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WO2022029431A1 - Récepteur chimérique liant le tgf-bêta - Google Patents

Récepteur chimérique liant le tgf-bêta Download PDF

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WO2022029431A1
WO2022029431A1 PCT/GB2021/052017 GB2021052017W WO2022029431A1 WO 2022029431 A1 WO2022029431 A1 WO 2022029431A1 GB 2021052017 W GB2021052017 W GB 2021052017W WO 2022029431 A1 WO2022029431 A1 WO 2022029431A1
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polypeptide
cell
nucleic acid
cells
endodomain
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Shaun CORDOBA
Matteo Righi
Martin PULÉ
Marco DELLA PERUTA
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Autolus Ltd
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Autolus Ltd
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4204—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a cell which expresses a chimeric receptor which binds TGF ⁇ .
  • Adoptive immunotherapy of cancer involves the ex vivo generation of cancer-antigen specific cells and their administration.
  • Adoptively transferred immune effector cells also activate existing adaptive and innate immune cells within the tumour once they activate and start causing inflammation.
  • the native specificity of immune effector cells can be exploited in adoptive immunotherapy - for example during the generation of melanoma specific T-cells from expansion of tumour infiltrating lymphocytes in tumour resections. Otherwise a specificity can be grafted onto a T- cell using genetic engineering. Two common methods for achieving this are using chimeric antigen receptors or transgenic T-cell receptors. Different kinds of immune effector cells can also be used. For example, alpha/beta T-cells, NK cells, gamma delta T-cells or macrophages can be used.
  • B-ALL B-cell Acute Lymphoblastic Leukaemia
  • DLBCL Diffuse Large B-cell Lymphoma
  • MM Multiple Myeloma
  • One of the main inhibitory mechanisms within the tumour microenvironment is transforming growth factor beta (TGF ⁇ ).
  • TGF ⁇ Transforming growth factor beta
  • TGF ⁇ plays an important role in the establishment of a tumour. Under normal conditions TGF ⁇ plays a tumour suppressor role by causing cell-cycle arrest, apoptosis and suppression of tumorigenic inflammation. But, as tumours progress, there is an increased concentration ofTGF ⁇ present within the tumour microenvironment (TME). Produced by cells recruited to the tumour, TGF ⁇ within the milieu serves to promote tumour progression and suppress the cytotoxic function of infiltrating lymphocytes (Mariathasan et al., 2018 Nature 554, 544-548; Polanczyk et al., 2019, J. Transl. Med. 17, 219).
  • TGF ⁇ limits the efficacy of the CAR T cells produced.
  • Tumour derived TGF ⁇ can cause inhibition of T cell proliferation, differentiation into cytotoxic lymphocytes (CTLs) and helper T cells and inhibit the T cell stimulatory functions of antigen presenting cells (APCs).
  • CTLs cytotoxic lymphocytes
  • APCs antigen presenting cells
  • Fresolimumab is a neutralizing antibody which blocks TGF ⁇ 1-3.
  • Fresolimumab has been tested in metastatic melanoma and high-grade glioma. This showed some effectiveness in the enhancement of a tumour-specific immune response but failed to eradicate the tumour.
  • tumour-unique antigen Another major challenge for T cell therapies is the identification of a tumour-unique antigen.
  • Many of the tumour associated antigens (TAAs) targeted are also expressed at low levels in healthy cells, causing on-target off-tumour toxicity. Solid tumours also tend to display a large degree of heterogeneity. Many tumours have only a subset of cells which express the target antigen. And even in the case of a uniformly expressed TAA, antigen escape or loss is possible, making the target antigen unavailable for the CAR T cell to bind to.
  • ERBB2 On-target off-tumour toxicity has been borne out by clinical trials. For example, an approach targeting ERBB2 caused death to a patient with colon cancer metastatic to the lungs and liver. ERBB2 is over-expressed in colon cancer in some patients, but it is also expressed on several normal tissues, including heart and normal vasculature.
  • the challenges of solid tumour targeting may be overcome including on- target off-tumour toxicity.
  • FIG. 1 Schematic diagram illustrating a TGF ⁇ soluble AND gate of the first embodiment of the invention in which the second polypeptide comprises a CD3 ⁇ endodomain.
  • A) In the absence of both the tumour associated antigen (TAA) and TGF ⁇ .
  • B) In the presence of both TAA and TGF ⁇ .
  • TGF ⁇ binding to extracellular TBRII of the first polypeptide recruits TBRI and the endodomain of the second polypeptide, while TAA binding to the antigen binding domain of the first polypeptide initiates T cell signalling via the endodomain of the chimeric receptor.
  • TGF ⁇ In the presence of TGF ⁇ but not TAA. Lack of a TAA bound to the antigen binding domain of the first polypeptide means there is no signal to initiate T cell activation.
  • Figure 2 Graph showing results of plate bound assay measuring CD25 MFI to indicate level of activation of cells expressing the constructs shown in Figure 4, after 48 hours.
  • Figure 3 Graph showing result of co-culture assay showing cytotoxic effect of cells expressing the constructs shown in Figure 4 to target SUPT 1-EGFR cells after 24 hour incubation.
  • Figure 4 Summary of constructs tested in Examples 1 and 2.
  • FIG. 5 Schematic diagram illustrating an alternative TGF ⁇ soluble AND gate of the first embodiment of the invention in which the second polypeptide comprises a CD3 ⁇ endodomain, together with a co-stimulatory domain, for example the endodomain from CD28 or 41 BB.
  • the second polypeptide comprises a CD3 ⁇ endodomain, together with a co-stimulatory domain, for example the endodomain from CD28 or 41 BB.
  • TGF ⁇ target antigen
  • Figure 6 Graph showing result of co-culture assay showing cytotoxic effect of cells expressing the CD28-expressing constructs shown in Figure 8 to target SUPT1-EGFR cells after 24 hour incubation, in the presence of varying concentrations of TGF ⁇ .
  • Figure 7 Graph showing result of co-culture assay showing cytotoxic effect of cells expressing the 4-1 BB-expressing constructs shown in Figure 8 to target SUPT1-EGFR cells after 24 hour incubation, in the presence of varying concentrations of TGF ⁇ .
  • Figure 8 Summary of constructs tested in Example 3.
  • Figure 9 Schematic diagram illustrating an enhancer system of the second embodiment of the invention in which the first polypeptide comprises a CD3 ⁇ endodomain and the endodomain of the second polypeptide comprises a co-stimulatory domain.
  • A) In the absence of both the tumour associated antigen (TAA) and TGF ⁇ .
  • B) In the presence of both TAA and TGF ⁇ .
  • TGF ⁇ binding to extracellular TBRII of the first polypeptide recruits the TBRI and the endodomain of the second polypeptide, while TAA binding to the antigen binding domain of the first polypeptide initiates a second generation CAR signal via the endodomain of the chimeric receptor.
  • TGF ⁇ binding to TBRII of the first polypeptide means the co-stimulatory endodomain of the second polypeptide is not brought into proximity of the CD3z endodomain of the first polypeptide, so the first polypeptide alone initiates a first generation CAR signal D) In the presence of TGF ⁇ but not TAA. Lack of a TAA bound to the antigen binding domain of the first polypeptide means there is no signal to initiate T cell activation.
  • Figure 10 Schematic diagram illustrating an alternative enhancer system of the second embodiment of the invention in which the first polypeptide comprises a CD3 ⁇ endodomain and a co-stimulatory domain and the endodomain of the second polypeptide comprises an additional co-stimulatory domain.
  • the first polypeptide comprises a CD3 ⁇ endodomain and a co-stimulatory domain
  • the endodomain of the second polypeptide comprises an additional co-stimulatory domain.
