SPECIFIC BINDING MOLECULES FOR HTERT

Abstract

The present invention provides a nucleic acid molecule encoding a specific binding molecule capable of binding an h TERT peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 when the peptide is presented by a Class II Major Histocompatibility Complex (MHC), wherein the specific binding molecule comprises a first polypeptide comprising a variable region of an α-chain and a second polypeptide comprising a variable region of a β-chain of a T-cell receptor (TCR), and wherein: (a) the variable region of an α-chain comprises CDR sequences CDR1, CDR2 and CDR3 which respectively comprise the amino acid sequences set forth in SEQ ID NOs: 2, 3 and 4; and (b) the variable region of a β-chain comprises CDR sequences CDR1, CDR2 and CDR3 which respectively comprise the sequences set forth in SEQ ID NOs: 5, 6 and 7. Recombinant constructs, vectors and host cells comprising such nucleic acid molecules are also provided, as are specific binding molecules encoded by such nucleic acid molecules. The present invention has utility in the treatment of cancer.

Claims

1. A nucleic acid molecule encoding a specific binding molecule capable of binding an hTERT peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 when the peptide is presented by an HLA-DP4 Class II Major Histocompatibility Complex (MHC), wherein the specific binding molecule comprises a first polypeptide comprising a variable region of an α-chain and a second polypeptide comprising a variable region of a β-chain of a T-cell receptor (TCR), and wherein: (a) the variable region of an α-chain comprises CDR sequences CDR1, CDR2 and CDR3 which respectively comprise the amino acid sequences set forth in SEQ ID NOs: 2, 3 and 4; and (b) the variable region of a β-chain comprises CDR sequences CDR1, CDR2 and CDR3 which respectively comprise the sequences set forth in SEQ ID NOs: 5, 6 and 7.

2. (canceled)

3. (canceled)

4. The nucleic acid molecule of claim 1, wherein the variable region of an α-chain comprises the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 95% sequence identity thereto; and the variable region of a β-chain comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 95% sequence identity thereto.

5. The nucleic acid molecule of claim 1, wherein the specific binding molecule is encoded as a single chain comprising the first polypeptide linked to the second polypeptide.

6. The nucleic acid molecule of claim 5, wherein the first and second polypeptides of the specific binding molecule are joined by a self-splicing linker, wherein the self-splicing linker is a 2A peptide comprising the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having at least 80% sequence identity thereto.

7. (canceled)

8. (canceled)

9. The nucleic acid molecule of claim 1, wherein the first polypeptide further comprises a constant region of an α-chain and the second polypeptide further comprises a constant region of a β-chain.

10. The nucleic acid molecule of claim 9, wherein the specific binding molecule is a TCR molecule which, when expressed by an immune effector cell, is located on the surface of the cell, and wherein, the constant region of an α-chain comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 95% sequence identity thereto; and the constant region of a β-chain comprises the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 95% sequence identity thereto.

11. (canceled)

12. The nucleic acid molecule of claim 10, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 38, or an amino acid sequence having at least 95% sequence identity thereto; and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 40, or an amino acid sequence having at least 95% sequence identity thereto.

13. The nucleic acid molecule of claim 12, wherein the TCR molecule comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17.

14. (canceled)

15. The nucleic acid molecule of claim 9, wherein the specific binding molecule is: (i) a soluble TCR; (ii) a TCR-CAR, wherein the first polypeptide or the second polypeptide comprises a transmembrane domain and an intracellular signaling domain; or (iii) a TCR-antibody construct, wherein the first polypeptide or the second polypeptide further comprises an Fc domain of an antibody; and the constant region of an α-chain comprises the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence having at least 95% sequence identity thereto; and the constant region of a β-chain comprises the amino acid sequence set forth in SEQ ID NO: 22, or an amino acid sequence having at least 95% sequence identity thereto.

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. The nucleic acid molecule of claim 5, wherein the specific binding molecule is a chimeric TCR comprising: (i) an scFv-TCR comprising the variable region of an α-chain and the variable region of a β-chain; (ii) a transmembrane domain; and (iii) an intracellular signalling domain.

27. (canceled)

28. (canceled)

29. (canceled)

30. The nucleic acid molecule of claim 1, wherein the nucleic acid is DNA or RNA.

31. (canceled)

32. A recombinant construct comprising the nucleic acid molecule of claim 1 linked to a heterologous nucleic acid sequence.

33. A vector comprising the nucleic acid molecule of claim 1, or the recombinant construct comprising said nucleic acid molecule linked to a heterologous nucleic acid sequence.

