THERAPEUTIC AGENTS

Abstract

An immunoresponsive cell, such as a T-cell expressing (i) a second generation chimeric antigen receptor comprising: (a) a signalling region; (b) a co-stimulatory signalling region; (c) a transmembrane domain; and (d) a binding element that specifically interacts with a first epitope on a target antigen; and (ii) a chimeric costimulatory receptor comprising (e) a co-stimulatory signalling region which is different to that of (b); (f) a transmembrane domain; and g) a binding element that specifically interacts with a second epitope on a target antigen.

This arrangement is referred to as parallel chimeric activating receptors (pCAR). Cells of this type are useful in therapy, and kits and methods for using them as well as methods for preparing them are described and claimed.

Claims

1. An immuno-responsive cell, expressing: (i) a first single-pass transmembrane fusion protein comprising means for delivering an activation signal and means for delivering a first co-stimulatory signal upon a first antigen-specific engagement; and (ii) a second single-pass transmembrane fusion protein comprising means for delivering a second co-stimulatory signal upon a second antigen-specific engagement, wherein the first co-stimulatory signal is different from the second co-stimulatory signal.

2. The immuno-responsive cell of claim 1, wherein the means for delivering the first co-stimulatory signal and the means for delivering the second co-stimulatory signal are costimulatory regions separately selected from B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2, PDCD6, LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, LILRB4, 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha, TNF RII, 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A, SLAM, TIM-1, TIM-3, TIM-4, CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, and TSLP R.

3. The immuno-responsive cell of claim 1, wherein the means for delivering the activation signal is selected from human CD3? or a variant thereof.

4. The immune-responsive cell of claim 1, wherein the immune-responsive cell is a T-cell or a Natural Killer (NK) cell.

5. An isolated immuno-responsive cell expressing: (i) a second generation chimeric antigen receptor (CAR); and (ii) a modified second generation CAR that lacks a signal activation region (a chimeric costimulatory receptor, CCR), wherein the co-stimulatory signaling region of the CAR is different from the co-stimulatory signaling region of the CCR.

6. The isolated immuno-responsive cell of claim 5, wherein the CAR comprises a signal activation region selected from human CD3? or a variant thereof.

7. The isolated immuno-responsive cell of claim 5, wherein the CAR and CCR each has a co-stimulatory signaling region separately selected from B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2, PDCD6, LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, LILRB4, 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha, TNF RII, 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A, SLAM, TIM-1, TIM-3, TIM-4, CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, and TSLP R.

8. The isolated immuno-responsive cell of claim 7, wherein the CAR has a co-stimulatory signaling region selected from CD28, CD27, ICOS, 4-1 BB, OX40, CD30, GITR, HVEM, DR3, and CD40.

9. The isolated immuno-responsive cell of claim 5, wherein the CAR has a transmembrane domain selected from CD8?, CD28, CD4, or CD3? transmembrane domains or a variant thereof; and the CCR has a transmembrane domain selected from CD8?, CD28, CD4, or CD3? transmembrane domains or a variant thereof.

10. The isolated immuno-responsive cell of claim 9, wherein the CAR transmembrane domain and the CCR transmembrane domain are the same.

11. The isolated immuno-responsive cell of claim 9, wherein the CAR transmembrane domain and the CCR transmembrane domain are different.

12. The isolated immuno-responsive cell of claim 5, wherein the immuno-responsive cell is a T-cell or a Natural Killer (NK) cell.

13. In an immuno-responsive cell expressing a second generation chimeric antigen receptor (CAR), the improvement comprising: expressing a modified second generation CAR that lacks a signal activation region (chimeric costimulatory receptor, CCR), wherein the CCR comprises a co-stimulatory signaling region that is different from the co-stimulatory signaling region of the CAR.

14. An immuno-responsive cell comprising one or more vectors collectively encoding: (i) a second generation chimeric antigen receptor (CAR); and (ii) a modified second generation CAR that lacks a signal activation region (chimeric costimulatory receptor, CCR), wherein each of the CAR and CCR comprise a co-stimulatory signaling region and the co-stimulatory signaling region of the CAR is different from the co-stimulatory signaling region of the CCR.

