Patent Publication Number: US-2021187026-A1

Title: Cell

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a Continuation of U.S. application Ser. No. 15/037,391, filed May 18, 2016, which is a U.S. National Phase of International Application No. PCT/GB2014/053451, filed Nov. 21, 2014, which claims priority to Great Britain Application No. 1410934.2, filed Jun. 19, 2014 and Great Britain Application No. 1320573.7, filed Nov. 21, 2013. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a cell which comprises more than one chimeric antigen receptor (CAR). The cell may be capable of specifically recognising a target cell, due to a differential pattern of expression (or non-expression) of two or more antigens by the target cell. 
     BACKGROUND TO THE INVENTION 
     A number of immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), immunoconjugated mAbs, radioconjugated mAbs and bi-specific T-cell engagers. 
     Typically these immunotherapeutic agents target a single antigen: for instance, Rituximab targets CD20; Myelotarg targets CD33; and Alemtuzumab targets CD52. 
     However, it is relatively rare for the presence (or absence) of a single antigen effectively to describe a cancer. 
     A particular problem in the field of oncology is provided by the Goldie-Coldman hypothesis: which describes that the sole targeting of a single antigen may result in tumour escape by modulation of said antigen due to the high mutation rate inherent in most cancers. This modulation of antigen expression may reduce the efficacy of known immunotherapeutics. 
     Tumour heterogeneity describes the observation that different tumour cells can show distinct morphological and phenotypic profiles, including cellular morphology, gene expression, metabolism, proliferation and metastatic potential. This phenomenon occurs both between tumours and within tumours. Tumour heterogeneity has been observed in leukaemias, breast, prostate, colon, brain, head and neck, bladder and gynecological cancers, for example. Tumour heterogeneity may result in the expression of different antigens on the surface of cells within a tumour or between tumours. This heterogeneity of cancer cells introduces significant challenges in designing effective treatment strategies. 
     There is thus a need for immunotherapeutic agents which are capable of more targeting to reflect the complex pattern of marker expression that is associated with many cancers. 
     Chimeric Antigen Receptors (CARs) 
     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 ). 
     The most common form 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 signaling 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. 
     It has been observed that using a CAR approach for cancer treatment, tumour heterogeneity and immunoediting can cause escape from CAR treatment. For example, in the study described by Grupp et al (2013; New Eng. J. Med 368:1509-1518) CAR-modified T cell approach was used for the treatment of acute lymphoid leukemia. It was found that one patient of the anti-CD19 CAR trial relapsed with CD19-negative disease two months after treatment. 
     There is thus a need for alternative CAR treatment approaches which address the problems of cancer escape and tumour heterogeneity. 
     Expression of Two CAR Binding Specificities 
     Bispecific CARs known as tandem CARs or TanCARs have been developed in an attempt to target multiple cancer specific markers simultaneously. In a TanCAR, the extracellular domain comprises two antigen binding specificities in tandem, joined by a linker. The two binding specificities (scFvs) are thus both linked to a single transmembrane portion: one scFv being juxtaposed to the membrane and the other being in a distal position. 
     Grada et al (2013, Mol Ther Nucleic Acids 2:e105 describes a TanCAR which includes a CD19-specific scFv, followed by a Gly-Ser linker and then a HER2-specific scFv. The HER2-scFv was in the juxta-membrane position, and the CD19-scFv in the distal position. The Tan CAR was shown to induce distinct T cell reactivity against each of the two tumour restricted antigens. This arrangement was chosen because the respective lengths of HER2 (632 aa/125 Å) and CD19 (280aa, 65 Å) lends itself to that particular spatial arrangement. It was also known that the HER2 scFv bound the distal-most 4 loops of HER2. 
     The problem with this approach is that the juxta-membrane scFv may be inaccessible due to the presence of the distal scFv, especially which it is bound to the antigen. In view of the need to choose the relative positions of the two scFvs in view of the spatial arrangement of the antigen on the target cell, it may not be possible to use this approach for all scFv binding pairs. Moreover, it is unlikely that the TanCar approach could be used for more than two scFvs, a TanCAR with three or more scFvs would be a very large molecule and the scFvs may well fold back on each other, obscuring the antigen-binding sites. It is also doubtful that antigen-binding by the most distal scFv, which is separated from the transmembrane domain by two or more further scFvs, would be capable of triggering T cell activation. 
     There is thus a need for an alternative approach to express two CAR binding specificities on the surface of a cell such as a T cell. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1A-1D :  FIG. 1A . Generalized architecture of a CAR: A binding domain recognizes antigen; the spacer elevates the binding domain from the cell surface; the trans-membrane domain anchors the protein to the membrane and the endodomain transmits signals.  FIG. 1B-1D . Different generations and permutations of CAR endodomains:  FIG. 1B . initial designs transmitted ITAM signals alone through FcεR1-γ or CD3ζ endodomain, while later designs transmitted additional  FIG. 1C . one or  FIG. 1D . two co-stimulatory signals in cis. 
         FIG. 2 : Schematic diagram illustrating the invention 
       The invention relates to engineering T-cells to respond to logical rules of target cell antigen expression. This is best illustrated with an imaginary FACS scatter-plot. Target cell populations express both, either or neither of antigens “A” and “B”. Different target populations (marked in red) are killed by T-cells transduced with a pair of CARs connected by different gates. With OR gated receptors, both single-positive and double-positive cells will be killed. With AND gated receptors, only double-positive target cells are killed. With AND NOT gating, double-positive targets are preserved while single-positive targets 
         FIG. 3 : Creation of target cell populations 
       SupT1 cells were used as target cells. These cells were transduced to express either CD19, CD33 or both CD19 and CD33. Target cells were stained with appropriate antibodies and analysed by flow cytometry. 
         FIG. 4 : Cassette design for an OR gate 
       A single open reading frame provides both CARs with an in-frame FMD-2A sequence resulting in two proteins. Signal1 is a signal peptide derived from IgG1 (but can be any effective signal peptide). scFv1 is the single-chain variable segment which recognizes CD19 (but can be a scFv or peptide loop or ligand or in fact any domain which recognizes any desired arbitrary target). STK is the CD8 stalk but may be any suitable extracellular domain. CD28tm is the 
       CD28 trans-membrane domain but can by any stable type I protein transmembrane domain and CD3Z is the CD3 Zeta endodomain but can be any endodomain which contains ITAMs. Signal2 is a signal peptide derived from CD8 but can be any effective signal peptide which is different in DNA sequence from signal1. scFv recognizes CD33 but as for scFv1 is arbitrary. HC2CH3 is the hinge-CH2-CH3 of human IgG1 but can be any extracellular domain which does not cross-pair with the spacer used in the first CAR. CD28tm′ and CD3Z′ code for the same protein sequence as CD28tm and CD3Z but are codon-wobbled to prevent homologous recombination. 
         FIG. 5A-5B : Schematic representation of the chimeric antigen receptors (CARs) for an OR gate 
       Stimulatory CARs were constructed consisting of either an N-terminal  FIG. 5A . anti-CD19 scFv domain followed by the extracellular hinge region of human CD8 or  FIG. 5B . anti-CD33 scFv domain followed by the extracellular hinge, CH2 and CH3 (containing a pvaa mutation to reduce FcR binding) region of human IgG1. Both receptors contain a human CD28 transmembrane domain and a human CD3 Zeta (CD247) intracellular domain. “S” depicts the presence of disulphide bonds. 
         FIG. 6 : Expression data showing co-expression of both CARs on the surface of one T-cell. 
         FIG. 7 : Functional analysis of the OR gate 
       Effector cells (5×10{circumflex over ( )}4 cells) expressing the OR gate construct were co-incubated with a varying number of target cells and IL-2 was analysed after 16 hours by ELISA. The graph displays the average maximum IL-2 secretion from a chemical stimulation (PMA and lonomycin) of the effector cells alone and the average background IL-2 from effector cells without any stimulus from three replicates. 
         FIG. 8 : Cartoon showing both versions of the cassette used to express both AND gates Activating and inhibiting CARs were co-expressed once again using a FMD-2A sequence. Signal1 is a signal peptide derived from IgG1 (but can be any effective signal peptide). scFv1 is the single-chain variable segment which recognizes CD19 (but can be a scFv or peptide loop or ligand or in fact any domain which recognizes any desired arbitrary target). STK is the CD8 stalk but may be any non-bulky extracellular domain. CD28tm is the CD28 trans-membrane domain but can by any stable type I protein transmembrane domain and CD3Z is the CD3 Zeta endodomain but can be any endodomain which contains ITAMs. Signal2 is a signal peptide derived from CD8 but can be any effective signal peptide which is different in DNA sequence from signal1. scFv recognizes CD33 but as for scFv1 is arbitrary. HC2CH3 is the hinge-CH2-CH3 of human IgG1 but can be any bulky extracellular domain. CD45 and CD148 are the transmembrane and endodomains of CD45 and CD148 respectively but can be derived from any of this class of protein. 
         FIG. 9 : Schematic representation of the protein structure of chimeric antigen receptors (CARs) for the AND gates 
       The stimulatory CAR consisting of an N-terminal anti-CD19 scFv domain followed by the extracellular stalk region of human CD8, human CD28 transmembrane domain and human CD3 Zeta (CD247) intracellular domain. Two inhibitory CARs were tested. These consist of an N-terminal anti-CD33 scFv domain followed by the extracellular hinge, CH2 and CH3 (containing a pvaa mutation to reduce FcR binding) region of human IgG1 followed by the transmembrane and intracellular domain of either human CD148 or CD45. “S” depicts the presence of disulphide bonds. 
         FIG. 10 : Co-expression of activation and inhibitory CARs 
       BW5147 cells were used as effector cells and were transduced to express both the activation anti-CD19 CAR and one of the inhibitory anti-CD33 CARs. Effector cells were stained with CD19-mouse-Fc and CD33-rabbit-Fc and with appropriate secondary antibodies and analysed by flow cytometry. 
         FIG. 11A-11B : Functional analysis of the AND gates 
       Effector cells (5×10{circumflex over ( )}4 cells) expressing activation anti-CD19 CAR and the inhibitory anti-CD33 CAR with the  FIG. 11A . CD148 or  FIG. 11B . CXD45 intracellular domain were co-incubated with a varying number of target cells and IL-2 was analysed after 16 hours by ELISA. The graph displays the maximum IL-2 secretion from a chemical stimulation (PMA and lonomycin) of the effector cells alone and the background IL-2 from effector cells without any stimulus from three replicates. 
         FIG. 12 : Cartoon showing three versions of the cassette used to generate the AND NOT gate 
       Activating and inhibiting CARs were co-expressed once again using a FMD-2A sequence. Signal1 is a signal peptide derived from IgG1 (but can be any effective signal peptide). scFv1 is the single-chain variable segment which recognizes CD19 (but can be a scFv or peptide loop or ligand or in fact any domain which recognizes any desired arbitrary target). STK is the human CD8 stalk but may be any non-bulky extracellular domain. CD28tm is the CD28 trans-membrane domain but can by any stable type I protein transmembrane domain and CD3Z is the CD3 Zeta endodomain but can be any endodomain which contains ITAMs. Signal2 is a signal peptide derived from CD8 but can be any effective signal peptide which is different in DNA sequence from signal1. scFv recognizes CD33 but as for scFv1 is arbitrary. muSTK is the mouse CD8 stalk but can be any spacer which co-localises but does not cross-pair with that of the activating CAR. dPTPN6 is the phosphatase domain of PTPN6. LAIR1 is the transmembrane and endodomain of LAIR1. 2Aw is a codon-wobbled version of the FMD-2A sequence. SH2-CD148 is the SH2 domain of PTPN6 fused with the phosphatase domain of CD148. 
         FIG. 13A-13D : Schematic representation of the chimeric antigen receptors (CARs) for the NOT AND gates 
         FIG. 13A . A stimulatory CAR consisting of an N-terminal anti-CD19 scFv domain followed by the stalk region of human CD8, human CD28 transmembrane domain and human CD247 intracellular domain.  FIG. 13B . An inhibitory CAR consisting of an N-terminal anti-CD33 scFv domain followed by the stalk region of mouse CD8, transmembrane region of mouse CD8 and the phosphatase domain of PTPN6.  FIG. 13C . an inhibitory CAR consisting of an N-terminal anti-CD33 scFv domain followed by the stalk region of mouse CD8 and the transmembrane and intracellular segments of LAIR1.  FIG. 13D . An inhibitory CAR identical to previous CAR except it is co-expressed with a fusion protein of the PTPN6 SH2 domain and the CD148 phosphatase domain. 
         FIG. 14A-14B : Functional analysis of the NOT AND gate 
       Effector cells (5×10{circumflex over ( )}4 cells) expressing the  FIG. 14A . full length SHP-1 or  FIG. 14B . truncated form of SHP-1 were co-incubated with a varying number of target cells and IL-2 was analysed after 16 hours by ELISA. The graph displays the average maximum IL-2 secretion from a chemical stimulation (PMA and lonomycin) of the effector cells alone and the average background IL-2 from effector cells without any stimulus from three replicates. 
         FIG. 15 : Amino acid sequence of an OR gate 
         FIG. 16 : Amino acid sequence of a CD148 and a CD45 based AND gate 
         FIG. 17 : Amino acid sequence of three AND NOT gates 
         FIG. 18A-18C : Dissection of AND gate function 
         FIG. 18A . The prototype AND gate is illustrated on the right and its function in response to CD19, CD33 single and CD19, CD33 double positive targets is shown on the left.  FIG. 18B . The scFvs are swapped so the activating endodomain is triggered by CD33 and the inhibitory endodomain is activated by CD19. This AND gate remains functional despite this scFv swap.  FIG. 18C . The CD8 mouse stalk replaced Fc in the spacer of the inhibitory CAR. With this modification, the gate fails to respond to either CD19 single positive or CD19, CD33 double positive targets. 
         FIG. 19A-19D : Expression of target antigens on artificial target cells 
         FIG. 19A . Shows flow cytometry scatter plots CD19 vs CD33 of the original set of artificial target cells derived from SupT1 cells. From left to right: double negative SupT1 cells, SupT1 cells positive for CD19, positive for CD33 and positive for both CD19 and CD33.  FIG. 19B . Shows flow cytometry scatter plots CD19 vs GD2 of the artificial target cells generated to test the CD19 AND GD2 gate: From left to right: negative SupT1 cells, SupT1 cells expressing CD19, SupT1 cells transduced with GD2 and GM3 synthase vectors which become GD2 positive and SupT1 cells transduced with CD19 as well as GD2 and GM3 synthase which are positive for both GD2 and CD19.  FIG. 19C . Shows flow cytometry scatter plots of CD19 vs EGFRvIII of the artificial targets generated to test the CD19 AND EGFRvIII gate. From left to right: negative SupT1 cells, SupT1 cells expressing CD19, SupT1 cells transduced with EGFRvIII and SupT1 cells transduced with both CD19 and EGFRvIII.  FIG. 19D . Shows flow cytometry scatter plots of CD19 vs CD5 of the artificial targets generated to test the CD19 AND CD5 gate. From left to right: negative 293T cells, 293T cells transduced with CD19, 293T cells transduced with CD5, 293T cells transduced with both CD5 and CD19 vectors. 
         FIG. 20A-20D : Generalizability of the AND gate 
         FIG. 20A . Cartoon of AND gate modified so the second CAR&#39;s specificity is changed from the original specificity of CD33, to generate 3 new CARs: CD19 AND GD2, CD19 AND EGFRvIII, CD19 AND CD5.  FIG. 20B . CD19 AND GD2 AND gate: Left: expression of AND gate is shown recombinant CD19-Fc staining (x-axis) for the CD19 CAR, versus anti-human-Fc staining (Y-axis) for the GD2 CAR. Right: function in response to single positive and double positive targets.  FIG. 20C . CD19 AND EGFRvIII AND gate: Left: expression of AND gate is shown recombinant CD19-Fc staining (x-axis) for the CD19 CAR, versus anti-human-Fc staining (Y-axis) for the EGFRvIII CAR. Right: function in response to single positive and double positive targets.  FIG. 20D . CD19 AND CD5 AND gate: Left: expression of AND gate is shown recombinant CD19-Fc staining (x-axis) for the CD19 CAR, versus anti-human-Fc staining (Y-axis) for the CD5 CAR. Right: function in response to single positive and double positive targets. 
         FIG. 21A-21C : Function of the AND NOT Gates 
       Function of the three implementations of an AND NOT gate is shown. A cartoon of the gates tested is shown to the right, and function in response to single positive and double positive targets is shown to the left.  FIG. 21A . PTPN6 based AND NOT gate whereby the first CAR recognizes CD19, has a human CD8 stalk spacer and an ITAM containing activating endodomain; is co-expressed with a second CAR that recognizes CD33, has a mouse CD8 stalk spacer and has an endodomain comprising of a PTPN6 phosphatase domain.  FIG. 21B . ITIM based AND NOT gate is identical to the PTPN6 gate, except the endodomain is replaced by the endodomain from LAIR1.  FIG. 21C . CD148 boosted AND NOT gate is identical to the ITIM based gate except an additional fusion between the PTPN6 SH2 and the endodomain of CD148 is expressed. All three gates work as expected with activation in response to CD19 but not in response to CD19 and CD33 together. 
         FIG. 22A-22C : Dissection of PTPN6 based AND NOT gate function 
       The original PTPN6 based AND NOT gate is compared with several controls to demonstrate the model. A cartoon of the gates tested is shown to the right, and function in response to single positive and double positive targets is shown to the left.  FIG. 22A . Original AND NOT gate whereby the first CAR recognizes CD19, has a human CD8 stalk spacer and an ITAM containing activating endodomain; is co-expressed with a second CAR recognizes CD33, has a mouse CD8 stalk spacer and has an endodomain comprising of a PTPN6 phosphatase domain.  FIG. 22B . AND NOT gate modified so the mouse CD8 stalk spacer is replaced with an Fc spacer.  FIG. 22C . AND NOT gate modified so that the PTPN6 phosphatase domain is replaced with the endodomain from CD148. Original AND NOT gate ( FIG. 22A ) functions as expected triggering in response to CD19, but not in response to both CD19 and CD33. The gate in  FIG. 22B . triggers both in response to CD19 along or CD19 and CD33 together. The gate in  FIG. 22C . does not trigger in response to one or both targets. 
         FIG. 23A-23B : Dissection of LAIR1 based AND NOT gate 
       Functional activity against CD19 positive, CD33 positive and CD19, CD33 double-positive targets is shown.  FIG. 23A . Structure and activity of the original ITIM based AND NOT gate. This gate is composed of two CARs: the first recognizes CD19, has a human CD8 stalk spacer and an ITAM containing endodomain; the second CAR recognizes CD33, has a mouse CD8 stalk spacer and an ITIM containing endodomain.  FIG. 23B . Structure and activity of the control ITIM based gate where the mouse CD8 stalk spacer has been replaced by an Fc domain. This gate is composed of two CARs: the first recognizes CD19, has a human CD8 stalk spacer and an ITAM containing endodomain; the second CAR recognizes CD33, has an Fc spacer and an ITIM containing endodomain. Both gates respond to CD19 single positive targets, while only the original gate is inactive in response to CD19 and CD33 double positive targets. 
         FIG. 24A-24D : Kinetic segregation model of CAR logic gates 
       Model of kinetic segregation and behaviour of AND gate, NOT AND gate and controls. CARs recognize either CD19 or CD33. The immunological synapse can be imagined between the blue line, which represents the target cell membrane and the red line, which represents the T-cell membrane. ‘45’ is the native CD45 protein present on T-cells. ‘H8’ is a CAR ectodomain with human CD8 stalk as the spacer. ‘Fc’ is a CAR ectodomain with human HCH2CH3 as the spacer. ‘M8’ is a CAR ectodomain with murine CD8 stalk as the spacer. ‘19’ represents CD19 on the target cell surface. ‘33’ represents CD33 on the target cell surface. The symbol ‘⊕’ represents an activating endodomain containing ITAMS. The symbol ‘⊖’ represents a phosphatase with slow kinetics—a ‘ligation on’ endodomain such as one comprising of the catalytic domain of PTPN6 or an ITIM. The symbol ‘0’ represents a phosphatase with fast kinetics—a ‘ligation off’ endodomain such as the endodomain of CD45 or CD148. This symbol is enlarged in the figure to emphasize its potent activity. 
         FIG. 24A . Shows the postulated behaviour of the functional AND gate which comprises of a pair of CARs whereby the first CAR recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; and the second CAR recognizes CD33, has an Fc spacer and a CD148 endodomain; 
         FIG. 24B . Shows the postulated behaviour of the control AND gate. Here, the first CAR recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; and the second CAR recognizes CD33, but has a mouse CD8 stalk spacer and a CD148 endodomain; 
         FIG. 24C . Shows the behaviour of a functional AND NOT gate which comprises of a pair of CARs whereby the first CAR recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; and the second CAR recognizes CD33, has a mouse CD8 stalk spacer and a PTPN6 endodomain; 
         FIG. 24D . Shows the postulated behaviour of the control AND NOT gate which comprises of a pair of CARs whereby the first CAR recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; and the second CAR recognizes CD33, but has an Fc spacer and a PTPN6 endodomain; In the first column, target cells are both CD19 and CD33 negative. In the second column, targets are CD19 negative and CD33 positive. In the third column, target cells are CD19 positive and CD33 negative. In the fourth column, target cells are positive for both CD19 and CD33. 
         FIG. 25A-25C : Design of APRIL-based CARs. 
       The CAR design was modified so that the scFv was replaced with a modified form of A proliferation-inducing ligand (APRIL), which interacts with interacts with BCMA, TACl and proteoglycans, to act as an antigen binding domain: APRIL was truncated so that the proteoglycan binding amino-terminus is absent.  FIG. 25A . signal peptide was then attached to truncated APRIL amino-terminus to direct the protein to the cell surface. Three CARs were generated with this APRIL based binding domain:  FIG. 25A . In the first CAR, the human CD8 stalk domain was used as a spacer domain. B. In the second CAR, the hinge from IgG1 was used as a spacer domain.  FIG. 25C . In the third CAR, the hinge, CH2 and CH3 domains of human IgG1 modified with the pva/a mutations described by Hombach et al (2010 Gene Ther. 17:1206-1213) to reduce Fc Receptor binding was used as a spacer (henceforth referred as Fc-pvaa). In all CARs, these spacers were connected to the CD28 transmembrane domain and then to a tripartite endodomain containing a fusion of the CD28, OX40 and the CD3-Zeta endodomain (Pule et al, Molecular therapy, 2005: Volume 12; Issue 5; Pages 933-41). 
         FIG. 26A-26C : Annotated Amino acid sequence of the above three APRIL-CARS 
         FIG. 26A . Shows the annotated amino acid sequence of the CD8 stalk APRIL CAR;  FIG. 26B . Shows the annotated amino acid sequence of the APRIL IgG1 hinge based CAR;  FIG. 26C . Shows the annotated amino acid sequence of the APRIL Fc-pvaa based CAR. 
         FIG. 27A-27C : Expression and ligand binding of different APRIL based CARs 
         FIG. 27A . The receptors were co-expressed with a marker gene truncated CD34 in a retroviral gene vector. Expression of the marker gene on transduced cells allows confirmation of transduction.  FIG. 27B . T-cells were transduced with APRIL based CARs with either the CD8 stalk spacer, IgG1 hinge or Fc spacer. To test whether these receptors could be stably expressed on the cell surface, T-cells were then stained with anti-APRIL-biotin/Streptavidin APC and anti-CD34. Flow-cytometric analysis was performed. APRIL was equally detected on the cell surface in the three CARs suggesting they are equally stably expressed.  FIG. 27C . Next, the capacity of the CARs to recognize TACl and BCMA was determined. The transduced T-cells were stained with either recombinant BCMA or TACl fused to mouse IgG2a Fc fusion along with an anti-mouse secondary and anti-CD34. All three receptor formats showed binding to both BCMA and TACl. A surprising finding was that binding to BCMA seemed greater than to TACl. A further surprising finding was that although all three CARs were equally expressed, the CD8 stalk and IgG1 hinge CARs appeared better at recognizing BCMA and TACl than that with the Fc spacer. 
         FIG. 28A-28C : Function of the different CAR constructs. 
       Functional assays were performed with the three different APRIL based CARs. Normal donor peripheral blood T-cells either non-transduced (NT), or transduced to express the different CARs. Transduction was performed using equal titer supernatant. These T-cells were then CD56 depleted to remove non-specific NK activity and used as effectors. SupT1 cells either non-transduced (NT), or transduced to express BCMA or TACl were used as targets. Data shown is mean and standard deviation from 5 independent experiments.  FIG. 28A . Specific killing of BCMA and TACl expressing T-cells was determined using Chromium release.  FIG. 28B . Interferon-μ release was also determined. Targets and effectors were co-cultured at a ratio of 1:1. After 24 hours, Interferon-μ in the supernatant was assayed by ELISA. FIG.  28 C. Proliferation/survival of CAR T-cells were also determined by counting number of CAR T-cells in the same co-culture incubated for a further 6 days. All 3 CARs direct responses against BCMA and TACl expressing targets. The responses to BCMA were greater than for TACl. 
         FIG. 29 : AND gate functionality in primary cells. 
       PBMCs were isolated from blood and stimulated using PHA and IL-2. Two days later the cells were transduced on retronectin coated plates with retro virus containing the CD19:CD33 AND gate construct. On day 5 the expression level of the two CARs translated by the AND gate construct was evaluated via flow cytometry and the cells were depleted of CD56+ cells (predominantly NK cells). On day 6 the PBMCs were placed in a co-culture with target cells at a 1:2 effector to target cell ratio. On day 8 the supernatant was collected and analysed for IFN-gamma secretion via ELISA 
         FIG. 30A-30B :  FIG. 30A . DNA alignment of the CD28 transmembrane and cytosolic domain of TCRz in the OR gate platform  FIG. 30B . Protein alignment of the CD28 transmembrane and cytosolic domain of TCRz in the OR gate platform. 
         FIG. 31 : Design rules for building logic gated CAR T-cells. OR, AND NOT and AND gated CARs are shown in cartoon format with the target cell on top, and the T-cell at the bottom with the synapse in the middle. Target cells express arbitrary target antigens A, and B. 
       T-cells express two CARs which comprise of anti-A and anti-B recognition domains, spacers and endodomains. An OR gate requires (1) spacers simply which allow antigen recognition and CAR activation, and (2) both CARs to have activatory endodomains; An AND NOT gate requires (1) spacers which result in co-segregation of both CARs upon recognition of both antigens and (2) one CAR with an activatory endodomain, and the other whose endodomain comprises or recruits a weak phosphatase; An AND gate requires (1) spacers which result in segregation of both CARs into different parts of the immunological synapse upon recognition of both antigens and (2) one CAR with an activatory endodomain, and the other whose endodomain comprises of a potent phosphatase. 
     
