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WO2018229492A1 - Récepteur antigénique chimérique - Google Patents

Récepteur antigénique chimérique Download PDF

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Publication number
WO2018229492A1
WO2018229492A1 PCT/GB2018/051636 GB2018051636W WO2018229492A1 WO 2018229492 A1 WO2018229492 A1 WO 2018229492A1 GB 2018051636 W GB2018051636 W GB 2018051636W WO 2018229492 A1 WO2018229492 A1 WO 2018229492A1
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WIPO (PCT)
Prior art keywords
car
domain
nucleic acid
acid sequence
cells
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Ceased
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PCT/GB2018/051636
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English (en)
Inventor
Evangelia KOKALAKI
Maria STAVROU
Shaun CORDOBA
Martin Pule
Simon Thomas
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Autolus Ltd
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Autolus Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • 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
    • A61K40/4231—Cytokines
    • A61K40/4232—Tumor necrosis factors [TNF] or CD70
    • 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]
    • A61K2239/22—Intracellular domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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

Definitions

  • the present invention relates to chimeric antigen receptor (CAR) system comprising a CAR which binds the B cell maturation antigen (BCMA).
  • CAR chimeric antigen receptor
  • BCMA B cell maturation antigen
  • Myeloma is a bone-marrow malignancy of plasma cells. Collections of abnormal plasma cells accumulate in the bone marrow, where they interfere with the production of normal blood cells. Myeloma is the second most common hematological malignancy in the U.S. (after non-Hodgkin lymphoma), and constitutes 13% of hematologic malignancies and 1 % of all cancers. The disease is burdensome in terms of suffering as well as medical expenditure since it causes pathological fractures, susceptibility to infection, renal and then bone-marrow failure before death.
  • myeloma is currently incurable.
  • Standard chemotherapy agents used in lymphoma are largely ineffective for myeloma.
  • CD20 expression is lost in plasma cells, Rituximab cannot be used against this disease.
  • New agents such as Bortezamib and Lenolidomide are partially effective, but fail to lead to long-lasting remissions.
  • Chimeric antigen receptors are proteins which, in their usual format, graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell.
  • mAb monoclonal antibody
  • Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activation signals (see Figure 2).
  • scFv single-chain variable fragments
  • monoclonal antibodies to recognize a target antigen.
  • the scFv is fused via a spacer and a transmembrane domain to a signaling endodomain.
  • Such molecules result in activation of the T-cell in response to recognition by the scFv of its target.
  • T cells express such a CAR, they recognize and kill target cells that express the target antigen.
  • CARs have been developed against tumour associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trial for the treatment of various cancers.
  • BCMA B-cell maturation antigen
  • Carpenter et al demonstrate that T cells transduced to express the anti-BCMA CAR are capable of specifically killing myeloma cells from a plasmacytoma of a myeloma patient.
  • MAS macrophage activation syndrome
  • On-target off-tumour toxicity i.e. recognition of the target antigen on normal tissues.
  • MAS is presumed to be caused by persistent antigen-driven activation and proliferation of T-cells which in turn release copious inflammatory cytokines leading to hyper-activation of macrophages and a feed-forward cycle of immune activation.
  • a large spike in serum IL-6 is characteristic and the syndrome can result in a severe systemic illness requiring ICU admission.
  • Suicide genes are genetically expressed elements which can conditionally destroy cells which express them. Examples include Herpes-simplex virus thymidine kinase, which renders cells susceptible to Ganciclovir; inducible Caspase 9, which renders cells susceptible to a small molecular homodimerizer and CD20 and RQR8, which renders cells susceptible to Rituximab.
  • This technology adds a certain amount of safety, however there are limitations. Firstly, it is a binary approach wherein all the CAR T-cells are destroyed upon addition of the suicide agent. In addition, medicinal therapeutics often have a therapeutic window. With a suicide gene the potency of the product cannot be tuned such that efficacy with tolerable toxicity can be achieved.
  • BAFF B-cell-activating factor
  • BAFF-R BAFF-Receptor
  • BCMA B-cell membrane antigen
  • TACI transmembrane activator and calcium modulator and cyclophilin ligand interactor
  • APRIL A proliferation-inducing ligand
  • BAFF-R activation affects peripheral B-cell survival
  • BCMA may affect plasma cell survival.
  • APRIL interaction with proteoglycans involves acidic sulphated glycol-saminoglycan side-chain containing amino-terminus of APRIL.
  • FIG 4 Schematic diagram illustrating and APRIL CAR and various APRIL CAR systems
  • Figure 5 Graphs showing the cytotoxic capacity of APRIL CAR and various APRIL CAR systems using flow cytometry
  • Percentage survival of MM1S target cells is shown following 72hr culture of CAR expressing T cells with target cell lines at either a 1 :2 ( Figure 5a) or 1 :4 cell ratio in the presence or absence of tetracycline.
  • Percentage survival of BCMA-expressing SKOV3 cells is shown over time following culture of CAR expressing T cells with target cells at an 1 :8 ratio in the presence or absence of tetracycline.
  • FIG. 7 Graphs showing the cytotoxic capacity of APRIL CAR and various APRIL CAR systems using an IncuCyte® assay at 72 hours
  • Percentage survival of BCMA-expressing SKOV3 cells is shown following 72hr culture of CAR expressing T cells with target cells at a 1 :8 cell ratio in the presence or absence of tetracycline.
  • FIG. 8 Histograms showing T cell proliferation following co-culture of T cells epresiing APRIL CAR or one of various APRIL CAR systems with MM1 S target cells. Dilution of the Cell Trace Violet (CTV), which occurs as the T-cells divide, is shown following co-culture of CAR expressing T cells with MM1S target cell lines.
  • CTV Cell Trace Violet
  • Figure 9 Schematic diagram showing two APRIL CARs designed to ensure trimeric stoichiometry of binding with BCMA or TACI target antigen.
  • a - An APRIL CAR with a trimerising spacer such as a coiled-coil spacer derived from Tenascin C
  • APRIL CAR having three APRIL-derived BCMA/TACI binding domains in the antigen-binding domains.
  • the APRIL domains in this "Triple APRIL" antigen-binding domain may be linked by linkers (shown as black lines).
  • the present inventors have developed a CAR system which is made up of a CAR having a BCMA-binding domain which comprises at least part of a proliferation- inducing ligand (APRIL), and separate signalling component.
  • APRIL proliferation- inducing ligand
  • signalling can be rapidly inhibited/terminated despite continued binding of the CAR to BCMA.
  • This inhibition of signalling may occur, for example, in the presence of an agent, such as a small molecule, meaning that CAR activity can be controlled reversibly, without destroying the CAR-expressing cells.
  • the present invention provides a chimeric antigen receptor (CAR) system comprising: a CAR comprising:
  • BCMA B cell maturation antigen-binding domain which comprises at least part of a proliferation-inducing ligand (APRIL);
  • an intracellular signaling molecule comprising:
  • a second heterodimerization domain which dimerizes to (i.e. is capable of binding to) the first heterodimerization domain
  • the BCMA-binding domain may comprise a truncated APRIL which comprises the BCMA binding site but lacks the amino terminal portion of APRIL responsible for proteoglycan binding.
  • APRIL truncated APRIL which comprises the BCMA binding site but lacks the amino terminal portion of APRIL responsible for proteoglycan binding.
  • Such a molecule may comprise the sequence shown as SEQ ID No. 2.
  • the molecule may comprise a variant of that sequence having at least 80% sequence identity, provided that the variant retains the capacity to bind BCMA.
  • the intracellular domain of the CAR may comprise a co-stimulatory domain.
  • the co- stimulatory domain may comprise one or more of the following endodomain(s): CD28 endodomain, 41 BB endodomain and OX40 endodomain.
  • the CAR may comprise two co-stimulatory domains: one which transits proliferation signals and one which transmits survival signals.
  • the first heterodimerization domain may comprise a Tet Repressor Protein (TetRB), and the second heterodimerization domain may comprise a Transcription inducing peptide (TiP).
  • TetRB Tet Repressor Protein
  • TiP Transcription inducing peptide
  • the first heterodimerization domain may comprise a Transcription inducing peptide (TiP)
  • the second heterodimerization domain may comprise aTet Repressor Protein (TetRB).
