cells into a population of modified T cells.
  • TCR T cell receptor
  • CCL19 C-C motif chemokine ligand 19
  • a method of the eighth aspect may comprise introducing a nucleic acid construct or a vector according to the second or third aspects into the population of progenitor cells.
  • Progenitor cells for use in methods of the eighth aspect may include induced pluripotent stem cells (iPSCs), mesoderm cells (MCs), haematopoietic progenitor cells (HPCs) or progenitor T cells.
  • iPSCs induced pluripotent stem cells
  • MCs mesoderm cells
  • HPCs haematopoietic progenitor cells
  • a ninth aspect of the invention provides a method of treating cancer in an individual in need thereof comprising; producing a population of modified T cells by a method of the seventh or eighth aspect, and administering the population of modified T cells to a recipient individual.
  • a tenth aspect of the invention provides a method of treating cancer in an individual in need thereof comprising; introducing a nucleic acid construct or a vector according to the second or third aspects into a population of T cells obtained from a donor individual to produce a population of modified T cells, and administering the population of modified T cells to a recipient individual.
  • the donor individual and the recipient individual may be the same (i.e. autologous treatment; the modified T cells are obtained from an individual who is subsequently treated with the modified T cells) or the donor individual and the recipient individual may be different (i.e. allogeneic treatment; the modified T cells are obtained from one individual and subsequently used to treat a different individual).
  • Suitable cancers for treatment in accordance with the ninth or tenth aspects include solid tumours.
  • Figure 1 shows the change in T-cell counts following antigen stimulation.
  • ADP-A2M4, ADP- A2M4N7X19 and ntd (non-transduced) T-cells from 3 donors were stimulated on a weekly basis with irradiated A375 (MAGE-A4 antigen-positive) cells. Cell counts were performed every 7-days and used to calculate fold-change from the known density of cells seeded.
  • Figure 2 shows the change in CD8 and TCR-positive and TCR-negative fractions of ADP-A2M4N7X19 following antigen stimulation.
  • ADP-A2M4N7X19 were stimulated on a weekly basis with irradiated A375 (MAGE-A4 antigen-positive) cells.
  • CD8 and anti-TCR antibodies were used to determine all possible combinations of expression patterns for the two markers on CD45+ T-cells. Each subset frequency was calculated by multiplying TCR+/- frequency with the parental CD8+/- frequency. The sum of these was assumed to be 100 % and each frequency was expressed as a percentage of this (normalized frequency).
  • absolute cell counts were performed every 7 days (CD45+ T-cells/mL) and used to calculate fold change from the known density of cells seeded (0.5 x 106 CD45+ T-cells/mL).
  • FIG 4 shows IFN ⁇ production.
  • ADP-A2M4, ADP-A2M4N7X19 and ntd (non-transduced) T- cells from 3 donors were stimulated on a weekly basis with irradiated A375 (MAGE-A4 antigen-positive) cells.
  • Supernatants were collected at day 1, 7, 8, 14, 15, 21, 22, and 28 and cytokine levels (pg/mL) determined on supernatants. Each point represents the mean from duplicate wells from a single condition. Closed circles represent points falling within the range of the standard curve. Open circles represent data above or below the range of the standard curve.
  • Figure 5 shows IL-7 production.
  • ADP-A2M4, ADP-A2M4N7X19 and ntd (non-transduced) T- cells from 3 donors were stimulated on a weekly basis with irradiated A375 (MAGE-A4 antigen-positive) cells.
  • Supernatants were collected at day 1, 7, 8, 14, 15, 21, 22, and 28 and cytokine levels (pg/mL) determined on supernatants. Each point represents the mean from duplicate wells from a single condition. Closed circles represent points falling within the range of the standard curve. Open circles represent data above or below the range of the standard curve. Figure 6 shows CCL19 production.
  • ADP-A2M4, ADP-A2M4N7X19 and ntd (non-transduced) T-cells from 3 donors were stimulated on a weekly basis with irradiated A375 (MAGE-A4 antigen-positive) cells.
  • Supernatants were collected at day 1, 7, 8, 14, 15, 21, 22, and 28 and cytokine levels (pg/mL) determined on supernatants. Each point represents the mean from duplicate wells from a single condition. Closed circles represent points falling within the range of the standard curve. Open circles represent data above or below the range of the standard curve.
  • Figure 7 shows mDC migration towards activated ADP-A2M4N7X19.
  • ADP-A2M4, ADP- A2M4N7X19, ADP-A2M4IL7 and ntd (non-transduced) T-cells from 3 donors were incubated with NCI-H1755 (MAGE-A4 antigen-positive) cells in the lower chamber of a transwell plate for 48 hours.
  • the upper chamber pre-seeded with primary dermal microvascular endothelial cells, had fluorescently stained mDCs added followed by measurement of migration in a plate reader over 6 hours.
  • a positive control of rh-CCL19 was added to independent wells.
  • Figure 8 shows the cytotoxic activity of ADP-A2M4N7X19 in response to tumor cell lines.
  • the tumor target cell lines, A375 (HLA-A2+/MAGE-A4 + ), and Colo205 (HLA-A2 + /MAGE-A4-) were cultured in the absence or presence of MAGE-A4 peptide (10 -6 and 10 -8 M), were co- incubated with afamitresgene autoleucel (red), ADP-A2M4N7X19 (blue) or ntd (non- transduced; grey) from 3 donors (Wave266, Wave267 and Wave268).
  • Wells containing targets cells only (black) were a control for background target cell death.
  • IncuCyte® Caspase-3/7 Green Dye reagent was added to all the wells, followed by peptide to the relevant wells. A single image was taken of each well every 3h for a period of 72h. The number of green (caspase3/7 + ) objects/mm 2 , a measure of target cells undergoing apoptosis, was determined and plotted against time. Data shows mean +/- SEM of triplicate wells. Figure 9 shows the survival rate of P1A-TCR in vivo study.
  • This invention relates to T cells that express a heterologous T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4.
  • TCR heterologous T cell receptor
  • the T cells are further modified to express heterologous interleukin 7 (IL-7) and heterologous C-C motif chemokine ligand 19 (CCL19).
  • IL-7 and CCL19 are shown herein to improve the anti-tumour activity of the T cells described herein.
  • the T cells may show improved or enhanced survival and proliferation and/or reduced T-cell exhaustion relative to T cells that do not express IL-7 and CCL19.
  • the T cells may increase the survival and proliferation of other immune cells, such as tumour infiltrating lymphocytes (TILs), in the tumour microenvironment of a patient, for example through paracrine effects; and/or the T cells may increase and/or accelerate the migration of other immune cells, such as dendritic cells (DCs), into the solid tumour of a patient.
  • TILs tumour infiltrating lymphocytes
  • DCs dendritic cells
  • T cells described herein stimulated on a weekly basis with irradiated MAGE-A4 antigen-positive cells as described herein may secrete 10-fold or more, 20-fold or more, 30- fold or more, 50-fold or more or 100-fold or more IFN ⁇ at day 22 relative to T cells that do not express heterologous IL-7 and CCL19.
  • T cells described herein that are stimulated on a weekly basis with irradiated MAGE-A4 antigen-positive cells as described herein may show an increase in cell count of 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more or 100-fold or more at day 28 relative to T cells that do not express heterologous IL-7 and CCL19.
  • T cells as described herein that are stimulated with MAGE-A4 antigen-positive cells. for example in a transwell assay described herein may increase the migration of dendritic cells by 2-fold or more, 3-fold or more, or 4-fold or more after 6 hours relative to T cells that do not express heterologous IL-7 and CCL19.
  • T cells also called T lymphocytes
  • T cells are white blood cells that play a central role in cell- mediated immunity. T cells can be distinguished from other lymphocytes by the presence of a T cell receptor (TCR) on the cell surface.
  • T-cells do not express endogenous IL-7 or CCL19.
  • TCR T cell receptor
  • T helper cells are known as CD4 + T cells because they express the CD4 surface glycoprotein.
  • CD4 + T cells play an important role in the adaptive immune system and help the activity of other immune cells by releasing T cell cytokines and helping to suppress or regulate immune responses. They are essential for the activation and growth of cytotoxic T cells.
  • Cytotoxic T cells TC cells, CTLs, killer T cells
  • CD8 + T cells act to destroy virus-infected cells and tumour cells.
  • Most CD8 + T cells express TCRs that can recognise a specific antigen displayed on the surface of infected or damaged cells by a class I MHC molecule.
  • T cells for use as described herein may be CD4 + T cells; CD8 + T cells; or CD4 + T cells and CD8 + T cells.
  • the T cells may be a mixed population of CD4 + T cells and CD8 + T cells.
  • Suitable T cells for use as described herein are human T cells and may be obtained, for example from a donor individual.
  • the donor individual may be the same person as the recipient individual to whom the T cells will be administered following modification and expansion as described herein (autologous treatment).
  • the donor individual may be a different person to the recipient individual to whom the T cells will be administered following modification and expansion as described herein (allogeneic treatment).
  • the donor individual may be a healthy individual who is human leukocyte antigen (HLA) matched (either before or after donation) with a recipient individual suffering from cancer.
  • HLA human leukocyte antigen
  • Other suitable T cells may be obtained by the directed differentiation or forward programming of pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). Suitable methods are described in for example WO2021/032855, WO2021/032851, WO2021/032852 and WO2021/032836.
  • a method described herein may comprise the step of obtaining T cells from a donor individual and/or isolating T cells from a sample obtained from a donor individual with cancer.
