WO2022200782A1 - Signalling system - Google Patents

Signalling system Download PDF

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WO2022200782A1
WO2022200782A1 PCT/GB2022/050720 GB2022050720W WO2022200782A1 WO 2022200782 A1 WO2022200782 A1 WO 2022200782A1 GB 2022050720 W GB2022050720 W GB 2022050720W WO 2022200782 A1 WO2022200782 A1 WO 2022200782A1
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signalling
domain
binding
car
binding domain
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PCT/GB2022/050720
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French (fr)
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Shaun CORDOBA
Shimobi ONUOHA
Simon Thomas
Evangelia KOKALAKI
Martin PULÉ
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Autolus Limited
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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 A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • 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
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a chimeric antigen receptor signalling system.
  • Chimeric antigen receptors are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. 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 1A).
  • scFv single-chain variable fragments
  • toxicities include immunological toxicity caused by sustained intense activation of the CAR T-cells resulting in a macrophage activation syndrome (MAS) and "On- target off-tumour" toxicity i.e. recognition of the target antigen on normal tissues.
  • MAS macrophage activation syndrome
  • 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.
  • On-target off-tumour toxicity has been reported with other CARs, for example a group of patients treated with a CAR against the renal cell carcinoma antigen CAIX developed unexpected and treatment limiting biliary toxicity.
  • Two fatalities have been reported with CAR studies: one patient died of a respiratory distress syndrome which occurred immediately post infusion of a large dose of 3rd generation anti-ERBB2 CAR T-cells; a further patient died in a different study after a possible cytokine storm following treatment of CLL with a second generation anti-CD19 CAR.
  • CAR T-cells do not have a half- life and one cannot cease administration and wait for the agent to breakdown/become excreted. CAR T-cells are autonomous and can engraft and proliferate. Toxicity can therefore be progressive and fulminant.
  • 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 to CAR T-cell therapy, 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. Secondly, it is not clear whether a suicide gene would help with some of the immune-toxicities described above: for instance by the time a macrophage activation syndrome had been triggered, it may well no longer need the CAR T- cells to perpetuate and the suicide gene would no longer be helpful. The more acute cytokine release syndromes probably occur too quickly for the suicide gene to work. There is thus a need for alternative methods for controlling CAR T-cells that are not associated with the disadvantages and problems mentioned above.
  • Figure 1 - a Schematic diagram illustrating a classical CAR.
  • FIG. 2 Figure 2 - Structures of TetR and TiP.
  • TiP can be seen engaged deep within the TetR homodimer associating with many of the residues tetracycline associates with.
  • Figure 6 - 0X40 Fab Tet-CAR Cytotoxicity time course. In each case 3 donors were used, at an E:T ratio of 8:1. Target cells were SKOV Red cells expressing CD19. 1600 nM tetracycline was added at 0 hours.
  • Figure 12 - CD28-41 BB Fab Tet CAR Cytotoxicity time course. In each case 3 donors were used, at an E:T ratio of 8:1. Target cells were SKOV Red cells expressing CD19. 1600 nM tetracycline was added at 48 hours.
  • FIG. 15 Comparison of FMC63-BBz, FMC63-Tet-BBz, and TIP-less-Tet-BBz
  • A) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Bars show mean ( ⁇ SD), n 4 donors from 1 experiment.
  • B) Median fluorescent intensity of CAR expression on the surface of RQR8+ cells as measured by staining with soluble, Fc-tagged CD19 protein. Bars show mean ( ⁇ SD), n 4 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with Tukey’s multiple comparisons between each group.
  • CARs contain an anti human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41 BB- ⁇ 3z endodomain.
  • TetCARs have a TetRB endodomain with 41 BB- ⁇ 3z as a separate protein, with or without TIP.
  • B) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ⁇ SD, n 5 donors from 2 independent experiments.
  • C) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8+ cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ⁇ SD, n 3 donors from 1 experiment. Unpaired T tests were used for statistical analysis.
  • FIG 19 A) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41 BB or CD28 endodomains, with a TIR-O ⁇ 3z or TIP-41 BB-O ⁇ 3z domains.
  • B) Killing of SupT1-CD19-GFP after 24 hours co-culture with CAR-T cells at 1 :1 E:T ratio. 100nM of minocycline was added to relevant wells. Data shows mean ⁇ SD, n 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group iminocycline (with Sidak’s multiple comparisons).
  • Figure 20 -IFN-y and IL-2 release after 24 hours of co-culture with SupT1-CD19 at 1 :1 E:T ratio ( ⁇ 100nM minocycline). Data shows mean ⁇ SD, n 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group iminocycline (with Sidak’s multiple comparisons).
  • FIG. 21 Overview of the standard CAR and Fab-TetCAR constructs used.
  • An ‘inert’ TetCAR was constructed through removal of the costimulation domains (41 BB and CD28) and inclusion of a ‘TIP-less’ O ⁇ 3z domain.
  • Figure 22 Killing of SupT1-CD19-GFP after 24 hours of co-culture with CAR-T cells at 1:4 E:T ratio.
  • a range of minocycline doses from 0.02-1600nM was added to relevant wells.
  • FIG. 24 - IFN-g and IL-2 release after 24 hours of co-culture with SupT1-CD19 at various doses of tetracycline or tigecycline. Data shows mean ⁇ SD, n 4 donors from 1 experiment.
  • Figure 26 - 28BB-Tet-z CARs were incubated overnight with 100nM minocycline then plated in 96-well plates.
  • FIG. 27 - A) Killing of SupT1-CD19-GFP after 24 hours or NALM6 after 48 hours of co culture with CAR-T cells at 1:1 - 1:32 E:T ratio. Data shows mean ⁇ SD, n 4 donors from 2 independent experiments.
  • Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group iminocycline (with Sidak’s multiple comparisons).
  • mice were randomly assigned on the basis of tumor burden to receive 5x106 non-transduced (NT), FMC-BBz or 28BB-Tet-z CAR T cells. Groups were further divided with some to receive 0.4mg minocycline i.p. every 1-2 days, starting either on day 0 or day 3.
  • C Geometric mean radiance (photons/s/cm2/sr) of NALM6-FLuc cells, in mice in all groups treated with NT, FMC-BBz (iminocycline) or 28BB-Tet-z CAR T cells (iminocycline).
  • n 4 mice group from 1 experiment. Table shows statistical analysis through one-way ANOVA with multiple comparisons between groups at each time point.
  • the present inventors have found that it is possible to separate the antigen-recognition and signalling components of a CAR to produce a system in which signalling can be rapidly inhibited/terminated despite continued binding of antigen to an antigen-recognition component of the CAR system.
  • This inhibition of signalling occurs in the presence of an agent, such as a small molecule, which inhibits the co-localisation and interaction which would otherwise occur between an extracellular antigen-binding component (referred to herein as the receptor component) and an intracellular signalling component of the CAR.
  • the present inventors have found that the use of a Fab format in the receptor component leads to improved stability and cytokine production. Furthermore, incorporation of a signalling domain in the receptor component has been found to improve cytokine production by transduced cells.
  • a chimeric antigen receptor (CAR) system comprising:
  • a first receptor component comprising:
  • a second receptor component comprising either:
  • an intracellular signalling component comprising:
  • binding of the first and second binding domains is disrupted by the presence of an agent, such that in the absence of the agent the heterodimeric receptor component and the signalling component heterodimerize and binding of the antigen binding domain to antigen results in signalling through the signalling domain, whereas in the presence of the agent the heterodimeric receptor component and the signalling component do not heterodimerize and binding of the antigen binding domain to antigen does not result in signalling through the signalling domain.
  • the second receptor component may further comprise a transmembrane domain and a first binding domain.
  • the first receptor component and/or the second receptor component may comprise a linker between the transmembrane domain and the first binding domain.
  • the linker may comprise or consist of a signalling domain.
  • the linker may comprise or consist of CD28 endodomain, 41 BB endodomain or 0X40 endodomain. In some cases the linker may be derived from the sequence of CD4.
  • the linker may comprise or consist of the sequence shown as SEQ ID NO: 3.
  • the first binding domain may comprise Tet Repressor Protein (TetR) or a variant thereof and the second binding domain may comprise TetR inducing Peptide (TiP, as described by Klotzsche et a/; The Journal of biological chemistry; 2005; 280(26); 24591-9) (TiP); or vice versa.
  • TetR Tet Repressor Protein
  • TiP TetR inducing Peptide
  • the agent may be tetracycline, doxycycline or minocycline or an analogue thereof.
  • the first binding domain may comprise two TetR domains.
  • the two TetR domains may be separated by a linker.
  • Each TetR domain may have a different affinity for the agent.
  • the first binding domain may comprise a single domain binder which binds both the binding domain on the other component and the agent.
  • heterodimerisation occurs via the binding of a single domain binder to a binding domain on the other component. Since the single domain binder also binds the agent, in the presence of agent the receptor component and intracellular signalling component disassociate and signalling cannot occur.
  • the single domain binder may be or comprise: 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.
  • the single domain binder may be or comprise a domain antibody (dAb), such as a VH or VL dAb.
  • dAb domain antibody
  • the second binding domain may be or comprise a peptide which binds to the single domain binder of the first binding domain, which binding is competitively inhibited by the agent.
  • the peptide may be identified as having the required binding affinities by for example peptide array or phage display using the single domain binder.
  • the peptide may be eluted by adding the particular agent.
  • the single domain binder may alternatively be positioned on the intracellular signalling component, i.e. , it may be part of the second binding domain.
  • the first binding domain may be or may comprise a peptide which binds to the single domain binder of the second binding domain, which binding is competitively inhibited by the agent as described above.
  • the single domain binder prefferably be positioned on the extracellular side of the cell membrane, meaning that two further configurations are possible: one with the single binding domain on the extracellular side of the receptor component and one with the single binding domain on the extracellular side of a transmembrane domain-containing signalling component.
  • the signalling domain of the signalling component may comprise a single endodomain selected from CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and 0X40 endodomain.
  • the signalling domain of the signalling component may comprise at least one of CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and 0X40 endodomain.
  • the CAR system of the first aspect of the invention may comprise a plurality of signalling components, each comprising a signalling domain and a second binding domain, wherein the second binding domains each recognise the same first binding domain but the signalling domains comprise different endodomains.
  • the plurality of signalling components may comprise a plurality of second binding domains, each of which independently recognises the first binding domain with different affinities.
  • the present invention provides a first or second receptor component suitable for use in the CAR system of the first aspect of the invention which comprises:
  • the present invention provides a nucleic acid sequence encoding the first or second receptor component according to the second aspect of the invention.
  • the present invention provides a nucleic acid sequence encoding a CAR system of the first aspect of the invention, wherein the first receptor component, second receptor component, and signalling component are co-expressed by means of self-cleaving peptides which are cleaved between the receptor components and the signalling component after translation.
  • the present invention provides a vector comprising a nucleic acid sequence according to the third to fourth aspects of the invention.
  • the present invention provides a retroviral vector or a lentiviral vector or a transposon comprising a vector according to the fifth aspect of the invention.
  • the present invention provides a T cell or NK cell comprising a nucleic acid according to the third to fourth aspects of the invention or a vector according to the fifth or sixth aspect of the invention.
  • the present invention provides a pharmaceutical composition comprising a plurality of T cells or NK cells according to the seventh aspect of the invention.
  • the present invention relates to a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the eighth aspect of the invention to a subject.
  • the method according to the ninth aspect of the invention may comprise the following steps: (i) isolation of a T cell or NK containing sample;
  • the method may involve administration of T cells/NK cells to a subject, which T cells/NK cells have been previously isolated from the subject and transduced/transfected with a nucleic acid sequence according to any of the third to fourth aspects of the invention or a vector according to the fifth or sixth aspect of the invention.
  • the method according to the ninth aspect of the invention may involve monitoring toxic activity in the subject and comprise the step of administering an agent for use in the CAR system of the first aspect of the invention to the subject to reduce adverse toxic effects.
  • 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 for use in the CAR system of the first aspect of the invention to the subject to provide acceptable levels of disease progression and/or toxic activity.
  • the disease may be cancer.
  • the present invention relates to the use of a pharmaceutical composition according to the eighth aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the present invention provides a kit which comprises a nucleic acid according to the third to fourth aspects of the invention or a vector according to the fifth or sixth aspect of the invention.
  • the present invention relates to a method for making a T or NK cell according to the seventh aspect of the invention, which comprises the step of introducing a nucleic acid sequence according to third to fourth aspect of the invention or the vector according to the fifth or sixth aspect of the invention into a T or NK cell.
  • the T or NK cell may be from a sample isolated from a subject.
  • the present invention relates to a method for inhibiting the CAR system according to the first aspect of the invention in a subject which comprises a T or NK cell according to the seventh aspect of the invention which method comprises the step of administering the agent to the subject.
  • the present invention therefore provides a 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 receptor component and signalling component.
  • an agent for example a small molecule, which prevents co-localisation of the receptor component and signalling component.
  • 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 toxi cities.
  • Classical CARs which are shown schematically in Figure 1, 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.
  • the binder is based upon a Fab fragment derived from a monoclonal antibody (mAb).
  • 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 41 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 present inventors outlined a CAR system in which signalling could be “switched-off” by the addition of an agent.
  • the present application describes an improvement to this system which leads to improved CAR stability and improved cytokine production upon activation of the CAR system. This improvement is based upon redesigning the receptor component to include a Fab-based antigen binding domain and the optional use of a signalling domain in the receptor component.
  • the present invention relates to a CAR system in which an antigen recognizing/antigen binding domain based on a Fab light chain or Fab heavy chain and transmembrane domain are provided on a first molecule (termed herein ‘first receptor component’), which localizes to the cell membrane.
  • the other Fab chain i.e. , heavy or light
  • second receptor component is provided on a second molecule (termed herein ‘second receptor component’) which heterodimerises to the first receptor component to form a heterodimeric receptor component comprising an antigen binding domain.
  • the intracellular signalling domain is provided on a second, intracellular molecule (termed herein ‘signalling component’).
  • the first receptor component comprises a first binding domain and the signalling component comprises a second binding domain which specifically binds to the first binding domain of the receptor component.
  • binding of the first binding domain to the second binding domain causes heterodimerization and co-localization of the receptor component and the signalling component.
  • antigen binds to the antigen binding domain of the receptor component there is signalling through the signalling component.
  • the second receptor component may also comprise a transmembrane domain and a first binding domain.
  • the first or second binding domain is also capable of binding a further agent in addition to the reciprocal binding domain.
  • the further agent may be, for example, a small molecule.
  • the binding between the agent and the first or second binding domain is of a higher affinity than the binding between the first binding domain and the second binding domain.
  • the agent when it is present it preferentially binds to the first or second binding domain and inhibits/disrupts the heterodimerization between the receptor component and the signalling component.
  • antigen binds to the antigen binding domain of the receptor component in the presence of the further agent there is no signalling through the signalling component.
  • the receptor component and signalling component 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 through the signaling component.