  • the present inventors have found that it is possible to exploit the natural heterodimerization of the TGF ⁇ receptor complex a produce a chimeric receptor which is only fully activated in the presence of both TGF ⁇ and the target antigen, thereby reducing on-target off-tumour toxicity.
  • the present invention provides a cell which expresses a chimeric receptor comprising (a) a first polypeptide and (b) a second polypeptide, wherein the first polypeptide (a) comprises: an antigen binding domain, a linker, an extracellular region of a first transforming growth factor beta receptor (TBR), a first transmembrane domain and a first endodomain, and wherein the second polypeptide (b) comprises: an extracellular region of a second TBR, a second transmembrane domain and a second endodomain.
  • TBR transforming growth factor beta receptor
  • the extracellular region of the first TBR and the extracellular region of the second TBR are capable of heterodimerization, such that in the presence of TGF ⁇ ligand, the first TRB recruits the second TBR forming a complex, and, bring the first and second polypeptides of the chimeric receptor into proximity with each other.
  • TGF ⁇ may be a soluble TGF ⁇ , mutant TGF ⁇ or a truncated TGF ⁇ polypeptide.
  • the linker may be a flexible linker.
  • the linker may be a glycine/serine flexible linker.
  • the linker may be a spacer.
  • the first TBR may be TRBII and the second TRB may be TBRI.
  • the first transmembrane domain may be a TRBII transmembrane domain and when the second TRB is TRBI, the second transmembrane domain may be a TRBI transmembrane domain.
  • the first TBR may be TBRI and the second TRB may be TRBII.
  • the first transmembrane domain When the first TBR is TRBI, the first transmembrane domain may be a TRBI transmembrane domain and when the second TRB is TRBII, the second transmembrane domain may be a TRBII transmembrane domain.
  • the endodomain of the second polypeptide comprises an intracellular signaling domain such as a CD3 ⁇ endodomain.
  • the endodomain of the second polypeptide may also comprise a co-stimulatory domain.
  • endodomain of the first polypeptide lacks an intracellular signalling domain such as a CD3 ⁇ endodomain, but it may comprise a co-stimulatory domain. In the absence of TGF ⁇ no signalling occurs, but in the presence of TGF ⁇ , the first and second polypeptides are brought together and signalling occurs in the presence of target antigen.
  • the endodomain of the first polypeptide comprises an intracellular signalling domain such as a CD3 ⁇ endodomain.
  • the first polypeptide alone provides an activation signal in the presence of target antigen and in the absence of TGF ⁇ .
  • the endodomain of the second polypeptide comprises a co-stimulatory domain, such that, in the presence of TGF ⁇ , a complex is formed and signalling is enhanced.
  • the endodomain of the first polypeptide comprises an intracellular signalling domain alone and the endodomain of the second polypeptide comprises a co-stimulatory domain.
  • the first polypeptide provides a first-generation activation signal.
  • second-generation signalling can occur in the presence of target antigen.
  • the first polypeptide comprises a signalling domain and a first co-stimulatory domain, such that first polypeptide alone provides a second generation activation signal in the presence of target antigen and in the absence of TGF ⁇ .
  • a complex is formed and third-generation signalling occurs in the presence of target antigen.
  • the co-stimulatory domain may comprise a TNFR endodomain.
  • the co-stimulatory domain may be selected from the group consisting of CD28, ICOS, 41 BB, CD40L, 0X40, CD27 and ICOS endodomains.
  • the antigen binding domain may be capable of binding to a tumour associated antigen (TAA) present on a solid tumour cell.
  • TAA tumour associated antigen
  • the TAA may be selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), GD2, ROR1 and B7H3.
  • the present invention provides a nucleic acid construct encoding the first polypeptide and the second polypeptide of the chimeric receptor of the cell as defined in the first aspect.
  • nucleic acid construct of the second aspect having the following structure:
  • ABD is a nucleic acid sequence encoding the antigen-binding domain of the first polypeptide
  • linker is a nucleic acid sequence encoding the linker of the first polypeptide
  • 1TBR is a nucleic acid sequence encoding the transforming growth factor beta receptor (TBR) of the first polypeptide
  • 1TM is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide
  • 1endo is a nucleic acid sequence encoding the endodomain of the first polypeptide coexpr is a nucleic acid sequence enabling co-expression of both the first and second polypeptides;
  • 2TBR is a nucleic acid sequence encoding the transforming growth factor beta receptor (TBR) of the second polypeptide
  • 2TM is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide
  • 2endo is a nucleic acid sequence encoding second endodomain of the second polypeptide.
  • the endodomain of the first polypeptide is "inert" in that sense that it lacks an intracellular signalling domain or a costimulatory domain; and the endodomain of the second polypeptide comprises an intracellular signalling domain.
  • the endodomain of the first polypeptide is "inert" in that sense that it lacks an intracellular signalling domain or a costimulatory domain; and the endodomain of the second polypeptide comprises an intracellular signalling domain and a costimulatory domain.
  • the endodomain of the first polypeptide is "inert" in that sense that it lacks an intracellular signalling domain or a costimulatory domain; and the endodomain of the second polypeptide comprises an intracellular signalling domain, a first costimulatory domain and a second costimulatory domain, such that the receptor complex produces a third generation activation signal.
  • the first co-stimulatory domain may, for example, be from 4-1 BB or 0X40 and the second endodomain may be from CD28.
  • the endodomain of the first polypeptide comprises an intracellular signalling domain and the endodomain of the second polypeptide comprises a co-stimulatory domain.
  • the endodomain of the first polypeptide comprises an intracellular signalling domain and a first co-stimulatory domain and the endodomain of the second polypeptide comprises a second co-stimulatory domain.
  • the first co-stimulatory domain may, for example, be from 4-1 BB or 0X40 and the second endodomain may be from CD28, or vice versa.
  • the nucleic acid construct When the nucleic acid construct is expressed in a cell such as a T cell, the first and second polypeptides are co-expressed at the cell surface.
  • coexpression sequence "coexpr” may encode a sequence comprising a self-cleaving peptide.
  • the present invention provides a vector comprising a nucleic acid construct according to the second aspect of the invention.
  • the vector of the third aspect of the invention may be retroviral vector or a lentiviral vector or a transposon.
  • the present invention provides a kit which comprises: i) a vector comprising a nucleic acid sequence encoding a first polypeptide of the chimeric receptor, as defined in the first aspect of the invention, and ii) a vector comprising a nucleic acid sequence encoding a second polypeptide of the chimeric receptor, as defined in the first aspect of the invention.
  • the present invention provides a method for making a cell according to the first aspect of the invention, which comprises the step of introducing: a nucleic acid construct according to the second aspect of the invention; a vector according to the third aspect of the invention; or a kit of vectors according to the fourth aspect of the invention.
  • the cell may be from a sample isolated from a subject.
  • the present invention provides a pharmaceutical composition comprising a plurality of cells according to the first aspect of the invention.
  • the invention provides a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition of the sixth aspect of the invention to a subject.
  • the method may comprise the following steps: (i) isolation of a cell-containing sample from a subject; (ii) transduction or transfection of the cells with a nucleic acid construct according to the second aspect of the invention; a vector according to the third aspect of the invention; or a kit of vectors according to the fourth aspect of the invention; and administering the cells from (ii) to the subject.
  • the sample may be a T-cell containing sample.
  • the disease may be a cancer.
  • the invention provides a pharmaceutical composition according to the sixth aspect of the invention, for use in treating and/or preventing a disease.
  • the present invention provides use of a cell according to the first aspect of the invention, in the manufacture of a medicament for treating and/or preventing a disease.
  • both embodiments of the invention exploit the natural heterodimerization of the TGF ⁇ receptor complex such that the chimeric receptor of the cell is only fully activated in the presence of both TGF ⁇ and the target antigen.