34. The vector of claim 33, wherein the vector is: (i) an expression vector or a cloning vector; and/or (ii) a viral vector; and/or (iii) an mRNA vector.

35. (canceled)

36. A host cell comprising: (i) the nucleic acid molecule of claim 1; (ii) a recombinant construct comprising said nucleic acid molecule linked to a heterologous nucleic acid sequence; (iii) a vector comprising said nucleic acid molecule or recombinant construct; or (iv) a first vector comprising a nucleic acid molecule encoding a first polypeptide as defined in claim 1, and a second vector comprising a nucleic acid molecule encoding a second polypeptide as defined in claim 1, wherein the specific binding molecule is heterologous to the cell.

37. The host cell of claim 36, wherein the host cell is an immune effector cell, said nucleic acid molecule, construct, vector or pair of vectors encodes a TCR, TCR-CAR or a chimeric TCR, and the TCR, TCR-CAR or chimeric TCR is expressed on the surface of the immune effector cell.

38. (canceled)

39. The host cell of claim 37, wherein the immune effector cell is a T-cell or an NK cell.

40. (canceled)

41. (canceled)

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

49. A composition comprising the host cell of claim 37 and at least one physiologically-acceptable diluent, carrier or excipient.

50. (canceled)

51. (canceled)

52. (canceled)

53. (canceled)

54. (canceled)

55. (canceled)

56. (canceled)

57. (canceled)

58. (canceled)

59. (canceled)

60. A kit comprising a first vector and a second vector, wherein the first vector comprises a nucleic acid molecule encoding a first polypeptide as defined in claim 1 and the second vector comprises a nucleic acid molecule encoding a second polypeptide as defined in claim 1.

Description

FIGURE LEGENDS

[0288] FIG. 1 shows that the original hTERT-TCR-1-expressing T-cell clone recognised autologous cancer cells in ascites. The clone was incubated with empty antigen-presenting cells (i.e. antigen-presenting cells (APCs) with no exogenous peptide added), APCs loaded with the GV1001 peptide and autologous ascites containing hTERT-expressing cancer cells. Target recognition was analysed by measurement of T-cell proliferation; proliferation demonstrates target recognition. Error bars indicate one standard deviation.

[0289] FIG. 2 shows that TCR-negative Jurkat T-cells expressed hTERT-TCR-1 following transduction with a vector encoding the TCR. Jurkat T-cells become CD3+ when expressing a functional TCR; CD3 staining demonstrated successful hTERT-TCR-1 expression.

[0290] FIG. 3 shows that primary T-cells expressing hTERT-TCR-1 recognise GV1001 peptide-loaded target cells. T-cells were incubated with target cells, and target cell recognition determined based on cytokine production by the T-cells. Recognition by both CD4+ and CD8+ T-cells was demonstrated. Part A shows the proportion of CD4+ (left-hand side) and CD8+ (right-hand side) T-cells which produced the IFNγ and/or TNFα following challenge with the various target cells. Part B presents the same data in a different format, demonstrating the total proportion of CD4+ and CD8+ T-cells which produced cytokines following challenge with target cells loaded with the defined concentrations of GV1001 peptide. Error bars indicate one standard deviation.

[0291] FIG. 4 shows that primary T-cells expressing hTERT-TCR-1 can specifically kill target melanoma cells (ESTDAB-039, DP4+ hTERT+), whereas control (mock-transfected) T-cells had no activity against the melanoma cells. Error bars indicate one standard deviation. Effector:target ratios are indicated.

[0292] FIG. 5 shows the identification of the minimal hTERT-TCR-1 epitope. EBV-LCL (Epstein-Barr virus-transformed lymphoblastoid cell line) cells were used as APCs, and loaded with hTERT peptides. The peptides are defined by their sequence locations within hTERT (x-axis), and target recognition determined based on T-cell proliferation. Error bars indicate one standard deviation.

[0293] FIG. 6 shows the results of xenograft studies in mice. Mice were injected with ESTDAB-039 tumour cells engineered with a retroviral vector, SW1000, to express the hTERT-TCR-1 antigen (GV1001), firefly luciferase and EGFP. SW1000 encoded these three genes within a single open reading frame, the structure and amino acid sequence of which (SEQ ID NO: 68) is shown in part A. Part B is a schematic diagram showing the time-line for the xenograft experiments. The time-line shows the days of the study, when mice were injected and when they were imaged. As shown, mice were injected with SW1000-engineered ESTDAB-039 tumour cells at the beginning of the experiment. Mice were injected with hTERT-TCR-1-expressing T-cells at the points shown (the arrows pointing from “T-cell injection” to the timeline) and imaged in the IVIS in vivo imaging system at the point shown (the arrows pointing from “IVIS” to the timeline).