  • the present invention provides a nucleic acid construct which encodes a CAR system of the first aspect of the invention
  • the nucleic acid construct may comprise the following structure:
  • TM is a nucleic acid sequence encoding the transmembrane domain of the CAR
  • Ht1 is a nucleic acid sequence encoding the first heterodimerization domain of the CAR
  • coexpr is a nucleic acid sequence enabling co-expression of both the CAR of the CAR system and the intracellular signaling molecule of the CAR system;
  • Ht2 is a nucleic acid sequence encoding the second heterodimerization domain of the intracellular signaling molecule
  • SD is a nucleic acid sequence encoding the signaling domain of the intracellular signaling molecule.
  • the "coexpr” may encode a sequence comprising a self-cleaving peptide.
  • Alternative codons may be used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination.
  • the present invention provides a kit of nucleic acid sequences comprising: (i) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) of the CAR system as defined in the first aspect of the invention comprises; and ii) a second nucleic acid sequence encoding the intracellular signaling molecule of the CAR system as defined in the first aspect of the invention.
  • the first nucleic acid sequence may have the following structure:
  • TM is a nucleic acid sequence encoding the transmembrane domain of the CAR
  • Ht1 is a nucleic acid sequence encoding the first heterodimerization domain of the CAR
  • the second nucleic acid sequence may have the following structure:
  • Ht2 is a nucleic acid sequence encoding a second heterodimerization domain of the intracellular signaling molecule
  • SD is a nucleic acid sequence encoding the signaling domain of the intracellular signaling molecule.
  • the present invention provides a kit comprising a first vector which comprises the first nucleic acid sequence as defined above; and a second vector which comprises the second nucleic acid sequence as defined above.
  • the invention provides a vector which comprises a nucleic acid construct according to the second aspect of the invention.
  • the or each vector may, for example, be an integrating viral vector, such as a retroviral vector or a lentiviral vector, or a transposon.
  • an integrating viral vector such as a retroviral vector or a lentiviral vector, or a transposon.
  • the present invention provides a cell which expresses a chimeric antigen receptor (CAR) system of the first aspect of the invention.
  • the cell may comprises a nucleic acid construct according to the second aspect of the invention, a first and second nucleic acid sequence as defined in the third aspect of the invention or a vector according to the fourth aspect of the invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a plurality of cells according to the sixth aspect of the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.
  • composition according to the seventh aspect of the invention for use in treating or preventing a disease.
  • the present invention provides a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the seventh aspect of the invention to a subject.
  • the method may comprise the following steps:
  • the method may involve monitoring toxic activity in the subject and comprises the step of administering an agent which disrupts dimerizing of the first and second dimerization domain to the subject.
  • the method may involve monitoring the progression of disease and / or monitoring toxic activity in the subject and comprise the step of administering an agent, which disrupts dimerizing of the first and second dimerization domain, to provide acceptable levels of disease progression and toxic activity.
  • the disease may be a plasma cell disorder, for example a plasma cell disorder selected from: 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 and smoldering multiple myeloma.
  • a plasma cell disorder selected from: 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 and smoldering multiple myeloma.
  • the plasma cell disorder may be multiple myeloma.
  • the present invention provides use of a pharmaceutical composition according to the sixth aspect of the invention in the manufacture of a medicament for the treatment and/ or prevention of a disease.
  • the present invention provides a method of inhibiting a CAR system in a subject which comprises a cell according to fifth aspect of the invention, which method comprises the step of administering an agent to the subject, wherein the agent disrupts dimerization of the first and second heterodimerization domain.
  • the present invention therefore provides an APRIL CAR system in which signalling can be inhibited in the presence of an agent, for example a small molecule, which prevents co-localisation of the CAR and intracellular signalling molecule.
  • an agent for example a small molecule
  • This allows CAR signalling and thus the potency of CAR cells to be reversibly terminated in a controllable manner in order to avoid potential toxic effects associated with unabated CAR signalling.
  • the present system also allows the potency of CAR cells to be controlled pharmacologically and tuned to an acceptable balance between achieving the desired therapeutic effect and avoiding unwanted toxicities.
  • a particular consideration when targeting BCMA or TACI is the particularly low density of these antigens on myeloma cells, in comparison for instance with CD19 on a lymphoma cell.
  • the ligand, APRIL binds to BCMA as a trimer (Hymowitz et al (2005) 280:7218- 7227). It also binds TACI as a trimer.
  • the present inventors have developed two strategies to ensure that an APRIL-containing CAR binds BCMA/TACI with the same binding stoichiometry as the natural ligand.
  • the first strategy involves expressing three copies of APRIL, or truncated APRIL which comprises the BCMA/TACI binding site, in the CAR.
  • the CAR may, for example, comprises three copies of APRIL, joined by linking sequences, followed by the spacer, transmembrane and endodomain (see Figure 9B).
  • the second strategy involves using a spacer which forms trimers at the cell surface.
  • a trimerising coiled-coil spacer may be used, such as one derived from Tenascin C ( Figure 9A). Further aspects of the invention relating to these two embodiments as provided in the following numbered paragraphs.
  • a chimeric antigen receptor comprising the B cell maturation antigen (BCMA)-binding domain from a proliferation-inducing ligand (APRIL) as antigen binding domain, which binds BCMA as a trimer.
  • a CAR according to paragraph 1 wherein the BCMA-binding domain comprises a truncated APRIL which comprises the BCMA binding site but lacks the amino terminal portion of APRIL responsible for proteoglycan binding.
  • a CAR according to paragraph 2 which comprises the sequence shown as SEQ ID No. 2.
  • linkers Sp is a spacer
  • TM is a transmembrane domain
  • Endo is an endodomain. 5.
  • a CAR according to paragraph 5 which comprises a trimerising coiled-coil spacer.
  • a method for making a cell according to paragraph 11 which comprises the step of introducing a nucleic acid according to paragraph 9 into a cell.
  • a pharmaceutical composition which comprises a cell according to paragraph 11 , together with a pharmaceutically acceptable carrier, diluent or excipient.
  • a method for treating a plasma cell disorder which comprises the step of administering a cell according to paragraph 11 to a subject.
  • the plasma cell disorder is selected from 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 and smoldering multiple myeloma.
  • a cell according to paragraph 11 for use in treating a plasma cell disorder 17.
  • CARS CHIMERIC ANTIGEN RECEPTORS
  • Chimeric antigen receptors also known as chimeric T cell receptors, artificial T cell receptors and chimeric immunoreceptors, are engineered receptors, which graft an arbitrary specificity onto an immune effector cell.
  • CAR Figure 2
  • the specificity of a monoclonal antibody is grafted on to a T cell or NK cell.
  • CAR- encoding nucleic acids may be introduced into T cells or NK cells using, for example, retroviral vectors. In this way, a large number of cancer-specific T cells or NK cells can be generated for adoptive cell transfer.
  • the target-antigen binding domain of a CAR is commonly fused via a spacer and transmembrane domain to a signaling endodomain. When the CAR binds the target- antigen, this results in the transmission of an activating signal to the T-cell it is expressed on.
  • the first aspect of the invention relates to a CAR system which comprises a CAR component comprising a B cell maturation antigen (BCMA)-binding domain which comprises at least part of a proliferation-inducing ligand (APRIL).
  • BCMA B cell maturation antigen
  • APRIL proliferation-inducing ligand
  • BCMA also known as TNFRSF17, is a plasma cell specific surface antigen which is expressed exclusively on B-lineage haemopoietic cells or dendritic cells. It is a member of the TNF receptor family. BCMA is not expressed on naive B cells but is up-regulated during B-cell differentiation into plasmablasts, and is brightly expressed on memory B cells, plasmablasts and bone marrow plasma cells. BCMA is also expressed on the majority of primary myeloma cells. Unlike other CAR targets such as CD19, BCMA is expressed at low density.
  • BCMA functions within a network of interconnected ligands and receptors which is shown schematically in Figure 1.
  • Two other TNF receptors share the ligands APRIL and BAFF with BCMA - TACI (TNFRSF13B), which is found on activated T-cells and all B-cells and BAFF-R (TNFRSF13C) which is predominantly expressed on B- lymphocytes.
  • BCMA - TACI TNFRSF13B
  • BAFF-R TNFRSF13C
  • Multiple myeloma cells express TACI in some cases and BCMA in most cases, but not BAFF-R.
  • the BCMA-binding domain of the CAR of the invention comprises at least part of a proliferation-inducing ligand (APRIL).
