WO2024194355A1 - Récepteur antigénique chimérique - Google Patents

Récepteur antigénique chimérique Download PDF

Info

Publication number
WO2024194355A1
WO2024194355A1 PCT/EP2024/057453 EP2024057453W WO2024194355A1 WO 2024194355 A1 WO2024194355 A1 WO 2024194355A1 EP 2024057453 W EP2024057453 W EP 2024057453W WO 2024194355 A1 WO2024194355 A1 WO 2024194355A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
domain
cell
set forth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2024/057453
Other languages
English (en)
Inventor
Elmar JAECKEL
Matthias HARDTKE-WOLENSKI
Michael Hust
Tom PIEPER
Tobias RIET
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universitaet Braunschweig
Medizinische Hochschule Hannover
Original Assignee
Technische Universitaet Braunschweig
Medizinische Hochschule Hannover
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP23162911.4A external-priority patent/EP4434539A1/fr
Application filed by Technische Universitaet Braunschweig, Medizinische Hochschule Hannover filed Critical Technische Universitaet Braunschweig
Priority to CN202480033542.0A priority Critical patent/CN121586582A/zh
Priority to EP24711920.9A priority patent/EP4683660A1/fr
Publication of WO2024194355A1 publication Critical patent/WO2024194355A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • 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
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain

