WO2021205173A1 - Method - Google Patents

Method Download PDF

Info

Publication number
WO2021205173A1
WO2021205173A1 PCT/GB2021/050863 GB2021050863W WO2021205173A1 WO 2021205173 A1 WO2021205173 A1 WO 2021205173A1 GB 2021050863 W GB2021050863 W GB 2021050863W WO 2021205173 A1 WO2021205173 A1 WO 2021205173A1
Authority
WO
WIPO (PCT)
Prior art keywords
calcineurin
cells
car
seq
cell
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/GB2021/050863
Other languages
English (en)
French (fr)
Inventor
Martin PULÉ
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.)
Autolus Ltd
Original Assignee
Autolus Ltd
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
Application filed by Autolus Ltd filed Critical Autolus Ltd
Priority to US17/915,737 priority Critical patent/US20230338534A1/en
Priority to EP21719220.2A priority patent/EP4132567A1/en
Publication of WO2021205173A1 publication Critical patent/WO2021205173A1/en
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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • 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
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • 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
    • 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
    • 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
    • A61K40/418—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A61K40/4252—Phosphatases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4271—Melanoma antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10002—Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03016—Phosphoprotein phosphatase (3.1.3.16), i.e. calcineurin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]
    • 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
    • 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]
    • A61K2239/23—On/off switch
    • A61K2239/25—Suicide switch
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0087—Purging against subsets of blood cells, e.g. purging alloreactive T cells