[0294] Part C shows the results of the experiments. Tumour load was measured by IVIS based on total bioluminescence at the days indicated (following SW1000-engineered ESTDAB-039 cell injection). Tumour load of mice injected with T-cells transfected with hTERT-TCR-1 is compared to the tumour load of mock-transfected T-cells.

[0295] FIG. 7 shows that hTERT-TCR-1 T-cells promote specific tumour apoptosis. Part A shows the lysis kinetics obtained by bioluminescence (BLI) assay of effector T-cells co-cultured with two different tumour cell lines stably transduced with Ii-hTERT (Granta-519 and HLA-DP04+ EBV-LCL). Data represent mean±standard deviation (SD) of quadruplicates. Statistics were calculated on the 9.5-hour time point. Part B shows the density of Annexin V+ cells (i.e. apoptotic cells) after co-culture of effector T-cells and patient ascites cells is shown. Data represent mean±SD of dodecaplicates. Statistics were calculated on the 10-hour time point.

[0296] FIG. 8 shows that hTERT-TCR-1 T-cells control tumour load in vivo. NSG mice were engrafted with GFP/Luc+ ESTDAB-1000 tumors intra-peritoneally (i.p.) and 3 days after tumor inoculation, mice were randomized and received i.p. injections of mock T-cells, hTERT-TCR-1 transduced T-cells or medium (n=10 for each group) for a total of 4 injections. This experiment timeline is shown in Part A.

[0297] Part B shows luminescence images obtained from IVIS of mice inoculated with ESTDAB-1000 and treated with mock or hTERT-TCR-1 transduced T-cells.

[0298] Part C shows Kaplan-Meier survival curves of the mice shown in Part B. Survival curves were analysed by Mantel-Cox (log-rank) test. Data represent mean±SD.

[0299] FIG. 9 shows that hTERT-TCR-1 T-cells do not react against bone marrow. Healthy donor bone marrow progenitor cells were co-cultured with hTERT-TCR-1 transduced T-cells or mock-transfected T-cells for 6 hours at an Effector:Target ratio (E:T) of 10:1. The cells were then plated in semisolid methylcellulose progenitor culture for 14 days and scored for the presence of red (CFU-E), white (CFU-GM) and mixed (CFU-GEMM) colonies. For each set of three columns, the left-hand column represents CFU-E colonies, the middle column CFU-GM colonies and the right-hand column CFU-GEMM colonies. Data represent mean±SD of triplicates.

[0300] FIG. 10 shows that hTERT-TCR-1-expressing T-cells do not show alloreactivity against PBMCs isolated from a panel of 30 donors. hTERT-TCR-1 transduced T-cells or non-transduced T-cells were incubated with PBMCs at an E:T ratio of 1:5. Controls included incubation of T-cells with titrating concentrations of hTERT peptide with HLA-DPB1*0401 PBMCs. Following overnight incubation at 37° C., IFNγ release was measured by ELISPOT. The figure shows mean IFNγ release and error bars represent SD.

EXAMPLES

[0301] Methods

[0302] Cell Lines, Media and Reagents

[0303] T-cells were isolated from blood from healthy donors. Epstein Barr Virus-transformed lymphoblastoid cell lines (EBV-LCLs) used as target cells were generated by transformation of B-cells from HLA-A2+ donors as previously described (Gjertsen et al., Int. J. Cancer 72: 784-790, 1997). Melanoma cell line ESTDAB-039 was a kind gift from Graham Pawelec (University of Tubingen, Germany). J76 cells were a kind gift from Mirjam Heemskerk (Leiden University, Netherlands). Cell lines were cultured in RPMI-1640 (Gibco, Thermo Fisher Scientific, USA) supplemented with gentamicin and 10% heat-inactivated FCS (Life Technologies, Thermo Fisher Scientific). All T-cells were grown in CelIGro DC medium (CellGenix GmbH, Germany) supplemented with 5% heat-inactivated human pooled serum (PAN-Biotech GmbH, Germany), 10 mM N-acetylcysteine (Mucomyst 200 mg/ml, AstraZeneca AS, UK), 0.01 M HEPES (Life Technologies, Thermo Fisher Scientific) gentamycin 0.05 mg/ml (Garamycin, Schering-Plough Europe, Belgium), and 100 U/ml IL-2 (Chiron, USA), denoted complete medium hereafter, unless otherwise stated.