    
    
     SUMMARY OF ASPECTS OF THE INVENTION 
     The present inventors have developed a panel of “logic-gated” chimeric antigen receptor pairs which, when expressed by a cell, such as a T cell, are capable of detecting a particular pattern of expression of at least two target antigens. If the at least two target antigens are arbitrarily denoted as antigen A and antigen B, the three possible options are as follows: 
     “OR GATE”—T cell triggers when either antigen A or antigen B is present on the target cell “AND GATE”—T cell triggers only when both antigens A and B are present on the target cell “AND NOT GATE”—T cell triggers if antigen A is present alone on the target cell, but not if both antigens A and B are present on the target cell 
     Engineered T cells expressing these CAR combinations can be tailored to be exquisitely specific for cancer cells, based on their particular expression (or lack of expression) of two or more markers. 
     Thus in a first aspect, the present invention provides a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising:
         (i) an antigen-binding domain;   (ii) a spacer   (iii) a trans-membrane domain; and   (iv) an intracellular T cell signaling domain (endodomain)       

     wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first or second CARs is an activating CAR comprising an activating intracellular T cell signaling domain. 
     The cell may be an immune effector cell, such as a T-cell or natural killer (NK) cell. Features mentioned herein in connection with a T cell apply equally to other immune effector cells, such as NK cells. 
     The spacer of the first CAR may be different to the spacer of the second CAR. such that the first and second CARs do not form heterodimers 
     The spacer of the first CAR may have a different length and/or size and/or configuration from the spacer of the second CAR, such that each CAR is tailored for recognition of its respective target antigen. 
     The spacer of the first CAR may have a different length and/or charge and/or shape and/or configuration and/or glycosylation to the spacer of the second CAR, such that when the first CAR and the second CAR bind their respective target antigens, the first CAR and second CAR become spatially separated on the T cell. Ligation of the first and second CARs to their respective antigens causes them to be compartmentalized together or separately in the immunological synapse resulting in control of activation. This may be understood when one considers the kinetic separation model of T-cell activation (see below). 
     The first spacer or the second spacer may comprise a CD8 stalk and the other spacer may comprise the hinge, CH2 and CH3 domain of an IgG1. 
     In the present invention, relating to the “OR” gate, both the first and second CAR are activating CARs. An activating CAR comprises an activating endodomain, such that it causes T cell activation upon antigen binding. Since the OR gate comprises first and second CARs which are both activating, T cell activation occurs when a target cell expresses either or both target antigens. 
     Either the first or the second CAR may bind CD19 and the other CAR may bind CD20. This is advantageous because some lymphomas and leukemias become CD19 negative after CD19 targeting, so it gives a “back-up” antigen, should this occur. 
     In a second aspect, the present invention provides a nucleic acid sequence encoding both the first and second chimeric antigen receptors (CARs) as defined in the first aspect of the invention. 
     The nucleic acid sequence according may have the following structure: AgB1-spacer1-TM1-endo1-coexpr-AgB2-spacer2-TM2-endo2 
     in which 
     AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR; spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR; 
     TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; 
     coexpr is a nucleic acid sequence allowing co-expression of two CARs (e.g. a cleavage site); 
     AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
     spacer 2 is a nucleic acid sequence encoding the spacer of the second CAR; 
     TM2 is a a nucleic acid sequence encoding the transmembrane domain of the second CAR; 
     endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR; 
     which nucleic acid sequence, when expressed in a T cell, encodes a polypeptide which is cleaved at the cleavage site such that the first and second CARs are co-expressed at the T cell surface. 
     The nucleic acid sequence allowing co-expression of two CARs may encode a self-cleaving peptide or a sequence which allows alternative means of co-expressing two CARs such as an internal ribosome entry sequence or a 2 nd  promoter or other such means whereby one skilled in the art can express two proteins from the same vector. 
     Alternative codons may be used in regions of sequence encoding the same or similar amino acid sequences, such as the transmembrane and/or intracellular T cell signalling domain (endodomain) in order to avoid homologous recombination. An example of such “codon wobbling” is shown in  FIG. 30 . 
     In a third aspect, the present invention provides a kit which comprises
         (i) a first nucleic acid sequence encoding the first chimeric antigen receptor (CAR) as defined in the first aspect of the invention, which nucleic acid sequence has the following structure:       

       AgB1-spacer1-TM1-endo1 
     in which 
     AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR; 
     spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR; 
     TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; 
     endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; and
         (ii) a second nucleic acid sequence encoding the second chimeric antigen receptor       

     (CAR) as defined in the first aspect of the invention, which nucleic acid sequence has the following structure: 
       Ag B2-spacer2-TM2-endo2 
     AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
     spacer 2 is a nucleic acid sequence encoding the spacer of the second CAR; 
     TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; 
     endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR. 
     In a fourth aspect, the present invention provides a kit comprising: a first vector which comprises the first nucleic acid sequence as defined above; and a second vector which comprises the first nucleic acid sequence as defined above. 
     The vectors may be plasmid vectors, retroviral vectors or transposon vectors. The vectors may be lentiviral vectors. 
     In a fifth aspect, the present invention provides a vector comprising a nucleic acid sequence according to the second aspect of the invention. The vector may be a lentiviral vector. 
     The vector may be a plasmid vector, a retroviral vector or a transposon vector. 
     In a sixth aspect, the present invention involves co-expressing more than two CARs in such a fashion that a complex pattern of more than two antigens can be recognized on the target cell. 
     In a seventh aspect, the present invention provides a method for making a T cell according to the first aspect of the invention, which comprises the step of introducing one or more nucleic acid sequence (s) encoding the first and second CARs; or one or more vector(s) as defined above into a T cell. 
     The T cell may be from a sample isolated from a patient, a related or unrelated haematopoietic transplant donor, a completely unconnected donor, from cord blood, differentiated from an embryonic cell line, differentiated from an inducible progenitor cell line, or derived from a transformed T cell line. 
     In an eighth aspect, the present invention provides a pharmaceutical composition comprising a plurality of T cells according to the first aspect of the invention. 
     In a ninth aspect, the present invention provides a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the eighth aspect of the invention to a subject. 
     The method may comprise the following steps:
         (i) isolation of a T cell as listed above.   (ii) transduction or transfection of the T cells with one or more nucleic acid sequence(s) encoding the first and second CAR or one or more vector(s) comprising such nucleic acid sequence(s); and   (iii) administering the T cells from (ii) to the subject.       

     The disease may be a cancer. 
     In a tenth aspect, the present invention provides a pharmaceutical composition according to the eighth aspect of the invention for use in treating and/or preventing a disease. 
     The disease may be a cancer. 
     In an eleventh aspect, the present invention provides use of a T cell according to the first aspect of the invention in the manufacture of a medicament for treating and/or preventing a disease. 
     The disease may be a cancer. 
     The present invention also provides a nucleic acid sequence which comprises: 
     a) a first nucleotide sequence encoding a first chimeric antigen receptor (CAR); 
     b) a second nucleotide sequence encoding a second CAR; 
     c) a sequence encoding a self-cleaving peptide positioned between the first and second nucleotide sequences, such that the two CARs are expressed as separate entities. 
     Alternative codons may be used in one or more portion(s) of the first and second nucleotide sequences in regions which encode the same or similar amino acid sequence(s). 
     The present invention also provides a vector and a cell comprising such a nucleic acid. 
     By providing two or more CARs on the surface of the T cell, it is possible to target multiple cancer markers simultaneously, providing better therapeutic efficacy for heterogeneic tumours and avoiding the problem of cancer escape. 
     Because the CARs are expressed on the surface of the T cell as separate molecules, this approach overcomes the spatial and accessibility issues associated with TanCARs. T-cell activation efficiency is also improved. As each CAR has its own spacer, it is possible to tailor the spacer and therefore the distance that the binding domain projects from the T cell surface and its flexibility etc to the particular target antigen. This choice is unfettered by the design considerations associated with TanCARs, i.e. that one CAR needs to be juxta-posed to the T cell membrane and one CAR needs to be distal, positioned in tandem to the first CAR. 
     By providing a single nucleic acid which encodes the two CARs separated by a cleavage site, it is possible to engineer T cells to co-express the two CARs using a simple single transduction procedure. A double transfection procedure could be used with CAR-encoding sequences in separate constructs, but this would be more complex and expensive and requires more integration sites for the nucleic acids. A double transfection procedure would also be associated with uncertainty as to whether both CAR-encoding nucleic acids had been transduced and expressed effectively. This is especially true for a multiple CAR approach where three or more CARs are introduced to the cell. 
     The two or more CARs will have portions of high homology, for example the transmembrane and/or intracellular signalling domains are likely to be highly homologous. If the same or similar linkers are use for the two CARs, then they will also be highly homologous. This would suggest that an approach where both CARs are provided on a single nucleic acid sequence would be inappropriate, because of the likelihood of homologous recombination between the sequences. However, the present inventors have found that by “codon wobbling” the portions of sequence encoding areas of high homology, it is possible to express two CARs from a single construct with high efficiency. Codon wobbling involves using alternative codons in regions of sequence encoding the same or similar amino acid sequences. 
     FURTHER ASPECTS OF THE INVENTION 
     The present invention also relates to the aspects listed in the following numbered paragraphs: 
     1. A T cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising:
         (i) an antigen-binding domain;   (ii) a spacer   (iii) a trans-membrane domain; and   (iv) an endodomain       

     wherein the antigen binding domains of the first and second CARs bind to different antigens, wherein the spacer of the first CAR is different to the spacer of the second CAR and wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is either an activating CAR comprising an activating endodomain or an inhibitory CAR comprising a ligation-on or ligation-off inhibitory endodomain. 
     2. A T cell according to paragraph 1, wherein the spacer of the first CAR has a different length and/or charge and/or size and/or configuration and/or glycosylation of the spacer of the second CAR, such that when the first CAR and the second CAR bind their respective target antigens, the first CAR and second CAR become spatially separated on the T cell membrane. 
     3. A T cell according to paragraph 2, wherein either the first spacer or the second spacer comprises a CD8 stalk and the other spacer comprises the hinge, CH2 and CH3 domain of IgG1. 
     4. A T cell according to paragraph 1, wherein both the first and second CARs are activating CARs. 
     5. A T cell according to paragraph 4, wherein one CAR binds CD19 and the other CAR binds CD20. 
     6. A T cell according to paragraph 2 or 3, wherein one of the first or second CARs is an activating CAR comprising an activating endodomain, and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain, which inhibitory CAR inhibits T-cell activation by the activating CAR in the absence of inhibitory CAR ligation, but does not significantly inhibit T-cell activation by the activating CAR when the inhibitory CAR is ligated. 
     7. A T cell according to paragraph 6, wherein the inhibitory endodomain comprises all or part of the endodomain from CD148 or CD45. 
     8. A T cell according to paragraph 6 or 7, wherein the antigen-binding domain of the first CAR binds CD5 and the antigen-binding domain of the second CAR binds CD19. 
     9. A T cell according to paragraph 1 wherein the first and second spacers are sufficiently different so as to prevent cross-pairing of the first and second CARs but are sufficiently similar to result in co-localisation of the first and second CARs following ligation. 
     10. A T cell according to paragraph 9, wherein one of the first or second CARs in an activating CAR comprising an activating endodomain, and the other CAR is an inhibitory CAR comprising a ligation-on inhibitory endodomain, which inhibitory CAR does not significantly inhibit T-cell activation by the activating CAR in the absence of inhibitory CAR ligation, but inhibits T-cell activation by the activating CAR when the inhibitory CAR is ligated. 
     11. A T cell according to paragraph 10, wherein the ligation-on inhibitory endodomain comprises at least part of a phosphatase. 
     12. A T cell according to paragraph 11, wherein the ligation-on inhibitory endodomain comprises all or part of PTPN6. 
     13. A T cell according to paragraph 10, wherein the ligation-on inhibitory endodomain comprises at least one ITIM domain. 
     14. A T cell according to paragraph 13, wherein activity of the ligation-on inhibitory endodomain is enhanced by co-expression of a PTPN6-CD45 or -CD148 fusion protein. 
     15. A T cell according to any of paragraphs 10 to 14, wherein the CAR comprising the activating endodomain comprises an antigen-binding domain which binds CD33 and the CAR which comprises the ligation-on inhibitory endodomain comprises an antigen-binding domain which binds CD34. 
     16. A T cell which comprises more than two CARs as defined in the preceding paragraphs such that it is specifically stimulated by a cell, such as a T cell, bearing a distinct pattern of more than two antigens. 
     17. A nucleic acid sequence encoding both the first and second chimeric antigen receptors (CARs) as defined in any of paragraphs 1 to 16. 
     18. A nucleic acid sequence according to paragraph 17, which has the following structure: 
       AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 
     in which 
     AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR; 
     spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR; 
     TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; 
     endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; 
     coexpr is a nucleic acid sequence enabling co-expression of both CARs 
     AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; 
     spacer 2 is a nucleic acid sequence encoding the spacer of the second CAR; 
     TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; 
     endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR; 
     which nucleic acid sequence, when expressed in a T cell, encodes a polypeptide which is cleaved at the cleavage site such that the first and second CARs are co-expressed at the T cell surface. 
     19. A nucleic acid sequence according to paragraph 18, wherein coexpr encodes a sequence comprising a self-cleaving peptide. 
     20. A nucleic acid sequence according to paragraph 18 or 19, wherein alternative codons are used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination. 
     21. A kit which comprises
         (i) a first nucleic acid sequence encoding the first chimeric antigen receptor (CAR) as defined in any of paragraphs 1 to 16, which nucleic acid sequence has the following structure:       

       AgB1-spacer1-TM1-endo1 
     in which 
     AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR; 
     spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR; 
     TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; 
     endo 1 is a nucleic acid sequence encoding the endodomain of the first CAR; and
         (ii) a second nucleic acid sequence encoding the second chimeric antigen receptor (CAR) as defined in any of paragraphs 1 to 16, which nucleic acid sequence has the following structure:       

       AgB2-spacer2-TM2-endo2 
     AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR; spacer 2 is a nucleic acid sequence encoding the spacer of the second CAR; 
     TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; 
     endo 2 is a nucleic acid sequence encoding the endodomain of the second CAR. 
     22. A kit comprising: a first vector which comprises the first nucleic acid sequence as defined in paragraph 21; and a second vector which comprises the first nucleic acid sequence as defined in paragraph 21. 
     23. A kit according to paragraph 22, wherein the vectors are integrating viral vectors or transposons. 
     24. A vector comprising a nucleic acid sequence according to any of paragraphs 17 to 20. 
     25. A retroviral vector or a lentiviral vector or a transposon according to paragraph 24. 
     26. A method for making a T cell according to any of paragraphs 1 to 16, which comprises the step of introducing: a nucleic acid sequence according to any of paragraphs 17 to 20; a first nucleic acid sequence and a second nucleic acid sequence as defined in paragraph 21; and/or a first vector and a second vector as defined in paragraph 22 or a vector according to paragraph 24 or 25, into a T cell. 
     27. A method according to paragraph 24, wherein the T cell is from a sample isolated from a subject. 
     28. A pharmaceutical composition comprising a plurality of T cells according to any of paragraphs 1 to 16. 
     29. A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to paragraph 28 to a subject. 
     30. A method according to paragraph 29, which comprises the following steps:
         (i) isolation of a T cell-containing sample from a subject;   (ii) transduction or transfection of the T cells with: a nucleic acid sequence according to any of paragraphs 17 to 20; a first nucleic acid sequence and a second nucleic acid sequence as defined in paragraph 21; a first vector and a second vector as defined in paragraph 22 or 23 or a vector according to paragraph 24 or 25; and   (iii) administering the T cells from (ii) to a the subject.       

