WO2017137759A1 - Système de signalisation - Google Patents

Système de signalisation Download PDF

Info

Publication number
WO2017137759A1
WO2017137759A1 PCT/GB2017/050341 GB2017050341W WO2017137759A1 WO 2017137759 A1 WO2017137759 A1 WO 2017137759A1 GB 2017050341 W GB2017050341 W GB 2017050341W WO 2017137759 A1 WO2017137759 A1 WO 2017137759A1
Authority
WO
WIPO (PCT)
Prior art keywords
signalling
component
car
cells
domain
Prior art date
Application number
PCT/GB2017/050341
Other languages
English (en)
Inventor
Martin PULÉ
Shaun CORDOBA
Simon Thomas
Shimobi ONUOHA
Maria STAVROU
Original Assignee
Autolus Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Autolus Limited filed Critical Autolus Limited
Publication of WO2017137759A1 publication Critical patent/WO2017137759A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • 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
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present invention relates to a chimeric antigen receptor signalling system. BACKGROUND TO THE INVENTION
  • 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
  • MAS macrophage activation syndrome
  • On- target off-tumour toxicity i.e. recognition of the target antigen on normal tissues.
  • MAS is presumed to be caused by persistent antigen-driven activation and proliferation of T- cells which in turn release copious inflammatory cytokines leading to hyper-activation of macrophages and a feed-forward cycle of immune activation.
  • a large spike in serum IL-6 is characteristic and the syndrome can result in a severe systemic illness requiring ICU admission.
  • 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.
  • PCT/GB2015/052494 describes a CAR system which comprises separate antigen- recognition and signalling components.
  • 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 the extracellular antigen-binding component and the intracellular signalling component of the CAR.
  • PCT/GB2015/052494 describes a CAR system which comprises;
  • a receptor component comprising an antigen binding domain, a transmembrane domain and a first binding domain which comprises a tetracycline repressor protein (TetR); and (ii) an intracellular signalling component comprising a signalling domain and a second binding domain which comprises a TetR interacting peptide (TiP) which specifically binds TetR of the receptor component.
  • TetR tetracycline repressor protein
  • the Tet operon is a well-known biological operon which has been adapted for use in mammalian cells.
  • the TetR binds tetracycline as a homodimer and undergoes a conformational change which then modulates the DNA binding of the TetR molecules.
  • Klotzsche et al. 2007 J. Biol. Chem. 280, 24591-24599 (2005); Luckner et al.; J. Mol. Biol. 368, 780-790
  • TetR interacting protein/TiP has a binding site in TetR which overlaps, but is not identical to, the tetracycline binding site (Luckner et al.; as above). Thus TiP and tetracycline compete for binding of TetR.
  • binding of the TetR on the receptor component and TiP on the intracellular signalling component is disrupted by the presence of tetracycline, such that in the absence of tetracycline the 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 tetracycline the 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 activity of CAR-expressing cells in a patient can be dimmed or turned-off using a small-molecule agent (tetracycline), without eliminating the CAR-expressing cells from the patient.
  • a small-molecule agent tetracycline
  • TetR/TiP based CAR signalling system is that, because TetR is a prokaryotic protein, it may be immunogenic when expressed on a cell which is introduced to a patient.
  • 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.
  • a membrane spanning receptor component comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular linker to TetR.
  • a separate molecule, the signalling component comprises an intracellular protein which is generated by fusion of TiP to one or several T-cell signalling domains.
  • the receptor and the signalling components interact and in the presence of cognate antigen the system signals.
  • TiP is displaced from TetR and the receptor can not transmit signals even in the presence of cognate antigen.
  • Figure 4 Intracellular linker domain derived from CD4.
  • Figure 5 Test construct with eGFP to demonstrate function of the system, (a) a bicistronic construct expressed as a single transcript which self-cleaves at the 2A site to yield: TiP fused to eGFP; and a CAR with TetR as its endodomain. (b) Fluorescent micrograph of SupT1 cells expressing this construct in the absence of tetracycline. The eGFP fluorescence can clearly be seen at the cell membrane; (c) Fluorescent micrograph of the same cells but now in the presence of tetracycline. Here, the eGFP is cytoplasmic showing that tetracycline has displaced TiP.
  • FIG. 6 Initial TetCAR construct and control
  • a bicistronic construct expressed as a single transcript which self-cleaves at the 2A site to yield: a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD3-Zeta; and a receptor component which comprises a CD33 recognizing scFv, a spacer derived from the Fc domain of lgG1 , a CD4 derived transmembrane and intracellular domain; and TetR.
  • a control was also constructed which was identical except TiP was absent from the signalling component.
  • annotated amino-acid sequence of the basic TetCAR is shown.
  • TetCAR was expressed in BW5 T-cells. These T-cells were challenged with wild-type SupT1 cells or SupT1 cells engineered to express CD33 in the absence of tetracycline or in the presence of increasing concentrations of tetracycline.