[0304] .sup.3H-Thymidine Incorporation Assays for Measuring Proliferation (FIGS. 1 and 5)

[0305] The peptide-specific proliferative response was determined by seeding T-cells in 96-well plates at 0.5×10.sup.5 cells/well in CelIGro DC medium (CellGenix GmbH, Germany) supplemented with HEPES buffer, N-acetyl cysteine and gentamycin. Autologous antigen-presenting cells (APCs), either autologous peripheral blood mononuclear cells (PBMCs) or EBV-LCLs irradiated at 3000 rad or 10 000 rad, respectively, to prevent proliferation, were washed and seeded at 0.5×10.sup.5 cells/well. All conditions were tested in triplicate: T-cells and irradiated APCs alone (included as a control), T cells and irradiated APCs with GV1001 peptide (FIG. 1) or truncated peptides with indicated amino acid sequences (FIG. 5), at 10-25 μM peptide making a total volume of 200 μl/well. T-cells were also tested for reactivity against autologous cancer cells isolated from patient ascites (FIG. 1). On day 2 the cells were subjected to .sup.3H-thymidine, (20 μl/well, a total of 0.037 MBq/well) overnight (between 16-20 hours). The proliferation is shown as mean counts per minute (cpm).

[0306] In Vitro mRNA Transcription of TCR Targeting hTERT

[0307] A telomerase (hTERT)-specific, HLA-DP4-restricted TCR was identified in a T-cell clone from a GV1001 peptide-vaccinated pancreatic cancer patient (Bernhardt, S. L. et al., supra) and named hTERT-TCR-1. The in vitro mRNA synthesis was performed essentially as previously described (Almåsbak, H. et al., Cytotherapy 13(5): 629-640, 2011). Anti-Reverse Cap Analog (Trilink Biotechnologies Inc., San Diego, Calif., USA) was used to cap the RNA. The mRNA was assessed by agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific, Waltham, Mass., USA).

[0308] In Vitro Expansion of Human T-Cells

[0309] T-cells from healthy donors were expanded using a protocol adapted for GMP production of T-cells employing Dynabeads CD3/CD28 essentially as previously described (Almåsbak H., et al., supra). In brief, PBMCs were isolated from buffy coats by density gradient centrifugation and cultured with Dynabeads (Dynabeads® ClinExVivo™ CD3/CD28, kindly provided by Dynal, Thermo Fisher Scientific) at a 3:1 ratio in complete CelIGro DC Medium with 100 U/ml recombinant human interleukin-2 (IL-2) (Proleukin, Prometheus Laboratories, USA) for 10 days. The cells were frozen and aliquots were thawed and rested in complete medium before transfection.

[0310] Electroporation of J76 Jurkat Cells and Expanded T-Cells

[0311] Expanded T-cells were washed twice and resuspended in CelIGro DC medium (CellGenix GmbH) and resuspended to 70×10.sup.6 cells/m. The mRNA was mixed with the cell suspension at 100 μg/ml, and electroporated in a 4 mm gap cuvette at 500 V and 2 ms using a BTX 830 Square Wave Electroporator (BTX Technologies Inc., Hawthorne, N.Y., USA). Immediately after transfection, T-cells were transferred to complete culture medium at 37° C. in 5% CO.sub.2 overnight to allow TCR expression. The same protocol was used to electroporate J76 Jurkat cells.

[0312] In Vitro Functional Assay, Antibodies and Flow Cytometry (FIGS. 2 and 3)

[0313] For extracellular staining only, cells were washed in staining buffer (SB) consisting of phosphate buffered saline (PBS) containing 2% FCS before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1% paraformaldehyde.

[0314] Peptide GV1001, EARPALLTSRLRFIPK (SEQ ID NO: 52) from the hTERT protein (hTERT sequence, GenBank accession number: AB085628) was provided by ProImmune Ltd, UK. For intracellular staining, T-cells were stimulated for 6 hours with APCs loaded with GV1001 peptide or a 173 amino acid (563-735) recombinant hTERT protein fragment (GenScript, USA) at the indicated concentrations, at an effector to target (E:T) ratio of 2:1 and in the presence of BD GolgiPlug and BD Golgistop at recommended concentrations. Cells were stained extracellularly and intracellularly using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc, USA). The following antibodies were used: CD3-APC, CD4-BV421 (BioLegend), CD8-PE-Cy7, IFN-γ-FITC, TNF-α-PE (BD Biosciences, USA). Antibodies were purchased from eBioscience, USA, except where noted. Cells were acquired on a BD FACSCanto10 flow cytometer and the data analysed using FlowJo software (Treestar Inc., Ashland, Oreg., USA).