  • co-localization or ‘heterodimerization’ of the receptor and signalling components is analogous to ligation/recruitment of the signalling component to the receptor component via binding of the first binding domain of the receptor component and the second binding domain of the signalling component.
  • Antigen binding by the receptor component in the presence of the agent may be termed as resulting in ‘non-productive’ signalling through the signalling component. Such signalling does not result in cell activation, for example T cell activation. Antigen binding by the receptor component in the absence of the agent may be termed as resulting in ‘productive’ signalling through the signalling component. This signalling results in T-cell activation, triggering for example target cell killing and T cell activation.
  • Antigen binding by the receptor component in the absence of the agent may result in signalling through the signalling component which is 2, 5, 10, 50, 100, 1,000 or 10,000-fold higher than the signalling which occurs when antigen is bound by the receptor component in the presence of the agent.
  • Signalling through the signalling component 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.
  • PTKs protein tyrosine kinases
  • PIP2 protein tyrosine kinases
  • PKC protein kinase C
  • elevation of intracellular calcium ion concentration for example assaying levels of specific protein tyrosine kinases (PTKs), breakdown of phosphatidylinositol
  • IL-2 interleukin-2
  • the first binding domain, second binding domain and agent of the present CAR system may be any combination of molecules/peptides/domains which enable the selective co-localization and dimerization of the receptor component and signalling component in the absence of the agent.
  • the first binding domain and second binding domain are capable of specifically binding.
  • the signalling system of the present invention is not limited by the arrangement of a specific dimerization system.
  • the receptor component may comprise either the first binding domain or the second binding domain of a given dimerization system so long as the signalling component comprises the corresponding, complementary binding domain which enables the receptor component and signalling component to co-localize in the absence of the agent.
  • the first binding domain and second binding 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 is a molecule, for example a small molecule, which is capable of specifically binding to the first binding domain or the second binding domain at a higher affinity than the binding between the first binding domain and the second binding domain.
  • the binding system may be based on a peptide: peptide binding domain system.
  • the first or second binding 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 binding domain or the second binding domain with at least 10, 20, 50, 100, 1000 or 10000-fold greater affinity than the affinity between the first binding domain and the second binding domain.
  • the agent may be any pharmaceutically acceptable molecule which preferentially binds the first binding domain or the second binding domain with a higher affinity than the affinity between the first binding domain and the second binding 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.
  • TetR Tet repressor
  • TiP TetR interacting protein
  • tetracycline system Klotzsche et a/.; J. Biol. Chem. 280, 24591-24599 (2005); Luckner et ai; 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 protein also referred to as TetRB herein
  • Klotzsche etal. (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 ai; as above).
  • the first binding domain of the receptor component may be TetR or TiP, providing that the second binding domain of the signalling component is the corresponding, complementary binding partner.
  • the second binding domain of the signalling component is TiP.
  • the first binding domain of the receptor component is TiP
  • the second binding domain of the signalling component is TetR.
  • first binding domain or second binding domain may comprise the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2:
  • the receptor component may comprise a linker between the transmembrane domain and the first binding domain (TetR).
  • the linker enables TetR to homodimerize with a TetR from a neighbouring receptor component and orient in the correct direction.
  • the linker may be the sequence shown as SEQ ID NO: 3.
  • the linker may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as the sequence shown as SEQ ID NO: 3.
  • the linker may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 3 providing it provides the function of enabling TetR to homodimerize with a TetR from a neighbouring receptor component and orient in the correct direction.
  • the linker may comprise or consist of a signalling domain, such as the CD28 endodomain, 41 BB endodomain or 0X40 endodomain.
  • TetR is xenogenic and immunogenic.
  • the TetR sequence may therefore be a variant which is less immunogenic but retains the ability to specifically bind TiP.
  • the agent may be tetracycline, doxycycline, minocycline or an analogue thereof.
  • An analogue refers to a variant of tetracycline, doxycycline or minocycline which retains the ability to specifically bind to TetR.
  • binding domains and agents which may be used in the present CAR system are known in the art.
  • the CAR system may use a streptavidin/biotin- based binding system.
  • 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 heterodimerisation between the streptavidin domain and the biotin mimic domain.
  • the biotin mimic may bind streptavidin with for example with a Kd of 1nM 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. Table 1. Biotin mimicking peptides.
  • the biotin mimic may be selected from the following group: Streptagl I , Flankedccstreptag and ccstreptag.
  • the streptavidin domain may comprise streptavidin having the sequence shown as SEQ ID No. 11 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 b-strands, which fold to give an antiparallel beta barrel tertiary structure.
  • a biotin binding-site is located at one end of each b-barrel.
  • Four identical streptavidin monomers i.e. four identical b-barrels
  • the biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from neighbouring subunit. In this way, each subunit contributes to the binding site on the neighbouring subunit, and so the tetramer can also be considered a dimer of functional dimers.
  • the 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. 11 , 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. 11 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.
  • Variany streptavidin may, for example, comprise all 8 of these residues.
  • variant streptavidin is present in the binding domain as a dimer orteramer, it may also comprise Trp120 which is involved in biotin binding by the neighbouring subunit.
  • Small molecules agents which disrupt protein-protein interactions have long been developed for pharmaceutical purpose (reviewed by Vassilev et a/; 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 binding 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.
  • Second binding domains which competitively bind to the same first binding domain as the agents described above, and thus may be used to co-localise the receptor component and signalling component of the present signalling system in the absence of the agent, may be identified using techniques and methods which are well known in the art. For example such second binding domains may be identified by display of a single domain VHH library.
  • the first binding domain and/or second binding domain of the present 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 receptor component and signalling component.
  • 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 the CAR system of the first aspect of the invention, which method comprises the step of administering the agent.
  • administration of the agent results in a disruption of the co-localization between the receptor component and the signalling component, such that signalling through the signalling component is inhibited even upon binding of antigen to the antigen binding domain.
  • the first and second binding 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 binding domain or the second binding domain with a higher affinity than binding affinity between the first and second binding domains
  • co-localization of the receptor and signalling components 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 antigen without completely inhibiting it.
  • the specific concentrations of agent will differ depending on the level of signalling required and the specific binding 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 first or second binding domain of the CAR system of the present invention may comprise a single domain binder.
  • a “single domain binder” 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 binder may have an antibody-like binding site which binds to the agent.
  • the single domain binder may comprise one or more complementarity determining regions (CDRs).
  • the single domain binder may comprise three CDRs
  • the single domain binder may lack disulphide bonds.
  • the single domain binder 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 domain binder 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 binder used in the CAR system of the invention may be or comprise such a VHH domain or derivative thereof.
  • non-immunoglobulin single domain binders have also been designed and characterised, including those based on natural and synthetis protein scaffolds.
  • fibronectin-derived Adnectins/monobodies are characterized by an Ig-like b-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 binder 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 binder may be less than 150 amino acids in length, for example, less than 140, 130 or 120 amino acids in length.
  • the single domain binder may be approximately 110 amino acids in length, for example from 105-115 amino acids in length
  • the single domain binder 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
  • abdurin/ nanoantibody a centyrin
  • centyrin an alphabody or a nanofitin.
  • 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 antibodies found in camelids; i.e. VHH fragments.
  • Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained.
  • 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.
  • single domain antibodies can be made from common murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens.
  • a problem with this approach is that the binding region of common IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity.
  • An Affibody molecule consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa.
  • the original Affibody protein scaffold was designed based on the Z domain (the immunoglobulin G binding domain) of protein A. I n contrast to antibodies, Affibody molecules are composed of alpha helices and lack disulfide bridges.
  • Affibody molecules with unique binding properties are acquired by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain. Lately, amino acids outside of the binding surface have been substituted in the scaffold to create a surface entirely different from the ancestral protein A domain.
  • Specific affibody molecules binding a desired target protein can be “fished out” from libraries of variants, using phage display.
  • Fibronectin artificial antibody scaffold are antibody mimics based on the scaffold of the fibronectin type III domain.
  • Anticalins are derived from human lipocalins, a family of naturally binding proteins. Anticalins have a size of about 180 amino acids and a mass of about 20 kDa.
  • Affilin proteins are structurally derived from human ubiquitin (historically also from gamma-B crystallin). Affilin proteins are constructed by modification of surface-exposed amino acids of these proteins and isolated by display techniques such as phage display and screening. Like other antibody mimetics they resemble antibodies in their affinity and specificity to antigens but not in structure,
  • Designed ankyrin repeat proteins are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin proteins, one of the most common classes of binding proteins in nature, which are responsible for diverse functions such as cell signalling, regulation and structural integrity of the cell. DARPins consist of at least three repeat motifs proteins, and usually consist of four or five. Their molecular mass is about 14 or 18 kDa (kilodaltons) for four- or five-repeat DARPins, respectively. iBodies are modular synthetic antibody mimetics based on hydrophilic polymers.
  • An affimer is a small, highly stable protein engineered to display peptide loops which provide a high affinity binding surface for a specific target protein. It is a protein of low molecular weight, 12-14 kDa, derived from the cysteine protease inhibitor family of cystatins.
  • Fynomers are small binding proteins derived from the human Fyn SH3 domain. Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. Fynomers have neither have cysteine residues nor disulfide bonds and are approximately 7 kDa in size.
  • Abdurins are a new class of antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Abdurins are small (12.5kDa) proteins which retain a portion of the native Fc receptor binding motif which binds to the neonatal Fc receptor to increase protein half-life and tumour uptake.
  • Centyrins are a new class of alternative scaffold protein based on a consensus fibronectin domain.
  • Alphabodies also known as Cell-Penetrating Alphabodies or CPAB, are small 10 kDa antibody mimetic proteins engineered to bind to a variety of antigens. Alphabodies are different from many other antibody mimetics in their ability to reach and bind to intracellular protein targets. Their single chain alpha-helical structure is designed by computer modelling, inspired by naturally existing coiled-coil protein structures.
  • Affitins are antibody mimetics structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaeal domain. By randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards the target of interest.
  • heterodimerisation of the receptor and signalling component may occur through the binding of the single domain binder with a single domain binder-interacting peptide (sdbiP).
  • the sdbiP may, for example, be between 8-30, for example 10-20 amino acids in length.
  • Suitable sdbiPs may be generated and identified using peptide display methods such as phage display, CIS display, ribosome display and mRNA display (Ullman et al (2011) 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 sdbiP 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 sdbiP may be incorporated into the receptor molecule (first and third embodiments) or the signalling molecule (second and fourth embodiments) and tested to make sure the binding properties of the sdbiP are retained.
  • sdbiP which specifically bind to a methotrexate dAb or a caffeine dAb are described in WO2017/137758 (which is incorporated herein by reference).
  • the agent may be a small molecule such as: a steroid, methotrexate, caffeine, cocaine or an antibiotic.
  • a steroid is an organic compound with four “fused” carbon rings.
  • examples of steroids include the dietary lipid cholesterol, the sex hormones estradiol and testosterone and the anti inflammatory drug dexamethasone.
  • the steroid core structure is composed of seventeen carbon atoms, bonded in four "fused" rings: three six-member cyclohexane rings (rings A, B and C in the first illustration) and one five-member cyclopentane ring (the D ring).
  • Steroids vary by the functional groups attached to this four-ring core and by the oxidation state of the rings.
  • Sterols are forms of steroids with a hydroxyl group at position three and a skeleton derived from cholestane.
  • Methotrexate formerly known as amethopterin, is an antimetabolite and antifolate drug.
  • Caffeine is a purine, a methylxanthine alkaloid. It is a stimulant of the central nervous system, but is generally recognised as safe (GRAS) by the Food and Dru Administration. Toxic doses, over 10 grams per day for an adult, are much higher than typical dose of under 500 milligrams per day. A cup of coffee contains 80-175 mg of caffeine.
  • Cocaine also known as benzoylmethylecgonine or coke
  • Cocaine is a strong stimulant.
  • Various analogs of cocaine are known including stereoisomers; 3b-phenyl ring substituted analogues; 2b-substituted analogues; N-modified analogues of cocaine; 3b- carbamoyl analogues; 3b-alkyl-3-benzyl tropanes; 6/7-substituted cocaines; 6-alkyl-3-benzyl tropanes; and piperidine homologues.
  • Antibiotics or antibacterials are a type of antimicrobial used in the treatment and prevention of bacterial infection.
  • Antibacterial antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity. Most target bacterial functions or growth processes. Those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins), or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones, and sulfonamides) have bactericidal activities. Those that target protein synthesis (macrolides, lincosamides and tetracyclines) are usually bacteriostatic (with the exception of bactericidal aminoglycosides).
  • “Narrow-spectrum” antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad-spectrum antibiotics affect a wide range of bacteria.
  • cyclic lipopeptides such as daptomycin
  • glycylcyclines such as tigecycline
  • oxazolidinones such as linezolid
  • lipiarmycins such as fidaxomicin
  • the agent may, for example, be an antibiotic such as tetracycline, or a derivative thereof such as doxycycline or minocycline.
  • the present invention provides a receptor component composed of a first receptor component comprising a Fab light chain and a second receptor component comprising a Fab heavy chain, such that the first and second receptor components heterodimerise to form a receptor component comprising an antigen-binding domain.
  • the first receptor component may instead comprise a Fab heavy chain
  • the receptor component further comprises, an optional spacer domain, a transmembrane domain and a first binding domain.
  • the receptor component localises to the cell membrane.
  • the antigen-binding domain of the molecule is orientated on the extracellular side of the membrane and the first binding domain is localised to the intracellular side of the membrane.
  • the receptor component therefore provides the antigen-binding function of the CAR system of the present invention.
  • the receptor component may also include one or more co-stimulatory domains, such as CD28 endodomain, 4-1 BB endodomain or 0X40 endodomain.
  • the co stimulatory domain will be intracellular, to allow for signalling.
  • the co-stimulatory domain may be positioned such that it is proximal to the internal surface of the membrane. The position of the domain relative to the membrane may be adjusted via any suitable method, for example by the use of spacer sequences.
  • the antigen-binding domain is the portion of a classical CAR which recognizes antigen.
  • the antigen-binding is located within the receptor component.
  • the antigen binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid (dAb); VHH antigen binding domains; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • the antigen binding comprises a Fab fragment of, for example, a monoclonal antibody.
  • a FabCAR comprises two chains: one having an antibody-like light chain variable region (VL) and constant region (CL)(also referred to herein as a Fab light chain); and one having a heavy chain variable region (VH) and constant region (CH) (also referred to herein as a Fab heavy chain). Association between the CL and CH causes assembly of the receptor component. In some cases, association between the two chains results in the formation of disulphide bonds.
  • Either chain may also comprise a transmembrane domain and a first binding domain as defined herein. In some circumstances, both chains my comprise a transmembrane domain and a first binding domain as defined herein.
  • the first binding domain may be connected to the transmembrane domain via a linker.
  • the linker may comprise or consist of a co-stimulatory domain, such as CD28 endodomain, 4-1 BB endodomain or 0X40 endodomain. In some cases the linker may be derived from the sequence of CD4.