Definitions

  • the invention relates to methods using cells which co-express a chimeric antigen receptor (CAR) and a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • CAR chimeric antigen receptor
  • the invention provides a method for treating a disease in a subject, which comprises the step of administering such cells to a subject.
  • the subject may be receiving or have received treatment with a calcineurin inhibitor.
  • the CAR-expressing cells may be administered prior to, following, simultaneously with or in combination with a calcineurin inhibitor.
  • HSCT hematopoietic stem cell transplants
  • GVHD graft-vs-host disease
  • Alloreactive T-cells that recognise mismatches HLA via their T-cell receptor (TOR) are major mediators of rejection and GVHD.
  • CD8+ T cell specificity is dictated by the clonotypic TOR which recognises short antigenic peptides presented on MHC class I molecules.
  • MHC class I molecules are non-covalent heterodimers made up of the membrane-integral, highly polymorphic a-chain and the non-membrane attached non- polymorphic b2 microglobulin ( ⁇ 2m ).
  • Adoptive cell therapy involves administrating disease-relevant immune cells to a subject.
  • ACT may involve administering immune cells with direct anticancer activity.
  • ACT using naturally occurring tumour-reactive lymphocytes has mediated durable, complete regressions in patients with melanoma and has also been used in the treatment of epithelial cancers.
  • the ability to genetically engineer lymphocytes to express conventional T cell receptors (TCRs) or chimeric antigen receptors (CARs) has further extended the successful application of ACT for cancer treatment. Graft rejection by the host is also a problem for ACT, particularly for CAR-T cells.
  • CARs are artificial proteins which are typically composed of a targeting domain, a spacer domain, a transmembrane domain and a signaling domain.
  • the targeting domain is typically derived from an scFv which may be murine. While this scFv can be human or humanized and other components individually are derived from self proteins, the junctions between them can still be immunogenic. For instance, within the scFv there are junctions between the heavy chain and the linker and the linker and the light chain. There is then a junction between the scFv and the spacer domain. If the transmembrane domain is not continuous with the spacer there is a further junction there. Similarly, if the transmembrane domain is not continuous with the amino-terminal portion of the endodomain, there is a further junction there. Finally, most endodomains have at least two components and sometimes more with junctions subsequently between each component.
  • CAR T-cells are often engineered with further components.
  • these components include suicide genes (e.g. the HSV-TK enzyme). This enzyme was found to be highly immunogenic and caused a cellular immune depletion of CAR T- cells outside of the context of the profound immunosuppression of haploidentical haematopoietic stem cell transplantation.
  • Other less immunogenic suicide genes may still provide some immunogenicity, as almost every kind of engineered component which involves a fusion between two proteins or use of a xenogeneic protein can be immunogenic.
  • CAR T-cell therapy An important concern in CAR T-cell therapy is the production of antibodies and CTLs against these non-natural components which may result in CAR T-cell rejection.
  • Several studies using murine CAR scFvs have reported that anti-murine scFv CAR antibodies and CTLs are generated after CAR T-cell infusion.
  • the problem of CAR-T cell rejection is particularly acute where the treatment involves multiple rounds of CAR T cell reinfusion.
  • CAR T-cells have been primarily generated from autologous T-cells.
  • T-cells from an allogeneic donor are used. This can occur if for instance the patient has had an allogeneic haematopoietic stem cell transplant. In this case, harvested T-cells will be allogeneic. Otherwise, a patient may have insufficient T-cells to generate a CAR T-cell product due to chemotherapy induced lymphopenia.
  • Rejection of allogeneic cells can be due to minor mismatch or major mismatch.
  • Minor mismatch occurs in the setting where allogeneic T-cells are human leukocyte antigen (HLA)-matched to the recipient.
  • HLA human leukocyte antigen
  • rejection occurs due to minor histocompatibility antigens which are non-HLA differences between individuals which result in presentation of non-self (donor) epitopes / immunogeneic peptides on HLA.
  • donor and recipient are mismatched, or are only partially matched.
  • T-cell receptors (TCR) on endogenous T-cells of a recipient can interact in a non specific way with a mismatched HLA and cause rejection consequently. Both minor and major forms of allogeneic rejection are caused by HLA interacting with TCR.
  • ACT has multiple advantages compared with other forms of cancer immunotherapy which rely on the active in vivo development of sufficient numbers of anti-tumour cells with the function necessary to mediate cancer regression.
  • large numbers of antitumor lymphocytes up to 10 11
  • In vitro activation allows such cells to be released from the inhibitory factors that exist in vivo.
  • ACT enables the manipulation of the host before cell transfer to provide a favourable microenvironment that better supports antitumor immunity.
  • preconditioning a patient with one or more immunosuppressive chemotherapy drugs prior to T cell infusion can increase the effectiveness of the transplanted T cells.
  • patients may receive cyclophosphamide and fludarabine as preconditioning to decrease immunosuppressive cells prior to T cell infusion.
  • Pre-conditioning patients prior to T cell therapies with cyclophosphamide and fludarabine improves the efficacy of the T cell therapy by reducing the number of endogenous lymphocytes and increasing the serum level of homeostatic cytokines and/or pro-immune factors present in the patient.
  • Fludarabine is a nucleoside analog which induces cellular cytotoxicity via multiple pathways that ultimately lead to an inhibition of DNA synthesis.
  • a rate-limiting step in this process is the activity of deoxycytidine kinase, which is abundant in lymphocytes, making them susceptible to accumulation of F-ara-ATP, the active metabolite of fludarabine, and hence giving fludarabine particularly potent lymphodepleting properties.
  • fludarabine A disadvantage of using fludarabine is the associated myelosuppression which can be profound and sometimes even fatal. Fludarabine also has known associations with neurotoxicity. In a recent clinical trial using CAR-T cells engineered to target the CD19 B-cell antigen to treat acute lymphoblastic leukemia, JCAR-015 (NCT02535364), two patient deaths due to cerebral edema led to trial suspension and fludarabine was suggested as the causative agent.
  • Calcineurin inhibitors are also commonly used in the treatment of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.
  • CNIs Commonly used calcineurin inhibitors
  • CsA cyclosporine A
  • tacrolimus also known as FK506
  • Cyclosporine is a lipophilic cyclic peptide of 11 amino acids
  • tacrolimus is a macrolide antibiotic. Both drugs have been isolated from fungi and possess similar suppressive effects on cell-mediated and humoral immune responses.
  • Calcineurin is formed by two subunits: A, which is a catalytic subunit (CnA) responsible for its phosphatase activity, and B, a regulatory subunit (CnB) that is responsive to intracellular calcium and regulates CnA activation.
  • A catalytic subunit
  • CnB regulatory subunit
  • T cell activation through TCR stimulation elevates intracellular calcium concentration and activates CnB, which unleashes the phosphatase activity of CnA.
  • Activated CnA dephosphorylates cytoplasmic NFATc, a transcription factor, which causes its translocation, along with the activated calcineurin, into the nucleus where it upregulates the expression of multiple cytokines and costimulatory molecules necessary for full activation of T cells.
  • NFAT1, NFAT2, and NFAT4 are involved in the transcriptional activation of genes encoding cytokines, including IL-2 and IL-4, and CD40 ligand. Production of IL-2, in particular, stimulates the growth and differentiation of T cells.
  • CNI post-transplant lymphoproliferative disorders
  • melanomas melanomas
  • FIG. 1 The role of calcineurin in T cell activation.
  • TOR recognition of the alloantigen leads to an increase in the intracellular calcium concentration of T cells (step 2), activating CnB (step 3). Once activated, the CnB unleashes the phosphatase activity of CnA (step 4).
  • Activated CnA dephosphorylates cytoplasmic NFATc (step 5), a transcription factor, allowing for its translocation with activated calcineurin into the nucleus (step 6) where it upregulates the expression of multiple cytokines and costimulatory molecules necessary for full activation of T cells (step 7).
  • Generated IL-2 binds to the IL-2 receptors and induces cell activation and proliferation (step 8).
  • Figure 2 Graphs to show the (A) percentage and (B) number of CAR-expressing (RQR8-positive) cells proliferating after 96 hours co-culture with Jurkat KO, Jurkat TRBC1 and Jurkat TRBC2 target cells, in the absence of Tacrolimus
  • Figure 3 Graphs to show the (A) percentage and (B) number of CAR-expressing (RQR8-positive) cells proliferating after 96 hours co-culture with Jurkat KO, Jurkat TRBC1 and Jurkat TRBC2 target cells, in the presence of 20ng/ml of Tacrolimus.
  • Figure 4 Graphs to show the number of CAR-expressing (RQR8-positive) cells in each division following co-culture with Jurkat KO, Jurkat TRBC1 and Jurkat TRBC2 target cells, in the absence of Tacrolimus. Proliferation analysis was calculated on single/live/CellTrace Violet -positive cells using FlowJoTM proliferation tool and the CD19 CAR used as the negative control for all the conditions. Cell number in each division is plotted for each CAR + target combination.