     31. A method according to paragraph 29 or 30, wherein the disease is a cancer. 
     32. A pharmaceutical composition according to paragraph 28 for use in treating and/or preventing a disease. 
     33. The use of a T cell according to any of paragraphs 1 to 16 in the manufacture of a medicament for treating and/or preventing a disease. 
     DETAILED DESCRIPTION 
     Chimeric Antigen Receptors (CARs) 
     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 is usually 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 CD8a 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. 
     Early CAR designs had endodomains derived from the intracellular parts of either the γ chain of the FcεR1 or CD3ζ. 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 CD3ζ 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 41 BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals. 
     CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. Lentiviral vectors may be employed. In this way, a large number of cancer-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 tumour cells expressing the targeted antigen. 
     The first aspect of the invention relates to a T-cell which co-expresses a first CAR and a second CAR such that a T-cell can recognize a desired pattern of expression on target cells in the manner of a logic gate as detailed in the truth tables: table 1, 2 and 3. 
     Both the first and second (and optionally subsequent) CARs comprise: 
     (i) an antigen-binding domain; 
     (ii) a spacer; 
     (iii) a transmembrane domain; and 
     (iii) an intracellular domain. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Truth Table for CAR OR GATE 
               
            
           
           
               
               
               
               
            
               
                   
                 Antigen A 
                 Antigen B 
                 Response 
               
               
                   
                   
               
               
                   
                 Absent 
                 Absent 
                 No activation 
               
               
                   
                 Absent 
                 Present 
                 Activation 
               
               
                   
                 Present 
                 Absent 
                 Activation 
               
               
                   
                 Present 
                 Present 
                 Activation 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Truth Table for CAR AND GATE 
               
            
           
           
               
               
               
               
            
               
                   
                 Antigen A 
                 Antigen B 
                 Response 
               
               
                   
                   
               
               
                   
                 Absent 
                 Absent 
                 No activation 
               
               
                   
                 Absent 
                 Present 
                 No Activation 
               
               
                   
                 Present 
                 Absent 
                 No Activation 
               
               
                   
                 Present 
                 Present 
                 Activation 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Truth Table for CAR AND NOT GATE 
               
            
           
           
               
               
               
               
            
               
                   
                 Antigen A 
                 Antigen B 
                 Response 
               
               
                   
                   
               
               
                   
                 Absent 
                 Absent 
                 No activation 
               
               
                   
                 Absent 
                 Present 
                 No Activation 
               
               
                   
                 Present 
                 Absent 
                 Activation 
               
               
                   
                 Present 
                 Present 
                 No Activation 
               
               
                   
                   
               
            
           
         
       
     
     The first and second CAR of the T cell of the present invention may be produced as a polypeptide comprising both CARs, together with a cleavage site. 
     SEQ ID No. 1 to 5 give examples of such polypeptides, which each comprise two CARs. The CAR may therefore comprise one or other part of the following amino acid sequences, which corresponds to a single CAR. 
     SEQ ID No 1 is a CAR OR gate which recognizes CD19 OR CD33 
     SEQ ID No 2 Is a CAR AND gate which recognizes CD19 AND CD33 using a CD148 phosphatase 
     SEQ ID No 3 Is an alternative implementation of the CAR AND GATE which recognizes CD19 AND CD33 which uses a CD45 phosphatase 
     SEQ ID No 4 Is a CAR AND NOT GATE which recognizes CD19 AND NOT CD33 based on PTPN6 phosphatase 
     SEQ ID No 5 Is an alternative implementation of the CAR AND NOT gate which recognizes CD19 AND NOT CD33 and is based on an ITIM containing endodomain from LAIR1 
     SEQ ID No 6. Is a further alternative implementation of the CAR AND NOT gate which recognizes CD19 AND NOT CD33 and recruits a PTPN6-CD148 fusion protein to an ITIM containing endodomain. 
     
       
         
           
               
            
               
                 SEQ ID No. 1 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS 
               
               
                   
               
               
                 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL 
               
               
                   
               
               
                 TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR 
               
               
                   
               
               
                 QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSL 
               
               
                   
               
               
                 QTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPT 
               
               
                   
               
               
                 PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVL 
               
               
                   
               
               
                 ACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRRE 
               
               
                   
               
               
                 EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK 
               
               
                   
               
               
                 GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCG 
               
               
                   
               
               
                 DVEENPGPMAVPIQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRV 
               
               
                   
               
               
                 TITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSG 
               
               
                   
               
               
                 SGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGG 
               
               
                   
               
               
                 SGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTL 
               
               
                   
               
               
                 SNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAK 
               
               
                   
               
               
                 STLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDP 
               
               
                   
               
               
                 AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCV 
               
               
                   
               
               
                 VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL 
               
               
                   
               
               
                 HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD 
               
               
                   
               
               
                 ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
               
               
                 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDP 
               
               
                   
               
               
                 KFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQ 
               
               
                   
               
               
                 NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK 
               
               
                   
               
               
                 DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP 
               
               
                   
               
               
                 R 
               
               
                   
               
               
                 SEQ ID No. 2 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS 
               
               
                   
               
               
                 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL 
               
               
                   
               
               
                 TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR 
               
               
                   
               
               
                 QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSL 
               
               
                   
               
               
                 QTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPT 
               
               
                   
               
               
                 PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVL 
               
               
                   
               
               
                 ACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRRE 
               
               
                   
               
               
                 EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK 
               
               
                   
               
               
                 GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCG 
               
               
                   
               
               
                 DVEENPGPMAVPIQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRV 
               
               
                   
               
               
                 TITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSG 
               
               
                   
               
               
                 SGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGG 
               
               
                   
               
               
                 SGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTL 
               
               
                   
               
               
                 SNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAK 
               
               
                   
               
               
                 STLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDP 
               
               
                   
               
               
                 AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCV 
               
               
                   
               
               
                 VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL 
               
               
                   
               
               
                 HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD 
               
               
                   
               
               
                 ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
               
               
                 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDP 
               
               
                   
               
               
                 KAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLI 
               
               
                   
               
               
                 RVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGK 
               
               
                   
               
               
                 NRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGP 
               
               
                   
               
               
                 LPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYG 
               
               
                   
               
               
                 DITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVP 
               
               
                   
               
               
                 DTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLI 
               
               
                   
               
               
                 YQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQK 
               
               
                   
               
               
                 DSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA 
               
               
                   
               
               
                 SEQ ID No. 3 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS 
               
               
                   
               
               
                 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL 
               
               
                   
               
               
                 TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR 
               
               
                   
               
               
                 QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSL 
               
               
                   
               
               
                 QTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPT 
               
               
                   
               
               
                 PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVL 
               
               
                   
               
               
                 ACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRRE 
               
               
                   
               
               
                 EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK 
               
               
                   
               
               
                 GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCG 
               
               
                   
               
               
                 DVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRV 
               
               
                   
               
               
                 TITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSG 
               
               
                   
               
               
                 SGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGG 
               
               
                   
               
               
                 SGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTL 
               
               
                   
               
               
                 SNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAK 
               
               
                   
               
               
                 STLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDP 
               
               
                   
               
               
                 AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCV 
               
               
                   
               
               
                 VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL 
               
               
                   
               
               
                 HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD 
               
               
                   
               
               
                 ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 
               
               
                   
               
               
                 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDP 
               
               
                   
               
               
                 KALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDD 
               
               
                   
               
               
                 EKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIK 
               
               
                   
               
               
                 EARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFK 
               
               
                   
               
               
                 EPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAE 
               
               
                   
               
               
                 YWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREV 
               
               
                   
               
               
                 THIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVG 
               
               
                   
               
               
                 RTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILI 
               
               
                   
               
               
                 HQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLP 
               
               
                   
               
               
                 SYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEH 
               
               
                   
               
               
                 DSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDF 
               
               
                   
               
               
                 WQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDT 
               
               
                   
               
               
                 DKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISM 
               
               
                   
               
               
                 IQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLE 
               
               
                   
               
               
                 SAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNG 
               
               
                   
               
               
                 QVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGS 
               
               
                   
               
               
                 EPTSGTEGPEHSVNGPASPALNQGS 
               
               
                   
               
               
                 SEQ ID No. 4 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS 
               
               
                   
               
               
                 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLT 
               
               
                   
               
               
                 ISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGG 
               
               
                   
               
               
                 GSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPP 
               
               
                   
               
               
                 RKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDD 
               
               
                   
               
               
                 TAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTI 
               
               
                   
               
               
                 ASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLL 
               
               
                   
               
               
                 VTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 
               
               
                   
               
               
                 RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG 
               
               
                   
               
               
                 HDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPM 
               
               
                   
               
               
                 AVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDI 
               
               
                   
               
               
                 YFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISS 
               
               
                   
               
               
                 LQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSG 
               
               
                   
               
               
                 GGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPG 
               
               
                   
               
               
                 KGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAED 
               
               
                   
               
               
                 TAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVH 
               
               
                   
               
               
                 PTGTSQPQRPEDCRPRGSVKGTGLDFACDIYWAPLAGICVALLLSLII 
               
               
                   
               
               
                 TLICYHRSRKRVCKSGGGSFWEEFESLQKQEVKNLHQRLEGQRPENKG 
               
               
                   
               
               
                 KNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKT 
               
               
                   
               
               
                 YIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEV 
               
               
                   
               
               
                 GMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLS 
               
               
                   
               
               
                 WPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTII 
               
               
                   
               
               
                 VIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAI 
               
               
                   
               
               
                 AQFIETTKKKL 
               
               
                   
               
               
                 SEQ ID No. 5 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS 
               
               
                   
               
               
                 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL 
               
               
                   
               
               
                 TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR 
               
               
                   
               
               
                 QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSL 
               
               
                   
               
               
                 QTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPT 
               
               
                   
               
               
                 PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVL 
               
               
                   
               
               
                 ACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRRE 
               
               
                   
               
               
                 EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK 
               
               
                   
               
               
                 GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCG 
               
               
                   
               
               
                 DVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRV 
               
               
                   
               
               
                 TITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSG 
               
               
                   
               
               
                 SGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGG 
               
               
                   
               
               
                 SGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTL 
               
               
                   
               
               
                 SNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAK 
               
               
                   
               
               
                 STLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDP 
               
               
                   
               
               
                 ATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDILI 
               
               
                   
               
               
                 GVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVD 
               
               
                   
               
               
                 VLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQR 
               
               
                   
               
               
                 TARAVSPQSTKPMAESITYAAVARH 
               
               
                   
               
               
                 SEQ ID No. 6 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRAS 
               
               
                   
               
               
                 QDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSL 
               
               
                   
               
               
                 TISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR 
               
               
                   
               
               
                 QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSL 
               
               
                   
               
               
                 QTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPT 
               
               
                   
               
               
                 PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVL 
               
               
                   
               
               
                 ACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRRE 
               
               
                   
               
               
                 EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK 
               
               
                   
               
               
                 GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCG 
               
               
                   
               
               
                 DVEENPGPMAVPIQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRV 
               
               
                   
               
               
                 TITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSG 
               
               
                   
               
               
                 SGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGG 
               
               
                   
               
               
                 SGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTL 
               
               
                   
               
               
                 SNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAK 
               
               
                   
               
               
                 STLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDP 
               
               
                   
               
               
                 ATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDILI 
               
               
                   
               
               
                 GVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVD 
               
               
                   
               
               
                 VLERTADKATVNGLPEKDRETDTSALAAGSSQEVIYAQLDHWALTQR 
               
               
                   
               
               
                 TARAVSPQSTKPMAESITYAAVARHRAEGRGSLLTCGDVEENPGPWY 
               
               
                   
               
               
                 HGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGP 
               
               
                   
               
               
                 GSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGA 
               
               
                   
               
               
                 FVYLRQPYSGGGGSFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKY 
               
               
                   
               
               
                 AAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHS 
               
               
                   
               
               
                 KKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEY 
               
               
                   
               
               
                 WPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFH 
               
               
                   
               
               
                 FTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRT 
               
               
                   
               
               
                 GTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQ 
               
               
                   
               
               
                 CVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIASGS 
               
            
           
         
       
     
     The CAR may comprise a variant of the CAR-encoding part of the sequence shown as SEQ ID No. 1, 2, 3, 4, 5 or 6 having at least 80, 85, 90, 95, 98 or 99% sequence identity, provided that the variant sequence is a CAR having the required properties. 
     Methods of sequence alignment are well known in the art and are accomplished using suitable alignment programs. The % sequence identity refers to the percentage of amino acid or nucleotide residues that are identical in the two sequences when they are optimally aligned. Nucleotide and protein sequence homology or identity may be determined using standard algorithms such as a BLAST program (Basic Local Alignment Search Tool at the National Center for Biotechnology Information) using default parameters, which is publicly available at http://blast.ncbi.nlm.nih.gov. Other algorithms for determining sequence identity or homology include: LALIGN (http://www.ebi.ac.uk/Tools/psa/Ialign/ and http://www.ebi.ac.uk/Tools/psa/Ialign/nucleotide.html), AMAS (Analysis of Multiply Aligned Sequences, at http://www.compbio.dundee.ac.uk/Softwar/Amas/amas.html), FASTA (http://ebi.ac.uk/Tools/sss/fasta/) Clustal Omega (http://www.ebi.ac.uk.Tools/msa/clustalo/), SIM (http://web.expasy.org/sim/), and EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.ht,l). 
     Car Logical or Gate 
     In this embodiment, the antigen binding domains of the first and second CARs of the present invention bind to different antigens and both CARs comprise an activating endodomain. The spacer domains may be the same, or sufficiently different to prevent cross-pairing of the two different receptors. A T cell can hence be engineered to activate upon recognition of either or both antigens. This is useful in the field of oncology as indicated by the Goldie-Coldman hypothesis: sole targeting of a single antigen may result in tumour escape by modulation of said antigen due to the high mutation rate inherent in most cancers. By simultaneously targeting two antigens, the probably of such escape is exponentially reduced. 
     Various tumour associated antigens are known as shown in the following Table 4. For a given disease, the first CAR and second CAR may bind to two different TAAs associated with that disease. In this way, tumour escape by modulating a single antigen is prevented, since a second antigen is also targeted. For example, when targeting a B-cell malignancy, both CD19 and CD20 can be simultaneously targeted. In this embodiment, it is important that the two CARs do not heterodimerize. 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Cancer type 
                 TAA 
               
               
                   
               
             
            
               
                 Diffuse Large B-cell Lymphoma 
                 CD19, CD20 
               
               
                 Breast cancer 
                 ErbB2, MUC1 
               
               
                 AML 
                 CD13, CD33 
               
               
                 Neuroblastoma 
                 GD2, NCAM 
               
               
                 B-CLL 
                 CD19, CD52 
               
               
                 Colorectal cancer 
                 Folate binding protein, CA-125 
               
               
                   
               
            
           
         
       
     
     Kinetic Segregation Model 
     Subsequent pairing of CARs to generate the AND gate and the AND NOT gate are based on the kinetic segregation model (KS) of T-cell activation. This is a functional model, backed by experimental data, which explains how antigen recognition by a T-cell receptor is converted into down-stream activation signals. Briefly: at the ground state, the signalling components on the T-cell membrane are in dynamic homeostasis whereby dephosphorylated ITAMs are favoured over phosphorylated ITAMs. This is due to greater activity of the transmembrane CD45/CD148 phosphatases over membrane-tethered kinases such as Ick. When a T-cell engages a target cell through a T-cell receptor (or CAR) recognition of cognate antigen, tight immunological synapses form. This close juxtapositioning of the T-cell and target membranes excludes CD45/CD148 due to their large ectodomains which cannot fit into the synapse. Segregation of a high concentration of T-cell receptor associated ITAMs and kinases in the synapse, in the absence of phosphatases, leads to a state whereby phosphorylated ITAMs are favoured. ZAP70 recognizes a threshold of phosphorylated ITAMs and propagates a T-cell activation signal. This advanced understanding of T-cell activation is exploited by the present invention. In particular, the invention is based on this understanding of how ectodomains of different length and/or bulk and/or charge and/or configuration and/or glycosylation result in differential segregation upon synapse formation. 
     The Car Logical and Gate 
     In this embodiment, one CAR comprises an activating endodomain and one CAR comprises an inhibitory endodomain whereby the inhibitory CAR constitutively inhibits the first activating CAR, but upon recognition of its cognate antigen releases its inhibition of the activating CAR. In this manner, a T-cell can be engineered to trigger only if a target cell expresses both cognate antigens. This behaviour is achieved by the activating CAR comprising an activating endodomain containing ITAM domains for example the endodomain of CD3 Zeta, and the inhibitory CAR comprising the endodomain from a phosphatase able to dephosphorylate an ITAM (e.g. CD45 or CD148). Crucially, the spacer domains of both CARs are significantly different in size and/or shape and/or charge etc. When only the activating CAR is ligated, the inhibitory CAR is in solution on the T-cell surface and can diffuse in and out of the synapse inhibiting the activating CAR. When both CARs are ligated, due to differences in spacer properties, the activating and inhibiting CAR are differentially segregated allowing the activating CAR to trigger T-cell activation unhindered by the inhibiting CAR. 
     This is of considerable utility in the field of cancer therapy. Currently, immunotherapies typically target a single antigen. Most cancers cannot be differentiated from normal tissues on the basis of a single antigen. Hence, considerable “on-target off-tumour” toxicity occurs whereby normal tissues are damaged by the therapy. For instance, whilst targeting CD20 to treat B-cell lymphomas with Rituximab, the entire normal B-cell compartment is depleted. For instance, whilst targeting CD52 to treat chronic lymphocytic leukaemia, the entire lymphoid compartment is depleted. For instance, whilst targeting CD33 to treat acute myeloid leukaemia, the entire myeloid compartment is damaged etc. By restricting activity to a pair of antigens, much more refined targeting, and hence less toxic therapy can be developed. A practical example is targeting of CLL which expresses both CD5 and CD19. Only a small proportion of normal B-cells express both antigens, so the off-target toxicity of targeting both antigens with a logical AND gate is substantially less than targeting each antigen individually. 
     The design of the present invention is a considerable improvement on previous implementation as described by Wilkie et al. ((2012).  J. Clin. Immunol.  32, 1059-1070) and then tested in vivo (Kloss et al (2013)  Nat. Biotechnol.  31, 71-75). In this implementation, the first CAR comprises of an activating endodomain, and the second a co-stimulatory domain. This way, a T-cell only receives an activating and co-stimulatory signal when both antigens are present. However, the T-cell still will activate in the sole presence of the first antigen resulting in the potential for off-target toxicity. Further, the implementation of the present invention allows for multiple compound linked gates whereby a cell can interpret a complex pattern of antigens. 
     