  • T-cells challenged with wild-type SupT1 cells do not activate in either the presence or absence of Tetracyline; T-cells challenged with SupT1 cells expressing CD33 activate in the absence of Tetracycline, but activation is rapidly inhibited in the presence of tetracycline with activation fully inhibited in the presence of 100nM of Tetracycline, (b) Control TetCAR which lacks the TiP domain was transduced into BW5.
  • T-cells were challenged with wild-type SupT1 cells or SupT1 cells engineered to express CD33 in the absence or in the presence of increasing concentration of Tetracycline. A lack of TiP element in the signalling component resulted in no signalling in any conditions.
  • FIG 8 Dual tetR domain tetCARs.
  • tetR is expressed as a single-chain with two TetRs attached together. If tetR domains with differing affinity for tetracycline (and hence TiP) are used, the kinetics of Tetracycline mediated displacement of TiP can modulate the levels of signalling.
  • FIG. 9 A tetCAR signalling system utilising a plurality of signalling components containing single endodomains.
  • a single CAR is expressed with many different signalling components all of which comprise TiP at their amino terminus but a different individual signalling domain, in contrast to a compound signalling domain. These randomly interact with the receptor component. Lack of steric interaction between the different signalling domains and their second messengers improves their function.
  • FIG 10 A tetCAR signalling system utilising a plurality of signalling components containing single endodomains and different TiP domains.
  • Each signalling component comprises of an individual signalling domain.
  • Each signalling component also comprises of a TiP, however each TiP has different affinities to the TetR domain. Hence the stoichiometry of the interactions between the CAR and the signalling domains can be varied.
  • the signalling system is constructed such that OX40 > CD3Zeta > CD28.
  • Figure 11 A tetCAR signalling system utilising a plurality of receptor components and a plurality of signalling components, each signalling component containing a single endodomain.
  • Figure 12 TetCAR signalling in primary cells
  • Figure 13 Interferon-Gamma release from non-transduced T-cells, and T-cells transduced with the different CAR construct challenged ((i) Classical first generation CAR, (ii) tetCAR and (iii) control tetCAR), with SupT1 cells (red), SupT1.CD19 cells (blue) in different concentrations of Tetracyline.
  • Figure 14 Killing of target cells.
  • a chromium release assay was used to demonstrate killing of target cells (SupT1.CD19) in the absence of tetracycline. Key: (i) - regular CAR; (ii) - control tetCAR (no TiP on endodomain); and (iii) - tetCAR.
  • Figure 15 Annotated sequence of wild-type TetR showing secondary structure.
  • FIG 16 Investigating the expression of receptor components comprising truncated versions of TetR.
  • TetR variants were created lacking successive alpha helices from the N terminus of the molecule and expressed in PBMCs from two different donors. These data show the effect of removing 1 , 2, 3, 4, 5 or 6 alpha helices from the N-terminus of TetR in the receptor component of a split CAR system.
  • the present inventors have previously 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 original system was based on the interaction between the tetracycline repressor protein (TetR) and the TetR interacting peptide (TiP).
  • TetR on the antigen-recognition component and TiP on the signalling component (or vice versa)
  • the present inventors have now developed truncated versions of TetR which retain the capacity to bind TiP and tetracycline and its derivatives, but have reduced immunogenicity when expressed or introduced in vivo in a mammal.
  • the present invention provides a chimeric antigen receptor (CAR) system comprising; (i) a receptor component comprising an antigen binding domain, a transmembrane and a first binding domain which comprises a truncated Tet Repressor protein (TetR); and
  • an intracellular signalling component comprising a signalling domain and a second binding domain which specifically binds the truncated TetR of the first binding domain of the receptor component; 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 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 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.
  • truncated TetR may alternatively be positioned on the intracellular signalling component.
  • a chimeric antigen receptor (CAR) system comprising;
  • an intracellular signalling component comprising a signalling domain and a first binding domain which comprises a truncated Tet Repressor protein (TetR);
  • a receptor component comprising an antigen binding domain, a transmembrane and a second binding domain which specifically binds the truncated TetR of the first binding domain of the signalling component; and 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 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 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.
  • TetR Tet Repressor protein
  • the truncated Tet Repressor protein may comprise helices eight, nine and ten from the N- terminus, compared to wild-type TetR.
  • the truncated Tet Repressor protein may comprise the sequence shown as SEQ ID No. 15.
  • the truncated Tet Repressor protein may lack the first N-terminal alpha helix, compared to wild type TetR.
  • the CAR system may comprise the sequence shown as SEQ ID No. 11.
  • the truncated Tet Repressor protein may lack the first and second N-terminal alpha helices, compared to wild type TetR.
  • the CAR system may comprise the sequence shown as SEQ ID No. 12.