Definitions

  • the present disclosure relates generally to the field of chimeric antigen receptors (CARs) and related therapies, such as the treatment of autoimmune or inflammatory diseases, particularly type 1 diabetes. More particularly, the disclosure provides CARs comprising an antigen recognition domain that binds to GLP1 R and that are expressed in immune cells, particularly Tregs. Such immune cells have therapeutic uses in diseases and conditions associated with cells that express GLP1 R on their surface. The disclosure further provides nucleic acid molecules encoding such CARs and vectors containing them that may be used to modify host cells, e.g., immune cells, to express the CARs.
  • Immunotherapy is emerging as a beneficial tool for the treatment of many conditions, ranging from cancer, autoimmune and inflammatory diseases, to the prevention of solid organ transplant rejection.
  • ACT adoptive cellular immunotherapy
  • Treg regulatory T cell
  • CD4+FOXP3+ regulatory T cells are a lymphocyte subset that is essential for the maintenance of dominant immunological tolerance by inhibiting the function of various effector immune cell subsets such as T effector cells.
  • Tregs are also known to promote tissue repair and regeneration. Tregs are able to confer immune tolerance through multiple contact-dependent and independent mechanisms. These include production of anti-inflammatory soluble mediators such as IL-10, TGF-p and IL-35, consumption of IL-2, expression of negative regulatory cell surface receptors, such as CTLA- 4, and targeting T cells directly or indirectly through APCs.
  • Tregs can suppress immune responses in a non-antigen specific fashion (bystander suppression), i.e., once activated, Tregs have the ability to modulate the local immune micro-environment and suppress inflammation. Furthermore, they can confer a suppressive phenotype on other cells of the immune system, a process called “infectious tolerance”.
  • Type 1 diabetes is a chronic autoimmune disease in which pancreatic beta cells, which are responsible for the production of insulin, are destroyed by the immune system. This is triggered by genetic and environmental factors. Destruction of beta cells reduces or eliminates production of insulin by the body and results in inflammation in the pancreatic islets. Insulin is a hormone required to regulate glucose levels in the bloodstream and, before treatment, subjects with type 1 diabetes will have excessively high blood sugar levels (hyperglycaemia). Type 1 diabetes is a serious and lifelong condition. Patients with type 1 diabetes currently need to closely monitor their blood glucose levels and take an appropriate dose of insulin (e.g. by injection or pump). This therapy is not a cure and must be continuously administered.
  • Type 1 diabetes thus imposes a substantial burden on health systems worldwide. Further, epidemiological data show a steady increase in the prevalence of autoimmune diseases such as type 1 diabetes in Western society over the last decades. The prevalence of type 1 diabetes in Western Europe and North America is approximately 0.5% or approximately 2 million people with a steady upward trend.
  • Treg-mediated suppression of effector T cells contributes to the development of disease (see Visperas and Vignali, J Immunol. 2016; 197(10): 3762-3770).
  • Treg phenotype and suppressive capacity have been observed in samples obtained from type 1 diabetes patients.
  • depletion of Tregs has been shown to accelerate autoimmune diabetes development.
  • Treg-based immunotherapies The prospect of ameliorating immunopathology and re-establishing tolerance in inflammatory diseases has prompted a growing interest in the clinical development of Treg-based immunotherapies.
  • Treg immunotherapy it is beneficial to develop strategies that promote the trafficking of Tregs to the site of tissue damage and induce their activation in situ.
  • T regs from a transgenic mouse expressing a TCR specific for an islet antigen have been used to prevent or reverse diabetes in NOD mice (see Tang et al, J Exp Med. 2004; 199: 1455-65).
  • this required activation of the Tregs through a TCR and, due to MHC restriction, this transgenic model is not transferable to patients since different patients would require different TCRs.
  • CARs Artificial chimeric antigen receptors
  • CARs have been used to confer antigen specificity on a cell.
  • CARs are generally composed of an extracellular antigen-binding domain (e.g. a scFv specific for the target antigen), a transmembrane domain, and an intracellular signalling domain which sends signals into the cell and activates it upon binding of the antigen to the extracellular antigen-binding domain.
  • CAR-Teff cell therapy has been approved for the treatment of certain blood cancers.
  • CARs are artificial molecules engineered into cells and are less sensitive than TCRs, partly due to the number of molecules involved in the TCR machinery, i.e., CD4/CD8 co-receptors, immunoreceptor tyrosinase-rich activation motifs (ITAMs), and subunits within the receptor complex. It is therefore difficult to select an appropriate target for CAR-T cell therapy which will allow trafficking and subsequent activation of the CAR-T cells to have a therapeutic effect. For example, although insulin-specific CAR-T regs proliferated in response to insulin and were suppressive in vitro, they did not prevent spontaneous diabetes in NOD mice (Tenspolde et al, J Autoimmunity. 2019; 103: 102289).
  • a therapy for the treatment of an autoimmune or inflammatory disease may be developed by providing immune cell subsets with a chimeric antigen receptor (CAR) comprising an antigen recognition domain that is specific for GLP1 R.
  • CAR chimeric antigen receptor
  • the expression of such a CAR on the surface of Tregs provides a generic therapy that can be used for the treatment of autoimmune disease or inflammatory disease, where GLP1 R is expressed locally at the site of disease, in view of the well-known bystander effect of Tregs and their ability, once activated, to reduce the immune response and modulate the activation status of other immune cell subsets.
  • the inventors have developed a Treg expressing an anti-GLP1 R CAR for the treatment of type 1 diabetes.
  • GLP1 R as a surprisingly effective target for CAR-Treg therapy, particularly for type 1 diabetes and other pancreatic autoimmune or inflammatory disorders.
  • the inventors have found that cells expressing CARs specific for GLP1 R are activated in the presence of antigen.
  • the inventors have identified both ligand-based and scFv-based CARs that are activated in the presence of antigen.
  • T-effector cells expressing CARs specific for GLP1 R are able to induce the onset of type 1 diabetes, indicating that they traffic to the pancreas.
  • GLP1 R Whilst GLP1 R has been reported to be expressed on pancreatic tissue, expression of GLP1 R in other tissues (brain, heart, abdominal organs) has been reported in the art, making selection of GLP1 R as a target for specific trafficking (thus allowing activation of T regs to sufficient levels to treat type I diabetes) unintuitive. Particularly, RNA expression of GLP1 R in many other cell types and tissues has been reported in the art. The inventors’ finding that GLP1 R protein is an effective pancreatic target is therefore particularly surprising.
  • the present invention provides a regulatory T cell (Treg) comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition domain that specifically binds to GLP1 R (e.g. specifically binds to human GLP1 R and/or murine GLP1 R).
  • Treg regulatory T cell
  • CAR chimeric antigen receptor
  • the CAR may comprise: a. an exodomain comprising the antigen recognition domain; b. a transmembrane domain; and c. an endodomain comprising an intracellular signalling domain.
  • the CAR may further comprise a hinge domain and/or one or more co-stimulatory domains.
  • hinge domain typically refers to the portion of the exodomain that connects the antigen recognition domain with the transmembrane domain.
  • the hinge domain may be selected from the hinge regions of CD28, CD8a, CD4, CD7, CH2CH3, an immunoglobulin, or a part or variant thereof.
  • the CAR may comprise a CD8a or CH2CH3 hinge domain.
  • the co-stimulatory domain may be selected from the intracellular domains of CD28, ICOS, CD134 (0X40), CD137 (4-1 BB), CD27, or TNFRSF25, or a part or variant thereof.
  • the CAR may comprise a CD28 co-stimulatory domain.
  • the CAR may comprise one or more transmembrane domains, which may be selected from the transmembrane domains of CD28, ICOS, CD8a, CD4, CD134 (0X40), CD137 (4-1 BB), CD3 zeta, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or a part or variant thereof.
  • the CAR comprises a CD4, CD28 or CD8a transmembrane domain.
  • the CAR (specifically the endodomain of the CAR) may comprise one or more intracellular signalling domains selected from the group consisting of the CD3 zeta signalling domain or any of its homologs, a CD3 polypeptide, a syk family tyrosine kinase, a src family tyrosine kinase, CD2, CD5, and CD28, or a part or variant thereof.
  • the CAR may comprise the CD3 zeta signalling domain.
  • the CAR may comprise a CD8a or CH2CH3 hinge domain (i.e. a hinge domain derived from CD8a or CH2CH3); a CD8a or CD28 transmembrane domain (i.e. a transmembrane domain derived from CD8a or CD28); a CD28 co-stimulatory domain (i.e. a co-stimulatory domain derived from CD28); and the CD3 zeta signalling domain (i.e.
  • the CAR may comprise a CD8a hinge domain, a CD8a transmembrane domain, a CD28 co-stimulatory domain, and the CD3 zeta signalling domain.
  • the CAR may comprise a CH2CH3 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and the CD3 zeta signalling domain. Further, the CAR may comprise a CD28 transmembrane domain (i.e. a transmembrane domain derived from CD28), particularly in combination with a CD28 co-stimulatory domain.
  • a CD28 transmembrane domain i.e. a transmembrane domain derived from CD28
  • the CAR may comprise a signal peptide and/or a reporter peptide.
  • the polynucleotide sequence encoding a CAR may comprise a further polynucleotide sequence encoding a reporter peptide linked by a self-cleaving or cleavage domain.
  • the antigen recognition domain of the CAR may be an antibody, an antibody fragment, or derived from an antibody.
  • the antigen recognition domain is a single chain antibody (scFv).
  • the antigen recognition domain of the CAR may be a ligand for GLP1 R.
  • the ligand for GLP1 R may be a GLP1 R agonist.
  • the CAR may, for example, comprise an antigen recognition domain which comprises:
  • VH CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 151 , 20 and 21 respectively or (b) SEQ ID NOs: 152, 20 and 21 respectively or (c) SEQ ID NOs: 153, 20 and 21 respectively, and VL CDR1 , 2 and 3 sequences set forth in SEQ ID NOs: 22, 23 and 24 respectively; or
  • the CAR may, for example, comprise an antigen recognition domain which comprises:
  • the antigen recognition domain of the CAR may comprise:
  • VH domain comprising the sequence set forth in SEQ ID NO: 31 , or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 32, or a sequence having at least 70% identity thereto;
  • VH domain comprising the sequence set forth in SEQ ID NO: 33, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 34, or a sequence having at least 70% identity thereto
  • VH domain comprising the sequence set forth in SEQ ID NO: 35, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 36, or a sequence having at least 70% identity thereto
  • VH domain comprising the sequence set forth in SEQ ID NO: 37, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 38, or a sequence having at least 70% identity thereto;
  • VH domain comprising the sequence set forth in SEQ ID NO: 39, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 40, or a sequence having at least 70% identity thereto.
  • the antigen recognition domain may, for example, comprise:
  • VH domain comprising the sequence encoded by SEQ ID NO: 41 , or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 41
  • VL domain comprising the sequence encoded by SEQ ID NO: 42, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 42;
  • VH domain comprising the sequence encoded by SEQ ID NO: 43, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 43, and a VL domain comprising the sequence encoded by SEQ ID NO: 44, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 44;
  • VH domain comprising the sequence encoded by SEQ ID NO: 45, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 45, and a VL domain comprising the sequence encoded by SEQ ID NO: 46, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 46;
  • VH domain comprising the sequence encoded by SEQ ID NO: 47, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 47
  • VL domain comprising the sequence encoded by SEQ ID NO: 48, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 48;
  • VH domain comprising the sequence encoded by SEQ ID NO: 49, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:
  • VL domain comprising the sequence encoded by SEQ ID NO: 50, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:
  • the antigen recognition domain of the CAR may comprise or consist of:
  • the antigen recognition domain may, for example, comprise or consist of:
  • the antigen recognition domain may, for example, comprise or consist of the sequence set forth in SEQ ID NO: 61 or a sequence having at least 70% sequence identity thereto.
  • the antigen recognition domain may, for example, comprise or consist of the sequence encoded by the sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63 or a sequence having at least about 70% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63 respectively.
  • the invention provides a CAR comprising an antigen recognition domain that specifically binds to GLP1 R.
  • the CAR may be in accordance with any of the embodiments described herein in relation to the first aspect of the invention.
  • the invention provides a CAR comprising an antigen recognition domain that specifically binds to GLP1 R, wherein the antigen recognition domain is a ligand for GLP1 R.
  • the ligand is a GLP1 R agonist.
  • the antigen recognition domain of the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 61 or a sequence differing by up to 10 amino acids, for example by 1 to 5 amino acids.
  • the invention provides a CAR comprising an antigen recognition domain that specifically binds to GLP1 R, wherein the antigen recognition domain comprises:
  • the antigen recognition domain may comprise one of the other VH and/or VL sequences or be encoded by one of the nucleotide sequences defined herein
  • the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a CAR according to the invention.
  • the invention provides a vector comprising the nucleic acid molecule according to the invention.
  • the vector may further comprise a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 polypeptide, or a derivative or variant thereof.
  • the invention provides a cell comprising the CAR, nucleic acid molecule or vector according to the invention.
  • the cell may further comprise an exogenous FOXP3 polypeptide or an exogenous nucleic acid encoding FOXP3.
  • the cell may be an immune cell or a progenitor or precursor thereof.
  • the cell may be a T cell, or a precursor thereof, or a stem cell.
  • the cell may be a Treg, or a precursor thereof, or an iPSC cell.
  • the cell may be a production host cell.
  • the invention provides a T-effector cell (Teff) comprising a CAR, wherein the antigen recognition domain of the CAR specifically binds to GLP1 R.
  • Teff T-effector cell
  • the cell may be provided in a cell population, which forms a further aspect of the invention.
  • the cell population may comprise a plurality of cells according to the invention, particularly a plurality of T cells (e.g. a plurality of Tregs or a plurality of Teffs) according to the invention.
  • the plurality of T cells (particularly Tregs) according to the invention may have TCRs that are polyclonal.
  • the plurality of T cells (particularly Tregs) according to the invention may have polyclonal endogenous TCRs.
  • the clonality of the TCRs of the plurality of T cells (particularly Tregs) according to the invention may not have been modified ex vivo.
  • the invention also provides a pharmaceutical composition comprising the cell, cell population or vector according to the invention.
  • the invention provides a cell, cell population or pharmaceutical composition according to the invention for use in therapy (e.g., for use in treating and/or preventing an autoimmune or inflammatory disease, or for use in inducing immunosuppression, or for use in promoting tissue repair and/or tissue regeneration).
  • the therapy may be adoptive cell transfer therapy.
  • the invention provides a method for treating and/or preventing an autoimmune or inflammatory disease, or for inducing immunosuppression, or for promoting tissue repair and/or tissue regeneration, wherein the method comprises administering a cell, particularly a Treg cell, a cell population, or a pharmaceutical composition, particularly comprising a Treg, according to the invention.
  • the method may comprise the following steps:
  • the invention also provides use of a cell, cell population or pharmaceutical composition according to the invention in the manufacture of a medicament for treating and/or preventing an autoimmune or inflammatory disease, or for inducing immunosuppression, or for promoting tissue repair and/or tissue regeneration in a subject, particularly wherein the cell is a Treg cell.
  • the autoimmune or inflammatory disease may particularly be type 1 diabetes (T1 D), for example recent-onset type 1 diabetes.
  • T1 D type 1 diabetes
  • the invention provides a method of making a cell according to the invention, which comprises the step of introducing into the cell (e.g., transducing or transfecting a cell with) the nucleic acid molecule or vector according to the invention.
  • the cell may be a Treg cell, and the method may comprise isolating or providing a cell-containing sample comprising Tregs, and/or enriching Tregs or generating Tregs from the cell-containing sample prior to or after the step of introducing the nucleic acid molecule or vector into the cell.
  • the invention also provides a cell obtainable by this method, which forms a further aspect of the invention.
  • the invention provides the use of a CAR-Treg to reduce death (e.g., reduce the rate of death) or prevent death of pancreatic beta cells in a subject.
  • the invention provides the use of a CAR-Treg to maintain or increase fasting blood insulin levels in a subject.
  • the invention provides the use of a CAR-Treg to maintain or increase fasting C-peptide levels in a subject.
  • the invention provides the use of a CAR-Treg to reduce or prevent hyperglycaemia in a subject.
  • the invention provides the use of a CAR-Treg to maintain or decrease fasting blood glucose levels in a subject.
  • the invention provides the use of a CAR-Treg to maintain or reduce HbA1 c levels in a subject.
  • the CAR is the CAR of the present invention, i.e., it comprises an antigen recognition domain that specifically binds to GLP1 R (e.g. to human GLP1 R and/or murine GLP1 R), and it may have any of the features of the CAR as disclosed herein.
  • the subject may, for example, have or be at risk of developing type 1 diabetes, particularly the subject may have recent-onset type 1 diabetes.
  • the subject may, for example, not be being administered exogenous insulin.
  • the subject may be being administered a reduced dose of insulin compared to the required dose of insulin prior to administration of the CAR-Tregs.
  • Figure 1 shows binding of various scFvs to HEK293T cells expressing either human GLP1 R (first row) or murine GLP1 R (second row).
  • Figure 2 shows immunohistochemistry staining of various GLP1 R-specific scFvs to murine pancreas sections.
  • Figure 3 shows activation of murine hybridoma cells expressing a GLP1 R-specific CAR by human GLP1 R protein, HEK cells expressing human GLP1 R antigen and HEK cells expressing murine GLP1 R antigen.
  • Figure 4 shows activation of murine hybridoma cells expressing a GLP1 R-specific CAR by human GLP1 R protein, HEK cells expressing human GLP1 R antigen and HEK cells expressing murine GLP1 R antigen.
  • Figure 5 shows an overview of the experimental protocol used to observe induction of diabetes using a GLP1 R-specific CAR.
  • Figure 6 shows diabetes induction in C57BI/6.RAG mice using T-effector cells expressing a GLP1 R- specific CAR.
  • Figure 7 shows the configuration of the scFv CARs used in the Examples -
  • A shows the schematic structure of CARs with a mutated Fc-IgG hinge and a CD8 hinge
  • B shows the schematic structure of the constructs used in gamma retroviral vectors for transduction of cells.
  • Figure 8 shows the configuration of the ligand CAR used in the Examples -
  • A shows the schematic structure of CARs with a CD8 hinge
  • B shows the sequence of the GLP1 R ligand used in the antigenrecognition domain
  • C shows the schematic structure of the constructs used in gamma retroviral vectors for transduction of cells.
  • Figure 9 shows an overview of the experimental protocol used to observe prevention of diabetes onset in NOD.SCID mice administered Teffs from BDC2.5 NOD mice and NOD Tregs expressing a ligandbased GLP1 R-specific CAR.
  • Figure 10 shows the fraction of NOD.SCID mice that are diabetes free ( Figure 10A) and survive ( Figure 10B) following administration of Teffs from BDC2.5.NOD mice and NOD Tregs expressing a ligandbased GLP1 R-specific CAR.
  • Figure 11 shows activation of CAR-Tregs exposed to target (GLP1 R) compared to control antigen and anti-CD3/CD28 beads.
  • Figure 12 shows proliferation of CAR-Tregs exposed to target (GLP1 R) compared to control antigen and anti-CD3/CD28 beads.
  • the present invention provides CAR-Tregs specific for GLP1 R that are activated in the presence of GLP1 R antigen which is particularly expressed on pancreatic beta cells.
  • these CAR-Tregs have therapeutic potential in treating autoimmune and inflammatory disorders where GLP1 R is expressed locally at the site of disease.
  • these CAR-Tregs have therapeutic potential for type 1 diabetes.
  • the antigen may be simply present and/or expressed at the site of inflammation or disease.
  • a “chimeric antigen receptor”, "CAR” or “CAR construct” refers to engineered receptors which can confer an antigen specificity onto cells (e.g., immune cells, such as Tregs).
  • a CAR enables a cell to bind specifically to a particular antigen, e.g., a target molecule such as a target protein, whereupon a signal is generated by the endodomain (comprising an intracellular signalling domain) of the CAR, e.g., a signal resulting in activation of the cell.
  • CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.
  • CARs The structure of CARs is well-known in the art and several generations of CARs have been produced.
  • a CAR may contain an extracellular antigen-specific targeting region, antigen binding domain, target binding domain or ligand binding domain, which is or forms part of the exodomain (also known as the extracellular domain or ectodomain) of the CAR, a transmembrane domain, and an intracellular signalling domain (which is, or is comprised within, an endodomain).
  • the CAR may contain further domains to improve its functionality, e.g., one or more costimulatory domains to improve T cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence.
  • a chimeric receptor or CAR construct generally comprises a binding domain (which may be viewed as an antigen (i.e., target) or ligand binding domain and the terms binding domain, antigen recognition domain, antigen binding domain and ligand binding domain are used interchangeably herein), optionally a hinge domain, which functions as a spacer to extend the binding domain away from the plasma membrane of the cell (e.g., immune cell) on which it is expressed, a transmembrane domain, an intracellular signalling domain (e.g., the signalling domain from the zeta chain of the CD3 molecule (CD3Q of the TCR complex, or an equivalent) and optionally one or more co-stimulatory domains, which may assist in signalling or functionality of the cell expressing the CAR.
  • a binding domain which may be viewed as an antigen (i.e., target) or ligand binding domain and the terms binding domain, antigen recognition domain, antigen binding domain and ligand binding domain are used interchangeably herein
  • a CAR may also comprise a signal or leader sequence or domain which functions to target the protein to the membrane and may form part of the exodomain of the CAR.
  • the different domains may be linked directly or by linkers, and/or may occur within different polypeptides, e.g., within two polypeptides which associate with one another.
  • the CAR When the CAR binds its target antigen (i.e., GLP1 R), this results in the transmission of an activating signal to the cell in which it is expressed.
  • GLP1 R target antigen
  • the CAR directs the specificity of the engineered cells towards GLP1 R, particularly towards cells expressing GLP1 R.
  • the CAR recognises the GLP1 R target molecule. Accordingly, it is meant that the CAR is capable of binding specifically to GLP1 R.
  • the antigen-binding domain of the CAR is capable of binding specifically to GLP1 R (more particularly when the CAR is expressed on the surface of a cell, notably an immune effector cell). Specific binding may be distinguished from non-specific binding to a non-target molecule or antigen.
  • a cell expressing the CAR is directed, or re-directed, to bind specifically to a target cell, expressing GLP1 R, particularly a target cell expressing GLP1 R on its cell surface.
  • “specific” binding means that binding only or mainly occurs to GLP1 R and not to other proteins or polypeptides (i.e., binding to other proteins or polypeptides is insignificant or weaker). Some cross-reaction with other proteins may occur but this level of binding can be considered as background.
  • the CAR is capable of binding to GLP1 R and of transducing a signal into a cell in which it is expressed. The cell may then be activated and may exert a suppressive effect within the local environment. Activation of a cell expressing a CAR after antigen binding can be determined by an increased level of CD69 as compared to the same cells expressing a CAR in the absence of antigen.
  • an increase of at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% in CD69 can be determined using standard techniques, for example FACS, using commercially available antibodies (e.g. FITC anti-human CD69 antibody, Biolegend).
  • CAR function within a cell can be determined by activation status of the cell in which the CAR is expressed, e.g., by determining CD69 expression.
  • GLP1 R Glucagon-Like Peptide-1 Receptor
  • GLP1 R is a member of the glucagon receptor family of G protein- coupled receptors. GLP1 R is expressed on pancreatic beta cells and is involved in the control of blood sugar level by enhancing insulin secretion.
  • the amino acid sequence of human GLP1 R is shown in SEQ ID NO: 64 and the amino acid sequence of mouse GLP1 R is shown in SEQ ID NO: 65.
  • the antigen-binding domain of a CAR may be derived or obtained from any protein or polypeptide which binds (i.e., has affinity for) GLP1 R (e.g. which binds to any region or part of GLP1 R, or alternatively viewed which binds to any epitope within GLP1 R, either in an isolated protein form or when expressed on cells).
  • the antigen-binding domain of a CAR may be derived or obtained from any protein or polypeptide which binds (i.e., has affinity for) the extracellular domain of GLP1 R. This may be for example, a ligand of GLP1 R, or a physiological binding protein for GLP1 R, or a part thereof, or a synthetic or derivative protein.
  • the target molecule (i.e., GLP1 R) may commonly be expressed on the surface of a cell, for example a target cell (e.g., a pancreatic beta cell), or a cell in the vicinity of a target cell (for a bystander effect), but need not be.
  • a target cell e.g., a pancreatic beta cell
  • a cell in the vicinity of a target cell for a bystander effect
  • the antigen-binding domain of a CAR is most commonly derived from antibody variable chains (for example it commonly takes the form of a scFv), but may also be generated from other molecules, such as ligands or other binding molecules.