[0315] Bioluminescence-Based Cytotoxicity Assay (FIGS. 4 & 7)

[0316] Luciferase-expressing tumour cells were counted and resuspended at a concentration of 3×10.sup.5 cells/ml. Cells were given Xenolight D-Luciferin potassium salt (75 μg/ml; Perkin Elmer) and were placed in 96-well white round bottomed plates at 100 μl cells/well in triplicate. Effector T-cells were added at indicated E:T ratios. In order to determine spontaneous and maximal killing, wells with target cells only or with target cells in 1% Triton™ X-100 (Sigma-Aldrich), respectively, were seeded. Cells were left at 37° C. and the bioluminescence (BLI) was measured with a luminometer (VICTOR Multilabel Plate Reader) as relative light units (RLU) at indicated time points. Target cells that were incubated without any effector cells were used to determine baseline spontaneous death RLU in each time point. Triplicate wells were averaged and lysis percentage was calculated using the following equation: % specific lysis=100× (spontaneous cell death RLU−sample RLU)/(spontaneous death RLU−maximal killing RLU). Sigmoid curves (no Hill equation) were fitted for every set of points (using Igor Pro 6.36 or 8.1) as guide for the eye with standard deviation as weighting factor, base hold to 0 and max lysis kept below 100.

[0317] Mouse Xenograft Studies (FIGS. 6 & 8)

[0318] NOD.Cg-Prkdc.sup.scid II2rg.sup.tm1WjI/SzJ (NSG) mice were bred in-house under an approved institutional animal care protocol and maintained under pathogen-free conditions. 6-8 week-old mice were injected intra-peritoneally (i.p.) with 1-1.5×10.sup.6 ESTDAB-039 tumour cells. The ESTDAB-039 cells were engineered with a retroviral vector, SW1000, to express the hTERT-TCR-1 antigen (for TCR testing) in combination with firefly luciferase (for in vivo analysis) and EGFP (to detect the transfected cells and sort them). The structure of the construct is shown in FIG. 6A; it consists of the coding sequence of the invariant chain (CD74) containing a CLIP peptide replacement in order to load MHCI (Wälchli et al., Eur. J. Immunol. 44: 774-784, 2014) and MHCII (Mensali et al., in preparation) molecules. The peptide used to replace the CLIP sequence is GV1001 (SEQ ID NO: 52). This sequence was introduced after opening the vector using unique restriction sites at both ends of the CLIP region and using an oligonucleotide fusion method. Invariant chain was then fused with a Luciferase-GFP module through a picornavirus 2A ribosome skipping sequence after removing its natural STOP codon by PCR. The Luciferase-GFP module was extracted from an initial construct (Löw et al., BMC Biotechnol. 20:81, 2010) and rendered compatible to the fusion with invariant chain-2A fragment by adding unique restriction sites by PCR, then subcloned into the pENTR vector (Invitrogen). The full construct was sequence verified and finally subcloned into the pMP71 retroviral vector. The antigen construct is referred to as Ii-hTERT.

[0319] The sequence of the whole fusion protein is set out in SEQ ID NO: 68 and provided in FIG. 6A, with an indication of the different regions of interest. Retroviral particles were produced as depicted in Walchli et al. (PLoS One, 6(11): e27930, 2011) and used to transduce ESTDAB-039 cells. Cells were sorted on the basis of their GFP expression and a pure GFP-positive population was expanded and stocked. The reactivity of the hTERT-TCR-1-redirected T-cells was checked against these cells before they were injected into animals.

[0320] Tumour growth was monitored by bioluminescent imaging using the Xenogen Spectrum system and Living Image v3.2 software. Anaesthetised mice were injected i.p. with 150 mg/kg body weight of D-luciferin (Caliper Life Sciences, Hopkinton, Mass.). Animals were imaged 10 minutes after luciferin injection. 8-10×10.sup.6 hTERT-TCR-1 mRNA-electroporated T-cells (FIG. 6) or 10.sup.7 hTERT-TCR-1 transduced T-cells (FIG. 8), or 10.sup.7 mock-transfected T-cells as a control were injected i.p. as indicated. Schematic diagrams showing the timelines for animal injections for the two experiments are shown in FIGS. 6B and 8B.