  • the linker may comprise or consist of the sequence shown as SEQ ID NO: 3.
  • the two chains of the receptor component may have the general structure:
  • VL - CL spacer- transmembrane domain - first binding domain
  • VH - CH - spacer - transmembrane domain - first binding domain VL - CL - spacer - transmembrane domain - first binding domain
  • VL - CL - spacer- transmembrane domain - first binding domain VL - CL - spacer- transmembrane domain - first binding domain
  • the antigen binding domain is made up of a VH from one polypeptide chain and a VL from another polypeptide chain.
  • the two chains may also include a spacer between the CH/CL domains and the transmembrane domain.
  • the polypeptide chains may comprise a linker between the VH/VL domain and the CH/CL domains.
  • the linker may be flexible and serve to spatially separate the VH/VL domain from the CH/CL domain.
  • tumour associated antigens are known, as shown in the following Table 1.
  • the antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.
  • the transmembrane domain is the sequence of a classical CAR that spans the membrane.
  • the transmembrane domain is located in the receptor component. It may comprise a hydrophobic alpha helix.
  • the transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the receptor component of the CAR system of the present invention may comprise a signal peptide so that when the receptor component 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 signal peptide may be at the amino terminus of the molecule.
  • the signal peptide may comprise the sequence shown as SEQ ID NO: 12, 13 or 14 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR.
  • SEQ ID NO: 12 MGTSLLCWMALCLLGADHADG
  • the signal peptide of SEQ ID NO: 12 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.
  • SEQ ID NO: 13 MSLPVTALLLPLALLLHAARP
  • the signal peptide of SEQ ID NO: 13 is derived from lgG1.
  • SEQ ID NO: 14 MAVPTQVLGLLLLWLTDARC
  • the signal peptide of SEQ ID NO: 14 is derived from CD8.
  • the CAR system described herein may comprise a spacer sequence to connect the CH/CL domains with the transmembrane domain in the first and second receptor components.
  • 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.
  • amino acid sequences for these spacers are given below:
  • SEQ ID NO: 17 (human lgG1 hinge):
  • the receptor component may comprise a plurality of first binding domains and thus be capable of recruiting more than one signalling component.
  • the plurality of first binding domains may be present in a single intracellular domain of the receptor component.
  • the receptor component may comprise an appropriate number of transmembrane domains such that each first binding domain is orientated on the intracellular side of the cell membrane.
  • the receptor component may comprise 3, 5, 7, 9, 11, or more transmembrane domains. In this way, a single receptor component may recruit multiple signalling components amplifying signalling in response to antigen.
  • the first binding domains may each be variants which have a different affinity for the second binding domain of the signalling component.
  • the CAR system may comprise two or more receptor components each recognizing different antigens but comprising of the same intracellular first binding domain.
  • Such a CAR system would be capable of recognizing multiple antigens. This might be useful for instance in avoiding tumour escape.
  • the first binding domains of the receptor components differ in residues which dictate their affinity for the second binding domain of the signalling component. In this way, a CAR system can be tuned such that signalling in response to one antigen is greater or lesser than the response to another. This might be useful for instance when targeting two tumour antigens simultaneously but one is expressed at a higher density than the other. Response to this antigen could be tuned down to avoid toxicity caused by over-stimulation.
  • Methods suitable for altering the amino acid residues of the first or second binding domain such that the binding affinity between the two domains is altered are known in the art and include substitution, addition and removal of amino acids using both targeted and random mutagenesis.
  • Methods for determining the binding affinity between a first binding domain and a second binding domain are also well known in the art and include bioinformatics prediction of protein-protein interactions, affinity electrophoresis, surface plasma resonance, bio-layer interferometry, dual polarisation interferometry, static light scattering and dynamic light scattering.
  • the present invention also provides a signalling component comprising a signalling domain and a second binding domain.
  • the signalling component is a soluble molecule and thus localises to the cytoplasm when it is expressed in a cell, for example a T cell.
  • the intracellular signalling domain is the signal-transmission portion of a classical CAR.
  • the intracellular signalling domain (signalling domain) is located in the signalling component.
  • the membrane- bound, receptor component and the intracellular signalling component are brought into proximity. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell.
  • the signalling domain of the signalling component is analogous to the endodomain of a classical CAR molecule.
  • CD3-zeta endodomain 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.
  • chimeric CD28 and 0X40 can be used with CD3- Zeta to transmit a proliferative / survival signal, or all three can be used together (illustrated in Figure 1B).
  • the signalling component described herein comprises a signalling domain, it may comprise the CD3-Zeta endodomain alone, the CD3-Zeta endodomain with that of either CD28 or 0X40 or the CD28 endodomain and 0X40 and CD3-Zeta endodomain.
  • the signalling component of a CAR system may comprise the sequence shown as SEQ ID NO: 20, 21 or 22 or a variant thereof having at least 80% sequence identity.
  • a variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 20, 21 or 22, provided that the sequence provides an effective intracellular signalling domain.
  • the signalling system according to the first aspect of the present invention may comprise a plurality of signalling components, each comprising a signalling domain and a second binding domain, wherein each second binding domain is bound by the same first binding domain of the receptor component but the signalling domains comprise different endodomains. In this way, multiple different endodomains can be activated simultaneously. This is advantageous over a compound signalling domain since each signalling domain remains unencumbered from other signalling domains.
  • each signalling component comprises a second binding domain which differs in residues which alter their affinity to the first binding domain of the receptor component
  • the signalling components comprising different signalling domains ligate to the first binding domain with differing kinetics. This allows greater control over the signalling in response to antigen-binding by the receptor component as different signalling components are recruited to the receptor component in varying kinetics/dynamics. This is advantageous since rather than a fixed equal ratio of signal transmitted by a compound endodomain, an optimal T-cell activation signal may require different proportions of different immunological signals.
  • the present invention further provides a nucleic acid encoding the first or second receptor component of the second aspect and/or a nucleic acid encoding a signalling component.
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
  • 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.
  • oligonucleotides 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.
  • 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.
  • the nucleic acid of the invention may be a nucleic acid which encodes both the receptor component and the signalling component.
  • the nucleic acid may produce a polypeptide which comprises the receptor component and the signalling component 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 receptor component and the signalling component without the need for any external cleavage activity.
  • FMDV Foot-and-Mouth disease virus
  • the nucleic acid may produce a polypeptide which comprises the sequence shown as SEQ ID NO: 25.
  • SEQ ID NO: 25 The sequence shown as SEQ ID NO: 25.
  • a polypeptide sequence comprising the sequence shown as SEQ ID NO: 25 may further comprise an antigen binding domain sequence positioned immediately after, i.e., C-terminally to, the methionine at position 652 of SEQ ID NO: 25 (shown in bold).
  • the antigen binding domain sequence may be positioned between the methionine at position 652 and the serine at position 653 of SEQ ID NO: 25.
  • Any antigen binding domain may be included, for example an scFv, as described herein.
  • the co-expressing 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 a nucleic acid encoding the receptor component of the second aspect and/or a nucleic acid encoding a signalling component.
  • the present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) encoding a first and/or second receptor component of the second aspect and/or signalling component.
  • a vector may be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses the receptor component and signalling component of the CAR system according to the first aspect of the invention.
  • 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 or a NK cell.
  • the present invention also relates to an immune cell comprising the CAR system according to the first aspect of the invention.
  • the cytolytic immune cell may comprise a nucleic acid or a vector of the present invention.
  • the cytolytic immune cell may comprise a receptor component and a signalling component of the present invention.
  • the cytolytic immune cell may comprise at least one signalling component of the present invention.
  • the cytolytic immune cell may comprise one, two, three, four, five, up to a plurality of signalling components of the present invention.
  • the cytolytic immune cell may comprise at least one receptor component of the present invention.
  • the cytolytic immune cell may comprise one, two, three, four, five, up to a plurality of receptor components of the present invention.
  • Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. 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.
  • Cytolytic T cells TC cells, or CTLs 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. 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 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.
  • Natural Killer Cells are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.
  • LGL large granular lymphocytes
  • the CAR cells of the invention may be any of the cell types mentioned above.
  • T or NK cells expressing the molecules of the CAR system according to the first aspect of 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).
  • T or NK cells expressing the molecules of the CAR system according to the first aspect of the invention may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells.
  • an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.
  • CAR cells are generated by introducing DNA or RNA coding for the receptor component and signalling component by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • the CAR cell of the invention may be an ex vivo T or NK cell from a subject.
  • the T or NK cell may be from a peripheral blood mononuclear cell (PBMC) sample.
  • PBMC peripheral blood mononuclear cell
  • T or NK cells may be activated and/or expanded prior to being transduced with nucleic acid encoding the molecules providing the CAR system according to the first aspect of the invention, for example by treatment with an anti-CD3 monoclonal antibody.
  • the T or NK cell of the invention may be made by:
  • transduction or transfection of the T or NK cells with one or more a nucleic acid sequence(s) encoding the first and/or second receptor components according to the second aspect and/or a signalling component of the CAR system.
  • the T or NK cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide.
  • the present invention also provides a kit which comprises a T or NK cell comprising the CAR system according to the first aspect of the invention.
  • the present invention also relates to a pharmaceutical composition containing a plurality of cytolytic immune cells expressing the components of the CAR system of the first aspect of the invention.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a method for treating and/or preventing a disease which comprises the step of administering the cytolytic immune 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 cytolytic immune 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 cytolytic immune cells of the present invention.
  • cytolytic immune cells of the present invention 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 T or NK cell-containing sample may be isolated from a subject or from other sources, for example as described above.
  • the T or NK cells may be isolated from a subject’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and any toxic activity and administering an agent suitable for use in the 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, biliary toxicity and respiratory distress syndrome.
  • the level of signalling through the signalling system of the first aspect of the invention may be adjusted by altering the amount of agent present, or the amount of time the agent is 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.
  • 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 suitable for use in the CAR system according to the first aspect 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, 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.
  • the disease to be treated and/or prevented by the methods of the present invention may be an infection, such as a viral infection.
  • the methods of the invention may also be for the control of pathogenic immune responses, for example in autoimmune diseases, allergies and graft-vs-host rejection.
  • the methods may be for the treatment of 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 CAR cells 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 CAR cells and pharmaceutical compositions of present invention may be for use in the treatment and/or prevention of the diseases described above.
  • the CAR cells and pharmaceutical compositions of present invention may be for use in any of the methods described above.
  • a bicistronic construct was expressed in donor T cells as a single transcript which self-cleaves at two 2A sites to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of 0X40 and the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and T etR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab ( Figure 3).
  • constructs were expressed comprising (separated by 2A sites):
  • a signalling component which comprises TiP fused via a flexible linker to the endodomain of 0X40 and the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19- binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, a CD4L domain, and TetR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19- binding Fab;
  • a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, an 0X40 endodomain, and TetR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab;
  • a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, the endodomain of 0X40 and the endodomain of CD3-Zeta; and a second receptor component which comprises the VL and CL domains of the HD37 CD19- binding Fab.
  • a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of 0X40 and the endodomain of CD3-Zeta; and a receptor component which comprises an scFv based on the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and TetR.
  • T-cells were challenged with SKOV Red cells engineered to express CD19 in the absence of tetracycline or in the presence of 1600 nM of tetracycline, at an effectortarget (E:T) ratio of 8:1.
  • E:T effectortarget
  • T-cells challenged with SKOV Red cells expressing CD19 were activated in the absence of tetracycline, but activation is rapidly inhibited in the presence of tetracycline with activation fully inhibited in the presence of 1600nM of tetracycline ( Figures 6 and 7). Cytokine production was also investigated ( Figure 8). Locating the 0X40 domain on the receptor component resulted in
  • a bicistronic construct was expressed in donor T cells as a single transcript which self-cleaves at two 2A sites to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD28 and the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and T etR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, a five amino acid spacer, and TetR (Figure 9).
  • constructs comprising the following in addition to a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD28 and the endodomain of CD3-Zeta:
  • a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, 41 BB endodomain, and TetR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, 41 BB endodomain, and TetR;
  • a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, the endodomain of 41 BB and the endodomain of CD3-Zeta; and a second receptor component which comprises the VL and CL domains of the HD37 CD19- binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, the endodomain of 41 BB and the endodomain of CD3-Zeta.
  • a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD28 and the endodomain of CD3-Zeta; and a receptor component which comprises an scFv based on the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and TetR.
  • T-cells were challenged with SKOV Red cells engineered to express CD19 in the absence of tetracycline or in the presence of 1600 nM of tetracycline, at an effectortarget (E:T) ratio of 8:1.
  • E:T effectortarget
  • T-cells challenged with SKOV Red cells expressing CD19 were activated in the absence of Tetracycline, but activation is rapidly inhibited in the presence of tetracycline with activation fully inhibited in the presence of 1600nM of tetracycline ( Figure 12 and 13). Cytokine production was also investigated (Figure 8).
  • Example 3 - Fab format and TetR attached CD28 co-stimulation improves TetCAR activity.
  • TetCAR variants were tested as before. All TetCAR variants induced similar cytotoxicity, although here the inhibition of cytotoxicity upon addition of minocycline was less pronounced and only BB-Tet-z CAR was significantly inhibited (Figure 19B). However, when using SupT 1.CD19-eGFP as targets, repositioning of the co-stimulatory domains efficiently restored cytokine secretion to the levels seen in the control CAR ( Figure 20): for instance, IFN-y secretion with 28BB-Tet-z was 27,966pg/ml ( ⁇ 17323), matching the IFN-g secreted by the control CAR (26,803pg/ml ⁇ 14667).
  • IL-2 secretion was also highest in the 28BB-Tet-z CAR (12,178pg/ml ⁇ 6615), reaching a higher level than even the control CAR (7984pg/ml ⁇ 3577).
  • the BB-Tet-z CAR produced diminished but still detectable IFN-g
  • IL-2 secretion was almost undetectable in these CARs compared to the variants containing CD28-TetR.
  • the addition of the extra 41 BB domain to the TIP-tail did not further increase IFN-g or IL-2 secretion.
  • both TetCARs showed a similar dose-dependent reduction in both IFN-g and IL-2 with increasing concentrations of minocycline, fully inhibiting cytokine secretion at concentrations >6.25nM.
  • the IC-'SO for 28- Tet-z and 28BB-Tet-z were 0.21 nM and 0.24nM for IFN-g and 0.34nM and 0.44nM for IL-2 secretion.
  • secretion of a number of other cytokines by 28BB-Tet- z was assessed from 2 representative donors.
  • Minocycline induces rapid and reversible inhibition of TetCAR signalling
  • IL-2 secretion was assessed 1-5 hours after co-culture with SupT1-CD19 targets. Each hour, 100nM of minocycline was added to relevant wells to determine the time scale necessary for inhibition of cytokine secretion (Figure 25). As expected, the control CAR was unaffected by minocycline addition and induced detectable IL-2 secretion after 3 hours. This was mirrored by 28BB-Tet-z in the absence of minocycline, however addition of minocycline at different time points was able to inhibit further cytokine secretion within 2-3 hours.