  • the CAR is typically expressed as a polypeptide also comprising a signal sequence (also known as a leader sequence), and in particular a signal sequence which targets the CAR to the plasma membrane of the cell. This will generally be positioned next to or close to the antigen binding domain, generally upstream ofthe antigen binding domain.
  • the extracellular domain, or ectodomain, of the CAR maythus comprise, consist essentially of or consist of a signal sequence and an antigen binding domain.
  • the antigen binding domain may be any protein or peptide that possesses the ability to specifically recognize and bind to GLP1 R.
  • the antigen binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for GLP1 R.
  • Illustrative antigen-specific targeting domains include antibodies or antibody fragments or derivatives, or ligands for soluble or membrane bound GLP1 R.
  • the antigen recognition domain comprises or consists of a ligand for GLP1 R.
  • the ligand may be a natural ligand for GLP1 R (i.e., a ligand that occurs in nature and may be referred to as a physiological ligand) or an artificial ligand for GLP1 R (i.e., a synthetic ligand that does not occur in nature and is not a physiological ligand).
  • the artificial ligand for GLP1 R may be derived from a natural ligand for GLP1 R.
  • the artificial ligand for GLP1 R may have at least about 70%, for example at least about 75%, 80%, 85%, 90%, 95% or 96% identity to a natural ligand for GLPI R.
  • the artificial ligand for GLP1 R may differ from a natural ligand for GLP1 R by up to 10 amino acids, for example up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids.
  • the artificial ligand for GLP1 R may differ from a natural ligand by at least 1 , 2 or 3 amino acids.
  • the ligand for GLP1 R from which the antigen recognition domain of the CAR may be derived may be an agonist or antagonist. However, activation of GLP1 R results in increased insulin synthesis and release. Therefore, the ligand for GLP1 R may particularly be an GLP1 R agonist.
  • Glucagon-like peptide-1 (GLP1) and glucagon are natural ligands for GLPI R.
  • the antigen recognition domain may comprise or consist of GLP1 or glucagon or a peptide derived from GLP1 or glucagon.
  • the peptide derived from GLP1 or glucagon may have at least about 70%, for example at least about 75%, 80%, 85%, 90%, 95% or 96% identity to GLP1 or glucagon respectively.
  • the peptide derived from GLP1 or glucagon may differ from GLP1 or glucagon respectively by up to 10 amino acids, for example up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids.
  • the peptide derived from GLP1 or glucagon may differ from GLP1 or glucagon by at least 1 , 2 or 3 amino acids.
  • any antigen recognition domain comprising or consisting of a peptide derived from GLP1 or glucagon retains its ability to bind GLP1 R.
  • GLP1 analogues are discussed in Gupta, Indian J Endocrinol Metab., 2013; 17(3): 413-421 , which is incorporated herein by reference. Any of the GLP1 analogues discussed therein, or derivatives thereof, may be used in the antigen recognition domain of a CAR.
  • GLP1 and glucagon are both produced from proglucagon which is encoded by the GCG gene.
  • the amino acid sequence of human proglucagon is shown in SEQ ID NO: 66.
  • Proglucagon is post- translationally processed in a tissue-specific manner in pancreatic a cells and intestinal L cells. In pancreatic a cells, glucagon is produced as a result of cleavage by PCSK2/PC2.
  • the amino acid sequence of human glucagon is shown in SEQ ID NO: 67.
  • the antigen recognition domain may therefore comprise or consist of the sequence of SEQ ID NO: 67 or a sequence differing by 1 , 2, 3 or more amino acids, for example up to 10 amino acids, for example from 1 to 5 amino acids.
  • GLP1 is further N-terminally truncated by post-translational processing in the intestinal L cells producing the biologically active forms GLP1 (7-37) and GLP1 (7- 36)amide.
  • the amino acid sequence of the initial product GLP1 (1-37) is shown in SEQ ID NO: 68.
  • the amino acid sequence of GLP1 (7-37) is shown in SEQ ID NO: 69.
  • the antigen recognition domain may therefore comprise or consist of the sequence of SEQ ID NO: 68 or SEQ ID NO: 69 or a sequence differing to SEQ ID NO: 68 or SEQ ID NO: 69 by 1 , 2, 3 or more amino acids, for example up to 10 amino acids, for example from 1 to 5 amino acids.
  • the peptide bond between the Ala residue at position 8 and the Glu residue at position 9 of GLP1 is cleaved by dipeptidyl peptidase-4 (DPP-4) in vivo, which significantly decreases the amount of GLP1 in circulation. Therefore, modifying one or more of the Ala at position 8 and the Glu at position 9 of SEQ ID NO: 68 (corresponding to positions 2 and 3 of SEQ ID NO: 69) may be beneficial in preventing cleavage of the antigen recognition domain of the CAR.
  • DPP-4 dipeptidyl peptidase-4
  • the antigen recognition domain may comprise or consist of a sequence derived from SEQ ID NO: 68 or SEQ ID NO: 69 wherein one or both of the Ala at position 8 of SEQ ID NO: 68 (position 2 of SEQ ID NO: 69) and the Glu at position 9 of SEQ ID NO: 68 (position 3 of SEQ ID NO: 69) are modified, for example by substitution, deletion or insertion.
  • the Ala at position 8 of SEQ ID NO: 68 (position 2 of SEQ ID NO: 69) may be modified, for example by substitution.
  • the Ala at position 8 of SEQ ID NO: 68 (position 2 of SEQ ID NO: 69) may be substituted with Gly.
  • Other residues that may be modified e.g.
  • substitution, insertion or deletion to assist in preventing cleavage include the His at position 7, Gly at position 10, Phe at position 12, Thr at position 13, and Asp at position 15 of SEQ ID NO: 68. Any one or more of these residues may be modified (e.g. by substitution, insertion or deletion), for example in addition to modification of one or both of Ala at position 8 and Glu at position 9 of SEQ ID NO: 68.
  • the ligand for GLP1 R that may make up part or all of the antigen recognition domain of the CARs described herein may include one or more modifications (e.g. substitutions, insertions or deletions) to alter its affinity for GLP1 R.
  • the ligand for GLP1 R may include one or more modifications (e.g. substitutions, insertions or deletions) to increase its affinity for GLP1 R.
  • the ligand for GLP1 R may include one or more modifications (e.g. substitutions, insertions or deletions) to increase its affinity by 10%, 20%, 30%, 40%, 50% or more.
  • the affinity of a ligand for its receptor e.g.
  • the equilibrium-binding affinity (Kd)) can be determined by methods known in the art, for example by flow cytometry.
  • the antigen recognition domain may comprise or consist of a sequence derived from SEQ ID NO: 68 or SEQ ID NO: 69 wherein one or both of the Gly at position 22 of SEQ ID NO: 68 (position 16 of SEQ ID NO: 69) and the Arg at position 36 of SEQ ID NO: 68 (position 30 of SEQ ID NO: 69) are modified.
  • the Arg at position 36 of SEQ ID NO: 68 (position 30 of SEQ ID NO: 69) may be substituted, for example with Gly.
  • 68 (position 16 of SEQ ID NO: 69) may be substituted, for example with Glu.
  • the antigen recognition domain of the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 61 , or a sequence differing by 1 , 2, 3 or more amino acids, for example up to 10 amino acids or up to 5 amino acids or up to 3 amino acids.
  • the sequence may differ by from 1 to 5 amino acids.
  • a modification at position 2 and/or position 16 and/or position 30 relative to SEQ ID NO: 69 may be retained.
  • amino acid sequence differs to SEQ ID NO: 61 by one or more amino acids
  • at least the Gly at position 2 and/or the Glu at position 16 and/or the Gly at position 30 of SEQ ID NO: 61 may be retained.
  • at least the Gly at position 2 may be retained.
  • the antigen recognition domain of the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 61 , or a sequence having at least about 70% identity thereto.
  • the antigen recognition domain of the CAR may have at least about 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO: 61.
  • the amino acid sequence differs to SEQ ID NO: 61 in certain embodiments at least the Gly at position 2 and/or the Glu at position 16 and/or the Gly at position 30 of SEQ ID NO: 61 may be retained. Particularly, at least the Gly at position 2 may be retained.
  • the CAR construct may comprise, or consist of, a sequence as set forth in any one of SEQ ID Nos: 126 or 150, or a variant thereof, e.g., a sequence having at least 70% identity thereto.
  • the antigen binding domain is, or is derived from, an antibody.
  • antibody refers broadly to any immunological binding agent or molecule that comprises an antigen binding domain, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, whole antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG and IgM and the antibodies as described herein may be in any one of these classes. Several of these are further divided into subclasses or isotypes such as lgG1 , lgG2, lgG3, lgG4 and the like. Generally, IgG or IgM antibodies are the most common antibodies utilised in physiological settings. As will be understood by those in the art, the term “antibody” extends to all antibodies including whole antibodies, dimeric, trimeric and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies and fragments thereof.
  • An antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen.
  • examples include a variable region (Fv), a complementarity determining region (CDR), Fab or F(ab’)2, or the light and heavy chain variable regions can be joined together in a single chain (e.g. as a scFv) and in either orientation (e.g. VL-VH or VH-VL).
  • the VL and/or VH sequences may be modified.
  • the framework regions may be modified (e.g., substituted, for example to humanise the antigen-binding domain).
  • VHH heavy chain variable region
  • VL light chain variable region
  • sdAb single domain antibody
  • HCAb camelid heavy-chain antibody
  • the antigen-binding domain is a single chain antibody (scFv).
  • the scFv may be murine, human or humanized scFv.
  • the antigen-binding domain is derived from a camelid heavychain antibody (HCAb), for example the antigen-binding domain may be a VHH domain of a HCAb.
  • HCAb camelid heavychain antibody
  • VHHs have a similar structure to that of VH domains from conventional IgGs and include three variable CDRs, although CDRs 1 and 3 often have more amino acids than those of VH domains.
  • the VHH domain may comprise one or more CDRs comprising or consisting of the sequence(s) set forth in SEQ ID NOs: 1 to 30 specified herein, or sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the VHH domain may comprise one, two or three of the VH CDRs comprising or consisting of the sequence(s) set forth in SEQ ID NOs: 1 to 30 specified herein, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the VHH domain comprises two or three VH CDRs of SEQ ID NOs: 1 to 30 or 151 to 163, the CDRs may be from the same binder described herein (e.g. all from the A2, A4, A9 binder etc).
  • CDR complementarity determining region
  • the heavy chain variable region and the light chain variable region each contain 3 CDRs.
  • Heavy chain variable region or “VH” refers to the fragment of the heavy chain of an antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • Light chain variable region or “VL” refers to the fragment of the light chain of an antibody that contains three CDRs interposed between framework regions.
  • Fv refers to the smallest fragment of an antibody to bear the complete antigen binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another, in either orientation, directly or via a peptide linker sequence.
  • Antibodies that specifically bind a predetermined antigen, i.e., GLP1 R can be prepared using methods well known in the art. Such methods include phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target molecule, i.e., to GLP1 R. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined.
  • the antigen recognition domain may bind, suitably specifically bind, one or more regions or epitopes within GLP1 R.
  • An epitope also known as antigenic determinant, is the part of an antigen that is recognised by an antigen recognition domain (e.g., an antibody).
  • an antigen recognition domain e.g., an antibody
  • the epitope is the specific piece of the antigen to which an antibody binds.
  • the antigen recognition domain binds, suitably specifically binds, to one region or epitope within GLP1 R.
  • the antigen recognition domain may comprise at least one CDR (e.g. CDR3), which can be predicted from an antibody which binds to an antigen, i.e., GLP1 R (or a variant of such a predicted CDR (e.g. a variant with one, two or three amino acid substitutions)).
  • CDR e.g. CDR3
  • GLP1 R or a variant of such a predicted CDR (e.g. a variant with one, two or three amino acid substitutions)
  • molecules containing three or fewer CDR regions e.g. a single CDR or even a part thereof
  • Molecules containing two CDR regions are described in the art as being capable of binding to a target antigen, e.g.
  • the antigen binding domain may comprise one or more variable heavy chain CDRs, e.g., one, two or three variable heavy chain CDRs.
  • the antigen binding domain may comprise one or more variable light chain CDRs, e.g. one, two or three variable light chain CDRs.
  • the antigen binding domain may comprise three heavy chain CDRs and/or three light chain CDRs (and more particularly a heavy chain variable region comprising three CDRs and/or a light chain variable region comprising three CDRs) wherein at least one CDR, preferably all CDRs, may be from an antibody which binds to GLP1 R.
  • the antigen binding domain may comprise any combination of variable heavy and light chain CDRs, e.g. one variable heavy chain CDR together with one variable light chain CDR, two variable heavy chain CDRs together with one variable light chain CDR, two variable heavy chain CDRs together with two or three variable light chain CDRs, three variable heavy chain CDRs together with one or two variable light chain CDRs, one variable heavy chain CDR together with two or three variable light chain CDRs, or three variable heavy chain CDRs together with three variable light chain CDRs.
  • the antigen binding domain comprises three variable heavy chain CDRs (CDR1 , CDR2 and CDR3) and/or three variable light chain CDRs (CDR1 , CDR2 and CDR3).
  • the one or more CDRs present within the antigen binding domain may not all be from the same antibody, as long as the domain has the desired binding activity.
  • one CDR may be predicted from the heavy or light chains of an antibody which binds to GLP1 R whilst another CDR present may be predicted from a different antibody which binds to GLP1 R.
  • a combination of CDRs may be used from different antibodies, particularly from antibodies that bind to the same desired region or epitope.
  • the antigen binding domain comprises three CDRs predicted from the variable heavy chain sequence of an antibody which binds to GLP1 R and/or three CDRs predicted from the variable light chain sequence of an antibody which binds to GLP1 R (preferably the same antibody).
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 152, 2 and 3 respectively or (b) SEQ ID NOs: 153, 2 and 3 respectively or (c) SEQ ID NOs: 154, 2 and 3 respectively, and VL CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 156, 161 and 162 respectively or (b) SEQ ID NOs: 156, 161 and 163 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 1 , 2 and 3 respectively and VL CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 4, 5 and 6 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence as set forth in SEQ ID NO: 31 , or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 32, or a sequence having at least 70% sequence identity thereto.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence encoded by SEQ ID NO: 41 , or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 41 , and a VL domain comprising the sequence encoded by SEQ ID NO: 42, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 42.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences set forth in SEQ ID NOs: 7, 8 and 9 respectively and VL CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 156, 161 and 12 respectively or (b) SEQ ID NOs: 157, 161 and 12 respectively or (c) 158, 161 and 12 respectively or (d) 160, 161 and 12 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 7, 8 and 9 respectively and VL CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 10, 11 and 12 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence as set forth in SEQ ID NO: 33, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 34, or a sequence having at least 70% sequence identity thereto.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence encoded by SEQ ID NO: 43, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 43, and a VL domain comprising the sequence encoded by SEQ ID NO: 44, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 44.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences set forth in SEQ ID NOs: 13, 14 and 155 respectively and VL CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 157, 161 and 162 respectively or (b) SEQ ID NOs: 158, 161 and 162 respectively or (c) SEQ ID NOs: 159, 161 and 162 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 13, 14 and 15 respectively and VL CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 16, 17 and 18 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence as set forth in SEQ ID NO: 35, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 36, or a sequence having at least 70% sequence identity thereto.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence encoded by SEQ ID NO: 45, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 45, and a VL domain comprising the sequence encoded by SEQ ID NO: 46, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 46.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 151 , 20 and 21 respectively or (b) SEQ ID NOs: 152, 20 and 21 respectively or (c) SEQ ID NOs: 153, 20 and 21 respectively, and VL CDR1 , 2 and 3 sequences set forth in SEQ ID NOs: 22, 23 and 24 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 19, 20 and 21 respectively and VL CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 22, 23 and 24 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence as set forth in SEQ ID NO: 37, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 38, or a sequence having at least 70% sequence identity thereto.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence encoded by SEQ ID NO: 47, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 47, and a VL domain comprising the sequence encoded by SEQ ID NO: 48, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 48.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 151 , 26 and 155 respectively or (b) SEQ ID NOs: 154, 26 and 155 respectively, and VL CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 159, 161 and 163 respectively or (b) 160, 161 and 163 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen-binding domain comprises VH CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 25, 26 and 27 respectively and VL CDR1 , 2 and 3 sequences as set forth in SEQ ID NOs. 28, 29 and 30 respectively, or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence as set forth in SEQ ID NO: 39, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence as set forth in SEQ ID NO: 40, or a sequence having at least 70% sequence identity thereto.
  • the antigen binding domain of the CAR comprises a VH domain comprising the sequence encoded by SEQ ID NO: 49, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 49, and a VL domain comprising the sequence encoded by SEQ ID NO: 50, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 50.
  • a CDR does contain an amino acid sequence modification
  • this may be a deletion, addition, or substitution of an amino acid residue of the CDR sequence as set out in the above-mentioned SEQ ID NOs.
  • the modification may be an amino acid substitution, for example a conservative amino acid substitution, e.g., as set out above.
  • a longer CDR may tolerate more amino acid residue modifications.
  • the modifications may be of 0, 1 , 2 or 3 residues, e.g. 2 residues.
  • CDRs 1 and 2 may be modified, and CDR3 may be unmodified.
  • the antigen binding domain may be in the form of a scFv comprising the VH and VL domain sequences as set out above, in either order, for example VH-VL.
  • the VH and VL sequences may be linked by a linker sequence.
  • Suitable linkers can be readily selected and can be of any of a suitable length, such as from 1 amino acid (e.g. Gly) to 30 amino acids, e.g. from any one of 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids to any one of 12, 15, 18, 20, 21 , 25, 30 amino acids, for example, 5-30, 5-25, 6-25, 10-15, 12-25, 15 to 25 etc.
  • 1 amino acid e.g. Gly
  • 30 amino acids e.g. from any one of 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids to any one of 12, 15, 18, 20, 21 , 25, 30 amino acids, for example, 5-30, 5-25, 6-25, 10-15, 12-25, 15 to 25 etc.
  • Exemplary linkers include glycine polymers (G), glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, as discussed above.
  • the linker may comprise 1 or more “GS” domains as discussed above.
  • the linker sequence may be a flexible linker sequence.
  • Flexible linkers are a category of linker sequences well known and described in the art. Linker sequences are generally known as sequences which may be used to link, or join together, proteins or protein domains, to create for example fusion proteins or chimeric proteins, or multifunctional proteins or polypeptides. They can have different characteristics, and for example may be flexible, rigid or cleavable. Protein linkers are reviewed for example in Chen et al., 2013, Advanced Drug Delivery Reviews 65, 1357-1369, which compares the category of flexible linkers with those of rigid and cleavable linkers.
  • a flexible linker is a linker which allows a degree of movement between the domains, or components, which are linked. They are generally composed of small non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acid residues. The small size of the amino acids provides flexibility and allows for mobility of the connected parts (domains or components). The incorporation of polar amino acids can maintain the stability of the linker in aqueous environments by forming hydrogen bonds with water molecules.
  • GS linkers The most commonly used flexible linkers have sequences primarily composed of Ser and Gly residues (so-called “GS linkers”). However, many other flexible linkers have also been described (see Chen et al,. 2013, supra, for example), which may contain additional amino acids such as Thr and/or Ala, and/or Lys and/or Glu which may improve solubility. Any flexible linker known and reported in the art may be used.
  • the length of the linker is not critical, it may in some embodiments be desirable to have a shorter linker sequence.
  • the linker sequence may have a length of no more than 25, preferably no more than 24, 23, 22 or 21 amino acids.
  • a longer linker sequence may be desired, for example composed of, or comprising, multiple repeats of a GS domain.
  • the linker may be from any one of 2, 3, 4, 5 or 6 to any one of 24, 23, 22 or 21 amino acids in length. In other embodiments it may be from any one of 2, 3, 4, 5 or 6 to any one of 21 , 20, 19, 18, 17, 16, or 15 amino acids in length. In other embodiments it may be intermediate between these ranges, from example from 6 to 21 , 6 to 20, 7 to 20, 8-20, 9-20, 10-20, 8-18, 9-18, 10-18, 9-17, 10-17, 9-16, 10-16 etc. It may accordingly be in a range made up from any of the integers listed above.
  • GS linkers or more particularly GS (“Gly-Ser”) domains in linkers
  • Gly-Ser GS
  • linkers may allow the length of the linker readily to be varied by varying the number of GS domain repeats, and so such linkers represent one preferred class of linkers.
  • flexible linkers are not limited to those based on “GS” repeats, and other linkers comprising Ser and Gly residues dispersed throughout the linker sequence have been reported, including in Chen et al., supra.
  • the linker sequence may comprise at least 40% Gly or Gly and Ser residues.
  • the linker sequence may comprise Ser and/or Gly residues, and no more than 15 other amino acid residues, preferably no more than 14, 13, 12, 11 , 10, 9, 8, 6, 7, 5, or 4 other amino acid residues. It will be understood than an “other” amino acid residue may be any amino acid which is not Ser or Gly.
  • linker sequence tend to confer rigidity and so in one embodiment the linker sequence does not comprise any Pro residues. However, this is not absolute, as depending on the sequence context, a flexible linker sequence may contain one or more Pro residues.
  • the linker sequence comprises at least one Gly-Ser domain composed solely of Ser and Gly residues.
  • the linker may contain no more than 15 other amino acid residues, preferably no more than 14, 13, 12, 11 , 10, 9, 8, 6, 7, 5, or 4 other amino acid residues.
  • the Gly-Ser domain may have the formula:
  • a representative sequence GGGS is shown in SEQ ID NO: 70.
  • Gly-Ser domains may flank a stretch of other amino acids in the linker sequence.
  • GS domains need not be repeated, and G and/or S residues, or a short domain such as GS, may simply be distributed along the length or the sequence.
  • GGGSI- 5 (where GGGS is SEQ ID NO. 70)
  • G 8 (SEQ ID NO. 82) KESGSVSSEQLAQFRSLD (SEQ ID NO. 83)
  • GSSSSSS (SEQ ID NO. 94)
  • the linker has the sequence (GGGGS)s (SEQ ID NO: 76).
  • linker sequences of the polypeptides of the invention are flexible sequences
  • present disclosure includes also other polypeptides, including those which comprise linkers which are not flexible, and/or which do not meet the definitions and requirements set out above.
  • a further example of a linker that may be used to join the VH and VL domains is KLEEGEFSEARV (SEQ ID NO: 98) or a sequence having at least about 60% identity thereto, for example a sequence having at least about 65% or at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% identity thereto.
  • the linker may have the sequence of SEQ ID NO: 98 or a sequence that differs by no more than 6, for example no more than 5 or no more than 4 or no more than 3 or no more than 2 or no more than 1 amino acid.
  • this linker may be truncated or elongated at the N terminus and/or the C terminus by one or more amino acids (e.g. see Schmiedl A. et al (Protein Eng. 2000 Oct; 13(10): 725-34 where this linker is shortened by one amino acid at each terminus compared to SEQ ID NO: 98).
  • the linker may be modified by exchanging lysine (K) against isoleucine (I), arginine (R) against glycine (G) and valine (V) against cysteine (C).
  • the resulting linker has the amino acid sequence ILEEGEFSEAGC (SEQ ID NO: 99). Any peptide linker carrying the consensus amino acid sequence X1LEEGEFSEAX2X3 (SEQ ID NO: 100) wherein Xi is K or I, X2 is R or G and X3 is V or C, may also be used.
  • the antigen binding domain may comprise, or consist of, a VH sequence as set forth in SEQ ID NO: 31 linked via a linker of sequence (X)n, where X is any amino acid and n is an integer of between 15 and 25, to the VL sequence of SEQ ID NO: 32.
  • the antigen binding domain may comprise, or consist of, a VH sequence as set forth in SEQ ID NO: 33 linked via a linker of sequence (X)n, where X is any amino acid and n is an integer of between 15 and 25, to the VL sequence of SEQ ID NO: 34.
  • the antigen binding domain may comprise, or consist of, a VH sequence as set forth in SEQ ID NO: 35 linked via a linker of sequence (X)n, where X is any amino acid and n is an integer of between 15 and 25, to the VL sequence of SEQ ID NO: 36.
  • the antigen binding domain may comprise, or consist of, a VH sequence as set forth in SEQ ID NO: 37 linked via a linker of sequence (X)n, where X is any amino acid and n is an integer of between 15 and 25, to the VL sequence of SEQ ID NO: 38.
  • the antigen binding domain may comprise, or consist of, a VH sequence as set forth in SEQ ID NO: 39 linked via a linker of sequence (X)n, where X is any amino acid and n is an integer of between 15 and 25, to the VL sequence of SEQ ID NO: 40.
  • the antigen binding domain of the CAR comprises or consists of the sequence as set forth in SEQ ID NO: 51 or a sequence having at least 80% identity thereto.
  • the antigen binding domain of the CAR comprises or consists of the sequence as set forth in SEQ ID NO: 52 or a sequence having at least 80% identity thereto.