  • Figure 5 Graphs to show the number of CAR-expressing (RQR8-positive) cells in each division following co-culture with Jurkat KO, Jurkat TRBC1 and Jurkat TRBC2 target cells, in the presence of 20ng/ml of Tacrolimus. Proliferation analysis was calculated on single/live/CellTrace Violet -positive cells using FlowJoTM proliferation tool and the CD 19 CAR used as the negative control for all the conditions. Cell number in each division is plotted for each CAR + target combination.
  • Figure 6 Histogram plots showing the proliferation of CAR-expressing (RQR8- positive) cells following co-culture with Jurkat KO, Jurkat TRBC1 and Jurkat TRBC2 target cells, with or without the addition of 20ng/ml of Tacrolimus. Proliferation analysis was calculated on single/live/CellTrace Violet -positive cells using FlowJoTM proliferation tool and the CD19 CAR used as the negative control for all the conditions. Results are shown using cells from two separate donors.
  • Figure 7 Graph showing the cell count of non-transduced cells (NT) and TRBC2 CAR-expressing (RQR8-positive) cells before (day 0) and after (day 4) co-culture with TRBC2 targets with or without the addition of 20ng/ml of Tacrolimus.
  • Figure 8 Graph showing the percentage of TRBC2 CAR-expressing (RQR8-positive) cells before (day 0) and after (day 4) co-culture with TRBC2 targets with or without the addition of 20ng/ml of Tacrolimus.
  • the present invention relates to a new approach which facilitates the simultaneous use of calcineurin inhibitors (CNIs) and adoptive T-cell therapy, such as the use of CAR-T cells.
  • CNIs calcineurin inhibitors
  • This approach has a variety of different clinical applications, for example: it enables a patient to be preconditioned with a CNI prior to adoptive cell therapy, thereby potentially avoiding the use of alternative preconditioning agents such as fludarabine which may have undesirable side effects; it enables CNIs to be used to prevent rejection of the grafted T cells by the host, which is particularly useful for allogeneic grafted T cells and therapies involving repeated doses of engineered T cells; and it enables adoptive T-cell therapies to be used on a patient who is already being treated with a CNI, for example following transplant or to treat and/or prevent autoimmune disease.
  • alternative preconditioning agents such as fludarabine which may have undesirable side effects
  • CNIs to be used to prevent rejection of the grafted T cells by the host, which is particularly useful for allogeneic grafted T cells and therapies involving repeated doses of engineered T cells
  • adoptive T-cell therapies to be used on a patient who is already being treated with a CNI, for example following transplant or to
  • the invention provides a method for treating and/or preventing a disease in a subject who is receiving or has received treatment with a calcineurin inhibitor, which method comprises the step of administering to the subject a plurality of cells which express:
  • CAR chimeric antigen receptor
  • the invention also provides a plurality of cells for use in a method of treating and/or preventing a disease in a subject who is receiving or has received treatment with a calcineurin inhibitor, wherein the plurality of cells express:
  • CAR chimeric antigen receptor
  • a plurality of cells in the manufacture of a pharmaceutical composition for use in the a method of treating and/or preventing a disease in a subject who is receiving or has received treatment with a calcineurin inhibitor, wherein the plurality of cells express:
  • CAR chimeric antigen receptor
  • the subject may be receiving or have received treatment with a calcineurin inhibitor in order to treat and/or prevent an autoimmune disease.
  • the CAR may bind an autoantigen associated with the autoimmune disease.
  • the subject may be post-transplant and be receiving or have received treatment with a calcineurin inhibitor in order to prevent transplant rejection.
  • the CAR may binds a cancer antigen.
  • the method may be to treat and/or prevent a transplant-associated cancer such as post-transplant lymphoproliferative disorder (PTLD) or a melanoma.
  • PTLD post-transplant lymphoproliferative disorder
  • the invention provides a method for treating a disease in a subject, which comprises the following steps:
  • CAR chimeric antigen receptor
  • the invention also provides a plurality of cells for use in a method of treating a disease in a subject, wherein the plurality of cells express:
  • CAR chimeric antigen receptor
  • CAR chimeric antigen receptor
  • the CAR-expressing cells may be allogeneic.
  • the method may involve administering a plurality of doses of the CAR-expressing cells to the subject.
  • the calcineurin inhibitor may be selected from cyclosporine A (CsA), and tacrolimus.
  • the CAR may bind to a B-cell antigen or a plasma cell antigen.
  • the CAR may bind CD19, CD20, CD22 or BCMA.
  • the CAR may bind CD19 and have a CD19-binding domain which comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the cells may express one of the following mutant versions of calcineurin A and/or calcineurin B: calcineurin A comprising mutations T351E and L354A with reference to the shown as SEQ ID No. 30; calcineurin A comprising mutations V314R and Y341F and with reference to shown as SEQ ID No 30; or calcineurin B comprising mutation L124T and K-125-LA-lns with reference to shown as SEQ ID No. 31.
  • the cells may express mutant calcineurin B having the sequence shown as SEQ ID No. 32.
  • a cell which expresses a chimeric antigen receptor (CAR) and a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • CAR chimeric antigen receptor
  • CAR comprises a CD19-binding domain which comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:
  • a nucleic acid construct which comprises:
  • a vector comprising a nucleic acid construct according to paragraph 8.
  • a kit of vectors comprising:
  • a second vector comprising a nucleic acid sequence which encodes a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • a pharmaceutical composition comprising a plurality of cells according to any of paragraphs 1 to 7.
  • a pharmaceutical composition according to paragraph 11 which also comprises a calcineurin inhibitor.
  • calcineurin inhibitor is selected from cyclosporine A (CsA), and tacrolimus.
  • a method for treating and/or preventing a disease which comprises the step of administering a pharmaceutical composition according to any of paragraphs 11 to 13 to a subject.
  • step (ii) involves regular administration of the calcineurin inhibitor to the subject for a period of about 6 weeks following administration of the cells.
  • a pharmaceutical composition according to any of paragraphs 11 to 13 for use in treating a disease is provided.
  • step (iii) adding a calcineurin inhibitor to the cell population from step (ii) in order to enrich for transduced/transfected cells.
  • the present invention relates to cells which express one or more chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • Classical CARs are chimeric type I trans-membrane proteins which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain).
  • the binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site (such as a Fab) or on a ligand for the target antigen.
  • scFv single-chain variable fragment
  • mAb monoclonal antibody
  • a spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation.
  • a common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen.
  • a trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.
  • TNF receptor family endodomains such as the closely related 0X40 and 4-1 BB which transmit survival signals.
  • CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.
  • CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors.
  • retroviral vectors In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer.
  • the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on.
  • the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.
  • the antigen-binding domain is the portion of a CAR which recognizes antigen.
  • the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a wild-type ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • a single domain binder such as a camelid
  • an artificial binder single as a Darpin or a single-chain derived from a T-cell receptor.
  • TAA tumour associated antigens
  • the antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.
  • the CAR may bind a B-cell antigen or a plasma cell antigen.
  • the CAR may bind CD19, CD20, CD22 or BCMA
  • CD19-targeting CAR is described in WO2016/139487.
  • This CAR comprises a CD19-binding domain which comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:
  • CDR3 - SLLYGDYLDY (SEQ ID No. 3); and b) a light chain variable region (VL) having CDRs with the following sequences: CDR1 - SASSSVSYMH (SEQ ID No. 4);
  • the CDRs may be in the format of a single-chain variable fragment (scFv), which is a fusion protein of the heavy variable region (VH) and light chain variable region (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids.
  • the scFv may be in the orientation VH-VL, i.e. the VH is at the amino-terminus of the CAR molecule and the VL domain is linked to the spacer and, in turn the transmembrane domain and endodomain.
  • the CDRs may be grafted on to the framework of a human antibody or scFv.
  • the CAR may comprise the following VH sequence:
  • the CAR may comprise the following VL sequence:
  • the CAR may comprise the following scFv sequence:
  • the CAR of the invention may comprise a variant of the sequence shown as SEQ ID No. 7, 8 or 9 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD19 (when in conjunction with a complementary VL or VH domain, if appropriate).
  • the percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http://blast.ncbi.nlm.nih.gov.
  • the CAR may comprise the sequence shown as SEQ ID No. 10, which comprises:
  • T-cell receptor beta chain V region signal peptide (underlined)
  • the CAR of the invention may comprise a variant of the sequence shown as SEQ ID No. 10 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD19 and induce a T-cell activation signal.
  • a CD22-targeting CAR is described in WO2019/220109.