  • APRIL is also known as TNFSF13.
  • APRIL The wild-type sequence of APRIL is available at UNIPROT/075888 and is show below (SEQ ID No. 1). It is not a classical secreted protein in that it has no signal peptide. It has a furin cleavage site "KQKKQK” (underlined in SEQ ID No. 1). The amino terminus is involved in proteoglycan binding.
  • the BCMA-binding domain may comprise the BCMA-binding site of APRIL.
  • the BCMA-binding domain may comprise a fragment of APRIL which comprises the BCMA-binding site.
  • the BCMA-binding domain may comprise a truncated APRIL, which lacks the amino terminal end of the molecule.
  • the truncated APRIL may retain BCMA and TACI binding but lose proteoglycan binding.
  • Truncated APRIL can be cleaved at or immediately after the furin cleavage site.
  • Truncated APRIL may lack the amino terminal 1 16 amino acids from the wild-type APRIL molecule shown as SEQ ID No. 1.
  • Truncated APRIL may comprise the sequence shown as SEQ ID No. 2 (which corresponds to the portion of SEQ ID No. 1 shown in bold) or a variant thereof. This corresponds to the portion of the molecule which is needed for BCMA and TACI binding.
  • SEQ ID No. 1 SEQ ID No. 2
  • VPINATSKDD SDVTEVMWQP ALRRGRGLQA QGYGVRIQDA GVYLLYSQVL FQDVTFTMGQ
  • the CAR of the present invention may comprise a variant of the truncated APRIL molecule shown as SEQ ID No. 2 which has at least 80% amino acid sequence identity and which has the same or improved BCMA binding capabilities.
  • the variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 2.
  • the present invention relates to a CAR system, composed of two components: (i) a CAR comprising a B cell maturation antigen (BCMA)-binding domain which comprises at least part of a proliferation-inducing ligand (APRIL) described above; a transmembrane domain, which is described in more detail below; and an intracellular domain which comprises a first heterodimerization domain, and (ii) a separate intracellular signalling molecule which comprises a second heterodimerization domain which dimerizes with the first heterodimerization domain, and a signalling domain.
  • BCMA B cell maturation antigen
  • APRIL proliferation-inducing ligand
  • the present invention further provides a cell comprising a CAR system in which the BCMA- binding domain and transmembrane domain of the tunable CAR are provided on a first molecule which localizes to the cell membrane.
  • the signalling domain is provided on a separate intracellular signalling molecule.
  • the intracellular domain of the CAR comprises a first heterodimerzation domain and the signalling domain of the separate intracellular signalling molecule comprises a second heterodimerzation domain which specifically binds to the first heterodimerzation domain of the CAR.
  • binding of the first heterodimerzation domain to the second heterodimerzation domain causes heterodimerization and co- localization of the CAR and the signalling domain of the separate intracellular signalling molecule.
  • an antigen such as BCMA binds to the BCMA binding domain of the CAR component there is signalling through the signalling domain of the intracellular signalling molecule.
  • Dimerisation of the first and second heterodimerization domains may occur spontaneously, for example as described in WO2016/124930.
  • dimerization may occur only in the presence of an agent, known as a chemical inducer of dimerization (CID).
  • CID chemical inducer of dimerization
  • dimerization may occur only in the absence of an agent, such as a small molecule.
  • agent such as a small molecule.
  • the agent acts to disrupt binding of the first and second heterodimerization domains.
  • the first or second heterodimerzation domain may also be capable of binding the agent in addition to its reciprocal domain (the first heterodimerzation domain dimerizes with the second heterodimerization domain or the second heterodimerzation domain dimerizes with the first heterodimerization domain).
  • the binding between the agent and the first or second heterodimerzation domain may be of a higher affinity than the binding between the first heterodimerzation domain and the second heterodimerzation domain.
  • the agent when it preferentially binds to the first or second heterodimerzation domain and inhibits/disrupts the dimerization between the CAR and the signalling domain of the separate intracellular signalling molecule of the CAR system.
  • BCMA binds to the BCMA binding domain of the CAR in the presence of the agent there is no signalling through the signalling domain of the separate intracellular signalling molecule.
  • the CAR and signalling domain of the intracellular signalling molecule are located in a stochastically dispersed manner and binding of antigen by the antigen-binding domain of the receptor component does not result in signalling.
  • BCMA binding by the CAR in the presence of the agent may be termed as 'non- productive' as it does not result in signalling through the signalling domain of the separate intracellular signalling molecule.
  • BCMA-binding by the CAR in the absence of the agent may be termed as resulting in 'productive' as it results in signalling through the signalling component. This signalling results in T-cell activation, triggering for example target cell killing and T cell activation.
  • BCMA binding by the BCMA binding domain of the CAR in the absence of the agent may result in signalling through the signalling domain of the intracellular signalling molecule which is 2, 5, 10, 50, 100, 1 ,000 or 10,000-fold higher than the signalling which occurs when BCMA is bound by the CAR in the presence of the agent.
  • Signalling through the signalling domain of the separate intracellular signalling molecule may be determined by a variety of methods known in the art. Such methods include assaying signal transduction, for example assaying levels of specific protein tyrosine kinases (PTKs), breakdown of phosphatidylinositol 4,5-biphosphate (PIP2), activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration.
  • Functional readouts such as clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells and induction of cytotoxicity or cytokine secretion may also be utilised.
  • the inventors determined percentage of cell survival (cytotoxicity) of T-cells expressing CAR and a separate intracellular signalling molecule of a tunable CAR system upon binding of antigen BCMA to the CAR in the presence or absence of an agent.
  • the CAR system of the present invention may, for example, comprise one of the following amino acid sequences which represent three possible CAR configurations associated with three possible intracellular signalling molecule configurations:
  • the first heterodimerization domain, second heterodimerization domain and agent of the tunable CAR system may be any combination of molecules/peptides/domains which enable the selective co-localization and dimerization of the CAR and signalling domain of the separate intracellular signalling molecule in the absence of the agent.
  • the first heterodimerization domain and second heterodimerization domain are capable of specifically dimerizing.
  • the CAR system of the present invention is not limited by the arrangement of a specific dimerization system.
  • the receptor component may comprise either the first heterodimerization domain or the second heterodimerization domain of a given dimerization system so long as the signalling domain comprises the corresponding, complementary domain which enables the CAR and the signalling domain of the intracellular signalling molecule to co-localize in the absence of the agent.
  • the first heterodimerization domain and second heterodimerization domain may be a peptide domain and a peptide binding domain; or vice versa.
  • the peptide domain and peptide binding domain may be any combination of peptides/domains which are capable of specific binding.
  • the agent may be a molecule, for example a small molecule, which is capable of specifically binding to the first heterodimerization domain or the second heterodimerization domain at a higher affinity than the binding between the first heterodimerization domain and the second heterodimerization domain.
  • the heterodimerization domains may be based on a peptide:peptide binding domain system.
  • the first or second heterodimerization domain may comprise the peptide binding domain and the other binding domain may comprise a peptide mimic which binds the peptide binding domain with lower affinity than the peptide.
  • the use of peptide as agent disrupts the binding of the peptide mimic to the peptide binding domain through competitive binding.
  • the peptide mimic may have a similar amino acid sequence to the "wild-type" peptide, but with one of more amino acid changes to reduce binding affinity for the peptide binding domain.
  • the agent may bind the first heterodimerization domain or the second heterodimerization domain with at least 10, 20, 50, 100, 1000 or 10000-fold greater affinity than the affinity between the first heterodimerization domain and the second heterodimerization domain.
  • the agent may be any pharmaceutically acceptable molecule which preferentially binds the first heterodimerization domain or the second heterodimerization domain with a higher affinity than the affinity between the first heterodimerization domain and the second heterodimerization domain.
  • the agent is capable of being delivered to the cytoplasm of a target cell and being available for intracellular binding.
  • the agent may be capable of crossing the blood-brain barrier.
  • Small molecule systems for controlling the co-localization of peptides are known in the art, for example the Tet repressor (TetR), TetR interacting protein (TiP), tetracycline system (Klotzsche et al.; J. Biol. Chem. 280, 24591-24599 (2005); Luckner et al.; J. Mol. Biol. 368, 780-790 (2007)).
  • TetR Tet repressor
  • the Tet operon is a well-known biological operon which has been adapted for use in mammalian cells.