  • the first TRB recruits the second TRB upon binding to TGF ⁇ , forming the TGF ⁇ complex and bringing the first polypeptide of the chimeric receptor is brought into proximity with the second polypeptide.
  • the second polypeptide comprises an intracellular signalling domain, such as a CD3 ⁇ endodomain.
  • the T cell When the antigen binding domain of the first polypeptide binds to an antigen present on the surface of a target cell, without presence of TGF ⁇ , the T cell is not capable of effecting a T cell signalling response via the endodomain of the second polypeptide because the first and second polypeptides are not in proximity with one another. Conversely, when the antigen binding domain binds to a target antigen in the presence of TGF ⁇ , the T cell is capable of effecting a T cell response.
  • the presence of TGF ⁇ is a positive marker of a tumour microenvironment, inducing T cell activation only in the presence of a cancerous cell. This reduces the risk of on-target off-tumour toxicity. Engagement of TGF ⁇ in the receptor complex of the invention also reduces its concentration in the tumour microenvironment thereby reducing the suppressive effects of TGF ⁇ on adoptive immunotherapy.
  • the first embodiment of the present invention provides cells, such as T cells, which express a chimeric receptor having a first and second polypeptide.
  • the antigen binding domain of the first polypeptide of the chimeric receptor is capable of binding to an antigen present on the surface of a target cell but is only capable of effecting a cell signalling response via the endodomain when the first and second polypeptides are brought into proximity of each other.
  • first TRB of the first polypeptide recruits a second TRB of the second polypeptide upon binding to TGF ⁇ ligand and forming the TGF ⁇ complex
  • the first polypeptide of the chimeric receptor is brought into proximity with the second polypeptide. This results in cell signalling via the endodomain that can occur only in the presence of TGF ⁇ , thereby reducing the risk of on-target off-tumour toxicity as well as the reducing the suppressive effects of TGF ⁇ on adoptive immunotherapy.
  • the first polypeptide comprises an intracellular signalling domain, such as a CD3 ⁇ endodomain and the endodomain of the second polypeptide comprises a costimulatory domain.
  • the first TRB recruits the second TRB upon binding to TGF ⁇ , forming the TGF ⁇ complex, the first polypeptide of the chimeric receptor is brought into proximity with the second polypeptide so that the co- stimulation signals provided by the second polypeptide can occur only in the presence of TGF ⁇ .
  • the presence of the second polypeptide in the complex can either turn a first generation signal into a second generation signal (as illustrated schematically in Figure 9) or turn a second generation signal into a third generation signal (as illustrated schematically in Figure 10).
  • the cell When CD3 ⁇ is present on the first polypeptide chain and the antigen binding domain of the first polypeptide binds to an antigen present on the surface of a target cell, without presence of TGF ⁇ , the cell does not receive additional co-stimulation signals present on the endodomain of the second polypeptide chain because the first and second polypeptides are not in proximity with one another. Conversely, when the antigen binding domain binds to its target antigen in the presence of TGF ⁇ , the T cell does receive co-stimulation signals present on the endodomain of the second polypeptide chain.
  • the presence of TGF ⁇ is a positive marker of a tumour microenvironment, enhancing T cell activation through co-stimulation signals only in the presence of a cancerous cell as well as TGF ⁇ .
  • engagement of TGF ⁇ in the receptor complex of the second embodiment of the invention also reduces its concentration in the tumour microenvironment thereby reducing the suppressive effects of TGF ⁇ on adoptive immunotherapy.
  • the cells of the present invention are also able to modulate the activity other cells within the tumour which respond to TGF ⁇ , such as stromal cells which are involved in fibrosis and the survival of cancer stem cells.
  • the cell of the present invention expresses a chimeric receptor having first and second polypeptides, in which the first polypeptide comprises an antigen binding domain.
  • the second polypeptide has an endodomain comprising a signal transmission portion, whereas the first polypeptide lacks an intracellular signalling domain.
  • the chimeric receptor of the cell described herein comprises a first polypeptide comprising: an antigen-binding domain, a linker, an extracellular region of a first TRB, a first transmembrane domain and a first endodomain; and a second polypeptide comprising: an extracellular region of a second TBR, second transmembrane domain and a second endodomain, wherein the extracellular regions of the first and second TBRs are capable of heterodimerisation upon binding to TGF ⁇ .
  • hetero-tetrameric receptor complex brings together the first and second polypeptides such that they are in proximity with one another in the presence of tumour microenvironment secreting factor, TGF ⁇ . This structural change aligns the endodomain of the second polypeptide more closely with the antigen binding domain of the first polypeptide, which, in the first embodiment of the invention, then permits cell signalling to occur.
  • the transmission of an activating signal to the T-cell is triggered.
  • the chimeric receptor-encoding nucleic acids may be transferred to cells such as T cells using, for example, retroviral vectors. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer.
  • the chimeric receptor described herein directs not only the specificity and cytotoxicity of the T cell towards cells expressing the target antigen but is also only inducible in a TGF ⁇ - secreting tumour microenvironment. This arrangement is particularly advantageous in the treatment of solid tumour cancers, reducing on target, off tumour toxicity.
  • Table 1a provides a truth table for the TGF ⁇ -activated chimeric receptor of the first embodiment of the invention:
  • the first polypeptide has an endodomain comprising a signal transmission portion, whereas the first polypeptide has an endodomain comprising a co-stimulatory domain.
  • Table 1b provides a truth table for the TGF ⁇ -activated chimeric receptor of the second embodiment of the invention: Table 1 b:
  • TGF ⁇ plays an important role in the establishment of a tumour. Under normal conditions TGF ⁇ plays a tumour suppressor role by causing cell-cycle arrest, apoptosis and suppression of tumorigenic inflammation. However, in more advanced cancers, TGF ⁇ can promote immune evasion and angiogenesis. In the context of adaptive immunotherapy, TGF ⁇ limits the efficacy of the T cells produced. Tumour derived TGF ⁇ can cause inhibition of T cell proliferation, differentiation into cytotoxic lymphocytes (CTLs) and helper T cells and inhibit the T cell stimulatory functions of antigen presenting cells (APCs). Tumour cells become resistant to the anti-mitogenic effects and TGF ⁇ becomes a tumour promoter. TGF ⁇ is a cytokine belonging to the transforming growth factor superfamily. TGF ⁇ 1 and 2 are implicated in cancer, where they may stimulate the cancer stem cell, increase fibrosis I desmoplastic reactions and suppress immune recognition of the tumour.
  • CTLs cytotoxic lymphocytes
  • APCs antigen presenting cells
  • the ligand TGF ⁇ 1 binds to cell surface transforming growth factor beta receptor, type-two (TBRII), which in turn recruits and phosphorylates transforming growth factor beta receptor, type-one (TBRI) to form a hetero-tetrameric receptor complex.
  • TGF ⁇ type-two
  • the main signalling pathway for TGF ⁇ is the SMAD pathway in which TBRI phosphorylates SMAD2 and SMAD3.
  • SMAD2/3 along with SMAD4 translocate to the cell nucleus and recruit several co-factors and transcription factors to activate target genes such as COL1A1/2 CTGF and ASMA which are mainly used in wound healing, migration, proliferation and angiogenesis (Neuzillet et al., 2015, Pharmacol. Ther. 147, 22-31).
  • TGF ⁇ is a soluble factor present at high concentrations and has an immunosuppressive effect on adaptive immunotherapy, limiting the efficacy of the treatment in solid tumours.
  • a variety of cancerous tumour cells are known to produce TGF ⁇ directly.