       
         
           
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Cancer Type 
                 Antigens 
               
               
                   
               
             
            
               
                 Chronic Lymphocytic Leukaemia 
                 CD5, CD19 
               
               
                 Neuroblastoma 
                 ALK, GD2 
               
               
                 Glioma 
                 EGFR, Vimentin 
               
               
                 Multiple myeloma 
                 BCMA, CD138 
               
               
                 Renal Cell Carcinoma 
                 Carbonic anhydrase IX, G250 
               
               
                 T-ALL 
                 CD2, N-Cadherin 
               
               
                 Prostate Cancer 
                 PSMA, hepsin (or others) 
               
               
                   
               
            
           
         
       
     
     The Car Logical and not Gate 
     In this embodiment, one CAR comprises an activating endodomain and one CAR comprises an inhibitory endodomain such that this inhibitory CAR is only active when it recognizes its cognate antigen. Hence a T-cell engineered in this manner is activated in response to the sole presence of the first antigen but is not activated when both antigens are present. This invention is implemented by inhibitory CARs with a spacer that co-localise with the first CAR but either the phosphatase activity of the inhibitory CAR should not be so potent that it inhibits in solution, or the inhibitory endodomain in fact recruits a phosphatase solely when the inhibitory CAR recognizes its cognate target. Such endodomains are termed “ligation-on” or semi-inhibitory herein. 
     This invention is of use in refining targeting when a tumour can be distinguished from normal tissue by the presence of tumour associated antigen and the loss of an antigen expressed on normal tissue. The AND NOT gate is of considerable utility in the field of oncology as it allows targeting of an antigen which is expressed by a normal cell, which normal cell also expresses the antigen recognised by the CAR comprising the activating endodomain. An example of such an antigen is CD33 which is expressed by normal stem cells and acute myeloid leukaemia (AML) cells. CD34 is expressed on stem cells but not typically expressed on AML cells. A T-cell recognizing CD33 AND NOT CD34 would result in destruction of leukaemia cells but sparing of normal stem cells. 
     Potential antigen pairs for use with AND NOT gates are shown in Table 6. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                   
                 Antigen expressed 
               
               
                   
                   
                 Normal cell which 
                 by normal cell but 
               
               
                 Disease 
                 TAA 
                 expresses TAA 
                 not cancer cell 
               
               
                   
               
             
            
               
                 AML 
                 CD33 
                 stem cells 
                 CD34 
               
               
                 Myeloma 
                 BCMA 
                 Dendritic cells 
                 CD1c 
               
               
                 B-CLL 
                 CD160 
                 Natural Killer cells 
                 CD56 
               
               
                 Prostate cancer 
                 PSMA 
                 Neural Tissue 
                 NCAM 
               
               
                 Bowel cancer 
                 A33 
                 Normal bowel 
                 HLA class I 
               
               
                   
                   
                 epithelium 
               
               
                   
               
            
           
         
       
     
     Compound Gates 
     The kinetic segregation model with the above components allows compound gates to be made e.g. a T-cell which triggers in response to patterns of more than two target antigens. For example, it is possible to make a T cell which only triggers when three antigens are present (A AND B AND C). Here, a cell expresses three CARs, each recognizing antigens A, B and C. One CAR is excitatory and two are inhibitory, which each CAR having spacer domains which result in differential segregation. Only when all three are ligated, will the T-cell activate. A further example: (A OR B) AND C: here, CARs recognizing antigens A and B are activating and have spacers which co-localise, while CAR recognizing antigen C is inhibitory and has a spacer which results in different co-segregation. A further example (A AND NOT B) AND C: Here CAR against antigen A has an activating endodomain and co-localises with CAR against antigen B which has a conditionally inhibiting endodomain. CAR against antigen C has a spacer who segregates differently from A or B and is inhibitory. In fact, ever more complex boolean logic can be programmed with these simple components of the invention with any number of CARs and spacers. 
     Signal Peptide 
     The CARs of the T cell of the present invention may comprise a signal peptide so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. 
     The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. 
     The signal peptide may be at the amino terminus of the molecule. 
     The signal peptide may comprise the SEQ ID No. 7, 8 or 9 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR. 
     
       
         
           
               
               
            
               
                   
                 SEQ ID No. 7: 
               
               
                   
                 MGTSLLCWMALCLLGADHADG  
               
            
           
         
       
     
     The signal peptide of SEQ ID No. 7 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase. 
     
       
         
           
               
               
            
               
                   
                 SEQ ID No. 8: 
               
               
                   
                 MSLPVTALLLPLALLLHAARP  
               
            
           
         
       
     
     The signal peptide of SEQ ID No. 8 is derived from IgG1. 
     
       
         
           
               
               
            
               
                   
                 SEQ ID No. 9: 
               
               
                   
                 MAVPTQVLGLLLLWLTDARC  
               
            
           
         
       
     
     The signal peptide of SEQ ID No. 9 is derived from CD8. 
     The signal peptide for the first CAR may have a different sequence from the signal peptide of the second CAR (and from the 3 rd  CAR and 4 th  CAR etc). 
     Antigen Binding Domain 
     The antigen binding domain is the portion of the CAR which recognizes antigen. 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 antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-chain derived from a T-cell receptor. 
     The antigen binding domain may comprise a domain which is not based on the antigen binding site of an antibody. For example the antigen binding domain may comprise a domain based on a protein/peptide which is a soluble ligand for a tumour cell surface receptor (e.g. a soluble peptide such as a cytokine or a chemokine); or an extracellular domain of a membrane anchored ligand or a receptor for which the binding pair counterpart is expressed on the tumour cell. 
     Examples 11 to 13 relate to a CAR which binds BCMA, in which the antigen binding doaimn comprises APRIL, a ligand for BCMA. 
     The antigen binding domain may be based on a natural ligand of the antigen. 
     The antigen binding domain may comprise an affinity peptide from a combinatorial library or a de novo designed affinity protein/peptide. 
     Spacer Domain 
     CARs comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding. 
     In the T cell of the present invention, the first and second CARs comprise different spacer molecules. For example, 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. 
     Examples of amino acid sequences for these spacers are given below: 
     
       
         
           
               
            
               
                 (hinge-CH2CH3 of human IgG1) 
               
               
                 SEQ ID No. 10 
               
               
                 AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVD 
               
               
                   
               
               
                 VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 
               
               
                   
               
               
                 GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL 
               
               
                   
               
               
                 TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS 
               
               
                   
               
               
                 RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD 
               
               
                   
               
               
                 (human CD8 stalk): 
               
               
                 SEQ ID No. 11 
               
               
                 ITTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHIRGLDFACDI 
               
               
                   
               
               
                 (human IgG1 hinge): 
               
               
                 SEQ ID No. 12 
               
               
                 AEPKSPDKTHTCPPCPKDPK 
               
               
                   
               
               
                 (CD2 ectodomain) 
               
               
                 SEQ ID No. 13 
               
               
                 KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKE 
               
               
                   
               
               
                 KETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDL 
               
               
                   
               
               
                 KIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITH 
               
               
                   
               
               
                 KWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD 
               
               
                   
               
               
                 (CD34 ectodomain) 
               
               
                 SEQ ID No. 14 
               
               
                 SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNE 
               
               
                   
               
               
                 ATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPE 
               
               
                   
               
               
                 TTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIR 
               
               
                   
               
               
                 EVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSL 
               
               
                   
               
               
                 LLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA 
               
               
                   
               
               
                 SHQSYSQKT 
               
            
           
         
       
     
     Since CARs are typically homodimers (see  FIG. 1 a   ), cross-pairing may result in a heterodimeric chimeric antigen receptor. This is undesirable for various reasons, for example: (1) the epitope may not be at the same “level” on the target cell so that a cross-paired CAR may only be able to bind to one antigen; (2) the VH and VL from the two different scFv could swap over and either fail to recognize target or worse recognize an unexpected and unpredicted antigen. For the “OR” gate and the “AND NOT” gate, the spacer of the first CAR may be sufficiently different from the spacer of the second CAR in order to avoid cross-pairing. The amino acid sequence of the first spacer may share less that 50%, 40%, 30% or 20% identity at the amino acid level with the second spacer. 
     In other aspects of the invention (for example the AND gate) it is important that the spacer of the first CAR has a different length, and/or charge and/or shape and/or configuration and/or glycosylation, such that when both first and second CARs bind their target antigen, the difference in spacer charge or dimensions results in spatial separation of the two types of CAR to different parts of the membrane to result in activation as predicted by the kinetic separation model. In these aspects, the different length, shape and/or configuration of the spacers is carefully chosen bearing in mind the size and binding epitope on the target antigen to allow differential segregation upon cognate target recognition. For example the IgG1 Hinge, CD8 stalk, IgG1 Fc, ectodomain of CD34, ectodomain of CD45 are expected to differentially segregate. 
     Examples of spacer pairs which differentially segregate and are therefore suitable for use with the AND gate are shown in the following Table: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Stimulatory CAR spacer 
                 Inhibitory CAR spacer 
               
               
                   
                   
               
             
            
               
                   
                 Human-CD8STK 
                 Human-IgG-Hinge-CH2CH3 
               
               
                   
                 Human-CD3z ectodomain 
                 Human-IgG-Hinge-CH2CH3 
               
               
                   
                 Human-IgG-Hinge 
                 Human-IgG-Hinge-CH2CH3 
               
               
                   
                 Human-CD28STK 
                 Human-IgG-Hinge-CH2CH3 
               
               
                   
                 Human-CD8STK 
                 Human-IgM-Hinge-CH2CH3CD4 
               
               
                   
                 Human-CD3z ectodomain 
                 Human-IgM-Hinge-CH2CH3CD4 
               
               
                   
                 Human-IgG-Hinge 
                 Human-IgM-Hinge-CH2CH3CD4 
               
               
                   
                 Human-CD28STK 
                 Human-IgM-Hinge-CH2CH3CD4 
               
               
                   
                   
               
            
           
         
       
     
     In other aspects of the invention (for example the AND NOT gate), it is important that the spacer be sufficiently different as to prevent cross-pairing, but to be sufficiently similar to co-localise. Pairs of orthologous spacer sequences may be employed. Examples are murine and human CD8 stalks, or alternatively spacer domains which are monomeric—for instance the ectodomain of CD2. 
     Examples of spacer pairs which co-localise and are therefore suitable for use with the AND NOT gate are shown in the following Table: 
     
       
         
           
               
               
             
               
                   
               
               
                 Stimulatory CAR spacer 
                 Inhibitory CAR spacer 
               
               
                   
               
             
            
               
                 Human-CD8aSTK 
                 Mouse CD8aSTK 
               
               
                 Human-CD28STK 
                 Mouse CD8aSTK 
               
               
                 Human-IgG-Hinge 
                 Human-CD3z ectodomain 
               
               
                 Human-CD8aSTK 
                 Mouse CD28STK 
               
               
                 Human-CD28STK 
                 Mouse CD28STK 
               
               
                 Human-IgG-Hinge-CH2CH3 
                 Human-IgM-Hinge-CH2CH3CD4 
               
               
                   
               
            
           
         
       
     
     All the spacer domains mentioned above form homodimers. However the mechanism is not limited to using homodimeric receptors and should work with monomeric receptors as long as the spacer is sufficiently rigid. An example of such a spacer is CD2 or truncated CD22. 
     Transmembrane Domain 
     The transmembrane domain is the sequence of the CAR that spans the membrane. 
     A transmembrane domain may be any protein structure which is thermodynamically stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the invention. The presence and span of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that the transmembrane domain of a protein is a relatively simple structure, i.e. a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain may also be used (U.S. Pat. No. 7,052,906 B1 describes synthetic transmembrane components). 
     The transmembrane domain may be derived from CD28, which gives good receptor stability. 
     Activating Endodomain 
     The endodomain is the signal-transmission portion of the CAR. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling 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. 
     In the “OR gate”, the cell of the present invention comprises two CARs, each with an activating endodomain. The activating endodomain is capable of transmitting both immunological signal 1 and immunological signal 2. An endodomain is not considered “activating” if it just comprises CD3ζ, which is sufficient to trigger T-cell killing of cognate target cells does not fully activate the T-cell to proliferate and survive. An “activating endodomain” is a compound endodomains: in which the intracellular part of a T-cell co-stimulatory molecule is fused to that of CD3ζ resulting in a “second generation” receptor 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. In addition to a co-stimulatory domain, the activating endodomain may also include TNF receptor family endodomains, such as the closely related OX40 and 41 BB which transmit survival signals. 
     An “activating” endodomain.may therefore not comprise the CD3-Zeta endodomain or the CD28 endodomain alone. An activating endodomain may, for example, comprise the CD3-Zeta endodomain with that of either CD28 or OX40 or the CD28 endodomain and OX40 and CD3-Zeta endodomain. 
     Each of the CARs in the OR gate is independently capable of activating the T cell. The T cell is thus activated by the presence of either antigen alone. The two CARs are not “complementary” in the sense that activation of both CARs is necessary to provide activation and co-stimulatory signals. 
     A endodomain which contains an ITAM motif can act as an activation endodomain in this invention. Several proteins are known to contain endodomains with one or more ITAM motifs. Examples of such proteins include the CD3 epsilon chain, the CD3 gamma chain and the CD3 delta chain to name a few. The ITAM motif can be easily recognized as a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/I. Typically, but not always, two of these motifs are separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/Ix(6-8)YxxL/I). Hence, one skilled in the art can readily find existing proteins which contain one or more ITAM to transmit an activation signal. Further, given the motif is simple and a complex secondary structure is not required, one skilled in the art can design polypeptides containing artificial ITAMs to transmit an activation signal (see WO 2000063372, which relates to synthetic signalling molecules). 
     The transmembrane and intracellular T-cell signalling domain (endodomain) of a CAR with an activating endodomain may comprise the sequence shown as SEQ ID No. 16 or 17 or a variant thereof having at least 80% sequence identity. 
     
       
         
           
               
            
               
                 comprising CD28 transmembrane domain and CD3 
               
               
                 Z endodomain  
               
               
                 SEQ ID No. 15 
               
               
                 FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYN 
               
               
                   
               
               
                 ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS 
               
               
                   
               
               
                 EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 
               
               
                   
               
               
                 comprising CD28 transmembrane domain and CD28 
               
               
                 and CD3 Zeta endodomains  
               
               
                 SEQ ID No. 16 
               
               
                 FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT 
               
               
                   
               
               
                 RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY 
               
               
                   
               
               
                 DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR 
               
               
                   
               
               
                 GKGHDGLYQGLSTATKDTYDALHMQALPPR 
               
               
                   
               
               
                 comprising CD28 transmembrane domain and CD28, 
               
               
                 OX40 and CD3 Zeta endodomains.  
               
               
                 SEQ ID No. 17 
               
               
                 FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT 
               
               
                   
               
               
                 RKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHST 
               
               
                   
               
               
                 LAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG 
               
               
                   
               
               
                 GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA 
               
               
                   
               
               
                 TKDTYDALHMQALPPR 
               
            
           
         
       
     
     A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 16 or 17, provided that the sequence provides an effective trans-membrane domain and an effective intracellular T cell signaling domain. 
     “Ligation-Off” Inhibitory Endodomain 
     In the embodiment referred above as the AND gate, one of the CARs comprises an inhibitory endodomain such that the inhibitory CAR inhibits T-cell activation by the activating CAR in the absence of inhibitory CAR ligation, but does not significantly inhibit T-cell activation by the activating CAR when the inhibitory CAR is ligated. This is termed a “ligation-off” inhibitory endodomain. 
     In this case, the spacer of the inhibitory CAR is of a different length, charge, shape and/or configuration and/or glycosylation from the spacer of the activating CAR, such that when both receptors are ligated, the difference in spacer dimensions results in isolation of the activating CARs and the inhibitory CARs in different membrane compartments of the immunological synapse, so that the activating endodomain is released from inhibition by the inhibitory endodomain. 
     The inhibitory endodomains for use in a ligation-off inhibitory CAR may therefore comprise any sequence which inhibits T-cell signaling by the activating CAR when it is in the same membrane compartment (i.e. in the absence of the antigen for the inhibitory CAR) but which does not significantly inhibit T cell signaling when it is isolated in a separate part of the membrane from the inhibitory CAR. 
     The ligation-off inhibitory endodomain may be or comprise a tyrosine phosphatase, such as a receptor-like tyrosine phosphatase. An inhibitory endodomain may be or comprise any tyrosine phosphatase that is capable of inhibiting the TCR signalling when only the stimulatory receptor is ligated. An inhibitory endodomain may be or comprise any tyrosine phosphatase with a sufficiently fast catalytic rate for phosphorylated ITAMs that is capable of inhibiting the TCR signalling when only the stimulatory receptor is ligated. 
     For example, the inhibitory endodomain of an AND gate may comprise the endodomain of CD148 or CD45. CD148 and CD45 have been shown to act naturally on the phosphorylated tyrosines up-stream of TCR signalling. 
     CD148 is a receptor-like protein tyrosine phosphatase which negatively regulates TCR signaling by interfering with the phosphorylation and function of PLCγ1 and LAT. 
     CD45 present on all hematopoetic cells, is a protein tyrosine phosphatase which is capable of regulating signal transduction and functional responses, again by phosphorylating PLCγ1. 
     An inhibitory endodomain may comprise all of part of a receptor-like tyrosine phosphatase. The phospatase may interfere with the phosphorylation and/or function of elements involved in T-cell signalling, such as PLCγ1 and/or LAT. 
     The transmembrane and endodomain of CD45 and CD148 is shown as SEQ ID No. 18 and No. 19 respectively. 
     
       
         
           
               
            
               
                 CD45 trans-membrane and endodomain sequence  
               
               
                 SEQ ID 18 
               
               
                 ALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDDEKQL 
               
               
                   
               
               
                 MNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFN 
               
               
                   
               
               
                 QNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQG 
               
               
                   
               
               
                 PRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFG 
               
               
                   
               
               
                 DVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPED 
               
               
                   
               
               
                 PHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAEN 
               
               
                   
               
               
                 KVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHP 
               
               
                   
               
               
                 YLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNV 
               
               
                   
               
               
                 IPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYW 
               
               
                   
               
               
                 KPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWG 
               
               
                   
               
               
                 EGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSV 
               
               
                   
               
               
                 EQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTG 
               
               
                   
               
               
                 IFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIAS 
               
               
                   
               
               
                 TYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQ 
               
               
                   
               
               
                 AEGSEPTSGTEGPEHSVNGPASPALNQGS 
               
               
                   
               
               
                 CD148 trans-membrane and endodomain sequence  
               
               
                 SEQ ID 19 
               
               
                 AVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVEN 
               
               
                   
               
               
                 FEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVL 
               
               
                   
               
               
                 PYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWR 
               
               
                   
               
               
                 MVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLP 
               
               
                   
               
               
                 EWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDY 
               
               
                   
               
               
                 MKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLR 
               
               
                   
               
               
                 MHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAP 
               
               
                   
               
               
                 VTTFGKTNGYIA 
               
            
           
         
       
     
     An inhibitory CAR may comprise all or part of SEQ ID No 18 or 19 (for example, it may comprise the phosphatase function of the endodomain). It may comprise a variant of the sequence or part thereof having at least 80% sequence identity, as long as the variant retains the capacity to basally inhibit T cell signalling by the activating CAR. 
     Other spacers and endodomains may be tested for example using the model system exemplified herein. Target cell populations can be created by transducing a suitable cell line such as a SupT1 cell line either singly or doubly to establish cells negative for both antigens (the wild-type), positive for either and positive for both (e.g. CD19−CD33−, CD19+CD33−, CD19−CD33+ and CD19+CD33+). T cells such as the mouse T cell line BW5147 which releases IL-2 upon activation may be transduced with pairs of CARs and their ability to function in a logic gate measured through measurement of IL-2 release (for example by ELISA). For example, it is shown in Example 4 that both CD148 and CD45 endodomains can function as inhibitory CARs in combination with an activating CAR containing a CD3 Zeta endodomain. These CARs rely upon a short/non-bulky CD8 stalk spacer on one CAR and a bulky Fc spacer on the other CAR to achieve AND gating. When both receptors are ligated, the difference in spacer dimensions results in isolation of the different receptors in different membrane compartments, releasing the CD3 Zeta receptor from inhibition by the CD148 or CD45 endodomains. In this way, activation only occurs once both receptors are activated. It can be readily seen that this modular system can be used to test alternative spacer pairs and inhibitory endodomains. If the spacers do not achieve isolation following ligation of both receptors, the inhibition would not be released and so no activation would occur. If the inhibitory endodomain under test is ineffective, activation would be expected in the presence of ligation of the activating CAR irrespective of the ligation status of the inhibitory CAR. 
     “Ligation-On” Endodomain 
     In the embodiment referred above as the AND NOT gate, one of the CARs comprises a “ligation-on” inhibitory endodomain such that the inhibitory CAR does not significantly inhibit T-cell activation by the activating CAR in the absence of inhibitory CAR ligation, but inhibits T-cell activation by the activating CAR when the inhibitory CAR is ligated. 
     The “ligation-on” inhibitory endodomain may be or comprise a tyrosine phosphatase that is incapable of inhibiting the TCR signalling when only the stimulatory receptor is ligated. 
     The “ligation-on” inhibitory endodomain may be or comprise a tyrosine phosphatase with a sufficiently slow catalytic rate for phosphorylated ITAMs that is incapable of inhibiting the TCR signalling when only the stimulatory receptor is ligated but it is capable of inhibiting the TCR signalling response when concentrated at the synapse. Concentration at the synapse is achieved through inhibitory receptor ligation. 
     If a tyrosine phosphatase has a catalytic rate which is too fast for a “ligation-on” inhibitory endodomain, then it is possible to tune-down the catalytic rates of phosphatase through modification such as point mutations and short linkers (which cause steric hindrance) to make it suitable for a “ligation-on” inhibitory endodomain. 
     In this first embodiment the endodomain may be or comprise a phosphatase which is considerably less active than CD45 or CD148, such that significant dephosphorylation of ITAMS only occurs when activating and inhibitory endodomains are co-localised. Many suitable sequences are known in the art. For example, the inhibitory endodomain of a NOT AND gate may comprise all or part of a protein-tyrosine phosphatase such as PTPN6. 
     Protein tyrosine phosphatases (PTPs) are signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. The N-terminal part of this PTP contains two tandem Src homolog (SH2) domains, which act as protein phospho-tyrosine binding domains, and mediate the interaction of this PTP with its substrates. This PTP is expressed primarily in hematopoietic cells, and functions as an important regulator of multiple signaling pathways in hematopoietic cells. 
     The inhibitor domain may comprise all of PTPN6 (SEQ ID No. 20) or just the phosphatase domain (SEQ ID No. 21). 
     