  • the antigen binding domain of the CAR comprises or consists of the sequence as set forth in SEQ ID NO: 53 or a sequence having at least 80% identity thereto.
  • the antigen binding domain of the CAR comprises or consists of the sequence as set forth in SEQ ID NO: 54 or a sequence having at least 80% identity thereto.
  • the antigen binding domain of the CAR comprises or consists of the sequence as set forth in SEQ ID NO: 55 or a sequence having at least 80% identity thereto.
  • the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by a sequence as set forth in SEQ ID NO: 56 or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 56.
  • the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining afterthe sequence encoding the signal sequence and/or any tag sequence (e.g. myc tag and/or his-tag) is removed from SEQ ID NO: 56 or variant thereof.
  • the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by a sequence as set forth in SEQ ID NO: 57 or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 57.
  • the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining afterthe sequence encoding the signal sequence and/or any tag sequence (e.g. myc tag and/or his-tag) is removed from SEQ ID NO: 57 or variant thereof.
  • the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by a sequence as set forth in SEQ ID NO: 58 or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 58.
  • the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining afterthe sequence encoding the signal sequence and/or any tag sequence (e.g. myc tag and/or his-tag) is removed from SEQ ID NO: 58 or variant thereof.
  • the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by a sequence as set forth in SEQ ID NO: 59 or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 59.
  • the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining afterthe sequence encoding the signal sequence and/or any tag sequence (e.g. myc tag and/or his-tag) is removed from SEQ ID NO: 59 or variant thereof.
  • the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by a sequence as set forth in SEQ ID NO: 60 or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 60.
  • the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining afterthe sequence encoding the signal sequence and/or any tag sequence (e.g. myc tag and/or his-tag) is removed from SEQ ID NO: 60 or variant thereof.
  • the CAR construct may comprise, or consist of, a sequence as set forth in any one of SEQ ID Nos: 101 to 105 or 145 to 149, or a variant thereof, e.g., a sequence having at least 70% identity thereto.
  • variant sequences disclosed and described herein, including the variant CAR, VH, VL and antigen binding domain sequences, may have at least 75, 80, 85, 90, 92, 95, 96, 97, 98, or 99% sequence identity to the specified SEQ ID NOs.
  • the CAR also preferably comprises a hinge domain to hold the extracellular domain, particularly the antigen binding domain, away from the cell surface, and further comprises a transmembrane domain.
  • the hinge and transmembrane domains may comprise the hinge and transmembrane sequences from any protein which has a hinge domain and/or a transmembrane domain, including any of the type I, type II or type III transmembrane proteins.
  • the hinge domain may be selected from the hinge regions of CD28, CD8alpha, CD4, CD7, CH2CH3, an immunoglobulin, or a part or variant thereof.
  • the hinge may be derived from CD8, particularly, CD8alpha, or from CH2CH3.
  • the hinge may comprise one or more cysteine residues e.g., to allow disulphide bonding.
  • the CD8 hinge may comprise one or more cysteine residues e.g., one cysteine residue, two cysteine residues or three cysteine residues.
  • the transmembrane domain of the CAR may also comprise an artificial hydrophobic sequence.
  • the transmembrane domains of the CAR may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those of skill in the art.
  • Examples of transmembrane (TM) regions used in CAR constructs are: 1) The CD28 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.); 2) The 0X40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41); 3) The 4-1 BB TM region (Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35); 4).
  • the CD3 zeta TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.); 5) The CD8a TM region (Maher et al, Nat Biotechnol, 2002, Jan;20(1):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Milone et al, Mol Ther, 2009, Aug ; 17(8): 1453- 64.).
  • transmembrane domains which may be used include those from ICOS, CD4, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86 or CD154.
  • the transmembrane domain may be derived from CD4, CD28 or CD8a.
  • the CAR may not comprise a dimerisation domain which binds to a regulating molecule.
  • a regulating molecule is any molecule that can bind to at least one dimerisation domain in a CAR and can prevent the interaction, or cause the interaction, of a pair of dimerisation domains. Examples of regulating molecules include soluble proteins (e.g., cytokines, TGF-beta, VEGF) or small molecules.
  • soluble proteins e.g., cytokines, TGF-beta, VEGF
  • dimerisation with other CAR molecules it may not be controlled.
  • monovalent binding of the CAR to an antigen may allow activation of the cell in which the CAR is expressed.
  • a hinge domain may conveniently be obtained from the same protein as the transmembrane domain.
  • the hinge domain is derived from the CD8a hinge domain.
  • the hinge domain may be obtained from a different protein to the transmembrane domain.
  • the hinge domain may be derived from the CH2CH3 hinge domain and the transmembrane domain may be derived from the CD28 transmembrane domain.
  • the hinge domain may be derived from the CD8a hinge domain and may comprise the amino acid sequence shown in SEQ ID NO: 106, or a variant thereof which is at least 80% identical to SEQ ID NO; 106.
  • the variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO: 106.
  • An example of a modified CD8a hinge domain is shown in SEQ ID NO: 107.
  • the hinge domain may comprise the amino acid sequence shown in SEQ ID NO: 108 or encoded by the nucleotide sequence shown in SEQ ID NO: 109, or a variant thereof which is at least 80% identical to SEQ ID NO: 108 or the sequence encoded by SEQ ID NO: 109.
  • the variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO: 108 or the sequence encoded by SEQ ID NO: 109.
  • the transmembrane domain may be derived from the CD8a transmembrane domain and may comprise the amino acid sequence shown as SEQ ID NO: 110 which represents amino acids 183 to 203 of human CD8a, or a variant which is at least 80% identical to SEQ ID NO: 110.
  • the variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO: 110.
  • the CD8a transmembrane domain may be combined with a CD8a hinge domain.
  • the CAR comprises a combined CD8a hinge and transmembrane domain sequence as shown in SEQ ID NO. 111 or SEQ ID NO: 112, or a variant thereof which has at least 80% sequence identity thereto.
  • the variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO: 1 11 or SEQ ID NO: 112 respectively.
  • SEQ ID NO. 112 comprises a modified hinge domain comprising 2 amino acid modifications of cysteine residues relative to the wild-type CD8a hinge sequence.
  • the modified CD8a hinge domain sequence is shown in SEQ ID NO. 107.
  • the wildtype CD8a hinge and transmembrane domain sequence is shown in SEQ ID NO. 111.
  • the 6 amino acids at the end of SEQ ID NO. 111 and 112, when present, are not located in the membrane and form part of the endodomain of the CAR.
  • the hinge domain may be derived from the CH2CH3 hinge domain and may comprise the sequence shown in SEQ ID NO. 113 or SEQ ID NO. 114 or a variant thereof which is at least 80% identical to SEQ ID NO. 113 or 114 respectively.
  • the variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO. 113 or 114 respectively.
  • an example of a CD28 hinge and transmembrane sequence which may be used is SEQ ID NO: 115 or a variant thereof which is at least 80% identical to SEQ ID NO: 115.
  • the variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO: 115.
  • the CAR may comprise a native or modified CD8a hinge domain and a CD28 transmembrane domain, or a CD28 hinge domain and CD8a transmembrane domain, for example based on the sequences given above.
  • a CH2CH3 hinge sequence comprising one or more cysteine residues may be used.
  • Other hinge domains which may be used include those from CD4, CD7, or an immunoglobulin, or a part or variant thereof. These hinge domains may comprise one or more cysteine residues, for example one, two, three, four or more cysteine residues.
  • the transmembrane domain may be derived from the CD4 transmembrane domain and may comprise the sequence shown in SEQ ID NO: 116 or a variant thereof which is at least 80% identical to SEQ ID NO: 116.
  • the variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO: 116.
  • the CAR may further comprise a signal (or alternatively termed, leader) sequence which targets it to the endoplasmic reticulum pathway for expression on the cell surface.
  • a signal/leader sequence is MALPVTALLLPLALLLHAAAP as shown in SEQ ID NO. 117. This comprises a single amino acid substitution compared to the wild type CD8a sequence MALPVTALLLPLALLLHAARP as shown in SEQ ID NO. 118. Either sequence, or a variant sequence having at least 70% sequence identity thereto may be used. For example, a variant sequence may have at least 75, 80, 85, 90, 95, 97, 98 or 99% sequence identity thereto.
  • the endodomain of a CAR as described herein comprises motifs necessary to transduce the effector function signal and direct a cell expressing the CAR to perform its specialized function upon antigen binding.
  • the endodomain may comprise one or more (e.g. two or three) Immunoreceptor tyrosine-based activation motifs (ITAMs), typically comprising the amino acid sequence of YXXL/I, where X can be any amino acid.
  • ITAMs Immunoreceptor tyrosine-based activation motifs
  • intracellular signaling domains include, but are not limited to, chain endodomain of the T-cell receptor or any of its homologs (e.g., q chain, FceR1 y and p chains, MB1 (Iga) chain, B29 (Igp) chain, etc.), CD3 polypeptide domains (A, 6 and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.
  • chain endodomain of the T-cell receptor or any of its homologs e.g., q chain, FceR1 y and p chains, MB1 (Iga) chain, B29 (Igp) chain, etc.
  • CD3 polypeptide domains A, 6 and e
  • the intracellular signaling domain may comprise human CD3 zeta chain endodomain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the intracellular signaling domain comprises the intracellular signaling domain of a human CD3 zeta chain.
  • the sequence of the intracellular signaling domain of human CD3 zeta chain is set out in SEQ ID NO. 119.
  • the CAR may comprise a CD3 signalling domain comprising or consisting of a sequence as set out in SEQ ID NO. 119 or a sequence having at least 80, 85, 90, 95, 97, 98 or 99% identity to SEQ ID NO: 119.
  • the signaling domain comprises or consists of SEQ ID NO. 119.
  • the present CAR may not comprise a costimulatory domain derived from 4-1 BB within the endodomain.
  • the present CAR may comprise a compound endodomain comprising a fusion of the intracellular part of a T-cell co-stimulatory molecule to that of e.g. CD3 .
  • Such a compound endodomain may be referred to as a second-generation CAR which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition.
  • the co-stimulatory domain most commonly used is that of CD28.
  • the CAR endodomain may also comprise one or more TNF receptor family signalling domain, such as the signalling domain of ICOS, (CD134) 0X40, 4-1 BB, CD27 or TNFRSF25, or a part or variant thereof, although preferably the CAR may not comprise an endodomain comprising the signalling domains of both CD28 and 4-1 BB.
  • TNF receptor family signalling domain such as the signalling domain of ICOS, (CD134) 0X40, 4-1 BB, CD27 or TNFRSF25, or a part or variant thereof, although preferably the CAR may not comprise an endodomain comprising the signalling domains of both CD28 and 4-1 BB.
  • An intracellular signaling domain of CD28 which may be used as a co-stimulatory domain is shown in SEQ ID NO. 121 .
  • Illustrative sequences for 0X40, 4-1 BB, ICOS and TNFRSF25 signalling domains are shown in SEQ ID NO: 122 to 125.
  • the CAR may comprise one or more co-stimulatory domains comprising or consisting of the sequence of any one of SEQ ID NO: 121 , 122, 123, 124, 125, or a variant thereof having at least 80, 85, 90, 95, 97, 98 or 99% sequence identity thereto.
  • the transmembrane domain and the intracellular signalling domain derived from a T-cell co-stimulatory molecule may be derived from the same protein.
  • the transmembrane domain and intracellular signalling domain derived from a T-cell co- stimulatory molecule may be derived from CD28.
  • the CAR may comprise a combined CD28 transmembrane and CD28 intracellular signalling domain as shown in SEQ ID NO: 120, or a variant thereof having at least 80% sequence identity thereto. The variant may be at least 85, 90, 95, 97, 98 or 99% identical to SEQ ID NO: 120.
  • the CAR comprises a human CD8 hinge domain or a variant thereof and a human CD8 transmembrane domain.
  • the CAR comprises an endodomain comprising, consisting essentially of or consisting of a human CD28 co-stimulatory domain and a human CD3 zeta signalling domain.
  • the CAR comprises a hinge, transmembrane, and intracellular (or endo) domains as follows:
  • a CH2CH3 hinge sequence comprising or consisting of the sequence as set forth in SEQ ID NO. 113 or SEQ ID NO. 114, or a sequence having at least 80% sequence identity thereto;
  • a CD28 transmembrane and co-stimulatory domain comprising or consisting of the sequence as set forth in SEQ ID NO. 120, or a sequence having at least 80% sequence identity thereto;
  • a CD3 signalling domain comprising or consisting of the sequence as set forth in SEQ ID NO. 1 19, or a sequence having at least 80% sequence identity thereto.
  • the CAR comprises a hinge, transmembrane, and intracellular (or endo) domains as follows:
  • a CD8a hinge and transmembrane domain sequence comprising or consisting of the sequence as set forth in SEQ ID NO. 1 11 , or a sequence having at least 80% sequence identity thereto;
  • a CD28 co-stimulatory domain comprising or consisting of the sequence as set forth SEQ ID NO. 121 , or a sequence having at least 80% sequence identity thereto;
  • a CD3 signalling domain comprising or consisting of the sequence as set forth in SEQ ID NO. 119, or a sequence having at least 80% sequence identity thereto.
  • the CAR comprises a hinge, transmembrane, and intracellular (or endo) domains as follows:
  • a CD8a hinge and transmembrane domain sequence comprising or consisting of the sequence as set forth in SEQ ID NO. 1 12, or a sequence having at least 80% sequence identity thereto;
  • a CD28 co-stimulatory domain comprising or consisting of the sequence as set forth in SEQ ID NO. 121 , or a sequence having at least 80% sequence identity thereto;
  • a CD3 signalling domain comprising or consisting of the sequence as set forth in SEQ ID NO. 119, or a sequence having at least 80% sequence identity thereto.
  • the CAR may further comprise a leader sequence comprising or consisting of a sequence as set out in SEQ ID NO. 1 17 or SEQ ID NO: 1 18, or a sequence having at least 80% sequence identity thereto.
  • the antigen binding domain of the CAR may comprise or consist of a sequence as set out in SEQ ID NO. 51 , 52, 53, 54, 55, 61 or a sequence having at least 80% sequence identity thereto which is capable of binding to GLP1 R.
  • one preferred representative CAR may comprise: i.a leader sequence comprising or consisting of a sequence as set out in SEQ ID NO. 117 or 118, or a sequence having at least 80% sequence identity thereto; ii.an antigen binding domain comprising or consisting of a sequence as set out in SEQ ID NO. 51 , 52, 53, 54, 55, 61 or a sequence having at least 80% sequence identity thereto; iii. a CH2CH3 hinge sequence comprising or consisting of the sequence as set forth in SEQ ID NO.
  • a CD28 transmembrane and co-stimulatory domain comprising or consisting of the sequence as set forth in SEQ ID NO. 120, or a sequence having at least 80% sequence identity thereto
  • v. a CD3 signalling domain comprising or consisting of the sequence as set forth in SEQ ID NO. 119, or a sequence having at least 80% sequence identity thereto.
  • a CD8 hinge and transmembrane domain sequence comprising or consisting of the sequence as set forth in SEQ ID NO. 1 11 or 112, or a sequence having at least 80% sequence identity thereto;
  • a CD28 co-stimulatory domain comprising or consisting of the sequence as set forth in SEQ ID NO. 121 , or a sequence having at least 80% sequence identity thereto;
  • v.a CD3 signalling domain comprising or consisting of the sequence as set forth in SEQ ID NO. 119, or a sequence having at least 80% sequence identity thereto.
  • the CAR of the present invention may comprise, or consist of, any one or more of the sequences as set out in SEQ ID NOs. 101 to 105, 126 and 145 to 150, or a variant thereof having at least about 80% identity thereto.
  • any variant may have at least about 85, 90, 95, 97, 98 or 99% identity to SEQ ID NOs 101 to 105, 126 or 145 to 150.
  • the CARs of SEQ ID NOs 101 to 105, 126, 145 to 150, or variants thereof may further comprise a signal sequence, for example a signal sequence having the sequence set out in SEQ ID NO: 117 or 1 18.
  • the CAR is capable of binding to GLP1 R and of transducing a signal into a cell in which it is expressed.
  • the cell may express only one type of CAR, i.e., where the cell expresses more than one CAR molecule, the amino acid sequences of each of these expressed molecules are identical to one another.
  • the endodomain of a CAR herein may contain further domains.
  • the CAR may comprise domains that confer the ability on the CAR to provide a productive IL signal to the cell in which it is expressed in an antigen specific manner, without requiring exogenous IL to be administered.
  • it may comprise a domain comprising a STAT5 association motif, a JAK1 and/or JAK 2 binding motif and optionally a JAK 3 binding motif.
  • the endodomain may comprise one or more sequences from an endodomain of a cytokine receptor, for example an interleukin receptor (IL) receptor.
  • IL interleukin receptor
  • Exemplary amino acid sequences derived from IL-2 receptor beta that include STAT5 association motif and JAK binding motifs are provided in SEQ ID NOs: 127-129.
  • the inclusion of such domains confers the ability on the CAR to provide a productive IL signal to the cell in which it is expressed in an antigenspecific manner, without requiring exogenous IL to be administered.
  • IL-2 is important for the survival, proliferation and in vivo persistence of Treg cells, but IL-2 levels may frequently be low or impaired in patients needing treatment.
  • a CAR may thus comprise a sequence corresponding to all or part of a p chain endodomain of an IL receptor or a variant thereof, such as the IL2 receptor, optionally in combination with the y chain endodomain of an IL receptor or a variant thereof, such as the IL2 receptor.
  • nucleic acid sequences or polypeptides may be introduced into the cell or population of cells in order to improve in vivo persistence or survival of the cells, for example to provide a productive IL signal to the cell without requiring exogenous IL to be administered.
  • IL signal may be constitutive or inducible.
  • Exemplary technologies may, for example, involve the use of engineered or chimeric receptors that can transmit an IL signal without requiring exogenous IL to be administered.
  • inducible engineered receptors such as those described in WO 2018/111834, WO 2019/169290 and WO 2020/264039; or constitutive engineered receptors such as those described in WO 2018/038954, WO 2019/102207, WO 2019/053420, WO 2020/180694 and WO 2017/218850; chimeric cytokine receptors such as those described in WO 2020/183131 , WO 2017/029512, WO 2012/138858, WO 2014/172584, WO 2017/068360, WO 2021/023987, WO 2020/180664 and WO 2020/044239; or engineered receptors having a tethered activation molecule such as those described in WO 2017/201432 and WO 2019/183389.
  • the cell or cell population of the invention may further comprise additional polypeptides, particularly exogenous polypeptides, such as a FOXP3 and/or safety switch polypeptide.
  • additional polypeptides such as a FOXP3 and/or safety switch polypeptide.
  • the polypeptides of the present invention e.g., the CAR, FOXP3 and safety switch, may be encoded by a single nucleic acid molecule.
  • the nucleic acid molecule may comprise nucleotide sequences encoding self-cleavage sequences in between the encoded polypeptides, allowing the polypeptides to be expressed and/or produced as separate, or discrete components.
  • polypeptides are encoded by a single nucleic acid molecule, through “cleavage” during or after translation at the encoded cleavage sites, they may be expressed or produced as separate polypeptides, and thus at the end of the protein production process in the cell, they may be present in the cell as separate entities, or separate polypeptide chains.
  • additional exogenous polypeptides may be encoded by separate nucleic acid molecules or vectors.
  • the polypeptides are not linked to one another and are physically distinct. Indeed, following expression, they are located in different, or separate cellular locations.
  • the CAR, FOXP3 and safety switch polypeptide are thus ultimately expressed as single and separate components.
  • the CAR is expressed as a cell surface molecule.
  • the safety switch polypeptide may be expressed inside a cell, or on the cell surface. In a particular embodiment, the safety switch polypeptide and the CAR are expressed on the surface of a cell which is intended for ACT.
  • the FOXP3 is expressed inside the cell, where it can exert its effect as a transcription factor to regulate cell development and/or activity, as described further below.
  • the safety switch polypeptide provides a cell in or on which it is expressed with a suicide moiety. This is useful as a safety mechanism which allows a cell which has been administered to a subject to be deleted should the need arise, or indeed more generally, according to desire or need, for example once a cell has performed or completed its therapeutic effect.
  • a suicide moiety possesses an inducible capacity to lead to cellular death, or more generally to elimination or deletion of a cell.
  • An example of a suicide moiety is a suicide protein, encoded by a suicide gene, which may be expressed in or on a cell alongside a desired transgene, in this case the CAR, which when expressed allows the cell to be deleted to turn off expression of the transgene (CAR).
  • a suicide moiety herein is a suicide polypeptide that is a polypeptide that under permissive conditions, namely conditions that are induced or turned on, is able to cause the cell to be deleted.
  • the suicide moiety may be a polypeptide, or amino acid sequence, which may be activated to perform a cell-deleting activity by an activating agent which is administered to the subject, or which is active to perform a cell-deleting activity in the presence of a substrate which may be administered to a subject.
  • the suicide moiety may represent a target for a separate cell-deleting agent which is administered to the subject.
  • the cell-deleting agent By binding to the suicide moiety, the cell-deleting agent may be targeted to the cell to be deleted.
  • the suicide moiety may be recognised by an antibody, and binding of the antibody to the safety switch polypeptide, when expressed on the surface of a cell, causes the cell to be eliminated, or deleted.
  • the suicide moiety may be HSV-TK or iCasp9. However, it is preferred for the suicide moiety to be, or to comprise, an epitope which is recognised by a cell-deleting antibody or other binding molecule capable of eliciting deletion of the cell. In such an embodiment, the safety switch polypeptide is expressed on the surface of a cell.
  • cell deletion as used herein in the context of cell deletion is synonymous with “remove” or “ablate” or “eliminate”.
  • the term is used to encompass cell killing, or inhibition of cell proliferation, such that the number of cells in the subject may be reduced. 100% complete removal may be desirable but may not necessarily be achieved. Reducing the number of cells, or inhibiting their proliferation, in the subject may be sufficient to have a beneficial effect.
  • the suicide moiety may be a CD20 epitope which is recognised by the antibody Rituximab.
  • the suicide moiety may comprise a minimal epitope based on the epitope from CD20 that is recognised by the antibody Rituximab.
  • Biosimilars for Rituximab are available and may be used. A person of skill in the art is readily able to use routine methods to prepare an antibody having the binding specificity of Rituximab using the available amino acid sequences therefor.
  • CAR-cells specific for GLP1 R which also express a safety switch polypeptide comprising this sequence can be selectively killed using the antibody Rituximab, or an antibody having the binding specificity of Rituximab.
  • the safety switch polypeptide is expressed on the cell surface and when the expressed polypeptide is exposed to or contacted with Rituximab, or an antibody with the same binding specificity, death of the cell ensues.
  • Rituximab or an antibody having the binding specificity thereof, may be provided for use in ACT in combination with a cell of the invention.
  • the cell or nucleic acid or vector or construct for production of the cell and the Rituximab or equivalent antibody may be provided in a kit, or as a combination product.
  • the suicide constructs of WO2013/153391 or WO2021/239812 may be used in a cell or cell population (e.g., Treg or Treg population) as described herein.
  • the nucleic acid molecule of the present invention may be designed to increase FOXP3 expression in cells (e.g., Tregs) by introducing into the cells a nucleotide sequence encoding FOXP3, which term is synonymous with the term “a FOXP3 polypeptide”.
  • the nucleic acid molecule, and constructs and vectors containing it, thus provide a means for increasing FOXP3 in a cell, e.g., in a Treg or a CD4+ cell.
  • a single nucleic acid molecule may encode the CAR of the invention and a FOXP3 polypeptide or the CAR and FOXP3 may be encoded by separate or discrete nucleic acid molecules.
  • the invention provides a cell comprising a nucleic acid molecule comprising a nucleotide sequence encoding a CAR and a nucleic acid molecule comprising a nucleotide sequence encoding FOXP3, particularly a pluripotent cell (e.g. an iPSC), an HPC cell (e.g. expressing CD34), a CD4+ T cell or a Treg cell.
  • a pluripotent cell e.g. an iPSC
  • an HPC cell e.g. expressing CD34
  • CD4+ T cell e.g. a CD4+ T cell or a Treg cell.
  • FOXP3 is the abbreviated name of the forkhead box P3 protein.
  • FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of the regulatory pathway in the development and function of regulatory T cells.
  • FOXP3 as used herein encompasses variants, isoforms, and functional fragments of FOXP3.
  • Increasing FOXP3 expression means to increase the levels of FOXP3 mRNA and/or protein in a cell (or population of cells) in comparison to a corresponding cell which has not been modified (or population of cells) by introduction of the nucleic acid molecule, construct or vector.
  • the level of FOXP3 mRNA and/or protein in a cell modified according to the present invention may be increased to at least 1 .5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 150-fold greater than the level in a corresponding cell which has not been modified according to the present invention (or population of such cells).
  • the cell is a Treg or the population of cells is a population of Tregs.
  • the level of FOXP3 mRNA and/or protein in a modified cell may be increased to at least 1 .5-fold greater, 2-fold greater, or 5-fold greater than the level in a corresponding cell which has not been so modified (or population of such cells).
  • the cell is a Treg or the population of cells is a population of Tregs.
  • mRNA levels in a population of cells may be measured by techniques such as the Affymetrix ebioscience prime flow RNA assay, Northern blotting, serial analysis of gene expression (SAGE) or quantitative polymerase chain reaction (qPCR).