15. A method of making a construct capable of expressing a parallel chimeric antigen receptor (pCAR), the method comprising: cloning into an expression vector polynucleotides encoding (i) a second generation chimeric antigen receptor (CAR); and (ii) a modified second generation CAR that lacks a signal activation region (chimeric costimulatory receptor, CCR), wherein each of the CAR and CCR comprise a co-stimulatory signaling region and the co-stimulatory signaling region of the CAR is different from the co-stimulatory signaling region of the CCR.

16. A method of enhancing cytotoxic activity of second generation CAR-T cells, comprising: expressing a chimeric costimulatory receptor (CCR) in the second generation CAR-T cells, wherein the CCR comprises a co-stimulatory signaling region that is different from the co-stimulatory region of the CAR.

17. A viral vector, comprising: (i) a coding sequence of a signaling region; (ii) a coding sequence of a co-stimulatory signaling region, and (iii) a coding sequence of a transmembrane domain; (iv) a coding region of an extracellular domain; (v) a first ribosomal skip peptide; (vi) a coding sequence of a co-stimulatory signaling region which is different from that of (ii); (vi) a coding sequence of a transmembrane domain; and (vii) a coding region of an extracellular domain.

18. The viral vector of claim 17, further comprising: a second ribosomal skip peptide and a coding sequence of an IL-4 responsive 4?? receptor.

19. The viral vector of claim 17 or 18, wherein the viral vector is a retroviral vector.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0069] The invention will now be particularly described by way of example and with reference to the following Figures in which:

[0070] FIGS. 1A,B is a schematic diagram showing a panel of CARs and pCARs (named C34B and 34CB) embodying the invention. All CARs and pCARs were co-expressed in the SFG retroviral vector with 4??, a chimeric cytokine receptor in which the IL-4 receptor-? ectodomain has been fused to the transmembrane and endodomain of IL-2 receptor-?. Use of 4?? allows selective enrichment and expansion of gene-modified T-cells by culture in IL-4, since it recruits the gamma c (?c) chain.

[0071] FIGS. 2A,B shows the results of an experiment using CARs shown in FIG. 1A,B. T-cells (1?10.sup.6 cells) expressing these CARs and pCARs (or untransduced (UT) as control) were co-cultivated in vitro for 24 hours with T47D tumour cells that express (T47D-FMS) or lack (T47D) the cognate target antigen (Colony-stimulating factor-1 receptor (CSF-1R), encoded by c-fms). Residual viable tumour cells were then quantified by MTT assay.

[0072] FIG. 3 shows a representative experiment in which T-cells that express CARs and pCARs of FIG. 1A,B (or untransduced) T-cells as control) were subjected to successive rounds of Ag stimulation in the absence of exogenous cytokine. Stimulation was provided by weekly culture on T47D FMS monolayers and T-cell numbers were enumerated at the indicated intervals.

[0073] FIG. 4 shows pooled data from 7 similar replicate experiments to that shown in FIG. 3, indicating the fold expansion of CAR T-cells that occurred in the week after each cycle of stimulation.

[0074] FIG. 5 shows illustrative cytotoxicity assays performed at the time of stimulation cycles 2, 6, 9 and 12 in the experiment shown in FIG. 3. This follows from the testing of T-cells for their ability to kill T47D FMS and unmodified T47D monolayers (MTT assay), twenty four hours after the time of each re-stimulation cycle.

[0075] FIG. 6 shows the results of testing of supernatant, removed from cultures one day after each cycle of stimulation, for IL-2 and IFN-? content by ELISA.

[0076] FIGS. 7A-D demonstrates the establishment of an in vivo xenograft model of CSF-1R-expressing anaplastic large cell lymphoma, which allowed subsequent testing of anti-tumour activity of CAR and pCAR-engineered T-cells. The model was established using K299 cells, engineered to express firefly luciferase (luc) and red fluorescent protein (RFP). FIG. 7A shows tumour formation following the intravenous injection of the indicated doses of K299 luc cells, quantified by bioluminescence imaging (BLI). Representative BLI images are shown in FIG. 7B in mice that received 2 million tumour cells. Expression of RFP.sup.+ tumour cells (FIG. 7C) in the indicated tissues are shown, demonstrating that tumours only formed in lymph nodes in this model. Expression of the CSF-1R on five representative lymph node tumours is shown in FIG. 7D.

