CHIMERIC ANTIGEN RECEPTOR (CAR) SIGNALLING SYSTEM

Abstract

The present invention relates to a chimeric antigen receptor (CAR) signalling system comprising; (i) a receptor component comprising an extracellular antigen-binding domain, a transmembrane domain and a intracellular first chemical inducer of dimerization binding domain 1 (CBD1); and (ii) an intracellular signalling component comprising a signalling domain and a second chemical inducer of dimerization binding domain 2 (CBD2); wherein CBD1 and CBD2 are capable of simultaneously binding to a chemical inducer of dimerization (CID); wherein, in the absence of the CID, binding of the antigen-binding component to antigen does not result in signalling through the signalling component; whilst, in the presence of the CID, the receptor component and the signalling component heterodimerize and binding of the antigen-binding domain to antigen results in signalling through the signalling domain.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to an antigen receptor signalling system.

BACKGROUND TO THE INVENTION

[0002] 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.

[0003] 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 FIG. 1A).

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] These toxicities are very difficult to predict even with detailed animal studies or non-human primate work. Crucially, unlike small molecules and biologics, 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.

[0009] 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.

[0010] 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 entity. 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. Finally, suicide genes are not "fail-safe", i.e. the default status is for the CAR T-cells to be active.

[0011] 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

[0012] FIG. 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 Fc.epsilon.R1-.gamma. or CD3.zeta. endodomain, while later designs transmitted additional (c) one or (d) two co-stimulatory signals in the same compound endodomain.

[0013] FIG. 2--Structures of Rapamycin analogues.

[0014] FIG. 3--Illustration of an example of an inducible CAR signalling system in its simplest incarnation. (a) Receptor component comprising of an extracellular scFv, a spacer, a trans-membrane domain and an intracellular FKBP12. (b) Signalling component: intracellular fusion between FRB fragment of mTOR and the endodomains of CD28, OX40 and CD3 Zeta. (c) A chemical inducer of dimerization (CID), in this case is Rapamycin or an analogue. (d) Receptor component. In the presence of CID, the receptor component and the signalling components dimerize allowing signalling in the presence of cognate antigen.

[0015] FIG. 4--Function of an inducible CAR system. T-cells were transduced with a CAR system as illustrated in FIG. 3 and with SEQ ID No. 19. This CAR recognizes CD19. CAR expressing T-cells were challenged with CD19+ targets at 1:1 and 2:1 effector to target ratios in the presence of increasing concentration of CID. T-cells respond to target cells only in the presence of CID. Stimulation with PMA/Ionomycin is also show. Response to CD19+ targets of T-cells expressing a standard CD19 CAR (MP15577) to different effector target ratios is also shown.

[0016] FIG. 5--Illustration of an inducible CAR system with two receptor components. (a) Two receptor components are co-expressed along with a single signalling component. The two receptor components each have a different single chain which recognizes a different target. The receptor components have endodomains comprising of the FRB fragment of mTOR. A single signalling component comprises of FKBP12 and a fusion of CD28, OX40 and CD3 Zeta endodomains. In (a), the FRB sequences are identical and this CAR will signal equally in response to either target. In (b), the FRB sequences differ such that each has a different affinity for the CID. With this implementation, the CAR will signal with different strength in response to each cognate antigen in the presence of CID.

[0017] FIG. 6--Illustration of an example of a multiple transmembrane receptor domain receptor component for use in an inducible CAR signalling system. The receptor component is a chimera of a type I CAR linked to the multi-span protein. A scFv is connected to an Fc spacer domain and a TM domain. This in turn is connected to the CD20 molecule with truncated amino and carboxy termini. Each intracellular entry is attached to an FRB domain. The signalling component comprises of FKBP12 and a fusion of CD28, OX40 and CD3 Zeta endodomains. Addition of the CID allows the stochastic ligation of multiple signalling domains to the multiple endodomains which recognize a single antigen receptor component, resulting in amplified signalling in response to antigen.

[0018] FIG. 7--Illustration of an inducible CAR system with multiple signalling component inducible CAR. This CAR system has a single receptor component comprising of a scFv, an Fc spacer, a TM domain and a FKBP12 endodomain. The three different signalling domains comprise of a fusion between the FRB fragment of mTOR and the CD28 endodomain, a fusion between the FRB fragment of mTOR and the 41BB endodomain and a fusion between the FRB fragment of mTOR and the CD3 Zeta endodomain. In (a), the FRB domains are identical so that each signalling component is recruited equally to the receptor component and the CAR transmits an equal CD28, 41BB and CD3 Zeta signal in the presence of CID and upon recognition of the cognate antigen. In (b), the FRB domains are different so that each signalling component can be recruited differently to the receptor component so that the CAR transmits more or less of each of CD28, 41BB and CD3 Zeta signals depending on the optimum needed for the application.

[0019] FIG. 8--Illustration of an inducible CAR with signalling component which comprises of two CID binding domains. In this example, there is one receptor component which comprises of a scFv, and Fc spacer, a transmembrane domain and an FKBP12 endodomain. The signalling domain comprises of the FRB domain of mTOR, a fusion of CD28, OX40 and Zeta endodomains and an FKBP12 domain. In the presence of CID, the signalling components multimerize with each other and with the receptor component. Upon antigen recognition, a signal equivalent to the sum of the signalling component multimer is transmitted.

[0020] FIG. 9--Chemical structure of RSL1, the synthetic diacylhydrazine (N-(2-ethyl-3-methoxybenzoyl)-N'-(3,5-dimethylbenzoyl)-N'-tert-butylhydra- zine. RSL1 is one of a family of compounds that have been found to act as non-steroidal ecdysone agonists and can function as gene inducers.

[0021] FIG. 10--RSL1 inducible CAR (a) an RSL1 inducible CAR was constructed by using retinoic acid receptor domains and ecodysone receptor domains to constitute the heterodimerization motifs for the antigen recognition component and the signalling component respectively. The antigen recognition component recognizes CD19. (b) T-cells expressing this CAR along with non-transduced T-cells were stained with anti-Fc which recognizes the CAR spacer. The transduced T-cells expressed the CAR well. (c) Transduced T-cells were next challenged with increasing concentration of RSL1 and challenged with either CD19 negative or CD19 positive target cells. T-cells challenged with CD19 positive targets signal only in the presence of RSL1.

[0022] FIG. 11--AP1903 homodimerizer inducible CAR. (a) AP1903 homodimerizer inducible CAR was constructed by using modified FKBP12 domains to constitute the heterodimerization motifs for both the antigen recognition component and the signalling component. The antigen recognition component recognizes CD19. (b) T-cells expressing this CAR along with non-transduced T-cells were stained with anti-Fc which recognizes the CAR spacer. The transduced T-cells expressed the CAR well. (c) Transduced T-cells were next challenged with increasing concentration of CID dimerizer AP1903 and challenged with either CD19 negative or CD19 positive target cells. T-cells challenged with CD19 positive targets signal only in the presence of CID. At high CID concentrations, signalling is reduced likely to saturation of heterodimerization domains with an excess of CID reducing the formation of heterodimers.

SUMMARY OF ASPECTS OF THE INVENTION

[0023] The present inventors have found that it is possible to separate the antigen-recognition and signalling components of a CAR to produce an inducible system, where signalling only takes place in the presence of an agent such as a small molecule which can induce dimerization (henceforth referred to as chemical inducer of dimerization or CID).

[0024] The CID binds two protein domains simultaneously. By incorporating a CID binding domain into each of two non-functional halves of a CAR, the presence of CID will re-unite the halves constituting a functional whole.

[0025] Functional signalling by the CAR is hence dependent on the presence of the CID. This provides a mechanism for controlling the CAR T-cell after it has been administered to a patient: The CAR T-cell can be remotely switched on by administering the CID to the patient, and in case of toxicity switched off by not administering further CID. If activity of the CAR is proportional to local concentration of the agent (for instance when CID concentration is not saturating) activity of the CAR T-cells can be "tuned" by altering CID dose administered to the patient.

[0026] In a first aspect, the present relates to a chimeric antigen receptor (CAR) signalling system comprising of two separate protein components:

[0027] (i) A membrane spanning receptor component comprising an extracellular antigen-binding domain, a transmembrane domain and a first intracellular CID binding domain (referred henceforth as CBD1); and

[0028] (ii) An intracellular signalling component comprising at a minimum a signalling domain and a second CID binding domain (henceforth referred to as CBD2);

[0029] wherein CBD1 and CBD2 are capable of simultaneously and specifically binding to the CID.

[0030] Thus, in a first aspect, the present invention provides a chimeric antigen receptor (CAR) signalling system comprising;

[0031] (i) a receptor component comprising an extracellular antigen-binding domain, a transmembrane domain and a intracellular first chemical inducer of dimerization (CID) binding domain (CBD1); and

[0032] (ii) an intracellular signalling component comprising a signalling domain and a second CID binding domain (CBD2);

[0033] wherein CBD1 and CBD2 are capable of simultaneously binding to a CID;

[0034] wherein, in the absence of CID, binding of the antigen-binding domain to antigen does not result in signalling through the signalling domain; whilst, in the presence of CID, the receptor component and the signalling component heterodimerize and binding of the antigen-binding domain to antigen results in signalling through the signalling domain.

[0035] CBD1 and CBD2 of the CAR signalling system according to the first aspect may comprise different CID binding domains and the CID may comprise two different binding moieties.

[0036] CDB1 may comprise the rapamycin binding domain of FK-binding protein 12 (FKBP12), CDB2 may comprise the FKBP12-Rapamycin Binding (FRB) domain of mTOR, or visa-versa. In this case, the CID may comprise rapamycin or a derivative thereof which is capable of causing the receptor component and the signalling component to heterodimerize.

[0037] CBD1 may comprise the FK506 (Tacrolimus) binding domain of FK-binding protein 12 (FKBP12), CBD2 may comprise the cyclosporin binding domain of cylcophilin A (or visa versa). In this case, the CID may comprise an FK506/cyclosporin fusion protein or a derivative thereof which is capable of causing the receptor component and the signalling component to heterodimerize.

[0038] CBD1 may comprise an oestrogen-binding domain (EBD), CBD2 may comprise a streptavidin binding domain, or visa-versa. In this case, the CID may comprise an estrone/biotin fusion protein or a derivative thereof which is capable of causing the receptor component and the signalling component to heterodimerize.

[0039] CBD1 may comprise a glucocorticoid-binding domain (GBD), CBD2 may comprise a dihydrofolate reductase (DHFR) binding domain, or visa-versa. In this case, the CID may comprise a dexamethasone/methotrexate fusion molecule or a derivative thereof which is capable of causing the receptor component and the signalling component to heterodimerize.

[0040] CBD1 may comprise an O.sup.6-alkylguanine-DNA alkyltransferase (AGT) binding domain, CBD2 may comprise a dihydrofolate reductase (DHFR) binding domain, or vice-versa. In this case, the CID may comprise an O.sup.6-benzylguanine derivative/methotrexate fusion molecule or a derivative thereof which is capable of causing the receptor component and the signalling component to heterodimerize.

[0041] CBD1 may comprise a retinoic acid receptor domain, CBD2 may comprise an ecodysone receptor domain, or vice-versa. In this case, the CID may comprise RSL1 or a derivative thereof which is capable of causing the receptor component and the signalling component to heterodimerize.

[0042] CBD1 and CBD2 of the CAR signalling system according to the first aspect of the invention may comprise the same CID binding domain and the CID may comprise two identical binding moieties.

[0043] CBD1 and CBD2 of the CAR signalling system according to the first aspect of the invention may comprise FK506 binding protein (FKBP12) binding domains comprising a F36V mutation and the CID may comprise AP1903 or a derivative thereof which is capable of causing the receptor component and the signalling component to heterodimerize.

[0044] In a second embodiment, the CAR may comprise a receptor component which spans the membrane multiple times with each intracellular portion constituting a CBD1. Hence a single receptor component may recruit multiple signalling components hence amplifying signal.

[0045] In a third embodiment, the CAR may comprise a set of separate receptor components each with an intracellular CBD1 such that in the presence of CID, signalling components connect to each receptor protein component so that the CAR can signal in response to a multiplicity of cognate antigens.

[0046] The CID binding domains may be altered such that affinity to the CID differs. For instance altering amino acids at Positions 2095, 2098, and 2101 of FRB can alter binding to Rapamycin: KTW has high, KHF intermediate and PLW is low (Bayle et al, Chemistry & Biology 13, 99-107, January 2006)

[0047] Hence, in the fourth embodiment, the CAR may comprise multiple receptor components such that each one has a CBD1 domain of different affinity to the CID. In this way, in the presence of CID, signalling protein components connect to each receptor protein component differentially and hence the CAR signal strengths differentially in response to each cognate antigen.

[0048] In the above embodiment, the signalling domain of the signalling protein component may comprise of a single endodomain selected from activating or co-stimulating T-cell signalling receptor such as CD3 zeta, CD28, 41BB or OX40.

[0049] In the above embodiments, the signalling domain of the signalling protein component may comprise of a multiplicity of endodomains selected from activating and co-stimulating T-cell signalling receptor such as CD3 Zeta, CD28, 41BB and OX40

[0050] If an inhibitory CAR is desired, in the above aspects, the signalling domain of the signalling protein may comprise the endodomain of an inhibitory T-cell signalling receptor or a phosphatase.

[0051] In a fifth embodiment, the signalling component according to the above embodiments of the invention may comprise a plurality of signalling components, each comprising a different signalling domain and a similar CBD2, wherein the CID agent binding domains each recognise the same CID, but the signalling domains comprise different endodomains. In this way, the CAR can transmit multiple signals but the signalling components being in trans rather than cis are sterically unencumbered from each other's signalling transmission molecules unlike a single signalling component containing a multiplicity of endodomains.

[0052] In a sixth embodiment, the CBD2 of the multiple signalling domains in the aspect described above may bind to the CID molecule with different affinities such that the relative contribution of each signalling component can be tuned. Hence the CAR can transmit a more complex signal rather than the fixed stoichiometry of a single signalling component containing a multiplicity of endodomains.

[0053] In a seventh embodiment, the CAR systems described above may comprise signalling domains which contains a CBD1, single or multiple endodomains as well as CBD2. Hence, in the presence of CID, concatenation of multiple signalling domains together to amplify signals.

[0054] In a second aspect, the present invention provides a receptor component suitable for use in the CAR signalling system of the first aspect of the invention which comprises an antigen-binding domain, a transmembrane domain and a CID binding domain.

[0055] In a third aspect, the present invention provides a signalling component suitable for use in the CAR signalling system according to the first aspect of the invention which comprises a signalling domain and a CID binding domain.

[0056] In a fourth aspect, the present invention provides a nucleic acid encoding the receptor component according to the second aspect of the invention.

[0057] In a fifth aspect, the present invention provides a nucleic acid encoding the signalling component according to the third aspect of the invention.

[0058] In a sixth aspect, the present invention provides a nucleic acid sequence encoding the receptor component according to the second aspect of the invention and the signalling component according to the third aspect of the invention, wherein the expressed molecule is a self-cleaving peptide which is cleaved between the receptor component and the signalling component.

[0059] In a seventh aspect, the present invention provides a vector comprising a nucleic acid sequence according to any of the fourth to sixth aspects of the invention.

[0060] In an eighth aspect, the present invention provides a retroviral vector or a lentiviral vector or a transposon comprising a vector according to the seventh aspect of the invention.