  • a population of T cells may be isolated from a blood sample. Suitable methods for the isolation of T cells are well known in the art and include, for example fluorescent activated cell sorting (FACS: see for example, Rheinherz et al (1979) PNAS 764061), cell panning (see for example, Lum et al (1982) Cell Immunol 72122) and isolation using antibody coated magnetic beads (see, for example, Gaudernack et al 1986 J Immunol Methods 90179).
  • FACS fluorescent activated cell sorting
  • cell panning see for example, Lum et al (1982) Cell Immunol 72122
  • isolation using antibody coated magnetic beads see, for example, Gaudernack et al 1986 J Immunol Methods 90179.
  • CD4 + and CD8 + T cells may be isolated from the population of peripheral blood mononuclear cells (PBMCs) obtained from a blood sample.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs may be extracted from a blood sample using standard techniques. For example, ficoll TM may be used in combination with gradient centrifugation (Böyum A. Scand J Clin Lab Invest.1968; 21(Suppl.97):77-89), to separate whole blood into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells and erythrocytes.
  • the PBMCs may be depleted of CD14 + cells (monocytes). Following isolation, the T cells may be activated.
  • Suitable methods for activating T cells are well known in the art.
  • the isolated T cells may be exposed to a T cell receptor (TCR) agonist.
  • TCR agonists include ligands, such as a peptide displayed on a class I or II MHC molecule on the surface of an antigen presenting cell, such as a dendritic cell, and soluble factors, such as anti-TCR antibodies.
  • An anti-TCR antibody may specifically bind to a component of the TCR, such as ⁇ CD3, ⁇ CD3 or ⁇ CD28.
  • Anti-TCR antibodies suitable for TCR stimulation are well-known in the art (e.g. OKT3) and available from commercial suppliers (e.g. eBioscience CO USA).
  • T cells may be activated by exposure to anti- ⁇ CD3 antibodies and IL2. More preferably, T cells are activated by exposure to anti- ⁇ CD3 antibodies and anti- ⁇ CD28 antibodies. The activation may occur in the presence or absence of CD14 + monocytes.
  • the T cells may be activated with anti-CD3 and anti-CD28 antibody coated beads.
  • PBMCs or T cell subsets including CD4 + and/or CD8 + cells may be activated, without feeder cells (antigen presenting cells) or antigen, using antibody coated beads, for example magnetic beads coated with anti-CD3 and anti-CD28 antibodies, such as Dynabeads® Human T-Activator CD3/CD28 (ThermoFisher Scientific).
  • T cells described herein express heterologous IL-7 and CCL19 in addition to a heterologous TCR that specifically binds to cancer cells that express MAGE A4.
  • Interleukin 7 is a haematopoietic cytokine that binds to the heterodimeric IL-7 receptor and is involved in T lymphopoiesis in the thymus, T-cell homeostasis, lymphopenia-driven proliferation and regulation of lymph node organogenesis.
  • IL-7 may be human IL-7 (Gene ID 3574) and may have the reference amino acid sequence of NP_00871.1 (SEQ ID NO: 2 or SEQ ID NO: 4) and may be encoded by the reference nucleotide sequence of NM_000880.4 (SEQ ID NO: 1).
  • a nucleotide sequence encoding IL-7 may be codon-optimised for expression in human T cells.
  • nucleotide sequence encoding IL-7 may have the nucleotide sequence of SEQ ID NO: 3.
  • CCL19 is a cytokine that binds to the chemokine receptor CCR7 and acts on migratory cells of the adaptive immune system, including na ⁇ ve T-cells, central memory T-cells, regulatory T-cells, na ⁇ ve B-cells, semi-mature/mature dendritic cells (DCs) and natural killer (NK) cells.
  • CCL19 may be human CCL19 (Gene ID 6363) and may have the reference amino acid sequence of NP_006265.1 (SEQ ID NO: 7) and may be encoded by the reference nucleotide sequence of NM_006274.3 (SEQ ID NO: 5; coding sequence residues 138-434).
  • a nucleotide sequence encoding CCL19 may be codon optimised for expression in human T cells.
  • nucleotide sequence encoding CCL19 may have the nucleotide sequence of SEQ ID NO: 6.
  • T cell receptor (TCRs) are disulphide-linked membrane anchored heterodimeric proteins, typically comprising highly variable alpha ( ⁇ ) and beta ( ⁇ ) chains expressed as a complex with invariant CD3 chain molecules. T cells expressing this type of TCR are commonly referred to as ⁇ : ⁇ (or ⁇ ) T cells.
  • ⁇ T cells A minority of T cells express an alternative TCR comprising variable gamma ( ⁇ ) and delta ( ⁇ ) chains and are referred to as ⁇ T cells.
  • the T cells described herein express a heterologous T cell receptor (TCR) that binds to cancer cells expressing melanoma-associated antigen A4 (MAGE-A4).
  • TCR heterologous T cell receptor
  • MHC major histocompatibility complex
  • the T cells may be modified to express a heterologous TCR that binds specifically to MHCs displaying peptide fragments of the tumour antigen MAGE A4 that are expressed by the cancer cells in a specific cancer patient.
  • the expression of MAGE A4 by cancer cells in the cancer patient may identified using standard techniques.
  • a heterologous TCR may be a synthetic or artificial TCR i.e.
  • a heterologous TCR may be engineered to increase its affinity or avidity for a tumour antigen (i.e. an affinity enhanced TCR).
  • the affinity enhanced TCR may comprise one or more mutations relative to a naturally occurring TCR, for example, one or more mutations in the hypervariable complementarity determining regions (CDRs) of the variable regions of the TCR ⁇ and ⁇ chains. These mutations alter the affinity of the TCR for MHCs that display a peptide fragment of the tumour antigen MAGE A4 expressed by cancer cells, such that the affinity is sufficient for activity without cross-reactivity.
  • a TCR bind to an MHC displaying a peptide fragment of the tumour antigen MAGE A4 with a dissociation constant of 0.05 ⁇ to 20.0 ⁇ ; for example, 0.1 ⁇ to 5 ⁇ or 0.2 ⁇ to 2 ⁇ .
  • Suitable methods of generated affinity enhanced TCRs include screening libraries of TCR mutants using phage or yeast display; and measuring binding affinity are well known in the art (see for example Robbins et al J Immunol (2008) 180(9):6116; San Miguel et al (2015) Cancer Cell 28 (3) 281-283; Schmitt et al (2013) Blood 122348-256; Jiang et al (2015) Cancer Discovery 5901). TCRs for use as described herein bind to cancer cells that express MAGE A4.
  • Expression of the heterologous TCR may alter the immunogenic specificity of the T cells so that they recognise or display improved recognition for one or more MAGE A4 derived tumour antigens that are present on the surface of the cancer cells of an individual with cancer.
  • the T cells may display reduced binding or no binding to cancer cells that express MAGE A4 in the absence of the heterologous TCR.
  • expression of the heterologous TCR may increase the affinity and/or specificity of the cancer cell binding of modified T cells relative to unmodified T cells.
  • MAGE A4 is a highly immunogenic member of the MAGE-A family of Cancer/Testis (CT) antigens and is expressed in testes and placenta but not in other types of healthy tissue.
  • CT Cancer/Testis
  • MAGE A4 is expressed in high percentages of cancer cells from a number of tumours, including melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma.
  • Melanoma-associated antigen A4 may be human MAGE A4 (Gene ID 4103) and may have the reference amino acid sequence of NP_001011548.1 (SEQ ID NO: 9) and may be encoded by the reference nucleotide sequence of NM_000880.4 (SEQ ID NO: 8).
  • An MHC is a set of cell-surface proteins which allow the acquired immune system to recognise ‘foreign’ molecules. Proteins are intracellularly degraded and presented on the surface of cells by the MHC.
  • MHCs displaying ‘foreign’ peptides are recognised by T cells with the appropriate TCRs, prompting cell destruction pathways.
  • MHCs on the surface of cancer cells may display peptide fragments of tumour antigen i.e. an antigen which is present on a cancer cell but not the corresponding non-cancerous cell. T cells which recognise these peptide fragments may exert a cytotoxic effect on the cancer cell.
  • TCRs for use as described herein bind to a peptide fragment of MAGE A4.
  • the peptide fragment of MAGE-A4 is GVYDGREHTV (residues 230- 239 of MAGE-A4; SEQ ID NO: 10).
  • the MHC is HLA-A*-201.
  • the peptide fragment of MAGE A4 may be HLA-A*-201 restricted.
  • a preferred TCR may specifically bind to HLA-A*-201 complexed with the MAGE A4 peptide GVYDGREHTV.
  • a TCR binds to GVYDGREHTV (SEQ ID NO: 10) in complex with HLA-A* 0201 with a dissociation constant of 0.05 ⁇ to 20.0 ⁇ . Dissociation may be measured using surface plasmon resonance at 25 o C and at a pH between 7.1 and 7.5 using a soluble form of the TCR.
  • the TCR may bind to an MHC displaying GVYDGREHTV (SEQ ID NO: 10) preferentially to an MHC displaying the MAGE-B2231240 GVYDGEEHSV (SEQ ID NO: 11) or may bind to an MHC displaying GVYDGREHTV but not to an MHC displaying the MAGE-B2231240 GVYDGEEHSV.