[0077] FIGS. 8A,B shows the results of therapeutic studies in which K299 luc cells were injected intravenously in SCID Beige mice (n=9 per group, divided over 2 separate experiments). After 5 days, mice were treated with CAR T-cells. Pooled bioluminescence emission from tumours is shown in FIG. 8A. Bioluminescence emission from individual mice is shown in FIG. 8B and survival of mice shown in FIG. 8A.

[0078] FIG. 9 shows the weights of animals used in the therapeutic study over time.

[0079] FIGS. 10-13 show the results of analysis of the expression of exhaustion markers from dual CAR (C34B) expressing T-cells of the invention where FIG. shows the results for PD1 analysis, FIG. 11 shows the results for TIM3 analysis, FIG. 12 shows the results of LAG3 analysis and FIG. 13 shows the results for 2B4 analysis.

[0080] FIG. 14 is a schematic diagram of a panel of CARs and constructs targeted to the integrin ?v?6 which have been prepared including a pCAR (named SFG TIE-41 BB/A20-28z) embodying the invention. A20-28z is a second generation CAR that is targeted using the A20 peptide derived from foot and mouth disease virus. A20 binds with high affinity to ?v?6 and with 85-1000 fold lower affinity to other RGD-binding integrins. C20-28z is a matched control in which key elements of A20 have been mutated to abrogate integrin binding activity. All CARs have been co-expressed with 4?? as described in FIG. 1A,B.

[0081] FIGS. 15A,B is a series of histograms obtained by flow cytometry illustrating integrin expression in A375 puro and Panc1 cells. Cells were stained with anti-?6 (Biogen Idec) followed by secondary anti-mouse PE, anti-?v? or anti-?v?5 (both APC conjugated, Bio-Techne). Gates were set based on secondary antibody alone or isotype controls.

[0082] FIGS. 16A,B is a series of graphs illustrating the cytotoxicity of CARs including the pCARs of the invention targeted to ?v?6. T-cells expressing the indicated CARs and pCARs were co-cultivated with ?v?6-negative (Panc1 and A375 puro) or ?v?6-positive (Bxpc3 and A375 puro ?6) tumour cells. Data show the mean?SEM of 2-7 independent experiments, each performed in triplicate. *p<0.05; **p<0.01; ***p<0.001.

[0083] FIGS. 17A,B is a series of graphs showing production of IFN-? by CARs including pCARs of the invention, targeted to ?v?6. T-cells expressing the indicated CARs and pCARs were co-cultivated with ?v?6-negative (Panc1 and A375 puro) or ?v?6-positive (Bxpc3 and A375 puro ?6) tumour cells. Data show the mean?SEM of 5-6 independent experiments, each performed in duplicate. *p<0.05; **p<0.01; ***p<0.001; nsnot significant.

[0084] FIGS. 18A,B shows the results of re-stimulation experiments using the CAR and pCAR-engineered T-cells described above and indicating the ability of A20-28z/T1E-41 BB pCAR T-cells to undergo repeated antigen stimulation, accompanied by expansion of T-cells and destruction of target cells that do (Bxpc3) or do not (Panc1) express the ?v?6 integrin.

[0085] FIGS. 19A,B shows the results of re-stimulation experiments using pCAR-engineered T-cells in which A20-28z was co-expressed with T1E-41 BB, T1E-CD27 or T1E-CD40, allowing the comparative evaluation of co-stimulation by additional members of the TNF receptor family. Control T-cells were non-transduced (NT) while CARs contained truncated (tr) endodomains. T-cells were re-stimulated on target cells that do (Bxpc3) or do not (Panc1) express the ?v?6 integrin, making comparison with unstimulated T-cells. In the case of Bxpc3 cells, superior expansion (FIG. 19A) accompanied by sustained cytotoxic activity (FIG. 19B) was observed with A20-28z/T1E-CD27 T-cells. By contrast, with Panc1 cells, superior expansion (FIG. 19A) accompanied by sustained cytotoxic activity (FIG. 19B) was observed with A20-28z/T1E-CD27 T-cells. These data demonstrate that additional members of the TNF receptor family can also deliver co-stimulation using the pCAR format.

