T-CELL RECEPTORS WHICH RECOGNISE FRAMESHIFT MUTANTS OF TGFBRII

Abstract

The present invention relates to TCR molecules which recognise neopeptides produced as a result of the cancer-associated ?1A frameshift mutation in human TGF?RII. The TCR molecules are capable of binding a peptide of SEQ ID NO: 1 when said peptide is presented by a Class I MHC, and comprise an ?-chain domain and a ?-chain domain, each chain domain comprising three CDR sequences, wherein a) CDRs 1, 2 and 3 of the ?-chain domain have the sequences of SEQ ID NOs: 2, 3 and 4 respectively; and b) CDRs 1, 2 and 3 of the ?-chain domain have the sequences of SEQ ID NOs: 5, 6 and 7 respectively, and wherein one or more of said CDR sequences may optionally be modified by substitution, addition or deletion of 1 or 2 amino acids. Nucleic acid molecules encoding such TCRs are provided, as are soluble TCR molecules with these CDR sequences. The nucleic acid molecules of the invention can be used to modify immune effector cells to express a TCR as defined herein, and such modified immune effector cells are useful in therapy for cancer, as are soluble TCRs as defined above.

Claims

1. A nucleic acid molecule encoding a T-cell receptor (TCR) molecule directed against a mutated TGF?RII protein which comprises the sequence of SEQ ID NO: 1, wherein said TCR molecule is capable of binding a peptide of SEQ ID NO: 1 when said peptide is presented by a Class I Major Histocompatibility Complex (MHC) comprising HLA-A2, and wherein said TCR molecule comprises an ?-chain domain and a ?-chain domain, each chain domain comprising three CDR sequences, wherein a) CDRs 1, 2 and 3 of the ?-chain domain have the sequences of SEQ ID NOs: 2, 3 and 4 respectively; and b) CDRs 1, 2 and 3 of the ?-chain domain have the sequences of SEQ ID NOs: 5, 6 and 7 respectively; and wherein said ?-chain domain comprises: i) a variable region comprising the amino acid sequence set forth in SEQ ID NO: 72; or an amino acid sequence with at least 95% sequence identity thereto; and ii) a constant region comprising the amino acid sequence set forth in SEQ ID NO: 9, or a modified version thereof comprising one or more amino acid substitutions or insertions relative to SEQ ID NO: 9 and having at least 95% sequence identity to SEQ ID NO: 9, or a murinised version of SEQ ID NO: 9; and said ?-chain domain comprises: i) a variable region comprising the amino acid sequence set forth in SEQ ID NO: 75; or an amino acid sequence with at least 95% sequence identity thereto; and ii) a constant region comprising the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 14, or a murinised version of SEQ ID NO: 14.

2. (canceled)

3. The nucleic acid molecule of claim 1, wherein the TCR molecule is encoded as a single-chain TCR (scTCR) comprising an ?-chain domain joined to a ?-chain domain by a self-splicing linker.

4-5. (canceled)

6. The nucleic acid molecule of claim 1, wherein the ?-chain domain and/or the ?-chain domain comprises a double Myc-tag with the amino acid sequence of SEQ ID NO: 19.

7-12. (canceled)

13. The nucleic acid molecule of claim 1, wherein the constant region of said ?-chain domain is modified by insertion of or substitution for a cysteine residue; and the constant region of said ?-chain domain is modified by insertion of or substitution for a cysteine residue.

14-15. (canceled)

16. The nucleic acid molecule of claim 1, wherein the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 11, and the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 16.

17. The nucleic acid molecule of claim 1, wherein the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 25, and the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 31.

18-23. (canceled)

24. The nucleic acid molecule of claim 13, wherein the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 12 and the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 17.

25. The nucleic acid molecule of claim 13, wherein the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 26 and the ?-chain domain comprises the amino acid sequence of SEQ ID NO: 32.

26. A nucleic acid molecule encoding a soluble T-cell receptor (TCR) molecule directed against a mutated TGF?RII protein which comprises the sequence of SEQ ID NO: 1, wherein said TCR molecule is capable of binding a peptide of SEQ ID NO: 1 when said peptide is presented by a Class I Major Histocompatibility Complex (MHC) comprising HLA-A2, and wherein said soluble TCR molecule comprises an ?-chain domain and a ?-chain domain, each chain domain comprising three CDR sequences, wherein a) CDRs 1, 2 and 3 of the ?-chain domain have the sequences of SEQ ID NOs: 2, 3 and 4 respectively; and b) CDRs 1, 2 and 3 of the ?-chain domain have the sequences of SEQ ID NOs: 5, 6 and 7 respectively; and wherein said ?-chain domain comprises: i) a variable region comprising the amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence with at least 95% sequence identity thereto; and ii) a constant region comprising the amino acid sequence set forth in SEQ ID NO: 60, or an amino acid sequence with at least 95% sequence identity thereto; and said ?-chain domain comprises: i) a variable region comprising the amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence with at least 95% sequence identity thereto; and ii) a constant region comprising the amino acid sequence set forth in SEQ ID NO: 62, or an amino acid sequence with at least 95% sequence identity thereto, wherein said TCR is soluble.