  • Protein levels in a population of cells may be measured by techniques such as flow cytometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), Western blotting or enzyme-linked immunosorbent assay (ELISA).
  • a “FOXP3 polypeptide” is a polypeptide having FOXP3 activity i.e., a polypeptide able to bind FOXP3 target DNA and function as a transcription factor regulating development and function of Tregs.
  • a FOXP3 polypeptide may have the same or similar activity to wildtype FOXP3 (SEQ ID NO. 130), e.g., may have at least 40, 50, 60, 70, 80, 90, 95, 100, 1 10, 120, 130, 140 or 150% of the activity of the wildtype FOXP3 polypeptide.
  • a FOXP3 polypeptide encoded by the nucleotide sequence in the nucleic acid, construct or vector described herein may have increased or decreased activity compared to wildtype FOXP3.
  • Techniques for measuring transcription factor activity are well known in the art.
  • transcription factor DNA-binding activity may be measured by ChlP.
  • the transcription regulatory activity of a transcription factor may be measured by quantifying the level of expression of genes which it regulates. Gene expression may be quantified by measuring the levels of mRNA and/or protein produced from the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC/MS, Western blotting or ELISA.
  • FOXP3 Genes regulated by FOXP3 include cytokines such as IL-2, IL-4 and IFN-y (Siegler et al. Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference).
  • FOXP3 or a FOXP3 polypeptide includes functional fragments, variants, and isoforms thereof, e.g., of SEQ ID NO. 130.
  • a “functional fragment of FOXP3” may refer to a portion or region of a FOXP3 polypeptide or a polynucleotide (i.e., nucleotide sequence) encoding a FOXP3 polypeptide that has the same or similar activity to the full-length FOXP3 polypeptide or polynucleotide.
  • the functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the full-length FOXP3 polypeptide or polynucleotide.
  • a person skilled in the art would be able to generate functional fragments based on the known structural and functional features of FOXP3.
  • a “FOXP3 variant” may include an amino acid sequence or a nucleotide sequence which may be at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, preferably at least 95% or at least 97% or at least 99% identical to a FOXP3 polypeptide or a polynucleotide encoding a FOXP3 polypeptide, e.g., to SEQ ID NO. 130.
  • FOXP3 variants may have the same or similar activity to a wildtype FOXP3 polypeptide or polynucleotide, e.g., may have at least 40, 50, 60, 70, 80, 90, 95, 100, 110, 120, 130, 140 or 150% of the activity of a wildtype FOXP3 polypeptide or polynucleotide.
  • a person skilled in the art would be able to generate FOXP3 variants based on the known structural and functional features of FOXP3 and/or using conservative substitutions.
  • FOXP3 variants may have similar or the same turnover time (or degradation rate) within a Treg cell as compared to wildtype FOXP3, e.g., at least 40, 50, 60, 70, 80, 90, 95, 99 or 100% of the turnover time (or degradation rate) of wildtype FOXP3 in a Treg.
  • Some FOXP3 variants may have a reduced turnover time (or degradation rate) as compared to wildtype FOXP3, for example, FOXP3 variants having amino acid substitutions at amino acid 418 and/or 422 of SEQ ID NO.
  • SEQ ID NO.s 132 to 134 represent the aa418, aa422 and aa418 and aa422 mutants respectively.
  • the FOXP3 polypeptide encoded by a nucleic acid molecule, construct or vector as described herein may comprise or consist of the polypeptide sequence of a human FOXP3, such as UniProtKB accession Q9BZS1 (SEQ ID NO: 130), or a functional fragment or variant thereof.
  • the FOXP3 polypeptide comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 130 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises or consists of an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 130 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises or consists of SEQ ID NO: 130 or a functional fragment thereof.
  • the FOXP3 polypeptide may comprise mutations at residues 418 and/or 422 of SEQ ID NO. 130, as set out in SEQ ID NO. 132, SEQ ID NO. 133, or SEQ ID NO. 134.
  • the FOXP3 polypeptide may be truncated at the N and/or C terminal ends, resulting in the production of a functional fragment.
  • an N and C terminally truncated functional fragment of FOXP3 may comprise or consist of an amino acid sequence of SEQ ID NO. 131 or a functional variant thereof having at least 80, 85, 90, 95 or 99% identity thereto.
  • the FOXP3 polypeptide may be a variant of SEQ ID NO: 130, for example a natural variant.
  • the FOXP3 polypeptide is an isoform of SEQ ID NO: 130.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 72-106 relative to SEQ ID NO: 130.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 246-272 relative to SEQ ID NO: 130.
  • the FOXP3 polypeptide comprises SEQ ID NO: 135 or a functional fragment thereof.
  • SEQ ID NO: 135 represents an illustrative FOXP3 polypeptide.
  • the FOXP3 polypeptide comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 135 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 135 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises or consists of SEQ ID NO: 135 or a functional fragment thereof.
  • the FOXP3 polypeptide may be a variant of SEQ ID NO: 135, for example a natural variant.
  • the FOXP3 polypeptide is an isoform of SEQ ID NO: 135 or a functional fragment thereof.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 72-106 relative to SEQ ID NO: 135.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 246-272 relative to SEQ ID NO: 135.
  • the polynucleotide encoding a FOXP3 polypeptide comprises or consists of a nucleotide sequence set forth in SEQ ID NO: 136, which represents an illustrative FOXP3 nucleotide sequence.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises nucleotide sequence which is at least 70% identical to SEQ ID NO: 136 or a fragment thereof which encodes a functional FOXP3 polypeptide.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 136 or a fragment thereof which encodes a functional FOXP3 polypeptide.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises or consists of SEQ ID NO: 136 or a fragment thereof which encodes a functional FOXP3 polypeptide.
  • the polynucleotide encoding a FOXP3 polypeptide comprises or consists of a polynucleotide sequence set forth in SEQ ID NO: 137, which represents another illustrative FOXP3 nucleotide.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises a nucleotide sequence which is at least 70% identical to SEQ ID NO: 137 or a fragment thereof which encodes a functional FOXP3 polypeptide.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 137 or a fragment thereof which encodes a functional FOXP3 polypeptide.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises or consists of SEQ ID NO: 137 or a fragment thereof which encodes a functional FOXP3 polypeptide.
  • FOXP3 expression within a Treg may be increased indirectly by introducing a polynucleotide into the cell which encodes a protein which increases transcription and/or translation of FOXP3 or which increases the half life (e.g. by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%) or function of FOXP3 (e.g. determined by suppressive ability of a transduced Treg, measured as previously discussed).
  • a polynucleotide into a Treg which increases transcription of endogenous FOXP3 by interacting with the endogenous FOXP3 promoter or non-coding sequences (CNS, e.g. CNS1 , 2 or 3) which are found upstream of the coding region.
  • CNS non-coding sequences
  • the polynucleotide encoding the FOXP3 polypeptide or functional fragment or variant thereof may be codon optimised.
  • the polynucleotide encoding the FOXP3 polypeptide or functional fragment or variant thereof may be codon optimised for expression in a human cell.
  • the nucleic acid molecule may comprise nucleotide sequences encoding selfcleavage sequences.
  • the self-cleaving sequences are self-cleaving peptides. Such sequences auto-cleave during protein production.
  • Self-cleaving peptides which may be used are 2A peptides or 2A-like peptides which are known and described in the art, for example in Donnelly et al., Journal of General Virology, 2001 , 82, 1027-1041 , herein incorporated by reference.
  • 2A and 2A-like peptides are believed to cause ribosome skipping and result in a form of cleavage in which a ribosome skips the formation of peptide bond between the end of a 2A peptide and the downstream amino acid sequence.
  • the "cleavage” occurs between the Glycine and Proline residues at the C-terminus of the 2A peptide meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline.
  • the term “cleavage” as used herein thus includes the skipping of peptide bond formation.
  • Suitable self-cleaving domains include P2A, T2A, E2A, and F2A sequences as shown in SEQ ID NO: 138 - 141 respectively.
  • the sequences may be modified to include the amino acids GSG at the N- terminus of the 2A peptides.
  • sequences corresponding to SEQ ID NOs. 138 - 141 are known and reported in the art.
  • Alternative 2A-like sequences which may be used are shown in Donnelly et al (supra), for example a TaV sequence.
  • the self-cleaving sequences included in the nucleic acid molecule may be the same or different. In an embodiment they are both 2A sequences, in particular P2A and/or T2A sequences.
  • the self-cleaving sequence may include an additional cleavage site, which may be cleaved by common enzymes present in the cell. This may assist in achieving complete removal of the 2A sequences after translation.
  • an additional cleavage site may for example comprise a Furin cleavage site RXXR (SEQ ID NO: 142), for example RRKR (SEQ ID NO: 143).
  • the nucleic acid molecule may comprise a nucleotide sequence encoding a CAR directed against GLP1 R having the sequence of any one of SEQ ID NOs. 101 to 105, 126, or 145 to 150 or a variant thereof as described herein, a nucleotide sequence encoding a safety switch and a nucleotide sequence encoding FOXP3.
  • the CAR may comprise:
  • leader sequence comprising or consisting of a sequence as set out in SEQ ID NO. 117 or 188, or a sequence having at least 80% sequence identity thereto;
  • an antigen binding domain comprising or consisting of a sequence as set out in SEQ ID NO. 51 , 52, 53, 54, 55, 61 , or a sequence having at least 80% sequence identity thereto;
  • a CD8a hinge and transmembrane domain sequence comprising or consisting of the sequence as set forth in SEQ ID NO. 11 1 or 112, or a sequence having at least 80% sequence identity thereto;
  • a CD28 co-stimulatory domain comprising or consisting of the sequence as set forth in SEQ ID NO. 121 , or a sequence having at least 80% sequence identity thereto;
  • a CD3 signalling domain comprising or consisting of the sequence as set forth in SEQ ID NO. 119, or a sequence having at least 80% sequence identity thereto.
  • the CAR may comprise:
  • leader sequence comprising or consisting of a sequence as set out in SEQ ID NO. 117 or 118, or a sequence having at least 80% sequence identity thereto;
  • an antigen binding domain comprising or consisting of a sequence as set out in SEQ ID NO. 51 , 52, 53, 54, 55, 61 , or a sequence having at least 80% sequence identity thereto;
  • a CH2CH3 hinge domain sequence comprising or consisting of the sequence as set forth in SEQ ID NO. 113 or 114, or a sequence having at least 80% sequence identity thereto;
  • a CD28 transmembrane and co-stimulatory domain comprising or consisting of the sequence as set forth in SEQ ID NO. 120, or a sequence having at least 80% sequence identity thereto;
  • a CD3 signalling domain comprising or consisting of the sequence as set forth in SEQ ID NO. 119, or a sequence having at least 80% sequence identity thereto.
  • variants, or derivatives and fragments thereof include any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide retains the desired function (for example, where the derivative or variant is an antigen binding domain, the desired function may be the ability of the antigen binding domain to bind its target antigen (for example, a variant of an antigen binding domain which binds to GLP1 R retains the ability to bind GLP1 R), where the derivative or variant is a signalling domain, the desired function may be the ability of that domain to signal (e.g.
  • variants or derivatives referred to herein are functional variants or derivatives.
  • variant or derivative may have at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% function compared to the corresponding, reference sequence.
  • the variant or derivative may have a similar or the same level of function as compared to the corresponding reference sequence or may have an increased level of function (e.g. increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%).
  • amino acid substitutions may be made, for example from 1 , 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains the required activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogues.
  • the variant or derivative may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% activity or ability compared to the corresponding, reference sequence.
  • the variant or derivative may have a similar or the same level of activity or ability as compared to the corresponding, reference sequence or may have an increased level of activity or ability (e.g., increased by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%).
  • Proteins or peptides may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
  • the derivative may be a homologue.
  • the term “homologue” as used herein means an entity having a certain homology with the wild type amino acid sequence and the wild type nucleotide sequence.
  • the term “homology” can be equated with “identity”.
  • a homologous or variant sequence may include an amino acid sequence which may be at least 70%, 75%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical to the subject sequence.
  • the variants will comprise the same active sites etc. as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties/functions), in the context herein it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percentage homology or identity between two or more sequences.
  • Percentage homology or sequence identity may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • “Fragment” typically refers to a selected region of the polypeptide or polynucleotide that is of interest functionally, e.g. is functional or encodes a functional fragment. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion (or part) of a full-length polypeptide or polynucleotide. Such variants, derivatives and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5' and 3' flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made.
  • flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut.
  • the DNA is then expressed in accordance with the invention to make the encoded protein.
  • Nucleic acid molecules and polynucleotides/nucleotides/nucleic acid sequences as defined herein may comprise DNA or RNA. They may be single-stranded or double-stranded. It will be understood by a skilled person that numerous different nucleic acid molecules/polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • nucleic acid molecules/polynucleotides/nucleotide sequences as defined herein may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecules/polynucleotides/nucleotide sequences as defined herein to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.
  • nucleic acid molecules/nucleotides/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 lifespan of the nucleic acid molecules/polynucleotides as defined herein.
  • Nucleic acid molecules/polynucleotides/nucleotide sequences such as DNA nucleic acid molecules/polynucleotides/sequences may be produced recombinantly, synthetically or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • PCR polymerase chain reaction
  • This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.
  • the present nucleic acid molecules/nucleotides/polynucleotides may further comprise a nucleic acid sequence encoding a selectable marker.
  • selectable markers are well known in the art and include, but are not limited to, fluorescent proteins - such as GFP.
  • the selectable marker may be a fluorescent protein, for example GFP, YFP, RFP, tdTomato, dsRed, or variants thereof.
  • the fluorescent protein is GFP or a GFP variant.
  • the nucleic acid sequence encoding a selectable marker may be provided in combination with a nucleic acid molecule herein in the form of a nucleic acid construct. Such a nucleic acid construct may be provided in a vector.
  • the selectable marker/reporter domain may be a luciferase-based reporter, a PET reporter (e.g. Sodium Iodide Symporter (NIS)), or a membrane protein (e.g. CD34, or Thy1 .1).
  • a PET reporter e.g. Sodium Iodide Symporter (NIS)
  • NIS Sodium Iodide Symporter
  • a membrane protein e.g. CD34, or Thy1 .
  • the nucleic acid sequences encoding one or more selectable markers may be separated from the present nucleic acid molecule, and/or from each other, by one or more co-expression sites which enables expression of each polypeptide as a discrete entity.
  • Suitable co-expression sites are known in the art and include, for example, internal ribosome entry sites (IRES) and self-cleaving sites such as those included in the present nucleic acid molecules, and as defined above. In an embodiment this may be a 2A cleavage sites, as discussed above.
  • a selectable marker is advantageous as it allows cells (e.g., Tregs) in which a nucleic acid molecule, construct or vector of the present invention has been successfully introduced (such that the encoded GLP1 R CAR and other modules, e.g., FOXP3 and safety switch polypeptide, are expressed) to be selected and isolated from a starting cell population using common methods, e.g., flow cytometry.
  • cells e.g., Tregs
  • a nucleic acid molecule, construct or vector of the present invention such that the encoded GLP1 R CAR and other modules, e.g., FOXP3 and safety switch polypeptide, are expressed
  • the nucleic acid molecules/nucleotides/polynucleotides used in the present invention may be codon- optimised. Codon optimisation has previously been described in WO1999/41397 and W02001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.
  • the constructs of the present invention may comprise one or more regulatory sequences, for example a promoter.
  • a “promoter” is a region of DNA that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5’ region of the sense strand). Any suitable promoter may be used, the selection of which may be readily made by the skilled person.
  • the promoter may be from any source, and may be a viral promoter, or a eukaryotic promoter, including mammalian or human promoters (i.e. a physiological promoter). In an embodiment the promoter is a viral promoter.
  • promoters include LTR promoters, EFS (or functional truncations thereof), SFFV, PGK, and CMV.
  • the promoter is SFFV or a viral LTR promoter.
  • a SFFV promoter may be used within a nucleic acid molecule, construct or vector of the invention to allow initiation of transcription of the nucleotide sequence(s). The promoter may thus control the expression of the CAR of the invention.
  • each sequence may be operably linked to the same promoter, e.g. nucleotide sequences encoding the CAR, FOXP3 and/or the safety switch
  • the SFFV promoter may comprise a nucleotide sequence as set out in SEQ ID NO. 144.
  • “Operably linked to the same promoter” means that transcription of the nucleic acid/nucleotide/polynucleotide sequences may be initiated from the same promoter (e.g., transcription of the first, second and third polynucleotide sequences is initiated from the same promoter) and that the nucleotide sequences are positioned and oriented for transcription to be initiated from the promoter. Nucleic acids/nucleotides/polynucleotides operably linked to a promoter are under transcriptional regulation of the promoter.
  • the nucleic acid/nucleotide/polynucleotide sequence is within an expression vector.
  • expression vector means a construct enabling expression of the CAR polypeptide and any additional polypeptides such as a FOXP3 polypeptide or safety switch polypeptide.
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g., a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell.
  • Vectors may be non-viral or viral.
  • examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses.
  • the vector may also be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, the vector may itself be a nucleotide of interest.
  • the vectors used herein may be, for example, plasmid, mRNA or virus vectors and may include a promoter (as described above) for the expression of a nucleic acid molecule/polynucleotide and optionally a regulator of the promoter.
  • the vector is a viral vector, for example a retroviral, e.g., a lentiviral vector or a gamma retroviral vector.
  • the vectors may further comprise additional promoters, for example, in one embodiment, the promoter may be a LTR, for example, a retroviral LTR or a lentiviral LTR.
  • LTRs Long terminal repeats
  • LTRs are identical sequences of DNA that repeat hundreds or thousands of times found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes. Signals of gene expression are found in LTRs: enhancer, promoter (can have both transcriptional enhancers or regulatory elements), transcription initiation (such as capping), transcription terminator and polyadenylation signal.
  • the vector may include a 5’LTR and a 3’LTR.
  • the vector may comprise one or more additional regulatory sequences which may act pre- or post- transcriptionally.
  • Regulatory sequences are any sequences which facilitate expression of the polypeptides, e.g., act to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory sequences include for example enhancer elements, post-transcriptional regulatory elements and polyadenylation sites. Suitably, the additional regulatory sequences may be present in the LTR(s).
  • the vector may comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), e.g., operably linked to the promoter.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • Vectors comprising the present nucleic acid molecules/polynucleotides may be introduced into cells using a variety of techniques known in the art, such as transformation and transduction.
  • techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation.
  • Non-viral delivery systems include but are not limited to DNA transfection methods.
  • transfection includes a process using a non-viral vector to deliver a gene to a target cell.
  • Non-viral delivery systems can include liposomal or amphipathic cell penetrating peptides, preferably complexed with a nucleic acid molecule or construct.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent- mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • nucleic acid molecules are designed to be used as single constructs, and this would be contained in a single vector, it is not precluded that they are introduced into a cell in conjunction with other vectors, for example encoding other polypeptides it may be desired also to introduce into the cell.
  • Engineered cells may be generated by introducing a nucleic acid molecule, construct, or vector as defined herein, by one of many means including transduction with a viral vector, and transfection with DNA or RNA.
  • the present cell may be made by: introducing to a cell (e.g. by transduction or transfection) the nucleic acid molecule/polynucleotide, construct or vector as defined herein. Suitable cells are discussed further below, but the cell may be from a sample isolated from a subject.
  • the subject may be a donor subject, or a subject for therapy (i.e., the cell may be an autologous cell, or a donor cell, for introduction to another recipient, e.g., an allogeneic cell).
  • the cell may be generated by a method comprising the following steps:
  • a target cell-enriched sample may be isolated from, enriched, and/or generated from the cell-containing sample priorto and/or after step (ii) of the method.
  • isolation, enrichment and/or generation of Tregs may be performed prior to and/or after step (ii) to isolate, enrich or generate a Treg-enriched sample.
  • Isolation and/or enrichment from a cell-containing sample may be performed after step (ii) to enrich for cells and/or Tregs (or other target cells) comprising the CAR, the nucleic acid molecule/polynucleotide, the construct and/or the vector as described herein.
  • a Treg-enriched sample may be isolated or enriched by any method known to those of skill in the art, for example by FACS and/or magnetic bead sorting.
  • a Treg-enriched sample may be generated from the cell-containing sample by any method known to those of skill in the art, for example, from Tcon cells by introducing DNA or RNA coding for FOXP3 and/or from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells. Methods for isolating and/or enriching other target cells are known in the art.
  • an engineered target cell may be generated by a method comprising the following steps:
  • the target cell may be a Treg cell, or precursor or a progenitor thereof.
  • An “engineered cell” means a cell which has been modified to comprise or express a polynucleotide which is not naturally encoded by the cell.
  • Methods for engineering cells 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, as discussed above. Any suitable method may be used to introduce a nucleic acid sequence into a cell. Non-viral technologies such as amphipathic cell penetrating peptides may be used to introduce nucleic acid.
  • a cell may also be genetically modified e.g. using any known gene editing technique to insert a nucleotide, polynucleotide or nucleic acid sequence as described herein into the genome, e.g. using CRISPR, Talens or Zn fingers.
  • an engineered cell is a cell which has been modified e.g., by transduction or by transfection.
  • an engineered cell is a cell which has been modified or whose genome has been modified e.g., by transduction or by transfection.
  • an engineered cell is a cell which has been modified or whose genome has been modified by retroviral transduction.
  • an engineered cell is a cell which has been modified or whose genome has been modified by lentiviral transduction.
  • the term “introduced” refers to methods for inserting foreign nucleic acid, e.g., DNA or RNA, into a cell.
  • the term introduced includes both transduction and transfection methods.
  • Transfection is the process of introducing nucleic acids into a cell by non-viral methods.
  • Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector.
  • Engineered cells may be generated by introducing a nucleic acid as described herein 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, or after, the introduction of a nucleic acid as described herein, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.
  • the cells may also be expanded in the presence of anti-CD3 and anti-CD28 monoclonal antibodies in combination with IL-2.
  • IL-2 may be substituted with IL-15.
  • Other components which may be used in a cell (e.g., Treg) expansion protocol include, but are not limited to rapamycin, all-trans retinoic acid (ATRA) and TGFp.
  • activated means that a cell has been stimulated, causing the cell to proliferate.
  • expansion 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 cell, or a precursor therefor.
  • a precursor cell may be a progenitor cell.
  • Representative immune cells thus include T-cells, in particular, cytotoxic T-cells (CTLs; CD8+ T-cells), helper Tcells (HTLs; CD4+ T-cells) and regulatory T cells (Tregs).
  • CTLs cytotoxic T-cells
  • HTLs helper Tcells
  • Tregs regulatory T cells
  • Other populations of T-cells are also useful herein, for example naive T-cells and memory T-cells.
  • Other immune cells include NK cells, NKT cells, tolerogenic NK or NKT cells, dendritic cells, MDSC, neutrophils, and macrophages.
  • Precursors of immune cells include pluripotent stem cells, e.g., induced PSC (iPSC), or more committed progenitors including multipotent stem cells (e.g. HPCs), or cells which are committed to a lineage.
  • Precursor cells can be induced to differentiate into immune cells in vivo or in vitro.
  • a precursor cell may be a somatic cell which is capable of being transdifferentiated to an immune cell of interest.
  • the immune cell may be an NK cell, a dendritic cell, a MDSC, or a T cell, such as a cytotoxic T lymphocyte (CTL), helper T cell or a Treg cell.
  • CTL cytotoxic T lymphocyte
  • the immune cell is a Treg cell.