  • TGF ⁇ can be produced by the wide variety of non- cancerous cells present at the tumour site. Specifically, tumour-associated T cells, natural killer (NK) cells, macrophages, epithelial cells and stromal cells have all been shown to produce TGF ⁇ in various tumour models.
  • TBRs transforming growth factor beta receptors
  • AKS activin receptor-like kinases
  • Auxiliary co-receptors also known as type III receptors
  • TGF ⁇ superfamily of ligands binds to type I and type II receptors.
  • TGF ⁇ superfamily receptors specific for TGF ⁇ there are three TGF ⁇ superfamily receptors specific for TGF ⁇ , the TBRs, which can be distinguished by their structural and functional properties.
  • TBRI ALK5
  • TBRII have similar ligand-binding affinities and can be distinguished from each other only by peptide mapping. Both TBRI and TBRII have a high affinity for TGF ⁇ 1 and low affinity for TGFP2.
  • TBR3 P-glycan
  • the TBRs also bind TGFP3.
  • the formation of the receptor complex composed is of TBRI and TBRII molecules symmetrically bound to the cytokine dimer and results in the phosphorylation and the activation of TRBI by the constitutively active TBRII.
  • TGF-beta receptor type-1 (TBRI) is available from UniProt accession P36897 is shown below as SEQ ID NO: 1.
  • TGF-beta receptor type-2 (TBRII) is available from UniProt accession P37173 is shown below as SEQ ID NO: 2.
  • HENILQFLTA EERKTELGKQ YWLITAFHAK GNLQEYLTRH VISWEDLRKLGSSLARGIAH LHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTA RYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREH PCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSE LEHLDRLSGR SCSEEKIPED GSLNTTK TGF ⁇ 1 , 2 and 3 signal via binding to receptors TpRI I and then association to TpRI and in the case of TGFP2 also to TpRI 11. This leads to subsequent signalling through SMADs via TpRI.
  • the present invention provides a cell expressing a chimeric receptor comprising a first polypeptide and a second polypeptide, each of which comprises an extracellular region of a first transforming growth factor receptor (TBR) and an extracellular region of a second TBR.
  • TBR transforming growth factor receptor
  • the extracellular region of the first TBR and extracellular region of the second TBR described herein, are capable of binding to TGF ⁇ , and forming the hetero-tetrameric receptor complex because the extracellular region is the TGF ⁇ binding portion of TRB.
  • the TGF ⁇ -induced immunosuppressive signal is inhibited, thereby rendering immune cells resistant to the immunosuppressive impacts of TGF ⁇ in the TME.
  • the lack of signal activation enables the extracellular TBRs to bind and sequester TGF ⁇ , preventing TGF ⁇ associated functions.
  • Dominant negative TBRs are TBRs which block TGFb signalling by blocking the kinase activity of the endodomain and thereby preventing signal transduction. They have been developed in a number of ways, for example, by mutations such as introducing a stop codon in the cytoplasmic tail of the TRB, completely removing the kinase domains (Weiser et al., 1993, Mol. Cell. Biol. 13, 7239-7247).
  • the first TBR described herein may be the extracellular region of TRBII and the second TRB described herein may be the extracellular region of TBRI.
  • the first TBR described herein may be the extracellular region of TBRI and the second TBR described herein may be the extracellular region of TRBII.
  • the extracellular region of TBRII may comprise SEQ ID NO: 3 or a variant thereof which has the ability to bind to TGF ⁇ and heterodimerize with TBRI to form the hetero- tetrameric receptor complex.
  • the SEQ ID NO: 3 variant may have at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:3 and have equivalent properties to SEQ ID NO: 3.
  • the extracellular region of TBRII may comprise SEQ ID NO: 4 or a variant thereof which has the ability to bind to TGF ⁇ and heterodimerize with TBRII to form the hetero- tetrameric receptor complex.
  • the SEQ ID NO: 4 variant may have at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:4 and have equivalent properties to SEQ ID NO: 4.
  • SEQ ID NO: 4 extracellular region of TRBI
  • variant TBRI polypeptide and “variant TRBII polypeptide” means the polypeptide has an amino acid sequence which has one, two, three or more additions, deletions and/or substitutions compared with the wild-type TBR polypeptide.
  • the percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST, which is freely available at http://blast.ncbi.nlm.nih.gov. Suitably, the percentage identity is determined across the entirety of the reference and/or the query sequence.
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Unless otherwise explicitly stated herein by way of reference to a specific, individual amino acid, amino acids may be substituted using conservative substitutions as recited below.
  • An aliphatic, non-polar amino acid may be a glycine, alanine, proline, isoleucine, leucine or valine residue.
  • An aliphatic, polar uncharged amino may be a cysteine, serine, threonine, methionine, asparagine or glutamine residue.
  • An aliphatic, polar charged amino acid may be an aspartic acid, glutamic acid, lysine or arginine residue.
  • An aromatic amino acid may be a histidine, phenylalanine, tryptophan or tyrosine residue.
  • the antigen-binding domain is the portion of a chimeric receptor which recognizes cell surface tumour associated antigen (TAA).
  • TAA cell surface tumour associated antigen
  • the antigen- binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a wild-type ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • the antigen-binding domain may bind to a tumour associated antigen (TAA).
  • TAA tumour associated antigen
  • Various cell surface TAAs are envisioned as being potentially suitable for targeting with the chimeric receptor described herein.
  • Tyrosine-protein kinase transmembrane receptor ROR1 also known as neurotrophic tyrosine kinase, receptor-related 1 (NTRKR1) is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family.
  • ROR1 has recently been shown to be expressed on ovarian cancer stem cell, on which it seems to play a functional role in promoting migration/invasion or spheroid formation in vitro and tumour engraftment in immune-deficient mice.
  • an illustrative antigen binding domain of the present invention is provided in SEQ ID NO: 5.
  • the antigen binding domain may comprise SEQ ID NO: 5 or a variant thereof which has the ability to bind to the TAA ROR1.
  • the chimeric receptor described herein is capable of directing a cytotoxic effect towards cells expressing ROR1.
  • the SEQ ID NO: 5 variant may have at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:5 and have equivalent properties to SEQ ID NO: 5.
  • PSMA Prostate-specific membrane antigen is also known as glutamate carboxypeptidase II (GCPII), as well as N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) or NAAG peptidase.
  • GCPII glutamate carboxypeptidase II
  • NAALADase I N-acetyl-L-aspartyl-L-glutamate peptidase I
  • NAAG peptidase NAAG peptidase.
  • Human PSMA contains 750 amino acids and weighs approximately 84 kDa.
  • PSMA is a zinc metalloenzyme that resides in membranes.
  • an illustrative antigen binding domain of the present invention is provided in SEQ ID NO: 6.
  • the antigen binding domain may comprise SEQ ID NO: 6 or a variant thereof which has the ability to bind to the TAA PSMA.
  • the chimeric receptor described herein is capable of directing a cytotoxic effect towards cells expressing PSMA.
  • the SEQ ID NO: 6 variant may have at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 6 and have equivalent properties to SEQ ID NO: 6.
  • SEQ ID NO: 6 (aPSMA VHH)
  • the epidermal growth factor receptor (EGFR; ErbB-1 ; HER1 in humans) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands.
  • an illustrative antigen binding domain of the present invention is provided in SEQ ID NO: 7.
  • the antigen binding domain may comprise SEQ ID NO: 7 or a variant thereof which has the ability to bind to the TAA EGFR.
  • the chimeric receptor described herein is capable of directing a cytotoxic effect towards cells expressing EGFR.
  • the SEQ ID NO: 7 variant may have at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 7 and have equivalent properties to SEQ ID NO: 7.
  • SEQ ID NO: 7 (aEGFR VHH)
  • the chimeric receptor described herein may comprise a linker sequence to connect the antigen-binding domain with the first TBR of the first polypeptide.