27. (canceled)

28. The nucleic acid molecule of claim 26, wherein the constant region of said ?-chain domain is modified by insertion of or substitution for a cysteine residue; and the constant region of said ?-chain domain is modified by insertion of or substitution for a cysteine residue.

29. (canceled)

30. The nucleic acid molecule of claim 26, wherein said ?-chain domain comprises the amino acid sequence set forth in SEQ ID NO: 73 and said ?-chain domain comprises the amino acid sequence set forth in SEQ ID NO: 76.

31-33. (canceled)

34. The nucleic acid molecule of claim 28, wherein said ?-chain domain comprises the amino acid sequence set forth in SEQ ID NO: 74, and said ?-chain domain comprises the amino acid sequence set forth in SEQ ID NO: 77.

35-36. (canceled)

37. The nucleic acid molecule of claim 3, wherein said self-splicing linker is a 2A peptide and comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 40% sequence identity thereto.

38. (canceled)

39. The nucleic acid molecule of claim 37, wherein the scTCR comprises the amino acid sequence of any one of SEQ ID NOs: 33, 34, 37 or 38.

40-44. (canceled)

45. The nucleic acid molecule of claim 1, wherein the nucleic acid is RNA.

46. A vector comprising the nucleic acid molecule of claim 1, wherein said vector is: i) an expression vector, optionally an mRNA expression vector, or a cloning vector; and/or ii) a viral vector, optionally a retroviral vector or a lentiviral vector.

47-49. (canceled)

50. A soluble TCR molecule as defined in claim 26.

51-53. (canceled)

54. The TCR molecule of claim 50, wherein the constant region of said ?-chain domain and the constant region of said ?-chain domain are both modified by insertion of or substitution for a cysteine residue.

55. The TCR molecule of claim 54, wherein said ?-chain domain comprises the sequence set forth in SEQ ID NO: 74, and said ?-chain domain comprises the sequence set forth in SEQ ID NO: 77.

56-59. (canceled)

60. The TCR molecule of claim 50, wherein said ?-chain domain comprises the amino acid sequence set forth in SEQ ID NO: 73, and said ?-chain domain comprises the sequence set forth in SEQ ID NO: 76.

61. A host cell comprising the nucleic acid molecule of claim 1, or a vector comprising said nucleic acid molecule, wherein said host cell is: i) an immune effector cell, optionally a T-cell or an NK cell; or ii) a cloning host cell.

62-65. (canceled)

66. A composition comprising the immune effector cell of claim 61, and at least one physiologically acceptable carrier or excipient.

67-71. (canceled)

72. A method of treating cancer, wherein said cancer expresses a mutated TGF?RII protein which comprises SEQ ID NO: 1, said method comprising administering to a subject in need thereof a composition as defined in claim 66.

73. A method of generating a TGF?RII frameshift mutant-specific immune effector cell, said method comprising introducing a nucleic acid molecule as defined in claim 1 or a vector comprising said nucleic acid molecule, into an immune effector cell, and, optionally, stimulating the cells and inducing them to proliferate before and/or after introducing the nucleic acid molecule or vector; wherein, optionally, said immune effector cell is a T-cell or an NK cell.

74-76. (canceled)

77. A composition comprising the soluble TCR molecule of claim 50, and at least one physiologically acceptable carrier or excipient.

78. A method of treating cancer, wherein said cancer expresses a mutated TGF?RII protein which comprises SEQ ID NO: 1, said method comprising administering to a subject in need thereof a composition as defined in claim 77.

Description

[0222] The present invention may be more fully understood from the non-limiting Examples below and in reference to the drawings, in which:

[0223] FIG. 1 shows that the originally-isolated T-cell clones are TGF?RII frameshift mutation-specific, CD8.sup.?/CD4.sup.?/CD56.sup.+ and kill target cells in a dose-dependent manner.

[0224] (a) shows that the original T-cell clone is CD8.sup.?CD4.sup.? and CD56.sup.+.

[0225] (b) shows the results of .sup.51Cr-release assays demonstrating specific lysis by T-cell clone 26 of colon cancer cell lines, loaded with 1 ?M p573 peptide (which has the sequence of SEQ ID NO: 1) or control peptide 1540 (which has the sequence of SEQ ID NO: 54), at various effector-to-target (E:T) ratios as indicated.