[0061] In a ninth aspect, the invention provides a T cell or NK cell which expresses a receptor component according to the second aspect of the invention and at least one signalling component according to the third aspect of the invention.

[0062] The T cell or NK cell according to the ninth aspect of the invention may comprise the nucleic acid according to any of the fourth to sixth aspects of the invention or the vector according to the seventh or eighth aspects of the invention.

[0063] In a tenth aspect, the present invention provides a pharmaceutical composition comprising a plurality of T cells or NK cells according to the ninth aspect of the invention.

[0064] In an eleventh aspect, the present invention provides a pharmaceutical composition according to the tenth 13.sup.th aspect of the invention for use in treating and/or preventing a disease.

[0065] In a twelfth aspect, the invention provides a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the tenth aspect of the invention to a subject.

[0066] The method according to the twelfth aspect of the invention may comprise the following steps:

[0067] (i) isolation of a T or NK cell-containing sample from a subject;

[0068] (ii) transduction or transfection of the T or NK cells with a nucleic acid sequence according to any the fourth to sixth aspects or a vector according to the seventh or eighth aspects; and

[0069] (iii) administering the cells from (ii) to the subject.

[0070] The method according to the twelfth aspect of the invention may further comprise the step of administering a CID suitable for use in the CAR signalling system according to the first aspect to the subject.

[0071] In a thirteenth, the present invention provides a method for treating and/or preventing a disease in a subject which subject comprises T cells according to the ninth aspect of the invention, which method comprises the step of administering a CIFD suitable for use in the CAR signalling system according to the first aspect of the invention to the subject.

[0072] The method according to the twelfth or thirteenth aspect of the invention may involve monitoring the progression of disease and/or monitoring toxic activity in the subject and adjusting the dose of the CID to provide acceptable levels of disease progression and/or toxic activity.

[0073] The disease to be prevented and/or treated according to the eleventh, twelfth or thirteenth aspect of the invention may be cancer.

[0074] In a fourteenth aspect, the present invention provides the use of a pharmaceutical composition according to the tenth aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

[0075] In a fifteenth aspect, the present invention provides a kit which comprises a nucleic acid according to any of the fourth to sixth aspects of the invention or a vector according to the seventh or eighth aspects of the invention.

[0076] The kit may also comprise a CID suitable for use in the CAR signalling system according to the first aspect of the invention.

[0077] In a sixteenth aspect, the invention provides a kit which comprises a T cell or NK cell according to the ninth aspect of the invention and a CID suitable for use in the CAR signalling system according to the first aspect of the invention.

[0078] In a seventeenth aspect, the present invention provides a method for making a T cell or NK cell according to claim ninth aspect of the invention, which comprises the step of introducing: a nucleic acid sequence according to any of the fourth to sixth aspects of the invention or a vector according to the seventh or eighth aspects of the invention, into a T or NK cell.

[0079] The T or NK cell may be from a sample isolated from a subject.

[0080] In an eighteenth aspect, the invention provides a method for activating the CAR signalling system according to the first aspect of the invention in a subject which comprises a T-cell according to the ninth aspect of the invention, which method comprises the step of administering the CID to the subject.

[0081] In a nineteenth aspect the invention provides a method for reducing the activity of the CAR signalling system according to the first aspect of the invention, in a subject which comprises a T-cell or NK cell according to the ninth aspect of the invention, which method comprises reducing or stopping administration of the CID to the subject.

[0082] In a twentieth aspect the present invention provides a method for activating signalling in a CAR signalling system according to the first aspect of the invention in a T cell or NK cell according to the ninth aspect of the invention in culture, which comprises the step of introducing the CID agent to the T or NK cell culture.

[0083] In a twenty-first aspect the present invention provides a method for reducing or stopping signalling via a CAR signalling system according to the first aspect of the invention in a T or NK cell according to the ninth aspect of the invention in culture, which comprises the step of removing the CID from the T or NK cell culture.

[0084] In a twenty-second aspect, there is provided a method for inducing dimerization in vivo between a receptor component according to the second aspect of the invention and a signalling component according to the third aspect of the invention, which comprises the step of administering a chemical inducer of dimerization (CID) to a subject comprising a cell according to the ninth aspect of the invention.

[0085] The present invention therefore provides a means of tuning the CAR activity to the presence of an agent, such as a small molecule. This allows the potency of CAR cells to be controlled pharmacologically and tuned to an acceptable balance between achieving the desired therapeutic effect, while avoiding CAR-associated toxicities.

DETAILED DESCRIPTION

[0086] Chimeric Antigen Receptors (CARs)

[0087] Classical CARs, which are shown schematically in FIG. 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. 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 IgG1. More compact spacers can suffice e.g. the stalk from CD8.alpha. and even just the IgG1 hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

[0088] Early CAR designs had endodomains derived from the intracellular parts of either the .gamma. chain of the Fc.epsilon.R1 or CD3.zeta.. 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 CD3.zeta. 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 OX40 and 41BB 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.

[0089] 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.

[0090] In a first aspect, the present invention relates to a CAR signalling 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 `signaling component`). Certain small molecules have been described which bind simultaneously to two protein domains. Such small molecules can hence act to dimerize proteins (termed herein "chemical inducer of dimerizeration" or "CID"). Both the receptor component and signaling components comprise a CID binding domain (termed herein `CID Binding Domain 1`, or `CBD1` and `CID Binding Domain 2` or `CBD2` respectively) which allows each receptor component and signalling component to bind simultaneously to the same CID molecule (FIG. 3).

[0091] When antigen binds to the antigen-binding domain of the receptor component in the absence of the CID, there is no signalling through the signaling component. When the CID is present, receptor binding of antigen to the antigen-binding domain of the receptor component results in signaling through the signalling component.

[0092] Specifically, in the absence of CID, 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. In the presence of the CID, both the receptor and signalling component of the CAR bind the CID via their CID binding domains, resulting in co-localization of the receptor component and signaling components (heterodimerization). This co-localisation allows signalling through the signalling domain of the signalling component when antigen is bound by the antigen-binding domain of the receptor component.

[0093] 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 the CID agent.

[0094] Antigen binding by the receptor component in the absence of the CID may be termed as resulting in `non-productive` signaling through the signaling component. Such signaling does not result in cell activation, for example T cell activation. Antigen binding by the receptor component in the presence of CID may be termed as resulting in `productive` signaling through the signaling component. This signaling results in T-cell activation, triggering for example target cell killing and T cell activation.

[0095] Antigen binding by the receptor component in the presence of CID 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 domain in the absence of CID.

[0096] Signaling through the signaling 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.sub.2), 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 signalling system according to the present invention upon binding of antigen to the receptor component in the presence of varying concentrations of CID.

[0097] Chemical Inducer of Dimerization & Binding Domains

[0098] The CID and CID binding domains of the CAR signalling system of the first aspect of the invention may be any combination of molecules/peptides/domains which enables the selective co-localisation and dimerization of the receptor component and signalling component in the presence of the CID.

[0099] As such, the CID agent is a molecule which is able to simultaneously bind to the receptor component and the signalling component. The CID agent therefore comprises at least two binding moieties.

[0100] The CID may be any pharmaceutically acceptable molecule which can simultaneously be bound by at least two binding domains.

[0101] The CID is capable being delivered to the cytoplasm of a target cell and being available for intracellular binding.

[0102] Small molecule dimerization systems for facilitating the co-localization of peptides are known in the art (Corson et al.; 2008; ACS Chemical Biology; 3(11); 667).

[0103] The binding moieties of the CID may interact with identical binding domains present on the receptor component and the signalling component. That is, the CID may comprise two identical binding moieties such that it can simultaneously interact with a binding domain on the receptor component and an identical binding domain on the signalling component.

[0104] The CID and CID binding domains may be the FK506 binding protein (FKBP) ligand dimerization system described by Clackson et al. (PNAS; 1998; 95; 10437-10442). This dimerization system comprises two FKBP-like binding domains with a F36V mutation in the FKBP binding domain and a dimerization agent (AP1903) with complementary amino acid substitutions. Exposing cells engineered to express FKBP-like binding domain fusion proteins to AP103 results in the dimerization of the proteins comprising the FKBP-like binding domains but no interactions involving endogenous FKBP.

[0105] The dimerization system described by Farrar et al., which utilises bacterial DNA gyrase B (GyrB) binding domains and the antibiotic coumermycin as CID may also be used in the signalling system of the present invention (Methods Enzymol; 2000; 327; 421-419 and Nature; 1996; 383; 178-181).

[0106] The binding moieties of the CID may interact with different binding domains on the receptor component and the signalling component. That is, the CID may comprise two different binding moieties which can simultaneously interact with a binding domain on the receptor component and a different binding domain on the signalling component.

[0107] The CID and CID binding domains may comprise the dimerization system described by Belshaw et al. (Nature; 1996; 93; 4604-4607), which utilises a FK506 (Tacrolimus)/cyclosporin fusion molecule as the CID agent with FK-binding protein 12 (FKBP12) and cylcophilin A as the binding domains.

[0108] The CID/CID binding domain may also be the rapamycin and FKBP12/FKBP12-Rapamycin Binding (FRB) domain of mTOR system described by Rivera et al. (Nature Med; 1996; 2; 1028-1032) or the non-immunosupressive rapamycin analogs (rapalogs) and FKBP12/FRB system described by Bayle et al. (Chem Bio; 2006; 13; 99-107). For example the CID may be C-20-methyllyrlrapamycin (MaRap) or C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap), as described by Bayle et al. and illustrated in FIG. 2 in combination with the corresponding binding domains. The CID may be C16-(S)-3-methylindolerapamycin (C16-iRap) or C16-(S)-7-methylindolerapamycin (AP21976/C16-AiRap) as described by Bayle et al., in combination with the respective complementary binding domains for each.

[0109] Other dimerization systems suitable for use in the signalling system of the present invention include an estrone/biotin CID in combination with an oestrogen-binding domain (EBD) and a streptavidin binding domain (Muddana & Peterson; Org. Lett; 2004; 6; 1409-1412; Hussey et al.; J. Am. Chem. Soc.; 125; 3692-3693); a dexamethasone/methotrexate CID in combination with a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain (Lin et al.; J. Am. Chem. Soc.; 2000; 122; 4247-4248); a similar system in which the methotrexate portion of the CID is replaced with the bacterial specific DHFR inhibitor trimethoprim (Gallagher et al.; Anal. Biochem.; 2007; 363; 160-162) and an O.sup.6-benzylguanine derivative/methotrexate CID in combination with an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain and a DHFR binding domain (Gendreizig et al.; J. Am. Chem. Soc.; 125; 14970-14971).

[0110] RSL1 is a synthetic non-steroidal analogue of 20-hydroxyecdysone (see FIG. 9). It is a member of a class of insecticides known as diacylhydrazines and can function to act as a non-steroidal ecdysone agonist. These molecules induce premature moulting and larvae death but are well tolerated in vertebrates. RSL1 has been used in artificial transcription switches in which a two-protein transcription switch is used consisting of a fusion between the ecdysone receptor (EcR) and GAL4, and the retinoid X receptor (RXR) and VPI6. In such systems, EcR is modified to interact specifically with RSL13, and RXR is chimeric comprising of helices 1-8 replaced with helices 1-8 of human RXR.beta., and helices 9-12 from Locusta migratoria RXR. The signalling system of the present invention may use EcR and RXR domains for heterdimerisation in the presence of RSL1 or a derivative thereof.

[0111] For example the CID binding domains may be or comprise the sequences shown as SEQ ID NO: 1 to SEQ ID NO: 5, SEQ ID No. 25 or 26.

TABLE-US-00001 FKBP12 domain SEQ ID No 1 MGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLG KQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVE LLKLE wild-type FRB segment of mTOR SEQ ID No 2 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKE TSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKLES FRB with T to L substitution at 2098 which allows binding to AP21967 SEQ ID No 3 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKE TSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLES FRB segment of mTOR with T to H substitution at 2098 and to W at F at residue 2101 of the full mTOR which binds Rapamycin with reduced affinity to wild type SEQ ID No 4 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKE TSFNQAYGRDLMEAQEWCRKYMKSGNVKDLHQAFDLYYHVFRRISKLES FRB segment of mTOR with K to P substitution at residue 2095 of the full mTOR which binds Rapamycin with reduced affinity SEQ ID No 5 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKE TSFNQAYGRDLMEAQEWCRKYMKSGNVPDLTQAWDLYYHVFRRISKLES EcR SEQ ID No. 25 MPVDRILEAELAVEQKSDQGVEGPGGTGGSGSSPNDPVTNICQAADKQLFT LVEWAKRIPHFSSLPLDDQVILLRAGWNELLIASFSHRSIDVRDGILLATG LHVHRNSAHSAGVGAIFDRVLTELVSKMRDMRMDKTELGCLRAIILFNPEV RGLKSAQEVELLREKVYAALEEYTRTTHPDEPGRFAKLLLRLPSLRSIGLK CLEHLFFFRLIGDVPIDTFLMEMLESPSDS RXR SEQ ID No. 26 RPECVVPETQCAMKRKEKKAQKEKDKLPVSITTVDDHMPPIMQCEPPPPEA ARIHEVVPRFLSDKLLETNRQKNIPQLTANQQFLIARLIWYQDGYEQPSDE DLKRITQTVVQQADDENEESDTPFRQITEMTILTVQLIVEFAKGLPGFAKI SQPDQITLLKACSSEVMMLRVARRYDAASDSILFANNQAYTRDNYRKAGMA EVIEDLLHFCRCMYSMALDNIHYALLTAVVIFSDRPGLEQPQLVEEIQRYY LNTLRIYILNQLSGSARSSVIYGKILSILSELRTLGMQNSNMCISLKLKNR KLPPFLEEIWDVADMSHTQPPPILESPTNL

[0112] Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 1 to 5, 25 or 26 provided that the sequences provide an effective dimerization system. That is, provided that the sequences facilitate sufficient co-localisation of the antigen-binding and intracellular signalling components, in the presence of the CID, for successful signaling to occur upon binding of the antigen-binding domain to antigen.

[0113] The signalling system according to the present invention is not limited by the arrangement of a specific dimerization system. The receptor component may comprise either 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 presence of the CID.

[0114] The present invention also relates to a method for activating the CAR signalling system of the first aspect which comprises the step of administering the CID. As described above, administration of the CID results in co-localization of the receptor component and signalling component, such that signalling through the signalling domain occurs upon binding of the antigen-binding domain to antigen.

[0115] The CID and CID binding domains may facilitate signalling through the CAR signalling system which is proportional to the concentration of CID present.

[0116] The present invention further provides a method for reducing the activity of the CAR system of the first aspect which comprises reducing or stopping the administration of the CID. Reducing the level of the CID results in fewer receptor: signalling component dimers and thus the total level of signalling through the CAR signaling system is decreased in the presence of the antigen. Stopping the administration of the CID results in the absence of receptor: signalling component dimers and termination of signalling despite the presence of antigen binding to the antigen-binding domain.

[0117] Another possibility of this system is to "tune" the signal strength the receptor transmits. If the affinity between one or both of the CID binding domains is lessened, less CAR systems will be active and hence signal propagation upon antigen binding is lessened. This may avoid toxicity.

[0118] Receptor Component

[0119] The present invention provides a receptor component comprising an antigen-binding domain, an optional spacer domain, a transmembrane domain and a CID biding domain (CBD1). 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 CBD is localised to the intracellular side of the membrane.

[0120] The receptor component therefore provides the antigen-binding function of the CAR signalling system of the present invention.