  • This CAR comprises a CD22-binding domain which comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:
  • CDR3 CDR3 - QRNYYDGSYDYEGYTMDA (SEQ ID No. 13); and b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the following sequences:
  • the CAR may comprise the following VH sequence:
  • the CAR may comprise the following VL sequence:
  • the CAR may comprise a variant of the sequence shown as SEQ ID No. 17 to 19 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD22 (when in conjunction with a complementary VL or VH domain, if appropriate).
  • the CAR may comprise the sequence shown as SEQ ID No. 20, which comprises:
  • the CAR may comprise a variant of the sequence shown as SEQ ID No. 20 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD22 and induce a T-cell activation signal.
  • a BCMA-targeting CAR is described in W02020/065330.
  • This CAR comprises a BCMA-binding domain which comprises a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1 - GFIFSDYN (SEQ ID No. 21)
  • CDR3 - ATRPGPFAY SEQ ID No. 23
  • VL light chain variable region having complementarity determining regions
  • the CAR may comprise the following VL sequence:
  • the CAR may comprise a variant of the sequence shown as SEQ ID No. 27 and/or 28 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind CD22 (when in conjunction with a complementary VL orVH domain, if appropriate).
  • the ant-BCMA CAR may be a FabCAR.
  • the CAR may comprise the sequence shown as SEQ ID No. 29, which comprises:
  • Murine Ig kappa signal peptide underlined
  • anti-BCMA VL sequence shown above as SEQ ID No.28
  • Murine Ig heavy chain V region 102 signal peptide shown in grey and underlined
  • anti-BCMA VH sequence shown above as SEQ ID No.27; in grey
  • the CAR comprise a variant of the sequence shown as SEQ ID No. 29 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence retain the capacity to bind BCMA and induce a T-cell activation signal.
  • the CAR may target an autoantigen.
  • the CAR may target....
  • the target antigen may be expressed on a solid cancer.
  • the target may be PSMA for the treatment of prostate cancer.
  • the CAR-expressing cell may target a cell which is not a T-cell, such as a B-cell, plasma cell or cancerous cell of non-T-cell origin such a malignant epithelial cell.
  • the CAR target antigen may not form part of the T-cell receptor complex.
  • the target antigen may be an antigen other than TRBC1 or TRBC2.
  • the transmembrane domain is the sequence of a CAR that spans the membrane. It may comprise a hydrophobic alpha helix.
  • the transmembrane domain may be derived from any transmembrane protein such as from the melanocyte protein Tyrpl, or from CD8 or CD28.
  • the CAR for use in the present invention may comprise a signal peptide so that when it is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • the CAR may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain.
  • a flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
  • the spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk.
  • the spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk.
  • a human lgG1 spacer may be altered to remove Fc binding motifs.
  • the endodomain is the signal-transmission portion of the CAR. It may be part of or associate with the intracellular domain of the CAR. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell.
  • the most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed. Co-stimulatory signals promote T-cell proliferation and survival.
  • co-stimulatory signals There are two main types of co-stimulatory signals: those that belong the Ig family (CD28, ICOS) and the TNF family (0X40, 41 BB, CD27, GITR etc).
  • CD28, ICOS the Ig family
  • TNF family the TNF family
  • chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together.
  • the endodomain may comprise:
  • an ITAM-containing endodomain such as the endodomain from CD3 zeta;
  • a co-stimulatory domain such as the endodomain from CD28 or ICOS;
  • a domain which transmits a survival signal for example a TNF receptor family endodomain such as OX-40, 4-1 BB, CD27 or GITR.
  • the CAR of the present invention may therefore comprise an antigen-binding component comprising an antigen-binding domain and a transmembrane domain; which is capable of interacting with a separate intracellular signalling component comprising a signalling domain.
  • the vector of the invention may express a CAR signalling system comprising such an antigen-binding component and intracellular signalling component.
  • the cells of the present invention co-express a CAR and a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • Calcineurin is a calcium and calmodulin dependent serine/threonine protein phosphatase involved in T-cell activation. Calcineurin is a heterodimer of a 61-kD calmodulin-binding catalytic subunit, calcineurin A (CnA) and a 19-kD Ca 2+ -binding regulatory subunit, calcineurin B (CnB).
  • T-cell receptor TCR
  • CnB T-cell receptor
  • CnA phosphatase activity of CnA
  • Activated CnA dephosphorylates cytoplasmic NFATc, a transcription factor, allowing for its translocation with activated calcineurin into the nucleus where it upregulates the expression of multiple cytokines (including IL2) and costimulatory molecules necessary for full activation of T cells.
  • Calcineurin is the target of a class of drugs called calcineurin inhibitors, which include cyclosporin, voclosporin, pimecrolimus and tacrolimus.
  • the calcineurin inhibitor may be cyclosporine A (CsA) or tacrolimus (also known as FK506).
  • Cyclosporine is a lipophilic cyclic peptide of 11 amino acids, while tacrolimus is a macrolide antibiotic. Both drugs have been isolated from fungi and possess similar suppressive effects on cell-mediated and humoral immune responses.
  • CNIs bind intracellular proteins called immunophilins: cyclophilins in the case of cyclosporine, and the FK-binding proteins in the case of tacrolimus.
  • the drug- receptor complex specifically and competitively binds to and inhibits calcineurin, leading to reduced transcriptional activation of cytokine genes and reduction in proliferation of T lymphocytes.
  • Cyclosporine and tacrolimus are the most commonly used CNIs in transplant recipients. Cyclosporine was initially approved in 1983 by the U.S. Food and Drug Administration (FDA) for immunosuppression following organ transplantation, and in 1995 a microemulsion formulation of cyclosporine (associated with better bioavailability and more consistent absorption) was approved. Cyclosporine formulations are usually administered twice daily. Tacrolimus received FDA approval in 1994 for liver transplant recipients, and in 1997 for kidney transplants. Tacrolimus is usually administered twice daily, but recently became available as an extended release once-daily formulation. FDA-approved generic equivalents are available for tacrolimus immediate release formulations, as well as modified and unmodified cyclosporine.
  • FDA U.S. Food and Drug Administration
  • the cell of the present invention may express calcineurin A and/or B comprising one or more mutations which increases its resistance to one or more immune suppressive drugs.
  • the cell may comprise one or more mutations which renders the cell resistant to tacrolimus and/or cyclosporin.
  • the cell may comprise a nucleic acid sequence encoding calcineurin (CN) with one or more mutations.
  • CN calcineurin
  • calcineurin is a heterodimer of a 61-kD calmodulin-binding catalytic subunit, calcineurin A and a 19-kD Ca2+-binding regulatory subunit, calcineurin B.
  • amino acid sequence for calcineurin A, alpha isoform is shown below as SEQ ID No. 30.
  • SEQ ID No. 30 (calcineurin A) MSEPKAIDPKLSTTDRWKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLMKEGRLE
  • Mutant calcineurin A may comprise a mutation at one or more of the following positions with reference to SEQ ID No. 30: V314; Y341; M347; T351; W352; S353; : L354; F356; and K360.
  • Mutant calcineurin A may comprise one or more of the following substitution mutations with reference to SEQ ID No. 30:
  • Mutant calcineurin A may comprise one or more of the following mutation combinations with reference to SEQ ID No. 30:
  • Mutant calcineurin B may comprise a mutation at one or more of the following positions with reference to SEQ ID No. 31: Q51; L116; M119; V120; G121; N122; N123; L124; K125; and K165.
  • Mutant calcineurin B may comprise one or more of the following substitution and optionally insertion mutations with reference to SEQ ID No. 31:
  • Mutant calcineurin B may comprise one or more of the following mutation combinations with reference to SEQ ID No. 31:
  • V120D and K125-LA-lns V120D and K125-LA-lns; and M119-F-lns and G121-LF-lns.
  • mutant calcineurin B may comprise the following mutation combination with reference to SEQ ID No. 31: L124T and K125-LA-lns. This is the module known as "CnB30" described in the Examples section.
  • the CnB30 has the amino acid shown as SEQ ID No. 32.
  • RVIDIFDTDGNG EVDFKEFIEGVSQFSVKGDKEQKLRFAFRIYDMDKDG
  • K-125-LA-lns K-125-LA-lns; and L124T and K-125-LA-lns.
  • the combination mutation T351E and L354A in CNa confers resistance to CsA but not FK506
  • the combination mutation V314R and Y341F in CNa confers resistance to FK506 but not CsA
  • the combination mutation L124T and K-125-LA-lns in CNb renders CTLs resistant to both calcineurin inhibitors.
  • the cell of the present invention may express a variant calcineurin A comprising one or more mutations in the CNa amino acid sequence and/or a variant calcineurin B comprising one or more mutations in the CNb amino acid sequence, which increases resistance of the effector immune cell to one or more calcineurin inhibitors.
  • the cell may express a variant calcineurin A and/or a variant calcineurin B as listed above which confers resistance to cyclosporin A and/or tacrolimus (FK506).
  • the invention provides a nucleic acid construct which comprises:
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
  • polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
  • the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular an immune effector cell as described herein.