  • the truncated Tet Repressor protein may lack the first, second and third N-terminal alpha helices, compared to wild type TetR.
  • the CAR system may comprise the sequence shown as SEQ ID No. 13.
  • the truncated Tet Repressor protein may lack the first, second, third and fourth N-terminal alpha helices, compared to wild type TetR.
  • the CAR system may comprise the sequence shown as SEQ ID No. 14.
  • the receptor component may comprise a linker between the transmembrane domain and the first binding domain.
  • the linker may, for example, comprise or consist of the sequence shown as SEQ ID NO: 16.
  • the second binding domain may comprises or consist of the TetR interacting peptide (TiP) or a variant thereof.
  • the agent may be tetracycline, doxycycline or minocycline or an analogue thereof.
  • the receptor component may comprise two first binding domains which are truncated TetR domains.
  • the two truncated TetR domains may be separated by a linker.
  • the signalling domain of the signalling component may comprise at least one endodomain selected from CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and OX40 endodomain.
  • the CAR system may comprise a plurality of signalling components, each comprising a signalling domain and binding domain, wherein the binding domains each recognise the same binding domain of the receptor component but the signalling domains comprise different endodomains.
  • the present invention provides a nucleic acid sequence encoding a CAR signalling system according to the first aspect of the invention, wherein the receptor component and signalling component are co-expressed by means of a self-cleaving peptide which is cleaved between the receptor component and the signalling component after translation.
  • a vector comprising a nucleic acid sequence according to the second aspect of the invention.
  • the vector may, for example, be a retroviral vector or a lentiviral vector or a transposon.
  • a cell which expresses a receptor component and a signalling component as defined in the first aspect of the invention.
  • the cell according may comprise a nucleic acid according to the second aspect of the invention or a vector according to the third aspect of the invention.
  • the cell may be an immune cell such as a T cell or an NK cell.
  • a pharmaceutical composition comprising a plurality of cells according to the fourth aspect of the invention.
  • composition according to the fifth aspect of the invention for use in treating and/or preventing a disease.
  • a method for treating and/or preventing a disease which comprises the step of administering a pharmaceutical composition according to the fifth aspect of the invention to a subject.
  • the method may comprise the following steps:
  • the method may involve monitoring toxic activity in the subject and comprises the step of administering an agent for use in the CAR signalling system according to 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 comprises the step of administering an agent for use in the CAR signalling system according to 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.
  • a pharmaceutical composition according to the fifth aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
  • a kit which comprises a first nucleic acid or vector encoding a receptor component as defined in the first aspect of the invention and a second nucleic acid or vector encoding a signalling component as defined in the first aspect of the invention.
  • a method for making a cell according to the fourth aspect of the invention which comprises the step of introducing a nucleic acid sequence according the second aspect of the invention, or a vector according to the second aspect of the invention, or a kit according to the ninth aspect of the invention into a cell.
  • the cell may be from a sample isolated from a 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 toxicities.
  • the system of the invention uses a truncated version of TetR, and is less likely to be immunogenic when expressed in or introduced to a patient than a CAR system based on full-length TetR.
  • Classical CARs which are shown schematically in Figure 1 , are chimeric type I transmembrane 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.
  • scFv single-chain variable fragment
  • mAb monoclonal antibody
  • a spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation.
  • a common spacer domain used is the Fc of IgGl More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen.
  • a transmembrane 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 ⁇ chain of the FceR1 or ⁇ 3 ⁇ . 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.
  • compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of ⁇ 3 ⁇ 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.
  • the CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors.
  • 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 invention relates to a CAR system in which the antigen- recognizing/antigen binding domain and transmembrane domain are provided on a first molecule (termed herein 'receptor component'), which localizes to the cell membrane.
  • the intracellular signalling domain is provided on a second, intracellular molecule (termed herein 'signalling component').
  • the 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 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 (PIP 2 ), activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration.
  • PTKs protein tyrosine kinases
  • PIP 2 protein kinase C
  • Functional readouts such as clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells and induction of cytotoxicity or cytokine secretion may also be utilised.