[0226] (c) shows the results of .sup.51Cr-release assays demonstrating specific lysis by the T-cell clone of autologous EBV-LCL (Epstein Barr Virus-transformed lymphoblastoid cell line) or T2 cells, loaded with titrated concentrations of p573 peptide. Blocking with anti-HLA class I at the highest peptide concentration is also shown. The E:T ratio was 25:1. The results shown are representative of three independent experiments.

[0227] FIG. 2 shows TGF?RII ?1A frameshift mutation (TGF?RII.sup.mut)-specific TCR expression in transfected in vitro-expanded T-cells. TGF?RII.sup.mut-specific TCR expression corresponds T-cells which are V?3.sup.+ (V?3=TCR ?-chain variable domain TRBV28). Mock transfected T-cells, TCR mRNA transfected T-cells and the original T-cell clone were all tested.

[0228] FIG. 3 shows that both TCR-transfected CD4.sup.+ and TCR-transfected CD8.sup.+ T-cells produce IFN-? and TNF-? in response to colon cancer cell lines harbouring the TGF?RII ?1A frameshift mutation. T-cells were transfected with TCR mRNA and left overnight before co-incubation with colon cancer cell lines LS174T and SW480 expressing mutated TGWU. LS174T is HLA Class I negative whereas SW480 expresses HLA-A2. SW480 cells were either loaded (+) or not loaded (?) with the TGF?RII frameshift peptide p573. After overnight stimulation, cells were stained intracellularly for cytokine production. Plots show CD4 or CD8 gated T-cells as indicated. The results shown are representative of three independent experiments.

[0229] FIG. 4 shows TGF?RII.sup.mut-TCR transfected CD8.sup.+ T-cells produce more IFN-? and degranulate more efficiently than the original T-cell clone in response to colon cancer cell lines loaded with peptide p573. TCR-transfected CD8.sup.+ T-cells and the original T-cell clone were incubated with colon cancer cell lines for 6 hrs before staining of CD107a and IFN-? was performed. The colon cancer cell lines all harbour the TGF?RII ?1A frameshift mutation. HCT116 and SW480 are both HLA-A2.sup.+, whereas colon cancer cell line LS174T is HLA-A2.sup.?. Plots are CD8 gated T-cells. The results shown are representative of three independent experiments.

[0230] FIG. 5 shows TGF?RII.sup.mut-TCR transfected T-cells kill target cells harbouring the TGF?RII ?1A frameshift mutation with comparable efficiency to the original T-cell clone. Colon cancer cell lines LS174 and HCT116 were loaded with .sup.51Cr. Half of the HCT116 cells were loaded with the TGF?RII frameshift peptide p573. The original patient T-cell clone, TCR-transfected CD8.sup.+ T-cells and mock-transfected CD8.sup.+ T-cells were added at E:T ratios as indicated. Cells were co-incubated for 6 hrs before .sup.51Cr-release was measured. The results shown are representative of two independent experiments.

[0231] FIG. 6 shows TGF?RII.sup.mut-TCR transduced T-cells are effective both in vitro and in vivo. Donor T-cells were transduced with TGF?RII.sup.mut-TCR or, as a negative control, DMF5 (MART-1-specific) TCR.

[0232] (a) shows that transduction efficiency was found to be around 60% for each of the TCRs when T-cells were stained with either anti-V?3 antibody (TGF?RII.sup.mut-TCR) or MART-1 dextramer (DMF5).

[0233] In (b) the transduced T-cells were tested for reactivity against the cognate antigen before infusion. HLA-A2.sup.+ EBV-LCLs were loaded with either a long TGF?RII ?1A frameshift mutation peptide covering the CD8.sup.+ T-cell epitope (p621, SEQ ID NO: 55), or the native MART-1 26-35 peptide (SEQ ID NO: 56). Transduced T-cells were co-incubated with the EBV-LCLs for 5 hours at an E:T ratio of 1:3 and stained for degranulation (CD107a) and TNF-?.

[0234] In (c) NSG mice (TGF?RII.sup.mut-TCR, n=10; MART-1 TCR, n=10) were injected intraperitoneally (i.p.) with 10.sup.6 HCT116 cells expressing firefly-luciferase two days before the intraperitoneal injection of 8?10.sup.6 TCR-transduced T-cells (injections on day 0 and day 2 respectively). Treatment was repeated on days 5 and 10 with 2?10.sup.7 TGF?RII.sup.mut-TCR.sup.+ T-cells. Tumour load was evaluated by bioluminescence imaging on days 2, 9, 16, 24 and 30.

[0235] In (d) bioluminescence signals (photons/sec) for all mice are shown in the scatter plot with mean indicated (+/?SD).