  • the TCR may bind to GVYDGEEHSV (SEQ ID NO: 11) in complex with HLA-A* 0201 with a dissociation constant of 30 ⁇ to 60 ⁇ when measured with surface plasmon resonance at 25 o C and at a pH between 7.1 and 7.5 using a soluble form of the TCR.
  • the dissociation constant may be above 50 ⁇ , such as 100 ⁇ or more, 200 ⁇ or more, or 500 ⁇ or more.
  • the TCR may comprise a TCR alpha chain variable domain and a TCR beta chain variable domain.
  • the TCR variable domains may form contacts with at least residues V2, Y3 and D4 of GVYDGREHTV (SEQ ID NO: 10).
  • the alpha chain variable domain of the TCR may comprise the amino acid sequence of residues 1 to 111 of SEQ ID NO: 12; SEQ ID NO: 14 or a variant thereof.
  • the alpha chain variable domain of the TCR may comprise the amino acid sequence of SEQ ID NO: 14, or residues 22 to 125 of SEQ ID NO: 16 or a variant of either of these.
  • the beta chain variable domain may comprise the amino acid sequence of residues 1 to 111 of SEQ ID NO: 13 a variant thereof.
  • the beta chain variable domain of the TCR may comprise the amino acid sequence of SEQ ID NO: 15 or residues 22 to 123 of SEQ ID NO: 17 or a variant of either of these. .
  • the TCR may comprise the TCR alpha chain amino acid sequence of of residues 22 to 276 of SEQ ID NO: 16 or a variant thereof and the TCR beta chain amino acid sequence of residues 22 to 311 of SEQ ID NO: 17 or a variant thereof.
  • the TCR may comprise the TCR alpha chain amino acid sequence (residues 22 to 125) and the TCR beta chain amino acid sequence (residues 327 to 624) shown in SEQ ID NO: 18.
  • the alpha and beta chain sequences may be separated by a self-cleaving peptide. This allows the chains of the TCR to be expressed as a single transcript which undergoes ribosomal skipping during translation to generate the two separate proteins.
  • Suitable self- cleaving peptides are well-known in the art and include 2A peptides, such as T2A, P2A, E2A and F2A.2A peptides include the P2A sequence of residues 284-305 of SEQ ID NO: 25.
  • the TCR may comprise the amino acid sequence shown in residues 22 to 617 of SEQ ID NO: 18.
  • a suitable TCR may be encoded by the nucleotide sequence of SEQ ID NO: 19.
  • Suitable TCRs for use as described herein are disclosed in WO2017/174824 and Sanderson et al. Oncoimmunol. (2019) 9 (1):e1682381, the contents of which are incorporated herein by reference.
  • the IL-7, CCL19 and TCR expressed in the modified T cell are recombinant proteins that are encoded by heterologous nucleic acid i.e. the IL-7, CCL19 and TCR are expressed from encoding nucleic acid that has been incorporated into the T cell by recombinant techniques.
  • a T cell described herein may comprise; (i) a first heterologous nucleotide sequence encoding a T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4, (ii) a second heterologous nucleotide sequence encoding C-C motif chemokine ligand 19 (CCL19); and (iii) a third heterologous nucleotide sequence encoding interleukin 7 (IL-7).
  • Modification of a T cell to express the IL-7, CCL19 and TCR may comprise introducing the first, second and third nucleotide sequences into the T cell.
  • the T cells may be modified to incorporate the first, second and third nucleotide sequences following isolation and activation.
  • progenitor cells such as pluripotent stem cells, may be modified to incorporate the first, second and third nucleotide sequences and then differentiated or forward programmed into T cells.
  • the first, second and third nucleotide sequences may be contained in a single nucleic acid construct that is introduced into the T cell or progenitor cell.
  • the first, second and third nucleotide sequences encoding IL-7, CCL19 and TCR respectively may be contained in a single nucleic acid construct and expressed from a single promoter (i.e. as a single transcript).
  • the first, second and third heterologous nucleotide sequences are arranged sequentially in the construct.
  • first nucleotide sequence (i) may be located adjacent the promoter, followed by the second nucleotide sequence (ii) and then the third nucleotide sequence (iii).
  • the first, second and third nucleotide sequences may be separated by nucleotide sequences encoding self-cleaving peptides.
  • a T cell may comprise; (iv) a fourth nucleotide sequence encoding a self-cleaving peptide, said fourth sequence being located between the first and second nucleotide sequences; and (v) a fifth nucleotide sequence encoding a self-cleaving peptide, said fifth sequence being located between the second and third nucleotide sequences.
  • a suitable construct comprising the first to the fifth nucleotide sequences is shown in SEQ ID NO: 24.
  • Suitable self-cleaving peptides are well-known in the art and include 2A peptides, such as T2A, P2A, E2A and F2A.
  • the fourth nucleotide sequence may encode a T2A peptide and the fifth nucleotide sequence may encode a F2A peptide.
  • the first to the fifth nucleotide sequences may be operably linked to a single promoter.
  • the promoter may be an inducible promoter or more preferably a constitutive promoter.
  • Suitable constitutive promoters are well known in the art and include mammalian promoters, such as Human elongation factor-1 alpha (EF1 ⁇ ).
  • heterologous nucleotide sequence from the constitutive promoter is shown herein to be increased in the T cells following stimulation with antigen.
  • minimal IL-7 and CCL19 may be produced when the cells are in a resting state, but the levels of both may increase to measurable levels upon T cell activation.
  • Suitable inducible promoters may comprise a nuclear factor of activated T cells (NFAT)/AP1 transcriptional response element (TRE). Upon recognition of the cognate peptide MHC1 complex, NFAT undergoes Ca2+ dependent translocation to the nucleus where it promotes transcription of genes which harbour an NFAT TRE.
  • Suitable NFAT TREs are well-known in the art and include the human IL2 promoter NFAT TRE (Macian et al (2001) Oncogene.2001 Apr 30; 20(19):2476-89) which has the sequence of SEQ ID NO: 20 or a variant thereof.
  • the inducible promoter may comprise one, two, three or more repeats of the NFAT TRE.
  • the inducible promoter may further comprise additional promoter elements, for example a minimal viral promoter such as CMV.
  • Suitable promoter elements are well known in the art and include the minimal CMV promoter of SEQ ID NO: 21 or a variant thereof.
  • a suitable inducible promoter sequence operably linked to a nucleotide sequence encoding IL-7 may comprise the nucleotide sequence of SEQ ID NO: 22, SEQ ID NO: 23 or a variant thereof
  • a T cell may inducibly express IL-7 and/or CCL19.
  • the second nucleotide sequence encoding CCL19 and the third nucleotide sequence encoding IL-7 may be operably linked to inducible promoters.
  • the nucleotide sequences may be operably linked the same or different inducible promoters. Expression of IL-7 and CCL19 from the inducible promoter(s) may be induced by T-cell activation.
  • the first nucleotide sequence encoding the TCR may be constitutively expressed from a constitutive promoter.
  • the coding sequences for the individual chains of the TCR e.g. TCR ⁇ and TCR ⁇ chains
  • Suitable cleavage recognition sequences are well known in the art and include 2A-furin sequence.
  • heterologous refers to a polypeptide or nucleic acid that is foreign to a particular biological system, such as a host cell, and is not naturally present in that system.
  • a heterologous polypeptide or nucleic acid may be introduced to a biological system by artificial means, for example using recombinant techniques.
  • heterologous nucleic acid encoding a polypeptide may be inserted into a suitable expression construct which is in turn used to transform a host cell to produce the polypeptide.
  • a heterologous polypeptide or nucleic acid may be synthetic or artificial or may exist in a different biological system, such as a different species or cell type.
  • An endogenous polypeptide or nucleic acid is native to a particular biological system, such as a host cell, and is naturally present in that system.
  • a recombinant polypeptide is expressed from heterologous nucleic acid that has been introduced into a cell by artificial means, for example using recombinant techniques.
  • a recombinant polypeptide may be identical to a polypeptide that is naturally present in the cell or may be different from the polypeptides that are naturally present in that cell.
  • a variant of a reference amino acid or nucleotide sequence set out herein may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence.
  • Particular amino acid sequence variants may differ from a repeat domain shown above by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.
  • Particular nucleotide sequence variants may differ from a reference sequence set out herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.
  • GAP GAP polypeptide sequence sequence
  • BLAST which uses the method of Altschul et al. (1990) J. Mol. Biol.215: 405-410
  • FASTA which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448
  • Smith-Waterman algorithm Smith and Waterman (1981) J. Mol Biol.147: 195-197
  • TBLASTN program of Altschul et al. (1990) supra, generally employing default parameters.
  • the psi-Blast algorithm Nucl. Acids Res. (1997) 253389-3402
  • Sequence comparison may be made over the full-length of the relevant sequence described herein.
  • the introduction of the heterologous nucleic acid sequences into T cells or progenitor cells and the subsequent expansion of the T cells or progenitor cells may be performed in vitro and/or ex vivo.
  • the first, second and third nucleotide sequences encoding TCR, CCL19 and IL-7, respectively, may be introduced into the T cells or progenitors thereof separately or more preferably in the same nucleic acid construct. This may increase the proportion of T cells or progenitor cells which express all three genes after transduction.
  • the nucleic acid construct may include one or more unique restriction sites to facilitate further manipulation.
  • the nucleic acid construct may be introduced directly into T cells or progenitor cells using gene editing techniques.
  • the nucleic acid construct may be incorporated into an expression vector.
  • Suitable vectors are well-known in the art and are described in more detail herein. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in mammalian cells.