  • the inventors determined 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.
  • IL-2 interleukin-2
  • the first binding domain and second binding domain of the CAR system 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 or comprise a truncated TetR and a TetR interacting peptide; or vice versa.
  • 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 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 is a molecule such as tetracycline or a derivative thereof which preferentially binds the truncated TetR with a higher affinity than the affinity between truncated TetR and its interacting peptide.
  • 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.
  • TRUNCATED TETR TetR is essentially made up of 10 alpha helical structures that can be divided into two domains, a DNA binding domain (first 4 n-terminal alpha helical structures) and a homodimerization domain (6 c-terminal alpha helical structures).
  • the TIP peptide and tetracycline binds to the interface between the homodimers, the most important region being alpha helices 8-10,
  • Wild-type TetR has the sequence shown as SEQ ID No. 17.
  • SEQ ID No. 17 SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRH HTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQ GFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEP AFLFGLELIICGLEKQLKC
  • This TetR protein sequence is completely conserved in gram negative bacteria. These include the species, Salmonella choleraesuis, Salmonella typhimurium, Escherichia Coli, Shigella flexneri and others.
  • Wild-type TetR has 10 distinct alpha helices as shown in Table 1.
  • the truncated TetR used in the CAR system of the present invention may lack one or more alpha helices.
  • it may lack helix 1 (the sequence shown as SEQ ID No. 1); helix 2 (the sequence shown as SEQ ID No. 2); helix 3 (the sequence shown as SEQ ID No. 3); helix 4 (the sequence shown as SEQ ID No. 4); helix 5 (the sequence shown as SEQ ID No. 5); helix 6 (the sequence shown as SEQ ID No. 6); helix 7 (the sequence shown as SEQ ID No. 7); helix 8 (the sequence shown as SEQ ID No. 8); helix 9 (the sequence shown as SEQ ID No. 9); helix 10 (the sequence shown as SEQ ID No. 10).
  • helix 1 the sequence shown as SEQ ID No. 1
  • helix 2 the sequence shown as SEQ ID No. 2
  • helix 3 the sequence shown as SEQ ID No. 3
  • helix 4 the sequence shown as S
  • SEQ ID 17 is for TetR class B or TetRB.
  • TetR sequence homologs for example form other classes of tetracycline resistant bacteria, which differ from the sequence given as SEQ ID No 17, the equivalent sequence may be derived by performing an alignment of the two sequences to determine the position of the equivalent a helix or helices in the variant sequence.
  • the truncated tetR may lack one or more helices from the N-or C-terminal end.
  • truncated TetR may lack helix 1 ; helices 1 and 2; helices 1 , 2 and 3; or helices 1 , 2, 3 and 4 from the N-terminal end.
  • TetR Truncation of TetR may have the effect of completely or partially removing the DNA binding domains but maintain the TIP peptide/tetracycline binding domain.
  • the truncated TetR may lack any one of more of helices 1-4 from the N-terminus, but may comprise helices 8 to 10.
  • the truncated TetR binds to an interacting peptide
  • the peptide may be or comprise TiP haing the sequence shown as SEQ ID No. 18
  • a different peptide may be selected, based on its ability to bind truncated TetR but be competitively inhibited by the agent such as tetracycline or a derivative thereof.
  • the interacting peptide may, for example, be between 8-30, for example 10-20 amino acids in length.
  • Suitable interacting peptides may be generated and identified using peptide display methods such as phage display, CIS display, ribosome display and mRNA display (Ullman et al (201 1) Brieifings 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 eg in a peptide array, so that the selection method reflects the properties of the interacting protein in the CAR signalling system, namely that it binds truncated TetR, but the binding is competitively inhibited by the presence of the tetracycline-like agent.
  • 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: 16.
  • 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 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.
  • 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 present invention provides a receptor component comprising an antigen-binding domain, an optional spacer domain, a transmembrane domain and a first biding domain which comprises truncated TetR.
  • the receptor component When expressed in a cell, 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 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; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • Various tumour associated antigens (TAA) are known, as shown in the following Table 3.
  • TAA tumour associated antigens
  • the antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.
  • the 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 CAR system described herein may comprise a spacer sequence to connect the antigen- binding domain with the transmembrane domain in the receptor component.
  • a flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
  • the spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk.
  • the spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk.
  • a human lgG1 spacer may be altered to remove Fc binding motifs.
  • the 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 ( Figure 11). 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 ( Figure 1 1). 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 which comprises truncated TetR.
  • 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.