[0236] In (e) Kaplan-Meyer analysis shows that TGF?RII.sup.mut-TCR.sup.+ T-cell treated mice had a significantly prolonged survival compared to control mice (p=0.038; unpaired t-test). In vivo experiments were repeated three times and one representative experiment is shown.

[0237] In (f) tumours were dissected from euthanized mice, single cell suspensions were made and stained for the presence of transduced T cells using anti-CD3 and either anti-V?3 antibody (TGF?RII.sup.mut-TCR) or MART-1 dextramer (DMF5). The percentage of MART-1-specific T-cells in the control group tumours was significantly lower than the percentage of TGF?RII.sup.mut-specific T-cells in tumours of mice treated with the TGF?RII.sup.mut-specific TCR (p=0.0038).

[0238] FIG. 7 shows CD107a expression of TCR-transfected CD8+ T-cells is slightly reduced following stimulation with peptide-loaded HLA-A2.sup.+ HEK cells unable to bind CD8. HEK293 cells were transfected with mRNA encoding HLA-A2 wt or mutant HLA-A2, unable to bind CD8. HEK293 cells were loaded with peptide p573 and used to stimulate for 5 hours CD8.sup.+ T-cells transfected with the TGF?RII.sup.mut-specific TCR.

[0239] FIG. 8 shows that the colon cancer cell HCT116 expresses TRAIL-receptor 4 (CD261). The left panel shows staining of HCT116 cells with isotype controls; the right panel shows the staining of CD261 and CCR6 on HCT116 cells.

[0240] FIG. 9 shows that both CD4+ and CD8+ T-cells redirected with the Radium-1 TCR directly kill target cells presenting cognate antigen. Purified CD4+ T-cells (A) or purified CD8+ T-cells (B) electroporated with Radium-1 mRNA could kill target cells carrying both the specific TGF?RII frameshift mutation and HLA-A2 (HCT 116, colon cancer cells) (No peptide). Addition of exogenous long TGF?RII frameshift peptide p621 (SEQ ID NO: 55), which contains SEQ ID NO:1 (also known as sequence p573), led to increased cell killing. Purified CD4+ T-cells (C) or purified CD8+ T-cells (D) were also able to kill HLA-A2 positive cell lines (Granta; B-cell lymphoma cell line, TGFbRII frameshift negative) loaded with exogenous peptide p621. All data are representative of at least two independent experiments.

[0241] FIG. 10 shows that T-cells electroporated with Radium-1 TCR mRNA reduce in vivo tumour growth in mice after multiple infusions. (A) shows the experimental timeline; (B) shows tumour load for the various mouse groups (n=10 for each group) as measured by bioluminescence. Bioluminescence signals are shown as total flux (photons/sec). The means for all mice are shown in the above plot; error bars indicate standard deviation.

[0242] FIG. 11 compares the EC.sub.50 of Radium-1 TCR-expressing CD8? T-cells relative to the EC.sub.50 of DMF5 TCR-expressing CD8? T-cells, each recognising their cognate antigen. All data are representative of two independent experiments.

[0243] FIG. 12 shows results of flow cytometry of SupT1 cells to identify soluble Radium-1 TCR binding. The soluble TCR does not bind HLA-A2-negative cells (A) or HLA-A2-positive cells presenting a non-specific peptide (B). However, the soluble TCR was shown to bind HLA-A2-positive cells on which the TGF?RII frameshift peptide p573 (SEQ ID NO: 1) was presented.

EXAMPLES

Example 1

[0244] Materials and Methods:

[0245] Cell Lines, Media and Reagents

[0246] A TGF?RII frameshift mutation-reactive, HLA-A2-restricted CTL (cytotoxic T-lymphocyte) clone was isolated from the blood of an MSI+ colon cancer patient and cloned by limiting dilution. The patient had been vaccinated with a 23-mer TGF?RII (?1A) frameshift peptide (a peptide with SEQ ID NO: 49). The clinical trial was approved by the Norwegian Medicines Agency, the Committee for Medical Research Ethics, Region South and the Hospital Review Board. The treatment was performed in compliance with the World Medical Association Declaration of Helsinki. Informed consent was obtained from the patient. The autologous Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) was generated by transformation of B-cells from the donor. The antigen processing-deficient T2 cell line was used as a T-cell target in flow cytometry and cytotoxicity assays. Colon cancer cell lines HCT116, SW480 and LS174T as well as Human Embryonic Kidney (HEK) 293 cells were obtained from the ATCC (Rockville, Md., USA). Hek-Phoenix (Hek-P, inventors' collection) were grown in DMEM (PAA, Paschung, Austria) supplemented with 10% HyClone FCS (HyClone, Logan, Utah, USA) and 1% antibiotic-antimicotic (penicillin/streptomycin, p/s, PAA).