  • Nucleic acids according to the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variant in relation to a nucleotide sequence or amino acid sequence includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.
  • the nucleic acid construct may have the general structure:
  • CAR is a nucleic acid sequence encoding a CAR
  • coexpr is a nucleic acid sequence enabling co-expression og the CAR and calcineurin mutant as separate polypeptdes;
  • CnM is a nucleic acid sequence which encodes a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • the co-expression site may be a cleavage site.
  • the cleavage site may be any sequence which enables the two polypeptides to become separated.
  • the cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
  • the term “cleavage” is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage.
  • FMDV Foot-and-Mouth disease virus
  • various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041).
  • the exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.
  • the cleavage site may be a furin cleavage site.
  • Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products.
  • Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor.
  • Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg') and is enriched in the Golgi apparatus.
  • the cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.
  • TSV Tobacco Etch Virus
  • TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin- like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo.
  • the consensus TEV cleavage site is ENLYFQ ⁇ S (where ‘V denotes the cleaved peptide bond).
  • Mammalian cells such as human cells, do not express TEV protease.
  • the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.
  • the cleavage site may encode a self-cleaving peptide.
  • a ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.
  • the self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
  • the primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus.
  • apthoviruses such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus
  • the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C-terminus (Donelly et al (2001) as above).
  • 2A-like sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al., 2001) as above.
  • the co-expression sequence may be an internal ribosome entry sequence (IRES).
  • the co-expressing sequence may be an internal promoter.
  • the present invention also provides a kit comprising of nucleic acid sequences which comprises: a first polynucleotide which encodes a chimeric antigen receptor (CAR); and a second polynucleotide which encodes which encodes a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • CAR chimeric antigen receptor
  • the present invention also provides a vector which comprises a nucleic acid construct(s) of the invention.
  • the invention also provides a kit of vectors comprising:
  • a second vector comprising a nucleic acid sequence which encodes a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • a vector or kit of vectors may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it co-expresses a CAR together with a mutant version of calcineurin A and/or calcineurin B.
  • the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.
  • the vector may be capable of transfecting or transducing a cell.
  • the present invention provides a cell which expresses a chimeric antigen receptor (CAR) and a mutant version of calcineurin A and/or calcineurin B which is resistant to a calcineurin inhibitor.
  • CAR chimeric antigen receptor
  • the cell may comprise a nucleic acid sequence, a nucleic acid construct or a vector of the present invention.
  • the cell may be a cytolytic immune cell such as a T cell or an NK cell.
  • T cells or T lymphocytes are a type of lymphocyte that play a central role in cell- mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface.
  • TCR T-cell receptor
  • Helper T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • TH cells express CD4 on their surface.
  • TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.
  • Cytolytic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection.
  • CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells.
  • MHC class I MHC class I
  • IL-10 adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.
  • Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections.
  • Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.
  • Treg cells Regulatory T cells
  • suppressor T cells are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell- mediated immunity toward the end of an immune reaction and to suppress auto reactive T cells that escaped the process of negative selection in the thymus.
  • Treg cells Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.
  • Naturally occurring Treg cells arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP.
  • Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.
  • Adaptive Treg cells may originate during a normal immune response.
  • the cell may be a Natural Killer cell (or NK cell).
  • NK cells form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.
  • LGL large granular lymphocytes
  • the cells of the invention may be any of the cell types mentioned above.
  • the present invention provides a method for making a cell, which comprises the step of introducing: a nucleic acid construct, a kit of nucleic acid sequences, a vector or a kit of vectors of the invention, into the cell.
  • the nucleic acid may be introduced to the cell ex vivo or in vitro.
  • the method may comprise the following steps:
  • step (iii) adding a calcineurin inhibitor to the cell population from step (ii).
  • step (iii) has the effect of enriching for transduced/transfected cells as cells which express the calcineurin mutant will be resistant to inhibition of proliferation by the calcineurin inhibitor, whereas untransduced cells will be susceptible to such inhibition
  • the calcineurin inhibitor may, for example, be cyclosporine or tacrolimus.
  • Cells according to the invention may either be created 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).
  • cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to, for example, T or NK cells.
  • an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.
  • chimeric polypeptide-expressing cells are generated by introducing DNA or RNA coding for the chimeric polypeptide by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • the cell of the invention may be an ex vivo cell from a subject.
  • the cell may be from a peripheral blood mononuclear cell (PBMC) sample.
  • PBMC peripheral blood mononuclear cell
  • the cells may be activated and/or expanded prior to being transduced with nucleic acid encoding the molecules providing the chimeric polypeptide according to the first aspect of the invention, for example by treatment with an anti-CD3 monoclonal antibody.
  • the present invention also relates to a pharmaceutical composition comprising a plurality of cells according to the present invention.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the pharmaceutical composition may also comprise a calcineurin inhibitor, such as cyclosporine A or tacrolimus.
  • a calcineurin inhibitor such as cyclosporine A or tacrolimus.
  • the present invention provides a method for treating and/or preventing a disease which comprises the step of administering the cells of the present invention (for example in a pharmaceutical composition as described above) to a subject.
  • a method for treating a disease relates to the therapeutic use of the cells of the present invention.
  • the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • the method for preventing a disease relates to the prophylactic use of the cells of the present invention.
  • the cells may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease.
  • the subject may have a predisposition for, or be thought to be at risk of developing, the disease.
  • the method may involve the steps of:
  • the cell-containing sample may be isolated from a subject or from other sources, as described above.
  • the plurality of cells may be administered in the form of a pharmaceutical composition.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds, for example one or more CNIs as described above.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a cell according to the present invention for use in treating and/or preventing a disease.
  • the present invention also relates to the use of a cell according to the present invention for the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the disease to be treated and/or prevented by the method of the present invention may be cancer.
  • the disease to be treated by the methods of the present invention may be a cancerous disease, such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.
  • the disease may be Multiple Myeloma (MM), B-cell Acute Lymphoblastic Leukaemia (B-ALL), Chronic Lymphocytic Leukaemia (CLL), Neuroblastoma, T-cell acute Lymphoblastic Leukaema (T-ALL) or diffuse large B-cell lymphoma (DLBCL).
  • MM Multiple Myeloma
  • B-ALL B-cell Acute Lymphoblastic Leukaemia
  • CLL Chronic Lymphocytic Leukaemia
  • Neuroblastoma T-cell acute Lymphoblastic Leukaema
  • DLBCL diffuse large B-cell lymphoma
  • the disease may be a plasma cell disorder such as plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain diseases, monoclonal gammopathy of undetermined significance or smoldering multiple myeloma.
  • plasmacytoma plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain diseases, monoclonal gammopathy of undetermined significance or smoldering multiple myeloma.
  • the cell of the present invention may be capable of killing target cells, such as cancer cells.
  • the target cell may be recognisable by expression of a TAA, for example the expression of a TAA provided above in Table 1.
  • the cancer may be a cancer listed in Table 1.
  • the disease may be a transplant-associated cancer, such as post-transplant lymphoproliferative disorder (PTLD) or a melanoma.
  • PTLD post-transplant lymphoproliferative disorder