  • 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.
  • chimeric CD28 and OX40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together (illustrated in Figure 1 B).
  • 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 OX40 or the CD28 endodomain and OX40 and CD3-Zeta endodomain ( Figure 3A).
  • the signalling component of a CAR system may comprise the sequence shown as SEQ ID NO: 19, 20 or 21 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: 19, 20 or 21 , 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 (Figure 9). 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 ( Figure 10).
  • Figure 10 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 receptor component of the second aspect and a nucleic acid encoding a signalling component of the third aspect.
  • 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. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variant in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • 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 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 of the third aspect.
  • the present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) encoding a receptor component of the second aspect and/or signalling component of the third aspect of the invention.
  • 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 cell may comprise a nucleic acid or a vector of the present invention.
  • the cell may comprise a receptor component and a signalling component of the present invention.
  • the cell may comprise at least one signalling component of the present invention.
  • the cell may comprise one, two, three, four, five, or more signalling components of the present invention.
  • the cell may comprise at least one receptor component of the present invention.
  • the cytolytic immune cell may comprise one, two, three, four, five, or more receptor components of the present invention.
  • the cell may be an immune cell, such as a cytolytic immune cell.
  • 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
  • These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.
  • Cytolytic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection.
  • CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells.
  • MHC class I MHC class I
  • IL-10 adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.
  • Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re- exposure to their cognate antigen, thus providing the immune system with "memory" against past infections.
  • Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.
  • Treg cells Regulatory T cells
  • suppressor T cells are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.
  • Treg cells Two major classes of CD4+ Treg cells have been described—natural occurring Treg cells and adaptive Treg cells.
  • Naturally occurring Treg cells arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD1 1c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP.
  • Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.
  • Adaptive Treg cells may originate during a normal immune response.
  • NK cells Natural Killer Cells
  • MHC Multiple Access to Cells
  • 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.
  • 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 (1 st 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:
  • 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.
  • 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:
  • 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.
  • 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 3.
  • 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 as a single transcript which self-cleaves at the 2A site to yield TiP fused to eGFP and a CAR with TetR as its endodomain (Figure 5a).
  • a bicistronic construct was expressed in BW5 T cells as a single transcript which self- cleaves at the 2A site to yield a signalling component which comprises TiP fused via a flexible linker to the endodomain of CD3-Zeta; and a receptor component which comprises a CD33 recognizing scFv, a spacer derived from the Fc domain of lgG1 , a CD4 derived transmembrane and intracellular domain; and TetR ( Figure 6a).
  • a control was also expressed which was identical except that TiP was absent from the signalling component (Figure 6b).
  • the BW5 T-cells were challenged with wild-type SupT1 cells or SupT1 cells engineered to express CD33 in the absence of tetracycline or in the presence of increasing concentrations of tetracycline.
  • T-cells challenged with wild-type SupT1 cells did not activate in either the presence or absence of Tetracyline; T-cells challenged with SupT1 cells expressing CD33 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 100nM of tetracycline (Figure 7a). Control TetCAR which lacks the TiP domain was also transduced into BW5.
  • SupT1 cells (which are CD19 negative), were engineered to be CD19 positive giving target negative and positive cell lines which were as similar as possible.
  • Primary human T-cells from 3 donors were transduced with three CAR constructs: (i) "Classical” 1st generation anti- CD"! 9 CAR; (ii) 1st generation anti-CD19 tetCAR; (iii) Control anti-CD19 tetCAR where TiP is missing from endodomain.
  • Non-transduced T-cells and T-cells transduced with the different CAR constructs were challenged 1 : 1 with either SupT1 cells or SupT1.CD19 cells in the presence of different concentrations of Tetracycline. Supernatant was sampled 48 hours after challenge.
  • SupT1 and SupT1.CD19 cells were loaded with 5 Cr and incubated with control and Tet-CAR T-cells for 4 hours in the presence or absence of tetracycline. Lysis of target cells was determined by counting 5 Cr in the supernatant. The results are shown in Figure 14. It was shown that Tet-CAR T-cells lysed SupT1.CD19 target cells only in the absence of Tetracycline.