Example 1

[0086] A panel of CARs targeted against the CSF-1 receptor (encoded by c-FMS), which is over-expressed in Hodgkin's lymphoma, anaplastic large cell lymphoma and some solid tumours such as triple negative breast cancer were prepared and are illustrated schematically in FIG. 1A,B. The panel of CARs included both second and third generation CARs with either of the two natural ligands, CSF-1 or IL-34, as the targeting moieties. Although both CSF-1 and IL-34 bind to CSF-1 receptor, IL-34 binds with much higher affinity (34-fold higher than CSF-1).

[0087] The constructs SFG C28? and SFG CTr were cloned in the SFG retroviral vector as Ncol/Xhol fragments, ensuring that their start codons are at the site of the naturally occurring Ncol site, previously occupied by the deleted env gene. Gene expression is achieved from the Moloney murine leukaemia virus (MoMLV) long terminal repeat (LTR), which has promoter activity and virus packaging of the RNA is ensured by the MoMLV ? packaging signal, which is flanked by splice donor and acceptor sites.

[0088] All other constructs were designed and cloned using the Polymerase Incomplete Primer Extension (PIPE) cloning method. PIPE cloning method is a PCR-based alternative to conventional restriction enzyme- and ligation-dependent cloning methods. It eliminates the need to incorporate restriction sites, which could encode additional unwanted residues into expressed proteins. The PIPE method relies on the inefficiency of the amplification process in the final cycles of a PCR reaction, possibly due to the decreasing availability of dNTPs, which results in the generation of partially single-stranded (PIPE) PCR products with overhanging 5 ends. A set of vector-specific primers was used for PCR vector linearization and another set of primers with 5-vector-end overlapping sequences then used for insert amplification, generating incomplete extension products by PIPE. In a following step, the PIPE products were mixed and the single-stranded overlapping sequences annealed and assembled as a complete SFG CAR construct. Successful cloning was confirmed by diagnostic restriction digestion. DNA sequencing was performed on all constructs to confirm that the predicted coding sequence was present, without any PCR-induced mutations (Source Bioscience, UK).

[0089] The panel included two dual targeted Chimeric Activating Receptors (pCARS) in which CSF-1 or IL-34 are coupled to 28z and 4-1 BB, or vice versa. The dual targeted pCAR combinations were then stoichiometrically co-expressed in the same T-cell population using a Thosea Asigna (T)2A-containing retroviral vector. One of these CARs was designated C34B (CSF1-28z plus IL34-41 BB) and the other was named 34CB (IL34-28z plus CSF1-41 BB).

[0090] In these dual targeted CAR T-cells, both co-stimulatory motifs (CD28/4-1 BB) are placed in their natural location, close to the membrane, physically separated from each other and co-expressed in the same T-cell.

[0091] All CARs were co-expressed with an IL-4 responsive 4?? receptor using an additional T2A element in the vector. This enables enrichment/expansion of T-cells using IL-4, making it easier to compare the function of these diverse cell populations after selection.

[0092] The main focus of the experiments was to test the behaviour of the T-cells on repeated re-stimulation with tumour target cells that either express or lack the FMS/CSF-1 receptor target. In each cycle, 1 million of the indicated IL-4 expanded CAR T-cells were suspended in RPMI+human AB serum and cultured with a confluent monolayer (24 well dish) of the antigen-expressing target (T47D FMS) or antigen null target (T47D).

[0093] Thereafter, if the CAR T-cells had persisted and destroyed the monolayer, 1 million T-cells were removed and re-stimulated in an identical manner each week. Total cell number was extrapolated at each time-point depending on the expansion of T-cells that occurred in each weekly cycle.

[0094] Throughout all of these experiments, T-cells were cultured in the absence of any exogenous cytokine such as IL-2 or IL-4so they had to make their own cytokines in order to persist and expand. Cytokine (IFN-? and IL-2) production was measured by ELISA in supernatants harvested from T-cell/tumour cell co-cultures, providing a second marker of effective co-stimulation.