  • Regulatory T cells (Treg) or T regulatory cells” are immune cells with immunosuppressive function that control cytopathic immune responses and are essential forthe maintenance of immunological tolerance.
  • Treg refers to a T cell with immunosuppressive function.
  • a T cell as used herein is a lymphocyte including any type of T cell, such as an alpha beta T cell (e.g., CD8 or CD4+), a gamma delta T cell, a memory T cell, a Treg cell.
  • alpha beta T cell e.g., CD8 or CD4+
  • gamma delta T cell e.g., CD8 or CD4+
  • memory T cell e.g., CD8 or CD4+
  • Treg cell e.g., a Treg cell.
  • immunosuppressive function may refer to the ability of the Treg to reduce or inhibit one or more of a number of physiological and cellular effects facilitated by the immune system in response to a stimulus such as a pathogen, an alloantigen, or an autoantigen.
  • effects include increased proliferation of conventional T cell (Tconv) and secretion of proinflammatory cytokines. Any such effects may be used as indicators of the strength of an immune response.
  • Tconv conventional T cell
  • cytokines secretion of proinflammatory cytokines.
  • Any such effects may be used as indicators of the strength of an immune response.
  • a relatively weaker immune response by Tconv in the presence of Tregs would indicate an ability of the Treg to suppress immune responses.
  • a relative decrease in cytokine secretion would be indicative of a weaker immune response, and thus indicative of the ability of Tregs to suppress immune responses.
  • Tregs can also suppress immune responses by modulating the expression of co-stimulatory molecules on antigen presenting cells (APCs), such as B cells, dendritic cells and macrophages.
  • APCs antigen presenting cells
  • CD80 and CD86 can be used to assess suppression potency of activated Tregs in vitro after coculture.
  • Assays are known in the art for measuring indicators of immune response strength, and thereby the suppressive ability of Tregs.
  • antigen-specific Tconv cells may be co-cultured with Tregs, and a peptide of the corresponding antigen added to the co-culture to stimulate a response from the Tconv cells.
  • the degree of proliferation of the Tconv cells and/or the quantity of the cytokine IL-2 they secrete in response to addition of the peptide may be used as indicators of the suppressive abilities of the co-cultured Tregs.
  • Antigen-specific Tconv cells co-cultured with Tregs as disclosed herein may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95% or 99% less than the same Tconv cells cultured in the absence of the Tregs.
  • antigen-specific Tconv cells co-cultured with the present Tregs may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95% or 99% less than the same Tconv cells cultured in the presence of non-engineered Tregs.
  • Treg cells are present in the peripheral blood, lymph nodes, and tissues and Tregs for use herein include thymus-derived, natural Treg (nTreg) cells, peripherally generated Tregs, and induced Treg (iTreg) cells.
  • the Treg may have a demethylated Treg-specific demethylated region (TSDR).
  • TSDR is an important methylation-sensitive element regulating Foxp3 expression (Polansky, J.K., et al., 2008. European journal of immunology, 38(6), pp.1654-1663).
  • Tregs include, but are not limited to, Tr1 cells (which do not express Foxp3, and have high IL-10 production); CD8 + FOXP3 + T cells; and y ⁇ 5 FOXP3 + T cells.
  • Tregs Different subpopulations of Tregs are known to exist, including naive Tregs (CD45RA + FoxP3 low ), effector/memory Tregs (CD45RA FoxP3 h ' 9h ) and cytokine-producing Tregs (CD45RA- FoxP3 low ).
  • “Memory Tregs” are Tregs which express CD45RO and which are considered to be CD45RO + . These cells have increased levels of CD45RO as compared to naive Tregs (e.g.
  • CD45RO CD45RA
  • naive Tregs e.g. at least 80, 90 or 95% less CD45RA as compared to naive Tregs
  • Cytokine-producing Tregs are Tregs which do not express or have very low levels of CD45RA (mRNA and/or protein) as compared to naive Tregs (e.g. at least 80, 90 or 95% less CD45RA as compared to naive Tregs), and which have low levels of FOXP3 as compared to Memory Tregs, e.g.
  • Cytokine-producing Tregs may produce interferon gamma and may be less suppressive in vitro as compared to naive Tregs (e.g., less than 50, 60, 70, 80 or 90% suppressive than naive Tregs).
  • Reference to expression levels herein may refer to mRNA or protein expression. Particularly, for cell surface markers such as CD45RA, CD25, CD4, CD45RO etc, expression may refer to cell surface expression, i.e., the amount or relative amount of a marker protein that is expressed on the cell surface. Expression levels may be determined by any known method of the art. For example, mRNA expression levels may be determined by Northern blotting/array analysis, and protein expression may be determined by Western blotting, or preferably by FACS using antibody staining for cell surface expression.
  • a naive Treg may express at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% more CD45RAthan a Treg cell which is not naive (e.g., a memory Treg cell).
  • a naive Treg cell may express at least 2, 3, 4, 5, 10, 50 or 100-fold the amount of CD45RA as compared to a non-naive Treg cell (e.g., a memory Treg cell).
  • the level of expression of CD45RA can be readily determined by methods of the art, e.g., by flow cytometry using commercially available antibodies.
  • non-naive Treg cells do not express CD45RA or low levels of CD45RA.
  • naive Tregs may not express CD45RO, and may be considered to be CD45RO-.
  • naive Tregs may express at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% less CD45RO as compared to a memory Treg, or alternatively viewed at least 2, 3, 4, 5, 10, 50 or 100 fold less CD45RO than a memory Treg cell.
  • naive Tregs express CD25 as discussed above, CD25 expression levels may be lower than expression levels in memory Tregs, depending on the origin of the naive Tregs. For example, for naive Tregs isolated from peripheral blood, expression levels of CD25 may be at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% lower than memory Tregs. Such naive Tregs may be considered to express intermediate to low levels of CD25. However, a skilled person will appreciate that naive Tregs isolated from cord blood may not show this difference.
  • a naive Treg as defined herein may be CD4 + , CD25 + , FOXP3 + , CD127
  • Low expression of CD127 refers to a lower level of expression of CD127 as compared to a CD4 + non-regulatory or Tcon cell from the same subject or donor.
  • naive Tregs may express less than 90, 80, 70, 60, 50, 40, 30, 20 or 10% CD127 as compared to a CD4 + non-regulatory or Tcon cell from the same subject or donor.
  • Levels of CD127 can be assessed by methods standard in the art, including by flow cytometry of cells stained with an anti-CD127 antibody.
  • naive Tregs do not express, or express low levels of CCR4, HLA-DR, CXCR3 and/or CCR6.
  • naive Tregs may express lower levels of CCR4, HLA-DR, CXCR3 and CCR6 than memory Tregs, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% lower level of expression.
  • Naive Tregs may further express additional markers, including CCR7 + and CD31 + .
  • Isolated naive Tregs may be identified by methods known in the art, including by determining the presence or absence of a panel of any one or more of the markers discussed above, on the cell surface of the isolated cells. For example, CD45RA, CD4, CD25 and CD127 low can be used to determine whether a cell is a naive Treg. Methods of determining whether isolated cells are naive Tregs or have a desired phenotype can be carried out as discussed below in relation to additional steps which may be carried out, and methods for determining the presence and/or levels of expression of cell markers are well-known in the art and include, for example, flow cytometry, using commercially available antibodies.
  • the cell such as a Treg
  • PBMCs peripheral blood mononuclear cells
  • the subject from whom the PBMCs are obtained is a mammal, preferably a human.
  • the cell is matched (e.g. HLA matched) or is autologous to the subject to whom the engineered cell is to be administered.
  • the subject to be treated is a mammal, preferably a human.
  • the cell may be generated ex vivo either 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).
  • the cell is autologous to the subject to whom the engineered cell is to be administered.
  • the Treg is part of a population of cells.
  • the population of Tregs comprises at least 70 % Tregs, such as at least 75, 85, 90, 95, 97, 98 or 99 % Tregs. Such a population may be referred to as an “enriched Treg population”.
  • the Treg may be derived from ex-vivo differentiation of inducible progenitor cells (e.g. iPSCs) or embryonic progenitor cells to the Treg.
  • inducible progenitor cells e.g. iPSCs
  • a nucleic acid molecule or vector as described herein may be introduced into the inducible progenitor cells or embryonic progenitor cells prior to, or after, differentiation to a Treg. Suitable methods for differentiation are known in the art and include that disclosed in Haque et al, J Vis Exp., 2016, 117, 54720 (incorporated herein by reference).
  • the term “conventional T cell” or Tcon or Tconv means a T lymphocyte cell which expresses an ap T cell receptor (TCR) as well as a co-receptor which may be cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8) and which does not have an immunosuppressive function.
  • TCR ap T cell receptor
  • CD4 cluster of differentiation 4
  • CD8 cluster of differentiation 8
  • Conventional T cells are present in the peripheral blood, lymph nodes, and tissues.
  • the engineered Treg may be generated from a Tcon by introducing the nucleic acid which includes a sequence coding for FOXP3.
  • the engineered Treg may be generated from a Tcon by in vitro culture of CD4+CD25-FOXP3- cells in the presence of IL-2 and TGF- P-
  • the target cell into which the nucleic acid molecule, construct or vector is introduced is not a cell intended for therapy.
  • the cell is a production host cell.
  • the cell may be for production of the nucleic acid, e.g., cloning, or vector, or polypeptides.
  • a cell population comprising a cell as defined or described herein. It will be appreciated that a cell population may comprise both cells of the invention which may comprise a nucleic acid molecule, expression construct or vector as defined herein, and cells which are not in accordance with the invention, for example cells which do not comprise a nucleic acid molecule, expression construct or vector as described herein, e.g., untransduced or untransfected cells.
  • all the cells in a population may be in accordance with the invention or may comprise a nucleic acid, expression construct or vector as described herein
  • cell populations having at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% of cells in accordance with the invention, for example comprising a nucleic acid, expression construct or vector as described herein are provided.
  • the population of cells may comprise more than one cell type, although in a preferred embodiment, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% of the cells are of the same type.
  • a cell population may comprise at least 70, 80, 90, 95 or 99% of T cells, more particularly Tregs.
  • at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% of the T cells, particularly Tregs may comprise a nucleic acid, expression construct or vector as described herein.
  • a cell population comprising a plurality of cells comprising a CAR comprising an antigen recognition domain that specifically binds to GLP1 R, or a nucleic acid molecule or vector encoding said CAR.
  • a cell population comprising a plurality of Tregs comprising a CAR comprising an antigen recognition domain that specifically binds to GLP1 R, or a nucleic acid molecule or vector encoding said CAR.
  • the CAR may be in accordance with any embodiment described herein. It is possible to “educate” or “reprogram” a population of T cells (e.g.
  • the T cells by exposing or contacting the T cells with one or more specific antigens (e.g. self or non-self antigens) either directly (e.g. by way of antigen presenting cells) or indirectly, such that the cells are activated by those specific antigens (e.g. have immunosuppressive or immune-tolerant properties towards those specific antigens in the case of Tregs), and proliferate.
  • specific antigens e.g. self or non-self antigens
  • those specific antigens e.g. have immunosuppressive or immune-tolerant properties towards those specific antigens in the case of Tregs
  • proliferate This may particularly take place ex vivo.
  • the endogenous TCRs of the T cells will bind those specific antigens resulting in activation preferential expansion such that the resulting population includes a larger proportion of T cells that are activated by those specific antigens (i.e.
  • the resulting population of cells has a larger proportion of cells that have TCRs that bind those specific antigens).
  • the diversity of the TCRs in the resulting population of cells is reduced.
  • the clonality of the cells or the TCR clonality of the cells has been modified or increased.
  • the clonality of the T cells (or alternatively viewed, TCR clonality or endogenous TCR clonality of the cells) is not modified ex vivo.
  • the clonality of the Tregs (or TCR clonality of the Tregs) is not modified ex vivo.
  • the population of T cells has not been selected to have TCRs that bind to pancreatic antigens.
  • “Clonality” as used herein refers to the diversity of antigens that the TCRs within a population of T cells can bind. An increase in clonality therefore results in a reduction in the diversity of antigens that can be bound and a decrease in clonality results in an increase in the diversity of antigens that can be bound.
  • the T cells are not exposed to or contacted with one or more specific antigens ex vivo, for example by way of antigen presenting cells such as dendritic cells.
  • the T cells are not exposed or contacted with one or more specific antigens ex vivo (e.g. self or non-self antigens) either directly (e.g. by way of antigen presenting cells) or indirectly, such that the cells are activated towards those specific antigens.
  • the T cells are not exposed to or contacted with one or more pancreatic antigens ex vivo.
  • One particular method that may be used to “educate” or “reprogram” Tregs may involve the steps of:
  • DC dendritic cell
  • IL-10 interleukin-10
  • the method may further comprise expanding said reprogrammed T reg cell, for example in the presence of IL-2.
  • the Tregs are not made by this method and/or the method for making a Treg (e.g. population of Tregs) does not comprise these steps, or any one of these steps. In other words, in some such embodiments tolerogenic dendritic cells are not used.
  • the population of T cells may therefore be referred to as polyclonal with respect to their endogenous TCRs, i.e., have TCRs that are not specific to a particular antigen, have activity toward multiple antigens, and/or have specificity to unknown antigens.
  • the TCRs may, for example, have specificity towards both pancreatic and non-pancreatic antigens.
  • Tregs having polyclonal TCRs may be immune tolerant to multiple antigens and/or immunosuppressive to multiple antigens, for example both pancreatic and non-pancreatic antigens. It is noted that the GLP1 R-specific CARs described herein will provide T cells having polyclonal TCRs with antigen-specificity.
  • compositions comprising a cell or cell population as defined or described herein, or a vector as defined herein.
  • the vector may be used for gene therapy.
  • a vector may be administered instead, to modify endogenous cells in the subject to express the introduced nucleic acid molecule.
  • Vectors suitable for use in gene therapy are known in the art, and include viral vectors.
  • the invention provides a cell, cell population or pharmaceutical composition as defined herein for use in therapy.
  • a pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent i.e., the cell (e.g., Treg), cell population or vector. It preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).
  • the formulation is sterile and pyrogen free.
  • the carrier, diluent, and/or excipient must be “acceptable” in the sense of being compatible with the cell or vector and not deleterious to the recipients thereof.
  • the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free, however, other acceptable carriers, diluents, and excipients may be used.
  • Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
  • the cells, cell population or pharmaceutical compositions may be administered in a manner appropriate for treating and/or preventing the desired disease or condition.
  • the quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease or condition, although appropriate dosages may be determined by clinical trials.
  • the pharmaceutical composition may be formulated accordingly.
  • the cell, cell population or pharmaceutical composition as described herein can be administered parenterally, for example, intravenously or intrathecally, or they may be administered by infusion techniques.
  • the cell, cell population or pharmaceutical composition may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9).
  • the pharmaceutical composition may be formulated accordingly.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • the pharmaceutical compositions may comprise cells in infusion media, for example sterile isotonic solution.
  • the pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the cell, cell population or pharmaceutical composition may be administered in a single or in multiple doses. Particularly, the cell, cell population or pharmaceutical composition may be administered in a single, one-off dose.
  • the pharmaceutical composition may be formulated accordingly.
  • the cell, cell population or pharmaceutical composition may be administered at a specific stage of disease.
  • pancreatic beta cells are destroyed such that they cannot produce insulin. Therefore, an optimum time to administerthe cell, cell population or pharmaceutical composition of the invention may be at an early stage of the disease before all pancreatic beta cells have been destroyed, in order to maintain at least some functioning pancreatic beta cells (with residual pancreatic beta cell function) and maintain production of insulin.
  • at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95% of pancreatic beta cells may be present at the time of administration of the cell, cell population or pharmaceutical composition described herein. Alternatively viewed, less than 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the pancreatic beta cells may have been destroyed before administration of the cell, cell population or pharmaceutical composition described herein.
  • the pharmaceutical composition may further comprise one or more active agents.
  • the pharmaceutical composition may further comprise one or more other therapeutic agents, such as lympho-depletive agents (e.g. thymoglobulin, campath-1 H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), inhibitors of mTOR (e.g. sirolimus, everolimus), drugs inhibiting costimulatory pathways (e.g. anti-CD40/CD40L, CTAL4lg), and/or drugs inhibiting specific cytokines (IL-6, IL-17, TNFalpha, IL18).
  • lympho-depletive agents e.g. thymoglobulin, campath-1 H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine
  • inhibitors of mTOR e.g. sirolimus, everolimus
  • the cell, cell population or pharmaceutical composition may be administered at varying doses (e.g. measured in cells/kg or cells/subject).
  • doses e.g. measured in cells/kg or cells/subject.
  • the physician in any event will determine the actual dosage which will be most suitable for any individual subject and it will vary with the age, weight and response of the particular subject.
  • doses of 5x10 7 to 3x10 9 cells, or 10 8 to 2x10 9 cells per subject may be administered.
  • the cell may be appropriately modified for use in a pharmaceutical composition.
  • cells may be cryopreserved and thawed at an appropriate time, before being infused into a subject.
  • kits comprising the cell, cell population and/or pharmaceutical composition herein.
  • kits are for use in the methods and uses as described herein, e.g., the therapeutic methods as described herein.
  • kits comprise instructions for use of the kit components.
  • the cell, cell population and pharmaceutical composition of the invention may find particular utility in the treatment of disorders associated with cells that express GLP1 R, or with disorders where GLP1 R is localised at or near the site of disease, particularly disorders that would benefit from the immunosuppressive activity or target killing activity of the cells of the invention.
  • the cells, cell populations, compositions and vectors herein may be for use in treating, preventing or reducing the risk of a disease or condition in a subject, notably a disease or condition which may be treated by or with the CAR.
  • the cells and compositions containing them are for adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • Various conditions may be treated by administration of cells, including particularly Treg cells, expressing a CAR according to the present disclosure. As noted above, this may be conditions responsive to immunosuppression, and particularly the immunosuppressive effects of Tregs cells.
  • the cells, cell populations, compositions and vectors described herein may thus be used for inducing, or achieving, immunosuppression in a subject.
  • the Treg cells administered, or modified in vivo may be targeted by expression of the CAR.
  • Conditions suitable for such treatment include autoimmune or inflammatory diseases (e.g. type I diabetes), or more broadly a condition associated with any undesired or unwanted or deleterious immune response. Additionally, the cells, cell populations, compositions and vectors herein may be for use in promoting tissue repair and/or tissue regeneration. Conditions to be treated or prevented include inflammation, or alternatively put, a condition associated with or involving inflammation. Inflammation may be chronic or acute. Furthermore, the inflammation may be low-level or systemic inflammation.
  • target cell refers to any cell expressing GLP1 R to which the cell of the invention is to be directed to exert its therapeutic effect.
  • the target cell functions as a marker of a disease site, i.e. to attract the cells of the invention to provide an immunosuppressive effect.
  • the target cell is killed or abrogated by the cells of the invention.
  • the target cell will be a pancreatic beta cell.
  • the disease or disorder to be treated may be type 1 diabetes.
  • Other diseases or disorders that may be treated using the CARs described herein include, for example, autoimmune pancreatitis and insulinomas.
  • the CARs may be expressed in cells having immunosuppressive function (e.g. CD4+ or CD8+ T-regulatory cells, tolerogenic NK or NKT cells, gamma-delta cells and immune regulatory 1 cells (Tr1) and other cells secreting immune modulatory cytokines such as IL-10, TGFbeta, IL-35 or amphiregulin) in order to treat type 1 diabetes or autoimmune pancreatitis.
  • the CARs may be expressed in cells having effector functions (e.g. T-effector cells, NK cells, NKT cells) in order to treat insulinomas, e.g. by eradicating or killing insulinoma cells.
  • the CARs described herein may be used to protect or prevent rejection of transplanted cells expressing GLP1 R (e.g. beta cell replacements including allogeneic islet transplants, xenogeneic islet transplants and stem cell derived beta cells).
  • the CARs may be expressed in cells having immunosuppressive function (e.g. CD4+ or CD8+ T-regulatory cells, tolerogenic NK or NKT cells, gamma-delta cells and immune regulatory 1 cells (Tr1) and other cells secreting immune modulatory cytokines such as IL-10, TGFbeta, IL-35 or amphiregulin) in order to protect or prevent rejection of transplanted cells expressing GLP1 R.
  • immunosuppressive function e.g. CD4+ or CD8+ T-regulatory cells, tolerogenic NK or NKT cells, gamma-delta cells and immune regulatory 1 cells (Tr1) and other cells secreting immune modulatory cytokines such as IL-10, TGFbe
  • the CARs described herein may be used for the targeting or homing of cells, e.g. cells having effector functions (e.g. T-effector cells, NK cells, NKT cells) or cells having immunosuppressive function (e.g. CD4+ or CD8+ T-regulatory cells, tolerogenic NK or NKT cells, gamma-delta cells and immune regulatory 1 cells (Tr1) and other cells secreting immune modulatory cytokines such as IL-10, TGFbeta, IL-35 or amphiregulin) to target cells or target sites which express GLP1 R, e.g. to islet cells.
  • effector functions e.g. T-effector cells, NK cells, NKT cells
  • immunosuppressive function e.g. CD4+ or CD8+ T-regulatory cells, tolerogenic NK or NKT cells, gamma-delta cells and immune regulatory 1 cells (Tr1) and other cells secreting immune modulatory cytokines such as IL-10
  • the engineered cells e.g., Tregs
  • the at least one symptom may be lessened, reduced, or improved by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or the at least one symptom may be completely alleviated.
  • the engineered cells e.g., Tregs may be administered to a subject with a disease in order to slow down, reduce, or block the progression of the disease.
  • the progression of the disease may be slowed down, reduced, or blocked by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject in which the engineered cells are not administered, or progression of the disease may be completely stopped.
  • the disease to be treated may be type 1 diabetes.
  • CAR-Tregs specific for GLP1 R may be able to traffic to sites of GLP1 R expression and control inflammation, via their bystander effect, to slow down the rate of pancreatic beta cell destruction.
  • Type 1 diabetes is a chronic autoimmune disease in which pancreatic beta cells, which are responsible for the production of insulin, are destroyed by the immune system. This is triggered by genetic and environmental factors. Destruction of beta cells reduces or eliminates production of insulin by the body and results in inflammation in the pancreatic islets. Insulin is a hormone required to regulate glucose levels in the bloodstream and, before treatment, subjects with type 1 diabetes will have excessively high blood sugar levels (hyperglycaemia). Type 1 diabetes is a serious and lifelong condition. Patients with type 1 diabetes currently need to closely monitor their blood glucose levels and take an appropriate dose insulin (e.g. by injection or pump). This therapy is not a cure and must be continuously administered.
  • glycaemic control is particularly poor in younger subjects, particularly subjects between the ages of approximately 16 and 25 years and the therapies described herein may therefore particularly be useful for this population.
  • Type 1 diabetes is a continuum that progresses sequentially at variable but predictable rates through distinct identifiable stages prior to the onset of symptoms. This is described in more detail in Drei et al, Diabetes Care. 2015; 38(10): 1964-1974. The ability to screen for risk and to stage type 1 diabetes prior to symptomatic type 1 diabetes provides an opportunity to intervene early to delay and ultimately prevent the onset of clinical symptoms.
  • HLA region on chromosome 6 accounts for about 30-50% of the genetic risk of type 1 diabetes, with the greatest association with HLA class II haplotypes RB1 *0301 -DQB1 *0201 (DR3-DQ2) and DRB1 *0401 -DQB1 *0302 (DR4-DQ8).
  • the remaining genetic risk for type 1 diabetes can be attributed to approximately 50 non-HLA genes or loci identified by candidate gene and genome-wide association study approaches. The highest non-HLA genetic contribution arises from the INS, PTPN22, CTLA4 and IL2RA genes.
  • Individuals who have been identified at increased risk of type 1 diabetes, but have not yet developed any signs of disease i.e. pre-stage 1 may be prophylactically administered CAR-Tregs as described herein.
  • Stage 1 represents individuals who have developed two or more type 1 diabetes-associated islet autoantibodies (to insulin, GAD65, IA-2 and/or ZnT8) but have normal blood glucose levels.
  • Stage 2 represents individuals who have developed two or more type 1 diabetes-associated islet autoantibodies (to insulin, GAD65, IA-2 and/or ZnT8) but whose disease has progressed to the development of glucose intolerance, or dysglycemia, from loss of functional pancreatic beta cell mass.