  • Example 5 Production of truncated versions TetR Truncated version of tetRB are generated in which successive alpha helices are removed from the N-terminal end of the molecule.
  • An N-terminal FLAG tag is included for protein quantification and a C-terminal dual His tag is included for immobilisation.
  • 293T cells are transfected with vector expressing the sequences and protein expression is quantitated.
  • a split CAR system similar to the one described in Example 2 was generated, which comprised a receptor component with a CD19-specific antigen-binding domain.
  • the intracellular domain also comprised TetR or truncated versions thereof in which successive alpha helices are removed from the N-terminal end of the molecule.
  • TetRB split CAR The construct expressing the full length TetRB split CAR system had the following structure:
  • This bicistronic construct is expressed as a single transcript which self-cleaves at the 2A site to yield a signalling component which comprises TiP fused via a flexible linker (L) to the endodomains of CD28 and CD3-Zeta; and a receptor component which comprises a CD19 recognizing scFv (aCD19 having a binder was based on FMC), a CD8 stalk spacer, a CD28 derived transmembrane domain, a linker and TetR.
  • a signalling component which comprises TiP fused via a flexible linker (L) to the endodomains of CD28 and CD3-Zeta
  • a receptor component which comprises a CD19 recognizing scFv (aCD19 having a binder was based on FMC), a CD8 stalk spacer, a CD28 derived transmembrane domain, a linker and TetR.
  • PBMCs from healthy donors were transduced with a vector expressing each of the split CAR systems.
  • cells were stained with soluble-CD19-Rb-Fc and anti-Rb-Fc-PE. Cells were also stained with 7-AAD to exclude dead cells.
  • CD3+ cells stained positively with sCD19 correspond to the transduced cells expressing the Tet-CAR constructs. Expression was investigated by flow cytometry on the basis of CD19 expression. The results are shown in Figure 16. Good expression of the receptor component was seen with the removal of 1 , 2, 3 or 4 alpha helices from the N-terminus of the molecule. Reduced expression of the receptor component was seen with the removal of 5 alpha helices from the N-terminus of the molecule.
  • TetR is essentially made up of 10 alpha helical structures that can be divided into two domains, a DNA binding domain (first 4 n-terminal alpha helical structures) and a homodimerization domain (6 c-terminal alpha helical structures). These data show that it is possible to remove all or part of the DNA binding domain without affecting the stability of the molecule. Removal of 5 or more helices, which is predicted to remove part of the homodimerisation domain, caused a reduction in the expression of the receptor component.
  • Example 7 Functional Validation of the truncated TetR variants in the split CAR system
  • PBMCs transduced with the Tet-CAR truncated versions, as well as the non-truncated Tet- CAR are co-cultured with target cells in the absence or presence of tetracycline and the ability of the CAR expressing cells to kill targets and produce cytokines is assessed.
  • Cells are expected to kill CD19+ targets and produce cytokines (IFN-gamma/IL-2) in response to the CD19+ targets in the absence of the drug, while in the presence of tetracycline, both target cell killing and cytokine production are expected to be compromised.
  • a FACS based toxicity assay is used in which PBMCs transduced with the different Tet- CAR constructs are cultured at ratios 1 : 1 and 4: 1 with the CD19-expressing SupTI target cells (SupT1-CD19); or negative control SupTI cells which do not express the target antigen (SupT1-NT).
  • PBMCs transduced with a 2nd generation CAR are also included as a positive control while non-transduced PBMCs serve as negative control.
  • Co-cultures are set up in the absence of tetracycline or in the presence of 1600nM tetracycline. Killing is assessed at 2 time points (24h and 72h).
  • 7-AAD allows gating to the live cell population while CD3 staining excludes T cells in order to gate to the target cell population.
  • CD3 staining excludes T cells in order to gate to the target cell population.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un système de récepteur antigénique chimérique (CAR) comprenant ; (i) un composant récepteur comprenant un domaine de liaison à l'antigène et un premier domaine de liaison ; et (ii) un composant de signalisation comprenant un domaine de signalisation et un deuxième domaine de liaison qui se lie spécifiquement au lieur de domaine unique du premier domaine de liaison du composant récepteur, le premier domaine ou le deuxième domaine de liaison comprenant TerR tronqué et la liaison des premier et deuxième domaines de liaison étant perturbée par la présence d'un agent, de telle sorte qu'en l'absence de l'agent, le composant récepteur et le composant de signalisation s'hétérodimérisent et la liaison du domaine de liaison de l'antigène à l'antigène résulte en la signalisation par le domaine de signalisation. Toutefois, en présence de l'agent, le composant récepteur et le composant de signalisation ne s'hétérodimérisent pas et la liaison du domaine de liaison de l'antigène à l'antigène ne résulte pas en la signalisation par le domaine de signalisation.
PCT/GB2017/050341 2016-02-12 2017-02-10 Système de signalisation WO2017137759A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1602571.0A GB201602571D0 (en) 2016-02-12 2016-02-12 Signalling system
GB1602571.0 2016-02-12