[0095] It was found (FIG. 2A,B) that on their first exposure to a tumour monolayer that expresses target (FMS encoded CSF-1 receptor), all CARs that are predicted to kill do so (pooled data from 12 expts). The controls are UT (untransduced), P4 (targets an irrelevant antigen, PSMA) and CT4 in which the endodomain is truncated. As expected, none of the CAR T-cells kill tumour cells that lack CSF-1 receptor (T47D).

[0096] A representative re-stimulation experiment is shown in FIG. 3. Pooled re-stimulation data from 7 experiments is shown in FIG. 4. In this case, proliferation on the first cycle was similar for most of the constructs, although the IL-34 targeted second and third generation constructs were poorer. This may be because the affinity of the IL-34 targeting moiety is too high.

[0097] In the later cycles however, the C34B dual pCAR combination (a CSF-1 targeted 28z second generation CAR co-expressed with an IL-34 targeted 4-1 BB co-stimulatory motif) consistently emerged as clearly superior.

[0098] In the experiment shown in FIG. 3, supernatant was collected 24 hours after the time of each re-stimulation cycle and was analysed for cytokine content (IFN-? and IL-2) by ELISA. The percentage of residual tumour cell viability was measured by MTT assay (representative examples shown in FIG. 5). The cytokine production results are shown in FIG. 6. It was found that only the C34B CAR T-cells retained the ability to make IL-2 throughout each cycle of stimulation. This was lost by all of the other CAR combinations after the first cycle. Sustained retention of the ability to make IL-2 through recursive re-stimulation is not usually seen with CAR T-cells and this suggests that this delivery of dual co-stimulation is fundamentally altering the differentiation of these cells in vitro, delaying the onset of anergy.

[0099] Number of viable T-cells post monolayer destruction on consecutive cycles of Ag-stimulation was also monitored and the results are shown in FIG. 5. After the second cycle of re-stimulation, all CARs except C34B begin to lose the ability to achieve CSF-1R-dependent tumour cell killing. By contrast, T-cells that express C34B retain antigen-dependent potency in this cytotoxic assay for up to 13 iterative cycles of re-stimulation, but never elicit cytotoxicity against unmodified T47D cells.

[0100] Also, so-called exhaustion markers on these T-cells (PD1, TIM3, 2B4 and LAG3) were also measured by flow cytometry. The results are shown in FIGS. 10-13. As expected, the percentage of T cells that expressed various exhaustion markers progressively increased on the re-stimulated T-cells, but this did not retard the proliferation, tumour cell destruction or cytokine release by the C34B cells, upon antigen stimulation. This suggests that the superior function of C34B is not the result of delayed upregulation of exhaustion markers.

[0101] In summary, the pCAR approach of the invention seems to maintain the cells in a state whereby they retain responsiveness to antigen through more cycles of re-stimulation. There are indications that it may retard differentiation beyond controlled memory state and it appears to delay the onset of anergy while retaining the ability of the cells to make IL-2 upon activation.

Example 2

[0102] Analysis of Effects In Vivo

[0103] A panel of CARs used in Example 1 above were tested for anti-tumour activity using a highly aggressive in vivo xenograft model in which the CSF-1 receptor target is expressed at low levels and in which disease is disseminated throughout lymph nodes (FIG. 7A-D). Tumour cells were tagged with firefly luciferase, allowing the non-invasive monitoring of disease burden.

[0104] SCID/Beige mice were randomised into 6 groups (9 animals per group combined over two independent experiments) and were inoculated intravenously (IV) with 2?10.sup.6 K299 tumour cells, re-suspended in 200 ?L PBS. On day 5, the groups were treated with one of the therapeutic regimens indicated below: [0105] C4B group: 20?10.sup.6 C4B T-cells IV [0106] C34B group: 20?10.sup.6 C34B T-cells IV [0107] 43428Bz: 20?10.sup.6 43428Bz T-cells IV [0108] 34CB group: 20?10.sup.6 34CB T-cells IV [0109] UT (Untransduced) group: 20?10.sup.6 untransduced T-cells IV [0110] NT (Non-treated) group: 200 ?L PBS IV

[0111] Tumour growth was monitored using bioluminescence imaging (BLI) at appropriate time-points for the duration of the study.