  • a flexible liner allows the antigen-binding domain to orient in different directions to facilitate binding. This is particularly advantageous when the TAA is a cell surface TAA present on a solid tumour.
  • a flexible linker is advantageous to easier facilitate binding of both the antigen binding domain to the cognate TAA and the first TRB to soluble TFGb. This is particularly important in a low density TAA presentation.
  • the linker sequence may, for example, comprise an lgG1 Fc region.
  • the linker may alternatively comprise a linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region.
  • the flexible linker sequence may be composed of glycine/serine amino acids of suitable length.
  • linker main may comprise SEQ ID NO: 8 or a variant thereof which has the ability to facilitate binding of both the antigen binding domain to the TAA and the first TRB to TGF ⁇ .
  • SEQ ID NO: 8 (Glycine /Serine flexible linker)
  • the transmembrane domain is the sequence of a chimeric receptor that spans the membrane. Since the chimeric receptor described herein comprises of two membrane spanning sites, the chimeric receptor comprises a first and second transmembrane domain arranged on the first and second polypeptides, respectively.
  • a transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues.
  • the transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention.
  • the presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs. dtu.dk/services/TMHMM-2.0/).
  • TMHMM algorithm http://www.cbs. dtu.dk/services/TMHMM-2.0/.
  • an artificially designed TM domain may also be used (US 7052906 B1 describes synthetic transmembrane components).
  • the transmembrane domain may be derived from TBR.
  • the transmembrane domain may be derived from TBRI or TBRII.
  • the first transmembrane domain of the first polypeptide derives from the same TBR as the first TBR of the first polypeptide.
  • the first transmembrane domain of the first polypeptide may be a TBRII derived transmembrane domain.
  • the second TBR of the second polypeptide is TBRI
  • the second transmembrane domain of the second polypeptide may be a TBRI derived transmembrane domain.
  • the first transmembrane domain of the first polypeptide may be a TBRI derived transmembrane domain.
  • the second TBR of the second polypeptide is TBRII
  • the second transmembrane domain of the second polypeptide may be a TBRII derived transmembrane domain.
  • first transmembrane domain of the present invention is provided in SEQ ID NO: 9.
  • the first transmembrane domain main may comprise SEQ ID NO: 9 or a variant thereof.
  • an illustrative second transmembrane domain of the present invention is provided in SEQ ID NO: 10.
  • the first transmembrane domain main may comprise SEQ ID NO: 10 or a variant thereof.
  • the first or second transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the chimeric receptor expressed by the cell of the present invention may comprise a signal peptide so that when it is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • signal peptide described herein is provided in SEQ ID NO: 12 or 13.
  • signal peptide may comprise SEQ ID NO: 12 or 13 or a variant thereof.
  • the endodomain is the intracellular portion of the first and second polypeptides of the chimeric receptor.
  • the endodomain may comprise an intracellular signalling domain and/or a co-stimulatory domain.
  • the endodomain may be "inert" in the sense that it does not comprise either an intracellular signalling domain and/or a co-stimulatory domain.
  • the second polypeptide comprises an intracellular signalling domain.
  • the first polypeptide may comprise an "inert" endodomain or one or more costimulatory domains.
  • Classical CARs are known as first, second or third generation CARs, based on their number of costimulatory domains.
  • a first generation CAR just has an intracellular signalling domain (such as a CD3 ⁇ endodomain) without any costimulation domains.
  • a second generation CAR has an intracellular signalling domain with one costimulation domain, for example CD28-CD3 ⁇ , OX40-CD3 or 41 BB-CD3 ⁇ .
  • a third generation CAR has an intracellular signalling domain with two costimulation domains, for example CD28-41 BB-CD3 ⁇ , CD28-OX40-CD3 ⁇
  • the chimeric receptor of the first embodiment of the invention may be first, second or third generation-like, as illustrated in the following table:
  • the first polypeptide comprises an intracellular signalling domain and the second polypeptide comprises one or more costimulatory domains.
  • the chimeric receptor of the second embodiment of the invention may be second or third generation-like, as illustrated in the following table:
  • the endodomain may comprise one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAM immunoreceptor tyrosine-based activation motifs
  • An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system.
  • the motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/l. Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/IX (6-8) YxxL/I).
  • ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of important cell signalling molecules such as the CD3 and ⁇ -chains of the T cell receptor complex, the CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors.
  • the tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the cell.
  • the intracellular signalling domain component may comprise, consist essentially of, or consist of the CD3 ⁇ endodomain, which contains three ITAMs.
  • the CD3 ⁇ endodomain transmits an activation signal after the cell surface TAA is bound.
  • the endodomain of the first and/or second polypeptide may comprise one or more costimulatory domains as outlined in the tables above.
  • the costimulatory domain may transmit a proliferative signal, such as the endodomain from CD28 or ICOS; it may transmit a survival signal, such as the endodomain from IL2R, or additional TCR signal, such as the endodomain of CD3 ⁇ .
  • Co-stimulatory domains which transmit a survival signal include those generated by TNF receptor family endodomains such as OX-40, 41 BB, CD27, CD2, CD40 or GITR.
  • the endodomain of the chimeric receptor described herein may comprise the signalling portion of any one or more of the endodomains listed in Table 4:
  • the endodomain of the first polypeptide may be "inert" in the sense that it does not contain an intracellular signalling domain or any co-stimulatory domains. It may simply be a stretch of amino acids added to the end of the transmembrane domain stabilise the first polypeptide molecule.
  • An inert endodomain may comprise a rigid linker.
  • An illustrative non signalling linker is provided in SEQ ID NO: 15.
  • the endodomain may comprise SEQ ID No. 15 or variant thereof which has the ability to facilitate protein stability.
  • the present invention relates to cell which expresses a chimeric receptor capable binding to a target antigen via the antigen binding domain of the first polypeptide and a TGF ⁇ via the first and second TRBs of the first and second polypeptides.
  • the cell is "engineered” in that it has been modified to comprise or express a nucleic acid sequence which is not naturally encoded by the cell.
  • Methods for engineering cells are known in the art and include but are not limited to genetic modification of cells e.g. by transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection - DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation. Any suitable method may be used to introduce a nucleic acid sequence into a cell.
  • the chimeric receptor which is capable of binding a TAA and TGF ⁇ is not naturally expressed by a corresponding, unmodified cell.
  • Engineered cells according to the present invention may be generated by introducing DNA or RNA coding a CAR, TCR and/or antibody by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • Cells may be activated and/or expanded prior to the transfection or transduction, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.
  • activated means that a cell has been stimulated, causing the cell to proliferate, differentiate or initiate an effector function.
  • Methods for measuring cell activation include, for example, measuring the expression of activation markers by flow cytometry, such as the expression of CD69, CD25, CD38 or HLA-DR or measuring intracellular cytokines.
  • expanded means that a cell or population of cells has been induced to proliferate.
  • the expansion of a population of cells may be measured for example by counting the number of cells present in a population.
  • the phenotype of the cells may be determined by methods known in the art such as flow cytometry.
  • the cell may be an “immune effector cell” which is a cell of the immune system which responds to a stimulus and effects a change.
  • Immune effector cells include T cells (such as an alpha-beta T cell or a gamma-delta T cell), a B cells (such as a plasma cell), a Natural Killer (NK) cells or a macrophages.
  • T cells such as an alpha-beta T cell or a gamma-delta T cell
  • B cells such as a plasma cell
  • NK cells Natural Killer cells or a macrophages.
  • Cytolytic immune cell as used herein is a cell which directly kills other cells. Cytolytic cells may kill cancerous cells; virally infected cells or other damaged cells. Cytolytic immune cells include T cells and Natural killer (NK) cells. Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a TCR on their cell surface.