  • a vector may also comprise sequences, such as origins of replication, promoter regions and selectable markers, which allow for its selection, expression and replication in bacterial hosts such as E. coli.
  • the nucleic acid construct is contained in a viral vector, most preferably a gamma retroviral vector or a lentiviral vector, such as a VSVg-pseudotyped lentiviral vector.
  • the T cells may be transduced by contact with a viral particle comprising the nucleic acid.
  • Viral particles for transduction may be produced according to known methods.
  • HEK293T cells may be transfected with plasmids encoding viral packaging and envelope elements as well as a lentiviral vector comprising the coding nucleic acid.
  • a VSVg- pseudotyped viral vector may be produced in combination with the viral envelope glycoprotein G of the Vesicular stomatitis virus (VSVg) to produce a pseudotyped virus particle.
  • a viral vector such as a lentivirus, may be contained in a viral particle comprising the nucleic acid vector encapsulated by one or more viral proteins.
  • a viral particle may be produced by a method comprising transducing mammalian cells with a viral vector as described herein and one or more viral packaging and envelope vectors and culturing the transduced cells in a culture medium, such that the cells produce lentiviral particles that are released into the medium. Following release of viral particles, the culture medium comprising the viral particles may be collected and, optionally the viral particles may be concentrated.
  • the viral particles may be stored, for example by freezing at -80°C ready for use in transducing T cells or progenitor cells.
  • the nucleic acid construct or vector may be introduced into the T cells or progenitor cells by any convenient method. Suitable methods for introducing or incorporating a heterologous nucleic acid into a T cell, certain considerations are well-known to those skilled in the art.
  • the nucleic acid to be inserted may be assembled within a construct or vector which contains effective regulatory elements which will drive transcription in the T cell.
  • Suitable techniques for transporting the construct or vector into the T cell include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome- mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or lentivirus.
  • solid-phase transduction may be performed without selection by culture on retronectin-coated, retroviral vector-preloaded tissue culture plates.
  • Many known techniques and protocols for manipulation and transformation of nucleic acid for example in preparation of nucleic acid constructs, introduction of DNA into cells and gene expression are described in detail in Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992.
  • the heterologous nucleic acid sequences may be introduced into T cells, for example T cells obtained from a donor individual, in order to produce a population of modified T cells expressing the heterologous nucleic acid sequences.
  • the heterologous nucleic acid sequences may be introduced into progenitor cells, such as induced pluripotent stem cells (iPSCs), mesoderm cells (MCs), haemogenic endothelial cells (HECs), haematopoietic progenitor cells (HPCs) or progenitor T cells.
  • iPSCs induced pluripotent stem cells
  • MCs mesoderm cells
  • HECs haemogenic endothelial cells
  • HPCs haematopoietic progenitor cells
  • the progenitor cells may then be differentiated into T cells, in order to produce a population of modified T cells expressing the heterologous nucleic acid sequences.
  • a method of producing a modified population of T cells may comprise (i) differentiating a population of induced pluripotent stem cells (iPSCs) into mesoderm cells (MCs), (ii) differentiating the MCs to produce a population of haemogenic endothelial cells (HECs), (iii) differentiating the HECs into a population of haematopoietic progenitor cells (HPCs), (iv) differentiating the population of HPCs into progenitor T cells; and (v) maturing the progenitor T cells to produce a population of double positive CD4+ CD8+ T cells, wherein the method comprises (i) introducing a first heterologous nucleotide sequence encoding a T cell receptor (TCR) that binds to cancer cells expressing MAGE-A4 into any one of the iPSCs, MCs, HECs, HPCs or progenitor T cells; (ii) introducing a
  • a method may comprise introducing the first, second and third nucleotide sequences into any one of the iPSCs, MCs, HECs, HPCs or progenitor T cells.
  • the method may further comprise; (vi) activating and expanding the double positive CD4+ CD8+ T cells to produce a population of CD8+ T cells or a population of CD4+ T cells.
  • Induced pluripotent stem cells are pluripotent cells which are derived from non- pluripotent, fully differentiated donor or antecedent cells.
  • iPSCs are capable of self-renewal in vitro and exhibit an undifferentiated phenotype and are potentially capable of differentiating into any foetal or adult cell type of any of the three germ layers (endoderm, mesoderm and ectoderm).
  • the population of iPSCs may be clonal i.e. genetically identical cells descended from a single common ancestor cell.
  • iPSCs may express one or more of the following pluripotency associated markers: POU5f1 (Oct4), Sox2, Alkaline Phosphatase, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF4 and c-myc, preferably one or more of POU5f1, NANOG and SOX2.
  • An iPSC may lack markers associated with specific differentiative fates, such as Bra, Sox17, FoxA2, ⁇ FP, Sox1, NCAM, GATA6, GATA4, Hand1 and CDX2.
  • an iPSC may lack markers associated with endodermal fates.
  • the iPSCs are human iPSCs (hiPSCs).
  • IPSCs may be derived or reprogramed from donor cells, which may be somatic cells or other antecedent cells obtained from a source, such as a donor individual.
  • the donor cells may be mammalian, preferably human cells. Suitable donor cells include adult fibroblasts and blood cells, for example peripheral blood cells, such as HPCs or mononuclear cells.
  • Suitable donor cells for reprogramming into iPSCs as described herein may be obtained from a donor individual.
  • the donor individual may be the same person as the recipient individual to whom the T cells will be administered following production as described herein (autologous treatment).
  • the donor individual may be a different person to the recipient individual to whom the T cells will be administered following production as described herein (allogeneic treatment).
  • the donor individual may be a healthy individual who is human leukocyte antigen (HLA) matched (either before or after donation) with a recipient individual suffering from cancer.
  • HLA human leukocyte antigen
  • the donor individual may not be HLA matched with the recipient individual.
  • the donor individual may be a neonate (new-born), for example the donor cells may be obtained from a sample of umbilical cord blood.
  • iPSCs may be differentiated into mesoderm cells by culturing the population of iPSCs under suitable conditions to promote mesodermal differentiation.
  • the iPSCs cells may be cultured sequentially in first, second and third mesoderm induction media to induce differentiation into mesoderm cells.
  • a suitable first mesoderm induction medium may stimulate SMAD2 and SMAD3 mediated signalling pathways.
  • the first mesoderm induction medium may comprise activin.
  • a suitable second mesoderm induction medium may (i) stimulate SMAD1, SMAD2, SMAD3, SMAD5 and SMAD9 and/or SMAD1, SMAD2, SMAD3, SMAD5 and SMAD9 mediated signalling pathways and (ii) have fibroblast growth factor (FGF) activity.
  • the second mesoderm induction medium may comprise activin, preferably activin A, BMP, preferably BMP4 and FGF, preferably bFGF.
  • a suitable third mesoderm induction medium may (i) stimulate SMAD1, SMAD2, SMAD3, SMAD5 and SMAD9 and/or SMAD1, SMAD2, SMAD3, SMAD5 and SMAD9 mediated signalling pathways (ii) have fibroblast growth factor (FGF) activity and (iii) inhibit glycogen synthase kinase 3 ⁇ .
  • the third mesoderm induction medium may comprise activin, preferably activin A, BMP, preferably BMP4, FGF, preferably bFGF, and a GSK3 inhibitor, preferably CHIR99021.
  • the first, second and third mesoderm induction media may be devoid of differentiation factors other than the differentiation factors set out above.
  • the first, second and third mesoderm induction media are chemically defined media.
  • the first mesoderm induction medium may consist of a chemically defined nutrient medium supplemented with an effective amount of activin, preferably activin A, for example 50ng/ml activin A
  • the second mesoderm induction medium may consist of a chemically defined nutrient medium supplemented with an effective amount of activin preferably activin A, for example 5ng/ml activin A, BMP, preferably BMP4, for example 10ng/ml BMP4
  • FGF preferably bFGF (FGF2), for example 5ng/ml bFGF
  • the third mesoderm induction medium may consist of a chemically defined nutrient medium supplemented with an effective amount of activin preferably activin A, for example 5ng/ml activin A, BMP, preferably BMP4, for example 10ng/ml BMP4
  • FGF preferably bFGF (FGF2), for example 5
  • a chemically defined medium is a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure.
  • a CDM is devoid of undefined components or constituents which include undefined components, such as feeder cells, stromal cells, serum, and complex extracellular matrices, such as matrigel TM
  • a CDM does not contain stromal cells, such as OP9 cells, expressing Notch ligands, such as DLL1 or DLL4.
  • the 21hemicallly defined nutrient medium may comprise a chemically defined basal medium.
  • Suitable chemically defined basal media include Iscove’s Modified Dulbecco’s Medium (IMDM), Ham’s F12, Advanced Dulbecco’s modified eagle medium (DMEM) (Price et al Focus (2003), 253-6), Williams E (Williams, G.M. et al Exp. Cell Research, 89, 139- 142 (1974)), RPMI-1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual.3, 503-508) and StemPro TM -34 PLUS (ThermoFisher Scientific).
  • the basal medium may be supplemented by serum-free culture medium supplements and/or additional components in the medium.
  • Suitable supplements and additional components are described above and may include L-glutamine or substitutes, such as GlutaMAX-1 TM , ascorbic acid, monothiolglycerol (MTG), antibiotics such as penicillin and streptomycin, human serum albumin, for example recombinant human serum albumin, such as Cellastim TM (Merck/Sigma) and Recombumin TM (albumedix.com), insulin, transferrin and 2- mercaptoethanol.