  • 28-Tet-z and 28BB-Tet-z CAR T cells are highly functional compared with a gold-standard CAR
  • a feature of CAR T cell activation is proliferation in response to relevant targets. This was assessed by culturing the CARs with mitomycin C-treated target cell lines for 7 days (Figure 27B).
  • Targets used in this assay were SupT1-CD19-GFP, NALM6 and Raji, which all express CD19, and lastly a Raji-cell line in which CD19 expression had been knocked out.
  • both Tet-CARs proliferated in response to SupT1-CD19 to the same extent as the control CAR.
  • proliferation of the TetCARs was significantly lower in response to NALM6 targets than SupT1-CD19-GFP.
  • 28BB-Tet-CAR can be functionally regulated by minocycline in vivo
  • NSG mice were engrafted with NALM6 engineered to express firefly luciferase (NALM6-Fluc); 4 days later, different cohorts were treated with either 5x10 6 NT, FMC63-BBz, or 28BB-Tet-z CAR T cells, with or without minocycline.
  • An additional cohort of 28BB-T et-z treated mice were treated with minocycline 3 days after T cell transfer, during the peak of the initial anti-tumour response (Figure 32A).
  • Minocycline was given at a dose of ⁇ 16mg/kg (0.4mg per mouse) i.p every 1-2 days.

Abstract

The present invention provides a chimeric antigen receptor (CAR) system comprising: (i) a first receptor component comprising: a Fab light chain or a Fab heavy chain; a transmembrane domain; and a first binding domain; (ii) a second receptor component comprising either: a Fab heavy chain when the first receptor component comprises a Fab light chain; or a Fab light chain when the first receptor component comprises a Fab heavy chain; such that the first and second receptor components heterodimerise to form a heterodimeric receptor component comprising an antigen binding domain; and (ii) an intracellular signalling component comprising: a signalling domain; and a second binding domain which specifically binds the first binding domain; wherein, binding of the first and second binding domains is disrupted by the presence of an agent, such that in the absence of the agent the heterodimeric receptor component and the signalling component heterodimerize and binding of the antigen binding domain to antigen results in signalling through the signalling domain, whereas in the presence of the agent the heterodimeric receptor component and the signalling component do not heterodimerize and binding of the antigen binding domain to antigen does not result in signalling through the signalling domain.

Description

SIGNALLING SYSTEM
FIELD OF THE INVENTION
The present invention relates to a chimeric antigen receptor signalling system.
BACKGROUND TO THE INVENTION
Traditionally, antigen-specific T-cells have been generated by selective expansion of peripheral blood T-cells natively specific for the target antigen. However, it is difficult and quite often impossible to select and expand large numbers of T-cells specific for most cancer antigens. Gene-therapy with integrating vectors affords a solution to this problem as transgenic expression of Chimeric Antigen Receptor (CAR) allows generation of large numbers of T cells specific to any surface antigen by ex vivo viral vector transduction of a bulk population of peripheral blood T-cells.
Chimeric antigen receptors are proteins which graft the specificity of a monoclonal antibody (mAb) to the effector function of a T-cell. 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 1A).
The most common forms of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies which recognize a target antigen, fused via a spacer and a trans-membrane domain to a signalling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several 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.
A number of toxicities have been reported from CAR studies, and additional theoretical toxicities exist. Such toxicities include immunological toxicity caused by sustained intense activation of the CAR T-cells resulting in a macrophage activation syndrome (MAS) and "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.
On-target off-tumour toxicity has been reported with other CARs, for example a group of patients treated with a CAR against the renal cell carcinoma antigen CAIX developed unexpected and treatment limiting biliary toxicity. Two fatalities have been reported with CAR studies: one patient died of a respiratory distress syndrome which occurred immediately post infusion of a large dose of 3rd generation anti-ERBB2 CAR T-cells; a further patient died in a different study after a possible cytokine storm following treatment of CLL with a second generation anti-CD19 CAR.
These toxicities are very difficult to predict even with detailed animal studies or non-human primate work. Crucially, unlike small molecules and biologies, CAR T-cells do not have a half- life and one cannot cease administration and wait for the agent to breakdown/become excreted. CAR T-cells are autonomous and can engraft and proliferate. Toxicity can therefore be progressive and fulminant.
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 to CAR T-cell therapy, 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. Secondly, it is not clear whether a suicide gene would help with some of the immune-toxicities described above: for instance by the time a macrophage activation syndrome had been triggered, it may well no longer need the CAR T- cells to perpetuate and the suicide gene would no longer be helpful. The more acute cytokine release syndromes probably occur too quickly for the suicide gene to work. There is thus a need for alternative methods for controlling CAR T-cells that are not associated with the disadvantages and problems mentioned above.
DESCRIPTION OF THE FIGURES
Figure 1 - a) Schematic diagram illustrating a classical CAR. (b) to (d): Different generations and permutations of CAR endodomains: (b) initial designs transmitted ITAM signals alone through FcsRI-g or ΰϋ3z endodomain, while later designs transmitted additional (c) one or (d) two co-stimulatory signals in the same compound endodomain.
Figure 2 - Structures of TetR and TiP. (a) sequence of TiP (SEQ ID NO: 26) attached at the amino-terminus of an arbitrary protein; (b) Crystallography derived structure of TiP interacting with TetR (from PDB 2NS8 and Luckner et al (J. Mol. Biol. 368, 780-790 (2007)). TiP can be seen engaged deep within the TetR homodimer associating with many of the residues tetracycline associates with.
Figure 3 - Schematic of 0X40 Fab Tet CAR
Figure 4 - Expression of 0X40 Fab Tet CAR
Figure 5 - Expression of 0X40 Fab Tet CAR - MFI Ratio
Figure 6 - 0X40 Fab Tet-CAR: Cytotoxicity time course. In each case 3 donors were used, at an E:T ratio of 8:1. Target cells were SKOV Red cells expressing CD19. 1600 nM tetracycline was added at 0 hours.
Figure 7 - 0X40 Fab Tet-CAR: Cytotoxicity
Figure 8 - 0X40 Fab Tet-CAR: Cytokine production
Figure 9 - Schematic of 0X40 Fab Tet CAR
Figure 10 - Expression of CD28-41BB Fab Tet CAR Figure 11 - Expression of CD28-41BB Fab Tet CAR - MFI Ratio
Figure 12 - CD28-41 BB Fab Tet CAR: Cytotoxicity time course. In each case 3 donors were used, at an E:T ratio of 8:1. Target cells were SKOV Red cells expressing CD19. 1600 nM tetracycline was added at 48 hours.
Figure 13 - CD28-41 BB Fab Tet CAR: Cytotoxicity
Figure 14 - CD28-41BB Fab Tet CAR: Cytokine production
Figure 15 - Comparison of FMC63-BBz, FMC63-Tet-BBz, and TIP-less-Tet-BBz A) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Bars show mean (±SD), n=4 donors from 1 experiment. B) Median fluorescent intensity of CAR expression on the surface of RQR8+ cells as measured by staining with soluble, Fc-tagged CD19 protein. Bars show mean (±SD), n=4 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with Tukey’s multiple comparisons between each group. C) Schematic of the CAR constructs with 41 BB-ΰϋ3z endodomains. CARs contain an anti human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41 BB- ΰϋ3z endodomain. TetCARs have a TetRB endodomain with 41 BB-ΰϋ3z as a separate protein, with or without TIP.
Figure 16 - A) Schematic overview of TetCAR constructs containing 41BB-z or OΌ28-z endodomains. Antigen recognition is provided by the FMC63 scFv or Fab fragment. B) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ±SD, n=5 donors from 2 independent experiments. C) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8+ cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ±SD, n=3 donors from 1 experiment. Unpaired T tests were used for statistical analysis.
Figure 17 - Killing of SupT1-CD19-GFP after 24 hours co-culture with CAR-T cells at a 1:1 effectortarget ratio. 100nM of minocycline was added to relevant wells. Data shows mean percentage (±SD) of live cells compared to non-transduced (NT) control, n=5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA with Sidak’s multiple comparisons within each group iminocycline.
Figure 18 - IFN-y and IL-2 release after 24 hours of co-culture with SupT1-CD19-GFP at 1 :1 E:T ratio. Data shows mean ±SD, n=5 donors from 2 independent experiments. Statistical analysis was through two-way ANOVAs between the T etCAR groups at OnM minocycline (with Tukey’s multiple comparisons) or within these groups iminocycline (with Sidak’s multiple comparisons).
Figure 19 - A) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41 BB or CD28 endodomains, with a TIR-Oϋ3z or TIP-41 BB-Oϋ3z domains. B) Killing of SupT1-CD19-GFP after 24 hours co-culture with CAR-T cells at 1 :1 E:T ratio. 100nM of minocycline was added to relevant wells. Data shows mean ±SD, n=5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group iminocycline (with Sidak’s multiple comparisons).
Figure 20 -IFN-y and IL-2 release after 24 hours of co-culture with SupT1-CD19 at 1 :1 E:T ratio (±100nM minocycline). Data shows mean ±SD, n=5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group iminocycline (with Sidak’s multiple comparisons).
Figure 21 - Overview of the standard CAR and Fab-TetCAR constructs used. An ‘inert’ TetCAR was constructed through removal of the costimulation domains (41 BB and CD28) and inclusion of a ‘TIP-less’ Oϋ3z domain.
Figure 22 - Killing of SupT1-CD19-GFP after 24 hours of co-culture with CAR-T cells at 1:4 E:T ratio. A range of minocycline doses from 0.02-1600nM was added to relevant wells. Data shows mean % of live targets relative to an inert TetCAR control, +SD. n=4 donors from 1 experiment.
Figure 23 - IFN-g and IL-2 release after 24 hours of co-culture with SupT1-CD19 at various minocycline doses. Data shows mean ±SD, n=4 donors from 1 experiment.
Figure 24 - IFN-g and IL-2 release after 24 hours of co-culture with SupT1-CD19 at various doses of tetracycline or tigecycline. Data shows mean ±SD, n=4 donors from 1 experiment.
Figure 25 - FMC63-BBz or 28BB-Tet-z CARs were co-cultured with SupT1-CD19-GFP for 5 hours at a 2:1 E:T ratio. Supernatants were harvested from relevant wells every hour, and 100nM minocycline was also added to separate wells every hour. Data shows the mean (±SD) secretion of IL-2 at each time-point in groups that received minocycline at the beginning of the experiment, or every hour afterwards. n=4 donors from 2 independent experiments. Figure 26 - 28BB-Tet-z CARs were incubated overnight with 100nM minocycline then plated in 96-well plates. At 48, 24 and 2 hours before addition of SupT1-CD19 targets, plates were spun and relevant wells were resuspended in fresh media. Wash steps are indicated by “[W]”. SupT1-CD19 targets were then added at a 1:1 E:T ratio. Data shows mean (±SD) % of live targets relative to NT T cells (E) or mean (±SD) IL-2 secretion (F) after 24 hours. n=3 donors from 1 experiment.
Figure 27 - A) Killing of SupT1-CD19-GFP after 24 hours or NALM6 after 48 hours of co culture with CAR-T cells at 1:1 - 1:32 E:T ratio. Data shows mean ±SD, n=4 donors from 2 independent experiments. B) SupT1-CD19, NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co-cultured with CAR-T cells at 1 :2 E:T ratio for 7 days. To relevant wells, 400nM of minocycline was added on day 0. Graphs show mean (±SD) number of RQR8+ T cells (filled bars) or total CD3+ T cells (white bars) for each target n = 4 donors (SupT1-CD19, Raji and Raji-CD19KO) or n=3 (NALM6) from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group iminocycline (with Sidak’s multiple comparisons).
Figure 28 - Mean fluorescent intensity of Tim3 and Lag3 after 7 days coculture with SupT1- CD19 targets, ±400nM minocycline. Data shows geometric mean (±SD) in CD3+ T cells n = 4 donors, from 2 independent experiments.
Figure 29 - NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co cultured with CAR-T cells at 1 :2 E:T ratio for 7 days. To relevant wells, 400nM of minocycline was added on day 0. Data shows geometric mean fluorescent intensity of Tim3 and Lag3 (±SD) in CD3+ T cells n = 3-4 donors, from 2 independent experiments.
Figure 30 - Percentage of naive (CD62L+, CD45RA+), Tern (CD62L+, CD45RA-), Tern (CD62L-, CD45RA-) or Temra (CD62L-, CD45RA+) memory T cell populations after 7 days coculture with SupT1-CD19 targets, ±400nM minocycline. Data shows mean (±SD) in CD3+ T cells n = 4 donors, from 2 independent experiments.
Figure 31 - Percentage of naive (CD62L+, CD45RA+), Tern (CD62L+, CD45RA-), Tern (CD62L-, CD45RA-) or Temra (CD62L-, CD45RA+) memory T cell populations, after 7 days coculture with NALM6, Raji or Raji-CD19KO, ±400nM minocycline. Data shows mean (±SD) in CD3+ T cells n = 3-4 donors, from 2 independent experiments. Figure 32 - A) Overview of experiment. NSG mice were injected i.v. with 0.5x106 NALM6- FLuc tumor cells. At day 0, mice were randomly assigned on the basis of tumor burden to receive 5x106 non-transduced (NT), FMC-BBz or 28BB-Tet-z CAR T cells. Groups were further divided with some to receive 0.4mg minocycline i.p. every 1-2 days, starting either on day 0 or day 3. B) Bioluminescence radiance (photons/s/cm2/sr) of NALM6-FLuc tumors in mice in select groups. C) Geometric mean radiance (photons/s/cm2/sr) of NALM6-FLuc cells, in mice in all groups treated with NT, FMC-BBz (iminocycline) or 28BB-Tet-z CAR T cells (iminocycline). n= 4 mice group from 1 experiment. Table shows statistical analysis through one-way ANOVA with multiple comparisons between groups at each time point.
SUMMARY OF ASPECTS OF THE INVENTION
The present inventors have found that it is possible to separate the antigen-recognition and signalling components of a CAR to produce a system in which signalling can be rapidly inhibited/terminated despite continued binding of antigen to an antigen-recognition component of the CAR system. This inhibition of signalling occurs in the presence of an agent, such as a small molecule, which inhibits the co-localisation and interaction which would otherwise occur between an extracellular antigen-binding component (referred to herein as the receptor component) and an intracellular signalling component of the CAR.
In particular, the present inventors have found that the use of a Fab format in the receptor component leads to improved stability and cytokine production. Furthermore, incorporation of a signalling domain in the receptor component has been found to improve cytokine production by transduced cells.