[0112] The results are shown in FIG. 8A,B. Again, the best performing system was that of the pCAR, C34B, indicated by lower average BLI emission (FIG. 8A-B), delayed tumour progression or tumour regression, leading to prolonged survival of mice (FIG. 8A).

[0113] Animals were weighed throughout the experiment and no significant toxicity was noted (FIG. 9).

Example 3

[0114] Selection of Targeting Moieties to Engineer pCARs that Elicit T-Cell Activation in an ?v?6-Dependent Manner.

[0115] A panel of CARs that target ?v?6 integrin alone or together with the extended ErbB family were prepared and are shown schematically in FIG. 14. The binding element used in this case was A20 peptide (SEQ ID NO 11) derived from the GH-loop of the capsid protein VP1 from Foot and Mouth Disease Virus (serotype 01 BFS)(U.S. Pat. No. 8,927,501). This was placed downstream of a CD124 signal peptide and fused to CD28 and CD3? endodomains to form A20-28?, a 2nd generation CAR. A control (C20-28?) was prepared comprising a similar construct but with a scrambled targeting peptide (named C20) in which the key RGDL motif was replaced with AAAA. A second control comprised A20 fused to a CD28 truncated endodomain (A20-Tr).

[0116] To create the pCAR of the invention (named TIE-41 BB/A20-28z), A20-28z was co-expressed with a chimeric co-stimulatory receptor comprising a pan-ErbB targeted peptide (T1E) fused to a CD8a transmembrane and a 41 BB endodomain.

[0117] Where indicated, CARs were co-expressed with the 4?? chimeric cytokine receptor to allow for IL-4-mediated enrichment in vitro. Equimolar co-expression of the IL-4-responsive 4?? chimeric cytokine receptor, in which the IL-4 receptor ? ectodomain is fused to the transmembrane and endodomain of the shared IL-2/15 receptor ?, was achieved using a Thosea Asigna (T)2A ribosomal skip peptide. These chimeric molecules were expressed in human T-cells by retroviral gene transfer.

[0118] The integrin expression pattern of cancer cell lines A375 was assessed using flow cytometry (FIG. 15A,B), and these were separated into ?v?6-negative (Panc1 and A375 puro) or ?v?6-positive (Bxpc3 and A375 puro ?6) tumour cells. These cells were co-cultured with CAR T-cells at an effector:target ratio of 1:1 for either 24, 28 or 72 hours, after which time, cytotoxicity was assessed by MTT assay and expressed relative to untreated tumour cells. The results are shown in FIGS. 16A,B.

[0119] These data show that A20-28z CAR T-cells kill all target cells that express ?v?6 integrin (Bxpc3 and A375 ?6 puro), but spare targets that lack this integrin (Panc1 and A375 puro). Secondly, the control CARs C20-28z and A20-Tr are inactive in these assays. Thirdly, T-cells that express the T1E-41 BB/A20-28z pCAR cause efficient killing of target cells that express ?v?6 integrin (Bxpc3 and A375 ?6 puro). All of these results are as expected. Notably however, T-cells that express the T1E-41 BB/A20-28z pCAR also cause the killing of target cells that lack ?v?6 (Panc1 and A375 puro). This indicates that, within a pCAR configuration, the ability of the A20 peptide to bind non-?v?6 integrins with low affinity is sufficient to trigger the activation of these engineered T-cells.

[0120] Production of IFN-? by the pCAR and control engineered T-cells was then assessed. Tumour cells that lacked ?v?6 (Panc1 and A375 puro) or expressed ?v?6 (Bxpc3 and A375 puro ?6) were co-cultured with genetically engineered T-cells at an effector:target ratio of 1:1 and supernatant was collected after 24, 48 or 72 hours. Levels of IFN-? were quantified by ELISA (eBioscience). The results are shown in FIG. 17A,B. As expected, the controls did not generate significant quantities of IFN-? while A20-28z CAR T-cells released IFN-? when cultured with ?v?6-positive (Bxpc3 and A375 puro ?6) tumour cells. Notably, T-cells that express the pCAR of the invention, TIE-41 BB/A20z, produce more IFN-? than A20-28z T-cells when cultured with ?v?6-positive (Bxpc3) tumour cells. In addition, TIE-41 BB/A20z.sup.+ T-cells produced IFN-? when cultured with ?v?6-negative (Panc1 and A375 puro) tumour cells. Once again, this demonstrates that, within a pCAR configuration, low affinity binding of the A20 peptide to non-?v?6 integrins is sufficient to trigger the activation of these engineered T-cells.