  • Cytolytic T cells destroy virally infected cells and tumour cells and are also implicated in transplant rejection.
  • CTLs express the CD8 at their surface.
  • CTLs may be known as CD8+ T cells. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells.
  • MHC class I MHC class I
  • IL-10 adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.
  • the cell of the present invention may be a T-cell.
  • the T cell may be an alpha- beta T cell.
  • the T cell may be a gamma-delta T cell.
  • Natural Killer Cells are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner.
  • NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.
  • LGL large granular lymphocytes
  • the cell of the present invention may be a wild-type killer (NK) cell.
  • the cell of the present invention may be a cytokine induced killer cell.
  • the cell may be derived from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).
  • T or NK cells for example, may be activated and/or expanded prior to being transduced with nucleic acid molecule(s) encoding the polypeptides of the invention, for example by treatment with an anti-CD3 monoclonal antibody.
  • the cell may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells.
  • an immortalized T-cell line which retains its lytic function may be used.
  • the cell may be a haematopoietic stem cell (HSC).
  • HSCs can be obtained for transplant from the bone marrow of a suitably matched donor, by leukapheresis of peripheral blood after mobilization by administration of pharmacological doses of cytokines such as G-CSF [peripheral blood stem cells (PBSCs)], or from the umbilical cord blood (UCB) collected from the placenta after delivery.
  • cytokines such as G-CSF [peripheral blood stem cells (PBSCs)]
  • PBSCs peripheral blood stem cells
  • URB umbilical cord blood
  • the marrow, PBSCs, or UCB may be transplanted without processing, or the HSCs may be enriched by immune selection with a monoclonal antibody to the CD34 surface antigen.
  • the present invention provides a nucleic acid sequence encoding the first polypeptide and/or second polypeptide of the chimeric receptor of the invention.
  • the present invention provides a nucleic acid construct which encodes first and second polypeptides of the chimeric receptor.
  • a nucleic acid construct encoding a chimeric receptor of the invention may have the structure:
  • ABD is a nucleic acid sequence encoding the antigen-binding domain of the first polypeptide
  • linker is a nucleic acid sequence encoding the linker of the first polypeptide
  • (iii) 1TBR is a nucleic acid sequence encoding the transforming growth factor beta receptor (TBR) of the first polypeptide;
  • (iv) 1TM is a nucleic acid sequence encoding the transmembrane domain of the first polypeptide
  • (v) 1endo is a nucleic acid sequence encoding the endodomain of the second polypeptide
  • coexpr is a nucleic acid sequence enabling co-expression of both the first and second polypeptides
  • (vii)2TBR is a nucleic acid sequence encoding the transforming growth factor beta receptor (TBR) of the second polypeptide
  • (viii) 2TM is a nucleic acid sequence encoding the transmembrane domain of the second polypeptide
  • (ix) 2endo is a nucleic acid sequence encoding the endodomain of the second polypeptide.
  • the co-expression sequence encodes a cleavage site or a self-cleaving peptide and the nucleic acid construct is expressed in a cell, it encodes a chimeric receptor which is cleaved at the cleavage site such that the first and second polypeptides are co-expressed at the cell surface.
  • the present invention provides a kit of nucleic acid sequences comprising: a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide of the chimeric receptor.
  • the kit may comprise a first nucleic acid sequence having the structure:
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
  • SEQ ID NO: 16 (SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGF ⁇ RII-RL-2A- dnTGF ⁇ RI-zeta)
  • MGWSCI I LFLVATATGVHSEVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAP GKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQI NSGNYNFKDYEYDYWGQGTQVTVSSRSGGGGSGGGGSGGGGST/PPWQKSVW/VDAW TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLL V7FQVTG/S/-/-PP/-GVA/SV7//FYCYRKKRLEAEAAAKEAAAKEAAAKEAAAKALERAEGRGS LLTCGDVEENPGPMGWSCIILFLVATATGVHSLQCFCHLCTKDNFTCVTDGLCFVSVTETTD
  • Buffer sequence is in bold aEGFR VHH is underlined
  • TRBII derived transmembrane is italicised and in bold
  • TRBI derived transmembrane is in bold and double underlined
  • CD3-zeta endodomain is bold and underlined and italicised
  • SEQ ID NO: 17 (SFGmR.HA8-2A-aROR1_VHH-9G8_16aaLinker-dnTGF ⁇ RII-RL-2A- dnTGF ⁇ RI-zeta)
  • MGWSCI I LFLVATATGVHSQVQLQESGGGSVQAGGSLRLSCAASGRTASISPMGWYRQPP GKQREFVARISSLGRTIYADSVKGRFTISRDNDKNTLYLQMNSLKFEDTGVYYCKAERTDAA DYYMMGQGTQYTYSSSGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFI LEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPP LGVA/SW//FyCYRKKBLEAEAAAKEAAAKEAAAKEAAAKALERAEGRGSLLTCGDVEENPG PMGWSCIILFLVATATGVHSLQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEID LIPRDRPFVCAPSSKTG
  • Buffer sequence is in bold aROR1 VHH is underlined
  • TRBII derived transmembrane is italicised and in bold Second signal peptide is dashed underlined
  • TRBI derived transmembrane is in bold and double underlined
  • CD3-zeta endodomain is bold and underlined and italicised
  • SEQ ID NO: 18 (SFGmR.HA8-2A-aPSMA_VHH-9G8_16aaLinker-dnTGF ⁇ RII-RL-2A- dnTGF ⁇ RI-zeta)
  • TRBII derived transmembrane is italicised and in bold
  • Second signal peptide is dashed underlined
  • TRBI derived transmembrane is in bold and double underlined
  • CD3-zeta endodomain is bold and underlined and italicised
  • the nucleic acid construct may comprise a plurality of nucleic acid sequences which encode a first polypeptide capable of binding a TGF ⁇ and an antigen; and encode a second polypeptide which encodes the endodomain comprising the signal transmission portion of, for example, CD3zeta.
  • the nucleic acid construct may comprise two nucleic acid sequences which encode different components of the invention.
  • the plurality of nucleic acid sequences may be separated by co-expression sites as defined herein.
  • polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
  • the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular an immune effector cell as described herein.
  • Nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variant in relation to a nucleotide sequence or amino acid sequence includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.
  • a co-expression site is used herein to refer to a nucleic acid sequence enabling co-expression of nucleic acid sequences encoding a chimeric receptor comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is capable of binding a transforming growth factor beta (TGFB) and an antigenaccording to the present invention.
  • TGFB transforming growth factor beta
  • the same co-expression site may be used.
  • the co-expression site may be a cleavage site.
  • the cleavage site may be any sequence which enables the two polypeptides to become separated.
  • the cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
  • cleavage is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage.
  • FMDV Foot-and-Mouth disease virus
  • various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041).
  • the exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.
  • the cleavage site may be a furin cleavage site.
  • Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products.
  • Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta- secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor.
  • Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg') and is enriched in the Golgi apparatus.
  • the cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.
  • TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of chimeric receptors both in vitro and in vivo.
  • the consensus TEV cleavage site is ENLYFQ ⁇ S (where ' ⁇ ' denotes the cleaved peptide bond).
  • Mammalian cells such as human cells, do not express TEV protease.
  • the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.
  • the cleavage site may encode a self-cleaving peptide.
  • a ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self- cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.
  • the self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
  • the primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus.
  • apthoviruses such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus
  • the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C-terminus (Donelly et al (2001) as above). This sequence is shown as SEQ ID NO: 18.
  • 2A-like sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al., 2001) as above.
  • the co-expression sequence may be an internal ribosome entry sequence (IRES).