  • the TetR binds tetracycline as a homodimer and undergoes a conformational change which then modulates the DNA binding of the TetR molecules.
  • Klotzsche et al. (as above), described a phage-display derived peptide which activates the TetR.
  • This protein (TetR interacting protein/TiP) has a binding site in TetR which overlaps, but is not identical to, the tetracycline binding site (Luckner et al.; as above).
  • TiP and tetracycline compete for binding of TetR.
  • the first heterodimerization domain of the CAR may be TetR or TiP, provided that the second heterodimerization domain of the intracellular signalling molecule is the corresponding, complementary dimerizing partner.
  • the second heterodimerization domain of the intracellular signalling molecule is TiP.
  • the first heterodimerization domain of the receptor component is TiP
  • the second heterodimerization domain of the intracellular signalling molecule is TetR.
  • first heterodimerization domain or second heterodimerization domain may comprise the sequence shown as SEQ ID NO: 6 or SEQ ID NO: 7:
  • the CAR may comprise a linker between the transmembrane domain and the first heterodimerization domain (TetR).
  • the intracellular signalling molecule may also comprise a linker between the second heterodimerization domain (TiP) and the signalling domain.
  • the first or second binding domain may comprise one or more streptavidin-binding epitope(s).
  • the other binding domain may comprise a biotin mimic.
  • Streptavidin is a 52.8 kDa protein from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have a very high affinity for biotin (vitamin B7 or vitamin H), with a dissociation constant (Kd) ⁇ 10-15 M.
  • the biotin mimic has a lower affinity for streptavidin than wild-type biotin, so that biotin itself can be used as the agent to disrupt or prevent heterodimerization between the streptavidin domain and the biotin mimic domain.
  • the biotin mimic may bind streptavidin with for example with a Kd of 1 nM to 100uM.
  • the 'biotin mimic' domain may, for example, comprise a short peptide sequence (for example 6 to 20, 6 to 18, 8 to 18 or 8 to 15 amino acids) which specifically binds to streptavidin.
  • the biotin mimic may comprise a sequence as shown in Table 1.
  • the biotin mimic may be selected from the following group: Streptagll, Flankedccstreptag and ccstreptag.
  • the streptavidin domain may comprise streptavidin having the sequence shown as SEQ ID No. 19 or a fragment or variant thereof which retains the ability to bind biotin.
  • Full length Streptavidin has 159 amino acids.
  • the N and C termini of the 159 residue full-length protein are processed to give a shorter 'core' streptavidin, usually composed of residues 13 - 139; removal of the N and C termini is necessary for the high biotin-binding affinity.
  • streptavidin exists in nature as a homo-tetramer.
  • the secondary structure of a streptavidin monomer is composed of eight antiparallel ⁇ -strands, which fold to give an antiparallel beta barrel tertiary structure.
  • a biotin binding-site is located at one end of each ⁇ -barrel.
  • Four identical streptavidin monomers i.e.
  • streptavidin domain of the CAR system of the present invention may consist essentially of a streptavidin monomer, dimer or tetramer.
  • sequence of the streptavidin monomer, dimer or tetramer may comprise all or part of the sequence shown as SEQ ID No. 19, or a variant thereof which retains the capacity to bind biotin.
  • a variant streptavidin sequence may have at least 70, 80, 90, 95 or 99% identity to SEQ ID No. 19 or a functional portion thereof.
  • Variant streptavidin may comprise one or more of the following amino acids, which are involved in biotin binding: residues Asn23, Tyr43, Ser27, Ser45, Asn49, Ser88, Thr90 and Asp128.
  • Variant streptavidin may, for example, comprise all 8 of these residues.
  • variant streptavidin is present in the binding domain as a dimer or tetramer, it may also comprise Trp120 which is involved in biotin binding by the neighbouring subunit.
  • the tunable CAR system may comprise a CAR comprising single domain dimerizer and the intracellular signalling molecule may comprise a dimerizing domain which binds the single domain dimerizer.
  • the intracellular signalling molecule may comprise a single domain dimerizer and the tunable CAR may comprise a dimerizing domain which binds the single domain dimerizer.
  • a “single domain dimerizer” is an entity which binds to an agent, such as a small molecule agent, and has a single domain.
  • a protein domain has a compact three- dimensional structure. It may be derivable from a larger protein, but the domain itself is independently stable and folds independently.
  • the single domain dimerizer may have an antibody-like binding site which binds to the agent.
  • the single domain dimerizer may comprise one or more complementarity determining regions (CDRs).
  • the single domain dimerizer may comprise three CDRs
  • the single domain dimerizer may lack disulphide bonds.
  • the single domain dimerizer may lack cysteine residues.
  • a conventional IgG molecule is comprised of two heavy and two light chains. Heavy chains comprise three constant domains and one variable domain (VH); light chains comprise one constant domain and one variable domain (VL).
  • VH variable domain
  • VL variable domain
  • the naturally functional antigen binding unit is formed by noncovalent association of the VH and the VL domain. This association is mediated by hydrophobic framework regions.
  • IgG can be derivatized to Fab, scFv, and single domain VH or VL binders.
  • the single dimerizer used in the CAR system of the invention may be or comprise such a single domain VH or VL binder.
  • Heavy chain antibodies are found in Camelidae, lack the light chain and the CH1 domain. They comprise a single, antigen binding domain, the VHH domain.
  • the single domain dimerizer used in the CAR system of the invention may be or comprise such a VHH domain or derivative thereof.
  • non-immunoglobulin single domain dimerizers have also been designed and characterised, including those based on natural and synthetic protein scaffolds.
  • fibronectin-derived Adnectins/monobodies are characterized by an Ig- like ⁇ -sandwich structure
  • anticalins are based on the lipocalin fold
  • affibodies derive from protein A and comprise three a helices
  • DARPins are designer proteins composed of ankyrin repeats.
  • Each design includes randomized residues that mediate ligand binding.
  • the single domain dimerizer may have a molecular weight (when considered separately from the rest of the receptor component or signalling component of less than 20kDa. It may, for example have a molecular weight of less than or equal to approximately 15 kDa, such as between 12-15kDa, the typical molecular weight of a single domain antibody.
  • Single chain variable fragments, which comprise two variable domains, VH and VL) typically have a molecular weight of about 25kDa.
  • the single domain dimerizer may be less than 150 amino acids in length, for example, less than 140, 130 or 120 amino acids in length.
  • the single domain dimerizer may be approximately 110 amino acids in length, for example from 105-115 amino acids in length
  • the single domain dimerizer used in the CAR system of the invention may be a single domain antibody (sdAb, also known as a nanobody), an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VNAR, an iBody, an affimer, a fynomer, a domain antibody (DAb), an abdurin/ nanoantibody, a centyrin, an alphabody or a nanofitin.
  • sdAb also known as a nanobody
  • an affibody also known as a nanobody
  • a fibronectin artificial antibody scaffold an anticalin
  • an affilin a DARPin
  • VNAR an iBody
  • an affimer a fynomer
  • DAb domain antibody
  • a single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain.
  • the first single-domain antibodies were engineered from heavy-chain
  • Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained.
  • IgNAR immunoglobulin new antigen receptor
  • An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers.
  • IgG immunoglobulin G
  • Nanobodies derived from light chains have also been shown to bind specifically to target epitopes.
  • a single-domain antibody can be obtained by immunization of dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies.
  • a gene library of single-domain antibodies may be produced. Screening techniques like phage display and ribosome display help to identify the clones binding the antigen.
  • Heterodimerisation of the tunable CAR and separate intracellular signalling molecule may occur through the dimerization of the single domain dimerizer with a single domain dimerizer-interacting peptide (sddiP).
  • the sddiP may, for example, be between 8-30, for example 10-20 amino acids in length.
  • Suitable sddiPs may be generated and identified using peptide display methods such as phage display, CIS display, ribosome display and mRNA display (Ullman et al (201 1) Briefings in Functional Genomics 10: 125-134). Peptides in a phage display peptide library may be selected using techniques such as biopanning (Miura et al (2004) Biochim. Et Biophys. Acta 1673: 131-138).
  • the agent itself may be used to elute the peptides, for example in a peptide array, so that the selection method reflects the properties of the sddiP in the CAR signalling system, namely that it binds the single domain binder, but the binding is competitively inhibited by the presence of the agent.