[0247] Where nothing else is indicated, cells were cultured in RPMI-1640 (PAA Laboratories, Pasching, Austria) supplemented with gentamicin, 10% heat-inactivated FCS (PAA Laboratories, Pasching, Austria). Colon cancer cell lines were treated with 500 U/ml IFN-? (PeproTech, Rocky Hill, N.J., USA) overnight before use as target cells.

[0248] All T-cells were grown in CellGro DC medium (CellGenix, Freiburg, Germany) supplemented with 5% heat-inactivated human serum (Trina Bioreactives AG, N?nikon, Switzerland), 10 mM N-acetylcysteine (Mucomyst, AstraZeneca AS, London, UK), 0.01 M HEPES (Life Technologies, Norway) and 0.05 mg/mL gentamycin (Garamycin, Schering-Plough Europe, Belgium), denoted complete medium hereafter, unless otherwise stated.

[0249] Generation of T-Cell Lines and Clones Specific for TGF?RII Frameshift Peptides

[0250] PBMCs collected pre- and post-vaccination were available for analysis. The PBMCs had been isolated and frozen as previously described (Brunsvig, P F et al. (2006), Cancer Immunol Immunother 55(12):1553-1564). Thawed PBMCs were stimulated one round in vitro with peptide for 10-12 days and then tested in triplicates in T-cell proliferation assays (3H-Thymidine) using autologous PBMCs as APCs. PBMCs from various time points were stimulated with TGF?RII frameshift peptides. This included peptides 573 (p573, SEQ ID NO: 1), and 621 (p621, SEQ ID NO: 55) from a TGF?RII frameshift protein resulting from a 1 bp-deletion (?1A) in an adenosine stretch (A10) from base number 709-718 of TGFBRII. (The GenBank sequence for wild type human TGFBRII is: NM 003242.) hTERT peptide I540 (SEQ ID NO: 54) was used as a negative control. Both peptides were provided by Norsk Hydro, ASA, Porsgrunn, Norway.

[0251] The MART-1 peptide with SEQ ID NO: 56 (amino acids 26-35 of native MART-1) was manufactured by ProImmune Ltd, UK. The stimulated T-cells were then tested in proliferation assays against peptide-loaded APCs, either autologous PBMC or EBV-LCL.

[0252] The Stimulation Index (SI) was defined as proliferation with peptide divided by proliferation without peptide and an SI?2 was considered a positive response. T-cell clones from responding T-cell lines were generated as previously described (Saeterdal, I et al. (2001), Cancer Immunol Immunother 50(9):469-476).

[0253] TCR and HLA-A2 Cloning

[0254] Frameshift-specific T-cell clones (26 and 30) were grown and total RNA was prepared. The cloning was performed using a modified 5-RACE method. Briefly, cDNA was synthesized using an oligo-dT primer and was tailed at the 5-end with a stretch of cytosines. A polyguanosine primer together with a constant domain-specific primer was used to amplify TCR chains. The amplicon was cloned and sequenced. The expression construct was prepared by amplifying TCR ?- and ?-chains separately with specific primers and a second PCR was performed to fuse the TCR chains as a TCR-2A construct. The TCR-2A reading frame was cloned into pENTR (Invitrogen) and subsequently recombined into other expression vectors.

[0255] For RNA synthesis the insert was sub-cloned into a Gateway modified version of pCIpA.sub.102 (Saeboe-Larssen, S et al. (2002), J Immunol Methods 259(1-2):191-203). A detailed method as well as the primer sequences can be found in (W?lchli, S et al. (2011), PloS one 6(11):e27930). For retroviral transduction the insert was sub-cloned into the pM71 vector. The HLA-A*0201-pCIpA.sub.102 construct was cloned as previously described (Stronen, E et al. (2009), Scand J Immunol 69(4):319-328). This construct was used as a template to generate a CD8 binding-deficient mutant by targeting the residues D227 and T228 and replacing them with K and A, respectively, as described in (Xu, X N et al. (2001), Immunity 14(5):591-602). A standard site-direct mutagenesis was performed using the following primers: 5-GAGGACCAGACCCAGAAGGCGGAGCTCGTGGAGAC-3 (SEQ ID NO: 57) and 5-GTCTCCACGAGCTCCGCCTTCTGGGTCTGGTCCTC-3 (SEQ ID NO: 58). HEK 293 cells were transfected with these constructs using FuGENE-6 (Roche, Switzerland) following the manufacturer's protocol.

[0256] In Vitro mRNA Transcription

[0257] In vitro mRNA synthesis was performed essentially as previously described (Almasbak, H et al. (2011), Cytotherapy 13(5):629-640). 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).