[0121] Antigen Binding Domain

[0122] 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.

[0123] 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; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.

[0124] 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-US-00002 TABLE 1 Cancer type TAA Diffuse Large B-cell Lymphoma CD19, CD20 Breast cancer ErbB2, MUC1 AML CD13, CD33 Neuroblastoma GD2, NCAM, ALK, GD2 B-CLL CD19, CD52, CD160 Colorectal cancer Folate binding protein, CA-125 Chronic Lymphocytic Leukaemia CD5, CD19 Glioma EGFR, Vimentin Multiple myeloma BCMA, CD138 Renal Cell Carcinoma Carbonic anhydrase IX, G250 Prostate cancer PSMA Bowel cancer A33

[0125] Transmembrane Domain

[0126] 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. The transmembrane domain may be from CD148, as shown in SEQ ID No. 23.

[0127] Signal Peptide

[0128] The receptor component of the CAR signalling 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 displayed.

[0129] 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.

[0130] The signal peptide may be at the amino terminus of the molecule.

[0131] The signal peptide may comprise the SEQ ID No, 6, 7 or 8 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.

TABLE-US-00003 SEQ ID No. 6: MGTSLLCWMALCLLGADHADG

[0132] The signal peptide of SEQ ID No. 6 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.

TABLE-US-00004 SEQ ID No. 7: MSLPVTALLLPLALLLHAARP

[0133] The signal peptide of SEQ ID No. 7 is derived from IgG1.

TABLE-US-00005 SEQ ID No. 8: MAVPTQVLGLLLLWLTDARC

[0134] The signal peptide of SEQ ID No. 8 is derived from CD8.

[0135] Spacer Domain

[0136] The CAR signalling 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.

[0137] The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 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 IgG1 Fc region, an IgG1 hinge or a CD8 stalk. A human IgG1 spacer may be altered to remove Fc binding motifs.

[0138] Examples of amino acid sequences for these spacers are given below:

TABLE-US-00006 SEQ ID No. 9 (hinge-CH2CH3 of human IgG1) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKKD SEQ ID No. 10 (human CD8 stalk): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI SEQ ID No. 11 (human IgG1 hinge): AEPKSPDKTHTCPPCPKDPK SEQ ID No. 12 (CD2 ectodomain) KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEK ETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKI QERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWT TSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD SEQ ID no. 13 (CD34 ectodomain) SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEA TTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETT LKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVK LTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQ SEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSY SQKT

[0139] Receptor Component Comprising Multiple Transmembrane Domains

[0140] The receptor component may comprise an appropriate number of transmembrane domains such that each CID binding domain is orientated on the intracellular side of the cell membrane (FIG. 6). 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 signalling system encoded by SEQ ID No. 22 is a multi-spanning receptor with 3 FRB domains.

[0141] Multiple Receptor Components

[0142] In another embodiment of the invention, the CAR signalling system may comprise of two or more receptor components each recognizing different antigens but comprising of the same intracellular CID binding domain. Such a CAR system would be capable of recognizing multiple antigens (FIG. 5a). This might be useful for instance in avoiding tumour escape. In a further related aspect of the invention, the CBD1 of the receptor components differ in residues which dictate their affinity to the CID. In this way, a CAR system can be tuned such that signalling in response to one antigen is greater or lesser than response to another (FIG. 5b). 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.

[0143] Methods suitable for altering the amino acid residues of the CID binding domain such that its binding affinity for CID is altered are known in the art and include substitution, addition and removal of amino acids using both targeted and random mutagenesis. For example, in the case of CID binding to FRB, a set of mutations have been published with a range of affinity (Bayle et al, Chemistry & Biology 13, 99-107, January 2006). Methods for determining the affinity of a CID for the CID 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.

[0144] The signalling system encoded by SEQ ID No. 20 comprises two receptor components and one signalling component.

[0145] Signalling Component

[0146] The present invention also provides a signalling component comprising a signalling domain and a CID binding domain (CBD2). 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.

[0147] 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 presence of the CID, as described above.

[0148] Intracellular Signalling Domain

[0149] 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 presence of the CID, 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.

[0150] As such the signalling domain of the signalling component is analogous to the endodomain of a classical CAR molecule.

[0151] 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 OX40 can be used with CD3-Zeta to transmit a proliferative/survival signal, or all three can be used together (illustrated in FIG. 1B).

[0152] 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 (FIG. 3A).

[0153] The signalling component of a CAR signalling system according to the present invention may comprise the sequence shown as SEQ ID No. 14, 15 or 16 or a variant thereof.

TABLE-US-00007 CD3 Z endodomain SEQ ID No. 14 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR CD28 and CD3 Zeta endodomains SEQ ID No. 15 SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD28, OX40 and CD3 Zeta endodomains. SEQ ID No. 16 SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHK PPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0154] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 14, 15 or 16, provided that the sequence provides an effective intracellular signalling domain.

[0155] Multiple Signalling Components

[0156] 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 CIDB2, wherein each signalling domain is bound by the same CID but the signalling domains comprise different endodomains (FIG. 7a). 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.

[0157] If each signalling component comprises of a CBD2 domains which differ in residues which alter their affinity to CID, the CID enables signalling components comprising different signalling domains to ligate to the CID with differing kinetics (FIG. 7b). 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.

[0158] The signalling system encoded by SEQ ID No. 21 has one receptor components and three signalling components.

[0159] Signalling Components with an Additional CID Binding Domain

[0160] In the case of very low density of target antigen expression, signalling strength may have to be amplified. Take for instance, the native T-cell receptor which is associated with multiple ITAM containing CD3 components. In such circumstances, signalling components can be modified to contain CBD2, a signalling domain and also a CBD1 (FIG. 8). In this way, addition of CID would lead to concatenation of multiple signalling components. The average size of the concatenation can be tuned by varying the amount of signalling components which have a CBD2. In the event of antigen recognition and in the presence of CID, signalling through the concatenation of multiple signalling components would be more potent than through individual signalling components.

[0161] Nucleic Acid

[0162] The third aspect of the invention relates to a nucleic acid encoding the receptor component of the second aspect and a nucleic acid encoding a signalling component of the third aspect.

[0163] The specific encoding nucleic acid sequences for four inducible CARs are given later in description, following the Examples section.

[0164] As used herein, the terms "polynucleotide", "nucleotide", and "nucleic acid" are intended to be synonymous with each other.

[0165] 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.

[0166] Nucleic acids according to the second aspect of 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.

[0167] 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.

[0168] The nucleic acid according to the third aspect of the invention may be a nucleic acid which encodes both the receptor component and the signalling component.

[0169] 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.

[0170] Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2a self-cleaving peptide, which has the sequence shown as SEQ ID No. 17 or SEQ ID No. 18:

TABLE-US-00008 SEQ ID No. 17 RAEGRGSLLTCGDVEENPGP. or SEQ ID No. 18 QCTNYALLKLAGDVESNPGP

[0171] Where the signalling system comprises a plurality of signalling components, each comprising a signalling domain and a CIDB2, the nucleic acid may have the general formula:

RC-coexpr1-SC1-coexpr2-SC2

[0172] wherein:

[0173] RC is a nucleic acid sequence encoding the receptor component;

[0174] Coexpr1 and coexpr2, which may be the same or different, are nucleic acid sequences allowing co-expression of the two flanking polypeptides (e.g. a sequence which encodes a cleavage site)

[0175] SC1 and SC2 are nucleic acid sequence encoding signalling components.

[0176] The signalling components encoded by SC1 and SC2 may bind the same CID, but have different endodomains. The nucleic acid construct may comprise nucleic acid sequences encoding more than two signalling components e.g. SC3, SC4 . . . up to SCx, each separated by a coexpression sequence, coexpr3, coexpr4 . . . up to coexprX.

[0177] The nucleic acid sequences encoding each component may be in any order, for example: RC-coexpr1-SC1-coexpr2-SC2; SC1-coexpr1-SC2-coexpr2-RC; or SC1-coexpr1-RC-coexpr2-SC2.

[0178] Where the signalling system comprises a plurality of receptor components, the nucleic acid may have the general formula:

RC1-coexpr1-RC2-coexpr2-SC

[0179] wherein:

[0180] RC1 and RC2 are nucleic acid sequences encoding receptor components;

[0181] Coexpr1 and coexpr2, which may be the same or different, are nucleic acid sequences allowing co-expression of the two flanking polypeptides (e.g. a sequence which encodes a cleavage site)

[0182] SC is a nucleic acid sequence encoding the signalling component.

[0183] The receptor components may bind to different antigens but the same CID. They may have the same CIDB1. The nucleic acid construct may comprise nucleic acid sequences encoding more than two receptor components e.g. RC3, RC4 . . . up to RCx, each separated by a coexpression sequence, coexpr3, coexpr4 . . . up to coexprX.

[0184] The nucleic acid sequences encoding each component may be in any order, for example: RC1-coexpr1-RC2-coexpr2-SC; RC1-coexpr1-SC-coexpr2-RC2; or SC-coexpr1-RC1-coexpr2-RC2.

[0185] The co-expressing sequence may be an internal ribosome entry sequence (IRES). The co-expressing sequence may be an internal promoter.

[0186] 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 kit may also comprise a heterodimerization agent which is suitable for use in the signalling system of the first aspect.

[0187] Vector

[0188] 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. 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 signalling system according to the first aspect of the invention. The kit may also comprise a CID which is suitable for use in the signalling system of the first aspect.

[0189] 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.

[0190] The vector may be capable of transfecting or transducing a T cell.

[0191] Cytolytic Immune Cell

[0192] The present invention also relates to an immune cell comprising the CAR signalling system according to the first aspect of the invention.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] Two major classes of CD4+ Treg cells have been described--naturally occurring Treg cells and adaptive Treg cells.

[0199] 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.

[0200] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

[0201] 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

[0202] 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.

[0203] The CAR cells of the invention may be any of the cell types mentioned above.

[0204] T or NK cells expressing the molecules of the CAR signalling system according to the first aspect of the invention may either be created ex vivo either from a patient's own peripheral blood (1.sup.st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2.sup.nd party), or peripheral blood from an unconnected donor (3.sup.rd party).

[0205] Alternatively, T or NK cells expressing the molecules of the CAR signalling 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.

[0206] 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.

[0207] 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 signalling system according to the first aspect of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

[0208] The T or NK cell of the invention may be made by:

[0209] (i) isolation of a T or NK cell-containing sample from a subject or other sources listed above; and

[0210] (ii) transduction or transfection of the T or NK cells with one or more a nucleic acid sequence(s) encoding the receptor component and/or signalling component of the CAR signalling system according to the second and third aspects of the invention.

[0211] 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.

[0212] The present invention also provides a kit which comprises a T or NK cell comprising the CAR signalling system according to the first aspect of the invention and a heterodimerization agent suitable for use in the signalling system.

[0213] Pharmaceutical Composition

[0214] The present invention also relates to a pharmaceutical composition containing a plurality of cytolytic immune cells expressing the molecules of the CAR signalling 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.

[0215] Methods

[0216] 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) and/or a CID suitable for use in a signalling system according to the first aspect of the invention to a subject.

[0217] 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.

[0218] 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.

[0219] The method may involve the steps of:

[0220] (i) isolating a T or NK cell-containing sample from a subject;

[0221] (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention;

[0222] (iii) administering the cells from (ii) to the subject.

[0223] The methods for treating a disease may further comprise the step of administering a CID suitable for use in the signalling system of the first aspect of the invention to the subject.

[0224] 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 a CID agent suitable for use in the CAR signalling system according to the first aspect to the subject. As such, this method involves administering a suitable CID agent to a subject which already comprises CAR cells of the present invention.

[0225] The CID 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.

[0226] The methods for treating a disease provided by the present invention may involve monitoring the progression of the disease and monitoring any toxic activity and adjusting the dose of the CID administered to the subject to provide acceptable levels of disease progression and toxic activity.

[0227] 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.

[0228] 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.

[0229] The level of signalling through the signalling system of the first aspect of the invention, and therefore the level of activation CAR cells expressing the signalling system, can be adjusted by altering the amount of CID present. In the present method the level of CAR cell activation may be augmented by increasing the dose of CID administered to the subject. Conversely, the level of CAR cell activation may be reduced by decreasing the dose of CID administered to the subject.

[0230] 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.

[0231] As such the dose of CID administered to a subject 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.

[0232] The present invention provides a CAR cell of the present invention for use in treating and/or preventing a disease.

[0233] 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.

[0234] The present invention also provides a CID agent suitable for activating a CAR signalling system according to the first aspect of the invention for use in treating and/or preventing a disease.

[0235] The present invention also provides a CID agent for use in activating a CAR signalling system according to the first aspect of the invention in a CAR cell.

[0236] The invention also provides the use of a CID suitable for activating a CAR signalling system according to the first aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

[0237] The invention also provides the use of a CID in the manufacture of a medicament for in activating a CAR signalling system according to the first aspect of the invention in a CAR cell.

[0238] The disease to be treated and/or prevented by the methods of the present invention may be an infection, such as a viral infection.

[0239] 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.

[0240] 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), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.

[0241] 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.

[0242] The CAR cells and pharmaceutical compositions of present invention may be for use in the treatment and/or prevention of the diseases described above.

[0243] The CAR cells and pharmaceutical compositions of present invention may be for use in any of the methods described above.

[0244] The present invention also provides a method of administering a cell according to the ninth aspect of the invention to a subject in need of same.

[0245] The present invention also provides a method of administering a CID as defined herein to a subject who comprises a cell according to the ninth aspect of the invention.

[0246] 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

A Rapamycin-Inducible CAR System

[0247] T-cells were transduced with a retroviral vector coding for an inducible CAR detailed in FIG. 3 and the sequence of which is shown as SEQ ID 19. This inducible CAR has a single receptor component and a single signalling component. The receptor component comprises of an anti-CD19 scFv, a spacer domain derived from human IgG1, a transmembrane-domain derived from CD28 and FKBP12 as endodomain. The signalling component comprises of the FRB domain from mTOR and a signalling domain derived from endodomains of CD28, OX40 and CD3-Zeta. Expression of the CAR was determined by staining the transduced cells with a conjugated antibody recognizing the spacer domain and analysing by flow cytometry. The T-cells were incubated with either target cells expressing the cognate target CD19 in ratio of 2:1, 1:1 and 0:1 (target:effector) in the absence of rapamycin or at increasing concentrations. Signalling only occurred in the presence of both the cognate antigen and rapamycin. A dose response to rapamycin and target antigen was observed.

Example 2

An RSL1-Inducible CAR System

[0248] RSL1 is a synthetic non-steroidal analogue of 20-hydroxyecdysone (FIG. 9). It is a member of a class of insecticides known as diacylhydrazines and can function to act as a non-steroidal ecdysone agonist. These molecules induce premature moulting and larvae death but are well tolerated in vertebrates (Dhadialla et al (1998) Annu. Rev. Entomol. 43, 545-569). RSL1 has been used in artificial transcription switches (Lessard et al (2007) The Prostate 67, 808-819) whereby a two-protein transcription switch consisting of a fusion between the ecdysone receptor (EcR) and GAL4, and the retinoid X receptor (RXR) and VP16. In such systems, EcR is modified to interact specifically with RSL1 (Kumar et al (2004) J. Biol. Chem. 279, 27211-27218), and RXR is chimeric comprising of helices 1-8 replaced with helices 1-8 of human RXR.beta., and helices 9-12 from Locusta migratoria RXR.