  • the agent for use with a single domain binder may be a small molecule such as: a steroid, methotrexate, caffeine, cocaine or an antibiotic.
  • Small molecules agents which disrupt protein-protein interactions have long been developed for pharmaceutical purpose (reviewed by Vassilev et al; Small-Molecule Inhibitors of Protein-Protein Interactions ISBN: 978-3-642-17082-9).
  • a CAR system as described may use such a small molecule.
  • the proteins or peptides whose interaction is disrupted can be used as the first and/or second heterodimerization domains and the small molecule may be used as the agent which inhibits CAR activation.
  • Such a system may be varied by altering the small molecule and proteins such the system functions as described but the small molecule is devoid of unwanted pharmacological activity (e.g. in a manner similar to that described by Rivera et al (Nature Med; 1996; 2; 1028-1032).
  • Second heterodimerization domains which competitively bind to the same first heterodimerization domain as the agents described above, and thus may be used to co-localise the CAR and the signalling domain on the separate intracellular signalling molecule in the absence of the agent, may be identified using techniques and methods which are well known in the art. For example such second heterodimerization domain may be identified by display of a single domain VHH library.
  • the first heterodimerization domain and/or heterodimerization domain of the signalling system may comprise a variant(s) which is able to specifically bind to the reciprocal binding domain and thus facilitate co-localisation of the CAR and intracellular signalling molecule.
  • Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the wild-type sequence, provided that the sequences provide an effective dimerization system. That is, provided that the sequences facilitate sufficient co- localisation of the receptor and signalling components, in the absence of the agent, for productive signalling to occur upon binding of the antigen-binding domain to antigen.
  • the present invention also relates to a method for inhibiting a tunable CAR system, which method comprises the step of administering the agent.
  • administration of the agent results in a disruption of the co-localization between the CAR and the intracellular signalling molecule such that signalling through the intracellular signalling molecule is inhibited even upon binding of BCMA to the BCMA- binding domain.
  • the first and second heterodimerization domains may facilitate signalling through the CAR system which is proportional to the concentration of the agent which is present.
  • the agent binds the first heterodimerization domain or the second heterodimerization domain with a higher affinity than binding affinity between the first and second heterodimerization domain
  • co-localization of the CAR and the signalling domain of the intracellular signalling molecule may not be completely ablated in the presence of low concentrations of the agent.
  • low concentrations of the agent may decrease the total level of signalling in response to BCMA antigen without completely inhibiting it.
  • the specific concentrations of agent will differ depending on the level of signalling required and the specific heterodimerization domains and agent. Levels of signalling and the correlation with concentration of agent can be determined using methods known in the art, as described above.
  • the transmembrane domain is the sequence of the CAR that spans the membrane.
  • a transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues.
  • the transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention.
  • the presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs. dtu.dk/services/TMHMM-2.0/).
  • transmembrane domain of a protein is a relatively simple structure, i.e a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane
  • an artificially designed TM domain may also be used (US 7052906 B1 describes synthetic transmembrane components).
  • the transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the transmembrane domain may be derived from human Tyrp-1.
  • the tyrp-1 transmembrane sequence is shown as SEQ ID No. 20. SEQ ID No. 20
  • the CAR may comprise a plurality of first heterodimerization domains and thus be capable of recruiting more than one intracellular signalling molecule.
  • the plurality of first heterodimerization domains may be present in a single intracellular domain of the CAR
  • the CAR of the CAR system may comprise an appropriate number of transmembrane domains such that each first heterodimerization domain is orientated on the intracellular side of the cell membrane.
  • the CAR may comprise 3, 5, 7, 9, 11 , or more transmembrane domains. In this way, a single CAR may recruit multiple intracellular signalling molecules, further amplifying signalling in response to antigen.
  • the first heterodimerization domains may each be variants which have a different affinity for the second heterodimerization domains of the intracellular signalling molecule.
  • the intracellular signalling molecule comprises a signalling domain and a second heterodimerization domain.
  • the intracellular signalling molecule may be a soluble molecule and which localises to the cytoplasm when it is expressed in a cell, for example a T cell.
  • the signalling molecule may be tethered to the membrane, for example, using a transmembrane domain or a myristoylation sequence, so that the signalling domain and second heterodimerisation domain are positioned proximal to the membrane on the intracellular side.
  • No signalling occurs through the signalling domain of the intracellular signalling molecule unless it is co-localised with a CAR of the CAR system. Such co- localisation occurs only in the absence of the agent, as described above.
  • the signalling domain is the signal-transmission portion of a classical CAR.
  • the signalling domain is located in the intracellular signalling molecule, and not in the CAR.
  • the membrane- bound CAR and the signaling domain of the intracellular signaling molecule are brought into proximity.
  • CD3-zeta endodomain which contains 3 ITAMs and has the sequence shown as SEQ ID No. 21. This transmits an activation signal to the T cell after antigen is bound.
  • CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signalling may be needed.
  • chimeric CD28 and OX40 can be used with CD3-zeta to transmit a proliferative / survival signal, or all three can be used together.
  • the signalling domain may comprise the CD3-zeta endodomain alone; or the CD3- zeta endodomain with one or more co-stimulatory domains, such as the endodomain CD28 or OX40 or the CD28 endodomain, OX40 endodomain and CD3-zeta endodomain.
  • the signalling domain of a CAR system of the invention may comprise the sequence shown as SEQ ID No. 22, 23 or 24 or a variant thereof having at least 80% sequence identity.
  • SEQ ID No. 22 comprising CD28 transmembrane domain and CD3-zeta endodomain FWVLVWGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGHDGLYQGLSTATKDTYDALHMQALPPR
  • SEQ ID No. 23 comprising CD28 transmembrane domain, CD28 endodomain and CD3-zeta endodomains
  • a variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 22, 23 or 24 provided that the sequence provides an effective trans-membrane domain and an effective intracellular T cell signaling domain.
  • the intracellular domain of the tunable CAR comprises one or more co-stimulatory domains (for example CD28-OX40, OX40-CD28, CD28- 41 BB, or 41 BB-CD28) and the intracellular signalling molecule comprises CD3-zeta only.
  • the intracellular domain of the CAR lacks CD3-zeta.
  • the CAR system may comprise a plurality of intracellular signalling molecules, each comprising a signalling domain and a second heterodimerization domain.
  • Each second heterodimerization domain may be bound by the same first heterodimerization domain of the CAR but the signalling domains may comprise different endodomains. In this way, multiple endodomains can be activated simultaneously. This is advantageous over a compound signalling domain since each signalling domain remains unencumbered from other signalling domains.
  • each intracellular signalling molecule comprises a second heterodimerization domain which differs in residues which alter their affinity to the first heterodimerization domain of the CAR
  • the intracellular signalling molecule comprising different signalling domains ligate to the first heterodimerization domain with differing kinetics.
  • This allows greater control over the signalling in response to BCMA-binding by the CAR of the CAR system as different intracellular signalling molecule are recruited to the CAR in varying kinetics/dynamics.
  • an optimal T-cell activation signal may require different proportions of different immunological signals.
  • a particular consideration when targeting BCMA or TACI is the particularly low density of this antigen on myeloma cells, in comparison for instance with CD19 on a lymphoma cell.
  • the ligand, APRIL binds to BCMA as a trimer (Hymowitz et al (2005) 280:7218- 7227). It also binds TACI as a trimer.
  • the present inventors have developed two strategies to ensure that an APRIL-containing CAR binds BCMA/TACI with the same binding stoichiometry as the natural ligand.
  • the first strategy involves expressing three copies of APRIL, or truncated APRIL which comprises the BCMA binding site, in the CAR ( Figure 9B).
  • the CAR may, for example, comprise three copies of APRIL, joined by linking sequences, followed by the spacer, transmembrane and endodomain.
  • the APRIL or truncated APRIL domains may be as defined above.
  • the domains may be attached by linkers, to spatially separate the domains and provide flexibility to enable trimeric BCMA binding.
  • the linker may, for example, by serine-glycine linkers of up to 10, such as 2-8 or 3-5 amino acids in length.
  • both or each linker may, for example have the sequence shown as SEQ ID No. 29.
  • the antigen-binding domain may have the general structure A1-L1-A2-L2-A3, in which:
  • L1 and L2 which may be the same or different, are linkers.