[0258] In Vitro Expansion of Human T-Cells

[0259] 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 (Almasbak, H. et al. (2011), Cytotherapy 13(5):629-640). In brief, PBMCs were isolated from buffy coats by density gradient centrifugation and cultured with Dynabeads (Dynabeads? ClinExVivo? CD3/CD28, kindly provided by Dynal Invitrogen, Oslo, Norway) at a 3:1 ratio in complete CellGro DC Medium with 100 U/mL recombinant human interleukin-2 (IL-2) (Proleukin, Novartis Healthcare, USA) for 10 days. The cells were frozen and aliquots were thawed and rested in complete medium before transfection.

[0260] Electroporation of Expanded T-Cells

[0261] Expanded T-cells were washed twice and resuspended in CellGro DC medium (CellGenix GmbH) and resuspended to 7?10.sup.7 cells/mL. The mRNA was mixed with the cell suspension at 100 ?g/mL, and electroporated in a 4-mm gap cuvette at 500 V and with a time constant of 2 msec 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.

[0262] Antibodies and Flow Cytometry

[0263] T-cells were washed in staining buffer (SB) consisting of phosphate buffered saline (PBS) containing 0.1% human serum albumin (HSA) and 0.1% sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1% paraformaldehyde. For intracellular staining, T-cells were stimulated for 6 hours or overnight with APCs, loaded or not with p573, at a T-cell to target ratio of 2:1 and in the presence of BD GolgiPlug and BD GolgiStop at a 1/1000 dilution. Cells were stained both extracellularly and intracellularly using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc, USA). The following antibodies were used: V?3-FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor 450, CD4-eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8-PE-Cy7, CD56-PE-Cy5.5 (BD Biosciences, USA) and CD107a-PE-Cy5 (BD Biosciences, USA), CXCR2-PE, IFN-?-FITC, IL-2-APC, TNF-?-PE (BD Biosciences, USA), CD261/TRAIL-R4-PE (BD Biosciences, USA). MART-1 (aa 26-35) specific TCR was detected with dextramer staining (Immudex, Denmark) following the manufacturer's recommendations. All antibodies were purchased from eBioscience, USA, except where noted. Cells were acquired on a BD LSR II flow cytometer and the data analysed using FlowJo software (Treestar Inc., Ashland, Oreg., USA).

[0264] .sup.51Cr-Release Assays

[0265] .sup.51Cr-release cytotoxicity assays were performed by labelling of 2?10.sup.6 target cells in 0.5 ml FCS with Na.sub.2.sup.51CrO.sub.4 (7.5 MBq) (Perkin Elmer, Waltham, Mass., USA), for 1 h with gentle mixing every 15 min. Cells were washed three times in cold RPMI-1640 and seeded at 2?10.sup.3 target cells in 96-well, U-bottomed microtitre plates. Autologous EBV-LCL, T2 target cells or colon cancer cell lines HCT116, SW480 and LS174T were pulsed with 10 ?M p573 or pI540 for 1 h at 37? C. The original T-cell clone, TCR-transfected T cells or mock-transfected T-cells were added at the effector-to-target (E:T) ratios indicated and the plate was left for 4 hours at 37? C. as indicated. The maximum and spontaneous .sup.51Cr release of target cells was measured after incubation with 5% Triton X-100 (Sigma-Aldrich, Oslo, Norway) or medium, respectively. Supernatants were harvested onto Luma Plates (Packard, Meriden, Conn.) and .sup.51Cr released from lysed cells was measured using a TopCount microplate scintillation counter (Packard Instrument Company, Meriden, USA). The percentage of specific chromium release was calculated by the formula: [(experimental release?spontaneous release)/(maximum release spontaneous release)]?100.

[0266] Retroviral Transduction

[0267] PBMCs isolated from healthy donors were cultured and activated in CellGro DC medium (CellGenix GmbH, Germany) supplemented with 5% human serum (HS) and 100 U/ml IL2 (Proleukin, Novartis Healthcare)) for 48 h in a 24-well plate precoated with anti-CD3 (OKT-3) and anti-CD28 antibodies (BD Biosciences, USA). After two days of culture PBMCs were harvested and transduced twice with retroviral supernatant. Spinoculation of PBMCs was performed with 1 volume of retroviral supernatant in a 12-well culture non-treated plate (Nunc A/S, Roskilde, Denmark) pre-coated with retronectin (20 ?g/mL, Takara Bio. Inc., Shiga, Japan). After two days, cells were harvested with PBS-EDTA (0.5 mM). Transduced T-cells were further expanded using Dynabeads CD3/CD28 as described above.