Retroviral Particle Production for T-Cell Transduction

[0321] 1.2×10.sup.6 HEK293T cells (Cellbiolabs, US) cells were plated in 6 cm plates. Transfection was performed using X-treme-GENE 9 transfection reagent (Roche) with a mix of DNA including the retroviral packaging vectors and the hTERT-TCR-1 expression vector to an equimolar ratio. After 24 hours, the medium was replaced with 1% HyClone FCS-containing DMEM and the cells were transferred to a 32° C. incubator. Supernatants were harvested after 24 h and 48 h incubation.

[0322] Retroviral Transduction of Expanded T-Cells

[0323] PBMCs were incubated for 2 days in a 24-well plate coated with CD3 and CD28 at 1×10.sup.6 cells/mL. A 24-well plate was coated with 50 μg/mL RetroNectin for 3 hours at room temperature before being washed with PBS and blocked with a solution of PBS supplemented with 0.1% FBS for 30 minutes. 1 mL virus solution was then deposited in each well, which was then topped with 500 μL activated T-cells at a concentration of 0.3×10.sup.5 cells/mL. The plate was then incubated for 30 minutes under a controlled atmosphere (37° C., 5% CO.sub.2) for 30 minutes, sealed and then spun down at 750×g at 32° C. for 60 minutes before being placed back in the incubator.

[0324] The same spinoculation step was repeated the following day before the cells were collected, spun down, washed and resuspended in complete X-Vivo 15 medium for 2 days before the expression of the TCR was checked and the cells expanded using the procedure described above.

[0325] Annexin V-Based Cytotoxicity Assay (FIG. 7)

[0326] 10.sup.4 tumour cells (patient ascites cells) were incubated in a 96-well flat-bottomed plate in 200 μL complete RPMI 1640 medium for 24 hours under a controlled atmosphere (37° C., 5% CO.sub.2). The following day, the plate was centrifuged at 100×g for 1 min and 100 μL supernatant was discarded. 50 μL of a 1:200 solution of IncuCyte Annexin V Red (Essen Biosciences, UK) diluted in complete RPMI 1640 was added to each well and the plate was then incubated at 37° C., 5% CO.sub.2 for 15 min.

[0327] Effector cells (hTERT-TCR-1 transduced or electroporated primary T-cells or patient T-cell clones, or mock-transfected T-cells) previously washed and resuspended in complete RPMI 1640 medium were introduced into each well at a final concentration of 5×10.sup.4 cells/mL (100 μL/well). The plate was then put into an IncuCyte S3 live cell analysis system (Essen Biosciences, UK) with the following settings: 12 images/day, 4 images/well, 2 channels (phase and red), 12 wells per condition. Analysis of cytotoxicity was performed using IncuCyte software. Metrics were then extracted and corrected using Igor Pro 8.1 (Wavemetrics, USA). Background was calculated on mock images and consecutively subtracted from all the other conditions.

[0328] Testing Against Bone Marrow in a Colony Forming Unit (CFU) Assay (FIG. 9)

[0329] Healthy HLA-DP04+ donor bone marrow (n=4 in total) progenitor cells were co-cultured with hTERT-TCR-1 transduced T-cells or mock-transfected T-cells for 6 hours at an E:T of 10:1. The cells were then plated in semisolid methylcellulose progenitor culture for 14 days and scored for the presence of red (CFU-E, i.e. erythroid), white (CFU-GM, i.e. granulocyte and monocyte) and mixed (CFU-GEMM, i.e. granulocyte, erythrocyte, monocyte and megakaryocyte) colonies. Data represent mean±SD of triplicates.

[0330] Alloreactivity Study (FIG. 10)

[0331] PBMCs were isolated from 30 donors as described above. hTERT-TCR-1 transduced T-cells or non-transduced controls were incubated with the PBMCs at an Effector:Target ratio of 1:5 (10000:50000). Controls included incubation of T-cells with titrating concentrations of hTERT peptide GV1001 (10 μM, 1 μM and 100 μM) with HLA-DPB1*0401 PBMC, and T-cells incubated with HLA-DPB1*0401 PBMC in the absence of hTERT peptide GV1001. The assay was incubated at 37° C. overnight and IFNγ release measured by ELISpot (R&D Systems, US) according to the manufacturer's protocol. Bars represent mean and error bars are SD (n=3).

[0332] Statistical Analysis

[0333] Continuous data were described with mean and standard deviation. Unless stated otherwise all statistics were obtained using the multi-variated bidirectional Student t-test. The Mantel-Haenszel test was used as log-rank estimator for survival curves. * p<0.05, ** p<0.01, *** p<0.001 All statistical analyses were performed using R software.