[0249] The present inventors have constructed a split CD19 CAR based on the CD19 system described in Example 1 with the heterodimerization motif on the antigen recognition component and signalling components being EcR and RXR respectively (FIG. 10a and SEQ ID No. 23). This CAR was transduced into T-cells and found to express well (FIG. 10b). These T-cells were challenged with targets which were CD19 negative and CD19 positive. Only CAR T-cells challenged with SupT1 targets in the presence of RSL1 released IL-2 (FIG. 10c).

Example 3

An AP1903-Inducible CAR System

[0250] AP1903 is a synthetic chemical inducer of dimerization which binds to two mutated FKB12 domains (Clackson et al (1998). Proc. Natl. Acad. Sci. U.S.A. 95, 10437-10442). An inducible CAR was developed based on this system, using the modified FKBP12 domains to constitute the heterodimerization domain of both the antigen recognition component and signalling component in the framework as described in Example 1 with the antigen recognition domain and the signalling domain co-expressed with a FMD-2A like sequence (FIG. 11a and SEQ ID No. 24). The FKBP12 domains were codon-wobbled to prevent homologous recombination. T-cells were transduced with this construct and challenged with target cells which were CD19 negative or positive.

[0251] T-cells responded only to CD19 positive targets in the presence of AP1903 (FIG. 11b). At very high concentration of dimerizer, signalling was inhibited likely due to saturating conditions where the stoichiometry favours one FKBP12 binding a single dimerizer molecule rather than one dimerizer molecule binding two FKBP12 domains (FIG. 11c).

[0252] Specific Inducible Car-Encoding Sequences

[0253] SEQ ID No. 19. Simple inducible CAR comprised of one receptor component and one signalling component. The receptor component comprises of a signal peptide, an anti-CD19 scFv, a spacer from the Fc domain of human IgG1, a CD28 transmembrane domain and FKB12 as endodomain. A FMD-2A peptide separates this receptor component from the signalling component. The signalling component comprises of FRB and a compound of endodomains from CD28, OX40 and CD3-Zeta.

TABLE-US-00009 SarAI --------- 1 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc gacatccaga tgacccagac >>...................signal peptide...................>> >>.....aCD19 heavy chain......> 81 caccagcagc ctgagcgcca gcctgggcga ccgggtgacc atcagctgca gagccagcca ggacatcagc aagtacctga >..................................aCD19 heavy chain..................................> 161 actggtacca gcagaagccc gacggcaccg tgaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc >..................................aCD19 heavy chain..................................> XcmI ---------------- 241 cggttcagcg gcagcggcag cggcaccgac tacagcctga ccatcagcaa cctggagcag gaggacatcg ccacctactt >......................................aCD19 heavy chain..............................> 321 ctgccagcag ggcaacaccc tgccctacac cttcggaggc ggcaccaagc tggagatcac caaggccgga ggcggaggct >..........................aCD19 heavy chain...........................>> >>..linker...> 401 ctggcggagg cggctctggc ggaggcggct ctggcggagg cggcagcgag gtgaagctgc aggagtctgg cccaggcctg >.....................linker.....................>> >>........aCD19 light chain........> 481 gtggccccaa gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac tacggcgtga gctggatcag >..................................aCD19 light chain..................................> 561 gcagccccca cggaagggcc tggagtggct gggcgtgatc tggggcagcg agaccaccta ctacaacagc gccctgaaga >..................................aCD19 light chain..................................> 641 gccggctgac catcatcaag gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc >..................................aCD19 light chain..................................> 721 atctactact gcgccaagca ctactactat ggcggcagct acgctatgga ctactggggc cagggcacca gcgtgaccgt >..................................aCD19 light chain..................................> BamHI FseI ------- -------- 801 gagctcggat cccgccgagc ccaaatctcc tgacaaaact cacacatgcc caccgtgccc agcacctccc gtggccggcc >.>> >>............................HCH2CH3pvaa..........................- ..> 881 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatcgcc cggacccctg aggtcacatg cgtggtggtg >.....................................HCH2CH3pvaa................- .....................> 961 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc >.....................................HCH2CH3pva.................- .....................> SacTI ------ 1041 gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg >.....................................HCH2CH3pvaa................- .....................> 1121 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga >.....................................HCH2CH3pvaa................- .....................> 1201 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg >.....................................HCH2CH3pvaa................- .....................> 1281 cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcaaccgg agaacaacta caagaccacg cctcccgtgc >.....................................HCH2CH3pvaa................- .....................> 1361 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca >.....................................HCH2CH3pvaa................- .....................> Ppu10I ------- NsiI ------- BfrBI ------- 1441 tgctccgtga tgcatgaggc cctgcacaat cactataccc agaaatctct gagtctgagc ccaggcaaga aggaccccaa >..............................HCH2CH3pvaa.......................- ........>> 1521 gttctgggtc ctggtggtgg tgggaggcgt gctggcctgt tactctctcc tggtgaccgt ggccttcatc atcttctggg >>......................................CD28tm...............- ........................> 1601 tgggagtgca ggtggaaacc atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac >> >>......................................FKBP12..............- ........................> 1681 accgggatgc ttgaagatgg aaagaaattc gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca >.......................................FKBP12...................- .....................> 1761 ggaggtgatc cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata tctccagatt >.......................................FKBP12...................- .....................> 1841 atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg >.......................................FKBP12...................- .....................> 1921 gaacgcgcag agggccgggg ctcattgctg acctgtggag atgtcgagga aaatcccggc ccaatggctt ctagaatcct >>> >>..............................2A.........................- .....>> >>..> 2001 ctggcatgag atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg >.........................................FRB....................- .....................> SphI ------- 2081 aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt >.........................................FRB....................- .....................> 2161 cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag gacctcctcc aagcctggga >.........................................FRB....................- .....................> XhoI ------ 2241 cctctattat catgtgttcc gacgaatctc aaagctcgag agtggcggag gaggcagttc aaggtccgcc gacgcgcctg >...............FRB................>> >>....................CD28OXZ....................> 2321 cataccagca ggggcagaat cagctgtaca acgagctcaa cctcggtagg cgcgaggaat acgatgtgct cgataagaga >.......................................CD28OXZ..................- .....................> NruI ------- 2401 agaggtcgcg atcccgagat gggaggaaaa cctcagcgcc gcaagaaccc tcaggagggg ctgtataacg aactgcagaa >.......................................CD28OXZ..................- .....................> SfiI -------------- 2481 ggataagatg gcagaggcct actccgagat tggcatgaag ggtgagagga gaagaggtaa aggccatgac ggcctctacc >.......................................CD28OXZ..................- .....................> 2561 aaggcctctc taccgcaacg aaagacactt atgacgctct gcatatgcag gctctccccc ctaggcagtg cactaattac >.......................................CD28OXZ..................- .....................> 2641 gcccttctga aacttgccgg cgatgtggag tctaaccctg gccctatcct gtggcacgag atgtggcatg agggtctgga >.......................................CD28OXZ..................- .....................> 2721 agaggccagc cggctgtact tcggagagag aaatgttaag ggtatgttcg aggtgctgga gccgcttcac gctatgatgg >.......................................CD28OXZ..................- .....................> 2801 agaggggccc ccagaccctc aaagaaacca gcttcaatca agcctacggg agggatctta tggaggcaca ggaatggtgt >.......................................CD28OXZ..................- .....................> 2881 cggaagtaca tgaagagcgg gaacgtcaag gacctgctcc aggcttggga tttgtattat cacgtcttta ggcggatcag >.......................................CD28OXZ..................- .....................> 2961 caagtctgga ggcggaggaa gtctgcacag cgattacatg aacatgaccc cccgaaggcc cggacccaca cgcaaacact >.......................................CD28OXZ..................- .....................> 3041 atcaacccta tgctccccca cgcgacttcg ccgcctaccg gtoacgcgcc gaggggcgcg gctotttgtt gacttgcggg >.......................................CD28OX2..................- .....................> 3121 gacgttgaag agaatcctgg ccccatcctt tggcacgaga tgtggcacga gggcctggag gaagcctccc ggctgtattt >.......................................CD28OXZ..................- .....................> 3201 cggagagcgc aacgtcaaag gaatgtttga ggtgctggag cctctccatg caatgatgga gagggggcct cagactctta >.......................................CD28OXZ..................- .....................> 3281 aagaaacatc ctttaatcag gcttacggta gagatttgat ggaggctcaa gaatggtgcc ggaaatacat gaagagtgga >.......................................CD28OXZ..................- .....................> 3361 aacgttaaag acctgctgca ggcatgggac ctgtactatc acgtattcag acggatctca aagtcagggg gaggtggctc

>.......................................CD28OXZ..................- .....................> 3441 cctttatatc ttcaagcagc ctttcatgag gccggtgcag accacacaag aagaggatgg gtgctcttgc cggttccccg >.......................................CD28OXZ..................- .....................> 3521 aggaggagga gggcggatgc gagctctga >..........CD28OXZ...........>>

[0254] SEQ ID No. 20. Inducible CAR comprised of two receptor component and one signalling component. The first receptor component comprises of a signal peptide, an anti-CD19 scFv, a spacer from the stalk region of CD8, a transmembrane domain from CD28, and FKB12 as an endodomain. The second signalling component comprises of an anti-CD29 scFv, spacer derived from the Fc domain of human IgG1, a CD28 transmembrane domain and FKB12 as endodomain. The signalling component comprises of FRB and a compound of endodomains from CD28, OX40 and CD3-Zeta. A foot-and-mouth disease 2A peptide separates the components.

TABLE-US-00010 1 atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccaga ccagacatcc agatgaccca >>...................signal peptide 1....................>> >>.......aCD19_scFv........> 81 gaccaccagc agcctgagcg ccagcctggg cgaccgggtg accatcagct gcagagccag ccaggacatc agcaagtacc >.....................................aCD19_scFv.................- .....................> 161 tgaactggta ccagcagaag cccgacggca ccgtgaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc >.....................................aCD19_scFv.................- .....................> 241 agccggttca gcggnagcgg cagcggcacc gactacagcc tgaccatcag caacctggag caggaggaca tcgccaccta >.....................................aCD19_scFv.................- .....................> 321 cttctgccag cagggcaaca ccctgcccta caccttcgga ggcggcacca agctggagat caccaaggcc ggaggcggag >.....................................aCD19_scFv.................- .....................> 401 gctctggcgg aggcggctct ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc tgcaggagtc tggcccaggc >.....................................aCD19_scFv.................- .....................> 481 ctggtggccc caagccagag cctgagcgtg acctgcaccg tgagcggcgt gagcctgccc gactacggcg tgagctggat >.....................................aCD19_scFv.................- .....................> 561 caggcagccc ccacggaagg gcctggagtg gctgggcgtg atctggggca gcgagaccac ctactacaac agcgccctga >.....................................aCD19_scFv.................- .....................> 641 agagccggct gaccatcatc aaggacaaca gcaagagcca ggtgttcctg aagatgaaca gcctgcagac cgacgacacc >.....................................aCD19_scFv.................- .....................> 721 gccatctact actgcgccaa gcactactac tatggcggca gctacgctat ggactactgg ggccagggca ccagcgtgac >.....................................aCD19_scFv.................- .....................> SgrAI --------- 801 cgtgagctca gatcccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc >....>> >>................................CD8STK...........................- .....> 881 tgcgcccaga ggcgtgccgg ccagoggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatcttttgg >....................................CD8STK......................- ..............>> >>...> 961 gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttc atcatcttct gggtgggagt >....................................CD28tmZ.....................- ................> >>..> 1041 gcaggtggaa accatctccc caggagacgg gcgcaccttc cccaagcgcg gccagacctg cgtggtgcac tacaccggga >.......................................FKBP12...................- .....................> 1121 tgcttgaaga tggaaagaaa ttcgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa gcaggaggtg >.......................................FKBP12...................- .....................> 1201 atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc caaactgact atatctccag attatgccta >.......................................FKBP12...................- .....................> 1281 tggtgccact gggcacccag gcatcatccc accacatgcc actctcgtct tcgatgtgga gcttctaaaa ctggaacgag >.....................................FKBP12.....................- ................>> >>.> NcoI ------ 1361 ccgagggcag gggaagtctt ctaacatgcg gggacgtgga ggaaaatccc gggcccatgg agaccgacac cctgctgctg >............................2A............................>&g- t; >>...signal peptide 2....> 1441 tgggtgctgc tgctgtgggt gcccggcagc accggccagg tgcagctgca gcagcccggc gccgagctgg tgaagcccgg >..........signal peptide 2..........>> >>.................aCD20_scFv..................> 1521 cgccagcgtg aagatgagct gcaaggccag cggctacacc ttcaccagct acaacatgca ctgggtgaag cagacccccg >.....................................aCD20_scFv.................- .....................> 1601 gccggggcct ggagtggatc ggcgccatct accccggcaa cggcgacacc agctacaacc agaagttcaa gggcaaggcc >.....................................aCD20_scFv.................- .....................> 1681 accctgaccg ccgacaagag cagcagcacc gcctacatgc agctgagcag cctgaccagc gaggacagcg ccgtgtacta >.....................................aCD20_scPv.................- .....................> 1761 ctgcgcccgg agcacctact acggcggcga ctggtacttc aacgtgtggg gcgccggcac caccgtgacc gtgagcggag >.....................................aCD20_scFv.................- .....................> 1841 gcggcggcag cggaggaggc ggctctgggg gaggcggatc tcagatcgtg ctgagccaga gccccgccat cctgagcgcc >.....................................aCD20_scFv.................- .....................> 1921 agcccoggcg agaaggtgac catgacctgc cgggccagca gcagcgtgag ctacatccac tggttccagc agaagcccgg >....................................aCD20_scFv..................- .....................> BstXI -------------- 2001 cagcagcccc aagccctgga tctacgccac cagcaacctg gccagcggcg tgcccgtgcg gttcagcggc agcggcagcg >.....................................aCD20_scFv.................- .....................> 2081 gcaccagcta cagcctgacc atcagccggg tggaggccga ggacgccgcc acctactact gccagcagtg gaccagcaac >.....................................aCD20_scFv.................- .....................> BamHI ------ 2161 ccacccacct tcggcggcgg caccaagctg gagatcaagc ggtcggatcc cgccgagccc aaatctcctg acaaaactca >................aCD20_scFv.................>> >>......HCH2CH3pvaa'.......> FseI -------- 2241 cacatgccca ccgtgcccag cacctcccgt ggccggcccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca >....................................HCH2CH3pvaa'................- .....................> 2321 tgatcgcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac >....................................HCH2CH3pvaa'................- .....................> SacII ------- 2401 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt >....................................HCH2CH3pvaa'................- .....................> 2481 cctcaccgtc ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca >....................................HCH2CH3pvaa'................- .....................> 2561 tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg >....................................HCH2CH3pvaa'................- .....................> 2641 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg >....................................HCH2CH3pvaa'................- .....................> 2721 gcaaccggag aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc aagctcaccg >....................................HCH2CH3pvaa'................- .....................> Ppu10I ------- NsiI ------- BfrBI ------- 2801 tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg catgaggccc tgcacaatca ctatacccag >....................................HCH2CH3pvaa'................- .....................> 2881 aaatctctga gtctgagccc aggcaagaag gaccccaagt tctgggtcct ggtggtggtg ggaggcgtgc tggcctgtta >.......HCH2CH3pvaa'.......>> >>..................CD28tm..................> 2961 ctctctcctg gtgaccgtgg ccttcatcat cttctgggtg ggagtgcagg tggaaaccat ctccccagga gacgggcgca >.................CD28tm.................>> >>.................FKBP12.................> 3041 ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt gaagatggaa agaaattcga ttcctcccgg >.......................................FKBP12...................- .....................> 3121 gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa gaaggggttg cccagatgag >.......................................FKBP12...................- .....................> 3201 tgtgggtcag agagccaaac tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc atcccaccac >.......................................FKBP12...................- .....................> 3281 atgccactct cgtcttcgat gtggagcttc taaaactgga acgcgcagag ggccggggct cattgctgac ctgtggagat >..................FKBP12..................>> >>..................2A...................> 3361 gtcgaggaaa atcccggccc aatggcttct agaatcctct ggcatgagat gtggcatgaa ggcctggaag aggcatctcg >.........2A.........>> >>......................FRB.......................>