  • a basal medium may be supplemented with a serum substitute, such as Knockout Serum Replacement (KOSR; Invitrogen).
  • the iPSCs may be cultured in the first mesoderm induction medium for 1 to 12 hours, for example any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours, preferably about 4 hours; then cultured in the second mesoderm induction medium for 30 to 54 hours, for example any of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 hours, preferably about 44 hours; and then cultured in the third mesoderm induction medium for 36 to 60 hours, , for example any of 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 hours, preferably about 48 hours to produce a population of mesodermal cells.
  • Mesoderm cells are partially differentiated progenitor cells that are committed to mesodermal lineages and are capable of differentiation under appropriate conditions into all cell types in the mesenchyme (fibroblast), muscle, bone, adipose, vascular and haematopoietic systems.
  • Mesoderm cells may express one or more mesodermal markers.
  • the mesoderm cells may express any one, two, three, four, five, six or all seven of Brachyury, Goosecoid, Mixl1, KDR, FoxA2, GATA6 and PDGF ⁇ R.
  • mesoderm cells may be differentiated into haemogenic endothelial cells (HECs) by culturing the population of mesoderm cells under suitable conditions to promote haemogenic endothelial (HE) differentiation.
  • the iPSCs cells may be cultured in an HE induction medium.
  • a suitable HE induction medium may (i) stimulate cKIT receptor (CD117) and/or cKIT receptor (CD117) mediated signalling pathways and (ii) stimulate VEGFR and/or VEGFR mediated signalling pathways.
  • the HE induction medium may comprise SCF and VEGF.
  • the HE induction medium is a chemically defined medium.
  • the HE induction medium may consist of a chemically defined nutrient medium supplemented with effective amounts of VEGF, for example 15ng/ml VEGF; and SCF, for example 100ng/ml SCF.
  • VEGF for example 15ng/ml VEGF
  • SCF for example 100ng/ml SCF.
  • Suitable chemically defined nutrient media are described above and include StemPro TM -34 (ThermoFisher Scientific).
  • the mesoderm cells may be cultured in the HE induction medium for 2 to 6 days or 3 to 5 days, preferably about 4 days, to produce a population of HE cells.
  • Haemogenic endothelial cells are partially differentiated endothelial progenitor cells that have hematopoietic potential and are capable of differentiation under appropriate conditions into haematopoietic lineages.
  • HE cells may express CD34.
  • HECs may not express CD73 or CXCR4 (CD184).
  • the HE cells may have the phenotype CD34+ CD73- or CD34+ CD73- CXCR4-.
  • haemogenic endothelial (HE) cells may be differentiated into haematopoietic progenitor cells (HPCs) by culturing the population of HE cells under suitable conditions to promote haematopoietic differentiation.
  • the HE cells may be cultured in a haematopoietic induction medium.
  • a suitable haematopoietic induction medium may stimulate the following (i) cKIT receptor (CD117) and/or cKIT receptor (CD117) mediated signalling pathways, (ii) VEGFR and/or VEGFR mediated signalling pathways, (iii) MPL (CD110) and/or MPL (CD110) mediated signalling pathways (iv) FLT3 and/or FLT3 mediated signalling pathways (v) IGF1R and/or IGF1R mediated signalling pathways (vi) SMAD1, 5 and 9 and/or SMAD1, 5 and 9 mediated signalling pathways (vii) Hedgehog and/or Hedgehog signalling pathways (viii) EpoR and/or EpoR mediated signalling pathway and (ix) AGTR2 and/or AGTR2 mediated signalling pathways.
  • a suitable haematopoietic induction medium may also inhibit the AGTR1 (angiotensin II type 1 receptor (AT1)) and/or AGTR1 (angiotensin II type 1 receptor (AT1)) mediated signaling pathway.
  • a suitable haematopoietic induction medium may also have interleukin (IL) activity and FGF activity.
  • a haematopoietic induction medium may comprise the differentiation factors: VEGF, SCF, Thrombopoietin (TPO), Flt3 ligand (Flt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMP, FGF, Sonic hedgehog (SHH), erythropoietin (EPO), angiotensin II, and an angiotensin II type 1 receptor (AT1) antagonist.
  • the haematopoietic induction medium is a chemically defined medium.
  • the haematopoietic induction medium may consist of a chemically defined nutrient medium supplemented with effective amounts of VEGF, for example 15ng/ml; SCF, for example 100ng/ml; thrombopoietin (TPO), for example 30ng/ml; Flt3 ligand (FLT3L), for example 25ng./ml; IL-3, for example 25ng/ml; IL-6, for example 10ng/ml; IL-7, for example 10 ng/ml; IL-11, for example 5 ng/ml; IGF-1, for example 25 ng/ml; BMP, for example BMP4 at 10ng/ml; FGF, for example bFGF at 5ng/ml; Sonic hedgehog (SHH), for example 25ng/ml; erythropoietin (EPO), for example 2 u/ml; angiotensin II, for example 10 ⁇ g/ml, and an angiotensin II type
  • a suitable haematopoietic induction medium be devoid of other differentiation factors.
  • a haematopoietic induction medium may consist of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of VEGF, SCF, Thrombopoietin (TPO), Flt3 ligand (Flt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMP, FGF, Sonic hedgehog (SHH), erythropoietin (EPO), angiotensin II, and an angiotensin II type 1 receptor (AT 1 ) antagonist (i.e.
  • the medium does not contain any differentiation factors other than VEGF, SCF, Thrombopoietin (TPO), Flt3 ligand (Flt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMP, FGF, Sonic hedgehog (SHH), erythropoietin (EPO), angiotensin II, and an angiotensin II type 1 receptor (AT 1 ) antagonist).
  • Suitable chemically defined nutrient media are described above and include StemPro TM -34 PLUS (ThermoFisher Scientific) or a basal medium such as IMDM supplemented with albumin, insulin, selenium transferrin, and lipids as described below.
  • the HE cells may be cultured in the haematopoietic induction medium for 8-21 days, for example any of about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days, preferably about 16 days, to produce the population of HPCs.
  • HPCs also called hematopoietic stem cells
  • HPCs are multipotent stem cells that are committed to a hematopoietic lineage and are capable of further hematopoietic differentiation into all blood cell types including myeloid and lymphoid lineages, including monocytes, B cells and T cells.
  • HPCs may express CD34.
  • HPCs may co-express CD133, CD45 and FLK1 (also known as KDR or VEGFR2) and may be negative for expression of CD38 and other lineage specific markers.
  • HPCs may display the phenotype CD34+ CD133+ CD45+ FLK1+ CD38-.
  • haematopoietic progenitor cells may be differentiated into progenitor T cells by culturing the population of HPCs under suitable conditions to promote lymphoid differentiation.
  • the haematopoietic progenitor cells may be cultured in a lymphoid expansion medium.
  • a lymphoid expansion medium is a cell culture medium that promotes the lymphoid differentiation of HPCs into progenitor T cells.
  • a suitable lymphoid expansion medium may (i) stimulate cKIT receptor (CD117; KIT receptor tyrosine kinase) and/or cKIT receptor (CD117; KIT receptor tyrosine kinase) mediated signalling pathways, (ii) stimulate MPL (CD110) and/or MPL (CD110) mediated signalling pathways (iii) FLT3 and/or FLT3 mediated signalling pathways and (iv) have interleukin (IL) activity.
  • a lymphoid expansion medium may comprise the differentiation factors SCF, FLT3L, TPO and IL7.
  • the lymphoid expansion medium is a chemically defined medium.
  • the lymphoid expansion medium may consist of a chemically defined nutrient medium supplemented with effective amounts of the above differentiation factors.
  • Suitable lymphoid expansion media are well-known in the art and include Stemspan TM SFEM II (Cat # 9605; StemCell Technologies Inc, CA).with Stemspan TM lymphoid expansion supplement (Cat # 9915; StemCell Technologies Inc, CA).
  • the HPCs may be cultured on a surface During differentiation into progenitor T cells.
  • the HPCs may be cultured on a surface of a culture vessel, bead or other biomaterial or polymer.
  • the surface may be coated with a factor that stimulates Notch signalling, for example a Notch ligand, such as Delta-like 1 (DLL1) or Delta-like 4 (DLL4).
  • a Notch ligand such as Delta-like 1 (DLL1) or Delta-like 4 (DLL4).
  • Suitable Notch ligands are well-known in the art and available from commercial suppliers.
  • the surface may also be coated with an extracellular matrix protein, such as fibronectin, vitronectin, laminin or collagen and/or one or more cell surface adhesion proteins, such as VCAM1.
  • the surface for HPC culture may have a coating that comprises a factor that stimulates Notch signalling, for example a Notch ligand, such as DLL4, without the extracellular matrix protein or cell surface adhesion protein.
  • the surface for HPC culture may have a coating that comprises a factor that stimulates Notch signalling, for example a Notch ligand, such as DLL4, an extracellular matrix protein, such as vitronectin, and a cell surface adhesion protein, such as VCAM1.
  • a factor that stimulates Notch signalling for example a Notch ligand, such as DLL4, an extracellular matrix protein, such as vitronectin, and a cell surface adhesion protein, such as VCAM1.
  • the surface may be coated with an extracellular matrix protein, factor that stimulates Notch signalling and cell surface adhesion protein by contacting the surface with a coating solution.
  • the coating solution may be incubated on the surface under suitable conditions to coat the surface. Conditions may, for example, include about 2 hours at room temperature.