[0334] Results

[0335] hTERT-TCR-1 is Functionally Expressed by T-Cells

[0336] To confirm expression of hTERT-TCR-1 following transfection of T-cells, Jurkat T-cells were transfected and expression of hTERT-TCR-1 detected. Successful hTERT-TCR-1 expression was determined based on surface CD3 expression. CD3 is only expressed on the surface of Jurkat cells when a TCR is co-expressed. Jurkat T-cells were transfected with hTERT-TCR-1 or mock-transfected, stained with allophycocyanin-conjugated anti-CD3 antibody and analysed by flow cytometry. As shown in FIG. 2, transfected T-cells were stained with the anti-CD3 antibody, while mock-transfected T-cells were not, demonstrating successful expression of the TCR,

[0337] To confirm TCR functionality in vitro, T-cells transfected to express hTERT-TCR-1 were incubated with antigen-presenting cells (APCs) with GV1001 peptide or autologous cancer cells from ascites; as a control, the same T-cells were also incubated with APCs without any exogenous peptide. T-cell activation was measured based on proliferation. Proliferation was analysed by .sup.3H-thymidine incorporation assays. The T-cells incubated with the APCs and GV1001 peptide proliferated more than four-fold more than the control T-cells (FIG. 1) demonstrating that T-cells expressing the hTERT-TCR-1 TCR are activated by exposure to APCs presenting the GV1001 peptide.

[0338] T-cells expressing hTERT-TCR-1 were also incubated with APCs loaded with GV1001 peptide at a series of concentrations. As a control, unloaded APCs were also tested. As shown in FIG. 3, both CD4+ and CD8+ T-cells transfected with hTERT-TCR-1 were stimulated to produce the cytokines IFNγ and/or TNFα in response to APCs loaded with GV1001 (FIG. 3A). As shown in FIG. 3B, the response to APCs loaded with increased concentrations of GV1001 rapidly plateaus for both CD4+ and CD8+ T-cells; a greater proportion of CD8+ T-cells were activated than CD4+ T-cells. This demonstrated that both CD4+ and CD8+ T-cells expressing hTERT-TCR-1 are stimulated to produce cytokines upon recognition of the hTERT-TCR-1 antigen GV1001 when presented by an APC.

[0339] To test target cell-killing by hTERT-TCR-1-expressing T-cells, transfected T-cells were incubated with hTERT+, HLA-DP4+ melanoma cells (ESTDAB-039 cell line) at two different effector:target ratios (as shown in FIG. 4). As a control, mock-transfected T-cells were also incubated with the same melanoma cell line. Target cell killing was measured based on bioluminescence. As shown in FIG. 4, T-cells transfected with hTERT-TCR-1 displayed approximately twice the level of specific killing of target cells as that displayed by mock-transfected T-cells. This demonstrates that the hTERT-TCR-1 TCR is able to activate T-cells to kill target cells expressing the antigen (hTERT).

[0340] To identify the minimal epitope recognised by hTERT-TCR-1, T-cells expressing hTERT-TCR-1 were incubated with APCs and GV1001 (as a positive control), APCs with no exogenous peptide (as a negative control) and APCs with a variety of shorter peptides located with GV1001. GV1001 corresponds to amino acids 611-626 of hTERT; it was found that the 4 N-terminal amino acids of GV1001 were not required for peptide recognition (removal of these residues did not affect T-cell activation), but removal of further cells from the N-terminus of GV1001, or of any amino acids from the C-terminus of GV1001, prevented recognition of the peptide by hTERT-TCR-1 (FIG. 5). Accordingly, the minimal sequence epitope was defined as amino acids 5-16 of GV1001, corresponding to amino acids 615-626 of hTERT.

[0341] T-Cells Expressing hTERT-TCR-1 Reduce Tumour Load In Vivo

[0342] As shown in FIG. 6A, a construct was generated which co-expressed a modified MHC II invariant chain and a luciferase-GFP fusion protein in a single ORF. The MHC II invariant chain was modified by replacement of the CLIP (Class II-associated invariant chain peptide) sequence with GV1001. The GV1001-modified invariant chain was encoded at the N-terminus of the ORF, followed by a luciferase-GFP fusion protein. The GV1001-modified invariant chain and luciferase-GFP fusion were separated by a self-cleaving 2A linker. Within the luciferase-GFP fusion protein, firefly luciferase was located at the N-terminus and GFP at the C-terminus, separated by a troponin-C linker. This is a highly flexible sequence, preventing disruption of the activity of firefly luciferase or GFP by the other.