SphI ------ 3441 tttgtacttt ggggaaagga acgtgaaagg catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc >.........................................FRB....................- .....................> 3521 agactctgaa ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag gaagtacatg >.........................................FRB....................- .....................> XhoI ------ 3601 aaatcaggga atgtcaagga cctcctccaa gcctgggacc tctattatca tgtgttccga cgaatctcaa agctcgagta >....................................FRB.........................- ............>> >> 3681 tagcggcggc ggcagcagga gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc cgccccgggc >....linker....>> >>..............................CD28OXZ............................- ..> 3761 ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc tatcgctcca gggaccagag gctgcceccc >.......................................CD28OXZ..................- .....................> 3841 gatgcccaca agccccctgg gggaggcagt ttccggaccc ccatccaaga ggagcaggcc gacgcccact ccaccctggc >.......................................CD28OXZ..................- .....................> 3921 caagatcaga gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat aacgagctca >.......................................CD28OXZ..................- .....................> 4001 atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg >.......................................CD28OXZ..................- .....................> 4081 aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg >.......................................CD28OXZ..................- .....................> 4161 cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc >.......................................CD28OXZ..................- .....................> 4241 acatgcaggc cctgcctcct cgctaa >.........CD28OXZ.........>>

[0255] SEQ ID No. 21. Inducible CAR comprised of one receptor component and three signalling components. The receptor component comprises of a signal peptide, an anti-CD19 scFv, a spacer derived from the Fc domain of human IgG1, a CD28 transmembrane domain and FKB12 as endodomain. The first signalling component comprises of FRB and endodomain from CD3 Zeta, the second comprises FRB and CD28 endodomain, the third FRB and 41BB endodomain. A foot-and-mouth disease 2A peptide separates the components.

TABLE-US-00011 SgrAI --------- 1 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc gacatccaga tgacccagac >>...................signal peptide...................>> >>.......scFv anti-CD19.......> 81 caccagcagc ctgagcgcca gcctgggcga ccgggtgacc atcagctgca gagccagcca ggacatcagc aagtacctga >...................................scFv anti-CD19....................................> 161 actggtacca gcagaagccc gacggcaccg tgaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc >...................................scFv anti-CD19....................................> XcmI ---------------- 241 cggttcagcg gcagcggcag cggcaccgac tacagcctga ccatcagcaa cctggagcag gaggacatcg ccacctactt >...................................scFv anti-CD19....................................> 321 ctgccagcag ggcaacaccc tgccctacac cttcggaggc ggcaccaagc tggagatcac caaggccgga ggcggaggct >...................................scFv anti-CD19....................................> 401 ctggcggagg cggctctggc ggaggcggct ctggcggagg cggcagcgag gtgaagctgc aggagtctgg cccaggcctg >...................................scFv anti-CD19....................................> 481 gtggccccaa gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac tacggcgtga gctggatcag >...................................scFv anti-CD19....................................> 561 gcagccccca cggaagggcc tggagtggct gggcgtgatc tggggcagcg agaccaccta ctacaacagc gccctgaaga >...................................scFv anti-CD19....................................> 641 gccggctgac catcatcaag gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc >...................................scFv anti-CD19....................................> 721 atctactact gcgccaagca ctactactat ggcggcagct acgctatgga ctactggggc cagggcacca gcgtgaccgt >...................................scFv anti-CD19....................................> BamHI FseI ------- -------- 801 gagctcggat cccgccgagc ccaaatctcc tgacaaaact cacacatgcc caccgtgccc agcacctccc gtggccggcc >.>> >>............................HCH2CH3pvaa..........................- ..> 881 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatcgcc cggacccctg aggtcacatg cgtggtggtg >.....................................HCH2CH3pvaa................- .....................> 961 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc >.....................................HCH2CH3pvaa................- .....................> SacII ------ 1041 gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg >.....................................HCH2CH3pvaa................- .....................> 1121 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga >.....................................HCH2CH3pvaa................- .....................> 1201 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg >.....................................HCH2CH3pvaa................- .....................> 1281 cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcaaccgg agaacaacta caagaccacg cctcccgtgc >.....................................HCH2CH3pvaa................- .....................> 1361 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca >.....................................HCH2CH3pvaa................- .....................> Ppu10I ------- NsiI ------- BfrBI ------- 1441 tgctccgtga tgcatgaggc cctgcacaat cactataccc agaaatctct gagtctgagc ccaggcaaga aggaccccaa >..............................HCH2CH3pvaa.......................- ........>> 1521 gttctgggtc ctggtggtgg tgggaggcgt gctggcctgt tactctctcc tggtgaccgt ggccttcatc atcttctggg >>......................................CD28tm...............- ........................> 1601 tgggagtgca ggtggaaacc atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac >> >>......................................FKBP12..............- ........................> 1681 accgggatgc ttgaagatgg aaagaaattc gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca >.......................................FKBP12...................- .....................> 1761 ggaggtgatc cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata tctccagatt >.......................................FKBP12...................- .....................> 1841 atgcctatgg tgccactggg cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg >.......................................FKBP12...................- .....................> 1921 gaacgcgcag agggccgggg ctcattgctg acctgtggag atgtcgagga aaatcccggc ccaatggctt ctagaatcct >>> >>............................FMD-2A.......................- .....>> >>..> 2001 ctggcatgag atgtggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg >.........................................FRB....................- .....................> SphI ------- 2081 aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt >.........................................FRB....................- .....................> 2161 cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag gacctcctcc aagcctggga >.........................................FRB....................- .....................> XhoI ------ 2241 cctctattat catgtgttcc gacgaatctc aaagctcgag agtggcggag gaggcagttc aaggtccgcc gacgcgcctg >...............FRB................>> >>....linker.....>> >>......CD3-Zeta.......> 2321 cataccagca ggggcagaat cagctgtaca acgagctcaa cctcggtagg cgcgaggaat acgatgtgct cgataagaga >......................................CD3-Zeta..................- .....................> NruI ------- 2401 agaggtcgcg atcccgagat gggaggaaaa cctcagcgcc gcaagaaccc tcaggagggg ctgtataacg aactgcagaa >......................................CD3-Zeta..................- .....................> SfiI -------------- 2481 ggataagatg gcagaggcct actccgagat tggcatgaag ggtgagagga gaagaggtaa aggccatgac ggcctctacc >......................................CD3-Zeta..................- .....................> 2561 aaggcctctc taccgcaacg aaagacactt atgacgctct gcatatgcag gctctccccc ctaggcagtg cactaattac >...............................CD3-Zeta.........................- .......>> >>..FMD-2A..> 2641 gcccttctga aacttgccgg cgatgtggag tctaaccctg gccctatcct gtggcacgag atgtggcatg agggtctgga >....................FMD-2A....................>> >>................FRB................> 2721 agaggccagc cggctgtact tcggagagag aaatgttaag ggtatgttcg aggtgctgga gccgcttcac gctatgatgg >.........................................FRB....................- .....................> 2801 agaggggccc ccagaccctc aaagaaacca gcttcaatca agcctacggg agggatctta tggaggcaca ggaatggtgt >.........................................FRB....................- .....................> 2881 cggaagtaca tgaagagcgg gaacgtcaag gacctgctcc aggcttggga tttgtattat cacgtcttta ggcggatcag >.........................................FRB....................- .....................> 2961 caagtctgga ggcggaggaa gtctgcacag cgattacatg aacatgaccc cccgaaggcc cggacccaca cgcaaacact >.>> >>.....linker.....>> >>............................CD28............................> 3041 atcaacccta tgctccccca cgcgacttcg ccgcctaccg gtcacgcgcc gaggggcgcg gctctttgtt gacttgcggg >....................CD28.....................>> >>...............FMD-2A...............> 3121 gacgttgaag agaatcctgg ccccatcctt tggcacgaga tgtggcacga gggcctggag gaagcctccc ggctgtattt >........FMD-2A.........>> >>...........................FRB............................> 3201 cggagagcgc aacgtcaaag gaatgtttga ggtgctggag cctctccatg caatgatgga gagggggcct cagactctta >.........................................FRB....................- .....................>

3281 aagaaacatc ctttaatcag gcttacggta gagatttgat ggaggctcaa gaatggtgcc ggaaatacat gaagagtgga >.........................................FRB....................- .....................> 3361 aacgttaaag acctgctgca ggcatgggac ctgtactatc acgtattcag acggatctca aagtcagggg gcggtggctc >...............................FRB..............................- ..>> >>....linker.....> 3441 cctttatatc ttcaagcagc ctttcatgag gccggtgcag accacacaag aagaggatgg gtgctcttgc cggttccccg > >>.......................................41BB................- ........................> 3521 aggaggagga gggcggatgc gagctctga >............41BB............>>

[0256] SEQ ID No. 22. Inducible CAR with multi-spanning receptor component. The receptor component comprises of a signal peptide, an anti-CD19 scFv, a spacer from the Fc domain of human IgG1, a CD28 transmembrane domain, a first FRB, then the first trans-membrane, minor extracellular loop and second transmembrane domain of CD20, then a second FRB, then the third transmembrane domain, the major extracellular loop and the fourth transmembrane domain of CD20, and a third FRB. The signalling component comprises of FRB and a compound of endodomains from CD28, OX40 and CD3-Zeta. A foot-and-mouth disease 2A peptide separates the two components.

TABLE-US-00012 SgrAI 1 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc gacatccaga tgacccagac >>...................signal peptide...................>> >>.......scFv anti-CD19.......> 81 caccagcagc ctgagcgcca gcctgggcga ccgggtgacc atcagctgca gagccagcca ggacatcagc aagtacctga >...................................scFv anti-CD19....................................> 161 actggtacca gcagaagocc gacggcaccg tgaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc >...................................scFv anti-CD19....................................> XcmI ---------------- 241 cggttcagcg gcagcggcag cggcaccgac tacagcctga ccatcagcaa cctggagcag gaggacatcg ccacctactt >...................................scFv anti-CD19....................................> 321 ctgccagcag ggcaacaccc tgccctacac cttcggaggc ggcaccaagc tggagatcac caaggccgga ggcggaggct >...................................scFv anti-CD19....................................> StuI ------ 401 ctggcggagg cggctctggc ggaggcggct ctggcggagg cggcagcgag gtgaagctgc aggagtctgg cccaggcctg >...................................scFv anti-CD19....................................> 481 gtggccccaa gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac tacggcgtga gctggatcag >...................................scFv anti-CD19....................................> 561 gcagccocca cggaagggcc tggagtggct gggcgtgatc tggggcagcg agaccaccta ctacaacagc gccctgaaga >...................................scFv anti-CD19....................................> 641 gccggctgac catcatcaag gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc >...................................scFv anti-CD19....................................> 721 atctactact gcgccaagca ctactactat ggcggcagct acgctatgga ctactggggc cagggcacca gcgtgaccgt <...................................scFv anti-CD19....................................> BamHI FseI ------- --------- 801 gagctcggat cccgccgagc ccaaatctcc tgacaaaact cacacatgcc caccgtgccc agcacctccc gtggccggcc >.>> >>............................HCH2CH3pvaa..........................- ..> 881 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatcgcc cggacccctg aggtcacatg cgtggtggtg >.....................................HCH2CH3pvaa................- .....................> 961 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc >.....................................HCH2CH3pvaa................- .....................> SacII ------ 1041 gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg >.....................................HCH2CH3pvaa................- .....................> 1121 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga >.....................................HCH2CH3pvaa................- .....................> 1201 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg >.....................................HCH2CH3pvaa................- .....................> 1281 cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcaaccgg agaacaacta caagaccacg cctcccgtgc >.....................................HCH2CH3pvaa................- .....................> 1361 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca >.....................................HCH2CH3pvaa................- .....................> Ppu10I ------- NsiI ------- BfrBI ------- 1441 tgctccgtga tgcatgaggc cctgcacaat cactataccc agaaatctct gagtctgagc ccaggcaaga aggaccccaa >..............................HCH2CH3pvaa.......................- ........>> 1521 gttctgggtc ctggtggtgg tgggaggcgt gctggcctgt tactctctcc tggtgaccgt ggccttcatc atcttctggg >>......................................CD28tm...............- ........................> StuI ------- 1601 tgatcctctg gcatgagatg tggcatgaag gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa cgtgaaaggc >> >>.......................................FRB................- ........................> StuI ------ 1681 atgtttgagg tgctggagcc cttgcatgct atgatggaac ggggccccca gactctgaag gaaacatcct ttaatcaggc >.........................................FRB....................- .....................> 1761 ctatggtcga gatttaatgg aggcccaaga gtggtgcagg aagtacatga aatcagggaa tgtcaaggac ctcctccaag >.........................................FRB....................- .....................> 1841 cctgggacct ctattatcat gtgttccgac gaatctcaaa gagcagcctg gtgggaccca cccagagctt cttcatgcgg >....................FRB...................>> >>.................CD20..................> StuI ------- 1921 gagagcaaga ccctgggagc cgtgcagatc atgaacggcc tgttccacat cgccctggga ggcctgctga tgatccctgc >........................................CD20....................- .....................> 2001 cggcatctac gccccaatct gcgtgaccgt gtggtaccca ctgtggggag gcatcatgta catcatcagc ggcagcctgc >........................................CD20....................- .....................> 2081 tggccgccac cgagaagaac agcggagggg gaagcatcct gtggcacgag atgtggcatg agggtctgga agaggccagc >.......CD20.......>> >>...linker...>> >>.....................FRB......................> 2161 cggctgtact tcggagagag aaatgttaag ggtatgttcg aggtgctgga gccgcttcac gctatgatgg agaggggccc >.........................................FRB....................- .....................> 2241 ccagaccctc aaagaaacca gcttcaatca agcctacggg agggatctta tggaggcaca ggaatggtgt cggaagtaca >.........................................FRB....................- .....................> 2321 tgaagagcgg gaacgtcaag gacctgctcc aggcttggga tttgtattat cacgtcttta ggcggatcag caagtctgga >.....................................FRB........................- ..............>> >>...> BclI ------ 2401 ggcggaggaa gtcggaagtg cctggtgaag ggcaagatga tcatgaacag cctgagcctg ttcgccgcca tcagcggcat >..linker..>> >>.................................CD20............................- ......> 2481 gatcctgagc atcatggaca tcctgaacat caagatcagc cacttcctga agatggagag cctgaacttc atccgggccc >........................................CD20....................- .....................> 2561 acaccccata catcaacatc tacaactgcg agcctgccaa ccccagcgag aagaacagcc ccagcaccca gtactgctac >........................................CD20....................- .....................> BsaBI ----------- 2641 agcatccaga gcctgttcct gggcatcctg agcgtgatgc tgatcttcgc cttcttccag gagctggtga tcgccggcat >........................................CD20....................- .....................> 2721 cgtggagaac gagtggaagc ggacctgcag ccggcccaag agcatcctgt ggcacgagat gtggcatgag ggtctggaag >....................CD20....................>> >>.................FRB.................> 2801 aggccagccg gctgtacttc ggagagagaa atgttaaggg tatgttcgag gtgctggagc cgcttcacgc tatgatggag >.........................................FRB....................- .....................> 2881 aggggccccc agaccctcaa agaaaccagc ttcaatcaag cctacgggag ggatcttatg gaggcacagg aatggtgtcg >.........................................FRB....................- .....................> 2961 gaagtacatg aagagcggga acgtcaagga cctgctccag gcttgggatt tgtattatca cgtctttagg cggatcagca >.........................................FRB....................- .....................> 3041 agcgcgcaga gggccggggc tcattgctga cctgtggaga tgtcgaggaa aatcccggcc caggagtgca ggtggaaacc >>> >>...........................FMD-2A........................- ....>> >>.....FKBP12.....> 3121 atctocccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg >.......................................FKBP12...................- .....................> 3201 aaagaaattc gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca

ggaggtgatc cgaggctggg >.......................................FKBP12...................- .....................> 3281 aagaaggggt tgcccagatg agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg tgccactggg >.......................................FKBP12...................- .....................> 3361 cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg gaaaggagta agaggagcag >..............................FKBP12............................- ..>> >>.....28OXZ.....> 3441 gctcctgcac agtgactaca tgaacatgac tccccgccgc ccogggccca cccgcaagca ttaccagccc tatgccccac >........................................28OXZ...................- .....................> 3521 cacgcgactt cgcagcctat cgctccaggg accagaggct gccccccgat gcccacaagc cccctggggg aggcagtttc >........................................28OXZ...................- .....................> 3601 cggaccccca tccaagagga gcaggccgac gcccactcca ccctggccaa gatcagagtg aagttcagca ggagcgcaga >........................................28OXZ...................- .....................> 3681 cgcccccgcg taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac gatgttttgg >........................................28OXZ...................- .....................> 3761 acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg >........................................28OXZ...................- .....................> 3841 cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct >........................................28OXZ...................- .....................> 3921 ttaccagggt ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gcctcctcgc tga >..................................28OXZ.........................- .........>>

[0257] SEQ ID No. 23. Simple inducible CAR comprised of one receptor component and one signalling component. The signalling component comprises an ecdysone receptor and the CD3-Zeta endodomain (italic). A FMD-2A peptide (shown in bold) separates this signalling component from the receptor component. The receptor component comprises an anti-CD19 scFv, a HCH2CH3 spacer (underlined), a CD148 transmembrane domain (highlighted in grey) and the retinoid X receptor as endodomain.

TABLE-US-00013 SEQ ID No. 23 MPVDRILEAELAVEQKSDQGVEGPGGTGGSGSSPNDPVTNICQAADKQLFTLVEWAKRIP HFSSLPLDDQVILLRAGWNELLIASFSHRSIDVRDGILLATGLHVHRNSAHSAGVGAIFD RVLTELVSKMRDMRMDKTELGCLRAIILFNPEVRGLKSAQEVELLREKVYAALEEYTRTT HPDEPGRFAKLLLRLPSLRSIGLKCLEHLFFERLIGDVPIDTFLMEMLESPSDSSGGGSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEE NPGPMETDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWY QQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGV SLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQT DDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPAEPKSPDKTHTCPPCPAPPVAGPSV FLFPPKPKDTLMIARTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG ##STR00001## ##STR00002## EKDKLPVSTTTVDDHMPPIMQCEPPPPEAARIHEVVPRFLSDKLLETNRQKNIPQLTANQ QFLIARLIWYQDGYEQPSDEDLKRITQTWQQADDENEESDTPFRQITEMTILTVQLIVEF AKGLPGFAKISQPDQITLLKACSSEVMMLRVARRYDAASDSILFANNQAYTRDNYRKAGM AEVIEDLLHFCRCMYSMALDNIHYALLTAVVIFSDRPGLEQPQLVEEIQRYYLNTLRIYI LNQLSGSARSSVIYGKILSILSELRTLGMQNSNMCISLKLKNRKLPPFLEEIWDVADMSH TQPPPILESPTNL

[0258] SEQ ID No. 24. Simple inducible CAR comprised of one receptor component and one signalling component. The signalling component comprises an FKBP12 domain (underlined) and the CD3-Zeta endodomain (italic). A FMD-2A peptide (shown in bold) separates this signalling component from the receptor component. The receptor component comprises an anti-CD19 scFv, a HCH2CH3 spacer (highlighted in grey), a CD148 transmembrane domain and the and FKBP12 domain (underlined).

TABLE-US-00014 SEQ ID No. 24 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPME TDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDG TVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGT KLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYG VSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIY ##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007## AQIKRVVSEKKTAQAPHRFQKTCSPISGGGGSGVQVETISPGDGRTFPKRGQTCVVHYTG MLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHP GIIPPHATLVFDVELLKLE

[0259] All publications mentioned in the above specification are herein incorporated by reference in their entirety, with special attention to the subject matter for which they are referred. 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 or related fields are intended to be within the scope of the following claims.

Sequence CWU 1

1

261108PRTArtificial SequenceChemical inducer of dimerization (CID) binding domain - FKBP12 domain 1Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe1 5 10 15Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp65 70 75 80Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 100 1052100PRTArtificial SequenceCID binding domain - wild-type FRB segment of mTOR 2Met Ala Ser Arg Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu1 5 10 15Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe 20 25 30Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr 35 40 45Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu 50 55 60Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp65 70 75 80Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser 85 90 95Lys Leu Glu Ser 1003100PRTArtificial SequenceCID binding domain - FRB with T to L substitution at 2098 which allows binding to AP21967 3Met Ala Ser Arg Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu1 5 10 15Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe 20 25 30Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr 35 40 45Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu 50 55 60Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp65 70 75 80Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser 85 90 95Lys Leu Glu Ser 1004100PRTArtificial SequenceCID binding domain - FRB segment of mTOR 4Met Ala Ser Arg Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu1 5 10 15Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe 20 25 30Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr 35 40 45Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu 50 55 60Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp65 70 75 80Leu His Gln Ala Phe Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser 85 90 95Lys Leu Glu Ser 1005100PRTArtificial SequenceCID binding domain - FRB segment of mTOR 5Met Ala Ser Arg Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu1 5 10 15Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe 20 25 30Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr 35 40 45Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu 50 55 60Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Pro Asp65 70 75 80Leu Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser 85 90 95Lys Leu Glu Ser 100621PRTArtificial SequenceSignal peptide 6Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala1 5 10 15Asp His Ala Asp Gly 20721PRTArtificial SequenceSignal peptide 7Met Ser Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro 20820PRTArtificial SequenceSignal peptide 8Met Ala Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr1 5 10 15Asp Ala Arg Cys 209234PRTArtificial SequenceSpacer (hinge-CH2CH3 of human IgG1) 9Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10 15Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp225 2301046PRTArtificial SequenceSpacer (human CD8 stalk) 10Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala1 5 10 15Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 35 40 451120PRTArtificial SequenceSpacer (human IgG1 hinge) 11Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5 10 15Lys Asp Pro Lys 2012185PRTArtificial SequenceSpacer (CD2 ectodomain) 12Lys Glu Ile Thr Asn Ala Leu Glu Thr Trp Gly Ala Leu Gly Gln Asp1 5 10 15Ile Asn Leu Asp Ile Pro Ser Phe Gln Met Ser Asp Asp Ile Asp Asp 20 25 30Ile Lys Trp Glu Lys Thr Ser Asp Lys Lys Lys Ile Ala Gln Phe Arg 35 40 45Lys Glu Lys Glu Thr Phe Lys Glu Lys Asp Thr Tyr Lys Leu Phe Lys 50 55 60Asn Gly Thr Leu Lys Ile Lys His Leu Lys Thr Asp Asp Gln Asp Ile65 70 75 80Tyr Lys Val Ser Ile Tyr Asp Thr Lys Gly Lys Asn Val Leu Glu Lys 85 90 95Ile Phe Asp Leu Lys Ile Gln Glu Arg Val Ser Lys Pro Lys Ile Ser 100 105 110Trp Thr Cys Ile Asn Thr Thr Leu Thr Cys Glu Val Met Asn Gly Thr 115 120 125Asp Pro Glu Leu Asn Leu Tyr Gln Asp Gly Lys His Leu Lys Leu Ser 130 135 140Gln Arg Val Ile Thr His Lys Trp Thr Thr Ser Leu Ser Ala Lys Phe145 150 155 160Lys Cys Thr Ala Gly Asn Lys Val Ser Lys Glu Ser Ser Val Glu Pro 165 170 175Val Ser Cys Pro Glu Lys Gly Leu Asp 180 18513259PRTArtificial SequenceSpacer (CD34 ectodomain) 13Ser Leu Asp Asn Asn Gly Thr Ala Thr Pro Glu Leu Pro Thr Gln Gly1 5 10 15Thr Phe Ser Asn Val Ser Thr Asn Val Ser Tyr Gln Glu Thr Thr Thr 20 25 30Pro Ser Thr Leu Gly Ser Thr Ser Leu His Pro Val Ser Gln His Gly 35 40 45Asn Glu Ala Thr Thr Asn Ile Thr Glu Thr Thr Val Lys Phe Thr Ser 50 55 60Thr Ser Val Ile Thr Ser Val Tyr Gly Asn Thr Asn Ser Ser Val Gln65 70 75 80Ser Gln Thr Ser Val Ile Ser Thr Val Phe Thr Thr Pro Ala Asn Val 85 90 95Ser Thr Pro Glu Thr Thr Leu Lys Pro Ser Leu Ser Pro Gly Asn Val 100 105 110Ser Asp Leu Ser Thr Thr Ser Thr Ser Leu Ala Thr Ser Pro Thr Lys 115 120 125Pro Tyr Thr Ser Ser Ser Pro Ile Leu Ser Asp Ile Lys Ala Glu Ile 130 135 140Lys Cys Ser Gly Ile Arg Glu Val Lys Leu Thr Gln Gly Ile Cys Leu145 150 155 160Glu Gln Asn Lys Thr Ser Ser Cys Ala Glu Phe Lys Lys Asp Arg Gly 165 170 175Glu Gly Leu Ala Arg Val Leu Cys Gly Glu Glu Gln Ala Asp Ala Asp 180 185 190Ala Gly Ala Gln Val Cys Ser Leu Leu Leu Ala Gln Ser Glu Val Arg 195 200 205Pro Gln Cys Leu Leu Leu Val Leu Ala Asn Arg Thr Glu Ile Ser Ser 210 215 220Lys Leu Gln Leu Met Lys Lys His Gln Ser Asp Leu Lys Lys Leu Gly225 230 235 240Ile Leu Asp Phe Thr Glu Gln Asp Val Ala Ser His Gln Ser Tyr Ser 245 250 255Gln Lys Thr14112PRTArtificial SequenceSignalling component CD3 Z endodomain 14Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 11015152PRTArtificial SequenceSignalling component CD28 and CD3 Zeta endodomains 15Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro1 5 10 15Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 20 25 30Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 35 40 45Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 50 55 60Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly65 70 75 80Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 85 90 95Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 100 105 110Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 115 120 125Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 130 135 140His Met Gln Ala Leu Pro Pro Arg145 15016188PRTArtificial SequenceSignalling component CD28, OX40 and CD3 Zeta endodomains 16Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro1 5 10 15Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 20 25 30Arg Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp 35 40 45Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu 50 55 60Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe65 70 75 80Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 85 90 95Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 100 105 110Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 115 120 125Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 130 135 140Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys145 150 155 160Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 165 170 175Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 180 1851720PRTFoot-and-mouth disease virus 17Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu1 5 10 15Asn Pro Gly Pro 201820PRTFoot-and-mouth disease virus 18Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser1 5 10 15Asn Pro Gly Pro 20193549DNAArtificial SequenceSimple inducible Chimeric Antigen Receptor (CAR) 19atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc 60gacatccaga tgacccagac caccagcagc ctgagcgcca gcctgggcga ccgggtgacc 120atcagctgca gagccagcca ggacatcagc aagtacctga actggtacca gcagaagccc 180gacggcaccg tgaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 240cggttcagcg gcagcggcag cggcaccgac tacagcctga ccatcagcaa cctggagcag 300gaggacatcg ccacctactt ctgccagcag ggcaacaccc tgccctacac cttcggaggc 360ggcaccaagc tggagatcac caaggccgga ggcggaggct ctggcggagg cggctctggc 420ggaggcggct ctggcggagg cggcagcgag gtgaagctgc aggagtctgg cccaggcctg 480gtggccccaa gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540tacggcgtga gctggatcag gcagccccca cggaagggcc tggagtggct gggcgtgatc 600tggggcagcg agaccaccta ctacaacagc gccctgaaga gccggctgac catcatcaag 660gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720atctactact gcgccaagca ctactactat ggcggcagct acgctatgga ctactggggc 780cagggcacca gcgtgaccgt gagctcggat cccgccgagc ccaaatctcc tgacaaaact 840cacacatgcc caccgtgccc agcacctccc gtggccggcc cgtcagtctt cctcttcccc 900ccaaaaccca aggacaccct catgatcgcc cggacccctg aggtcacatg cgtggtggtg 960gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1200gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320gggcaaccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440tgctccgtga tgcatgaggc cctgcacaat cactataccc agaaatctct gagtctgagc 1500ccaggcaaga aggaccccaa gttctgggtc ctggtggtgg tgggaggcgt gctggcctgt 1560tactctctcc tggtgaccgt ggccttcatc atcttctggg tgggagtgca ggtggaaacc 1620atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac 1680accgggatgc ttgaagatgg aaagaaattc gattcctccc gggacagaaa caagcccttt 1740aagtttatgc taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg 1800agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg tgccactggg 1860cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg 1920gaacgcgcag agggccgggg ctcattgctg acctgtggag atgtcgagga aaatcccggc 1980ccaatggctt ctagaatcct ctggcatgag atgtggcatg aaggcctgga agaggcatct 2040cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat 2100gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt 2160cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag 2220gacctcctcc aagcctggga cctctattat catgtgttcc gacgaatctc aaagctcgag 2280agtggcggag gaggcagttc aaggtccgcc gacgcgcctg cataccagca ggggcagaat 2340cagctgtaca acgagctcaa cctcggtagg cgcgaggaat acgatgtgct cgataagaga 2400agaggtcgcg atcccgagat gggaggaaaa cctcagcgcc gcaagaaccc tcaggagggg 2460ctgtataacg aactgcagaa ggataagatg gcagaggcct actccgagat tggcatgaag 2520ggtgagagga gaagaggtaa aggccatgac ggcctctacc aaggcctctc taccgcaacg 2580aaagacactt atgacgctct gcatatgcag gctctccccc ctaggcagtg cactaattac 2640gcccttctga aacttgccgg cgatgtggag tctaaccctg gccctatcct gtggcacgag 2700atgtggcatg agggtctgga agaggccagc cggctgtact tcggagagag aaatgttaag 2760ggtatgttcg aggtgctgga gccgcttcac gctatgatgg agaggggccc ccagaccctc 2820aaagaaacca gcttcaatca agcctacggg agggatctta tggaggcaca ggaatggtgt 2880cggaagtaca tgaagagcgg gaacgtcaag gacctgctcc aggcttggga tttgtattat 2940cacgtcttta ggcggatcag caagtctgga ggcggaggaa gtctgcacag cgattacatg 3000aacatgaccc cccgaaggcc cggacccaca cgcaaacact atcaacccta tgctccccca