  • the antigen-binding domain may optionally have a third linker, after the third APRIL domain, giving the general structure: A1-L1-A2-L2-A3-L3
  • the antigen-binding domain of the CAR may have the sequence shown as SEQ ID No. 30. In this sequence, the linkers are underlined.
  • the spacer, transmembrane domain and endodomain of the CAR may be as defined above.
  • the spacer may be comprise the stalk from CD8, for example, having the sequence shown as SEQ ID No. 31
  • the spacer may be monomeric, such that the CAR exists as a monomer at the cell surface.
  • the spacer may, for example, lack cysteine residues suitable for forming a disulphide bridge. Examples of such spacer include the ectodomain of CD2 or truncated CD22.
  • the transmembrane domain may be derivable from CD28, for example having the sequence shown as SEQ ID No. 32.
  • the endodomain may, for example, be a third generation endodomain comprising endodomains from CD28, OX40 and CD3zeta, for example having the sequence shown as SEQ ID No. 33 SEQ ID No. 33 - CD28-OX40-CD3z endodomain
  • the second strategy involves using a spacer which forms trimers at the cell surface.
  • a trimerising coiled-coil spacer may be used, such as one derived from Tenascin C ( Figure 9A). CARs having coiled-coil spacer domains are described in WO2016/151315.
  • a coiled coil is a structural motif in which two to seven alpha-helices are wrapped together like the strands of a rope. Many endogenous proteins incorporate coiled coil domains.
  • the coiled coil domain may be involved in protein folding (e.g. it interacts with several alpha helical motifs within the same protein chain) or responsible for protein-protein interaction. In the latter case, the coiled coil can initiate homo or hetero oligomer structures.
  • Coiled coils usually contain a repeated pattern, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeat.
  • the positions in the heptad repeat are usually labeled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, leucine, or valine. Folding a sequence with this repeating pattern into an alpha-helical secondary structure causes the hydrophobic residues to be presented as a 'stripe' that coils gently around the helix in left-handed fashion, forming an amphipathic structure.
  • the a-helices may be parallel or anti-parallel, and usually adopt a left-handed super- coil. Although disfavoured, a few right-handed coiled coils have also been observed in nature and in designed proteins. The relationship between the sequence and the final folded structure of a coiled coil domain are well understood in the art (Mahrenholz et al; Molecular & Cellular Proteomics; 2011 ; 10(5) :M1 10.004994). As such the coiled coil domain may be a synthetically generated coiled coil domain.
  • proteins which contain a coiled coil domain include kinesin motor protein, hepatitis D delta antigen, archaeal box C/D sRNP core protein, cartilage- oligomeric matrix protein (COMP), mannose-binding protein A, coiled-coil serine-rich protein 1 , polypeptide release factor 2, SNAP-25, SNARE, Lac repressor or apolipoprotein E.
  • a coiled-coil domain is used which forms a trimer.
  • Mannose-binding protein A parallel homotrimer (SEQ ID No. 34)
  • Coiled-coil serine-rich protein 1 parallel homotrimer (SEQ ID No. 35)
  • Polypeptide release factor 2 anti-parallel heterotrimer
  • Chain B VVDTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFR (SEQ ID No. 37)
  • the present invention further provides a nucleic acid encoding the CAR or CAR system as defined herein or part thereof, such as the CAR or intracellular signalling component.
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other. It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
  • 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 include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • NUCLEIC ACID CONSTRUCT The present invention also provides a nucleic acid construct which comprises the following nucleic acid sequences:
  • a nucleic acid sequence encoding an intracellular signalling molecule (ii) a nucleic acid sequence encoding an intracellular signalling molecule.
  • the nucleic acid may produce a polypeptide which comprises the CAR and intracellular signalling molecule joined by a cleavage site.
  • the cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the various components, without the need for any external cleavage activity.
  • Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2A peptide and similar sequence (Donnelly et al, Journal of General Virology (2001), 82, 1027-1041), for instance like the 2A-like sequence from Thosea asigna virus which has the sequence shown as SEQ ID No. 25:
  • FMDV Foot-and-Mouth disease virus
  • the co-expressing sequence may alternatively be an internal ribosome entry sequence (IRES) or an internal promoter.
  • the nucleic acid construct may have the following structure:
  • TM is a nucleic acid sequence encoding the transmembrane domain of the CAR
  • Ht1 is a nucleic acid sequence encoding the first heterodimerization domain of the CAR
  • coexpr is a nucleic acid sequence enabling co-expression of both the CAR of the CAR system and the intracellular signaling molecule of the CAR system;
  • Ht2 is a nucleic acid sequence encoding the second heterodimerization domain of the intracellular signaling molecule
  • SD is a nucleic acid sequence encoding the signaling domain of the intracellular signaling molecule.
  • the nucleic acid sequence of the CAR of the CAR system may have the following structure:
  • APRIL-spacer-TM-Ht1 in which APRIL is a nucleic acid sequence encoding the antigen-binding domain of the CAR;
  • spacer is a nucleic acid sequence encoding a spacer of the CAR
  • TM is a nucleic acid sequence encoding the transmembrane domain of the CAR
  • Ht1 is a nucleic acid sequence encoding the first heterodimerization domain of the CAR
  • the nucleic acid sequence of the intracellular signaling molecule of the CAR sytem may have the following structure:
  • Ht2 is a nucleic acid sequence encoding a second heterodimerization domain of the intracellular signaling molecule
  • SD is a nucleic acid sequence encoding the signaling domain of the intracellular signaling molecule.
  • nucleic acid construct encodes a APRIL CAR system comprising a CAR and an intracellular signalling molecule
  • it may have one of the following sequences:
  • SEQ ID NO: 28 TIP-CD28-OX40-z-2A-SP-APRIL-HNG-TM-L-TetRB
  • SEQ ID NO: 26 TIP-OX40-z-2A-SP-APRIL-HNG-TM-CD28-L-TetRB
  • SEQ ID NO: 27 (TIP-z-2A-SP-APRIL-HNG-TM-OX40-CD28-L-TetRB) ATGTGGACCTGGAACGCCTACGCCTTTGCCGCCCCTAGCGGAGGCGGATCTAG AGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC CAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCCGGAGGGGCAAGGGGCACGATGGCCT TTACCAGGGACTGAGCACCGCCACAAAGGATACCTACGACGCCCTGCACATGC AGGCCCTTCCACCTAGAGCTGAAGGCAGAGGCAGCCTTCTGACATGTGGCGAC GTGGAAGAACCCCGGATA
  • Alternative codons may be used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination. Due to the degeneracy of the genetic code, it is possible to use alternative codons which encode the same amino acid sequence. For example, the codons “ccg” and “cca” both encode the amino acid proline, so using “ccg” may be exchanged for "cca” without affecting the amino acid in this position in the sequence of the translated protein.
  • RNA codons which may be used to encode each amino acid are summarised in Table 3.
  • Alternative codons may be used in one or more co-stimulatory domains, such as the CD28 endodomain.
  • Alternative codons may be used in one or more domains which transmit survival signals, such as OX40 and 41 BB endodomains.
  • Alternative codons may be used in the portions of nucleic acid sequence encoding a CD3zeta endodomain and/or the portions of nucleic acid sequence encoding one or more costimulatory domain(s) and/or the portions of nucleic acid sequence encoding one or more domain(s) which transmit survival signals.
  • CELL The present inventors also describe a cell which comprises the CAR or CAR system of the invention.
  • the cell which co-expresses a CAR and intracellular signalling molecule.
  • the cell may be any eukaryotic cell capable of expressing a CAR at the cell surface, such as an immunological cell.
  • the cell may be an immune effector cell such as a T cell or a natural killer (NK) cell.
  • an immune effector cell such as a T cell or a natural killer (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
  • TH1 , TH2, TH3, TH17, Th9, or TFH which secrete different cytokines to facilitate different types of immune responses.
  • Cytotoxic T cells TC cells, or CTLs destroy virally infected cells and tumour 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. Through 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 autoreactive T cells that escaped the process of negative selection in the thymus.
  • Treg cells Two major classes of CD4+ Treg cells have been described—natural 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 (CD1 1c+) 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 also known as Tr1 cells or Th3 cells may originate during a normal immune response.
  • the T cell of the invention may be any of the T cell types mentioned above, in particular a CTL.
  • Natural killer (NK) cells are a type of cytolytic cell which forms 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
  • CAR expressing cells described herein may be any of the cell types mentioned above.