       
         
           
               
            
               
                 sequence of PTPN6  
               
               
                 SEQ ID 20 
               
               
                 MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVT 
               
               
                   
               
               
                 HIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPL 
               
               
                   
               
               
                 NCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVL 
               
               
                   
               
               
                 SDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIE 
               
               
                   
               
               
                 EASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFES 
               
               
                   
               
               
                 LQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSD 
               
               
                   
               
               
                 YINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMT 
               
               
                   
               
               
                 TREVEKGRNKCVPYWPEVGMQRAYGPYSVINCGEHDTTEYKLRTLQVSPL 
               
               
                   
               
               
                 DNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPII 
               
               
                   
               
               
                 VHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQ 
               
               
                   
               
               
                 TEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHA 
               
               
                   
               
               
                 KASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK 
               
               
                   
               
               
                 sequence of phosphatase domain of PTPN6  
               
               
                 SEQ ID 21 
               
               
                 FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRD 
               
               
                   
               
               
                 SNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQEN 
               
               
                   
               
               
                 SRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVINCGEHDTTEYKLR 
               
               
                   
               
               
                 TLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESL 
               
               
                   
               
               
                 PHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRA 
               
               
                   
               
               
                 QRSGMVQTEAQYKFIYVAIAQF 
               
            
           
         
       
     
     A second embodiment of a ligation-on inhibitory endodomain is an ITIM (Immunoreceptor Tyrosine-based Inhibition motif) containing endodomain such as that from CD22, LAIR-1, the Killer inhibitory receptor family (KIR), LILRB1, CTLA4, PD-1, BTLA etc. When phosphorylated, ITIMs recruits endogenous PTPN6 through its SH2 domain. If co-localised with an ITAM containing endodomain, dephosphorylation occurs and the activating CAR is inhibited. 
     An ITIM is a conserved sequence of amino acids (S/I/V/LxYxxI/V/L) that is found in the cytoplasmic tails of many inhibitory receptors of the immune system. One skilled in the art can easily find protein domains containing an ITIM. A list of human candidate ITIM-containing proteins has been generated by proteome-wide scans (Staub, et al (2004) Cell. Signal. 16, 435-456). Further, since the consensus sequence is well known and little secondary structure appears to be required, one skilled in the art could generate an artificial ITIM. 
     ITIM endodomains from PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 and KIR3DL3 are shown in SEQ ID 22 to 31 respectively 
     
       
         
           
               
            
               
                 PDCD1 endodomain 
               
               
                 SEQ ID 22 
               
               
                 CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPC 
               
               
                   
               
               
                 VPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL 
               
               
                   
               
               
                 BTLA4 
               
               
                 SEQ ID 23 
               
               
                 KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMM 
               
               
                   
               
               
                 EDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGD 
               
               
                   
               
               
                 YENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH 
               
               
                   
               
               
                 LILRB1 
               
               
                 SEQ ID 24 
               
               
                 LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENL 
               
               
                   
               
               
                 YAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLS 
               
               
                   
               
               
                 GEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPS 
               
               
                   
               
               
                 QEGPSPAVPSIYATLAIH 
               
               
                   
               
               
                 LAIR1 
               
               
                 SEQ ID 25 
               
               
                 HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRET 
               
               
                   
               
               
                 DTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVAR 
               
               
                   
               
               
                 H 
               
               
                   
               
               
                 CTLA4 
               
               
                 SEQ ID 26 
               
               
                 FLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPEC 
               
               
                   
               
               
                 EKQFQPYFIPIN 
               
               
                   
               
               
                 KIR2DL1 
               
               
                 SEQ ID 27 
               
               
                 GNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTV 
               
               
                   
               
               
                 NREDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPN 
               
               
                   
               
               
                 AESRSKVVSCP 
               
               
                   
               
               
                 KIR2DL4 
               
               
                 SEQ ID 28 
               
               
                 GIARHLHAVIRYSVAIILFTILPFFLLHRWCSKKKENAAVMNQEPAGHRT 
               
               
                   
               
               
                 VNREDSDEQDPQEVTYAQLDHCIFTQRKITGPSQRSKRPSTDTSVCIELP 
               
               
                   
               
               
                 NAEPRALSPAHEHHSQALMGSSRETTALSQTQLASSNVPAAGI 
               
               
                   
               
               
                 KIR2DL5 
               
               
                 SEQ ID 29 
               
               
                 TGIRRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRT 
               
               
                   
               
               
                 VNREDSDDQDPQEVTYAQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELP 
               
               
                   
               
               
                 NAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI 
               
               
                   
               
               
                 KIR3DL1 
               
               
                 SEQ ID 30 
               
               
                 KDPRHLHILIGTSVVIILFILLLFFLLHLWCSNKKNAAVMDQEPAGNRTA 
               
               
                   
               
               
                 NSEDSDEQDPEEVTYAQLDHCVFTQRKITRPSQRPKTPPTDTILYTELPN 
               
               
                   
               
               
                 AKPRSKVVSCP 
               
               
                   
               
               
                 KIR3DL3 
               
               
                 SEQ ID 31 
               
               
                 KDPGNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGN 
               
               
                   
               
               
                 RTVNREDSDEQDPQEVTYAQLNHCVFTQRKITRPSQRPKTPPTDTSV 
               
            
           
         
       
     
     A third embodiment of a ligation-on inhibitory endodomain is an ITIM containing endodomain co-expressed with a fusion protein. The fusion protein may comprise at least part of a protein-tyrosine phosphatase and at least part of a receptor-like tyrosine phosphatase. The fusion may comprise one or more SH2 domains from the protein-tyrosine phosphatase. For example, the fusion may be between a PTPN6 SH2 domain and CD45 endodomain or between a PTPN6 SH2 domain and CD148 endodomain. When phosphorylated, the ITIM domains recruit the fusion protein bring the highly potent CD45 or CD148 phosphatase to proximity to the activating endodomain blocking activation. 
     Sequences of fusion proteins are listed 32 and 33 
     
       
         
           
               
            
               
                 PTPN6-CD45 fusion protein 
               
               
                 SEQ ID 32 
               
               
                 WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPG 
               
               
                   
               
               
                 SPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYL 
               
               
                   
               
               
                 RQPYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRK 
               
               
                   
               
               
                 IADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVE 
               
               
                   
               
               
                 LSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKA 
               
               
                   
               
               
                 TVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQ 
               
               
                   
               
               
                 KLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFF 
               
               
                   
               
               
                 SGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLM 
               
               
                   
               
               
                 VQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLE 
               
               
                   
               
               
                 AEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVLKHELEMSKE 
               
               
                   
               
               
                 SEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDF 
               
               
                   
               
               
                 MIQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTY 
               
               
                   
               
               
                 TLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLP 
               
               
                   
               
               
                 QKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQ 
               
               
                   
               
               
                 VVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFD 
               
               
                   
               
               
                 NEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPAS 
               
               
                   
               
               
                 PALNQGS 
               
               
                   
               
               
                 PTPN6-CD148 fusion 
               
               
                 SEQ ID 33 
               
               
                 ETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVM 
               
               
                   
               
               
                 CEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYRKKRKDA 
               
               
                   
               
               
                 KNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGI 
               
               
                   
               
               
                 SQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGY 
               
               
                   
               
               
                 HSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYW 
               
               
                   
               
               
                 PSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSW 
               
               
                   
               
               
                 PDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAID 
               
               
                   
               
               
                 RLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQK 
               
               
                   
               
               
                 DSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA 
               
            
           
         
       
     
     A ligation-on inhibitory CAR may comprise all or part of SEQ ID No 20 or 21. It may comprise all or part of SEQ ID 22 to 31. It may comprise all or part of SEQ ID 22 to 31 co-expressed with either SEQ ID 32 or 33. It may comprise a variant of the sequence or part thereof having at least 80% sequence identity, as long as the variant retains the capacity to inhibit T cell signaling by the activating CAR upon ligation of the inhibitory CAR. 
     As above, alternative spacers and endodomains may be tested for example using the model system exemplified herein. It is shown in Example 5 that the PTPN6 endodomain can function as a semi-inhibitory CAR in combination with an activating CAR containing a CD3 Zeta endodomain. These CARs rely upon a human CD8 stalk spacer on one CAR and a mouse CD8 stalk spacer on the other CAR. The orthologous sequences prevent cross pairing. However, when both receptors are ligated, the similarity between the spacers results in co-segregation of the different receptors in the same membrane compartments. This results in inhibition of the CD3 Zeta receptor by the PTPN6 endodomain. If only the activating CAR is ligated the PTPN6 endodomain is not sufficiently active to prevent T cell activation. In this way, activation only occurs if the activating CAR is ligated and the inhibitory CAR is not ligated (AND NOT gating). It can be readily seen that this modular system can be used to test alternative spacer pairs and inhibitory domains. If the spacers do not achieve co-segregation following ligation of both receptors, the inhibition would not be effective and so activation would occur. If the semi-inhibitory endodomain under test is ineffective, activation would be expected in the presence of ligation of the activating CAR irrespective of the ligation status of the semi-inhibitory CAR. 
     Co-Expression Site 
     The second aspect of the invention relates to a nucleic acid which encodes the first and second CARs. 
     The nucleic acid may produce a polypeptide which comprises the two CAR molecules 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 first and second CARs without the need for any external cleavage activity. 
     Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2a self-cleaving peptide, which has the sequence shown as SEQ ID No. 34: 
     
       
         
           
               
               
            
               
                   
                 SEQ ID No. 34  
               
               
                   
                 RAEGRGSLLTCGDVEENPGP.  
               
            
           
         
       
     
     The co-expressing sequence may be an internal ribosome entry sequence (IRES). The co-expressing sequence may be an internal promoter. 
     Cell 
     The first aspect of the invention relates to a cell which co-expresses a first CAR and a second CAR at the cell surface. 
     The cell may be any eukaryotic cell capable of expressing a CAR at the cell surface, such as an immunological cell. 
     In particular the cell may be an immune effector cell such as a T cell or a natural killer (NK) cell. 
     T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below. 
     Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses. 
     Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis. 
     Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO. 
     Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. 
     Two major classes of CD4+ Treg cells have been described—naturally occurring Treg cells and adaptive Treg cells. 
     Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX. 
     Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response. 
     The T cell of the invention may be any of the T cell types mentioned above, in particular a CTL. 
     Natural killer (NK) cells are a type of cytolytic cell which forms part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner 
     NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation. 
     The CAR cells of the invention may be any of the cell types mentioned above. 
     CAR− expressing cells, such as CAR-expressing T or NK cells may either be created ex vivo either from a patient&#39;s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2 nd  party), or peripheral blood from an unconnected donor (3 rd  party). 
     The present invention also provide a cell composition comprising CAR expressing T cells and/or CAR expressing NK cells according to the present invention. The cell composition may be made by tranducing a blood-sample ex vivo with a nucleic acid according to the present invention. 
     Alternatively, CAR-expressing cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to the relevant cell type, such as T cells. Alternatively, an immortalized cell line such as a T-cell line which retains its lytic function and could act as a therapeutic may be used. 
     In all these embodiments, CAR cells are generated by introducing DNA or RNA coding for the CARs by one of many means including transduction with a viral vector, transfection with DNA or RNA. 
     A CAR T cell of the invention may be an ex vivo T cell from a subject. The T cell may be from a peripheral blood mononuclear cell (PBMC) sample. T cells may be activated and/or expanded prior to being transduced with CAR-encoding nucleic acid, for example by treatment with an anti-CD3 monoclonal antibody. 
     A CAR T cell of the invention may be made by:
         (i) isolation of a T cell-containing sample from a subject or other sources listed above; and   (ii) transduction or transfection of the T cells with one or more nucleic acid sequence(s) encoding the first and second CAR.       

     The T cells may then by purified, for example, selected on the basis of co-expression of the first and second CAR. 
     Nucleic Acid Sequences 
     The second aspect of the invention relates to one or more nucleic acid sequence(s) which codes for a first CAR and a second CAR as defined in the first aspect of the invention. 
     The nucleic acid sequence may comprise one of the following sequences, or a variant thereof 
     SEQ ID 35 OR gate 
     SEQ ID 36 AND gate using CD45 
     SEQ ID 37 AND gate using CD148 
     SEQ ID 38 AND NOT gate using PTPN6 as endodomain 
     SEQ ID 39 AND NOT gate using LAIR1 endodomain 
     SEQ ID 40 AND NOT gate using LAIR1 and PTPN6 SH2 fusion with CD148 phosphatase 
     
       
         
           
               
               
            
               
                 SEQ ID No. 35: 
                   
               
               
                 &gt;MP13974.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A- 
               
               
                 aCD33glx-HCH2CH3pvaa-CD28tmZw 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAG 
               
               
                   
               
               
                 ACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGG 
               
               
                   
               
               
                 TGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAG 
               
               
                   
               
               
                 AAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGT 
               
               
                   
               
               
                 GCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACC 
               
               
                   
               
               
                 TGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACC 
               
               
                   
               
               
                 TTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGG 
               
               
                   
               
               
                 CGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTG 
               
               
                   
               
               
                 GCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTG 
               
               
                   
               
               
                 AGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTG 
               
               
                   
               
               
                 GCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGC 
               
               
                   
               
               
                 TGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAG 
               
               
                   
               
               
                 ACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGC 
               
               
                   
               
               
                 TATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGC 
               
               
                   
               
               
                 CAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGC 
               
               
                   
               
               
                 CCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGC 
               
               
                   
               
               
                 CTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC 
               
               
                   
               
               
                 TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCA 
               
               
                   
               
               
                 GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG 
               
               
                   
               
               
                 AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA 
               
               
                   
               
               
                 AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG 
               
               
                   
               
               
                 GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA 
               
               
                   
               
               
                 TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC 
               
               
                   
               
               
                 AGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTG 
               
               
                   
               
               
                 GAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTG 
               
               
                   
               
               
                 GCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCAT 
               
               
                   
               
               
                 CTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTA 
               
               
                   
               
               
                 GTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCG 
               
               
                   
               
               
                 CTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTC 
               
               
                   
               
               
                 TAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAG 
               
               
                   
               
               
                 AATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGG 
               
               
                   
               
               
                 AGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTG 
               
               
                   
               
               
                 AGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTC 
               
               
                   
               
               
                 TCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGC 
               
               
                   
               
               
                 TCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACT 
               
               
                   
               
               
                 ATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTC 
               
               
                   
               
               
                 TACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA 
               
               
                   
               
               
                 GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCT 
               
               
                   
               
               
                 CGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGC 
               
               
                   
               
               
                 CCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC 
               
               
                   
               
               
                 CCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAG 
               
               
                   
               
               
                 ACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA 
               
               
                   
               
               
                 AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT 
               
               
                   
               
               
                 GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCC 
               
               
                   
               
               
                 CAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG 
               
               
                   
               
               
                 TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT 
               
               
                   
               
               
                 GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGG 
               
               
                   
               
               
                 AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC 
               
               
                   
               
               
                 AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT 
               
               
                   
               
               
                 GATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCA 
               
               
                   
               
               
                 AGAAGGACCCCAAGTTCTGGGTCCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCT 
               
               
                   
               
               
                 CTCCTGGTGACCGTGGCCTTCATCATCTTTTGGGTGCGCTCCCGGGTGAAGTTTTCTCG 
               
               
                   
               
               
                 CTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCAGCTGTACAATGAACTGAACC 
               
               
                   
               
               
                 TGGGCAGGCGGGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGACCCCGAGATG 
               
               
                   
               
               
                 GGCGGCAAACCACGGCGCAAAAATCCCCAGGAGGGACTCTATAACGAGCTGCAGAAGGA 
               
               
                   
               
               
                 CAAAATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGAAGACGCGGAAAGG 
               
               
                   
               
               
                 GCCACGACGGCCTGTATCAGGGATTGTCCACCGCTACAAAAGATACATATGATGCCCTG 
               
               
                   
               
               
                 CACATGCAGGCCCTGCCACCCAGATGA 
               
               
                   
               
               
                 SEQ ID No. 36 
               
               
                 &gt;MP14802.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ- 
               
               
                 2A-aCD33glx-HCH2CH3pvaa-dCD45 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAG 
               
               
                   
               
               
                 ACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGG 
               
               
                   
               
               
                 TGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAG 
               
               
                   
               
               
                 AAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGT 
               
               
                   
               
               
                 GCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACC 
               
               
                   
               
               
                 TGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACC 
               
               
                   
               
               
                 TTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGG 
               
               
                   
               
               
                 CGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTG 
               
               
                   
               
               
                 GCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTG 
               
               
                   
               
               
                 AGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTG 
               
               
                   
               
               
                 GCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGC 
               
               
                   
               
               
                 TGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAG 
               
               
                   
               
               
                 ACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGC 
               
               
                   
               
               
                 TATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGC 
               
               
                   
               
               
                 CAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGC 
               
               
                   
               
               
                 CCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGC 
               
               
                   
               
               
                 CTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC 
               
               
                   
               
               
                 TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCA 
               
               
                   
               
               
                 GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG 
               
               
                   
               
               
                 AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA 
               
               
                   
               
               
                 AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG 
               
               
                   
               
               
                 GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA 
               
               
                   
               
               
                 TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC 
               
               
                   
               
               
                 AGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTG 
               
               
                   
               
               
                 GAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTG 
               
               
                   
               
               
                 GCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCAT 
               
               
                   
               
               
                 CTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTA 
               
               
                   
               
               
                 GTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCG 
               
               
                   
               
               
                 CTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTC 
               
               
                   
               
               
                 TAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAG 
               
               
                   
               
               
                 AATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGG 
               
               
                   
               
               
                 AGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTG 
               
               
                   
               
               
                 AGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTC 
               
               
                   
               
               
                 TCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGC 
               
               
                   
               
               
                 TCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACT 
               
               
                   
               
               