  • Dysglycemia may be defined as fasting blood glucose levels equal to or greater than 5.6 mmol/L, or 2 hour plasma glucose with a 75g oral glucose tolerance test (OGTT) equal to or greater than 7.8 mmol/L, high glucose levels at intermediate time points on OGTT (30, 60, 90 minute levels equal to or greater than 11.1 mmol/L), and/or HbA1 c levels equal to or greater than 5.7% (39 mmol/mol).
  • OGTT oral glucose tolerance test
  • Stage 3 represents individuals who have typical clinical symptoms and signs of diabetes, which include, for example, polyuria, polydipsia, weight loss, fatigue and diabetic ketoacidosis (DKA).
  • DKA diabetic ketoacidosis
  • the CAR-Tregs described herein may, for example, be used to treat subjects at stage 1 , stage 2 and/or stage 3 of disease. Alternatively, the CAR-Tregs described herein may be used to treat a subject at risk of type 1 diabetes, i.e. before stage 1.
  • CAR-Treg treatment should begin as soon as possible to minimize pancreatic beta cell destruction and maximize residual pancreatic beta cell function. This may, for example, be determined by measuring blood insulin levels or blood C-peptide levels in the subject.
  • the subject may, for example, be a requirement that the subject has a minimum stimulated C-peptide of 0.2 pmol/mL, or for example a minimum stimulated C-peptide of 0.4 pmol/mL from a mixed meal tolerance test at the time of CAR-Treg administration. These subjects may be considered to have “recent-onset type 1 diabetes”.
  • CAR-Treg treatment may begin no more than about 24 weeks after the onset of symptoms or after diagnosis, for example no more than about 20 weeks or no more than about 16 weeks or no more than about 15 weeks or no more than about 14 weeks or no more than about 12 weeks or no more than about 8 weeks or no more than about 6 weeks after the onset of symptoms or after diagnosis.
  • CAR-Treg treatment may begin equal to or less than 100 days after the onset of symptoms or after diagnosis.
  • the subject may, for example, be at any age.
  • the subject may be under 30 years of age or under 25 years of age or under 20 years of age or under 18 years of age or under 16 years of age.
  • the subject may be from 8-30 years old, particularly from 8-25 years old, from 8-16 years old or from 16-25 years old.
  • pancreatic beta cell death may, for example, be monitored by a number of methods. For example, imaging of pancreatic beta cells can be used to highlight beta cell integrity.
  • the CAR- Tregs described herein may, for example, maintain or increase the number of pancreatic beta cells present in a subject after they are administered. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • insulin levels in a subject can be monitored.
  • Normal fasting blood insulin levels blood insulin levels after the subject has fasted (not eaten or drunk anything except water) for at least 8 hours
  • the CAR-Tregs described herein may, for example, maintain or increase fasting blood insulin levels in a subject after they are administered to the subject, for example within the normal range for fasting blood insulin. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • Insulin cell-free DNA cfDNA
  • unmethylated insulin may also be detected.
  • C-peptide levels in a subject can be monitored.
  • the beta cells of the pancreas first produce a protein called “proinsulin”.
  • proinsulin breaks down to one molecule of insulin and one molecule of C- peptide. Both are released when blood sugar levels are raised. Insulin and C-peptide are released in equal amounts but are broken down differently. Therefore, C-peptide can be employed as a surrogate marker of beta cell function.
  • the liver breaks down insulin at a variable rate, while the kidneys break down C-peptide at a fairly steady rate. C-peptide may therefore be a more reliable measure of insulin production and beta cell function. Normal fasting (i.e. after fasting for at least 8 hours) C-peptide levels may be considered to be around 0.8 to 3.85 ng/mL.
  • the CAR-Tregs described herein may, for example, maintain or increase fasting C-peptide levels in a subject after they are administered to the subject, for example within the normal range for fasting C-peptide. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • blood glucose levels in a subject can be monitored. Normal fasting blood glucose levels (i.e. after fasting for at least 8 hours). Normal fasting blood glucose levels may be considered to be around 3.9 to 6.9 mmol/L. Alternatively or additionally, an oral glucose tolerance test could be performed and blood glucose levels measured (e.g. using 75g oral glucose). 2 hours following oral glucose, normal blood glucose levels may be considered to be less than around 7.8 mmol/L. At intermediate time points prior to the 2 hours (e.g. 30 minutes, 60 minutes, 90 minutes), normal blood glucose levels may be considered to be less than around 11.1 mmol/L.
  • the CAR-Tregs described herein may, for example, maintain or reduce fasting blood glucose or OGTT blood glucose levels in a subject after they are administered to the subject, for example within the normal range for fasting blood glucose and/or OGTT blood glucose. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • Haemoglobin (Hb) HbA1 c levels in a subject can be monitored.
  • HbA1 c is created when glucose binds to haemoglobin and is a measure of average blood sugar levels over the preceding 2 to 3 months. Normal HbA1 c values are considered to be approximately 4.0 to 5.6% (20 - 38 mmol/mol).
  • the CAR- Tregs may, for example, maintain or reduce HbA1 c levels in a subject after they are administered to the subject, for example within the normal range for HbA1 c. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • the subject is a mammal.
  • the subject is a human.
  • the cell may be an engineered Treg cell and the cell population may be a population of engineered Treg cells, which have been engineered to express a CAR as described herein.
  • the CAR may comprise an antigen binding domain which is capable of specifically binding to GLP1 R, i.e., the antigen is GLP1 R.
  • a method for treating a disease or condition relates to the therapeutic use of the cells herein.
  • 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 or condition and/or to slow down, reduce or block the progression of the disease.
  • treating and/or preventing an autoimmune or inflammatory disease may refer to administering an effective amount of the cells (e.g., Tregs) such that the amount of existing medication (e.g. exogenous insulin) that a subject with said disease requires is reduced, or may enable the discontinuation of the subject’s existing medication.
  • an effective amount of the cells e.g., Tregs
  • existing medication e.g. exogenous insulin
  • the cells may be administered to a subject who has not yet contracted or developed the disease or condition and/or who is not showing any symptoms of the disease or condition to prevent the disease or condition or to reduce or prevent development of at least one symptom associated with the disease or condition.
  • the subject may have a predisposition for, or be thought to be at risk of developing, the disease or condition (e.g. pre-stage 1 type 1 diabetes).
  • treatment refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition.
  • An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.
  • an “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • An effective amount can be provided in one or more administrations.
  • a “therapeutically effective amount” is at least the minimum concentration required to affect a measurable improvement of a particular disease, disorder, or condition.
  • a therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the chimeric receptors to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the cell, cell population or pharmaceutic compositions are outweighed by the therapeutically beneficial effects.
  • subject refers to a mammal, preferably a human.
  • patient refers to a human having a disease or disorder as defined herein in need of treatment.
  • the patient may be subjected to other treatments prior to, contemporaneously with, or after the treatments of the present invention.
  • the patient may be treated with other procedures for the treatment of symptoms associated with the disease or disorder.
  • the cell may be a Treg as defined herein.
  • An enriched Treg population may be isolated and/or generated from the cell containing sample prior to, and/or after, step (ii) of the method. For example, isolation and/or generation may be performed prior to and/or after step (ii) to isolate and/or generate an enriched Treg sample. Enrichment may be performed after step (ii) to enrich for cells and/or Tregs comprising the CAR, the polynucleotide, and/or the vector as described herein.
  • the cell may be autologous.
  • the cell may be allogenic.
  • the cell e.g., the engineered Treg
  • the cell may be administered in combination with one or more other therapeutic agents, such as lympho-depletive agents (e.g., as discussed above).
  • the engineered cell, e.g., Treg may be administered simultaneously with or sequentially with (i.e., prior to or after) the one or more other therapeutic agents.
  • Cells e.g., Tregs
  • the cell e.g., Tregs
  • the cell may be washed after each step of the method, in particular after expansion.
  • the population of engineered cells, e.g., Treg cells may be further enriched by any method known to those of skill in the art, for example by FACS or magnetic bead sorting.
  • the steps of the method of production may be performed in a closed and sterile cell culture system.
  • the invention may also provide a method for increasing the stability and/or suppressive function of a cell comprising the step of introducing a nucleic acid molecule, an expression construct or vector as provided herein into the cell.
  • An increase in suppressive function can be measured as discussed above, for example by co-culturing activated antigen-specific Tconv cells with cells of the invention, and for example measuring the levels the cytokines produced by the Tconv cells.
  • An increase in suppressive function may be an increase of at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% as compared to a nonengineered Treg.
  • An increase in stability of a cell refers to an increase in the persistence or survival of those cells or to an increase in the proportion of cells retaining a Treg phenotype over a time period (e.g., to cells retaining Treg markers such as FOXP3 and Helios) as compared to a nonengineered Treg.
  • An increase in stability may be an increase in stability of at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%, and may be measured by techniques known in the art, e.g., staining of Treg cell markers within a population of cells, and analysis by FACS.
  • the invention also provides the use of a CAR-Treg to reduce the rate or prevent death of pancreatic beta cells, for example in a subject having or at risk of developing type 1 diabetes, particularly recentonset type 1 diabetes.
  • This may, for example, be determined by measuring number of pancreatic beta cells, blood insulin levels and/or blood C-peptide levels in a subject. Any maintenance or improvement in number of pancreatic beta cells, blood insulin levels and/or blood C-peptide levels may be considered to be reducing the rate or preventing death of pancreatic beta cells. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • the invention thus also provides the use of a CAR-Treg to maintain or increase fasting blood insulin levels and/or fasting blood C-peptide levels in a subject, for example in a subject having or at risk of developing type 1 diabetes, particularly recent-onset type 1 diabetes. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • the invention further provides the use of a CAR-T reg to reduce or prevent hyperglycaemia, for example in a subject having or at risk of developing type 1 diabetes, particularly recent-onset type 1 diabetes.
  • This may, for example, be determined by measuring fasting blood glucose and/or HbA1 c levels in a subject. Any maintenance or improvement in blood glucose levels and/or HbA1 c levels in a subject may be considered to be reducing or preventing hyperglycaemia in a subject. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • the invention thus also provides the use of a CAR-Treg to maintain or decrease fasting blood glucose and/or HbA1 c levels in a subject, for example in a subject having or at risk of developing type 1 diabetes, particularly recent-onset type 1 diabetes. This may, for example, be observed when the subject is receiving a reduced dose or no dose of exogenous insulin.
  • the CAR is the CAR of present invention, i.e., it comprises an antigen recognition domain that specifically binds to GLP1 R (e.g., to human GLP1 R and/or murine GLP1 R), and it may have any of the features of the CAR as disclosed herein.
  • GLP1 R e.g., to human GLP1 R and/or murine GLP1 R
  • an antibody, or an antibody fragment, which specifically binds to GLP1 R may particularly be an scFv.
  • Said antibody or antibody fragment, e.g. scFv may comprise the CDR sequences, the VH and VL sequences, or the scFv sequences as defined above with respect to the antigen binding domain of the CAR.
  • all sequences are set out above in relation to CDRs, VH and VL and scFvs may equally be comprised within antibodies or antibody fragments in this embodiment.
  • the antibody or antibody fragment may comprise:
  • VH CDR1 , 2 and 3 sequences set forth in (a) SEQ ID NOs: 151 , 20 and 21 respectively or (b) SEQ ID NOs: 152, 20 and 21 respectively or (c) SEQ ID NOs: 153, 20 and 21 respectively, and VL CDR1 , 2 and 3 sequences set forth in SEQ ID NOs: 22, 23 and 24 respectively;
  • the antibody or antibody fragment may comprise:
  • VH CDRs as set out in SEQ ID Nos 1-3 and VL CDRs as set out in SEQ ID Nos 4-6;
  • VH CDRs as set out in SEQ ID Nos 13-15 and VL CDRs as set out in SEQ ID Nos 16-18;
  • VH CDRs as set out in SEQ ID Nos 19-21 and VL CDRs as set out in SEQ ID Nos 22-24;
  • VH CDRs as set out in SEQ ID Nos 25-27 and VL CDRs as set out in SEQ ID Nos 28-30; or the CDRs may contain 1 to 3, or more particularly 1 or 2 amino acid sequence modifications in any of the aforementioned sequences.
  • An antibody or antibody fragment may be produced by any method known in the art, including by recombinant expression in a host cell transduced with a vector encoding said antibody or antibody fragment.
  • Suitable host cells include a wide variety of eukaryotic cells (e.g. yeast or mammalian cells) or prokaryotic cells (e.g. E.coli).
  • Antibodies or antibody fragments may alternatively be prepared by chemical synthesis using techniques well known in protein chemistry such as solid phase synthesis or synthesis in homogenous solution.
  • N-terminal or C-terminal fusion proteins comprising the antibody or antibody fragment as defined herein may be produced, or alternatively viewed, the antibody may be conjugated to another molecule, e.g., a therapeutic molecule or a detectable molecule.
  • the invention therefore further encompasses an antibody or antibody fragment as defined above conjugated to one or more additional molecules, e.g. to an immunoglobulin, hormone, growth factor, lectin, insulin, low density lipoprotein, glucagon, endorphins, transferrin, a tag, fluorochrome, radioactive isotope or a therapeutic molecule (e.g. an immunosuppressive drug).
  • Nucleic acid molecules comprising a nucleotide sequence encoding the antibody, antibody fragment or fusion protein as defined herein are also provided, as are vectors and cells comprising said nucleic acid molecules.
  • These antibodies, antibody fragments and fusion proteins/conjugates may be used to detect cells expressing GLP1 R for identification and/or imaging (e.g. islet mass imaging or insulinoma detection). They may also be used to deliver therapeutic molecules to a target site (e.g. cells expressing GLP1 R and/or pancreatic beta cells).
  • a target site e.g. cells expressing GLP1 R and/or pancreatic beta cells.
  • the invention provides a method for detecting or imaging a cell expressing GLP1 R, particularly an islet cell, comprising incubating a cell with an antibody, antibody fragment or fusion protein/conjugate comprising an antibody or antibody fragment as defined herein and determining whether said antibody, antibody fragment or fusion protein/conjugate is bound to said cell.
  • the invention provides a method for detecting or imaging a cell expressing GLP1 R in a subject comprising administering to said subject an antibody, antibody fragment or fusion protein/conjugate comprising an antibody or antibody fragment as defined herein and determining or detecting binding of said antibody, antibody fragment of fusion protein/conjugate to a cell.
  • detection may be carried out by fluorescence where the antibody, antibody fragment or fusion protein/conjugate may be fluorescently labelled. Other detectable labels may also be used.
  • a chimeric antigen receptor comprising an antigen recognition domain that specifically binds to GLP1 R.
  • the CAR according to paragraph 1 or 2 comprising: b. an exodomain comprising the antigen recognition domain; c. a transmembrane domain; and d. an endodomain comprising an intracellular signalling domain.
  • hinge domain is selected from the hinge regions of CD28, CD8a, CD4, CD7, CH2CH3, an immunoglobulin, or a part or variant thereof, preferably wherein the CAR comprises a CD8a or CH2CH3 hinge region.
  • the CAR comprises one or more transmembrane domains selected from the transmembrane domains of CD28, ICOS, CD8a, CD4, CD134 (0X40), CD137 (4-1 BB), CD3 zeta, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or a part or variant thereof, preferably wherein the CAR comprises a CD4, CD28 or CD8a transmembrane domain.
  • co-stimulatory domain is selected from the intracellular domains of CD28, ICOS, CD134 (0X40), CD137 (4-1 BB), CD27, or TNFRSF25, or a part or variant thereof, preferably wherein the CAR comprises a CD28 co-stimulatory domain.
  • the CAR comprises one or more intracellular signalling domains selected from the group consisting of the CD3 zeta signalling domain or any of its homologs, a CD3 polypeptide, a syk family tyrosine kinase, a src family tyrosine kinase, CD2, CD5, CD28, or a part or variant thereof, preferably wherein the CAR comprises the CD3 zeta signalling domain.
  • the CAR comprises a signal peptide and/or a reporter peptide.
  • the antigen recognition domain is an antibody, an antibody fragment, or derived from an antibody.
  • the antigen recognition domain is a single chain antibody (scFv).
  • the antigen recognition domain comprises:
  • the antigen recognition domain comprises:
  • VH domain comprising the sequence set forth in SEQ ID NO: 33, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 34, or a sequence having at least 70% identity thereto
  • VH domain comprising the sequence set forth in SEQ ID NO: 35, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 36, or a sequence having at least 70% identity thereto
  • VH domain comprising the sequence set forth in SEQ ID NO: 37, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 38, or a sequence having at least 70% identity thereto;
  • VH domain comprising the sequence set forth in SEQ ID NO: 39, or a sequence having at least 70% sequence identity thereto
  • VL domain comprising the sequence as set forth in SEQ ID NO: 40, or a sequence having at least 70% identity thereto.
  • VH domain comprising the sequence encoded by SEQ ID NO: 41 , or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 41
  • VL domain comprising the sequence encoded by SEQ ID NO: 42, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 42;
  • VH domain comprising the sequence encoded by SEQ ID NO: 43, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 43, and a VL domain comprising the sequence encoded by SEQ ID NO: 44, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 44;
  • a VH domain comprising the sequence encoded by SEQ ID NO: 45, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 45, and a VL domain comprising the sequence encoded by SEQ ID NO: 46, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 46;
  • a VH domain comprising the sequence encoded by SEQ ID NO: 47, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 47, and a VL domain comprising the sequence encoded by SEQ ID NO: 48, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 48;
  • VH domain comprising the sequence encoded by SEQ ID NO: 49, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:
  • VL domain comprising the sequence encoded by SEQ ID NO: 50, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:
  • the antigen recognition domain comprises or consists of:
  • the antigen recognition domain comprises or consists of the amino acid sequence encoded by SEQ ID NO: 62 or SEQ ID NO: 63 or an amino acid sequence having at least about 70% identity to the amino acid sequence encoded by SEQ ID NO: 62 or SEQ ID NO: 63.
  • a nucleic acid molecule comprising a nucleotide sequence encoding the CAR of any preceding paragraph.
  • a vector comprising the nucleic acid molecule of paragraph 27. 29.
  • the vector of paragraph 28 further comprising a nucleic acid molecule encoding a FOXP3 polypeptide.
  • a cell comprising the CAR of any of paragraphs 1 to 26, the nucleic acid molecule of paragraph 27, or the vector of paragraph 28 or 29.
  • the cell is an immune cell or a progenitor or precursor thereof, preferably a T cell, or a precursor thereof, or a stem cell.
  • the cell of paragraph 30 or 31 wherein the cell is a regulatory T cell (Treg), or a precursor thereof, or an iPSC cell, particularly wherein the cell further comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a FOXP3 polypeptide.
  • Treg regulatory T cell
  • iPSC iPSC cell
  • a cell population comprising a cell of any one of paragraphs 30, 32 or 33.
  • the cell population of paragraph 34 comprising a plurality of cells of any one of paragraphs 30, 32 or 33, particularly a plurality of T cells of paragraph 32, more particularly a plurality of Tregs of paragraph 33.
  • a pharmaceutical composition comprising a cell of any one of paragraphs 30, 32 or 33, a cell population of paragraph 34 or 35, or a vector of paragraph 28 or 29.
  • the cell, cell population or pharmaceutical composition for use of paragraph 41 wherein the autoimmune or inflammatory disease is type 1 diabetes, for example recent-onset type 1 diabetes.
  • a method of treating and/or preventing an autoimmune or inflammatory disease such as type 1 diabetes, or inducing immunosuppression, or promoting tissue repair and/or tissue regeneration comprising administering a cell of any one of paragraphs 30, 32 or 33, particularly a Treg, a cell population of paragraph 34 or 35, or a pharmaceutical composition of paragraph 38, particularly comprising a Treg.
  • the method according to paragraph 43 which comprises the following steps: i. isolation or provision of a Treg-enriched cell sample from a subject; ii. introduction into the Treg cells of a nucleic acid molecule of paragraph 27 or a vector of paragraph 28 or 29; and
  • a method of making a cell of any one of paragraphs 30, 32 or 33 which comprises the step of introducing into the cell (e.g. transducing or transfecting a cell with) the nucleic acid molecule of paragraph 27 or the vector of paragraph 28 or 29.
  • the method of paragraph 46 wherein the cell is a Treg cell, and the method comprises isolating or providing a cell-containing sample comprising Tregs, and/or Tregs are enriched and/or generated from the cell-containing sample prior to or after the step of introducing the nucleic acid molecule or vector into the cell.
  • a cell obtainable by the method of paragraph 46 or 47. 49.
  • GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSGYPKYFDYWGQGTLVTVSS SEQ ID NO: 38 - amino acid sequence of the VL domain of B11 (CDRs are underlined)
  • SEQ ID NO: 41 nucleotide sequence encoding the VH domain of A2 GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC
  • SEQ ID NO: 42 nucleotide sequence encoding the VL domain of A2
  • SEQ ID NO: 44 nucleotide sequence encoding the VL domain of A4
  • SEQ ID NO: 45 nucleotide sequence encoding the VH domain of A9
  • SEQ ID NO: 46 nucleotide sequence encoding the VL domain of A9
  • SEQ ID NO: 48 nucleotide sequence encoding the VL domain of B11
  • SEQ ID NO: 49 nucleotide sequence encoding the VH domain of E5
  • SEQ ID NO: 50 nucleotide sequence encoding the VL domain of E5
  • SEQ ID NO: 51 amino acid sequence of an antigen recognition domain including the VH and VL sequences of A2
  • SEQ ID NO: 52 amino acid sequence of an antigen recognition domain including the VH and VL sequences of A4
  • SEQ ID NO: 53 amino acid sequence of an antigen recognition domain including the VH and VL sequences of A9
  • SEQ ID NO: 54 amino acid sequence of an antigen recognition domain including the VH and VL sequences of B11
  • SEQ ID NO: 55 amino acid sequence of an antigen recognition domain including the VH and VL sequences of E5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQ
  • SEQ ID NO: 56 nucleotide sequence encoding A2 plus a signal sequence and a tag (the sequence encoding the signal sequence and tag are underlined)
  • SEQ ID NO: 60 nucleotide sequence encoding E5 plus a signal sequence and a tag (the sequence encoding the signal sequence and tag are underlined)
  • SEQ ID NO: 62 nucleotide sequence encoding the antigen recognition domain of the ligand CAR CACGGCGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTCGAGGAGCAGGCCGCCAAGGAA TTCATCGCCTGGCTGGTGAAGGGCGGCGGC
  • SEQ ID NO: 63 nucleotide sequence encoding the antigen recognition domain of the ligand CAR plus a signal sequence and a tag (the sequence encoding the signal sequence and tag are underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGG
  • X1LEEGEFSEAX2X3 wherein Xi is K or I, X 2 is R or G and X 3 is V or C
  • SEQ ID NO: 101 amino acid sequence of a CAR having an antigen binding domain derived from A2 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCARSSAGFDAFDIWGQGTMVTVSSGGGGSGGGGSGG GGSDIVMTQSPSSVSASVGDRVTVTCRASQGLSSWLAWYQQKPGKAPELLIYDASTLQSGVPSRFS GSGSGTDFTLTINSLQPEDFATYYCQQADSFPLTFGGGTKVEVKTRFVPVFLPAKPTTTPAPRPPTPA PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQN
  • SEQ ID NO: 103 amino acid sequence of a CAR having an antigen binding domain derived from A9 QIQLVQSGAGVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQ GRVTMTTDTSTSTAYLELRSLRSDDTAVYYCARVGSSGLLDYWGQGTLVTVSSGGGGSGGGGSGG GGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSG SGTDFTFTISSLQPEDIATYYCQQYDNLPYTFGQGTKLEIETRFVPVFLPAKPTTTPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQ
  • SEQ ID NO: 104 amino acid sequence of a CAR having an antigen binding domain derived from B11 QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSGYPKYFDYWGQGTLVTVSSGGGGSGGGGS GGGGSSSELTQDPAVSVALGQTVRITCQGDSLRGSYATWYQQKPGQAPVLVISAKNNRPSGIPDRF SVSSSGITASLTITGAQAEDEADYYCSSRDITTNHVIFGGGTKLTVLTRFVPVFLPAKPTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVR SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQ
  • SEQ ID NO: 105 amino acid sequence of a CAR having an antigen binding domain derived from E5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQ GRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVGNGGSLDYWGQGTLVTVSSGGGGSGGGGSG GGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKLLIYDASTLNAGVPSRFS GSGTDFTFTISSLQPEDIGTYYCQQFDDLPLTFGPGTKVDIKTRFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQQ
  • SEQ ID NO: 108 is the amino acid sequence of the CD8a hinge used in the CARs used in the examples
  • SEQ ID NO: 109 is the nucleotide sequence encoding the CD8a hinge used in the CARs used in the examples
  • SEQ ID NO: 110 amino acid sequence of the human CD8alpha transmembrane domain which represents amino acids 183 to 203 of human CD8a
  • SEQ ID NO: 127 is the amino acid sequence which represents amino acid numbers 266 to 551 of human IL-2 receptor beta chain
  • SEQ ID NO: 128 is the amino acid sequence which represents a truncated and sequence modified variant of SEQ ID NO: 127 (Y510)
  • SEQ ID NO: 129 is the amino acid sequence which represents a truncated and sequence modified variant of SEQ ID NO: 127 (Y510 and Y392)
  • SEQ ID NO: 135 amino acid sequence of an illustrative FOXP3 polypeptide MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMP

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Diabetes (AREA)
  • Organic Chemistry (AREA)
  • Endocrinology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Emergency Medicine (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un récepteur antigénique chimérique (CAR), une cellule (en particulier, une cellule immunitaire telle qu'une cellule T régulatrice) exprimant ledit CAR, un acide nucléique ou un vecteur codant pour ledit CAR et diverses utilisations desdits CAR, cellule, acide nucléique ou vecteur. En particulier, l'invention concerne un récepteur antigénique chimérique (CAR) comprenant un domaine de reconnaissance d'antigène qui se lie spécifiquement à GLP1R.
PCT/EP2024/057453 2023-03-20 2024-03-20 Récepteur antigénique chimérique Ceased WO2024194355A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202480033542.0A CN121586582A (zh) 2023-03-20 2024-03-20 嵌合抗原受体
EP24711920.9A EP4683660A1 (fr) 2023-03-20 2024-03-20 Récepteur antigénique chimérique

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP23162911.4 2023-03-20
EP23162911.4A EP4434539A1 (fr) 2023-03-20 2023-03-20 Récepteur antigénique chimérique
EP23174021.8 2023-05-17
EP23174021 2023-05-17

Publications (1)

Publication Number Publication Date
WO2024194355A1 true WO2024194355A1 (fr) 2024-09-26

Family

ID=90365362

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/057453 Ceased WO2024194355A1 (fr) 2023-03-20 2024-03-20 Récepteur antigénique chimérique

Country Status (3)

Country Link
EP (1) EP4683660A1 (fr)
CN (1) CN121586582A (fr)
WO (1) WO2024194355A1 (fr)

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041397A1 (fr) 1998-02-17 1999-08-19 Oxford Biomedica (Uk) Limited Vecteurs antiviraux
WO2001079518A3 (fr) 2000-04-19 2002-05-16 Oxford Biomedica Ltd Procede
WO2012138858A1 (fr) 2011-04-08 2012-10-11 Baylor College Of Medicine Inversion des effets du microenvironnement tumoral à l'aide de récepteurs de cytokine chimères
WO2013153391A1 (fr) 2012-04-13 2013-10-17 Ucl Business Plc Polypeptide utile dans la thérapie cellulaire adoptive
WO2014172584A1 (fr) 2013-04-17 2014-10-23 Baylor College Of Medicine Convertisseur de signal tgf-β immunosuppresseur
WO2017029512A1 (fr) 2015-08-20 2017-02-23 Autolus Ltd Récepteurs de cytokines chimériques
WO2017068360A1 (fr) 2015-10-23 2017-04-27 Autolus Ltd Récepteur
WO2017201432A2 (fr) 2016-05-19 2017-11-23 Jounaidi Youssef Interleukine-2 liée à son récepteur il-2rbêta, plate-forme pour améliorer l'activité des cellules tueuses naturelles et des lymphocytes t régulateurs
WO2017218850A1 (fr) 2016-06-16 2017-12-21 Memorial Sloan Kettering Cancer Center Cellules treg génétiquement modifiées
WO2018038954A1 (fr) 2016-08-25 2018-03-01 3M Innovative Properties Company Composition durcissable colorée destinée à des procédés de fabrication additive, article composite tridimensionnel et utilisation associée
WO2018111834A1 (fr) 2016-12-13 2018-06-21 Seattle Children's Hospital (dba Seattle Children's Research Institute) Méthodes d'activation de médicament exogène de complexes de signalisation induits par agents chimiques exprimés dans des cellules modifiées in vitro et in vivo
WO2019053420A1 (fr) 2017-09-13 2019-03-21 Autolus Limited Cellule
WO2019102207A1 (fr) 2017-11-24 2019-05-31 Autolus Limited Polypeptide
WO2019169290A1 (fr) 2018-03-02 2019-09-06 Allogene Therapeutics, Inc. Récepteurs de cytokines chimériques inductibles
WO2019183389A1 (fr) 2018-03-23 2019-09-26 Kite Pharma, Inc. Protéines transmembranaires chimériques et leurs utilisations
WO2019241549A1 (fr) 2018-06-15 2019-12-19 A2 Biotherapeutics, Inc. Lymphocytes t régulateurs-car exprimant le foxp3
WO2020044239A1 (fr) 2018-08-29 2020-03-05 National University Of Singapore Procédé permettant de stimuler de manière spécifique la survie et l'expansion de cellules immunitaires génétiquement modifiées
WO2020044055A1 (fr) 2018-08-31 2020-03-05 King's College London Lymphocyte t régulateur modifié
WO2020180694A1 (fr) 2019-03-01 2020-09-10 Allogene Therapeutics, Inc. Récepteurs de cytokines chimériques constitutivement actifs
WO2020180664A1 (fr) 2019-03-01 2020-09-10 Allogene Therapeutics, Inc. Récepteurs de cytokine chimériques portant un ectodomaine de pd-1
WO2020183131A1 (fr) 2019-03-08 2020-09-17 Autolus Limited Compositions et méthodes comprenant un récepteur d'antigène chimérique modifié et un modulateur de car
WO2020264039A1 (fr) 2019-06-27 2020-12-30 Seattle Children's Hospital (dba Seattle Children's Research Institute) Lymphocytes t artificiels immunorégulateurs spécifiques d'un antigène (airt)
WO2021023987A1 (fr) 2019-08-05 2021-02-11 Autolus Limited Récepteur de cytokine chimérique
WO2021239812A1 (fr) 2020-05-26 2021-12-02 Quell Therapeutics Limited Polypeptide utile dans la thérapie cellulaire adoptive
WO2022060854A1 (fr) * 2020-09-15 2022-03-24 City Of Hope Programmation de lymphocytes t régulateurs par des vésicules extracellulaires
WO2022240797A1 (fr) * 2021-05-14 2022-11-17 The University Of Toledo Cellules treg de récepteur antigénique chimérique spécifique d'un antigène immunosuppresseur pour la prévention et/ou le traitement de troubles auto-immuns et allo-immuns

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041397A1 (fr) 1998-02-17 1999-08-19 Oxford Biomedica (Uk) Limited Vecteurs antiviraux
WO2001079518A3 (fr) 2000-04-19 2002-05-16 Oxford Biomedica Ltd Procede
WO2012138858A1 (fr) 2011-04-08 2012-10-11 Baylor College Of Medicine Inversion des effets du microenvironnement tumoral à l'aide de récepteurs de cytokine chimères
WO2013153391A1 (fr) 2012-04-13 2013-10-17 Ucl Business Plc Polypeptide utile dans la thérapie cellulaire adoptive
WO2014172584A1 (fr) 2013-04-17 2014-10-23 Baylor College Of Medicine Convertisseur de signal tgf-β immunosuppresseur
WO2017029512A1 (fr) 2015-08-20 2017-02-23 Autolus Ltd Récepteurs de cytokines chimériques
WO2017068360A1 (fr) 2015-10-23 2017-04-27 Autolus Ltd Récepteur
WO2017201432A2 (fr) 2016-05-19 2017-11-23 Jounaidi Youssef Interleukine-2 liée à son récepteur il-2rbêta, plate-forme pour améliorer l'activité des cellules tueuses naturelles et des lymphocytes t régulateurs
WO2017218850A1 (fr) 2016-06-16 2017-12-21 Memorial Sloan Kettering Cancer Center Cellules treg génétiquement modifiées
WO2018038954A1 (fr) 2016-08-25 2018-03-01 3M Innovative Properties Company Composition durcissable colorée destinée à des procédés de fabrication additive, article composite tridimensionnel et utilisation associée
WO2018111834A1 (fr) 2016-12-13 2018-06-21 Seattle Children's Hospital (dba Seattle Children's Research Institute) Méthodes d'activation de médicament exogène de complexes de signalisation induits par agents chimiques exprimés dans des cellules modifiées in vitro et in vivo
WO2019053420A1 (fr) 2017-09-13 2019-03-21 Autolus Limited Cellule
WO2019102207A1 (fr) 2017-11-24 2019-05-31 Autolus Limited Polypeptide
WO2019169290A1 (fr) 2018-03-02 2019-09-06 Allogene Therapeutics, Inc. Récepteurs de cytokines chimériques inductibles
WO2019183389A1 (fr) 2018-03-23 2019-09-26 Kite Pharma, Inc. Protéines transmembranaires chimériques et leurs utilisations
WO2019241549A1 (fr) 2018-06-15 2019-12-19 A2 Biotherapeutics, Inc. Lymphocytes t régulateurs-car exprimant le foxp3
WO2020044239A1 (fr) 2018-08-29 2020-03-05 National University Of Singapore Procédé permettant de stimuler de manière spécifique la survie et l'expansion de cellules immunitaires génétiquement modifiées
WO2020044055A1 (fr) 2018-08-31 2020-03-05 King's College London Lymphocyte t régulateur modifié
WO2020180694A1 (fr) 2019-03-01 2020-09-10 Allogene Therapeutics, Inc. Récepteurs de cytokines chimériques constitutivement actifs
WO2020180664A1 (fr) 2019-03-01 2020-09-10 Allogene Therapeutics, Inc. Récepteurs de cytokine chimériques portant un ectodomaine de pd-1
WO2020183131A1 (fr) 2019-03-08 2020-09-17 Autolus Limited Compositions et méthodes comprenant un récepteur d'antigène chimérique modifié et un modulateur de car
WO2020264039A1 (fr) 2019-06-27 2020-12-30 Seattle Children's Hospital (dba Seattle Children's Research Institute) Lymphocytes t artificiels immunorégulateurs spécifiques d'un antigène (airt)
WO2021023987A1 (fr) 2019-08-05 2021-02-11 Autolus Limited Récepteur de cytokine chimérique
WO2021239812A1 (fr) 2020-05-26 2021-12-02 Quell Therapeutics Limited Polypeptide utile dans la thérapie cellulaire adoptive
WO2022060854A1 (fr) * 2020-09-15 2022-03-24 City Of Hope Programmation de lymphocytes t régulateurs par des vésicules extracellulaires
WO2022240797A1 (fr) * 2021-05-14 2022-11-17 The University Of Toledo Cellules treg de récepteur antigénique chimérique spécifique d'un antigène immunosuppresseur pour la prévention et/ou le traitement de troubles auto-immuns et allo-immuns

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING CO
ATSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
AUSUBEL ET AL., IBID, vol. 18, 1999, pages 7 - 60
BLUESTONE ET AL., SCI TRANSL MED., vol. 7, 2015, pages 315ra189
BRENTJENS ET AL., CCR, vol. 13, 15 September 2007 (2007-09-15), pages 5426 - 35
CASUCCI ET AL., BLOOD, vol. 122, no. 20, 14 November 2013 (2013-11-14), pages 3461 - 72
CHEN ET AL., ADVANCED DRUG DELIVERY REVIEWS, vol. 65, 2013, pages 1357 - 1369
CHICHILI ET AL., PROTEIN SCIENCE, vol. 22, 2013, pages 153 - 167
DEVEREUX ET AL., NUCLEIC ACIDS RES., vol. 12, 1984, pages 387
DONNELLY ET AL., JOURNAL OF GENERAL VIROLOGY, vol. 82, 2001, pages 1027 - 1041
FEMS MICROBIOL. LETT., vol. 177, 1999, pages 187 - 50
GUPTA, INDIAN J ENDOCRINOL METAB., vol. 17, no. 3, 2013, pages 413 - 421
HAQUE ET AL., J VIS EXP., vol. 117, 2016, pages 54720
IMAI C, LEUKEMIA, vol. 18, no. 4, April 2004 (2004-04-01), pages 676 - 84
INSEL ET AL., DIABETES CARE, vol. 38, no. 10, 2015, pages 1964 - 1974
JEM, vol. 7, no. 10, 2006, pages 1701 - 1711
KLEIN ET AL., PROTEIN ENGINEERING DESIGN AND SELECTION, vol. 27, no. 10, 2014, pages 325 - 330
LOPES, J.E. ET AL., THE JOURNAL OF IMMUNOLOGY, vol. 177, no. 5, 2006, pages 3133 - 3142
LOZANO, T. ET AL., FRONTIERS IN ONCOLOGY, vol. 3, 2013, pages 294
MAHER ET AL., NAT BIOTECHNOL, vol. 20, no. 1, January 2002 (2002-01-01), pages 70 - 5
MAREK-TRZONKOWSKI ET AL., CLIN IMMUNOL., vol. 153, 2014, pages 23 - 30
MILONE ET AL., MOL THER, vol. 17, no. 8, August 2009 (2009-08-01), pages 1453 - 64
NAT. BIOTECHNOL., vol. 14, 1996, pages 556
NICAISE ET AL., PROTEIN SCIENCE, vol. 13, 2004, pages 1882 - 91
POLANSKY, J.K. ET AL., EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 38, no. 6, 2008, pages 1654 - 1663
PULE ET AL., MOL THER, vol. 12, no. 5, November 2005 (2005-11-01), pages 933 - 41
SAVOLDO B, BLOOD, vol. 113, no. 25, 18 June 2009 (2009-06-18), pages 6392 - 402
SCHMIEDL A. ET AL., PROTEIN ENG., vol. 13, no. 10, October 2000 (2000-10-01), pages 725 - 34
SIEGLER ET AL., ANNU. REV. IMMUNOL., vol. 24, 2006, pages 209 - 26
SONG, X. ET AL., CELL REPORTS, vol. 1, no. 6, 2012, pages 665 - 675
TANG ET AL., J EXP MED., vol. 199, 2004, pages 1455 - 65
TENSPOLDE ET AL., J AUTOIMMUNITY, vol. 103, 2019, pages 102289
TENSPOLDE MICHEL ET AL: "Regulatory T cells engineered with a novel insulin-specific chimeric antigen receptor as a candidate immunotherapy for type 1 diabetes", JOURNAL OF AUTOIMMUNITY, LONDON, GB, vol. 103, 5 June 2019 (2019-06-05), XP085789057, ISSN: 0896-8411, [retrieved on 20190605], DOI: 10.1016/J.JAUT.2019.05.017 *
VAN ROSMALEN ET AL., BIOCHEMISTRY, vol. 56, 2017, pages 6565 - 6574
VAUGHANSOLLAZZO, COMBINATIONAL CHEMISTRY & HIGH THROUGHPUT SCREENING, vol. 4, 2001, pages 417 - 430
VISPERASVIGNALI, J IMMUNOL., vol. 197, no. 10, 2016, pages 3762 - 3770

Also Published As

Publication number Publication date
CN121586582A (zh) 2026-02-27
EP4683660A1 (fr) 2026-01-28

Similar Documents

Publication Publication Date Title
US20210338726A1 (en) Engineered regulatory t cell
US11827705B2 (en) Methods to protect transplanted tissue from rejection
TW202430546A (zh) 用靶向密連蛋白１８．２的嵌合抗原受體治療癌症之組成物和方法
US20250144137A1 (en) Nucleic acid constructs for expressing polypeptides in cells
US20230138428A1 (en) Chimeric receptors for use in engineered cells
KR20220051390A (ko) 조작된 조절 t 세포
WO2024110751A1 (fr) Récepteur antigénique chimérique constitutivement actif pour la survie et/ou la persistance de cellules treg
US20240342214A1 (en) Chimeric antigen receptor
EP4434539A1 (fr) Récepteur antigénique chimérique
EP4403580A1 (fr) Anti-entpd3 récepteur antigénique chimérique
EP4420676A1 (fr) Récepteur antigénique chimérique
EP4683660A1 (fr) Récepteur antigénique chimérique
CA3241186A1 (fr) Recepteurs de cytokines constitutifs
WO2024175805A1 (fr) Récepteur antigénique chimérique
WO2026027876A1 (fr) Récepteur antigénique chimérique
US20240052015A1 (en) Inducible signalling protein
WO2026008994A1 (fr) Construction d'expression
WO2022180152A1 (fr) Lymphocyte t régulateur modifié
WO2024261480A1 (fr) Récepteurs de cytokines constitutifs
WO2023099886A1 (fr) Protéine de signalisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24711920

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2024711920

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024711920

Country of ref document: EP

Effective date: 20251020

WWE Wipo information: entry into national phase

Ref document number: CN2024800335420

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 2024711920

Country of ref document: EP

Effective date: 20251020

ENP Entry into the national phase

Ref document number: 2024711920

Country of ref document: EP

Effective date: 20251020

ENP Entry into the national phase

Ref document number: 2024711920

Country of ref document: EP

Effective date: 20251020

ENP Entry into the national phase

Ref document number: 2024711920

Country of ref document: EP

Effective date: 20251020

ENP Entry into the national phase

Ref document number: 2024711920

Country of ref document: EP

Effective date: 20251020

WWP Wipo information: published in national office

Ref document number: 2024711920

Country of ref document: EP