Thus in a first aspect the present invention provides a chimeric antigen receptor (CAR) system comprising:
(i) a first receptor component comprising:
- a Fab light chain or a Fab heavy chain;
- a transmembrane domain; and
- a first binding domain;
(ii) a second receptor component comprising either:
- a Fab heavy chain when the first receptor component comprises a Fab light chain; or
- a Fab light chain when the first receptor component comprises a Fab heavy chain; such that the first and second receptor components heterodimerise to form a heterodimeric receptor component comprising an antigen binding domain; and (ii) an intracellular signalling component comprising:
- a signalling domain; and
- a second binding domain which specifically binds the first binding domain; wherein, binding of the first and second binding domains is disrupted by the presence of an agent, such that in the absence of the agent the heterodimeric receptor component and the signalling component heterodimerize and binding of the antigen binding domain to antigen results in signalling through the signalling domain, whereas in the presence of the agent the heterodimeric receptor component and the signalling component do not heterodimerize and binding of the antigen binding domain to antigen does not result in signalling through the signalling domain.
The second receptor component may further comprise a transmembrane domain and a first binding domain.
The first receptor component and/or the second receptor component may comprise a linker between the transmembrane domain and the first binding domain. The linker may comprise or consist of a signalling domain. The linker may comprise or consist of CD28 endodomain, 41 BB endodomain or 0X40 endodomain. In some cases the linker may be derived from the sequence of CD4. The linker may comprise or consist of the sequence shown as SEQ ID NO: 3.
The first binding domain may comprise Tet Repressor Protein (TetR) or a variant thereof and the second binding domain may comprise TetR inducing Peptide (TiP, as described by Klotzsche et a/; The Journal of biological chemistry; 2005; 280(26); 24591-9) (TiP); or vice versa. In this case the agent may be tetracycline, doxycycline or minocycline or an analogue thereof.
The first binding domain may comprise two TetR domains. The two TetR domains may be separated by a linker. Each TetR domain may have a different affinity for the agent.
The first binding domain may comprise a single domain binder which binds both the binding domain on the other component and the agent. In this system, heterodimerisation occurs via the binding of a single domain binder to a binding domain on the other component. Since the single domain binder also binds the agent, in the presence of agent the receptor component and intracellular signalling component disassociate and signalling cannot occur. The single domain binder may be or comprise: 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.
The single domain binder may be or comprise a domain antibody (dAb), such as a VH or VL dAb.
The second binding domain may be or comprise a peptide which binds to the single domain binder of the first binding domain, which binding is competitively inhibited by the agent. The peptide may be identified as having the required binding affinities by for example peptide array or phage display using the single domain binder. The peptide may be eluted by adding the particular agent.
The single domain binder may alternatively be positioned on the intracellular signalling component, i.e. , it may be part of the second binding domain. In this case, the first binding domain may be or may comprise a peptide which binds to the single domain binder of the second binding domain, which binding is competitively inhibited by the agent as described above.
It is also possible for the single domain binder to be positioned on the extracellular side of the cell membrane, meaning that two further configurations are possible: one with the single binding domain on the extracellular side of the receptor component and one with the single binding domain on the extracellular side of a transmembrane domain-containing signalling component.
The signalling domain of the signalling component may comprise a single endodomain selected from CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and 0X40 endodomain.
The signalling domain of the signalling component may comprise at least one of CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and 0X40 endodomain.
The CAR system of the first aspect of the invention may comprise a plurality of signalling components, each comprising a signalling domain and a second binding domain, wherein the second binding domains each recognise the same first binding domain but the signalling domains comprise different endodomains. The plurality of signalling components may comprise a plurality of second binding domains, each of which independently recognises the first binding domain with different affinities.
In a second aspect the present invention provides a first or second receptor component suitable for use in the CAR system of the first aspect of the invention which comprises:
- a Fab light chain or a Fab heavy chain;
- a transmembrane domain; and
- a first binding domain.
In a third aspect the present invention provides a nucleic acid sequence encoding the first or second receptor component according to the second aspect of the invention.
In a fourth aspect the present invention provides a nucleic acid sequence encoding a CAR system of the first aspect of the invention, wherein the first receptor component, second receptor component, and signalling component are co-expressed by means of self-cleaving peptides which are cleaved between the receptor components and the signalling component after translation.
In a fifth aspect the present invention provides a vector comprising a nucleic acid sequence according to the third to fourth aspects of the invention.
In a sixth aspect the present invention provides a retroviral vector or a lentiviral vector or a transposon comprising a vector according to the fifth aspect of the invention.
In a seventh aspect the present invention provides a T cell or NK cell comprising a nucleic acid according to the third to fourth aspects of the invention or a vector according to the fifth or sixth aspect of the invention.
In an eighth aspect the present invention provides a pharmaceutical composition comprising a plurality of T cells or NK cells according to the seventh aspect of the invention.
In a ninth aspect the present invention relates to a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the eighth aspect of the invention to a subject.
The method according to the ninth aspect of the invention may comprise the following steps: (i) isolation of a T cell or NK containing sample;
(ii) transduction or transfection of the T or NK cells with a nucleic acid sequence according to any of the third to fourth aspects of the invention or a vector according to the fifth or sixth aspect of the invention; and
(iii) administering the T cells or NK cells from (ii) to a subject.
The method may involve administration of T cells/NK cells to a subject, which T cells/NK cells have been previously isolated from the subject and transduced/transfected with a nucleic acid sequence according to any of the third to fourth aspects of the invention or a vector according to the fifth or sixth aspect of the invention.
The method according to the ninth aspect of the invention may involve monitoring toxic activity in the subject and comprise the step of administering an agent for use in the CAR system of the first aspect of the invention to the subject to reduce adverse toxic effects.
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 for use in the CAR system of the first aspect of the invention to the subject to provide acceptable levels of disease progression and/or toxic activity.
In the use of a pharmaceutical composition according to the eighth aspect of the invention or a method according to the ninth aspect of the invention, the disease may be cancer.
In a tenth aspect the present invention relates to the use of a pharmaceutical composition according to the eighth aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
In an eleventh aspect the present invention provides a kit which comprises a nucleic acid according to the third to fourth aspects of the invention or a vector according to the fifth or sixth aspect of the invention.
In a twelfth aspect the present invention relates to a method for making a T or NK cell according to the seventh aspect of the invention, which comprises the step of introducing a nucleic acid sequence according to third to fourth aspect of the invention or the vector according to the fifth or sixth aspect of the invention into a T or NK cell.
The T or NK cell may be from a sample isolated from a subject. In a thirteenth aspect the present invention relates to a method for inhibiting the CAR system according to the first aspect of the invention in a subject which comprises a T or NK cell according to the seventh aspect of the invention which method comprises the step of administering the agent to the subject.
The present invention therefore provides a 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 receptor component and signalling component. 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. Further 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 toxi cities.
DETAILED DESCRIPTION
CHIMERIC ANTIGEN RECEPTORS (CARs)
Classical CARs, which are shown schematically in Figure 1, 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. In the present invention, the binder is based upon a Fab fragment derived from a monoclonal antibody (mAb).
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.
Early CAR designs had endodomains derived from the intracellular parts of either the g chain of the FcsR1 or Oϋ3z. Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co stimulatory molecule to that of Oϋ3z results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co stimulatory domain most commonly used is that of CD28. This supplies the most potent co stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related 0X40 and 41 BB which transmit survival signals. Even more potent third generation 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. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.
In W02016/030691 the present inventors outlined a CAR system in which signalling could be “switched-off” by the addition of an agent. The present application describes an improvement to this system which leads to improved CAR stability and improved cytokine production upon activation of the CAR system. This improvement is based upon redesigning the receptor component to include a Fab-based antigen binding domain and the optional use of a signalling domain in the receptor component.
In a first aspect, the present invention relates to a CAR system in which an antigen recognizing/antigen binding domain based on a Fab light chain or Fab heavy chain and transmembrane domain are provided on a first molecule (termed herein ‘first receptor component’), which localizes to the cell membrane. The other Fab chain (i.e. , heavy or light) is provided on a second molecule (termed herein ‘second receptor component’) which heterodimerises to the first receptor component to form a heterodimeric receptor component comprising an antigen binding domain. The intracellular signalling domain is provided on a second, intracellular molecule (termed herein ‘signalling component’).
The first receptor component comprises a first binding domain and the signalling component comprises a second binding domain which specifically binds to the first binding domain of the receptor component. Thus binding of the first binding domain to the second binding domain causes heterodimerization and co-localization of the receptor component and the signalling component. When antigen binds to the antigen binding domain of the receptor component there is signalling through the signalling component.
In some cases, the second receptor component may also comprise a transmembrane domain and a first binding domain.
The first or second binding domain is also capable of binding a further agent in addition to the reciprocal binding domain. The further agent may be, for example, a small molecule. The binding between the agent and the first or second binding domain is of a higher affinity than the binding between the first binding domain and the second binding domain. Thus, when the agent is present it preferentially binds to the first or second binding domain and inhibits/disrupts the heterodimerization between the receptor component and the signalling component. When antigen binds to the antigen binding domain of the receptor component in the presence of the further agent there is no signalling through the signalling component.
Specifically, in the presence of the agent, the receptor component and signalling component 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 through the signaling component.
Herein ‘co-localization’ or ‘heterodimerization’ of the receptor and signalling components is analogous to ligation/recruitment of the signalling component to the receptor component via binding of the first binding domain of the receptor component and the second binding domain of the signalling component.
Antigen binding by the receptor component in the presence of the agent may be termed as resulting in ‘non-productive’ signalling through the signalling component. Such signalling does not result in cell activation, for example T cell activation. Antigen binding by the receptor component in the absence of the agent may be termed as resulting in ‘productive’ signalling through the signalling component. This signalling results in T-cell activation, triggering for example target cell killing and T cell activation.
Antigen binding by the receptor component in the absence of the agent may result in signalling through the signalling component which is 2, 5, 10, 50, 100, 1,000 or 10,000-fold higher than the signalling which occurs when antigen is bound by the receptor component in the presence of the agent. Signalling through the signalling component 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. As an illustration, in the present examples the inventors determined levels of interleukin-2 (IL-2) produced by T-cells expressing a receptor component and signalling component of the CAR system according to the present invention upon binding of antigen to the receptor component in the presence of varying concentrations of an agent.
FIRST BINDING DOMAIN, SECOND BINDING DOMAIN AND AGENT
The first binding domain, second binding domain and agent of the present CAR system may be any combination of molecules/peptides/domains which enable the selective co-localization and dimerization of the receptor component and signalling component in the absence of the agent.
As such, the first binding domain and second binding domain are capable of specifically binding.
The signalling system of the present invention is not limited by the arrangement of a specific dimerization system. The receptor component may comprise either the first binding domain or the second binding domain of a given dimerization system so long as the signalling component comprises the corresponding, complementary binding domain which enables the receptor component and signalling component to co-localize in the absence of the agent.
The first binding domain and second binding 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 is a molecule, for example a small molecule, which is capable of specifically binding to the first binding domain or the second binding domain at a higher affinity than the binding between the first binding domain and the second binding domain. For example, the binding system may be based on a peptide: peptide binding domain system. The first or second binding 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.
For example, the agent may bind the first binding domain or the second binding domain with at least 10, 20, 50, 100, 1000 or 10000-fold greater affinity than the affinity between the first binding domain and the second binding domain.
The agent may be any pharmaceutically acceptable molecule which preferentially binds the first binding domain or the second binding domain with a higher affinity than the affinity between the first binding domain and the second binding 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 a/.; J. Biol. Chem. 280, 24591-24599 (2005); Luckner et ai; J. Mol. Biol. 368, 780-790 (2007)).
The Tet repressor (TetR) system
The Tet operon is a well-known biological operon which has been adapted for use in mammalian cells. The TetR protein (also referred to as TetRB herein) binds tetracycline as a homodimer and undergoes a conformational change which then modulates the DNA binding of the TetR molecules. Klotzsche etal. (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 ai; as above). Thus TiP and tetracycline compete for binding of TetR. In the present CAR system the first binding domain of the receptor component may be TetR or TiP, providing that the second binding domain of the signalling component is the corresponding, complementary binding partner. For example if the first binding domain of the receptor component is TetR, the second binding domain of the signalling component is TiP. If the first binding domain of the receptor component is TiP, the second binding domain of the signalling component is TetR.
For example, the first binding domain or second binding domain may comprise the sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2:
SEQ ID NO: 1 - TetR
MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFC
PLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSA
VGH
SEQ ID NO: 2 - TiP
MWTWNAYAFAAPSGGGS
TetR must homodimerize in order to function. Thus when the first binding domain on the receptor component is TetR, the receptor component may comprise a linker between the transmembrane domain and the first binding domain (TetR). The linker enables TetR to homodimerize with a TetR from a neighbouring receptor component and orient in the correct direction.
The linker may be the sequence shown as SEQ ID NO: 3.
SEQ ID NO: 3 - modified CD4 endodomain
ALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMAQIKRVVSEKKTAQAPHRFQKTCSPI
The linker may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as the sequence shown as SEQ ID NO: 3.
The linker may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 3 providing it provides the function of enabling TetR to homodimerize with a TetR from a neighbouring receptor component and orient in the correct direction. In some cases the linker may comprise or consist of a signalling domain, such as the CD28 endodomain, 41 BB endodomain or 0X40 endodomain.
One potential disadvantage of the TetR/TiP system is TetR is xenogenic and immunogenic. The TetR sequence may therefore be a variant which is less immunogenic but retains the ability to specifically bind TiP.
Where the first and second binding domains are TetR or TiP or a variant thereof, the agent may be tetracycline, doxycycline, minocycline or an analogue thereof.
An analogue refers to a variant of tetracycline, doxycycline or minocycline which retains the ability to specifically bind to TetR.
Other combinations of binding domains and agents which may be used in the present CAR system are known in the art. For example, the CAR system may use a streptavidin/biotin- based binding system.
Strepta vidin-binding epitope
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 heterodimerisation between the streptavidin domain and the biotin mimic domain. The biotin mimic may bind streptavidin with for example with a Kd of 1nM 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. Table 1. Biotin mimicking peptides.
Figure imgf000019_0001
Figure imgf000020_0001
The biotin mimic may be selected from the following group: Streptagl I , Flankedccstreptag and ccstreptag.
The streptavidin domain may comprise streptavidin having the sequence shown as SEQ ID No. 11 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.
The sequence of “core” streptavidin (residues 13-139) is shown as SEQ ID No. 11 SEQ ID No. 11
EAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALG
WTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKV
KPSAAS
Streptavidin exists in nature as a homo-tetramer. The secondary structure of a streptavidin monomer is composed of eight antiparallel b-strands, which fold to give an antiparallel beta barrel tertiary structure. A biotin binding-site is located at one end of each b-barrel. Four identical streptavidin monomers (i.e. four identical b-barrels) associate to give streptavidin’s tetrameric quaternary structure. The biotin binding-site in each barrel consists of residues from the interior of the barrel, together with a conserved Trp120 from neighbouring subunit. In this way, each subunit contributes to the binding site on the neighbouring subunit, and so the tetramer can also be considered a dimer of functional dimers.
The streptavidin domain of the CAR system of the present invention may consist essentially of a streptavidin monomer, dimer or tetramer.