  • melanoma a transplant-associated cancer, such as post-transplant lymphoproliferative disorder (PTLD) or a melanoma.
  • the cell of the present invention may be capable of killing a cell implicated in the pathogenesis of an autoimmune disease.
  • a cell may be a B-cell which secretes auto-antibodies.
  • the autoimmune disease may be selected from rheumatoid arthritis, systemic lupus erythematosus, Sjogren's Syndrome, psoriasis, Experimental Autoimmune Encephalomyelitis (EAE), uveitis, pyoderma gangrenosum, membranous nephopathy, atopic dermatitis, Bechet's disease, refractory ulcerative colitis and chronic autoimmune urticara.
  • EAE Experimental Autoimmune Encephalomyelitis
  • the administration of a cell according to the present invention can be accomplished using any of a variety of routes, such as intraperitoneally, intravenously, subcutaneously, transcutaneously or intramuscularly.
  • the present invention provides a method which comprises the following steps:
  • Step (ii) subsequently administering a calcineurin inhibitor to the subject.
  • Step (ii) may involve regular administration of the calcineurin inhibitor to the subject for a period of about 6 weeks following administration of the cells.
  • the calcineurin inhibitor may be selected from cyclosporine A (CsA), and tacrolimus.
  • CsA cyclosporine A
  • tacrolimus The calcineurin inhibitor may be administered orally to the subject.
  • Tacrolimus is widely used for the prevention of transplant rejection
  • the present invention also provides a method for treating a disease in a subject who is receiving or has received treatment with a calcineurin inhibitor, which method comprises the step of administering to the subject a plurality of cells which express:
  • CAR chimeric antigen receptor
  • the subject may, for example, be receiving or have received treatment with a calcineurin inhibitor in order to treat and/or prevent an autoimmune disease or to prevent transplant rejection.
  • a calcineurin inhibitor in order to treat and/or prevent an autoimmune disease or to prevent transplant rejection.
  • Such a subject would normally be unsuitable for treatment with CAR-T cells, but may be treated with the cells of the invention which are resistant to immunosuppression with calcineurin inhibitors.
  • the patient may have recently received a transplanted organ, such as a kidney, heart, liver or other organ.
  • a transplanted organ such as a kidney, heart, liver or other organ.
  • Tacrolimus is routinely given for the prevention of rejection of a transplanted organ and is available is various formulations including capsules (Adoport®, Prograf®, Capexion®, Tacni® and Vivadex®); modified-release capsules (Advagraf®); Granules (Modigraf®): and liquid medicine.
  • Tacrolimus capsules are usually given twice each day, once in the morning and once in the evening; whereas modified-release capsules are given once each day.
  • the starting dose for capsules is usually between 0.05-0.2 mg/kg/day of body weight for adults (divided into 2 doses and taken every 12 hours) and 0.15-0.3 mg/kg/day of body weight for children (again, divided into 2 doses and taken every 12 hours).
  • the dose is usually between 0.15 to 0.2 mg/kg of body weight once a day before reperfusion (return of blood flow to the blocked organ) or within 48 hours after transplant.
  • the dose may be 0.3 g/kg of body weight given once a day within 24 hours of reperfusion.
  • Cyclosporine is available as an oral capsule, an oral solution and an injectable form. Cyclosporine oral capsule is used to treat inflammation in rheumatoid arthritis and psoriasis and also used to prevent the rejection of an organ transplant. There are various brand-name oral capsule versions of cyclosporine, including GengrafTM, NeoralTM, and SandimmuneTM.
  • cyclosporine may be taken at a dosage of between 2.5 to 4 mg/kg/day divided into two doses.
  • cyclosporine may be given at 10-15 mg/kg per day, with the first dose taken 4-12 hours following transplant. The same dosage may be taken for 1-2 weeks following transplant and then reduced by 5-10% to a maintenance dosage of 5-10 mg/kg/day.
  • the subject may have received treatment with a calcineurin inhibitor for days, weeks, months or even years prior to treatment with the CAR-expressing cells of the invention.
  • the present invention also provides a method which comprises the following steps:
  • CAR chimeric antigen receptor
  • the patient may be pre-conditioned with a composition or regimen which includes one or more other agents, such as cyclophosphamide.
  • Cyclophosphamide may, for example, be given at a dose of 300-500 mg/m 2 for 2 or 3 days, ending 2 to 4 days before administration of CAR-expressing cells.
  • the preconditioning composition or regimen may lack fludarabine.
  • the methods of the present invention are particularly suited to treatment approaches using allogeneic cells.
  • Allogeneic CAR-T therapies developed from cells of healthy donors, that could be available for patients immediately in a manner similar to other ‘off-the-shelf drugs. Allogeneic therapies could be envisioned to serve both as the primary therapy for a disease, once the desired patient efficacy is achieved, or as an additional line of treatment before they are ready for an autologous therapy, if efficacy in certain cases is limited.
  • allogeneic CAR-T therapies enable traditional economies of scale - compared to scale out for autologous therapies - through expansion of batch sizes and increased productivity, making it possible to reach a larger patient population sooner.
  • Clinical trials of allogeneic CAR-T therapies are continuing to increase with approximately 20 therapies currently in the early stages of development.
  • Rejection of allogeneic cells can be due to minor mismatch or major mismatch both of which are caused by HLA interacting with TCR.
  • calcineurin inhibitors such as cyclosporine and tacrolimus may be used to prevent rejection of allogeneic CAR-expressing cells by alloreactive host T-cells.
  • the CAR-expressing cells of the present invention express mutant calcineurin, the allogeneic CAR-T cells will be resistant to immunosuppression by treatment of the patient with e.g. tacrolimus whereas the alloreactive host T-cells will be inhibited.
  • Alloreactive responses are a particular problem for CAR-type therapies which involve multiple rounds of administration of CAR-T cells presumably due to triggering of a memory immune response.
  • the method of the invention provides an approach to avoid stimulation of such an immune response by selective immunosuppression of the host T-cells, precluding the generation of such a response.
  • the method of the invention may involve administering a plurality of doses of the CAR-expressing cells to a subject, in particular, it may involve administration of a plurality of doses of allogeneic CAR-expressing cells.
  • Example 1 Expression of a calcineurin mutant by CAR-T cells gives resistance to inhibition of proliferation by a calcineurin inhibitor
  • PBMCs were transduced with a vector expressing a CAR together with the sort- suicide gene RQR8 which is described in WO2013/153391.
  • the CARs tested are summarised below:
  • CD19 CAR A second generation CAR having an antigen binding domain derived from Fmc63, a hinge spacer and a 41BB/CD3z endodomain
  • TRBC1 CAR A second generation CAR having an antigen binding domain as described in WO2018/224844, a hinge spacer and a 41BB/CD3z endodomain
  • TRBC2 CAR A second generation CAR having an antigen binding domain as described in W02020/089644, a CD8 stalk spacer and a CD28/CD3z endodomain
  • One population of cells were transduced with a tricistronic vector expressing RQR8, the TRBC2 CAR and the CnB30 calcineurin mutant module described above having SEQ ID No. 32.
  • Transduced cells were co-cultured with one of the following target cell types:
  • Jurkat TRBC1 wild-type Jurkat cells which express TRBC1
  • Jurkat KO Jurkat cells engineered to lack TRBC1 expression
  • Jurkat TRBC2 Jurkat cells in which the TRBC1 gene is replaced by the TRBC2 gene using CRISPR-Cas9 technology so that the expression of TRBC2 is the same as that of TRBC1 on the wild-type cell.
  • Proliferation analysis was also calculated on single/live/CellTrace Violet-positive cells using the FlowJo proliferation tool using CD19 CAR as the negative control.
  • the cell number in each division was plotted for each CAR + target combination described above and the results are shown in Figures 4 (without Tacrolimus) and 5 (with tacrolimus).
  • the results for two separate donors are also shown in the histogram plots of Figure 6.
  • Tacrolimus only the cells co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant showed an increase in proliferation of effector cells following co-culture with TRBC2-expressing targets (Figure 5, bottom graph; and Figure 6).
  • Figure 8 shows the percentage of RQR8-expressing cells. While the percentage of CD19-expressing cells stayed constant, the percentage of cells expressing TRBC2 CAR increased following co-culture with TRBC2+ targets in the absence of Tacrolimus. This was true for cells expressing TRBC2 CAR alone or co-expressing TRBC2 CAR in combination with the calcineurin mutant. In the presence of Tacrolimus, the percentage of RQR8+ cells expressing TRBC2 CAR alone was reduced, showing that Tacrolimus inhibits proliferation of these cells. By contrast, the percentage of RQR8+ cells co-expressing TRBC2 CAR/CnB30 was the same as in the co-culture without Tacrolimus, indicating that these cells show resistance to calcineurin inhibition.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Rheumatology (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • External Artificial Organs (AREA)
  • Oncology (AREA)
PCT/GB2021/050863 2020-04-08 2021-04-08 Method Ceased WO2021205173A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/915,737 US20230338534A1 (en) 2020-04-08 2021-04-08 Method
EP21719220.2A EP4132567A1 (en) 2020-04-08 2021-04-08 Method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2005216.3 2020-04-08
GBGB2005216.3A GB202005216D0 (en) 2020-04-08 2020-04-08 Cell