                 ATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTC 
               
               
                   
               
               
                 TACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA 
               
               
                   
               
               
                 GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCT 
               
               
                   
               
               
                 CGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGC 
               
               
                   
               
               
                 CCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC 
               
               
                   
               
               
                 CCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAG 
               
               
                   
               
               
                 ACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA 
               
               
                   
               
               
                 AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT 
               
               
                   
               
               
                 GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCC 
               
               
                   
               
               
                 CAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG 
               
               
                   
               
               
                 TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT 
               
               
                   
               
               
                 GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGG 
               
               
                   
               
               
                 AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC 
               
               
                   
               
               
                 AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT 
               
               
                   
               
               
                 GATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCA 
               
               
                   
               
               
                 AGAAGGACCCCAAGGCACTGATAGCATTTCTGGCATTTCTGATTATTGTGACATCAATA 
               
               
                   
               
               
                 GCCCTGCTTGTTGTTCTCTACAAAATCTATGATCTACATAAGAAAAGATCCTGCAATTT 
               
               
                   
               
               
                 AGATGAACAGCAGGAGCTTGTTGAAAGGGATGATGAAAAACAACTGATGAATGTGGAGC 
               
               
                   
               
               
                 CAATCCATGCAGATATTTTGTTGGAAACTTATAAGAGGAAGATTGCTGATGAAGGAAGA 
               
               
                   
               
               
                 CTTTTTCTGGCTGAATTTCAGAGCATCCCGCGGGTGTTCAGCAAGTTTCCTATAAAGGA 
               
               
                   
               
               
                 AGCTCGAAAGCCCTTTAACCAGAATAAAAACCGTTATGTTGACATTCTTCCTTATGATT 
               
               
                   
               
               
                 ATAACCGTGTTGAACTCTCTGAGATAAACGGAGATGCAGGGTCAAACTACATAAATGCC 
               
               
                   
               
               
                 AGCTATATTGATGGTTTCAAAGAACCCAGGAAATACATTGCTGCACAAGGTCCCAGGGA 
               
               
                   
               
               
                 TGAAACTGTTGATGATTTCTGGAGGATGATTTGGGAACAGAAAGCCACAGTTATTGTCA 
               
               
                   
               
               
                 TGGTCACTCGATGTGAAGAAGGAAACAGGAACAAGTGTGCAGAATACTGGCCGTCAATG 
               
               
                   
               
               
                 GAAGAGGGCACTCGGGCTTTTGGAGATGTTGTTGTAAAGATCAACCAGCACAAAAGATG 
               
               
                   
               
               
                 TCCAGATTACATCATTCAGAAATTGAACATTGTAAATAAAAAAGAAAAAGCAACTGGAA 
               
               
                   
               
               
                 GAGAGGTGACTCACATTCAGTTCACCAGCTGGCCAGACCACGGGGTGCCTGAGGATCCT 
               
               
                   
               
               
                 CACTTGCTCCTCAAACTGAGAAGGAGAGTGAATGCCTTCAGCAATTTCTTCAGTGGTCC 
               
               
                   
               
               
                 CATTGTGGTGCACTGCAGTGCTGGTGTTGGGCGCACAGGAACCTATATCGGAATTGATG 
               
               
                   
               
               
                 CCATGCTAGAAGGCCTGGAAGCCGAGAACAAAGTGGATGTTTATGGTTATGTTGTCAAG 
               
               
                   
               
               
                 CTAAGGCGACAGAGATGCCTGATGGTTCAAGTAGAGGCCCAGTACATCTTGATCCATCA 
               
               
                   
               
               
                 GGCTTTGGTGGAATACAATCAGTTTGGAGAAACAGAAGTGAATTTGTCTGAATTACATC 
               
               
                   
               
               
                 CATATCTACATAACATGAAGAAAAGGGATCCACCCAGTGAGCCGTCTCCACTAGAGGCT 
               
               
                   
               
               
                 GAATTCCAGAGACTTCCTTCATATAGGAGCTGGAGGACACAGCACATTGGAAATCAAGA 
               
               
                   
               
               
                 AGAAAATAAAAGTAAAAACAGGAATTCTAATGTCATCCCATATGACTATAACAGAGTGC 
               
               
                   
               
               
                 CACTTAAACATGAGCTGGAAATGAGTAAAGAGAGTGAGCATGATTCAGATGAATCCTCT 
               
               
                   
               
               
                 GATGATGACAGTGATTCAGAGGAACCAAGCAAATACATCAATGCATCTTTTATAATGAG 
               
               
                   
               
               
                 CTACTGGAAACCTGAAGTGATGATTGCTGCTCAGGGACCACTGAAGGAGACCATTGGTG 
               
               
                   
               
               
                 ACTTTTGGCAGATGATCTTCCAAAGAAAAGTCAAAGTTATTGTTATGCTGACAGAACTG 
               
               
                   
               
               
                 AAACATGGAGACCAGGAAATCTGTGCTCAGTACTGGGGAGAAGGAAAGCAAACATATGG 
               
               
                   
               
               
                 AGATATTGAAGTTGACCTGAAAGACACAGACAAATCTTCAACTTATACCCTTCGTGTCT 
               
               
                   
               
               
                 TTGAACTGAGACATTCCAAGAGGAAAGACTCTCGAACTGTGTACCAGTACCAATATACA 
               
               
                   
               
               
                 AACTGGAGTGTGGAGCAGCTTCCTGCAGAACCCAAGGAATTAATCTCTATGATTCAGGT 
               
               
                   
               
               
                 CGTCAAACAAAAACTTCCCCAGAAGAATTCCTCTGAAGGGAACAAGCATCACAAGAGTA 
               
               
                   
               
               
                 CACCTCTACTCATTCACTGCAGGGATGGATCTCAGCAAACGGGAATATTTTGTGCTTTG 
               
               
                   
               
               
                 TTAAATCTCTTAGAAAGTGCGGAAACAGAAGAGGTAGTGGATATTTTTCAAGTGGTAAA 
               
               
                   
               
               
                 AGCTCTACGCAAAGCTAGGCCAGGCATGGTTTCCACATTCGAGCAATATCAATTCCTAT 
               
               
                   
               
               
                 ATGACGTCATTGCCAGCACCTACCCTGCTCAGAATGGACAAGTAAAGAAAAACAACCAT 
               
               
                   
               
               
                 CAAGAAGATAAAATTGAATTTGATAATGAAGTGGACAAAGTAAAGCAGGATGCTAATTG 
               
               
                   
               
               
                 TGTTAATCCACTTGGTGCCCCAGAAAAGCTCCCTGAAGCAAAGGAACAGGCTGAAGGTT 
               
               
                   
               
               
                 CTGAACCCACGAGTGGCACTGAGGGGCCAGAACATTCTGTCAATGGTCCTGCAAGTCCA 
               
               
                   
               
               
                 GCTTTAAATCAAGGTTCATAG 
               
               
                   
               
               
                 SEQ ID No. 37: 
               
               
                 &gt;MP14801.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A- 
               
               
                 aCD33glx-HCH2CH3pvaa-dCD148 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAG 
               
               
                   
               
               
                 ACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGG 
               
               
                   
               
               
                 TGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAG 
               
               
                   
               
               
                 AAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGT 
               
               
                   
               
               
                 GCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACC 
               
               
                   
               
               
                 TGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACC 
               
               
                   
               
               
                 TTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGG 
               
               
                   
               
               
                 CGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTG 
               
               
                   
               
               
                 GCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTG 
               
               
                   
               
               
                 AGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTG 
               
               
                   
               
               
                 GCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGC 
               
               
                   
               
               
                 TGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAG 
               
               
                   
               
               
                 ACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGC 
               
               
                   
               
               
                 TATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGC 
               
               
                   
               
               
                 CAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGC 
               
               
                   
               
               
                 CCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGC 
               
               
                   
               
               
                 CTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC 
               
               
                   
               
               
                 TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCA 
               
               
                   
               
               
                 GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG 
               
               
                   
               
               
                 AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA 
               
               
                   
               
               
                 AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG 
               
               
                   
               
               
                 GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA 
               
               
                   
               
               
                 TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC 
               
               
                   
               
               
                 AGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTG 
               
               
                   
               
               
                 GAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTG 
               
               
                   
               
               
                 GCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCAT 
               
               
                   
               
               
                 CTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTA 
               
               
                   
               
               
                 GTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCG 
               
               
                   
               
               
                 CTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTC 
               
               
                   
               
               
                 TAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAG 
               
               
                   
               
               
                 AATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGG 
               
               
                   
               
               
                 AGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTG 
               
               
                   
               
               
                 AGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTC 
               
               
                   
               
               
                 TCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGC 
               
               
                   
               
               
                 TCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACT 
               
               
                   
               
               
                 ATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTC 
               
               
                   
               
               
                 TACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA 
               
               
                   
               
               
                 GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCT 
               
               
                   
               
               
                 CGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGC 
               
               
                   
               
               
                 CCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC 
               
               
                   
               
               
                 CCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAG 
               
               
                   
               
               
                 ACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA 
               
               
                   
               
               
                 AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT 
               
               
                   
               
               
                 GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCC 
               
               
                   
               
               
                 CAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG 
               
               
                   
               
               
                 TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT 
               
               
                   
               
               
                 GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGG 
               
               
                   
               
               
                 AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC 
               
               
                   
               
               
                 AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT 
               
               
                   
               
               
                 GATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCA 
               
               
                   
               
               
                 AGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTG 
               
               
                   
               
               
                 GGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTT 
               
               
                   
               
               
                 TTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACT 
               
               
                   
               
               
                 TCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAG 
               
               
                   
               
               
                 CTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAA 
               
               
                   
               
               
                 TCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCC 
               
               
                   
               
               
                 ATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGAT 
               
               
                   
               
               
                 TTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTG 
               
               
                   
               
               
                 GGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCA 
               
               
                   
               
               
                 AATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCA 
               
               
                   
               
               
                 ATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATAT 
               
               
                   
               
               
                 CCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACG 
               
               
                   
               
               
                 GTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATG 
               
               
                   
               
               
                 AAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGAC 
               
               
                   
               
               
                 GGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGG 
               
               
                   
               
               
                 ATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAG 
               
               
                   
               
               
                 GACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTC 
               
               
                   
               
               
                 AAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGC 
               
               
                   
               
               
                 CCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAA 
               
               
                   
               
               
                 SEQ ID No. 38 
               
               
                 &gt;16076.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A- 
               
               
                 aCD33glx-muCD8STK-tm-dPTPN6 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAG 
               
               
                   
               
               
                 ACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGG 
               
               
                   
               
               
                 TGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAG 
               
               
                   
               
               
                 AAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGT 
               
               
                   
               
               
                 GCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACC 
               
               
                   
               
               
                 TGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACC 
               
               
                   
               
               
                 TTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGG 
               
               
                   
               
               
                 CGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTG 
               
               
                   
               
               
                 GCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTG 
               
               
                   
               
               
                 AGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTG 
               
               
                   
               
               
                 GCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGC 
               
               
                   
               
               
                 TGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAG 
               
               
                   
               
               
                 ACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGC 
               
               
                   
               
               
                 TATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGC 
               
               
                   
               
               
                 CAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGC 
               
               
                   
               
               
                 CCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGC 
               
               
                   
               
               
                 CTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC 
               
               
                   
               
               
                 TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCA 
               
               
                   
               
               
                 GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG 
               
               
                   
               
               
                 AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA 
               
               
                   
               
               
                 AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG 
               
               
                   
               
               
                 GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA 
               
               
                   
               
               
                 TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC 
               
               
                   
               
               
                 AGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTG 
               
               
                   
               
               
                 GAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTG 
               
               
                   
               
               
                 GCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCAT 
               
               
                   
               
               
                 CTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTA 
               
               
                   
               
               
                 GTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCG 
               
               
                   
               
               
                 CTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTC 
               
               
                   
               
               
                 TAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAG 
               
               
                   
               
               
                 AATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGG 
               
               
                   
               
               
                 AGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTG 
               
               
                   
               
               
                 AGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTC 
               
               
                   
               
               
                 TCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGC 
               
               
                   
               
               
                 TCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACT 
               
               
                   
               
               
                 ATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTC 
               
               
                   
               
               
                 TACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA 
               
               
                   
               
               
                 GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCT 
               
               
                   
               
               
                 CGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCAC 
               
               
                   
               
               
                 CCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAA 
               
               
                   
               
               
                 GGGCACCGGCCTGGACTTCGCCTGCGACATCTACTGGGCACCTCTGGCCGGAATATGCG 
               
               
                   
               
               
                 TGGCACTGCTGCTGAGCCTCATCATCACCCTGATCTGTTATCACCGAAGCCGCAAGCGG 
               
               
                   
               
               
                 GTGTGTAAAAGTGGAGGCGGAAGCTTCTGGGAGGAGTTTGAGAGTTTGCAGAAGCAGGA 
               
               
                   
               
               
                 GGTGAAGAACTTGCACCAGCGTCTGGAAGGGCAGCGGCCAGAGAACAAGGGCAAGAACC 
               
               
                   
               
               
                 GCTACAAGAACATTCTCCCCTTTGACCACAGCCGAGTGATCCTGCAGGGACGGGACAGT 
               
               
                   
               
               
                 AACATCCCCGGGTCCGACTACATCAATGCCAACTACATCAAGAACCAGCTGCTAGGCCC 
               
               
                   
               
               
                 TGATGAGAACGCTAAGACCTACATCGCCAGCCAGGGCTGTCTGGAGGCCACGGTCAATG 
               
               
                   
               
               
                 ACTTCTGGCAGATGGCGTGGCAGGAGAACAGCCGTGTCATCGTCATGACCACCCGAGAG 
               
               
                   
               
               
                 GTGGAGAAAGGCCGGAACAAATGCGTCCCATACTGGCCCGAGGTGGGCATGCAGCGTGC 
               
               
                   
               
               
                 TTATGGGCCCTACTCTGTGACCAACTGCGGGGAGCATGACACAACCGAATACAAACTCC 
               
               
                   
               
               
                 GTACCTTACAGGTCTCCCCGCTGGACAATGGAGACCTGATTCGGGAGATCTGGCATTAC 
               
               
                   
               
               
                 CAGTACCTGAGCTGGCCCGACCACGGGGTCCCCAGTGAGCCTGGGGGTGTCCTCAGCTT 
               
               
                   
               
               
                 CCTGGACCAGATCAACCAGCGGCAGGAAAGTCTGCCTCACGCAGGGCCCATCATCGTGC 
               
               
                   
               
               
                 ACTGCAGCGCCGGCATCGGCCGCACAGGCACCATCATTGTCATCGACATGCTCATGGAG 
               
               
                   
               
               
                 AACATCTCCACCAAGGGCCTGGACTGTGACATTGACATCCAGAAGACCATCCAGATGGT 
               
               
                   
               
               
                 GCGGGCGCAGCGCTCGGGCATGGTGCAGACGGAGGCGCAGTACAAGTTCATCTACGTGG 
               
               
                   
               
               
                 CCATCGCCCAGTTCATTGAAACCACTAAGAAGAAGCTGTGA 
               
               
                   
               
               
                 SEQ ID No. 39 
               
               
                 &gt;MP16091.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A- 
               
               
                 aCD33glx-muCD8STK-LAIR1tm-endo 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAG 
               
               
                   
               
               
                 ACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGG 
               
               
                   
               
               
                 TGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAG 
               
               
                   
               
               
                 AAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGT 
               
               
                   
               
               
                 GCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACC 
               
               
                   
               
               
                 TGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACC 
               
               
                   
               
               
                 TTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGG 
               
               
                   
               
               
                 CGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTG 
               
               
                   
               
               
                 GCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTG 
               
               
                   
               
               
                 AGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTG 
               
               
                   
               
               
                 GCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGC 
               
               
                   
               
               
                 TGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAG 
               
               
                   
               
               
                 ACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGC 
               
               
                   
               
               
                 TATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGC 
               
               
                   
               
               
                 CAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGC 
               
               
                   
               
               
                 CCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGC 
               
               
                   
               
               
                 CTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC 
               
               
                   
               
               
                 TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCA 
               
               
                   
               
               
                 GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG 
               
               
                   
               
               
                 AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA 
               
               
                   
               
               
                 AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG 
               
               
                   
               
               
                 GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA 
               
               
                   
               
               
                 TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC 
               
               
                   
               
               
                 AGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTG 
               
               
                   
               
               
                 GAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTG 
               
               
                   
               
               
                 GCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCAT 
               
               
                   
               
               
                 CTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTA 
               
               
                   
               
               
                 GTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCG 
               
               
                   
               
               
                 CTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTC 
               
               
                   
               
               
                 TAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAG 
               
               
                   
               
               
                 AATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGG 
               
               
                   
               
               
                 AGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTG 
               
               
                   
               
               
                 AGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTC 
               
               
                   
               
               
                 TCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGC 
               
               
                   
               
               
                 TCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACT 
               
               
                   
               
               
                 ATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTC 
               
               
                   
               
               
                 TACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA 
               
               
                   
               
               
                 GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCT 
               
               
                   
               
               
                 CGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCAC 
               
               
                   
               
               
                 CCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAA 
               
               
                   
               
               
                 GGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCTTCCTCT 
               
               
                   
               
               
                 TCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAGCAGGGG 
               
               
                   
               
               
                 CCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCTGGCTGTTGA 
               
               
                   
               
               
                 TGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAGAAGGACCGGG 
               
               
                   
               
               
                 AGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGACGTATGCTCAGCTG 
               
               
                   
               
               
                 GACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCCACAGTCCACAAAGCC 
               
               
                   
               
               
                 CATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAGACACTGA 
               
               
                   
               
               
                 SEQ ID no. 40 
               
               
                 &gt;MP16092.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A- 
               
               
                 aCD33glx-muCD8STK-LAIR1tm-endo-2A-PTPN6_SH2-dCD148 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAG 
               
               
                   
               
               
                 ACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGG 
               
               
                   
               
               
                 TGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAG 
               
               
                   
               
               
                 AAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGT 
               
               
                   
               
               
                 GCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACC 
               
               
                   
               
               
                 TGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACC 
               
               
                   
               
               
                 TTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGG 
               
               
                   
               
               
                 CGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTG 
               
               
                   
               
               
                 GCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTG 
               
               
                   
               
               
                 AGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTG 
               
               
                   
               
               
                 GCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGC 
               
               
                   
               
               
                 TGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAG 
               
               
                   
               
               
                 ACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGC 
               
               
                   
               
               
                 TATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGC 
               
               
                   
               
               
                 CAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGC 
               
               
                   
               
               
                 CCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGC 
               
               
                   
               
               
                 CTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC 
               
               
                   
               
               
                 TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCA 
               
               
                   
               
               
                 GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG 
               
               
                   
               
               
                 AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA 
               
               
                   
               
               
                 AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG 
               
               
                   
               
               
                 GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA 
               
               
                   
               
               
                 TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC 
               
               
                   
               
               
                 AGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTG 
               
               
                   
               
               
                 GAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTG 
               
               
                   
               
               
                 GCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCAT 
               
               
                   
               
               
                 CTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTA 
               
               
                   
               
               
                 GTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCG 
               
               
                   
               
               
                 CTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTC 
               
               
                   
               
               
                 TAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAG 
               
               
                   
               
               
                 AATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGG 
               
               
                   
               
               
                 AGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTG 
               
               
                   
               
               
                 AGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTC 
               
               
                   
               
               
                 TCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGC 
               
               
                   
               
               
                 TCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACT 
               
               
                   
               
               
                 ATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTC 
               
               
                   
               
               
                 TACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACA 
               
               
                   
               
               
                 GGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCT 
               
               
                   
               
               
                 CGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCAC 
               
               
                   
               
               
                 CCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAA 
               
               
                   
               
               
                 GGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCTTCCTCT 
               
               
                   
               
               
                 TCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAGCAGGGG 
               
               
                   
               
               
                 CCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCTGGCTGTTGA 
               
               
                   
               
               
                 TGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAGAAGGACCGGG 
               
               
                   
               
               
                 AGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGACGTATGCTCAGCTG 
               
               
                   
               
               
                 GACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCCACAGTCCACAAAGCC 
               
               
                   
               
               
                 CATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAGACACAGGGCAGAAGGAAGAGGTA 
               
               
                   
               
               