  • the co-expressing sequence may be an internal promoter.
  • the present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) or nucleic acid construct(s) of the invention.
  • a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it expresses a chimeric receptor as defined herein.
  • the vector may comprise a plurality of nucleic acid sequences which encode different components as provided by the present invention.
  • the vector may comprise two, three, four or more nucleic acid sequences which encode different components, such as the first polypeptide and the second polypeptide of the present invention.
  • the plurality of nucleic acid sequences may be separated by co-expression sites as defined herein.
  • the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.
  • the vector may be capable of transfecting or transducing a cell.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a cell according to the present invention or a cell obtainable (e.g. obtained) by a method according to the present invention.
  • the present invention also provides a pharmaceutical composition comprising a plurality of cells as defined herein.
  • the invention relates to a pharmaceutical composition containing a cell according to the present invention.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a method for treating and/or preventing a disease which comprises the step of administering a cell or plurality of cells or pharmaceutical composition according to the invention to a subject.
  • the present methods for treating and/or preventing a disease may comprise administering a cell according to the present invention (for example in a pharmaceutical composition as described above) to a subject.
  • the present invention also provides a method for treating and/or preventing a disease in a subject which subject comprises cells of the invention, which method comprises the step of administering an agent to the subject wherein the agent is capable of controlling the secretion or activity of secreted factor or antibody.
  • this method involves administering an agent to a subject which already comprises cells of the present invention.
  • the method for treating and/or preventing a disease may comprise the step of administering a plurality of cells expressing the chimeric receptor of the present invention.
  • a method for treating a disease relates to the therapeutic use of the cells of the present invention.
  • the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • the method for preventing a disease relates to the prophylactic use of the cells of the present invention.
  • the cells may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease.
  • the subject may have a predisposition for, or be thought to be at risk of developing, the disease.
  • the method may involve the steps of:
  • nucleic acid construct according to the present invention introducing the nucleic acid construct according to the present invention, a first and second nucleic acid sequence as defined herein, a vector according to the present invention or a first and second vector as defined herein to the cell;
  • the nucleic acid construct, vector(s) or nucleic acids may be introduced by transduction or transfection.
  • the cell may be autologous or allogenic.
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and/or any toxic activity.
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and monitoring any toxic activity and adjusting the dose of the agent administered to the subject to provide acceptable levels of disease progression and toxic activity.
  • “Monitoring the progression of the disease” means to assess the symptoms associated with the disease over time to determine if they are reducing/improving or increasing/worsening.
  • “Toxic activity” relates to adverse effects caused by the cells of the invention following their administration to a subject. Toxic activities may include, for example, immunological toxicity, biliary toxicity and respiratory distress syndrome.
  • the dose of the agent administered to a subject, or the frequency of administration may be altered in order to provide an acceptable level of both disease progression and toxic activity.
  • the specific level of disease progression and toxic activities determined to be ‘acceptable’ will vary according to the specific circumstances and should be assessed on such a basis.
  • the agent may be administered in the form of a pharmaceutical composition.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a cell or pharmaceutical composition according to the present invention, a nucleic acid construct according to the present invention for use in treating and/or preventing a disease.
  • the present invention also relates to the use of a cell according to the present invention for the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the disease to be treated and/or prevented by the method of the present invention may be cancer.
  • the cancer may be a cancer such as neuroblastoma, multiple myeloma, prostate cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, and thyroid cancer.
  • the cancer may be neuroblastoma.
  • the cancer may be multiple myeloma.
  • the cancer may be prostate cancer.
  • the cell of the present invention may be capable of killing target cells, such as cancer cells.
  • the target cell may be recognisable by expression of a TAA, for example the expression of a TAA provided above in Table 3.
  • the cancer may be a cancer listed in Table 3.
  • the administration of a cell or pharmaceutical composition according to the present invention can be accomplished using any of a variety of routes that make the active ingredient bioavailable.
  • the active ingredient can be administered by oral and parenteral routes, intranasally, intraperitoneally, intravenously, subcutaneously, transcutaneously or intramuscularly.
  • the cells of the present invention may be generated by introducing DNA or RNA coding for the secreted factor or antibody as defined herein, by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • the cell of the invention may be made by: introducing to a cell (e.g. by transduction or transfection) the polynucleotide according to the present invention, the nucleic acid construct or vector according to the present invention, a first and second nucleic acid sequence as defined herein, a vector or a first and second vector as defined herein.
  • the cell may be transduced or transfected in vitro or ex vivo.
  • the cell may be from a sample isolated from a subject.
  • Example 1 Investigating T cell activation in cells expressing an EGFR-specific chimeric receptor of the invention in the presence of rEGFR alone, rEGFR and TGF8, or TGF8 alone.
  • T cells were transduced with retroviral vectors expressing the constructs below:
  • SFGmR is a signal peptide
  • HA8 is a transduction marker aEGFR_VHH is an EGFR-binding VHH domain having the seguence shown as SEQ ID No. 7
  • 9G8_16aaLinker is a linker having the seguence shown as SEQ ID No. 8 dnTGFbRII is the extracellular domain of TGF ⁇ RII having the sequence shown as SEQ ID No.
  • 2A is an FMDV-like self-cleaving peptide sequence having the sequence shown as SEQ ID No. 19 dnTGFbRI is the extracellular domain of TGF ⁇ RI having the sequence shown as SEQ ID No. 4 zeta is the CD3 ⁇ endodomain having the sequence shown as SEQ ID No. 14
  • RL is a rigid linker having the sequence shown as SEQ ID No. 15.
  • SFGmR.HA8-2A-aEGFR_VHH- 9G8_16aaLinker-dnTGFbRII-RL-2A-dnTGFbRI-zeta is shown above as SEQ ID No. 16.
  • the first construct SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGFbRII-RL-2A- dnTGFbRI-zeta, produces a soluble AND gate as illustrated schematically in Figure 1.
  • the AND gate consists of two polypeptides: a first polypeptide which comprises an EGFR-specific VHH-type antigen binding domain, the extracellular region of TBRII and an inert rigid linker endodomain; and a second polypeptide which comprises the extracellular region of TBRI and a CD3z endodomain.
  • the second construct SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGFbRII-RL-2A- dnTGFbRI-RLw, is the same as the first except the second polypeptide has an inert rigid linker endodomain, rather than a CD3z endodomain.
  • This construct acts as a negative control as it is incapable of triggering T-cell activation.
  • the first polypeptide comprises both the anti-EGFR antigen-binding domain and a CD3 zeta endodomain, so this acts as a positive control as it will give a T-cell activation signal in the presence of EGFR regardless of the presence or absence of TGF ⁇ .
  • Transduced T cells were added to EGFR-coated plates in the presence of TGF ⁇ 1 at 20ng/ml or 5ng/mL concentration.
  • CD25 MFI mean fluorescence intensity
  • test construct (SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGF ⁇ RII-RL-2A- dnTGF ⁇ RI-zeta) showed a higher CD25 MFI when plated with both rEGFR and 5ng/ml TGF ⁇ 1 than when plated with rEGFR alone or 20ng/ml TGF ⁇ 1 alone.
  • the positive control construct (SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGF ⁇ RII- zeta-2A-dnTGF ⁇ RI-RLw), demonstrated a low level of CD25 MFI when incubated with TGF ⁇ alone.
  • this construct eliminates the need for TGF ⁇ 1 to bind to TBR, and therefore eliminates the need for the second polypeptide.
  • This construct allows the chimeric receptor- expressing cell to be activated in the presence of EGFR only, hence activation under these conditions is the highest.
  • the negative control construct (SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGF ⁇ RII- RL-2A-dnTGF ⁇ RI-RLw), which lacks a CD3zeta endodomain entirely, gave a lower level of CD25 MFI under each of the conditions tested.