[0268] Mouse Xenograft Studies

[0269] NOD.Cg-Prkdc.sup.scid II2rg.sup.tm1Wjl/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 i.p. with 1-1.5?10.sup.6 HCT116 tumour cells. The HCT116 cells were engineered with a retroviral vector (provided by Dr. Rainer L?w, EUFETS AG, Idar-Oberstein, Germany) to express firefly luciferase and EGFP. 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.

[0270] Statistical Analysis

[0271] Continuous data were described with median, mean and range. The Mann Whitney test was used for analysis of tumour load, while survival was calculated using the Kaplan Meier method with the unpaired t-test used for comparison of survival between groups. All p-values given are two-tailed values. A p-value below 0.05 was considered significant. All statistical analyses were performed using GraphPad Prism? (GraphPad Software, Inc. USA).

[0272] Results

[0273] Isolation of a TGF?RII frameshift Mutation-Specific T-Cell Clone

[0274] A TGF?RII frameshift mutation-reactive, HLA-A2-restricted CTL was isolated from the blood of an MSI+ colon cancer patient. The patient had been vaccinated with a 23-mer TGF?RII frameshift peptide of SEQ ID NO: 49. The CTL clones were shown to be CD8.sup.? CD4.sup.? and about 50% of the cells expressed CD56 (FIG. 1a). The CTL clones were previously suspected to be monoclonal since they were shown to express TCR V?3 (or TRBV 28, IMGT nomenclature) (Kyte, J A (2009), Expert Opin Investig Drugs 18(5):687-694). The molecular cloning revealed that they were indeed sister clones, harbouring the same pair of TCR chains. Specific lysis of the colon cancer cell lines HCT116 and SW480 in the absence of exogenously loaded peptide was observed. However, the E:T ratio required for lysis of cell lines with endogenous peptide was higher than if cell lines were loaded exogenously with TGF?RII frameshift peptide (p573, SEQ ID NO: 1). As a control, another colon cancer cell line, LS174T, which is HLA-A2 negative but expresses the TGF?RII mutation was not killed (FIG. 1b). Importantly, despite the expression of CD56 on the T-cell clone (FIG. 1a), the HLA-A2 negative LS174T cell line was not killed, indicating that the killing was not mediated by natural killer (NK)-cell-like activity, but by specific recognition of MHC molecules loaded with peptide.

[0275] To test the relative avidity of the T-cell clones, TAP-deficient T2 cells were loaded with titrated amounts of peptide (0.01-1.0 ?M). We observed that the killing activity followed the peptide concentration (FIG. 1c). The addition of HLA-specific blocking antibodies reduced the killing (FIG. 1c), supporting the HLA class I restriction of the TCR. Similar observations were made when autologous EBV-LCLs were used as APCs. Taken together, the data show that the TGF?RII.sup.mut-specific T-cells were co-receptor negative, peptide-specific and HLA class I-restricted.

[0276] TGF?RII.sup.mut-TCR Is Expressed and Active in Both CD4.sup.+ and CD8.sup.+ T-Cells Following mRNA Electroporation

[0277] The TCR ?- and ?-chains from the TGF?RII.sup.mut-reactive T-cell sister clones were identified and referred to hereafter as the Radium-1 TCR. We cloned the two chains into an mRNA expression vector (see Materials and Methods) and 10-day in vitro-expanded T-cells were electroporated in order to assess their ability to recognize their targets. Radium-1 TCR expression was measured in both CD4.sup.+ and CD8.sup.+ T-cells by surface staining using an anti-V?3 (TRBV 28) antibody (FIG. 2a). Around 70% of transfected T cells expressed the V?3 chain, with 42% of these T-cells being CD8 positive and 32% of T-cells expressing CD4, whereas less than 5% of the cells naturally express V?3.

[0278] We then monitored the activity of Radium-1-transfected T-cells by intracellular cytokine staining upon co-incubated with the colon cancer cell lines SW480 and LS174T. Colon cancer cell line SW480 was recognised by both CD8.sup.+ and CD4.sup.+ T-cells in the absence and presence of exogenously loaded peptide. The T-cells produced TNF-? and IFN-? (FIG. 3). As expected, the colon cancer cell line LS174T was not recognized. These data confirmed the HLA-peptide restriction of the Radium-1 TCR and its ability to efficiently redirect both CD4.sup.+ and CD8.sup.+ T-cells.