The sequence of the streptavidin monomer, dimer or tetramer may comprise all or part of the sequence shown as SEQ ID No. 11 , 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. 11 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. Variany streptavidin may, for example, comprise all 8 of these residues. Where variant streptavidin is present in the binding domain as a dimer orteramer, it may also comprise Trp120 which is involved in biotin binding by the neighbouring subunit. Small molecules agents which disrupt protein-protein interactions have long been developed for pharmaceutical purpose (reviewed by Vassilev et a/; 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 (or relevant fragments of these proteins) can be used as the first and/or second binding 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). A list of proteins/peptides whose interaction is disruptable using an agent such as a small molecule is given in Table 2. These disputable protein-protein interactions (PPI) may be used in the CAR system of the present invention. Further information on these PPIs is available from White et al 2008 (Expert Rev. Mol. Med. 10:e8). Table 2
Figure imgf000021_0001
Second binding domains which competitively bind to the same first binding domain as the agents described above, and thus may be used to co-localise the receptor component and signalling component of the present signalling system in the absence of the agent, may be identified using techniques and methods which are well known in the art. For example such second binding domains may be identified by display of a single domain VHH library.
The first binding domain and/or second binding domain of the present 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 receptor component and signalling component.
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 the CAR system of the first aspect of the invention, which method comprises the step of administering the agent. As described above, administration of the agent results in a disruption of the co-localization between the receptor component and the signalling component, such that signalling through the signalling component is inhibited even upon binding of antigen to the antigen binding domain.
The first and second binding domains may facilitate signalling through the CAR system which is proportional to the concentration of the agent which is present. Thus, whilst the agent binds the first binding domain or the second binding domain with a higher affinity than binding affinity between the first and second binding domains, co-localization of the receptor and signalling components may not be completely ablated in the presence of low concentrations of the agent. For example, low concentrations of the agent may decrease the total level of signalling in response to antigen without completely inhibiting it. The specific concentrations of agent will differ depending on the level of signalling required and the specific binding 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.
Single Domain Binders
The first or second binding domain of the CAR system of the present invention may comprise a single domain binder.
A “single domain binder” 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 binder may have an antibody-like binding site which binds to the agent. The single domain binder may comprise one or more complementarity determining regions (CDRs). The single domain binder may comprise three CDRs
The single domain binder may lack disulphide bonds. The single domain binder 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). 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 domain binder used in the CAR system of the invention may be or comprise such a single domain VH or VL binder.
Heavy chain antibodies (hcAb) 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 binder used in the CAR system of the invention may be or comprise such a VHH domain or derivative thereof.
A variety of non-immunoglobulin single domain binders have also been designed and characterised, including those based on natural and synthetis protein scaffolds. For example, fibronectin-derived Adnectins/monobodies are characterized by an Ig-like b-sandwich structure, anticalins are based on the lipocalin fold, affibodies derive from protein A and comprise three a helices, and DARPins are designer proteins composed of ankyrin repeats. Each design includes randomized residues that mediate ligand binding.
The single domain binder 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 binder may be less than 150 amino acids in length, for example, less than 140, 130 or 120 amino acids in length. The single domain binder may be approximately 110 amino acids in length, for example from 105-115 amino acids in length
The single domain binder 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.
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 antibodies found in camelids; i.e. VHH fragments. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, 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. By reverse transcription and polymerase chain reaction, 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. Alternatively, single domain antibodies can be made from common murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens. A problem with this approach is that the binding region of common IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity.
An Affibody molecule consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa. The original Affibody protein scaffold was designed based on the Z domain (the immunoglobulin G binding domain) of protein A. I n contrast to antibodies, Affibody molecules are composed of alpha helices and lack disulfide bridges.
Affibody molecules with unique binding properties are acquired by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain. Lately, amino acids outside of the binding surface have been substituted in the scaffold to create a surface entirely different from the ancestral protein A domain.
Specific affibody molecules binding a desired target protein can be “fished out” from libraries of variants, using phage display.
Fibronectin artificial antibody scaffold are antibody mimics based on the scaffold of the fibronectin type III domain.
Anticalins are derived from human lipocalins, a family of naturally binding proteins. Anticalins have a size of about 180 amino acids and a mass of about 20 kDa.
Affilin proteins are structurally derived from human ubiquitin (historically also from gamma-B crystallin). Affilin proteins are constructed by modification of surface-exposed amino acids of these proteins and isolated by display techniques such as phage display and screening. Like other antibody mimetics they resemble antibodies in their affinity and specificity to antigens but not in structure,
Designed ankyrin repeat proteins (DARPins) are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin proteins, one of the most common classes of binding proteins in nature, which are responsible for diverse functions such as cell signalling, regulation and structural integrity of the cell. DARPins consist of at least three repeat motifs proteins, and usually consist of four or five. Their molecular mass is about 14 or 18 kDa (kilodaltons) for four- or five-repeat DARPins, respectively. iBodies are modular synthetic antibody mimetics based on hydrophilic polymers.
An affimer is a small, highly stable protein engineered to display peptide loops which provide a high affinity binding surface for a specific target protein. It is a protein of low molecular weight, 12-14 kDa, derived from the cysteine protease inhibitor family of cystatins.
Fynomers are small binding proteins derived from the human Fyn SH3 domain. Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. Fynomers have neither have cysteine residues nor disulfide bonds and are approximately 7 kDa in size. Abdurins are a new class of antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Abdurins are small (12.5kDa) proteins which retain a portion of the native Fc receptor binding motif which binds to the neonatal Fc receptor to increase protein half-life and tumour uptake.
Centyrins are a new class of alternative scaffold protein based on a consensus fibronectin domain.
Alphabodies, also known as Cell-Penetrating Alphabodies or CPAB, are small 10 kDa antibody mimetic proteins engineered to bind to a variety of antigens. Alphabodies are different from many other antibody mimetics in their ability to reach and bind to intracellular protein targets. Their single chain alpha-helical structure is designed by computer modelling, inspired by naturally existing coiled-coil protein structures.
Affitins (or Nanofitins) are antibody mimetics structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaeal domain. By randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards the target of interest.
Specific single domain binders for minocycline or caffeine suitable for use in the present invention are described in WO2017/137758 (which is incorporated herein by reference).
Single Domain Binder-Interacting Peptide
In the CAR system of the present invention heterodimerisation of the receptor and signalling component may occur through the binding of the single domain binder with a single domain binder-interacting peptide (sdbiP).
The sdbiP may, for example, be between 8-30, for example 10-20 amino acids in length.
Suitable sdbiPs may be generated and identified using peptide display methods such as phage display, CIS display, ribosome display and mRNA display (Ullman et al (2011) 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 sdbiP 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.
Once identified, the sdbiP may be incorporated into the receptor molecule (first and third embodiments) or the signalling molecule (second and fourth embodiments) and tested to make sure the binding properties of the sdbiP are retained.
Examples of a sdbiP which specifically bind to a methotrexate dAb or a caffeine dAb are described in WO2017/137758 (which is incorporated herein by reference).
AGENT
The agent may be a small molecule such as: a steroid, methotrexate, caffeine, cocaine or an antibiotic.
A steroid is an organic compound with four “fused” carbon rings. Examples of steroids include the dietary lipid cholesterol, the sex hormones estradiol and testosterone and the anti inflammatory drug dexamethasone.
The steroid core structure is composed of seventeen carbon atoms, bonded in four "fused" rings: three six-member cyclohexane rings (rings A, B and C in the first illustration) and one five-member cyclopentane ring (the D ring). Steroids vary by the functional groups attached to this four-ring core and by the oxidation state of the rings. Sterols are forms of steroids with a hydroxyl group at position three and a skeleton derived from cholestane.
Methotrexate (MTX), formerly known as amethopterin, is an antimetabolite and antifolate drug.
Caffeine is a purine, a methylxanthine alkaloid. It is a stimulant of the central nervous system, but is generally recognised as safe (GRAS) by the Food and Dru Administration. Toxic doses, over 10 grams per day for an adult, are much higher than typical dose of under 500 milligrams per day. A cup of coffee contains 80-175 mg of caffeine.
Cocaine, also known as benzoylmethylecgonine or coke, is a strong stimulant. Various analogs of cocaine (methyl(1 R,2R,3S,5S)-3-(benzoyloxy)-8-methyl-8- azabicyclo[3.2.1]octane-2-carboxylate) are known including stereoisomers; 3b-phenyl ring substituted analogues; 2b-substituted analogues; N-modified analogues of cocaine; 3b- carbamoyl analogues; 3b-alkyl-3-benzyl tropanes; 6/7-substituted cocaines; 6-alkyl-3-benzyl tropanes; and piperidine homologues.
Antibiotics or antibacterials are a type of antimicrobial used in the treatment and prevention of bacterial infection. Antibacterial antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity. Most target bacterial functions or growth processes. Those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins), or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones, and sulfonamides) have bactericidal activities. Those that target protein synthesis (macrolides, lincosamides and tetracyclines) are usually bacteriostatic (with the exception of bactericidal aminoglycosides). Further categorization is based on their target specificity. "Narrow-spectrum" antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad-spectrum antibiotics affect a wide range of bacteria. Four new classes of antibacterial antibiotics have been brought into clinical use in the last ten years: cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), and lipiarmycins (such as fidaxomicin).
The agent may, for example, be an antibiotic such as tetracycline, or a derivative thereof such as doxycycline or minocycline.
FIRST AND SECOND RECEPTOR COMPONENT
The present invention provides a receptor component composed of a first receptor component comprising a Fab light chain and a second receptor component comprising a Fab heavy chain, such that the first and second receptor components heterodimerise to form a receptor component comprising an antigen-binding domain. It will be appreciated that the first receptor component may instead comprise a Fab heavy chain The receptor component further comprises, an optional spacer domain, a transmembrane domain and a first binding domain. When expressed in a cell, the receptor component localises to the cell membrane. Here, the antigen-binding domain of the molecule is orientated on the extracellular side of the membrane and the first binding domain is localised to the intracellular side of the membrane.
The receptor component therefore provides the antigen-binding function of the CAR system of the present invention. In some cases the receptor component may also include one or more co-stimulatory domains, such as CD28 endodomain, 4-1 BB endodomain or 0X40 endodomain. Typically, the co stimulatory domain will be intracellular, to allow for signalling. In particular, the co-stimulatory domain may be positioned such that it is proximal to the internal surface of the membrane. The position of the domain relative to the membrane may be adjusted via any suitable method, for example by the use of spacer sequences.
ANTIGEN BINDING DOMAIN
The antigen-binding domain is the portion of a classical CAR which recognizes antigen. In the signalling system of the present invention the antigen-binding is located within the receptor component.
Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid (dAb); VHH antigen binding domains; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
In the chimeric antigen receptors of the present invention, the antigen binding comprises a Fab fragment of, for example, a monoclonal antibody. A FabCAR comprises two chains: one having an antibody-like light chain variable region (VL) and constant region (CL)(also referred to herein as a Fab light chain); and one having a heavy chain variable region (VH) and constant region (CH) (also referred to herein as a Fab heavy chain). Association between the CL and CH causes assembly of the receptor component. In some cases, association between the two chains results in the formation of disulphide bonds. Either chain may also comprise a transmembrane domain and a first binding domain as defined herein. In some circumstances, both chains my comprise a transmembrane domain and a first binding domain as defined herein.
The first binding domain may be connected to the transmembrane domain via a linker. The linker may comprise or consist of a co-stimulatory domain, such as CD28 endodomain, 4-1 BB endodomain or 0X40 endodomain. In some cases the linker may be derived from the sequence of CD4. The linker may comprise or consist of the sequence shown as SEQ ID NO: 3. The two chains of the receptor component may have the general structure:
VH - CH - spacer - transmembrane domain - first binding domain; and VL - CL or
VL - CL - spacer- transmembrane domain - first binding domain; and VH - CH or
VH - CH - spacer - transmembrane domain - first binding domain; and VL - CL - spacer - transmembrane domain - first binding domain; or
VL - CL - spacer- transmembrane domain - first binding domain; and VH - CH - spacer - transmembrane domain - first binding domain;
The antigen binding domain is made up of a VH from one polypeptide chain and a VL from another polypeptide chain. The two chains may also include a spacer between the CH/CL domains and the transmembrane domain.
The polypeptide chains may comprise a linker between the VH/VL domain and the CH/CL domains. The linker may be flexible and serve to spatially separate the VH/VL domain from the CH/CL domain.
Various tumour associated antigens (TAA) are known, as shown in the following Table 1. The antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.
Table 1
Figure imgf000030_0001
Figure imgf000031_0001
TRANSMEMEBRANE DOMAIN
The transmembrane domain is the sequence of a classical CAR that spans the membrane. In the signalling system of the present invention the transmembrane domain is located in the receptor component. It may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which gives good receptor stability.
SIGNAL PEPTIDE
The receptor component of the CAR system of the present invention may comprise a signal peptide so that when the receptor component 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 signal peptide may be at the amino terminus of the molecule. The signal peptide may comprise the sequence shown as SEQ ID NO: 12, 13 or 14 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR.
SEQ ID NO: 12: MGTSLLCWMALCLLGADHADG
The signal peptide of SEQ ID NO: 12 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.
SEQ ID NO: 13: MSLPVTALLLPLALLLHAARP
The signal peptide of SEQ ID NO: 13 is derived from lgG1.
SEQ ID NO: 14: MAVPTQVLGLLLLWLTDARC
The signal peptide of SEQ ID NO: 14 is derived from CD8.
SPACER DOMAIN
The CAR system described herein may comprise a spacer sequence to connect the CH/CL domains with the transmembrane domain in the first and second receptor components. 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.
Examples of amino acid sequences for these spacers are given below:
SEQ ID NO: 15 (hinge-CH2CH3 of human lgG1)
AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCWVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD SEQ ID NO: 16 (human CD8 stalk):
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI
SEQ ID NO: 17 (human lgG1 hinge):
AEPKSPDKTHTCPPCPKDPK
SEQ ID NO: 18 (CD2 ectodomain)
KEITNALETWGALGQDI N LDI PSFQMSDDI DDI KWEKTSDKKKI AQFRKEKETFKEKDTYKLF KNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNG TDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD
SEQ ID NO: 19 (CD34 ectodomain)
SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKF
TSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATS
PTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQ
ADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA
SHQSYSQKT
RECEPTOR COMPONENT COMPRISING A PLURAILTY OF FIRST BINDING DOMAINS
The receptor component may comprise a plurality of first binding domains and thus be capable of recruiting more than one signalling component.
The plurality of first binding domains may be present in a single intracellular domain of the receptor component.
The receptor component may comprise an appropriate number of transmembrane domains such that each first binding domain is orientated on the intracellular side of the cell membrane. For example the receptor component may comprise 3, 5, 7, 9, 11, or more transmembrane domains. In this way, a single receptor component may recruit multiple signalling components amplifying signalling in response to antigen.
The first binding domains may each be variants which have a different affinity for the second binding domain of the signalling component.