[0121] Next, the CAR T-cell populations were re-stimulated bi-weekly in the absence of IL-2 support on Panc1 (?v?6 negative) or Bxpc3 tumour cells (?v?6 positive). Tumour cells were co-cultured with CAR T-cells derived from a patient with pancreatic ductal adenocarcinoma (PDAC) at an effector:target ratio of 1:1 (FIG. 18A,B). T-cells were initially added at 2?10.sup.5 cells/well and were counted 72 hrs after co-culture to assess expansion (top panels). Cytotoxicity was assessed at 72 hrs post-addition of T-cells by MTT assay (bottom panels). If there were a sufficient number of T-cells (2?10.sup.5), T-cells were re-stimulated on a fresh tumour monolayer and the process repeated a further 72 hrs later.

[0122] Results are shown in FIG. 18A,B. These illustrate that A20-28z/T1E-41BB.sup.+ T-cells undergo a number of rounds of expansion accompanied by IL-2 release (data not shown) and destruction of ?v?6.sup.+ Bxpc3 cells. Once again, they also underwent a number of rounds of expansion accompanied by IL-2 release and destruction of Panc1 tumour cells.

[0123] Overall, the results clearly showed that the pCAR comprising A20-28z/T1E-41 BB exhibits enhanced in vitro functionality compared to a 2.sup.nd generation CAR targeted against ?v?6. Furthermore, the A20-28z/T1E-41 BB.sup.+ T-cells also undergo activation by Panc1 or A375 puro cells, which express minimal to undetectable levels of this integrin. Taken with the findings obtained using the C34B pCAR (examples 1 and 2), this indicates that the pCAR configuration allows T-cell activation to occur upon serial re-stimulation when a high affinity binding interaction occurs with the 41 BB CCR while a lower affinity interaction occurs with the 28z 2.sup.nd generation CAR.

Example 4

[0124] Use of an Alternative TNF Receptor Family Member, CD27 to Engineer a Functional pCAR.

[0125] Using the A20-28z/T1E-41 BB pCAR as starting material, additional pCARs were engineered in which the 41 BB module was replaced by alternative members of the TNF receptor family, namely CD27 or CD40. Control pCARs were engineered in which endodomains were truncated (tr). Target cells that express (Bxpc3) or lack (Panc1) ?v?6 were plated at a density of 5?10.sup.4 cells per well of a 24 well plate. After 24 hours, 5?10.sup.4 pCAR T-cells were added to target cells or empty wells (unstimulated), without exogenous cytokine support. After a further 72 hours, T-cells were harvested from the wells and were counted (FIG. 19A). An MTT assay was performed to determine the percentage viability of the residual target cells, making comparison with control target cells that had been plated without addition of T-cells (FIG. 19B). If T-cells proliferated after each cycle of stimulation, they were re-stimulated on fresh target cells, exactly as described above. Proliferation of pCAR T-cells (FIG. 19A) and MTT assay (FIG. 19B) were performed after 72 hours as before. Iterative re-stimulation of pCAR T-cells and assessment of target cell killing was continued in this manner until T-cells no longer proliferated over the course of each 72 hour cycle.

[0126] These data once again confirm the superior functionality of the A20-28z/T1E-41 BB pCAR when T-cells are stimulated on Panc1 target cells, indicated by sustained T-cell proliferation and tumour cell killing. This provides further confirmation that low affinity binding of the A20 peptide to non-?v?6 integrins is sufficient to trigger the activation of these engineered T-cells. Notably however, the A20-28z/T1E-CD27 pCAR achieved the greatest level of proliferation (FIG. 19A) and sustained tumour cell killing (FIG. 19B) when re-stimulated on ?v?6-expressing Bxpc3 cells. By contrast, CD40-based pCARs exhibited modest function in these assays. Together, these data demonstrate that a number of TNF receptor family members can be employed to engineer pCARs that demonstrate superior functionality, exemplified by CD27 or 41 BB.