  • Coating solutions comprising an extracellular matrix protein and a factor that stimulates Notch signalling are available from commercial suppliers (StemSpanTM Lymphoid Differentiation Coating Material; Cat # 9925; Stem Cell Technologies Inc, CA).
  • the HPCs may be cultured in the lymphoid expansion medium on the substrate or surface for a time sufficient for the HPCs to differentiate into progenitor T cells.
  • the HPCs may be cultured for 2-6 weeks, 2 to 5 weeks or 2-4 weeks, preferably 3 weeks.
  • Progenitor T cells are multi-potent lymphopoietic progenitor cells that are capable of giving rise to ⁇ T cells, ⁇ T cells, tissue resident T cells and NK T cells.
  • Progenitor T cells may commit to the ⁇ T cell lineage after pre-TCR selection in the thymus.
  • Progenitor T cells may be capable of in vivo thymus colonization and may be capable of committing to the T cell lineage after pre-TCR selection in the thymus.
  • Progenitor T cells may also be capable of maturation into cytokine-producing CD3 + T-cells.
  • Progenitor T cells may express CD5 and CD7 i.e. the progenitor T cells may have a CD5+CD7+ phenotype.
  • Progenitor T cells may also co-express CD44, CD25 and CD2.
  • progenitor T cells may have a CD5+, CD7+ CD44+, CD25+ CD2+ phenotype.
  • progenitor T cells may also co-express CD45.
  • Progenitor T cells may lack expression of CD3, CD4 and CD8, for example on the cell surface.
  • progenitor T cells may be matured into T cells by culturing the population of progenitor T cells under suitable conditions to promote T cell maturation.
  • the progenitor T cells may be cultured in a T cell maturation medium.
  • a T cell maturation medium is a cell culture medium that promotes the maturation of progenitor T cells into mature T cells.
  • a suitable T cell maturation medium may (i) stimulate cKIT receptor (CD117; KIT receptor tyrosine kinase) and/or cKIT receptor (CD117; KIT receptor tyrosine kinase) mediated signalling pathways (ii) FLT3 and/or FLT3 mediated signalling pathways and (iii) have interleukin (IL) activity.
  • a T cell maturation medium may comprise the differentiation factors SCF, FLT3L, and IL7.
  • the T cell maturation medium is a chemically defined medium.
  • the T cell maturation medium may consist of a chemically defined nutrient medium supplemented with effective amounts of the above differentiation factors.
  • Suitable T cell maturation media are well-known in the art and include Stemspan TM SFEM II (Cat # 9605; StemCell Technologies Inc, CA) with Stemspan TM T cell maturation supplement (Cat # 9930; StemCell Technologies Inc, CA) and other media suitable for expansion of PBMCs and CD3+ cells, such as ExCellerate Human T cell expansion medium (R& D Systems, USA).
  • Other suitable T cell maturation media may include a basal medium such as IMDM, supplemented with ITS, albumin and lipids, as described elsewhere herein and further supplemented with effective amounts of the above differentiation factors.
  • the progenitor T cells may be cultured on a surface.
  • the progenitor T cells may be cultured on a surface of a culture vessel, bead or other biomaterial or polymer.
  • the surface may be coated with a factor that stimulates Notch signalling, for example a Notch ligand, such as Delta-like 1 (DLL1) or Delta-like 4 (DLL4).
  • Notch ligands are well-known in the art and available from commercial suppliers.
  • the surface may also be coated with an extracellular matrix protein, such as fibronectin, vitronectin, laminin or collagen and/or one or more cell surface adhesion proteins, such as VCAM1. Suitable coatings are well-known in the art and described elsewhere herein.
  • the progenitor T cells may be cultured in the T cell maturation medium on the substrate or surface for a time sufficient for the progenitor T cells to mature into T cells.
  • the progenitor T cells may be cultured for 1-4 weeks, preferably 2 or 3 weeks.
  • Suitable methods for the production of T cells from progenitor cells, such as iPSCs, are described in WO2021/032852, WO2021/032855, WO2021/032851, WO2021/032836, WO2022/175401 and WO2021/229212.
  • the initial population of modified T cells may be cultured in vitro such that the modified T cells proliferate and expand the population.
  • the modified T cell population may for example be expanded using magnetic beads coated with anti-CD3 and anti-CD28.
  • the modified T cells may be cultured using any convenient technique to produce the expanded population. Suitable culture systems include stirred tank fermenters, airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, in particular hollow fibre bioreactors. The use of such systems is well-known in the art. Numerous culture media suitable for use in the proliferation of T cells ex vivo are available, in particular complete media, such as AIM-V, Iscoves medium and RPMI-1640 (Invitrogen- GIBCO). The medium may be supplemented with other factors such as serum, serum proteins and selective agents.
  • RPMI-1640 medium containing 2 mM glutamine, 10% FBS, 25 mM HEPES, pH 7.2, 1% penicillin-streptomycin, and 55 ⁇ M ⁇ -mercaptoethanol and optionally supplemented with 20 ng/ml recombinant IL-2 may be employed.
  • the culture medium may be supplemented with the agonistic or antagonist factors described above at standard concentrations which may readily be determined by the skilled person by routine experimentation.
  • cells are cultured at 37°C in a humidified atmosphere containing 5% CO2 in a suitable culture medium.
  • T cells and other mammalian cells are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293, Ho WY et al J Immunol Methods. (2006) 310:40-52). In some embodiments, it may be convenient to isolate and/or purify the modified T cells from the population.
  • the population of modified T cells produced as described herein may be stored, for example by lyophilisation and/or cryopreservation, before use.
  • a population of modified T cells may be admixed with other reagents, such as buffers, carriers, diluents, preservatives and/or pharmaceutically acceptable excipients. Suitable reagents are described in more detail below.
  • a method described herein may comprise admixing the population of modified T cells with a pharmaceutically acceptable excipient.
  • Pharmaceutical compositions suitable for administration e.g.
  • aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti- oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient
  • aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer’s Solution, or Lactated Ringer’s Injection. Suitable vehicles can be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences, 18 th edition, Mack Publishing Company, Easton, Pa., 1990.
  • the modified T cells may be formulated into a pharmaceutical composition suitable for intravenous infusion into an individual.
  • pharmaceutically acceptable as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g., human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • a population of modified T cells expressing a nucleic construct or a vector as described herein and a population of T cells that express a heterologous TCR that binds to MAGE A4 expressing cancer cells and further express heterologous IL-7 and CCL19.
  • the T cells may bind specifically to cancer cells that express MAGE A4.
  • a suitable population may be produced by a method described above.
  • the population of modified T cells may be for use as a medicament.
  • a population of modified T cells as described herein may be used in cancer immunotherapy therapy, for example adoptive T cell therapy.
  • a population of modified T cells as described herein for the manufacture of a medicament for the treatment of cancer may comprise administering a population of modified T cells as described herein to an individual in need thereof.
  • Modified T cells that express IL-7 and CCL19 as described herein may display one or more of improved T-cell engraftment, functionality, and/or immune cell infiltration into a tumor relative to T cells that do not express IL-7 and CCL19.
  • the heterologous TCR expressed by the T cells may specifically bind to the cancer cells of a cancer patient. The cancer patient may be subsequently treated with the modified T cells.
  • Suitable cancer patients for treatment with the modified T cells may be identified by a method comprising; obtaining sample of cancer cells from an individual with cancer and; identifying one or more of the cancer cells in the sample to be MAGE A4 expressing cancer cells. Cancer cells identified as expressing MAGE A4 may bind to the TCR expressed by the modified T cells. Cancer cells may be identified as binding to the TCR encoded by the third nucleotide sequence by identifying the expression of MAGE A4 by the cancer cells. Methods of identifying antigens on the surface of cancer cells obtained from an individual with cancer are well-known in the art.
  • the cancer cells of an individual suitable for treatment as described herein may express MAGE A4 and may be of correct HLA type to bind the TCR expressed by the T cell (for example the cells may be HLA-A*-201). Cancer cells may be distinguished from normal somatic cells in an individual by the expression of MAGE A4 tumour antigen. Normal somatic cells in an individual may not express MAGE A4 or may express it in a different manner, for example at lower levels, in different tissue and/or at a different developmental stage.
  • the population of modified T cells may be autologous i.e. the modified T cells were originally obtained from the same individual to whom they are subsequently administered (i.e. the donor and recipient individual are the same).
  • a suitable population of modified T cells for administration to the individual may be produced by a method comprising providing an initial population of T cells obtained from the individual, modifying the T cells to inducibly express IL-7 and constitutively express an antigen receptor which binds specifically to cancer cells in the individual as described herein, and culturing the modified T cells.
  • the population of modified T cells may be allogeneic i.e. the modified T cells were originally obtained from a different individual to the individual to whom they are subsequently administered (i.e. the donor and recipient individual are different).
  • the donor and recipient individuals may be HLA matched to avoid GVHD and other undesirable immune effects.
  • a suitable population of modified T cells for administration to a recipient individual may be produced by a method comprising providing an initial population of T cells obtained from a donor individual, modifying the T cells to inducibly express IL-7 and constitutively express an antigen receptor which binds specifically to cancer cells in the recipient individual, as described herein, and culturing the modified T cells.
  • the recipient individual may exhibit a T cell mediated immune response against cancer cells in the recipient individual. This may have a beneficial effect on the cancer condition in the individual.