                 GCCTGCTGACTTGCGGGGACGTGGAAGAGAACCCAGGGCCATGGTATCATGGCCACATG 
               
               
                   
               
               
                 TCTGGCGGGCAGGCAGAGACGCTGCTGCAGGCCAAGGGCGAGCCCTGGACGTTTCTTGT 
               
               
                   
               
               
                 GCGTGAGAGCCTCAGCCAGCCTGGAGACTTCGTGCTTTCTGTGCTCAGTGACCAGCCCA 
               
               
                   
               
               
                 AGGCTGGCCCAGGCTCCCCGCTCAGGGTCACCCACATCAAGGTCATGTGCGAGGGTGGA 
               
               
                   
               
               
                 CGCTACACAGTGGGTGGTTTGGAGACCTTCGACAGCCTCACGGACCTGGTGGAGCATTT 
               
               
                   
               
               
                 CAAGAAGACGGGGATTGAGGAGGCCTCAGGCGCCTTTGTCTACCTGCGGCAGCCGTACA 
               
               
                   
               
               
                 GCGGTGGCGGTGGCAGCTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGT 
               
               
                   
               
               
                 GGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGC 
               
               
                   
               
               
                 AGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATA 
               
               
                   
               
               
                 TTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAAC 
               
               
                   
               
               
                 TACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAA 
               
               
                   
               
               
                 CACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGT 
               
               
                   
               
               
                 TGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAG 
               
               
                   
               
               
                 GCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATG 
               
               
                   
               
               
                 GACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGAC 
               
               
                   
               
               
                 AGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATC 
               
               
                   
               
               
                 AACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCT 
               
               
                   
               
               
                 GGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCA 
               
               
                   
               
               
                 TCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGA 
               
               
                   
               
               
                 ATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGT 
               
               
                   
               
               
                 TTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAA 
               
               
                   
               
               
                 CTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGT 
               
               
                   
               
               
                 TACATCGCCAGCGGTAGCTAA 
               
            
           
         
       
     
     The nucleic acid sequence may encode the same amino acid sequence as that encoded by SEQ ID No. 35, 36, 37, 38, 39 or 40, but may have a different nucleic acid sequence, due to the degeneracy of the genetic code. The nucleic acid sequence may have at least 80, 85, 90, 95, 98 or 99% identity to the sequence shown as SEQ ID No. 35, 36, 37, 38, 39 or 40, provided that it encodes a first CAR and a second CAR as defined in the first aspect of the invention. 
     Vector 
     The present invention also provides a vector, or kit of vectors which comprises one or more CAR-encoding nucleic acid sequence(s). Such a vector may be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses the first and second CARs. 
     The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA. 
     The vector may be capable of transfecting or transducing a T cell. 
     Pharmaceutical Composition 
     The present invention also relates to a pharmaceutical composition containing a plurality of CAR-expressing T cells or NK cells according to the first aspect of the invention. The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion. 
     Method of Treatment 
     The T cells of the present invention may be capable of killing target cells, such as cancer cells. The target cell may be recognisable by a defined pattern of antigen expression, for example the expression of antigen A AND antigen B; the expression of antigen A OR antigen B; or the expression of antigen A AND NOT antigen B or complex iterations of these gates. 
     T cells of the present invention may be used for the treatment of an infection, such as a viral infection. 
     T cells of the invention may also be used for the control of pathogenic immune responses, for example in autoimmune diseases, allergies and graft-vs-host rejection. 
     T cells of the invention may be used 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. 
     It is particularly suited for treatment of solid tumours where the availability of good selective single targets is limited. 
     T cells of the invention may be used to treat: cancers of the oral cavity and pharynx which includes cancer of the tongue, mouth and pharynx; cancers of the digestive system which includes oesophageal, gastric and colorectal cancers; cancers of the liver and biliary tree which includes hepatocellular carcinomas and cholangiocarcinomas; cancers of the respiratory system which includes bronchogenic cancers and cancers of the larynx; cancers of bone and joints which includes osteosarcoma; cancers of the skin which includes melanoma; breast cancer; cancers of the genital tract which include uterine, ovarian and cervical cancer in women, prostate and testicular cancer in men; cancers of the renal tract which include renal cell carcinoma and transitional cell carcinomas of the utterers or bladder; brain cancers including gliomas, glioblastoma multiforme and medullobastomas; cancers of the endocrine system including thyroid cancer, adrenal carcinoma and cancers associated with multiple endocrine neoplasm syndromes; lymphomas including Hodgkin&#39;s lymphoma and non-Hodgkin lymphoma; Multiple Myeloma and plasmacytomas; leukaemias both acute and chronic, myeloid or lymphoid; and cancers of other and unspecified sites including neuroblastoma. 
     Treatment with the T cells of the invention may help prevent the escape or release of tumour cells which often occurs with standard approaches. 
     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—Creation of Target Cell Populations 
     For the purposes of proving the principle of the invention, receptors based on anti-CD19 and anti-CD33 were arbitrarily chosen. Using retroviral vectors, CD19 and CD33 were cloned. These proteins were truncated so that they do not signal and could be stably expressed for prolonged periods. Next, these vectors were used to transduce the SupT1 cell line either singly or doubly to establish cells negative for both antigen (the wild-type), positive for either and positive for both. The expression data are shown in  FIG. 3 . 
     Example 2—Design and Function of the OR Gate 
     To construct the OR gate, a pair of receptors recognizing CD19 and CD33 were co-expressed. Different spacers were used to prevent cross-pairing. Both receptors had a trans-membrane domain derived from CD28 to improve surface stability and an endodomain derived from that of CD3 Zeta to provide a simple activating signal. In this way, a pair of independent 1st generation CARs were co-expressed. The retroviral vector cassette used to co-express the sequences utilizes a foot-and-mouth 2A self-cleaving peptide to allow co-expression 1:1 of both receptors. The cassette design is shown in  FIG. 4 , and the protein structures in  FIG. 5 . The nucleotide sequence of homologous regions was codon-wobbled to prevent recombination during retroviral vector reverse transcription. 
     Example 3—Testing the OR Gate 
     Expression of both CARs was tested on the T-cell surface by staining with cognate antigen fused to Fc. By using different species of Fc domains (mouse for CD19 and rabbit for CD33), co-expression of both CARs was determined on the cell surface by staining with different secondary antibodies conjugated with different fluorophores. This is shown in  FIG. 6 . 
     Functional testing was then carried out using the mouse T-cell line BW5147. This cell line releases IL2 upon activation allowing a simple quantitative readout. These T-cells were co-cultured with increasing amounts of the artificial target cells described above. T-cells responded to target cells expressing either antigen, as shown by IL2 release measured by ELISA. Both CARs were shown to be expressed on the cell surfaces and the T-cells were shown to respond to either or both antigens. These data are show in  FIG. 7 . 
     Example 4—Design and Function of the AND Gate 
     The AND gate combines a simple activating receptor with a receptor which basally inhibits activity, but whose inhibition is turned off once the receptor is ligated. This was achieved by combining a standard 1St generation CAR with a short/non-bulky CD8 stalk spacer and a CD3 Zeta endodomain with a second receptor with a bulky Fc spacer whose endodomain contained either CD148 or CD45 endodomains. When both receptors are ligated, the difference in spacer dimensions results in isolation of the different receptors in different membrane compartments, releasing the CD3 Zeta receptor from inhibition by the CD148 or CD45 endodomains. In this way, activation only occurs once both receptors are activated. CD148 and CD45 were chosen for this as they function in this manner natively: for instance, the very bulky CD45 ectodomain excludes the entire receptor from the immunological synapse. The expression cassette is depicted in  FIG. 8  and the subsequent proteins in  FIG. 9 . 
     Surface staining for the different specificity showed that both receptor pairs could be effectively expressed on the cell surface shown in  FIG. 10 . Function in BW5147 shows that the T-cell is only activated in the presence of both antigens ( FIG. 11 ). 
     Example 5: Demonstration of Generalizability of the AND Gate 
     To ensure that the observations were not a manifestation of some specific characteristic of CD19/CD33 and their binders which had been used, the two targeting scFvs were swapped such that now, the activation (ITAM) signal was transmitted upon recognition of CD33, rather than CD19; and the inhibitory (CD148) signal was transmitted upon recognition of CD19, rather than of CD33. Since CD45 and CD148 endodomains are considered to be functionally similar, experimentation was restricted to AND gates with CD148 endodomain. This should still result in a functional AND gate. T-cells expressing the new logic gate where challenged with targets bearing either CD19 or CD33 alone, or both. The T-cells responded to targets expressing both CD19 and CD33, but not to targets expressing only one or none of these antigens. This shows that the AND gate is still functional in this format ( FIG. 18B ). 
     On the same lines, it was sought to establish how generalizable our AND gate is: the AND gate should be generalizable across different targets. While there may be lesser or greater fidelity of the gate given relative antigen density, cognate scFv binding kinetics and precise distance of the scFv binding epitope, one would expect to see some AND gate manifestations with a wide set of targets and binders. To test this, three additional AND gates were generated. Once again, experimentation was restricted to the CD148 version of the AND gate. The second scFv from the original CD148 AND gate was replaced with the anti-GD2 scFv huK666 (SEQ ID 41 and SEQ ID 42), or with the anti-CD5 scFv (SEQ ID 43 and SEQ ID 44), or the anti-EGFRvIII scFv MR1.1 (SEQ ID 45 AND SEQ ID 46) to generate the following CAR AND gates: CD19 AND GD2; CD19 AND CD5; CD19 AND EGFRvIII. The following artificial antigen expressing cell lines were also generated: by transducing SupT1, and our SupT1.CD19 with GM3 and GD2 synthases SupT1.GD2 and SupT1.CD19.GD2 were generated. By transducing SupT1 and SupT1.CD19 with a retroviral vector coding for EGFRvIII SupT1.EGFRvIII and SupT1.CD19.EGFRvIII were generated. Since CD5 is expressed on SupT1 cells, a different cell line was used to generate the target cells: 293T cells were generated which express CD19 alone, CD5 alone and both CD5 and CD19 together. Expression was confirmed by flow-cytometry ( FIG. 19 ). T-cells expressing the three new CAR AND gates were challenged with SupT1.CD19 and respective cognate double positive and single positive target cells. All three AND gates demonstrated reduced activation by the double positive cell lines in comparison with the single positive targets ( FIG. 20 ). This demonstrates generalizability of the AND gate design to arbitrary targets and cognate binders. 
     Example 6: Experimental Proof of Kinetic Segregation Model of CAR AND Gate 
     The aim was to prove the model that differential segregation caused by different spacers is the central mechanism behind the ability to generate these logic CAR gates. The model is that if only the activating CAR is ligated, the potent inhibiting ‘ligation off’ type CAR is in solution in the membrane and can inhibit the activating CAR. Once both CARs are ligated, if both CAR spacers are sufficiently different, they will segregate within the synapse and not co-localize. Hence, a key requirement is that the spacers are sufficiently different. If the model is correct, if both spacers are sufficiently similar so they co-localize when both receptors are ligated, the gate will fail to function. To test this, the “bulky” Fc spacer in the original CAR we replaced with a murine CD8 spacer. It was predicted that this has the similar length, bulk and charge as human CD8 but so should not cross-pair with it. Hence, the new gate had a first CAR which recognizes CD19, a human CD8 stalk spacer and an activatory endodomain; while the second CAR recognizes CD33, has a mouse CD8 stalk spacer and a CD148 endodomain ( FIG. 18C ). T-cells were transduced to express this new CAR gate. These T-cells were then challenged with SupT1 cells expressing CD19 alone, CD33 alone or CD19 and CD33 together. T-cells did not respond to SupT1 cells expressing either antigen alone as per the original AND gate. However, CAR T-cells failed to respond to SupT1 cells expressing both antigens, thereby confirming the model ( FIG. 18C ). A functional AND gate requires both CARs to have spacers sufficiently different so that they do not co-localize within an immunological synapse ( FIGS. 23A  and B). 
     Example 7—Design and Function of an AND NOT Gate 
     Phosphatases such as CD45 and CD148 are so potent that even a small amount entering an immunological synapse can inhibit ITAM activation. This is the basis of inhibition of the logical AND gate. Other classes of phosphatases are not as potent e.g. PTPN6 and related phosphatases. It was predicted that a small amount of PTPN6 entering a synapse by diffusion would not inhibit activation. In addition, it was predicted that if an inhibitory CAR had a sufficiently similar spacer to an activating CAR, it could co-localize within a synapse if both CARs were ligated. In this case, large amounts of the inhibitory endodomain would be sufficient to stop the ITAMS from activating when both antigens were present. In this way, an AND NOT gate could be created. 
     For the NOT AND gate, the second signal needs to “veto” activation. This is done by bringing an inhibitory signal into the immunological synapse, for example by bringing in the phosphatase of an enzyme such as PTPN6. We hence generated an initial AND NOT gate as follows: two CARs co-expressed whereby the first recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; co-expressed with an anti-CD33 CAR with a mouse CD8 stalk spacer and an endodomain comprising of the catalytic domain of PTPN6 (SEQ ID 38,  FIG. 13  A with B). A suitable cassette is shown in  FIG. 12  and preliminary functional data are shown in  FIG. 14 . 
     In addition, an alternative strategy was developed for generating an AND NOT gate. Immune Tyrosinase Inhibitory Motifs (ITIMs) are activated in a similar manner to ITAMS, in that they become phosphorylated by Ick upon clustering and exclusion of phosphatases. Instead of triggering activation by binding ZAP70, phosphorylated ITIMs recruit phosphatases like PTPN6 through their cognate SH2 domains. An ITIM can function as an inhibitory endodomain, as long as the spacers on the activating and inhibiting CARs can co-localize. To generate this construct, an AND NOT gate was generated as follows: two CARs co-expressed—the first recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; co-expressed with an anti-CD33 CAR with a mouse CD8 stalk spacer and an ITIM containing endodomain derived from that of LAIR1 (SEQ ID 39,  FIG. 13  A with C). 
     A further, more complex AND NOT gate was also developed, whereby an ITIM is enhanced by the presence of an additional chimeric protein: an intracellular fusion of the SH2 domain of PTPN6 and the endodomain of CD148. In this design three proteins are expressed—the first recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; co-expressed with an anti-CD33 CAR with a mouse CD8 stalk spacer and an ITIM containing endodomain derived from that of LAIR1. A further 2A peptide, allows co-expression of the PTPN6-CD148 fusion (SEQ ID 40,  FIGS. 13  A and D). It was predicted that these AND NOT gates would have a different range of inhibition: PTPN6-CD148&gt;PTPN6&gt;&gt;ITIM. 
     T-cells were transduced with these gates and challenged with targets expressing either CD19 or CD33 alone, or both CD19 and CD33 together. All three gates responded to targets expressing only CD19, but not targets expressing both CD19 and CD33 together ( FIG. 21 ), confirming that all three of the AND NOT gates were functional. 
     Example 8: Experimental Proof of Kinetic Segregation Model of PTPN6 Based AND NOT Gate 
     The model of the AND NOT gate centres around the fact that the nature of the spacers used in both CARs is pivotal for the correct function of the gate. In the functional AND NOT gate with PTPN6, both CAR spacers are sufficiently similar that when both CARs are ligated, both co-localize within the synapse so the high concentration even the weak PTPN6 is sufficient to inhibit activation. If the spacers were different, segregation in the synapse will isolate the PTPN6 from the ITAM allowing activation disrupting the AND NOT gate. To test this, a control was generated replacing the murine CD8 stalk spacer with that of Fc. In this case, the test gate consisted of two CARs, the first recognizes CD19, has a human CD8 stalk spacer and an ITAM endodomain; while the second CAR recognizes CD33, has an Fc spacer and an endodomain comprising of the phosphatase from PTPN6. This gate activates in response to CD19, but also activates in response to CD19 and CD33 together ( FIG. 22B , where function of this gate is compared with that of the original AND NOT, and the control AND gate variant described in Example 6). This experimental data proves the model that for a functional AND NOT gate with PTPN6, co-localizing spacers are needed. 
     Example 9: Experimental Proof of Kinetic Segregation Model of ITIM Based AND NOT Gate 
     Similar to the PTPN6 based AND NOT gate, the ITIM based gate also requires co-localization in an immunological synapse to function as an AND NOT gate. To prove this hypothesis, a control ITIM based gate was generated as follows: two CARs co-expressed—the first recognizes CD19, has a human CD8 stalk spacer and an activating endodomain; co-expressed with an anti-CD33 CAR with an Fc spacer and an ITIM containing endodomain derived from that of LAIR1. The activity of this gate was compared with that of the original ITIM based AND NOT gate. In this case, the modified gate activated in response to targets expressing CD19, but also activated in response to cells expressing both CD19 and CD33. These data indicate that ITIM based AND NOT gates follow the kinetic segregation based model and a correct spacer must be selected to create a functional gate ( FIG. 23B ). 
     Example 10: Summary of Model of CAR Logic Gates Generated by Kinetic Segregation 
     Based on current understanding of the kinetic-segregation model and the experimental data described herein, a summary of the model for a two-CAR gate is presented in  FIG. 24 . The Figure shows a cell expressing two CARs, each recognizing a different antigen. When either or both CARs recognize a target antigen on a cell, a synapse forms and native CD45 and CD148 are excluded from the synapse due to the bulk of their ectodomain. This sets the stage for T-cell activation. In the case that the target cell bears only one cognate antigen, the cognate CAR is ligated and the cognate CAR segregates into the synapse. The unligated CAR remains in solution on the T-cell membrane and can diffuse in and out of the synapse so that an area of high local concentration of ligated CAR with low concentration of unligated CAR forms. In this case, if the ligated CAR has an ITAM and the non-ligated CAR has ‘ligation off” type inhibitory endodomain such as that of CD148, the amount of non-ligated CAR is sufficient to inhibit activation and the gate is off. In contrast, in this case, if the ligated CAR has an ITAM and the non-ligated CAR has a ‘ligation on’ type inhibitory endodomain such as PTPN6, the amount of non-ligated CAR is insufficient to inhibit and the gate is on. When challenged by a target cell bearing both cognate antigens, both cognate CARs are ligated and form part of an immunological synapse. Importantly, if the CAR spacers are sufficiently similar, the CARs co-localize in the synapse but if the CAR spacers are sufficiently different the CARs segregate within the synapse. In this latter case, areas of membrane form whereby high concentrations of one CAR are present but the other CAR is absent. In this case since segregation is complete, even if the inhibitory endodomain is a ‘ligation off’ type, the gate is on. In the former case, areas of membrane form with high concentrations of both CARs mixed together. In this case, since both endodomains are concentrated, even if the inhibitory endodomain is ‘ligation on’ type, the gate is off. By selecting the correct combination of spacer and endodomain logic can be programmed into a CAR T-cell. 
     Based on our work above, we have established a series of design rules to allow generation of logic-gated CARs (illustrated in  FIG. 31 ). To generate an “antigen A OR antigen B” gated CAR T-cell, anti-A and anti-B CARs must be generated such that (1) each CAR has a spacer which simply allows antigen access and synapse formation such that the CAR functions, and (2) Each CAR has an activating endodomain; To generate an “antigen A AND NOT B” gated CAR T-cell, anti-A and anti-B CARs must be generated such that (1) both CARs have spacers which do not cross-pair, but which will allow the CARs to co-segregate upon recognition of both cognate antigens on the target cell, (2) and one CAR has an activating endodomain, while the other CAR has an endodomain which comprises or recruits a weak phosphatase (e.g. PTPN6); (3) To generate an “antigen A AND antigen B” gated CAR T-cell, anti-A and anti-B CARs must be generated such that (1) one CAR has a spacer sufficiently different from the other CAR such that both CARs will not co-segregate upon recognition of both cognate antigens on the target cell, (2) one CAR has an activating endodomain, while the other car has an endodomain which comprises of a potent phosphatase (e.g. that of CD45 or CD148). The correct spacers to achieve the desired effect can be selected from a set of spacers with known size/shape etc as well as taking into consideration size/shape etc of the target antigen and the location of the cognate epitope on the target antigen. 
     