MULTIPLE RECEPTOR COMPONENTS In another embodiment of the invention, the CAR system may comprise two or more receptor components each recognizing different antigens but comprising of the same intracellular first binding domain. Such a CAR system would be capable of recognizing multiple antigens. This might be useful for instance in avoiding tumour escape. In a further related aspect of the invention, the first binding domains of the receptor components differ in residues which dictate their affinity for the second binding domain of the signalling component. In this way, a CAR system can be tuned such that signalling in response to one antigen is greater or lesser than the response to another. This might be useful for instance when targeting two tumour antigens simultaneously but one is expressed at a higher density than the other. Response to this antigen could be tuned down to avoid toxicity caused by over-stimulation.
Methods suitable for altering the amino acid residues of the first or second binding domain such that the binding affinity between the two domains is altered are known in the art and include substitution, addition and removal of amino acids using both targeted and random mutagenesis. Methods for determining the binding affinity between a first binding domain and a second binding domain are also well known in the art and include bioinformatics prediction of protein-protein interactions, affinity electrophoresis, surface plasma resonance, bio-layer interferometry, dual polarisation interferometry, static light scattering and dynamic light scattering.
SIGNALLING COMPONENT
The present invention also provides a signalling component comprising a signalling domain and a second binding domain. The signalling component is a soluble molecule and thus localises to the cytoplasm when it is expressed in a cell, for example a T cell.
No signalling occurs through the signalling domain of the signalling component unless it is co localised with the receptor component provided by the present invention. Such co-localisation occurs only in the absence of the agent, as described above.
INTRACELLULAR SIGNALLING DOMAIN
The intracellular signalling domain is the signal-transmission portion of a classical CAR. In the signalling system of the present invention the intracellular signalling domain (signalling domain) is located in the signalling component. In the absence of the agent, the membrane- bound, receptor component and the intracellular signalling component are brought into proximity. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell.
As such the signalling domain of the signalling component is analogous to the endodomain of a classical CAR molecule.
The most commonly used signalling domain component is that of CD3-zeta endodomain, 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. For example, chimeric CD28 and 0X40 can be used with CD3- Zeta to transmit a proliferative / survival signal, or all three can be used together (illustrated in Figure 1B).
The signalling component described herein comprises a signalling domain, it may comprise the CD3-Zeta endodomain alone, the CD3-Zeta endodomain with that of either CD28 or 0X40 or the CD28 endodomain and 0X40 and CD3-Zeta endodomain.
The signalling component of a CAR system according to the present invention may comprise the sequence shown as SEQ ID NO: 20, 21 or 22 or a variant thereof having at least 80% sequence identity.
SEQ ID NO: 20 - CD3 Z endodomain
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 21 - CD28 and CD3 Zeta endodomains
SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ
LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG
ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 22 - CD28, 0X40 and CD3 Zeta endodomains
SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFR TPIQEEQADAHSTLAKI RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 20, 21 or 22, provided that the sequence provides an effective intracellular signalling domain.
MULTIPLE SIGNALLING COMPONENTS
The signalling system according to the first aspect of the present invention may comprise a plurality of signalling components, each comprising a signalling domain and a second binding domain, wherein each second binding domain is bound by the same first binding domain of the receptor component but the signalling domains comprise different endodomains. In this way, multiple different endodomains can be activated simultaneously. This is advantageous over a compound signalling domain since each signalling domain remains unencumbered from other signalling domains.
If each signalling component comprises a second binding domain which differs in residues which alter their affinity to the first binding domain of the receptor component, the signalling components comprising different signalling domains ligate to the first binding domain with differing kinetics. This allows greater control over the signalling in response to antigen-binding by the receptor component as different signalling components are recruited to the receptor component in varying kinetics/dynamics. This is advantageous since rather than a fixed equal ratio of signal transmitted by a compound endodomain, an optimal T-cell activation signal may require different proportions of different immunological signals.
NUCLEIC ACID
The present invention further provides a nucleic acid encoding the first or second receptor component of the second aspect and/or a nucleic acid encoding a signalling component.
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.
The terms “variant”, “homologue” or “derivative” 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.
The nucleic acid of the invention may be a nucleic acid which encodes both the receptor component and the signalling component.
The nucleic acid may produce a polypeptide which comprises the receptor component and the signalling component 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 receptor component and the signalling component without the need for any external cleavage activity.
Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2a self-cleaving peptide, which has the sequence shown:
SEQ ID NO: 23
RAEGRGSLLTCGDVEENPGP. or
SEQ ID NO: 24 QCTNYALLKLAGDVESNPGP
The nucleic acid may produce a polypeptide which comprises the sequence shown as SEQ ID NO: 25. SEQ ID NO: 25
MWTWNAYAFAAPSGGGSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDV EENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGK APKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSG GGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEW VSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTL VTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPMSGGGGSMSRLDKSKVINSALELLNEVGI EGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCAL LSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEER ETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS
A polypeptide sequence comprising the sequence shown as SEQ ID NO: 25 may further comprise an antigen binding domain sequence positioned immediately after, i.e., C-terminally to, the methionine at position 652 of SEQ ID NO: 25 (shown in bold). In other words, the antigen binding domain sequence may be positioned between the methionine at position 652 and the serine at position 653 of SEQ ID NO: 25. Any antigen binding domain may be included, for example an scFv, as described herein.
The co-expressing 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 a nucleic acid encoding the receptor component of the second aspect and/or a nucleic acid encoding a signalling component.
VECTOR
The present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) encoding a first and/or second receptor component of the second aspect and/or signalling component. Such a vector may be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses the receptor component and signalling component of the CAR system according to the first aspect of the invention. 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 or a NK cell.
CYTOLYTIC IMMUNE CELL
The present invention also relates to an immune cell comprising the CAR system according to the first aspect of the invention.
The cytolytic immune cell may comprise a nucleic acid or a vector of the present invention.
The cytolytic immune cell may comprise a receptor component and a signalling component of the present invention.
The cytolytic immune cell may comprise at least one signalling component of the present invention. For example the cytolytic immune cell may comprise one, two, three, four, five, up to a plurality of signalling components of the present invention.
The cytolytic immune cell may comprise at least one receptor component of the present invention. For example the cytolytic immune cell may comprise one, two, three, four, five, up to a plurality of receptor components of the present invention.
Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. 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. There are various types of T cell, as summarised below.
Helper T helper cells (TH 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). 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 (TC cells, or CTLs) 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. 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.
Regulatory T cells (Treg cells), formerly known as 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.
Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.
Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ 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 (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.
Natural Killer Cells (or NK cells) are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.
The CAR cells of the invention may be any of the cell types mentioned above.
T or NK cells expressing the molecules of the CAR system according to the first aspect of 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).
Alternatively, T or NK cells expressing the molecules of the CAR system according to the first aspect of the invention may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells. Alternatively, an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.
In all these embodiments, CAR cells are generated by introducing DNA or RNA coding for the receptor component and signalling component by one of many means including transduction with a viral vector, transfection with DNA or RNA.
The CAR cell of the invention may be an ex vivo T or NK cell from a subject. The T or NK cell may be from a peripheral blood mononuclear cell (PBMC) sample. T or NK cells may be activated and/or expanded prior to being transduced with nucleic acid encoding the molecules providing the CAR system according to the first aspect of the invention, for example by treatment with an anti-CD3 monoclonal antibody.
The T or NK cell of the invention may be made by:
(i) isolation of a T or NK cell-containing sample from a subject or other sources listed above; and
(ii) transduction or transfection of the T or NK cells with one or more a nucleic acid sequence(s) encoding the first and/or second receptor components according to the second aspect and/or a signalling component of the CAR system.
The T or NK cells may then by purified, for example, selected on the basis of expression of the antigen-binding domain of the antigen-binding polypeptide. The present invention also provides a kit which comprises a T or NK cell comprising the CAR system according to the first aspect of the invention.
PHARMACEUTICAL COMPOSITION
The present invention also relates to a pharmaceutical composition containing a plurality of cytolytic immune cells expressing the components of the CAR system of the first aspect of the 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.
METHOD OF TREATMENT
The present invention provides a method for treating and/or preventing a disease which comprises the step of administering the cytolytic immune 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 cytolytic immune cells of the present invention. Herein the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease 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 cytolytic immune cells of the present invention. Herein such 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:
(i) isolating a T or NK cell-containing sample;
(ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention;
(iii) administering the cells from (ii) to a subject. The T or NK cell-containing sample may be isolated from a subject or from other sources, for example as described above. The T or NK cells may be isolated from a subject’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).
The methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and any toxic activity and administering an agent suitable for use in the 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, biliary toxicity and respiratory distress syndrome.
The level of signalling through the signalling system of the first aspect of the invention, and therefore the level of activation of CAR cells expressing the signalling system, may be adjusted by altering the amount of agent present, or the amount of time the agent is present. In the present method 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. Conversely, 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 CAR 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 suitable for use in the CAR system according to the first aspect to the subject. As such, this method involves administering a suitable agent to a subject which already comprises CAR cells of the present invention.
As such the dose of agent administered to a subject, or the frequency of administration, may be altered in order to provide an acceptable level of both disease progression and toxic activity. The specific level of disease progression and toxic activities determined to be ‘acceptable’ will vary according to the specific circumstances and should be assessed on such a basis. The 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.
The disease to be treated and/or prevented by the methods of the present invention may be an infection, such as a viral infection.
The methods of the invention may also be for the control of pathogenic immune responses, for example in autoimmune diseases, allergies and graft-vs-host rejection. The methods may be for the treatment of 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 CAR cells 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 CAR cells and pharmaceutical compositions of present invention may be for use in the treatment and/or prevention of the diseases described above.
The CAR cells and pharmaceutical compositions of present invention may be for use in any of the methods described above.
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.
EXAMPLES
Example 1 - Proof-of-Concept Study: 0X40 Fab-Tet-CARs
A bicistronic construct was expressed in donor T cells as a single transcript which self-cleaves at two 2A sites to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of 0X40 and the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and T etR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab (Figure 3).
In addition, constructs were expressed comprising (separated by 2A sites):
(i) a signalling component which comprises TiP fused via a flexible linker to the endodomain of 0X40 and the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19- binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, a CD4L domain, and TetR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19- binding Fab;
(ii) a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, an 0X40 endodomain, and TetR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab;
As controls, a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, the endodomain of 0X40 and the endodomain of CD3-Zeta; and a second receptor component which comprises the VL and CL domains of the HD37 CD19- binding Fab. Furthermore, a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of 0X40 and the endodomain of CD3-Zeta; and a receptor component which comprises an scFv based on the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and TetR.
Expression of these constructs is shown in Figures 4 and 5.
The engineered T-cells were challenged with SKOV Red cells engineered to express CD19 in the absence of tetracycline or in the presence of 1600 nM of tetracycline, at an effectortarget (E:T) ratio of 8:1. T-cells challenged with SKOV Red cells expressing CD19 were activated in the absence of tetracycline, but activation is rapidly inhibited in the presence of tetracycline with activation fully inhibited in the presence of 1600nM of tetracycline (Figures 6 and 7). Cytokine production was also investigated (Figure 8). Locating the 0X40 domain on the receptor component resulted in
Example 2 - Proof-of-Concept Study: CD28-41BB Fab-Tet CARs
A bicistronic construct was expressed in donor T cells as a single transcript which self-cleaves at two 2A sites to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD28 and the endodomain of CD3-Zeta; a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and T etR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, a five amino acid spacer, and TetR (Figure 9).
In addition, constructs were expressed comprising the following in addition to a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD28 and the endodomain of CD3-Zeta:
(i) a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, 41 BB endodomain, and TetR; and a second receptor component which comprises the VL and CL domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, 41 BB endodomain, and TetR;
As controls, a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a first receptor component which comprises the VH and CH1 domains of the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, the endodomain of 41 BB and the endodomain of CD3-Zeta; and a second receptor component which comprises the VL and CL domains of the HD37 CD19- binding Fab, a spacer derived from the hinge domain of lgG1 , a CD28 derived transmembrane domain, the endodomain of 41 BB and the endodomain of CD3-Zeta. Furthermore, a construct was expressed as a single transcript which self-cleaves at a 2A site to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD28 and the endodomain of CD3-Zeta; and a receptor component which comprises an scFv based on the HD37 CD19-binding Fab, a spacer derived from the hinge domain of lgG1, a CD28 derived transmembrane domain, a five amino acid spacer and TetR.
Expression of these constructs is shown in Figures 10 and 11.
The engineered T-cells were challenged with SKOV Red cells engineered to express CD19 in the absence of tetracycline or in the presence of 1600 nM of tetracycline, at an effectortarget (E:T) ratio of 8:1. T-cells challenged with SKOV Red cells expressing CD19 were activated in the absence of Tetracycline, but activation is rapidly inhibited in the presence of tetracycline with activation fully inhibited in the presence of 1600nM of tetracycline (Figure 12 and 13). Cytokine production was also investigated (Figure 8).
Example 3 - Fab format and TetR attached CD28 co-stimulation improves TetCAR activity.
Surface expression of an scFv-based TetCAR, as detected by soluble recombinant CD19 staining, was observed to be lower than that of monolithic CAR using the same scFv (Figure 15). Variant TetCARs were constructed in Fab CAR format with the aim of increasing stability, and with ΰϋ28-ΰ03z signalling domains with the aim of enhancing signalling. The function of these TetCAR variants (as outlined in figure 16A) was compared to the gold-standard monolithic FMC63 CAR with 41BB-ΰϋ3z endodomain.
The replacement of the scFv with the Fab fragment increased the MFI for CAR surface binding of CD19 protein in both TetCAR variants by 1.7-fold, however despite similar transduction efficiencies, these constructs still had significantly lower surface expression than the control CAR (3.8-fold lower for BBz and 4.4-fold lower for 28z) (figure 16B and C). To test whether these variants had improved function, transduced T cells were co-cultured with SupT1 cells engineered to express CD19-eGFP, with and without 100 nM minocycline (figure 17). No significant differences in cytotoxicity of any of the TetCAR variants compared to the control CARs were noted. Cytotoxicity of both the 41 BB and OΌ28-z variants of the scFv-TetCAR was significantly inhibited upon addition of minocycline (3.6 and 3.8-fold reduction respectively). In the Fab-TetCAR conformation, inhibition by minocycline was similar to that of the scFv- variants (3.3 and 4.3-fold reduction), although this was only significant in the OΌ28-z variant.
Whilst IFN-g and IL-2 secretion was still lower in all the TetCAR variants than the control CAR, the Fab-TetCAR variants with 41 BBz or OΌ28z endodomains secreted higher levels of IFN-y (1.9 and 3.1-fold increase) and IL-2 (4.5 and 5.7-fold increase) than their scFv-counterparts (figure 18). This increase was significant in both endodomain variants for IFN-g but was only significant in the OΌ28z TetCARs for IL-2 secretion. Despite the higher baseline levels of cytokine secretion in the Fab-TetCARs, addition of minocycline was still able to potently supress cytokine secretion, inducing a 29- and 83- fold reduction in IFN-g secretion and a 46- and 37-fold reduction in IL-2 secretion in the 41 BB and OΌ28-z variants respectively.