3060cgcgacttcg ccgcctaccg gtcacgcgcc gaggggcgcg gctctttgtt gacttgcggg 3120gacgttgaag agaatcctgg ccccatcctt tggcacgaga tgtggcacga gggcctggag 3180gaagcctccc ggctgtattt cggagagcgc aacgtcaaag gaatgtttga ggtgctggag 3240cctctccatg caatgatgga gagggggcct cagactctta aagaaacatc ctttaatcag 3300gcttacggta gagatttgat ggaggctcaa gaatggtgcc ggaaatacat gaagagtgga 3360aacgttaaag acctgctgca ggcatgggac ctgtactatc acgtattcag acggatctca 3420aagtcagggg gcggtggctc cctttatatc ttcaagcagc ctttcatgag gccggtgcag 3480accacacaag aagaggatgg gtgctcttgc cggttccccg aggaggagga gggcggatgc 3540gagctctga 3549204266DNAArtificial SequenceInducible CAR 20atgagcctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccaga 60ccagacatcc agatgaccca gaccaccagc agcctgagcg ccagcctggg cgaccgggtg 120accatcagct gcagagccag ccaggacatc agcaagtacc tgaactggta ccagcagaag 180cccgacggca ccgtgaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc 240agccggttca gcggcagcgg cagcggcacc gactacagcc tgaccatcag caacctggag 300caggaggaca tcgccaccta cttctgccag cagggcaaca ccctgcccta caccttcgga 360ggcggcacca agctggagat caccaaggcc ggaggcggag gctctggcgg aggcggctct 420ggcggaggcg gctctggcgg aggcggcagc gaggtgaagc tgcaggagtc tggcccaggc 480ctggtggccc caagccagag cctgagcgtg acctgcaccg tgagcggcgt gagcctgccc 540gactacggcg tgagctggat caggcagccc ccacggaagg gcctggagtg gctgggcgtg 600atctggggca gcgagaccac ctactacaac agcgccctga agagccggct gaccatcatc 660aaggacaaca gcaagagcca ggtgttcctg aagatgaaca gcctgcagac cgacgacacc 720gccatctact actgcgccaa gcactactac tatggcggca gctacgctat ggactactgg 780ggccagggca ccagcgtgac cgtgagctca gatcccacca cgacgccagc gccgcgacca 840ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatcttttgg 960gtgctggtgg tggttggtgg agtcctggct tgctatagct tgctagtaac agtggccttc 1020atcatcttct gggtgggagt gcaggtggaa accatctccc caggagacgg gcgcaccttc 1080cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga tggaaagaaa 1140ttcgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa gcaggaggtg 1200atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc caaactgact 1260atatctccag attatgccta tggtgccact gggcacccag gcatcatccc accacatgcc 1320actctcgtct tcgatgtgga gcttctaaaa ctggaacgag ccgagggcag gggaagtctt 1380ctaacatgcg gggacgtgga ggaaaatccc gggcccatgg agaccgacac cctgctgctg 1440tgggtgctgc tgctgtgggt gcccggcagc accggccagg tgcagctgca gcagcccggc 1500gccgagctgg tgaagcccgg cgccagcgtg aagatgagct gcaaggccag cggctacacc 1560ttcaccagct acaacatgca ctgggtgaag cagacccccg gccggggcct ggagtggatc 1620ggcgccatct accccggcaa cggcgacacc agctacaacc agaagttcaa gggcaaggcc 1680accctgaccg ccgacaagag cagcagcacc gcctacatgc agctgagcag cctgaccagc 1740gaggacagcg ccgtgtacta ctgcgcccgg agcacctact acggcggcga ctggtacttc 1800aacgtgtggg gcgccggcac caccgtgacc gtgagcggag gcggcggcag cggaggaggc 1860ggctctgggg gaggcggatc tcagatcgtg ctgagccaga gccccgccat cctgagcgcc 1920agccccggcg agaaggtgac catgacctgc cgggccagca gcagcgtgag ctacatccac 1980tggttccagc agaagcccgg cagcagcccc aagccctgga tctacgccac cagcaacctg 2040gccagcggcg tgcccgtgcg gttcagcggc agcggcagcg gcaccagcta cagcctgacc 2100atcagccggg tggaggccga ggacgccgcc acctactact gccagcagtg gaccagcaac 2160ccacccacct tcggcggcgg caccaagctg gagatcaagc ggtcggatcc cgccgagccc 2220aaatctcctg acaaaactca cacatgccca ccgtgcccag cacctcccgt ggccggcccg 2280tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatcgcccg gacccctgag 2340gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 2400gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 2460acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 2520tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 2580gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 2640accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 2700gtggagtggg agagcaatgg gcaaccggag aacaactaca agaccacgcc tcccgtgctg 2760gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 2820caggggaacg tcttctcatg ctccgtgatg catgaggccc tgcacaatca ctatacccag 2880aaatctctga gtctgagccc aggcaagaag gaccccaagt tctgggtcct ggtggtggtg 2940ggaggcgtgc tggcctgtta ctctctcctg gtgaccgtgg ccttcatcat cttctgggtg 3000ggagtgcagg tggaaaccat ctccccagga gacgggcgca ccttccccaa gcgcggccag 3060acctgcgtgg tgcactacac cgggatgctt gaagatggaa agaaattcga ttcctcccgg 3120gacagaaaca agccctttaa gtttatgcta ggcaagcagg aggtgatccg aggctgggaa 3180gaaggggttg cccagatgag tgtgggtcag agagccaaac tgactatatc tccagattat 3240gcctatggtg ccactgggca cccaggcatc atcccaccac atgccactct cgtcttcgat 3300gtggagcttc taaaactgga acgcgcagag ggccggggct cattgctgac ctgtggagat 3360gtcgaggaaa atcccggccc aatggcttct agaatcctct ggcatgagat gtggcatgaa 3420ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg catgtttgag 3480gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa ggaaacatcc 3540tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag gaagtacatg 3600aaatcaggga atgtcaagga cctcctccaa gcctgggacc tctattatca tgtgttccga 3660cgaatctcaa agctcgagta tagcggcggc ggcagcagga gtaagaggag caggctcctg 3720cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 3780ccctatgccc caccacgcga cttcgcagcc tatcgctcca gggaccagag gctgcccccc 3840gatgcccaca agccccctgg gggaggcagt ttccggaccc ccatccaaga ggagcaggcc 3900gacgcccact ccaccctggc caagatcaga gtgaagttca gcaggagcgc agacgccccc 3960gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 4020tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 4080aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 4140agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 4200ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgcctcct 4260cgctaa 4266213549DNAArtificial SequenceInducible CAR 21atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc 60gacatccaga tgacccagac caccagcagc ctgagcgcca gcctgggcga ccgggtgacc 120atcagctgca gagccagcca ggacatcagc aagtacctga actggtacca gcagaagccc 180gacggcaccg tgaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 240cggttcagcg gcagcggcag cggcaccgac tacagcctga ccatcagcaa cctggagcag 300gaggacatcg ccacctactt ctgccagcag ggcaacaccc tgccctacac cttcggaggc 360ggcaccaagc tggagatcac caaggccgga ggcggaggct ctggcggagg cggctctggc 420ggaggcggct ctggcggagg cggcagcgag gtgaagctgc aggagtctgg cccaggcctg 480gtggccccaa gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540tacggcgtga gctggatcag gcagccccca cggaagggcc tggagtggct gggcgtgatc 600tggggcagcg agaccaccta ctacaacagc gccctgaaga gccggctgac catcatcaag 660gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720atctactact gcgccaagca ctactactat ggcggcagct acgctatgga ctactggggc 780cagggcacca gcgtgaccgt gagctcggat cccgccgagc ccaaatctcc tgacaaaact 840cacacatgcc caccgtgccc agcacctccc gtggccggcc cgtcagtctt cctcttcccc 900ccaaaaccca aggacaccct catgatcgcc cggacccctg aggtcacatg cgtggtggtg 960gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1200gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320gggcaaccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440tgctccgtga tgcatgaggc cctgcacaat cactataccc agaaatctct gagtctgagc 1500ccaggcaaga aggaccccaa gttctgggtc ctggtggtgg tgggaggcgt gctggcctgt 1560tactctctcc tggtgaccgt ggccttcatc atcttctggg tgggagtgca ggtggaaacc 1620atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac 1680accgggatgc ttgaagatgg aaagaaattc gattcctccc gggacagaaa caagcccttt 1740aagtttatgc taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg 1800agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg tgccactggg 1860cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg 1920gaacgcgcag agggccgggg ctcattgctg acctgtggag atgtcgagga aaatcccggc 1980ccaatggctt ctagaatcct ctggcatgag atgtggcatg aaggcctgga agaggcatct 2040cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga gcccttgcat 2100gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca ggcctatggt 2160cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg gaatgtcaag 2220gacctcctcc aagcctggga cctctattat catgtgttcc gacgaatctc aaagctcgag 2280agtggcggag gaggcagttc aaggtccgcc gacgcgcctg cataccagca ggggcagaat 2340cagctgtaca acgagctcaa cctcggtagg cgcgaggaat acgatgtgct cgataagaga 2400agaggtcgcg atcccgagat gggaggaaaa cctcagcgcc gcaagaaccc tcaggagggg 2460ctgtataacg aactgcagaa ggataagatg gcagaggcct actccgagat tggcatgaag 2520ggtgagagga gaagaggtaa aggccatgac ggcctctacc aaggcctctc taccgcaacg 2580aaagacactt atgacgctct gcatatgcag gctctccccc ctaggcagtg cactaattac 2640gcccttctga aacttgccgg cgatgtggag tctaaccctg gccctatcct gtggcacgag 2700atgtggcatg agggtctgga agaggccagc cggctgtact tcggagagag aaatgttaag 2760ggtatgttcg aggtgctgga gccgcttcac gctatgatgg agaggggccc ccagaccctc 2820aaagaaacca gcttcaatca agcctacggg agggatctta tggaggcaca ggaatggtgt 2880cggaagtaca tgaagagcgg gaacgtcaag gacctgctcc aggcttggga tttgtattat 2940cacgtcttta ggcggatcag caagtctgga ggcggaggaa gtctgcacag cgattacatg 3000aacatgaccc cccgaaggcc cggacccaca cgcaaacact atcaacccta tgctccccca 3060cgcgacttcg ccgcctaccg gtcacgcgcc gaggggcgcg gctctttgtt gacttgcggg 3120gacgttgaag agaatcctgg ccccatcctt tggcacgaga tgtggcacga gggcctggag 3180gaagcctccc ggctgtattt cggagagcgc aacgtcaaag gaatgtttga ggtgctggag 3240cctctccatg caatgatgga gagggggcct cagactctta aagaaacatc ctttaatcag 3300gcttacggta gagatttgat ggaggctcaa gaatggtgcc ggaaatacat gaagagtgga 3360aacgttaaag acctgctgca ggcatgggac ctgtactatc acgtattcag acggatctca 3420aagtcagggg gcggtggctc cctttatatc ttcaagcagc ctttcatgag gccggtgcag 3480accacacaag aagaggatgg gtgctcttgc cggttccccg aggaggagga gggcggatgc 3540gagctctga 3549223993DNAArtificial SequenceInducible CAR 22atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc 60gacatccaga tgacccagac caccagcagc ctgagcgcca gcctgggcga ccgggtgacc 120atcagctgca gagccagcca ggacatcagc aagtacctga actggtacca gcagaagccc 180gacggcaccg tgaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 240cggttcagcg gcagcggcag cggcaccgac tacagcctga ccatcagcaa cctggagcag 300gaggacatcg ccacctactt ctgccagcag ggcaacaccc tgccctacac cttcggaggc 360ggcaccaagc tggagatcac caaggccgga ggcggaggct ctggcggagg cggctctggc 420ggaggcggct ctggcggagg cggcagcgag gtgaagctgc aggagtctgg cccaggcctg 480gtggccccaa gccagagcct gagcgtgacc tgcaccgtga gcggcgtgag cctgcccgac 540tacggcgtga gctggatcag gcagccccca cggaagggcc tggagtggct gggcgtgatc 600tggggcagcg agaccaccta ctacaacagc gccctgaaga gccggctgac catcatcaag 660gacaacagca agagccaggt gttcctgaag atgaacagcc tgcagaccga cgacaccgcc 720atctactact gcgccaagca ctactactat ggcggcagct acgctatgga ctactggggc 780cagggcacca gcgtgaccgt gagctcggat cccgccgagc ccaaatctcc tgacaaaact 840cacacatgcc caccgtgccc agcacctccc gtggccggcc cgtcagtctt cctcttcccc 900ccaaaaccca aggacaccct catgatcgcc cggacccctg aggtcacatg cgtggtggtg 960gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1200gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320gggcaaccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440tgctccgtga tgcatgaggc cctgcacaat cactataccc agaaatctct gagtctgagc 1500ccaggcaaga aggaccccaa gttctgggtc ctggtggtgg tgggaggcgt gctggcctgt 1560tactctctcc tggtgaccgt ggccttcatc atcttctggg tgatcctctg gcatgagatg 1620tggcatgaag gcctggaaga ggcatctcgt ttgtactttg gggaaaggaa cgtgaaaggc 1680atgtttgagg tgctggagcc cttgcatgct atgatggaac ggggccccca gactctgaag 1740gaaacatcct ttaatcaggc ctatggtcga gatttaatgg aggcccaaga gtggtgcagg 1800aagtacatga aatcagggaa tgtcaaggac ctcctccaag cctgggacct ctattatcat 1860gtgttccgac gaatctcaaa gagcagcctg gtgggaccca cccagagctt cttcatgcgg 1920gagagcaaga ccctgggagc cgtgcagatc atgaacggcc tgttccacat cgccctggga 1980ggcctgctga tgatccctgc cggcatctac gccccaatct gcgtgaccgt gtggtaccca 2040ctgtggggag gcatcatgta catcatcagc ggcagcctgc tggccgccac cgagaagaac 2100agcggagggg gaagcatcct gtggcacgag atgtggcatg agggtctgga agaggccagc 2160cggctgtact tcggagagag aaatgttaag ggtatgttcg aggtgctgga gccgcttcac 2220gctatgatgg agaggggccc ccagaccctc aaagaaacca gcttcaatca agcctacggg 2280agggatctta tggaggcaca ggaatggtgt cggaagtaca tgaagagcgg gaacgtcaag 2340gacctgctcc aggcttggga tttgtattat cacgtcttta ggcggatcag caagtctgga 2400ggcggaggaa gtcggaagtg cctggtgaag ggcaagatga tcatgaacag cctgagcctg 2460ttcgccgcca tcagcggcat gatcctgagc atcatggaca tcctgaacat caagatcagc 2520cacttcctga agatggagag cctgaacttc atccgggccc acaccccata catcaacatc 2580tacaactgcg agcctgccaa ccccagcgag aagaacagcc ccagcaccca gtactgctac 2640agcatccaga gcctgttcct gggcatcctg agcgtgatgc tgatcttcgc cttcttccag 2700gagctggtga tcgccggcat cgtggagaac gagtggaagc ggacctgcag ccggcccaag 2760agcatcctgt ggcacgagat gtggcatgag ggtctggaag aggccagccg gctgtacttc 2820ggagagagaa atgttaaggg tatgttcgag gtgctggagc cgcttcacgc tatgatggag 2880aggggccccc agaccctcaa agaaaccagc ttcaatcaag cctacgggag ggatcttatg 2940gaggcacagg aatggtgtcg gaagtacatg aagagcggga acgtcaagga cctgctccag 3000gcttgggatt tgtattatca cgtctttagg cggatcagca agcgcgcaga gggccggggc 3060tcattgctga cctgtggaga tgtcgaggaa aatcccggcc caggagtgca ggtggaaacc 3120atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac 3180accgggatgc ttgaagatgg aaagaaattc gattcctccc gggacagaaa caagcccttt 3240aagtttatgc taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg 3300agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg tgccactggg 3360cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg 3420gaaaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 3480cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 3540cgctccaggg accagaggct gccccccgat gcccacaagc cccctggggg aggcagtttc 3600cggaccccca tccaagagga gcaggccgac gcccactcca ccctggccaa gatcagagtg 3660aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 3720gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 3780cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 3840cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 3900ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 3960gcccttcaca tgcaggccct gcctcctcgc tga 3993231273PRTArtificial SequenceSimple inducible CAR 23Met Pro Val Asp Arg Ile Leu Glu Ala Glu Leu Ala Val Glu Gln Lys1 5 10 15Ser Asp Gln Gly Val Glu Gly Pro Gly Gly Thr Gly Gly Ser Gly Ser 20 25 30Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln 35 40 45Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe Ser Ser 50 55 60Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu65 70 75 80Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Asp Val Arg Asp Gly 85 90 95Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala His Ser 100 105 110Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser 115 120 125Lys Met Arg Asp Met Arg Met Asp Lys Thr Glu Leu Gly Cys Leu Arg 130 135 140Ala Ile Ile Leu Phe Asn Pro Glu Val Arg Gly Leu Lys Ser Ala Gln145 150 155 160Glu Val Glu Leu Leu Arg Glu Lys Val Tyr Ala Ala Leu Glu Glu Tyr 165 170 175Thr Arg Thr Thr His Pro Asp Glu Pro Gly Arg Phe Ala Lys Leu Leu 180 185 190Leu Arg Leu Pro Ser Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His 195 200 205Leu Phe Phe Phe Arg Leu Ile Gly Asp Val Pro Ile Asp Thr Phe Leu 210 215 220Met Glu Met Leu Glu Ser Pro Ser Asp Ser Ser Gly Gly Gly Ser Ala225 230 235 240Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 245 250 255Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 260 265 270Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 275 280 285Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 290 295 300Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly305 310 315 320Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 325 330 335His Met Gln Ala Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly Ser Leu 340 345 350Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Thr Asp 355 360 365Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly 370 375 380Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly385 390 395

400Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 405 410 415Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 420 425 430Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 435 440 445Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 450 455 460Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr465 470 475 480Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Lys Ala Gly Gly Gly 485 490 495Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 500 505 510Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser 515 520 525Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp 530 535 540Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp545 550 555 560Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu 565 570 575Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe 580 585 590Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys 595 600 605Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly 610 615 620Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro Ala Glu Pro Lys Ser625 630 635 640Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala 645 650 655Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 660 665 670Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 675 680 685Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 690 695 700His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr705 710 715 720Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 725 730 735Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 740 745 750Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 755 760 765Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 770 775 780Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu785 790 795 800Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 805 810 815Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 820 825 830Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 835 840 845His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 850 855 860Pro Gly Lys Lys Asp Pro Met Ala Leu Ile Val Leu Gly Gly Val Ala865 870 875 880Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val Arg Cys 885 890 895Arg His Arg Arg Arg Gln Ala Glu Arg Met Ala Gln Ile Lys Arg Val 900 905 910Val Ser Glu Lys Lys Thr Ala Gln Ala Pro His Arg Phe Gln Lys Thr 915 920 925Cys Ser Pro Ile Ser Gly Gly Gly Gly Ser Arg Pro Glu Cys Val Val 930 935 940Pro Glu Thr Gln Cys Ala Met Lys Arg Lys Glu Lys Lys Ala Gln Lys945 950 955 960Glu Lys Asp Lys Leu Pro Val Ser Thr Thr Thr Val Asp Asp His Met 965 970 975Pro Pro Ile Met Gln Cys Glu Pro Pro Pro Pro Glu Ala Ala Arg Ile 980 985 990His Glu Val Val Pro Arg Phe Leu Ser Asp Lys Leu Leu Glu Thr Asn 995 1000 1005Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala Asn Gln Gln Phe Leu 1010 1015 1020Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr Glu Gln Pro Ser 1025 1030 1035Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln Gln Ala Asp 1040 1045 1050Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile Thr Glu 1055 1060 1065Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys Gly 1070 1075 1080Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu 1085 1090 1095Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg 1100 1105 1110Arg Tyr Asp Ala Ala Ser Asp Ser Ile Leu Phe Ala Asn Asn Gln 1115 1120 1125Ala Tyr Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Glu Val 1130 1135 1140Ile Glu Asp Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala 1145 1150 1155Leu Asp Asn Ile His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe 1160 1165 1170Ser Asp Arg Pro Gly Leu Glu Gln Pro Gln Leu Val Glu Glu Ile 1175 1180 1185Gln Arg Tyr Tyr Leu Asn Thr Leu Arg Ile Tyr Ile Leu Asn Gln 1190 1195 1200Leu Ser Gly Ser Ala Arg Ser Ser Val Ile Tyr Gly Lys Ile Leu 1205 1210 1215Ser Ile Leu Ser Glu Leu Arg Thr Leu Gly Met Gln Asn Ser Asn 1220 1225 1230Met Cys Ile Ser Leu Lys Leu Lys Asn Arg Lys Leu Pro Pro Phe 1235 1240 1245Leu Glu Glu Ile Trp Asp Val Ala Asp Met Ser His Thr Gln Pro 1250 1255 1260Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 1265 127024919PRTArtificial SequenceSimple inducible CAR 24Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe1 5 10 15Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp65 70 75 80Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly 100 105 110Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 115 120 125Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 130 135 140Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro145 150 155 160Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 165 170 175Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 180 185 190Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 195 200 205Ala Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala Glu Gly Arg Gly 210 215 220Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu225 230 235 240Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser 245 250 255Thr Gly Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser 260 265 270Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser 275 280 285Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu 290 295 300Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe305 310 315 320Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu 325 330 335Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu 340 345 350Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Lys Ala Gly 355 360 365Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 370 375 380Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala385 390 395 400Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 405 410 415Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 420 425 430Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 435 440 445Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 450 455 460Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr465 470 475 480Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 485 490 495Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Asp Pro Ala Glu Pro 500 505 510Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro 515 520 525Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 530 535 540Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val545 550 555 560Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 565 570 575Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 580 585 590Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 595 600 605Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 610 615 620Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro625 630 635 640Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 645 650 655Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 660 665 670Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 675 680 685Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 690 695 700Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser705 710 715 720Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 725 730 735Leu Ser Pro Gly Lys Lys Asp Pro Met Ala Leu Ile Val Leu Gly Gly 740 745 750Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys Val 755 760 765Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met Ala Gln Ile Lys 770 775 780Arg Val Val Ser Glu Lys Lys Thr Ala Gln Ala Pro His Arg Phe Gln785 790 795 800Lys Thr Cys Ser Pro Ile Ser Gly Gly Gly Gly Ser Gly Val Gln Val 805 810 815Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln 820 825 830Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val 835 840 845Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys 850 855 860Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val865 870 875 880Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala 885 890 895Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp 900 905 910Val Glu Leu Leu Lys Leu Glu 91525234PRTArtificial SequenceCID binding domain - EcR 25Met Pro Val Asp Arg Ile Leu Glu Ala Glu Leu Ala Val Glu Gln Lys1 5 10 15Ser Asp Gln Gly Val Glu Gly Pro Gly Gly Thr Gly Gly Ser Gly Ser 20 25 30Ser Pro Asn Asp Pro Val Thr Asn Ile Cys Gln Ala Ala Asp Lys Gln 35 40 45Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His Phe Ser Ser 50 55 60Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly Trp Asn Glu65 70 75 80Leu Leu Ile Ala Ser Phe Ser His Arg Ser Ile Asp Val Arg Asp Gly 85 90 95Ile Leu Leu Ala Thr Gly Leu His Val His Arg Asn Ser Ala His Ser 100 105 110Ala Gly Val Gly Ala Ile Phe Asp Arg Val Leu Thr Glu Leu Val Ser 115 120 125Lys Met Arg Asp Met Arg Met Asp Lys Thr Glu Leu Gly Cys Leu Arg 130 135 140Ala Ile Ile Leu Phe Asn Pro Glu Val Arg Gly Leu Lys Ser Ala Gln145 150 155 160Glu Val Glu Leu Leu Arg Glu Lys Val Tyr Ala Ala Leu Glu Glu Tyr 165 170 175Thr Arg Thr Thr His Pro Asp Glu Pro Gly Arg Phe Ala Lys Leu Leu 180 185 190Leu Arg Leu Pro Ser Leu Arg Ser Ile Gly Leu Lys Cys Leu Glu His 195 200 205Leu Phe Phe Phe Arg Leu Ile Gly Asp Val Pro Ile Asp Thr Phe Leu 210 215 220Met Glu Met Leu Glu Ser Pro Ser Asp Ser225 23026335PRTArtificial SequenceCID binding domain - RXR 26Arg Pro Glu Cys Val Val Pro Glu Thr Gln Cys Ala Met Lys Arg Lys1 5 10 15Glu Lys Lys Ala Gln Lys Glu Lys Asp Lys Leu Pro Val Ser Thr Thr 20 25 30Thr Val Asp Asp His Met Pro Pro Ile Met Gln Cys Glu Pro Pro Pro 35 40 45Pro Glu Ala Ala Arg Ile His Glu Val Val Pro Arg Phe Leu Ser Asp 50 55 60Lys Leu Leu Glu Thr Asn Arg Gln Lys Asn Ile Pro Gln Leu Thr Ala65 70 75 80Asn Gln Gln Phe Leu Ile Ala Arg Leu Ile Trp Tyr Gln Asp Gly Tyr 85 90 95Glu Gln Pro Ser Asp Glu Asp Leu Lys Arg Ile Thr Gln Thr Trp Gln 100 105 110Gln Ala Asp Asp Glu Asn Glu Glu Ser Asp Thr Pro Phe Arg Gln Ile 115 120 125Thr Glu Met Thr Ile Leu Thr Val Gln Leu Ile Val Glu Phe Ala Lys 130 135 140Gly Leu Pro Gly Phe Ala Lys Ile Ser Gln Pro Asp Gln Ile Thr Leu145 150 155 160Leu Lys Ala Cys Ser Ser Glu Val Met Met Leu Arg Val Ala Arg Arg 165 170 175Tyr Asp Ala Ala Ser Asp Ser Ile Leu Phe Ala Asn Asn Gln Ala Tyr 180 185 190Thr Arg Asp Asn Tyr Arg Lys Ala Gly Met Ala Glu Val Ile Glu Asp 195 200 205Leu Leu His Phe Cys Arg Cys Met Tyr Ser Met Ala Leu Asp Asn Ile 210 215 220His Tyr Ala Leu Leu Thr Ala Val Val Ile Phe Ser Asp Arg Pro Gly225 230 235 240Leu Glu Gln Pro Gln Leu Val Glu Glu Ile Gln Arg Tyr Tyr Leu Asn 245 250 255Thr Leu Arg Ile Tyr Ile Leu Asn Gln Leu Ser Gly Ser Ala Arg Ser 260 265 270Ser Val Ile Tyr Gly Lys Ile Leu Ser Ile Leu Ser Glu Leu Arg Thr 275 280 285Leu Gly Met Gln Asn Ser Asn Met Cys Ile Ser Leu Lys Leu Lys Asn 290 295 300Arg Lys Leu Pro Pro Phe Leu Glu Glu Ile Trp Asp Val Ala Asp Met305 310 315 320Ser His Thr Gln Pro Pro Pro Ile Leu Glu Ser Pro Thr Asn Leu 325 330 335

Claims

1. A chimeric antigen receptor (CAR) signalling system comprising; (i) multiple receptor components comprising an extracellular antigen-binding domain, a transmembrane domain, and a intracellular first chemical inducer of dimerization (CID) binding domain (CBD1), wherein the antigen-binding domain of each receptor component binds to a different antigen; and (ii) an intracellular signalling component comprising a signalling domain and a second CID binding domain (CBD2); wherein CBD1 and CBD2 are capable of simultaneously binding to a CID; and wherein, in the absence of CID, binding of the antigen-binding domain to antigen does not result in signalling through the signalling domain; whilst, in the presence of CID, the receptor component and the signalling component heterodimerize and binding of the antigen-binding domain to antigen results in signalling through the signalling domain.

2-11. (canceled)

12. The CAR signalling system according to claim 1, wherein the CBD1 of the multiple receptor components differ in binding to CID such that each antigen propagates different signalling strengths.

13. The CAR signalling system according to claim 1, wherein the signalling domain of the signalling component comprises a single endodomain selected from CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and OX40 endodomain.

14. The CAR signalling system according to claim 1, wherein the signalling domain of the signalling component comprises at least one of CD3 zeta endodomain, CD28 endodomain, 41 BB endodomain and OX40 endodomain.

15-18. (canceled)

19. A nucleic acid comprising nucleotide sequences encoding the CAR signaling system receptor component according to claim 1.

20. (canceled)

21. A nucleic acid according to claim 19, 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.

22. A nucleic acid according to claim 21, wherein the multiple receptor components or multiple signalling components are co-expressed by means of a self-cleaving peptide which is cleaved between the receptor component(s) and the signalling component(s).

23. A vector comprising a nucleic acid according to claim 18.

24. A retroviral vector or a lentiviral vector or a transposon comprising a vector according to claim 23.

25. A T cell or NK cell which expresses a CAR signaling system according to claim 1.

26. (canceled)

27. A pharmaceutical composition comprising a plurality of T cells or NK cells according to claim 25.

28. (canceled)

29. A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 27 to a subject.

30. A method for treating and/or preventing a disease, which comprises the following steps: (i) isolation of a T cell or NK containing sample from a subject; (ii) transduction or transfection of the T or NK cells with a nucleic acid according to claim 18 or a vector comprising the nucleic acid sequence; and (iii) administering the T cells or NK cells from (ii) to the subject.

31. A method according to claim 29, which further comprises the step of administering the CID to which the CBD1 and CBD2 simultaneously bind to the subject.

32. A method for treating disease in a subject which subject comprises T cells or NK cells according to claim 25, which comprises the step of administering the CID to which the CBD1 and CBD2 simultaneously bind to the subject.

33. A method according to claim 29, which involves monitoring the progression of disease and/or monitoring toxic activity in the subject and adjusting the dose of the CID to provide acceptable levels of disease progression and/or toxic activity.

34. A method according to claim 29, wherein the disease is a cancer.

35-38. (canceled)

39. A method for modifying a T or NK cell, which comprises the step of introducing: a nucleic acid according to claim 21 or a vector comprising the nucleic add, into a T cell or NK cell.

40. A method according to claim 39, wherein the T or NK cell is from a sample isolated from a subject.

41. A method for activating the CAR signalling system in a subject comprising a T or NK cell according to claim 25, which method comprises the step of administering the CID to the subject.

42. A method for reducing the activity of the CAR signalling system, in a subject comprising a T or NK cell according to claim 25, which method comprises reducing or stopping administration of the CID to the subject.

43. (canceled)