[0343] Mice were injected with ESTDAB-039 tumour cells transfected with the SW1000 construct. These thus present the GV1001 peptide, and tumour load can be readily determined by bioluminescence measurements. Two days after injection of the mice with the tumour cells, they were injected with a first infusion of T-cells, which had either been transfected with hTERT-TCR-1 or mock-transfected. Six further T-cell infusions were given to the mice over the course of the next fortnight, and tumour load measured at regular time-points. As shown in FIG. 6C, mice administered T-cells transfected with hTERT-TCR-1 displayed, on average, a much lower tumour load by day 23 than did the mice administered mock transfected T-cells, demonstrating that in vivo T-cells expressing hTERT-TCR-1 can significantly slow progression of hTERT+ cancers, indicating the strong therapeutic potential of the TCR.

[0344] hTERT-TCR-1 T-cells Recognise and Specifically Kill Tumour Cells The ability of hTERT-TCR-1-expressing T-cells to lyse antigen-positive targets was evaluated using bioluminescence (BLI) cytotoxicity assays (FIG. 7A). In these investigations hTERT-TCR-1 TCR mRNA-electroporated or transduced T-cells as well as mock-transfected T-cells were incubated in the presence of HLA-DP04+ B-cell lymphoma (Granta-519) or Epstein Barr Virus-transformed lymphoblastoid cell lines (EBV-LCLs) stably transduced with the agonist Ii-hTERT. Both transduced and electroporated hTERT-TCR-1-expressing T-cells were able to kill the vast majority of the two target cells compared to the mock controls (FIG. 7A).

[0345] Additionally, the cytotoxic capabilities of hTERT-TCR-1 T-cells were evaluated through Annexin V real time assays. hTERT-TCR-1 transduced or electroporated donor T-cells, the original patient T-cell clone (from which the TCR was extracted) and mock-transfected T-cells were co-cultured with ascites cells extracted from the peritoneum of the pancreatic cancer patient from which the original TCR was sourced. hTERT-TCR-1 T-cells showed lysis of ascites cells compared to the mock control, demonstrating the recognition of the naturally occurring antigen by the TCR (FIG. 7B).

[0346] hTERT-TCR-1 T-Cells Improve Survival of Melanoma-Carrying Mice

[0347] NSG mice were injected intraperitoneally (i.p.) with the melanoma cancer cell line ESTDAB-1000, stably transduced with Ii-hTERT. After confirmation of tumour engraftment (at 3 days), mice were randomised and injected i.p. 4 times with 10.sup.7 effector cells every other day (FIG. 8A). Treatment with hTERT-TCR-1 transduced cells significantly reduced the tumour load compared to mock-transfected T-cells (FIG. 8B) and greatly enhanced survival of the treated mice (FIG. 8C).

[0348] hTERT-TCR-1 T-Cells do not React Against Bone Marrow

[0349] As hematological stem cells have been reported to express telomerase but exhibit relatively low levels of MHC class II compared to other cell types, potential reactivity of hTERT-TCR-1-expressing T-cells against this compartment was evaluated. This was investigated by probing the colony-forming ability of bone marrow progenitor cells in the presence of the hTERT-TCR-1 T-cells. A colony forming unit assay was used to demonstrate that myeloid and erythroid colony formation in HLA-DP04+ bone marrow samples was not affected by co-culture with hTERT-TCR-1-expressing T-cells at an Effector:Target (E:T) ratio of 10:1 (FIG. 9, exemplar donor). These observations demonstrated that hTERT-specific TCR redirected T-cells were not cytotoxic against autologous stem cells from bone marrow (4 donors showed similar results).

[0350] hTERT-TCR-1-Expressing T-Cells do not Show Alloreactivity Against a PBMC Panel Most human TCRs only recognise and bind effectively to specific self-HLA molecules loaded with peptides. A small proportion (less than 10%) of TCRs have the ability to recognise non-self HLA molecules. This phenomenon is termed “alloreactivity”. In a study evaluating the potential for an alloreactive response, hTERT-TCR-1-expressing T-cells were exposed to a range of MHC class I and MHC class II typed peripheral blood mononuclear cells (PBMCs) covering the majority (>90%) of HLA-types in the population (FIG. 10). Following exposure, T-cell target recognition was assessed according to whether exposure to the PBMCs led to activation (determined by intracellular cytokine production). The data show that hTERT-TCR-1 transduced T-cells do not recognise this panel of PBMCs, and thus do not demonstrate alloreactivity. As a positive control, hTERT-TCR-1-expressing T-cells were shown to be functional when exposed to specific peptide loaded onto HLA-DPB1*0401 PBMCs with sensitivity down to a concentration of at least 100 nM peptide.