  • CAR- expressing cells such as CAR-expressing T or NK cells 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).
  • a cell composition comprising CAR expressing T cells and/or CAR expressing NK cells of the CAR system described.
  • the cell composition may be made by transducing a blood-sample ex vivo with a nucleic acid according to the present invention.
  • CAR-expressing cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to the relevant cell type, such as T cells.
  • an immortalized cell line such as a T-cell line which retains its lytic function and could act as a therapeutic may be used.
  • CAR cells are generated by introducing DNA or RNA coding for the CARs by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • the present invention also provides a vector, or kit of vectors which comprises one or more CAR-encoding and/or intracellular signal molecule-encoding nucleic acid sequence(s).
  • a vector may be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses the first and second CARs.
  • 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 T cell.
  • the present invention also relates to a pharmaceutical composition containing a plurality of CAR-expressing cells, such as T cells or NK cells described above.
  • 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.
  • CAR system according to the first aspect of the invention to inhibit CAR signalling and thereby reduce or lessen any adverse toxic effects.
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and monitoring any toxic activity and adjusting the dose of the agent administered to the subject to provide acceptable levels of disease progression and toxic activity.
  • Monitoring the progression of the disease means to assess the symptoms associated with the disease over time to determine if they are reducing/improving or increasing/worsening.
  • Toxic activities relate to adverse effects caused by the CAR cells of the invention following their administration to a subject.
  • Toxic activities may include, for example, immunological toxicity such as cytokine release syndrome (CRS), neurotoxicity, biliary toxicity and/or respiratory distress syndrome.
  • immunological toxicity such as cytokine release syndrome (CRS)
  • CRS cytokine release syndrome
  • neurotoxicity biliary toxicity and/or respiratory distress syndrome.
  • the level of signalling through the CAR system and therefore the level of activation of CAR cells expressing the CAR(s), may be adjusted by altering the amount of agent(s) present, or the amount of time the agent(s) is/are present.
  • the level of CAR cell activation may be augmented by decreasing the dose of agent administered to the subject or decreasing the frequency of its administration.
  • the level of CAR cell activation may be reduced by increasing the dose of the agent, or the frequency of administration to the subject. Higher levels of cell activation are likely to be associated with reduced disease progression but increased toxic activities, whilst lower levels of CAR cell activation are likely to be associated with increased disease progression but reduced toxic activities.
  • the present invention also provides a method for treating and/or preventing a disease in a subject which subject comprises cells of the invention, which method comprises the step of administering an agent to the subject.
  • this method involves administering a suitable agent to a subject which already comprises CAR cells of the present invention.
  • the dose of agent administered to a subject, and/or the frequency of administration, may be altered in order to provide an acceptable level of both disease progression and toxic activity.
  • the specific level of disease progression and toxic activities determined to be 'acceptable' will vary according to the specific circumstances and should be assessed on such a basis.
  • the present invention provides a method for altering the activation level of the CAR cells in order to achieve this appropriate level.
  • the agent may be administered in the form of a pharmaceutical composition.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a CAR cell of the present invention for use in treating and/or preventing a disease.
  • the invention also relates to the use of a CAR cell of the present invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the present invention also provides an agent suitable for inhibiting a CAR system according to the first aspect of the invention for use in treating and/or preventing a disease.
  • the present invention also provides an agent for use in inhibiting a CAR system according to the first aspect of the invention in a CAR cell.
  • the invention also provides the use of an agent suitable for inhibiting a CAR system according to the first aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
  • METHOD OF TREATMENT T cells expressing a CAR and intracellular signalling molecule of the CAR system of the present invention are capable of killing cancer cells, such as multiple myeloma cells.
  • CAR- expressing T cells may either be created ex vivo either from a patient's own peripheral blood (1 st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2 nd party), or peripheral blood from an unconnected donor (3 rd party).
  • CAR T-cells may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T-cells.
  • CAR T-cells are generated by introducing DNA or RNA coding for the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • T cells expressing a CAR of the CAR of the present invention may be used for the treatment of a cancerous disease, in particular a plasma cell disorder or a B cell disorder which correlates with enhanced BCMA expression.
  • Plasma cell disorders include plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone plasmacytoma, extramedullar plasmacytoma, osteosclerotic myeloma (POEMS Syndrome) and heavy chain diseases as well as the clinically unclear monoclonal gammopathy of undetermined significance/smoldering multiple myeloma.
  • Plasma cell disorders include plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone plasmacytoma, extramedullar plasmacytoma, osteosclerotic myeloma (POEMS Syndrome) and heavy chain diseases as well as the clinically unclear monoclonal gammopathy of undetermined significance/smoldering multiple myeloma.
  • POEMS Syndrome osteosclerotic myeloma
  • the disease may be multiple myeloma.
  • B cell disorders which correlate with elevated BCMA expression levels are CLL (chronic lymphocytic leukemia) and non-Hodgkins lymphoma (NHL).
  • the bispecific binding agents of the invention may also be used in the therapy of autoimmune diseases like Systemic Lupus Erythematosus (SLE), multiple sclerosis (MS) and rheumatoid arthritis (RA).
  • SLE Systemic Lupus Erythematosus
  • MS multiple sclerosis
  • RA rheumatoid arthritis
  • the method of the present invention may be for treating a cancerous disease, in particular a plasma cell disorder or a B cell disorder which correlates with enhanced BCMA expression.
  • a method for the treatment of disease relates to the therapeutic use of a vector or T cell of the invention.
  • the vector or T cell 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 of the invention may cause or promote T-cell mediated killing of BCMA-expressing cells, such as plasma cells.
  • APRI L the extreme amino-terminus of APRI L was deleted to remove binding to proteoglycans.
  • a signal peptide was added to direct the nascent protein to the endoplasmic reticulum and hence the cell surface.
  • APRI L-based CAR systems For the generation of a APRI L-based CAR systems, three different configurations of the CAR were engineered as shown in Figure 4. All three CAR configurations had identical ecto- and transmembrane domains to the original APRI L CAR.
  • the original APRI L CAR is a third generation CAR with CD28 and OX40 co-stimulatory domain.
  • the first CAR system comprises a CAR (APRI L-HNG-TM-L-TetRB) having an intracellular domain bearing a linker and a first heterodimerization domain (TetRB).
  • the corresponding intracellular signalling molecule (TI P-L-CD28-OX40-z) of the CAR system comprises a second heterodimerization domain (TiP) fused via a linker to a signalling domain comprising two co-stimulatory domains (CD28, OX40) and ⁇ 3 ⁇ .
  • the second CAR system comprises a CAR (APRI L-HNG-TM-CD28-L-TetRB) having an intracellular domain comprising a CD28 costimulatory domain as well as a linker and a first heterodimerization domain (TetRB).
  • the corresponding intracellular signalling molecule (TI P-L-OX40-z) of the CAR system comprises a second heterodimerization domain (TiP) fused via a linker to a signalling domain comprising one co-stimulatory domain (OX40) and ⁇ 3 ⁇ .
  • the third CAR system comprises a CAR (APRIL-HNG-TM-CD28-OX40-L-TetRB) having an intracellular domain bearing both OX40 and CD28 costimulatory domains in addition to the linker and the first heterodimerization domain (TetRB).
  • the corresponding intracellular signalling molecule (TIP-L-z) of this CAR system comprises a second heterodimerization domain (TiP) fused via a linker to a signalling domain comprising ⁇ 3 ⁇ .
  • the CAR may comprise of a domain or a hinge, CH2 and CH3 domains of human lgG1 modified with the pva/a mutations described by Hombach et al (2010 Gene Ther. 17: 1206-1213).
  • the APRI L CAR and all APRIL CAR systems had the hinge from lgG1 as a spacer, connected to the CD28 transmembrane domain (CD28TM).
  • Example 2 Investigating the cytotoxic capacity of various APRI L CAR systems using flow cytometry
  • CD56-depleted CAR bearing T-cells were used to validate the cytotoxic capacity of the three CAR system configurations.
  • the flow cytometry assay was set-up six days after the T-cell transduction.
  • the T-cells were plated with the target cell lines at two effector-to-target cell ratios (1 :2 and 1 :4).
  • the target cell lines used for this experiment were SupT1 BCMA and MM 1 S, where 5x10 4 target cells were added per well.