       
         
           
               
               
            
               
                 SEQ ID No 41: SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148 
                   
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL 
               
               
                   
               
               
                 IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS 
               
               
                   
               
               
                 ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDP 
               
               
                   
               
               
                 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAF 
               
               
                   
               
               
                 IIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL 
               
               
                   
               
               
                 QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENP 
               
               
                   
               
               
                 GPMETDTLLLWVLLLWVPGSTGQVQLQESGPGLVKPSQTLSITCTVSGFSLASYNIHWVRQPPGKGLE 
               
               
                   
               
               
                 WLGVIWAGGSTNYNSALMSRLTISKDNSKNQVFLKMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLV 
               
               
                   
               
               
                 TVSSGGGGSGGGGSGGGGSENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKVWI 
               
               
                   
               
               
                 YSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPITFGQGTKVEIKRSDPAEPKS 
               
               
                   
               
               
                 PDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK 
               
               
                   
               
               
                 TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD 
               
               
                   
               
               
                 ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS 
               
               
                   
               
               
                 CSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPK 
               
               
                   
               
               
                 KSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKL 
               
               
                   
               
               
                 SVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEY 
               
               
                   
               
               
                 WPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRY 
               
               
                   
               
               
                 LVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQY 
               
               
                   
               
               
                 VFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA 
               
               
                   
               
               
                 SEQ ID No. 42: SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACAT 
               
               
                   
               
               
                 CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAG 
               
               
                   
               
               
                 CCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTG 
               
               
                   
               
               
                 ATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGA 
               
               
                   
               
               
                 CTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACA 
               
               
                   
               
               
                 CCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGC 
               
               
                   
               
               
                 GGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCC 
               
               
                   
               
               
                 AGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACT 
               
               
                   
               
               
                 ACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGC 
               
               
                   
               
               
                 GAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCA 
               
               
                   
               
               
                 GGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACT 
               
               
                   
               
               
                 ACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCC 
               
               
                   
               
               
                 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG 
               
               
                   
               
               
                 CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATA 
               
               
                   
               
               
                 TCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT 
               
               
                   
               
               
                 ATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA 
               
               
                   
               
               
                 GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG 
               
               
                   
               
               
                 GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG 
               
               
                   
               
               
                 CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG 
               
               
                   
               
               
                 GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG 
               
               
                   
               
               
                 CCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCC 
               
               
                   
               
               
                 GGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCCA 
               
               
                   
               
               
                 GGTGCAGCTGCAGGAGTCTGGCCCAGGCCTGGTGAAGCCCAGCCAGACCCTGAGCATCACCTGCACCG 
               
               
                   
               
               
                 TGAGCGGCTTCAGCCTGGCCAGCTACAACATCCACTGGGTGCGGCAGCCCCCAGGCAAGGGCCTGGAG 
               
               
                   
               
               
                 TGGCTGGGCGTGATCTGGGCTGGCGGCAGCACCAACTACAACAGCGCCCTGATGAGCCGGCTGACCAT 
               
               
                   
               
               
                 CAGCAAGGACAACAGCAAGAACCAGGTGTTCCTGAAGATGAGCAGCCTGACAGCCGCCGACACCGCCG 
               
               
                   
               
               
                 TGTACTACTGCGCCAAGCGGAGCGACGACTACAGCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTG 
               
               
                   
               
               
                 ACCGTGAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGAACCAGAT 
               
               
                   
               
               
                 GACCCAGAGCCCCAGCAGCTTGAGCGCCAGCGTGGGCGACCGGGTGACCATGACCTGCAGAGCCAGCA 
               
               
                   
               
               
                 GCAGCGTGAGCAGCAGCTACCTGCACTGGTACCAGCAGAAGAGCGGCAAGGCCCCAAAGGTGTGGATC 
               
               
                   
               
               
                 TACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTA 
               
               
                   
               
               
                 CACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCGGCT 
               
               
                   
               
               
                 ACCCCATCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAATCT 
               
               
                   
               
               
                 CCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTT 
               
               
                   
               
               
                 CCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG 
               
               
                   
               
               
                 TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG 
               
               
                   
               
               
                 ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA 
               
               
                   
               
               
                 GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA 
               
               
                   
               
               
                 AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAT 
               
               
                   
               
               
                 GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT 
               
               
                   
               
               
                 GGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG 
               
               
                   
               
               
                 GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA 
               
               
                   
               
               
                 TGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAA 
               
               
                   
               
               
                 GAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCA 
               
               
                   
               
               
                 TCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAA 
               
               
                   
               
               
                 AAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTG 
               
               
                   
               
               
                 TGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAAC 
               
               
                   
               
               
                 TGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTT 
               
               
                   
               
               
                 TCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAA 
               
               
                   
               
               
                 AGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGA 
               
               
                   
               
               
                 AAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTAT 
               
               
                   
               
               
                 TGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCC 
               
               
                   
               
               
                 GGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGT 
               
               
                   
               
               
                 TCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTAC 
               
               
                   
               
               
                 CTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGT 
               
               
                   
               
               
                 CGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGG 
               
               
                   
               
               
                 ATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTAT 
               
               
                   
               
               
                 GTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTA 
               
               
                   
               
               
                 CCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATG 
               
               
                   
               
               
                 GTTACATCGCCTAA 
               
               
                   
               
               
                 SEQ ID No. 43: SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL 
               
               
                   
               
               
                 IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS 
               
               
                   
               
               
                 ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDP 
               
               
                   
               
               
                 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAF 
               
               
                   
               
               
                 IIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL 
               
               
                   
               
               
                 QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENP 
               
               
                   
               
               
                 GPMETDTLLLWVLLLWVPGSTGQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKG 
               
               
                   
               
               
                 LEWLAHIWWDDDVYYNPSLKNQLTISKDASRDQVFLKITNLDTADTATYYCVRRRATGTGFDYWGQGT 
               
               
                   
               
               
                 TLTVSSGGGGSGGGGSGGGGSNIVMTQSHKFMSTSVGDRVSIACKASQDVGTAVAWYQQKPGQSPKLL 
               
               
                   
               
               
                 IYWTSTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCHQYNSYNTFGSGTRLELKRSDPAEPKS 
               
               
                   
               
               
                 PDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK 
               
               
                   
               
               
                 TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD 
               
               
                   
               
               
                 ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS 
               
               
                   
               
               
                 CSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPK 
               
               
                   
               
               
                 KSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKL 
               
               
                   
               
               
                 SVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEY 
               
               
                   
               
               
                 WPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRY 
               
               
                   
               
               
                 LVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQY 
               
               
                   
               
               
                 VFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA 
               
               
                   
               
               
                 SEQ ID No. 44: SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACAT 
               
               
                   
               
               
                 CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAG 
               
               
                   
               
               
                 CCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTG 
               
               
                   
               
               
                 ATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGA 
               
               
                   
               
               
                 CTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACA 
               
               
                   
               
               
                 CCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGC 
               
               
                   
               
               
                 GGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCC 
               
               
                   
               
               
                 AGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACT 
               
               
                   
               
               
                 ACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGC 
               
               
                   
               
               
                 GAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCA 
               
               
                   
               
               
                 GGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACT 
               
               
                   
               
               
                 ACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCC 
               
               
                   
               
               
                 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG 
               
               
                   
               
               
                 CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATA 
               
               
                   
               
               
                 TCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT 
               
               
                   
               
               
                 ATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA 
               
               
                   
               
               
                 GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG 
               
               
                   
               
               
                 GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG 
               
               
                   
               
               
                 CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG 
               
               
                   
               
               
                 GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG 
               
               
                   
               
               
                 CCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCC 
               
               
                   
               
               
                 GGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCA 
               
               
                   
               
               
                 GGTGACCCTGAAGGAGAGCGGTCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCT 
               
               
                   
               
               
                 TCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATTCGGCAGCCCAGCGGCAAGGGC 
               
               
                   
               
               
                 CTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACGTGTACTACAACCCCAGCCTGAAGAACCAGCT 
               
               
                   
               
               
                 GACCATCAGCAAGGACGCCAGCCGGGACCAGGTGTTCCTGAAGATCACCAACCTGGACACCGCCGACA 
               
               
                   
               
               
                 CCGCCACCTACTACTGCGTGCGGCGCCGGGCCACCGGCACCGGCTTCGACTACTGGGGCCAGGGCACC 
               
               
                   
               
               
                 ACCCTGACCGTGAGCAGCGGTGGCGGTGGCAGCGGCGGCGGCGGAAGCGGAGGTGGTGGCAGCAACAT 
               
               
                   
               
               
                 CGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGACCGGGTGAGCATCGCCTGCAAGG 
               
               
                   
               
               
                 CCAGCCAGGACGTGGGCACCGCCGTGGCCTGGTACCAGCAGAAGCCTGGCCAGAGCCCCAAGCTGCTG 
               
               
                   
               
               
                 ATCTACTGGACCAGCACCCGGCACACCGGCGTGCCCGACCGGTTCACCGGCAGCGGCAGCGGCACCGA 
               
               
                   
               
               
                 CTTCACCCTGACCATCACCAACGTGCAGAGCGAGGACCTGGCCGACTACTTCTGCCACCAGTACAACA 
               
               
                   
               
               
                 GCTACAACACCTTCGGCAGCGGCACCCGGCTGGAGCTGAAGCGGTCGGATCCCGCCGAGCCCAAATCT 
               
               
                   
               
               
                 CCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTT 
               
               
                   
               
               
                 CCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG 
               
               
                   
               
               
                 TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG 
               
               
                   
               
               
                 ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA 
               
               
                   
               
               
                 GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA 
               
               
                   
               
               
                 AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAT 
               
               
                   
               
               
                 GAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT 
               
               
                   
               
               
                 GGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG 
               
               
                   
               
               
                 GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA 
               
               
                   
               
               
                 TGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAA 
               
               
                   
               
               
                 GAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCA 
               
               
                   
               
               
                 TCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAA 
               
               
                   
               
               
                 AAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTG 
               
               
                   
               
               
                 TGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAAC 
               
               
                   
               
               
                 TGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTT 
               
               
                   
               
               
                 TCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAA 
               
               
                   
               
               
                 AGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGA 
               
               
                   
               
               
                 AAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTAT 
               
               
                   
               
               
                 TGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCC 
               
               
                   
               
               
                 GGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGT 
               
               
                   
               
               
                 TCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTAC 
               
               
                   
               
               
                 CTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGT 
               
               
                   
               
               
                 CGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGG 
               
               
                   
               
               
                 ATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTAT 
               
               
                   
               
               
                 GTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTA 
               
               
                   
               
               
                 CCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATG 
               
               
                   
               
               
                 GTTACATCGCCTAA 
               
               
                   
               
               
                 SEQ ID No. 45: SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- 
               
               
                 dCD148 
               
               
                 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLL 
               
               
                   
               
               
                 IYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSG 
               
               
                   
               
               
                 GGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS 
               
               
                   
               
               
                 ETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDP 
               
               
                   
               
               
                 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAF 
               
               
                   
               
               
                 IIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL 
               
               
                   
               
               
                 QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENP 
               
               
                   
               
               
                 GPMETDTLLLWVLLLWVPGSTGQVKLQQSGGGLVKPGASLKLSCVTSGFTFRKFGMSWVRQTSDKRLE 
               
               
                   
               
               
                 WVASISTGGYNTYYSDNVKGRFTISRENAKNTLYLQMSSLKSEDTALYYCTRGYSSTSYAMDYWGQGT 
               
               
                   
               
               
                 TVTVSSGGGGSGGGGSGGGGSDIELTQSPASLSVATGEKVTIRCMTSTDIDDDMNWYQQKPGEPPKFL 
               
               
                   
               
               
                 ISEGNTLRPGVPSRFSSSGTGTDFVFTIENTLSEDVGDYYCLQSFNVPLTFGDGTKLEIKRSDPAEPK 
               
               
                   
               
               
                 SPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 
               
               
                   
               
               
                 KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR 
               
               
                   
               
               
                 DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF 
               
               
                   
               
               
                 SCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKP 
               
               
                   
               
               
                 KKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVK 
               
               
                   
               
               
                 LSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEE 
               
               
                   
               
               
                 YWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFR 
               
               
                   
               
               
                 YLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQ 
               
               
                   
               
               
                 YVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA 
               
               
                   
               
               
                 SEQ ID No. 46: SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- 
               
               
                 dCD148 
               
               
                 ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACAT 
               
               
                   
               
               
                 CCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAG 
               
               
                   
               
               
                 CCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTG 
               
               
                   
               
               
                 ATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGA 
               
               
                   
               
               
                 CTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACA 
               
               
                   
               
               
                 CCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGC 
               
               
                   
               
               
                 GGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCC 
               
               
                   
               
               
                 AGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACT 
               
               
                   
               
               
                 ACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGC 
               
               
                   
               
               
                 GAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCA 
               
               
                   
               
               
                 GGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACT 
               
               
                   
               
               
                 ACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCC 
               
               
                   
               
               
                 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCG 
               
               
                   
               
               
                 CCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATA 
               
               
                   
               
               
                 TCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTT 
               
               
                   
               
               
                 ATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA 
               
               
                   
               
               
                 GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG 
               
               
                   
               
               
                 GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG 
               
               
                   
               
               
                 CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG 
               
               
                   
               
               
                 GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGG 
               
               
                   
               
               
                 CCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCC 
               
               
                   
               
               
                 GGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCA 
               
               
                   
               
               
                 GGTGAAGCTGCAGCAGAGCGGCGGAGGCCTGGTGAAGCCCGGCGCCAGCCTGAAGCTGAGCTGCGTGA 
               
               
                   
               
               
                 CCAGCGGCTTCACCTTCCGGAAGTTCGGCATGAGCTGGGTGCGGCAGACCAGCGACAAGCGGCTGGAG 
               
               
                   
               
               
                 TGGGTGGCCAGCATCAGCACCGGCGGCTACAACACCTACTACAGCGACAACGTGAAGGGCCGGTTCAC 
               
               
                   
               
               
                 CATCAGCCGGGAGAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAAGAGCGAGGACACCG 
               
               
                   
               
               
                 CCCTGTACTACTGCACCCGGGGCTACAGCAGCACCAGCTACGCTATGGACTACTGGGGCCAGGGCACC 
               
               
                   
               
               
                 ACCGTGACAGTGAGCAGCGGCGGAGGAGGCAGTGGTGGGGGTGGATCTGGCGGAGGTGGCAGCGACAT 
               
               
                   
               
               
                 CGAGCTGACCCAGAGCCCCGCCAGCCTGAGCGTGGCCACCGGCGAGAAGGTGACCATCCGGTGCATGA 
               
               
                   
               
               
                 CCAGCACCGACATCGACGACGACATGAACTGGTACCAGCAGAAGCCCGGCGAGCCCCCAAAGTTCCTG 
               
               
                   
               
               
                 ATCAGCGAGGGCAACACCCTGCGGCCCGGCGTGCCCAGCCGGTTCAGCAGCAGCGGCACCGGCACCGA 
               
               
                   
               
               
                 CTTCGTGTTCACCATCGAGAACACCCTGAGCGAGGACGTGGGCGACTACTACTGCCTGCAGAGCTTCA 
               
               
                   
               
               
                 ACGTGCCCCTGACCTTCGGCGACGGCACCAAGCTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAA 
               
               
                   
               
               
                 TCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCT 
               
               
                   
               
               
                 CTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGG 
               
               
                   
               
               
                 ACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC 
               
               
                   
               
               
                 AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA 
               
               
                   
               
               
                 CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG 
               
               
                   
               
               
                 AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG 
               
               
                   
               
               
                 GATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGC 
               
               
                   
               
               
                 CGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG 
               
               
                   
               
               
                 ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC 
               
               
                   
               
               
                 TCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGG 
               
               
                   
               
               
                 CAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCT 
               
               
                   
               
               
                 TCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCT 
               
               
                   
               
               
                 AAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAA 
               
               
                   
               
               
                 CTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAG 
               
               
                   
               
               
                 AACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAA 
               
               
                   
               
               
                 CTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAA 
               
               
                   
               
               
                 GAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGG 
               
               
                   
               
               
                 AGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAG 
               
               
                   
               
               
                 TATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCT 
               
               
                   
               
               
                 TCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGAC 
               
               
                   
               
               
                 AGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGG 
               
               
                   
               
               
                 TACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGG 
               
               
                   
               
               
                 GGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCG 
               
               
                   
               
               
                 TGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAG 
               
               
                   
               
               
                 TATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTAT 
               
               
                   
               
               
                 CTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCA 
               
               
                   
               
               
                 ATGGTTACATCGCCTAA 
               
            
           
         
       
     
     Example 11: Design and Construction of APRIL Based CARs 
     APRIL in its natural form is a secreted type II protein. The use of APRIL as a BCMA binding domain for a CAR requires conversion of this type II secreted protein to a type I membrane bound protein and for this protein to be stable and to retain binding to BCMA in this form. To generate candidate molecules, the extreme amino-terminus of APRIL was deleted to remove binding to proteoglycans. Next, a signal peptide was added to direct the nascent protein to the endoplasmic reticulum and hence the cell surface. Also, because the nature of spacer used can alter the function of a CAR, three different spacer domains were tested: an APRIL based CAR was generated comprising (i) a human IgG1 spacer altered to remove Fc binding motifs; (ii) a CD8 stalk; and (iii) the IgG1 hinge alone (cartoon in  FIG. 25  and amino acid sequences in  FIG. 26 ). These CARs were expressed in a bicistronic retroviral vector ( FIG. 27A ) so that a marker protein—truncated CD34 could be co-expressed as a convenient marker gene. 
     Example 12: Expression and Function of APRIL Based CARs 
     The aim of this study was to test whether the APRIL based CARs which had been constructed were expressed on the cell surface and whether APRIL had folded to form the native protein. T-cells were transduced with these different CAR constructs and stained using a commercially available anti-APRIL mAb, along with staining for the marker gene and analysed by flow-cytometry. The results of this experiment are shown in  FIG. 27B  where APRIL binding is plotting against marker gene fluorescence. These data show that in this format, the APRIL based CARs are expressed on the cell surface and APRIL folds sufficiently to be recognized by an anti-APRIL mAb. 
     Next, it was determined whether APRIL in this format could recognize BCMA and TACl. Recombinant BCMA and TACl were generated as fusions with mouse IgG2a-Fc. These recombinant proteins were incubated with the transduced T-cells. After this, the cells were washed and stained with an anti-mouse fluorophore conjugated antibody and an antibody to detect the marker gene conjugated to a different fluorophore. The cells were analysed by flow cytometry and the results are presented in  FIG. 27C . The different CARs were able to bind both BCMA and TACl. Surprisingly, the CARs were better able to bind BCMA than TACl. Also, surprisingly CARs with a CD8 stalk or IgG1 hinge spacer were better able to bind BCMA and TACl than CAR with an Fc spacer. 
     Example 13: APRIL Based Chimeric Antigen Receptors are Active Against BCMA Expressing Cells 
     T-cells from normal donors were transduced with the different APRIL CARs and tested against SupT1 cells either wild-type, or engineered to express BCMA and TACI. Several different assays were used to determine function. A classical chromium release assay was performed. Here, the target cells (the SupT1 cells) were labelled with 51Cr and mixed with effectors (the transduced T-cells) at different ratio. Lysis of target cells was determined by counting 51Cr in the co-culture supernatant ( FIG. 28A  shows the cumulative data). 
     In addition, supernatant from T-cells cultured 1:1 with SupT1 cells was assayed by ELISA for Interferon-gamma ( FIG. 28B  shows cumulative data). Measurement of T-cell expansion after one week of co-culture with SupT1 cells was also performed ( FIG. 28C ). T-cells were counted by flow-cytometry calibrated with counting beads. These experimental data show that APRIL based CARs can kill BCMA expressing targets. Further, these data show that CARs based on the CD8 stalk or IgG1 hinge performed better than the Fc-pvaa based CAR. 
     Example 14: Functional Analysis of the AND Gate in Primary Cells 
     PBMCs were isolated from blood and stimulated using PHA and IL-2. Two days later the cells were transduced on retronectin coated plates with retro virus containing the CD19:CD33 AND gate construct. On day 5 the expression level of the two CARs translated by the AND gate construct was evaluated via flow cytometry and the cells were depleted of CD56+ cells (predominantly NK cells). On day 6 the PBMCs were placed in a co-culture with target cells at a 1:2 effector to target cell ratio. On day 8 the supernatant was collected and analysed for IFN-gamma secretion via ELISA ( FIG. 29 ). 
     These data demonstrate that the AND gate functions in primary cells. 
     All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cell biology or related fields are intended to be within the scope of the following claims.