Publications (1)

Publication Number Publication Date
WO2017137759A1 true WO2017137759A1 (fr) 2017-08-17

Family

ID=55697641

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2017/050341 WO2017137759A1 (fr) 2016-02-12 2017-02-10 Système de signalisation

Country Status (2)

Country Link
GB (1) GB201602571D0 (fr)
WO (1) WO2017137759A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10588967B2 (en) 2014-04-01 2020-03-17 Ucl Business Ltd Chimeric antigen receptor (CAR) signalling system
US10604570B2 (en) 2015-02-05 2020-03-31 Ucl Business Ltd Chimeric antigen receptor signalling system comprising heterodimerization domains
US10654927B2 (en) 2014-08-29 2020-05-19 Ucl Business Ltd Signalling system
US11311576B2 (en) 2018-01-22 2022-04-26 Seattle Children's Hospital Methods of use for CAR T cells
US11649288B2 (en) 2017-02-07 2023-05-16 Seattle Children's Hospital Phospholipid ether (PLE) CAR T cell tumor targeting (CTCT) agents
US11759480B2 (en) 2017-02-28 2023-09-19 Endocyte, Inc. Compositions and methods for CAR T cell therapy

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014127261A1 (fr) * 2013-02-15 2014-08-21 The Regents Of The University Of California Récepteur d'antigène chimère et procédés d'utilisation de celui-ci
WO2015150771A1 (fr) * 2014-04-01 2015-10-08 Ucl Business Plc Système de signalisation de récepteur antigénique chimérique (car)
WO2016030691A1 (fr) * 2014-08-29 2016-03-03 Ucl Business Plc Système de signalisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014127261A1 (fr) * 2013-02-15 2014-08-21 The Regents Of The University Of California Récepteur d'antigène chimère et procédés d'utilisation de celui-ci
WO2015150771A1 (fr) * 2014-04-01 2015-10-08 Ucl Business Plc Système de signalisation de récepteur antigénique chimérique (car)
WO2016030691A1 (fr) * 2014-08-29 2016-03-03 Ucl Business Plc Système de signalisation