  • the amount of T-cells added to each well varied depending the ratio of each condition with 2.5x10 4 and 1 .25x10 4 transduced T-cells added for the ratios 1 :2 and 1 :4, respectively.
  • the cells from the co-culture well were labelled for CD3, QBendI O and viability dye and interrogated in the MACsquant.
  • the results were normalised to the NT T-cells, where the number of the alive target cells in this condition is set as 100% survival.
  • the CAR system in which the CAR comprised both the OX40 and CD28 costimulatory domains in the intracellular domain shows better killing in the absence of the agent tetracycline than both the CAR system in which the co-stimulatory domains are shared between the CAR and the intracellular signalling molecule and the CAR system in which both co-stimulatory domains are on the intracellular signalling molecule.
  • Example 3 Investigating the cytotoxic capacity of various APRIL CAR systems using an IncuCvte ® assay
  • CD56-depleted CAR bearing T-cells were used to validate the cytotoxic capacity for the IncuCyte ® assay, which was then also set-up six days after the T-cell transduction.
  • the T-cells were cultured in the presence of the adherent cell line SKOV3, which had been transduced to express the antigen BCMA and a nuclear fluorescent protein mKate.
  • SKOV3 cells are better suited to the IncuCyte ® assay because they attach to the plate as a monolayer and the target cells do not require harvesting if T cells are removed from the plate.
  • the ratios implemented in this experiment were 8: 1 and 4: 1 , with 10 4 target cells per well.
  • the APRIL-HNG-CD28OX40z CAR which lacks a first and second heterodimerization domain showed no difference in cytotoxic capacity in the presence or absence of the agent (1600 nM concentration of tetracycline).
  • This experiment showed decreased percentage of cell survival over time, both with and without the addition of the agent, and resulted in a sigmoidal curve which plateaued after about 75 hours.
  • the CAR system comprising the first CAR configuration APRIL-HNG-CD28TM-L- TetRB and the intracellular signalling molecule TIP-L-CD28-OX40-Z showed a difference in cytotoxic capacity in the presence and absence of the agent tetracycline.
  • the top line represents the CAR system in the presence of the agent, whereas the line which shows a decreased percentage of cell survival over time represents the CAR system without the agent.
  • the CAR system comprising the second CAR configuration APRIL-HNG-CD28TM- CD28-L-TetRB and the intracellular signalling molecule TIP-L-OX40-Z showed a difference in cytotoxic capacity in the presence of the agent tetracycline compared to the absence of the agent tetracycline.
  • the CAR system comprising the third CAR configuration APRIL-HNG-CD28TM- CD28-OX40-L-TetRB and the intracellular signalling molecule TIP-L-z also showed a cytotoxic capacity difference in the presence of tetracycline compared to the absence of tetracycline.
  • the IncuCyte ® assay supports the results of the flow cytometry assay, where the percentage of cell killing in the absence of tetracycline is greater in the CAR system in which the CAR comprised both the OX40 and CD28 costimulatory domains in the intracellular domain than both a) the CAR system in which the co-stimulatory domains are shared between the CAR and the intracellular signalling molecule and b) the CAR system in which both co-stimulatory domains are on the intracellular signalling molecule.
  • Example 4 Investigating the T cell proliferation of various APRIL CAR systems following co-culture with target cells
  • Proliferation is a key feature of CAR-mediated responses which is often used to measure the efficacy of a CAR alongside cytotoxicity and cytokine secretion. Although 1 st generation CARs display good levels of cytotoxicity, they do not display good proliferative responses in vitro and fail to persist well in vivo. Proliferation is enhanced by the inclusion of co-stimulatory domains such as CD28, OX40 or 4-1 BB into the CAR endodomain.
  • CTV Cell Trace Violet
  • the T-cells were resuspended at 2x10 6 cells per ml in PBS, and 1 ul/ml of CTV was added. The T-cells were incubated the CTV for 20 minutes at 37°C. Subsequently, the cells were quenched by adding 5m L of complete media. After a 5 minutes incubation, the T-cells were washed and resuspended in 2ml of complete media. An additional 10 minute incubation at room temperature allowed the occurrence of acetate hydrolysis and retention of the dye.
  • CTV Cell Trace Violet
  • T-cells were co-cultured with antigen-expressing or antigen-negative target cells for seven days.
  • the assay was carried out in a 96-well plate in 0.2 ml total volume using 5x10 4 transduced T-cells per well and an equal number of target cells (ratio 1 : 1).
  • the T-cells were analysed by flow cytometry to measure the dilution of the CTV which occurs as the T-cells divide.
  • Figure 8 shows that constructs comprising the CAR system comprising the third CAR configuration (APRIL-HNG-CD28TM-CD28-OX40-L-TetRB) with the intracellular signalling molecule (TIP-L-z) has the highest absolute number of transduced T cells where the area under the curve is the largest.
  • the proliferation for the third CAR configuration is similar to the control (APRIL-HNG-CD28OX40z) in both donors.
  • the results also show that all CAR configurations responded to addition of the agent tetracycline, whereby proliferation is reduced.

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Abstract

La présente invention concerne un système de récepteur antigénique chimérique (CAR) comprenant : a) un CAR comprenant : i) un domaine de liaison à l'antigène de maturation des lymphocytes B (BCMA) qui comprend au moins une partie d'un ligand induisant la prolifération (APRIL); ii) un domaine transmembranaire; et iii) un domaine intracellulaire qui comprend un premier domaine d'hétérodimérisation, et b) une molécule de signalisation intracellulaire comprenant : i) un second domaine d'hétérodimérisation qui se dimérise au premier domaine d'hétérodimérisation, et ii) un domaine de signalisation.
PCT/GB2018/051636 2017-06-15 2018-06-14 Récepteur antigénique chimérique Ceased WO2018229492A1 (fr)

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WO2020191346A1 (fr) 2019-03-21 2020-09-24 Regeneron Pharmaceuticals, Inc. Combinaison d'inhibiteurs de la voie il-4/il-13 et d'ablation de plasmocytes pour traiter une allergie
US10919951B2 (en) 2013-10-10 2021-02-16 Autolus Limited Chimeric antigen receptor
WO2023133509A3 (fr) * 2022-01-08 2023-08-31 Carogen Corporation Vaccins thérapeutiques à antigènes multiples pour traiter ou prévenir une infection chronique par le virus de l'hépatite b
WO2025160340A2 (fr) 2024-01-26 2025-07-31 Regeneron Pharmaceuticals, Inc. Immunosuppression combinée pour inhiber une réponse immunitaire et pour permettre une administration et une réadministration d'immunogène
WO2025160324A2 (fr) 2024-01-26 2025-07-31 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour utiliser des agents de déplétion des cellules plasmatiques et/ou des agents de déplétion des lymphocytes b pour supprimer une réponse d'anticorps anti-aav hôte et permettre la transduction et le redosage d'aav
WO2025184567A1 (fr) 2024-03-01 2025-09-04 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour redosage d'un aav à l'aide d'un anticorps antagoniste anti-cd40 pour supprimer une réponse d'anticorps anti-aav hôte

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10919951B2 (en) 2013-10-10 2021-02-16 Autolus Limited Chimeric antigen receptor
WO2020191346A1 (fr) 2019-03-21 2020-09-24 Regeneron Pharmaceuticals, Inc. Combinaison d'inhibiteurs de la voie il-4/il-13 et d'ablation de plasmocytes pour traiter une allergie
WO2023133509A3 (fr) * 2022-01-08 2023-08-31 Carogen Corporation Vaccins thérapeutiques à antigènes multiples pour traiter ou prévenir une infection chronique par le virus de l'hépatite b
WO2025160340A2 (fr) 2024-01-26 2025-07-31 Regeneron Pharmaceuticals, Inc. Immunosuppression combinée pour inhiber une réponse immunitaire et pour permettre une administration et une réadministration d'immunogène
WO2025160324A2 (fr) 2024-01-26 2025-07-31 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour utiliser des agents de déplétion des cellules plasmatiques et/ou des agents de déplétion des lymphocytes b pour supprimer une réponse d'anticorps anti-aav hôte et permettre la transduction et le redosage d'aav
WO2025184567A1 (fr) 2024-03-01 2025-09-04 Regeneron Pharmaceuticals, Inc. Procédés et compositions pour redosage d'un aav à l'aide d'un anticorps antagoniste anti-cd40 pour supprimer une réponse d'anticorps anti-aav hôte

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