Publications (1)

Publication Number Publication Date
WO2021205173A1 true WO2021205173A1 (en) 2021-10-14

Family

ID=70768915

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/GB2021/050863 Ceased WO2021205173A1 (en) 2020-04-08 2021-04-08 Method
PCT/GB2021/050862 Ceased WO2021205172A1 (en) 2020-04-08 2021-04-08 Cell

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/GB2021/050862 Ceased WO2021205172A1 (en) 2020-04-08 2021-04-08 Cell

Country Status (8)

Country Link
US (2) US20230148144A1 (https=)
EP (2) EP4132567A1 (https=)
JP (3) JP2023520205A (https=)
CN (1) CN115348869A (https=)
AU (1) AU2021251459A1 (https=)
CA (1) CA3174659A1 (https=)
GB (1) GB202005216D0 (https=)
WO (2) WO2021205173A1 (https=)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024027758A1 (zh) * 2022-08-03 2024-02-08 浙江大学医学院附属第一医院 一种通用型免疫效应细胞及其制备方法和应用
US12162940B2 (en) 2021-05-05 2024-12-10 Immatics Biotechnologies Gmbh BMA031 antigen binding polypeptides
US12398187B2 (en) 2019-03-05 2025-08-26 Nkarta, Inc. CD19-directed chimeric antigen receptors and uses thereof in immunotherapy
US12590148B2 (en) 2018-11-26 2026-03-31 Nkarta, Inc. Methods for the simultaneous expansion of multiple immune cell types, related compositions and uses of same in cancer immunotherapy

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115747166B (zh) * 2022-10-13 2024-12-06 上海怡豪生物科技有限公司 一种通用型car-t细胞及其制备方法与应用
EP4608856A1 (en) * 2022-10-27 2025-09-03 CARGO Therapeutics, Inc. Compositions and methods for improved immunotherapies
CN121586777A (zh) * 2023-07-26 2026-02-27 上海邦耀生物医药有限公司 用于治疗疾病的方法和组合物