  • Cancer conditions may be characterised by the abnormal proliferation of malignant cancer cells.
  • Preferred cancer conditions for treatment as described herein may include solid cancers or solid tumours, such as sarcomas, including synovial sarcomas and myxoid/round cell liposarcoma (MRCLS), skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, urothelial cancer, ovarian cancer, including metastatic ovarian cancer, prostate cancer, lung cancer, including metastatic lung cancer, lung carcinoid cancer, small cell lung cancer, and non- small cell lung cancer (NSCLC), such as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma and large cell carcinoma, and metastatic or advanced NSCLC, colorectal cancer, including colorectal carcinoma, colorectal adenocarcinoma and metastatic colorectal cancer, cervical cancer, liver cancer, including metastatic liver cancer, head and neck cancer, including head and neck SCC (squamous cell carcinoma), oesophageal cancer,
  • the cancer may also include haematologic malignancies such as acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML), myelodysplastic syndrome (MDS), acute lymphocytic leukaemia (ALL), chronic lymphocytic leukaemia (CLL), adult T-cell leukaemia (ATL), and lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma).
  • suitable cancers may express a tumour antigen, such as MAGE A4.
  • suitable cancers may also express a PD-1 ligand, such as PD-L1. Cancer cells within an individual may be immunologically distinct from normal somatic cells in the individual (i.e.
  • the cancerous tumour may be immunogenic).
  • the cancer cells may be capable of eliciting a systemic immune response in the individual against one or more antigens expressed by the cancer cells, such as MAGE A4.
  • the tumour antigens that elicit the immune response may be specific to cancer cells or may be shared by one or more normal cells in the individual.
  • An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g.
  • the individual is a human.
  • non- human mammals especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.
  • the individual may have minimal residual disease (MRD) after an initial cancer treatment.
  • An individual with cancer may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of cancer in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine such as Harrison’s Principles of Internal Medicine, 15 th Ed., Fauci AS et al., eds., McGraw- Hill, New York, 2001.
  • a diagnosis of a cancer in an individual may include identification of a particular cell type (e.g. a cancer cell) in a sample of a body fluid or tissue obtained from the individual.
  • Treatment may be any treatment and therapy, whether of a human or an animal (e.g.
  • treatment may also be prophylactic (i.e. prophylaxis).
  • an individual susceptible to or at risk of the occurrence or re-occurrence of cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of cancer in the individual.
  • Treatment may include inhibiting cancer growth, including complete cancer remission, and/or inhibiting cancer metastasis.
  • Cancer growth generally refers to any one of a number of indices that indicate change within the cancer to a more developed form.
  • indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumour volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumour growth, a destruction of tumour vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of T cells, and a decrease in levels of tumour-specific antigens.
  • CT computed tomographic
  • T cells modified as described herein may improve the capacity of the individual to resist cancer growth, in particular growth of a cancer already present the subject and/or decrease the propensity for cancer growth in the individual.
  • the modified T cells or the pharmaceutical composition comprising the modified T cells may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to; parenteral, for example, by infusion.
  • Infusion involves the administration of the T cells in a suitable composition through a needle or catheter.
  • T cells are infused intravenously or subcutaneously, although the T cells may be infused via other non-oral routes, such as intramuscular injections and epidural routes.
  • Suitable infusion techniques are known in the art and commonly used in therapy (see, e.g., Rosenberg et al., New Eng. J. of Med., 319:1676, 1988).
  • the number of cells administered is from about 10 5 to about 10 10 per Kg body weight, typically 2x10 8 to 2x10 10 cells per individual, typically over the course of 30 minutes, with treatment repeated as necessary, for example at intervals of days to weeks.
  • appropriate dosages of the modified T cells, and compositions comprising the modified T cells can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular cells, the route of administration, the time of administration, the rate of loss or inactivation of the cells, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient.
  • the amount of cells and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • modified T cells may be administered alone, in some circumstances the modified T cells may be administered in combination with the target antigen, APCs displaying the target antigen, and/or IL-2 to promote expansion in vivo of the population of modified T cells.
  • the population of modified T cells may be administered in combination with one or more other therapies, such as cytokines e.g. IL-2, cytotoxic chemotherapy, radiation and immuno- oncology agents, including checkpoint inhibitors, such as anti-B7-H3; anti-B7-H4; anti-TIM3; anti-KIR; anti-LAG3; PD-1 axis inhibitors, such as anti-PD-1, and anti-PD-L1; and anti- CTLA4 antibodies.
  • cytokines e.g. IL-2
  • cytotoxic chemotherapy e.g. IL-2
  • cytotoxic chemotherapy e.g. IL-2
  • immuno- oncology agents including checkpoint inhibitors, such as anti-B7-H3; anti-B7-H4; anti-
  • the population of modified T cells may be administered in combination with a PD-1 axis inhibitor.
  • Suitable PD-1 axis inhibitors may include anti-PD-1 and anti-PD-L1 antibodies, such as nivolumab and pembrolizumab.
  • the one or more other therapies may be administered by any convenient means, preferably at a site which is separate from the site of administration of the modified T cells. Administration of modified T cells can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment.
  • the modified T cells are administered in a single transfusion of a least 1 x 10 9 T-cells.
  • T-cells were thawed on the day of the assay and rested for at least 1 hour in R10 media at 37 0C/5 % CO2 prior inclusion in the assay.
  • the TCR constructs under investigation, along with the donor codes are listed in Table 1.
  • the HLA-A2 status of the donors is shown in Table 2.
  • Table 1 Plasmid/ADB code TCR/Construct Name Table 2 Target Cells
  • the HLA-A*02:01 and MAGEA4 antigen positive cell line NCI-H1755 was used to present antigen to T-cells.
  • Endothelial cells Primary human dermal microvascular endothelial cells (HDMEC7) were included in the Transwell assays to aid the migration of DCs from the upper to lower chamber.
  • Migratory Cells DCs were isolated from the whole blood of two donors.
  • the CD14+ fraction of PBMCs were isolated by magnetic bead separation and cultured in phenol red-free R10 for 6 days in the presence of IL-4 & GM-CSF, to generate immature DCs (iDCs) according to CBP 054v03.
  • immature DCs 48 hours before assay set-up, immature DCs were treated with a cytokine cocktail to induce maturation.
  • Restimulation Assay Target cells were cultured according to CBP 002v03 and thawed from assay ready vials on the day prior to co-culture set-up.
  • Target cells were washed and resuspended in 5 mL R10 prior to irradiation at 48 Gy. Target cells were resuspended at 1x10 6 cells/mL prior to plating for the assay.
  • 20 ng/mL of rhIL-7 was added at twice the concentration to the appropriate number of A375 cells, prior to plating.
  • ADP-A2M4, ADP-A2M4N7X19 and ntd T-cells from three different donors (Wave266, Wave267 and Wave 268; one per assay) were thawed and rested in 30 mL of pre-warmed R10 and rested for at least 2 hours.
  • T-cell counting was performed using flow cytometry. T-cells were resuspended to 1x10 6 cells/mL and plated out in an appropriate cell culture vessel with the irradiated targets at a ratio of 1:1. Co-cultures were incubated for 7 days. On the 7 th day, all conditions were harvested and counted by flow cytometry in the same way as before. After an accurate T-cell count was determined, they were then resuspended back to 1x10 6 cells/mL plated out in an appropriate cell culture vessel with a fresh batch of irradiated A375s (prepared as above).
  • T-cells were plated out as before but with an equivalent volume of R10 alone added. Stimulations in this manner were repeated until day 28.
  • Flow Cytometry and Cell Counting The Attune NxT Flow Cytometer was used to count and phenotype the T-cells over the course of the assay. Briefly, 60 ⁇ L of T-cells (PSPdev015) were transferred into a 96-well u- bottom plate and incubated with a cocktail of antibodies, as listed in Table 3. Cells were then incubated at 4°C for 15 minutes prior to the addition of the CountBrightTM counting beads.
  • CountBrightTM counting beads were warmed to room temperature and vortexed for 30 seconds to evenly resuspend them. Immediately after vortexing, 60 ⁇ L (PSPdev015) of the bead suspension was added to each sample to be counted. Tube volume was then topped up to 300 ⁇ L with FACS buffer and the samples analysed. A fluorescent minus one (FMO) panel was used to inform the gate voltages and gating was performed according to the strategies detailed in the appendix (section 7.3.1). Absolute T- cell counts were determined using lymphocyte common antigen (CD45) marker and counting beads.
  • CD45 lymphocyte common antigen
  • Absolute T-cell counts per ⁇ L were then calculated using the following formula: Immunophenotyping analysis assessing the frequency of expression of CD8, CCR7, CD45 RA and CD45 RO was performed using the same samples. Fold change calculations Fold change in T-cell number was calculated by dividing the concentration of cells after restimulation by the concentration before restimulation. Briefly, absolute cell counts were determined (CD45+ T cells/mL) by flow cytometry and used to seed T-cells at a final density of 0.5 x 10 6 CD45+ T cells/mL. After 7 days the samples were harvested, and an absolute count determined (CD45+ T cells/mL) in the same way. Fold change from day 0 was then inferred over the course of the assay by multiplying subsequent weeks together.
  • the fold change in T-cell subsets was determined by applying the phenotyping frequency data to the absolute counts of CD45+ T-cells as appropriate.