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
C.-Y. WU ET AL: "Remote control of therapeutic T cells through a small molecule-gated chimeric receptor", SCIENCE, vol. 350, no. 6258, 16 October 2015 (2015-10-16), pages aab4077 - aab4077, XP055260068, ISSN: 0036-8075, DOI: 10.1126/science.aab4077 *
JIE SUN ET AL: "The quest for spatio-temporal control of CAR T cells", CELL RESEARCH - XIBAO YANJIU, vol. 25, no. 12, 17 December 2015 (2015-12-17), GB, CN, pages 1281 - 1282, XP055361929, ISSN: 1001-0602, DOI: 10.1038/cr.2015.131 *
KLOTZSCHE ET AL., J. BIOL. CHEM., vol. 280, 2007, pages 24591 - 24599
LUCKNER ET AL., J. MOL. BIOL., vol. 368, 2007, pages 780 - 790
LUCKNER ET AL., J. MOL. BIOL., vol. 368, pages 780 - 790
LUCKNER ET AL: "How an Agonist Peptide Mimics the Antibiotic Tetracycline to Induce Tet-Repressor", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 368, no. 3, 6 April 2007 (2007-04-06), pages 780 - 790, XP022020037, ISSN: 0022-2836, DOI: 10.1016/J.JMB.2007.02.030 *
MIURA ET AL., BIOCHIM. ET BIOPHYS. ACTA, vol. 1673, 2004, pages 131 - 138
SERGEY REVERDATTO ET AL: "Peptide Aptamers: Development and Applications", CURR TOP MED CHEM, vol. 15, 1 January 2015 (2015-01-01), pages 1082 - 1101, XP055362079 *
ULLMAN ET AL., BRIEIFINGS IN FUNCTIONAL GENOMICS, vol. 10, 2011, pages 125 - 134
WESTROP ET AL., J. IMMUNOL. METHODS, vol. 341, 2009, pages 76 - 85
XUE ET AL., CLIN EXP IMMUNOL., vol. 183, 2016, pages 102 - 113

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10588967B2 (en) 2014-04-01 2020-03-17 Ucl Business Ltd Chimeric antigen receptor (CAR) signalling system
US10654927B2 (en) 2014-08-29 2020-05-19 Ucl Business Ltd Signalling system
US10604570B2 (en) 2015-02-05 2020-03-31 Ucl Business Ltd Chimeric antigen receptor signalling system comprising heterodimerization domains
US11649288B2 (en) 2017-02-07 2023-05-16 Seattle Children's Hospital Phospholipid ether (PLE) CAR T cell tumor targeting (CTCT) agents
US11759480B2 (en) 2017-02-28 2023-09-19 Endocyte, Inc. Compositions and methods for CAR T cell therapy
US11850262B2 (en) 2017-02-28 2023-12-26 Purdue Research Foundation Compositions and methods for CAR T cell therapy
US11311576B2 (en) 2018-01-22 2022-04-26 Seattle Children's Hospital Methods of use for CAR T cells
US11779602B2 (en) 2018-01-22 2023-10-10 Endocyte, Inc. Methods of use for CAR T cells

Also Published As

Publication number Publication date
GB201602571D0 (en) 2016-03-30

Similar Documents

Publication Publication Date Title
US20210032332A1 (en) Signalling system
EP3126380B1 (fr) Système de signalisation de récepteur antigénique chimérique (car)
EP3253783B1 (fr) Système de signalisation
CN107406518B (zh) 嵌合抗原受体
WO2017137759A1 (fr) Système de signalisation
US20180305433A1 (en) Cell
EP3414263B1 (fr) Système de signalisation
CN107075483A (zh) 用于过继细胞治疗的工程改造的细胞
US20200338124A1 (en) Cell
EP3612568A1 (fr) Cellule
JP2019535292A (ja) シグナル伝達改変タンパク質
KR20210143856A (ko) CAR을 발현하는 유전적으로 리프로그래밍된 Treg
WO2018229492A1 (fr) Récepteur antigénique chimérique
WO2020074868A1 (fr) Cellule
WO2019053420A1 (fr) Cellule
WO2022200782A1 (fr) Système de signalisation
CA3231382A1 (fr) Domaine de liaison

Legal Events

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

Ref document number: 17707103

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17707103

Country of ref document: EP

Kind code of ref document: A1