  • Example 2 Investigating killing of EGFR-expressing target cells after 24-hour incubation with cells expressing an EGFR-specific first-generation chimeric receptor of the invention.
  • T cells transduced with the constructs described in Example 1 were co-cultured with EGFR- expressing or non-transduced SupT1 target cells at a ratio of 1 :4 EffectorTarget (E:T) for 24 hours.
  • TGF ⁇ 1 was added to some wells at a concentration of 5ug/ml.
  • test construct (SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGF ⁇ RII-RL-2A- dnTGF ⁇ RI-zeta) gave no killing against EGFR-expressing target cells in the absence of TGF ⁇ . However, in the presence of added TGF ⁇ , killing of EGFR-expressing target cells was seen, with an average 50% reduction in percentage of live target cells
  • the negative control construct (SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGF ⁇ RII- RL-2A-dnTGF ⁇ RI-RLw), displayed no killing of EGFR-expressing target cells in either the absence or presence of TGF ⁇ .
  • the positive control construct (SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGF ⁇ RII- zeta-2A-dnTGF ⁇ RI-RLw), displayed killing of EGFR-expressing target cells in both the absence and presence of TGF ⁇ .
  • Example 3 Investigating killing of EGFR-expressing target cells after 24-hour incubation with cells expressing an EGFR-specific second-generation chimeric receptor of the invention
  • T cells were transduced with retroviral vectors expressing the constructs below:
  • SFGmR.HA8-2A-aEGFR_VHH-9G8_16aaLinker-dnTGFbRII- RL-2A-dnTGFbRI-RLw was the same as the one described in Examples 1 and 2.
  • T cells transduced with these constructs were co-cultured with EGFR-expressing SupT 1 target cells at a ratio of 1 :4 EffectorTarget (E:T) for 24 hours as described in Example 2.
  • E:T EffectorTarget
  • TGF ⁇ 1 was added to a various concentrations from 0 to 5ng/ml.
  • the results for the CD28-CD3z and 41 BB-CD3z second generation construct are shown in Figures 6 and 7 respectively.
  • test construct i.e. the TGF ⁇ -responsive AND gate
  • TGF ⁇ receptor complex it is therefore possible to exploit the natural heterodimerization of the TGF ⁇ receptor complex to produce a chimeric receptor which, when expressed on a cytolytic immune cell such as a T-cell, causes specific killing of cells expressing a target antigen and is only fully activated in the presence of both TGF ⁇ and the target antigen, thereby reducing on-target off-tumour toxicity.
  • T cells and SupT1 cells were cultured in complete RPMI 1640 media (Biowest), 10% fetal bovine serum (FBS, Biosera) and 1% L-Glutamine (GlutaMAX, Gibco) supplemented.
  • SupTls were purchased from ATCC and SupTls expressing EGFR were generated by transduction with retroviral vector.
  • HEK293T cells used for viral production were cultured in complete Iscove's Modified Dulbecco's Media (IMDM, HyClone), 10% FBS and 1% L-Glutamine supplemented.
  • Whole blood was obtained from the National Health Service Blood and Transplant (NHSBT, Colindale, UK) and PBMCs were isolated in house using Ficoll-Paque PLUS (GE Healthcare).
  • Transduced T cells were cultured in complete RPMI 1640, 10% FBS and 1% L-Glutamine supplemented with 100IU/ml interleukin-2 (IL-2).
  • IL-2 interleukin
  • TGF ⁇ induced soluble AND gate with VHH directed against EGFRvlll TGF ⁇ induced soluble AND gate with VHH directed against EGFRvlll.
  • the test construct is a first generation like CAR with CD3 ⁇ chain linked to TGF ⁇ RI, negative control has a double rigid linker and positive control is a first generation like CAR with CD3 ⁇ chain linked directly to the EGFRvlll- TGF ⁇ RII chain.
  • the HA marker gene added, recognised by anti-HA.11 Epitope Tag Antibody (Biolegend).
  • the retroviruses were produced by transfection of 70% confluent HEK 293T cells using GeneJuice (Millipore), RD114 envelope expression plasmid (RDF), gag-pol expression plasmid (pEQ-Pam3-E) and the retroviral transfer vectors described before.
  • the media was replaced 24 hours post transfection with fresh complete IMDM medium and the virus was harvested 48 hours post transfection.
  • Non TC treated 6-well plates were coated with Retronectin (Takara) and incubated overnight at 4°C. 3ml of retroviral supernatant and 1x10e6 T cells were added to the coated plates, spun at 1000g for 40 minutes, and incubated at 37°C, 5%CO2 for 72 hours. The transduction efficiency was assessed by Flow Cytometry (MACSQuant Analyser 10, Miltenyi) after harvesting the T cells and staining a small amount using anti-HA.11 Epitope Tag Antibody.
  • High binding 96-well plates (Corning) were coated overnight with EGFR at a concentration of 1ug/ml. The plate was washed with PBS and 100 ul of 50.000 transduced T cells were added in presence of 100 ul of recombinant human TGF ⁇ 1 (R&D Systems). 5 different concentrations of TGF ⁇ 1 starting from 20ng/ml diluted down to 1.25ng/ml in a ratio of 1 :2. Control conditions were in absence and presence of TGF ⁇ 1 in PBS coated wells with the same amount of T cells, as well as no addition of TGF ⁇ 1 in EGFR wells.
  • the plates were incubated for 24 and 48 hours and were then stained with CD25 and CD69 (Biolegend) to assess activation of the cells by Flow Cytometry.
  • the cells were also stained with HA to identify the CAR and fixable viability dye e fluor 780 (ThermoFisher) for the exclusion of dead cells.
  • the transduced T cells were co-cultured with effector cells at a ratio of 1 :4 EffectorTarget (E:T) for 24 hours in 96-well plates.
  • SupT1 cells expressing EGFR were used as target cells at 50.000 per well.
  • Non transduced (NT) targets and T cells were also added in all conditions as controls.
  • TGF ⁇ 1 at a concentration of 5ug/ml was also added so all conditions were co- cultures in the absence and presence of TGF ⁇ 1. Cytotoxicity was assessed using Flow Cytometry by staining the cells with CD3 to identify the T cells and CD2 to identify the targets, as well as e fluor 780 to exclude dead cells.

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Abstract

L'invention concerne une cellule qui exprime un récepteur chimérique comprenant (a) un premier polypeptide et (b) un second polypeptide, le premier polypeptide (a) comprenant : (i) un domaine de liaison à l'antigène, (ii) un lieur, (iii) une région extracellulaire d'un premier récepteur bêta du facteur de croissance transformant (TBR), (iv) un premier domaine transmembranaire et (v) un premier endodomaine ; et le second polypeptide (b) comprenant : (i) une région extracellulaire d'un second TBR, (ii) un second domaine transmembranaire et (iii) un second endodomaine.
PCT/GB2021/052017 2020-08-05 2021-08-04 Récepteur chimérique liant le tgf-bêta Ceased WO2022029431A1 (fr)

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WO2023077026A1 (fr) 2021-10-28 2023-05-04 Lyell Immunopharma, Inc. Procédés de culture de cellules exprimant une protéine de liaison à ror1
WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2024077174A1 (fr) 2022-10-05 2024-04-11 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
WO2025217398A1 (fr) 2024-04-10 2025-10-16 Lyell Immunopharma, Inc. Procédés de culture de cellules avec milieu de culture amélioré

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WO2024064952A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a surexprimant c-jun
WO2024064958A1 (fr) 2022-09-23 2024-03-28 Lyell Immunopharma, Inc. Procédés de culture de cellules déficientes en nr4a
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