Addition of a membrane-proximal non-inducible co-stimulatory domain further enhances performance
Whilst the replacement of the scFv with the Fab fragment had improved TetCAR function, a deficit still remained in cytokine secretion, even with the TetCAR incorporating a CD28 endodomain. We reasoned that a TIP-tethered co-stimulatory domain signals ineffectively and we explored further variants of the Fab-TetCARs with co-stimulatory domains between the cell membrane and the TetR protein. Several variants with co-41 BB and / or CD28 stimulatory domains present solely before TetR or both before TetR and after TIP were constructed (outlined in Figure 19A).
These variants were tested as before. All TetCAR variants induced similar cytotoxicity, although here the inhibition of cytotoxicity upon addition of minocycline was less pronounced and only BB-Tet-z CAR was significantly inhibited (Figure 19B). However, when using SupT 1.CD19-eGFP as targets, repositioning of the co-stimulatory domains efficiently restored cytokine secretion to the levels seen in the control CAR (Figure 20): for instance, IFN-y secretion with 28BB-Tet-z was 27,966pg/ml (±17323), matching the IFN-g secreted by the control CAR (26,803pg/ml ±14667). In a similar manner, IL-2 secretion was also highest in the 28BB-Tet-z CAR (12,178pg/ml ±6615), reaching a higher level than even the control CAR (7984pg/ml ±3577). Of note, whilst the BB-Tet-z CAR produced diminished but still detectable IFN-g, IL-2 secretion was almost undetectable in these CARs compared to the variants containing CD28-TetR. Furthermore, the addition of the extra 41 BB domain to the TIP-tail did not further increase IFN-g or IL-2 secretion. Despite inducing potent cytokine secretion, the optimal TetCAR construct (28BB-Tet-z), secreted 41 -fold lower IFN-g and 846-fold lower IL-2 secretion after addition of 100nM minocycline.
Tuneable control of optimised TetCAR activity through dose-dependent, minocycline inhibition 28-Tet-z and 28BB-Tet-z were taken forward for more detailed characterization. CAR inhibition over a range of minocycline concentrations was evaluated. CAR T cells were cocultured at a 1 :4 E:T ratio with SupT1-CD19-GFP with 4-fold increasing doses of minocycline from 0.02nM to 1600nM. Here cytotoxicity was compared relative to an inert FMC63-TetCAR, that could bind to CD19 but lacked any signalling capacity (CAR constructs outlined in Figure 21). The inhibition of cytotoxicity to SupT1-CD19-GFP (Figure 22) increased with minocycline concentration until 100nM, at which point a plateau was reached. 28BB-Tet- z was more potently inhibited by minocycline, reaching 99% live targets relative to the inert TetCAR (±31% SD), whereas 28-Tet-z inhibition peaked at 68% (±44% SD). The IC50 was 4.5nM for 28-Tet-z and 2.3nM for 28BB-Tet-z. Cytokine secretion over the same range of minocycline concentrations was also assessed (Figure 23). Overall, both TetCARs showed a similar dose-dependent reduction in both IFN-g and IL-2 with increasing concentrations of minocycline, fully inhibiting cytokine secretion at concentrations >6.25nM. The IC-'SO for 28- Tet-z and 28BB-Tet-z were 0.21 nM and 0.24nM for IFN-g and 0.34nM and 0.44nM for IL-2 secretion. In addition to IFN-g and IL-2, secretion of a number of other cytokines by 28BB-Tet- z was assessed from 2 representative donors. Inhibition of cytokine secretion by 28BB-Tet-z was also tested after addition of tetracycline and tigecycline, a glycylcycline derivative of tetracycline. Both small molecules induced inhibition of IFN-y and IL-2 secretion, however this required higher concentrations than minocycline (>100nM) and had no effect at lower doses (Figure 24).
Minocycline induces rapid and reversible inhibition of TetCAR signalling To further examine the kinetics of minocycline induced inhibition of TetCAR effector function, IL-2 secretion was assessed 1-5 hours after co-culture with SupT1-CD19 targets. Each hour, 100nM of minocycline was added to relevant wells to determine the time scale necessary for inhibition of cytokine secretion (Figure 25). As expected, the control CAR was unaffected by minocycline addition and induced detectable IL-2 secretion after 3 hours. This was mirrored by 28BB-Tet-z in the absence of minocycline, however addition of minocycline at different time points was able to inhibit further cytokine secretion within 2-3 hours.
To ensure that the inhibition of TetCAR was reversible and effector function could be restored upon removal of minocycline, 28BB-Tet-z cells were incubated overnight with 100nM minocycline then washed with media 48, 24 or 2 hours before activation with SupT1-CD19 targets. Removal of minocycline 48 hours before activation restored full TetCAR activity relative to a non-inhibited control, as measured by cytotoxicity and IL-2 secretion (Figure 26). Shorter timepoints (24 or 2 hours) before activation only partially restored effector function.
28-Tet-z and 28BB-Tet-z CAR T cells are highly functional compared with a gold-standard CAR
To ensure that 28-Tet-z and 28BB-Tet-z CAR T cells had no deficit of cell killing, we evaluated cytotoxicity at decreasing effector to target ratios ranging from 1 :1 to 1:32 to “stress” cytolytic function. There was no difference in cytotoxicity to SupT1-CD19-GFP or NALM6 at any E:T ratios in comparison to control CAR (Figure 27A).
A feature of CAR T cell activation is proliferation in response to relevant targets. This was assessed by culturing the CARs with mitomycin C-treated target cell lines for 7 days (Figure 27B). Targets used in this assay were SupT1-CD19-GFP, NALM6 and Raji, which all express CD19, and lastly a Raji-cell line in which CD19 expression had been knocked out. In the absence of minocycline, both Tet-CARs proliferated in response to SupT1-CD19 to the same extent as the control CAR. In line with our observations for cytokine secretion, proliferation of the TetCARs was significantly lower in response to NALM6 targets than SupT1-CD19-GFP. However, although the proliferative response of the TetCARs to Raji cells appeared slightly lower than the control CAR, this decrease was not significant. As expected, in the absence of CD19 on the Raji-CD19KO targets, none of the CARs proliferated above the NT T-cells. A single dose of 400nM minocycline on day 0 was sufficient to significantly reduce TetCAR proliferation in response to SupT1-CD19 and Raji cells. A similar trend was observed with NALM6, however this was not significant due to poor response seen in the absence of minocycline in this setting.
Although 0ϋ28-003z endodomains can drive potent CAR T cell activation, this can limit memory formation and skew populations towards short-lived effector cells. To show that Tet- CARs containing a CD28 endodomain were functionally equivalent to the 41 BBz gold standard and were not driving acquisition of an exhaustion phenotype, expression of Lag3 and Tim3 was also evaluated (Figure 28). After the 7 day co-culture with SupT1-CD19-GFP, both Lag3 and Tim3 expression on CD3+ T cells were similar in the T etCARs to the standard CAR. When inhibited with 400nM minocycline both TetCARs showed a significant reduction in Lag3 expression and a similar decreased expression of Tim3, although this was not significant. Lag3 and Tim3 expression was also similar between the TetCARs and the control CAR when co-cultured with other cell lines (Figure 29). Likewise, CAR T cell differentiation was also assessed after this co-culture by staining with antibodies to distinguish naive (CD62L+, CD45RA+), Tern (CD62L+, CD45RA-), Tern (CD62L-, CD45RA-) or Temra (CD62L-, CD45RA+) memory T cell populations. Proportions of these memory populations were similar between the CARs when co-cultured with SupT1-CD19-GFP (Figure 30) or with NALM6, Raji or Raji-CD19KO (Figure 31). Inhibition with minocycline also drove an accumulation of naive and Temra phenotypes at the expense of Tern and Tern populations in the TetCAR constructs when cultured with SupT1-CD19-GFP, but not the other target lines. Taken together these data show that even during maximal activation, TetCAR constructs containing CD28 endodomains did not drive an enhanced expression of exhaustion markers or skew differentiation of activated T cells.
28BB-Tet-CAR can be functionally regulated by minocycline in vivo
Lastly, we evaluated the activation and inhibition of 28BB-Tet-z CARs in vivo. NSG mice were engrafted with NALM6 engineered to express firefly luciferase (NALM6-Fluc); 4 days later, different cohorts were treated with either 5x106 NT, FMC63-BBz, or 28BB-Tet-z CAR T cells, with or without minocycline. An additional cohort of 28BB-T et-z treated mice were treated with minocycline 3 days after T cell transfer, during the peak of the initial anti-tumour response (Figure 32A). Minocycline was given at a dose of ~16mg/kg (0.4mg per mouse) i.p every 1-2 days. There was a significant reduction in tumour burden with 28BB-Tet-z and FMC63-BBz versus NT T Cells in the absence of minocycline (Figure 32B & C).. However, there was a non significant trend to shorter tumour control with 28BB-Tet-z which resembled the reduced activation and proliferation of the TetCAR in response to NALM6 targets in vitro. Whilst the addition of minocycline on day 0 had no effect on the FMC63-BBz group, early inhibition of 28BB-Tet-z completely abrogated the tumour control seen in the absence of minocycline. Injection of minocycline after initial tumour control (on day 3) was also able to inhibit subsequent TetCAR activity. Overall, these data show that although 28BB-Tet-z CARs are less potent than the FMC63- BBz CAR, they nevertheless provide significant tumour control, which can be regulated by treatment with minocycline in a relevant tumour model in vivo. Furthermore, inhibition of TetCAR function can be initiated during and after CAR T cell activation in vivo mirroring a clinical relevant application of this technology.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cellular immunology or related fields are intended to be within the scope of the following claims.

Claims

1. A chimeric antigen receptor (CAR) system comprising:
(i) a first receptor component comprising:
- a Fab light chain or a Fab heavy chain;
- a transmembrane domain; and
- a first binding domain;
(ii) a second receptor component comprising either:
- a Fab heavy chain when the first receptor component comprises a Fab light chain; or
- a Fab light chain when the first receptor component comprises a Fab heavy chain; such that the first and second receptor components heterodimerise to form a heterodimeric receptor component comprising an antigen binding domain; and
(ii) an intracellular signalling component comprising:
- a signalling domain; and
- a second binding domain which specifically binds the first binding domain; wherein, binding of the first and second binding domains is disrupted by the presence of an agent, such that in the absence of the agent the heterodimeric receptor component and the signalling component heterodimerize and binding of the antigen binding domain to antigen results in signalling through the signalling domain, whereas in the presence of the agent the heterodimeric receptor component and the signalling component do not heterodimerize and binding of the antigen binding domain to antigen does not result in signalling through the signalling domain.
2. The CAR system of claim 1 , wherein the second receptor component further comprises a transmembrane domain and a first binding domain.
3. The CAR system according to either claim 1 or claim 2, wherein the first receptor component and/or the second receptor component comprises a linker between the transmembrane domain and the first binding domain.
4. The CAR system according to claim 3 wherein the linker comprises or consists of CD28 endodomain, 41 BB endodomain or 0X40 endodomain.
5. The CAR system according to claim 3 wherein the linker comprises or consists of the sequence shown as SEQ ID NO: 3.
6. The CAR system according to any preceding claim wherein the first binding domain comprises Tet Repressor Protein (TetR) or a variant thereof and the second binding domain comprises Transcription inducing peptide (TiP) or a variant thereof; or wherein the first binding domain comprises TiP or a variant thereof and the second binding domain comprises TetR or a variant thereof; and the agent is tetracycline, doxycycline or minocycline or an analogue thereof.
7. The CAR system according to claim 6 wherein the first binding domain comprises two TetR domains.
8. The CAR system according to claim 7 wherein the two TetR domains are separated by a linker.
9. The CAR system according to claim 6 or 8 wherein each TetR domain has a different affinity for the agent.
10. The CAR system according to any of claims 1 to 9, wherein the signalling domain of the signalling component comprises a single endodomain selected from CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and 0X40 endodomain.
11. The CAR system according to any preceding claim, wherein the signalling domain of the signalling component comprises at least one of CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and 0X40 endodomain.
12. The CAR system according to any preceding claim which comprises a plurality of signalling components, each comprising a signalling domain and binding domain, wherein the binding domains each recognise the first binding domain but the signalling domains comprise different endodomains.
13. The CAR system according to claim 12 wherein the plurality of signalling components comprise a plurality of binding domains, each of which independently recognise the first binding domain with different affinities.
14. A first or second receptor component suitable for use in the CAR system according to any of claims 2 to 13 which comprises:
- a Fab light chain or a Fab heavy chain;
- a transmembrane domain; and - a first binding domain.
15. A nucleic acid sequence encoding the receptor component according to claim 14.
16. A nucleic acid sequence encoding a CAR signalling system according to any one of claims 1 to 13, wherein the first receptor component, second receptor component, and signalling component are co-expressed by means of self-cleaving peptides which are cleaved between the receptor components and the signalling component after translation.
17. A vector comprising a nucleic acid sequence according to any one of claims 15 to 16.
18. A retroviral vector or a lentiviral vector or a transposon comprising a vector according to claim 17.
19. A T cell or NK cell which comprises a nucleic acid according to any of claims 15 to 16 or a vector according to claim 17 or 18.
20. A pharmaceutical composition comprising a plurality of T cells or NK cells according to claim 19.
21. A pharmaceutical composition according to claim 20 for use in treating and/or preventing a disease.
22. A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 21 to a subject.
23. A method according to claim 22, which comprises the following steps:
(i) isolation of a T cell or NK containing sample;
(ii) transduction or transfection of the T or NK cells with a nucleic acid according to any of claims 15 to 16 or a vector according to claim 17 or 18; and
(iii) administering the T cells or NK cells from (ii) to a subject.
24. A method according to claim 22 or 23, which involves monitoring toxic activity in the subject and comprises the step of administering an agent for use in the CAR signalling system according to any of claims 1 to 13 to the subject to reduce adverse toxic effects.
25. A method according to claim 22 or 23, which involves monitoring the progression of disease and/or monitoring toxic activity in the subject and comprises the step of administering an agent for use in the CAR signalling system according to any of claims 1 to 13 to the subject to provide acceptable levels of disease progression and/or toxic activity.
26. The use of a pharmaceutical composition according to claim 21 or a method according to any one of claims 22 to 25 wherein the disease is cancer.
27. The use of a pharmaceutical composition according to claim 20 in the manufacture of a medicament for the treatment and/or prevention of a disease.
28. A kit which comprises a nucleic acid according to any of claims 15 to 16 or a vector according to claim 17 or 18.
29. A method for making a T or NK cell according to claim 19 or 20, which comprises the step of introducing a nucleic acid sequence according to any of claims 15 to 16 or the vector according to claim 17 or 18 into a T or NK cell.
30. A method according to claim 29 wherein the T or NK cell is from a sample isolated from a subject.
31. A method for inhibiting the CAR signalling system according to any of claims 1 to 13 in a subject which comprises a T or NK cell according to claim 19 or 20 which method comprises the step of administering the agent to the subject.
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