  • Supernatant Collection Supernatants were collected over the course of the assay, before restimulation and 24 hours afterwards (days: 1, 7, 814, 15, 21, 22 and 28). Where multiple wells for a single condition existed, supernatants were pooled prior to storage. Supernatants were stored at -80 °C prior to thawing for cytokine analysis by supernatant ELISA or MSDTM Multi Spot Assay.
  • Meso Scale DiscoveryTM Multi Spot Assay System Supernatants were analysed using the Meso Scale DiscoveryTM (MSDTM) Multi Spot Assay System according to the manufacturer’s instructions.
  • MSDTM Human Cytokine Panel 1 V-PLEX kit (10 analytes) and the Human Proinflammatory Panel 1 V-PLEX kit (10 analytes). Briefly, samples were thawed and diluted with MSDTM Diluent 100 prior to dilution with the appropriate Calibrator Diluent. Each sample was run in duplicate wells. Readings were acquired using a MESO QuickPlex SQ120 Electrochemiluminescence (ECL), using acquisition software Methodical Mind version 4.2. ECL data was exported and analysed within the Discovery Workbench software version 4.0. Readings falling below the respective lower standard curve limit were ascribed the minimal standard curve value for data plotting interpretation and analysis.
  • ECL Electrochemiluminescence
  • Immunophenotyping was performed by flow cytometry on day 7 (24 hours after the addition of the maturation cytokine cocktail, 24 hours before use in the Transwell migration assay) to confirm a mature status of dendritic cells using a panel of antibodies.
  • Transwell migration assay Transwell migration assays were performed to assess the chemotactic ability of ADP- A2M4N7X19 compared to ADP-A2M4 and ADP-A2M4IL7. Briefly, endothelial cells were grown on top of a light-impermeable 3 ⁇ M filter that separates two chambers.
  • CMFDA green fluorescent dye
  • mDCs mature DCs
  • rhCCL19 chemoattractant
  • target/effector co-culture was added to the lower chamber.
  • the fluorescence of mDCs that had migrated across the endothelial cells and through the filter was measured by a Fluostar plate reader as a measure of the number of cells migrating across the membrane. Inserts were coated with human dermal microvascular endothelial cells (HDMEC) that were grown to confluence for 72 hrs. Co-cultures containing targets and effectors were plated into the lower chamber 48 hrs before the assay.
  • HDMEC human dermal microvascular endothelial cells
  • Targets had been irradiated at 48 Gy and washed 1x in R10 prior to plating out. All three donors were tested, and each combination of effector/target was set up in triplicate. On assay day, mDCs were counted (as described in CBP 067v00) and resuspended in 2.5 ⁇ M CMFDA for 40 minutes at 37°C. They were then washed in PBS and resuspended to 10x106cells/mL in phenol red free R10 so that 5x105cells were dispensed in 50 ⁇ L.100 ng/mL rhCCL19 was included as a positive control.
  • Afamitresgene autoleucel (previously known as ADP-A2M4) T cells express a MAGEA4 specific TCR without IL-7 and CCL19.
  • ADP-A2M4N7X19 T cells were generated by additionally co-expressing recombinant IL-7 and CCL19 in the afamitresgene autoleucel T cells.
  • afamitresgene autoleucel ADP-A2M4
  • afamitresgene autoleucel ADP-A2M4N7X19
  • non-transduced T-cells were restimulated with irradiated A375 (MAGE-A4 antigen-positive) cells on a weekly basis for a total of 28 days.
  • irradiated A375 MAGE-A4 antigen-positive
  • ADP-A2M4N7X19 expanded over the course of the 28-day restimulation assay with some donor variation ( Figure 1).
  • afamitresgene autoleucel expanded to a lesser extent and only until day 14 before declining, while non- transduced T-cell products declined in number over the entire course of the assay.
  • the addition of exogenous rhIL-7 resulted in expansion of both afamitresgene autoleucel and ADP-A2M4N7X19 T-cells, with no expansion of the non-transduced T-cells ( Figure 1).
  • Cytokine Profiling At each time point for the assays detailed above, supernatants were taken both before and 24 hours after restimulation with antigen and stored for subsequent analysis for a range of cytokines (Table 6) by Meso Scale Discovery (MSD) multiplex assays. For each timepoint, the level of each cytokine detected in the presence of exogenous rhIL-7 was measured (data not shown). CCL19 secretion was measured by a separate ELISA. Comparable levels of IFN ⁇ were detected in the supernatants of both afamitresgene autoleucel and ADP-A2M4N7X19 at Day 1 and Day 7, with Day 1 values elevated compared to Day 7 ( Figure 4).
  • IFN ⁇ levels were consistently higher for ADP-A2M4N7X19 T-cells. Sustained IFN ⁇ production throughout the duration of the assay indicates that IL-7 reduces T-cell exhaustion from repeated stimulation. As expected, negligible IL-7 and CCL19 were detected with the non-transduced T-cells and afamitresgene autoleucel. In contrast, ADP-A2M4N7X19 had sustained IL-7 ( Figure 5) and CCL19 ( Figure 6) production throughout the duration of the restimulation assay, in line with IFN ⁇ data. Further cytokines were also evaluated and no responses of concern were observed.
  • ADP-A2M4N7X19 demonstrated: ⁇ An enhanced ability to respond to repeated antigen stimulations up to at least 2 weeks longer than afamitresgene autoleucel. ⁇ Expansion of IL-7-induced CD8+ TCR- population allowing the possibility of epitope spreading. ⁇ Secretion of higher levels of proinflammatory cytokines such as IFN ⁇ , than afamitresgene autoleucel. ⁇ Antigen-dependency of cytokine release and expansion.
  • transwell migration assay To confirm production of CCL19 by ADP- A2M4N7X19 has the intended effect, a transwell migration assay was developed. These assays were designed to mimic the gradient of CCL19 secretion that may be found in the tumor microenvironment.
  • the transwell migration assay measures the chemotactic ability of cells toward a chemo- attractant. In this case, the ability of CCL19 released by ADP-A2M4N7X19 T-cells to act as a chemo-attractant.
  • ADP-A2M4N7X19 were plated with NCI-H1755 (MAGE-A4 antigen- positive) cells in the bottom chamber of a transwell plate, with stimulation leading to the release of CCL19.
  • mDCs Mature dendritic cells
  • Controls included recombinant human (rh)-CCL19 as chemo-attractant control and ADP- A2M4IL7 (T-cells that express IL-7 under an inducible, rather than a constitutive, promoter as in ADP-A2M4N7X19).
  • Migration of fluorescently stained mDCs was quantified as mean fluorescent units measured in the lower chamber of the transwell. Only background migration was observed for non-transduced, afamitresgene autoleucel, and ADP-A2M4IL7 T-cells ( Figure 7).
  • the migration rate and peak migration of mDCs in the presence of ADP-A2M4N7X19 and rhCCL19 are very similar, even though the concentration of rhCCL19 used was ⁇ 10 fold greater than the CCL19 measured in the supernatants of ADP-A2M4N7X19 T-cells/targets
  • the lack of mDC migration towards activated ADP- A2M4IL7 indicates that the migration observed for ADP-A2M4N7X19 is due to expression of CCL19.
  • a crucial premise for co-expression of IL-7 and CCL19 is the capacity to engage other arms of the immune system (e.g. na ⁇ ve, central memory and regulatory T-cells, B-cells, and dendritic and NK cells) to increase SPEAR T-cell efficacy, which cannot be fully evaluated in the immunodeficient animal models required for these studies.
  • arms of the immune system e.g. na ⁇ ve, central memory and regulatory T-cells, B-cells, and dendritic and NK cells
  • mice were inoculated with the P815 mouse mastocytoma cell line which is positive for the mouse tumor antigen P1A. Treatments (Table3) were administered by single intravenous injection 14 days after P815 transplantation and 3 days after cyclophosphamide administration. Two mice from each group had serum IFN ⁇ , IL-7 and CCL19 measured at day 6.
  • Table 3 In Vivo Treatment Groups Study groups 3, 4 and 5 had significant decreases in tumor volume by the final day of the study, with 1 mouse each from Groups 2 and 5 and 3 mice in Group 3 fully clearing their tumor. In Groups 2 and 5, one mouse each survived on the final day of the observation period ( Figure 9). In Group 4, 2 mice survived, and the survival period was significantly longer than that of Group 1. In Group 3, no dead animals were observed until Day 32, 4 mice survived, and the survival period was significantly longer than that in Group 1. Despite clear evidence of the anti-tumor benefit from the addition of IL-7 and CCL19, neither were detectable in the serum of animals from any group. However, higher values of IFN ⁇ and IL-7 were found in Group 3 in the ELISA analysis of tumor tissues.

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Abstract

La présente invention concerne des lymphocytes T modifiés pour exprimer un récepteur de l'antigène des lymphocytes T (TCR) hétérologue qui se lie aux cellules cancéreuses exprimant MAGE-A4, et qui expriment en outre l'interleukine 7 (IL-7) hétérologue et le ligand 19 de la chimiokine à motif C-C (CCL19) hétérologue, ainsi que leur utilisation dans le traitement du cancer. L'invention concerne également des populations de lymphocytes T modifiés, des constructions d'acides nucléiques, des vecteurs et des méthodes de production et d'utilisation de lymphocytes T modifiés dans le traitement du cancer.
PCT/EP2023/059714 2022-04-14 2023-04-13 Lymphocytes t modifiés pour expression de l'interleukine 7 et du ligand 19 de la chimiokine à motif c-c WO2023198849A1 (fr)

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Citations (10)

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