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013153391A1 (en) 2012-04-13 2013-10-17 Ucl Business Plc Polypeptide useful in adoptive cell therapy
WO2015150771A1 (en) 2014-04-01 2015-10-08 Ucl Business Plc Chimeric antigen receptor (car) signalling system
WO2016030691A1 (en) 2014-08-29 2016-03-03 Ucl Business Plc Signalling system
WO2016124930A1 (en) 2015-02-05 2016-08-11 Ucl Business Plc Signalling system
WO2016139487A1 (en) 2015-03-05 2016-09-09 Ucl Business Plc Chimeric antigen receptor (car) comprising a cd19-binding domain
WO2018224844A1 (en) 2017-06-09 2018-12-13 Autolus Limited Anti trbc1 antigen binding domains
WO2019210280A1 (en) * 2018-04-27 2019-10-31 Casebia Therapeutics Limited Liability Partnership Anti-bcma car-t-cells for plasma cell depletion
WO2019220109A1 (en) 2018-05-15 2019-11-21 Autolus Limited Chimeric antigen receptor
WO2020065330A2 (en) 2018-09-27 2020-04-02 Autolus Limited Chimeric antigen receptor
WO2020089644A1 (en) 2018-10-31 2020-05-07 Autolus Limited Binding domain

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7381803B1 (en) 1992-03-27 2008-06-03 Pdl Biopharma, Inc. Humanized antibodies against CD3
WO2000063374A1 (en) 1999-04-16 2000-10-26 Celltech Therapeutics Limited Synthetic transmembrane components
CA2538141A1 (en) 2003-09-09 2005-03-17 Gpc Biotech Ag Therapeutic human anti-mhc class ii antibodies and their uses
DK3241561T3 (da) 2014-03-05 2025-06-30 Autolus Ltd Konjugeret antistof eller bispecifik t-celle-engager, som selektivt binder enten tcr-beta konstant region 1 (trbc1) eller trbc2
SG10201913782UA (en) * 2014-07-21 2020-03-30 Novartis Ag Treatment of cancer using a cll-1 chimeric antigen receptor
GB201504840D0 (en) 2015-03-23 2015-05-06 Ucl Business Plc Chimeric antigen receptor
US10294304B2 (en) * 2015-04-13 2019-05-21 Pfizer Inc. Chimeric antigen receptors targeting B-cell maturation antigen
US20200085871A1 (en) * 2017-03-17 2020-03-19 University Of Tennessee Research Foundation Methods of using cytotoxic t cells for treatment of autoimmune diseases
CN111315769A (zh) * 2017-09-06 2020-06-19 加州理工学院 信号传导和抗原呈递双功能受体(sabr)
GB201716728D0 (en) * 2017-10-12 2017-11-29 Autolus Ltd Cell
MX2021011816A (es) * 2019-03-29 2021-10-22 Univ Texas Metodos para produccion de celulas car-nk y uso de las mismas.

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013153391A1 (en) 2012-04-13 2013-10-17 Ucl Business Plc Polypeptide useful in adoptive cell therapy
WO2015150771A1 (en) 2014-04-01 2015-10-08 Ucl Business Plc Chimeric antigen receptor (car) signalling system
WO2016030691A1 (en) 2014-08-29 2016-03-03 Ucl Business Plc Signalling system
WO2016124930A1 (en) 2015-02-05 2016-08-11 Ucl Business Plc Signalling system
WO2016139487A1 (en) 2015-03-05 2016-09-09 Ucl Business Plc Chimeric antigen receptor (car) comprising a cd19-binding domain
WO2018224844A1 (en) 2017-06-09 2018-12-13 Autolus Limited Anti trbc1 antigen binding domains
WO2019210280A1 (en) * 2018-04-27 2019-10-31 Casebia Therapeutics Limited Liability Partnership Anti-bcma car-t-cells for plasma cell depletion
WO2019220109A1 (en) 2018-05-15 2019-11-21 Autolus Limited Chimeric antigen receptor
WO2020065330A2 (en) 2018-09-27 2020-04-02 Autolus Limited Chimeric antigen receptor
WO2020089644A1 (en) 2018-10-31 2020-05-07 Autolus Limited Binding domain

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Uniprot", Database accession no. Q96LZ3
BREWIN ET AL., BLOOD, vol. 114, 2009, pages 4792 - 4803
BREWIN JENNIFER ET AL: "Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 114, no. 23, 26 November 2009 (2009-11-26), pages 4792 - 4803, XP086510838, ISSN: 0006-4971, [retrieved on 20201119], DOI: 10.1182/BLOOD-2009-07-228387 *
DONNELLY ET AL., J. GEN. VIROL., vol. 82, 2001, pages 1027 - 1041

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12590148B2 (en) 2018-11-26 2026-03-31 Nkarta, Inc. Methods for the simultaneous expansion of multiple immune cell types, related compositions and uses of same in cancer immunotherapy
US12398187B2 (en) 2019-03-05 2025-08-26 Nkarta, Inc. CD19-directed chimeric antigen receptors and uses thereof in immunotherapy
US12162940B2 (en) 2021-05-05 2024-12-10 Immatics Biotechnologies Gmbh BMA031 antigen binding polypeptides
WO2024027758A1 (zh) * 2022-08-03 2024-02-08 浙江大学医学院附属第一医院 一种通用型免疫效应细胞及其制备方法和应用

Also Published As

Publication number Publication date
GB202005216D0 (en) 2020-05-20
JP2025107358A (ja) 2025-07-17
CA3174659A1 (en) 2021-10-14
US20230148144A1 (en) 2023-05-11
US20230338534A1 (en) 2023-10-26
JP2025122064A (ja) 2025-08-20
JP2023520205A (ja) 2023-05-16
CN115348869A (zh) 2022-11-15
EP4132567A1 (en) 2023-02-15
WO2021205172A1 (en) 2021-10-14
AU2021251459A1 (en) 2022-08-25
EP4132566A1 (en) 2023-02-15

Similar Documents

Publication Publication Date Title
US20230338534A1 (en) Method
US12187769B2 (en) Cell
US20220275052A1 (en) Receptor
US11903967B2 (en) Method of preparing T cells with increased activity
US10869911B2 (en) Chimeric protein
US20200239545A1 (en) Receptor
HK1243107A1 (en) Chimeric protein
US20240181056A1 (en) Chimeric antigen receptor (car)-t cells
CN111542547B (zh) 对bdca2抗原具有特异性的嵌合抗原受体
US12479903B2 (en) CD5 specific T cell receptor cell or gene therapy
US20230233606A1 (en) Cell

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: 21719220

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021719220

Country of ref document: EP

Effective date: 20221108