[0279] CD107a and IFN-? Production in Radium-1 TCR-Transfected CD8.sup.+ T-Cells

[0280] To determine the cytotoxic potential of TCR-transfected CD8.sup.+ T-cells against colon cancer cell lines, mRNA-electroporated T-cells were co-incubated with the colon cancer cell lines for 6 hrs and stained with antibodies against the degranulation marker CD107a and IFN-? (FIG. 4). Very low levels of IFN-? production and CD107a expression were detected in the absence of exogenously loaded peptide. Upon the addition of peptide p573 (SEQ ID NO: 1), both Radium-1 TCR-transfected T-cells and the original T-cell clone were strongly activated. Interestingly, TCR-transfected T-cells were more efficient IFN-? producers and also displayed higher levels of degranulation than the original T-cell clone, whereas mock-transfected T-cells were not activated. To test the co-receptor independency of the TCR HEK293 cells were transfected with either wild type (wt) HLA-A2 or mutant HLA-A2 unable to bind CD8, loaded with p573 and used to stimulate TCR-transfected CD8.sup.+ T-cells. The number of T-cells expressing CD107a was 36% (wt HLA-A2) and 26% (mutant HLA-A2), indicating that this TCR is at least partially co-receptor independent (FIG. 7).

[0281] Radium-1 TCR-Transfected T-cells Are Capable of Mediating Specific Tumour Cell Lysis

[0282] In addition to cytokine production, the main function required of adoptively transferred redirected T-cells is to specifically kill tumour cells. To investigate if the TCR-transfected T-cells were capable of target-cell lysis, they were tested against the colon cancer cell lines in 6-hr chromium-release assays (FIG. 5). TCR-transfected T-cells lysed HCT116 cells at levels comparable to the original patient clone. As expected, the lysis was further increased when exogenous p573 (SEQ ID NO: 1) was added. The lysis of HLA-A2 negative cell line LS174T was similar to that of mock-transfected T-cells, demonstrating low background lysis of HCT116 likely due to TRAIL-R expression on the target cells (FIG. 8). This cell line has been reported by others to be sensitive to TRAIL-mediated lysis (Tang, W et al. (2009), Febs J 276(2):581-593).

[0283] Radium-1-TCR-Transduced T-cells Are Effective In Vitro and In Vivo

[0284] We established a xenograft mouse model of colon cancer by intraperitoneal injection of HCT116 cells (Kishimoto, H et al. (2009), Proc Natl Acad Sci USA 106(34):14514-14517). T-cells were retrovirally transduced with TCR and tested for expression, which was around 60% for both the Radium-1 TCR and the MART-1-specific TCR (DMF5) used as a control (FIG. 6a). Prior to injection, T-cells were tested functionally against HLA-A2.sup.+ EBV-LCLs loaded with either a long TGF?RII frameshift peptide (p621, SEQ ID NO: 55) or low affinity (wt) MART-1 peptide (SEQ ID NO: 56) (FIG. 6b). The T-cells expressing the Radium-1 TCR all responded against EBV-LCLs loaded with the long TGF?RII frameshift peptide, while around half of the MART-1 TCR expressing cells responded against the low affinity MART-1 peptide. NSG mice were injected i.p. with 10.sup.6 HCT116 cells on day 0 and on d2, d5 and d10 mice were injected with 8?10.sup.6 (d2) and 2?10.sup.7 (d5 & d10) T-cells (FIG. 6c). Control mice were treated with T-cells expressing the MART-1 specific TCR.

[0285] In vivo live imaging of the mice showed that the tumour load was significantly lower (p=0.038) in mice that received the treatment with TGF?RII.sup.mut-specific T-cells compared to the MART-1-specific control T-cells (FIG. 6d). The mice receiving TGF?RII.sup.mut-specific T-cells also had enhanced survival compared to control mice (p=0.038, FIG. 6e). Tumours were dissected from mice that had to be euthanised due to high tumour load. Single cell suspensions of the tumours were made and stained with anti-human CD3 and anti-V?3 or MART-1 dextramer. The percentage of TCR-expressing T-cells in the tumour was found to be significantly higher in mice who received the treatment with TGF?RII.sup.mut-specific T-cells (p=0.0038, FIG. 6f) despite the transduction efficiency of the two T-cell populations being very similar, indicating that the TGF?RII.sup.mut-specific T-cells are either recruited to the tumour more efficiently or that they proliferate in vivo due to antigenic stimulation. Taken together, these data demonstrate the pre-clinical potency of Radium-1 TCR in vivo.

Example 2

[0286] To investigate target cell killing by CD4+ and CD8+ T-cells transduced with the Radium-1 TCR, target cells were stably-transduced to express luciferase. Two sets of target cells were used: the HCT116 cell line and the Granta cell line. HCT116 cells are described above; the Granta cell line is a human B-cell lymphoma cell line. Changes in bioluminescence were used to measure changes in target cell number during culture with the Radium-1-transduced T-cells, representing killing of the target cells by the T-cells.

[0287] Luciferase-transduced target cells were co-cultured with effector T-cells at an effector to target (E:T) ratio of 30:1, and bioluminscence measured. The cells were co-cultured for 24 hours, and bioluminescence measured at 1, 2, 3, 4, 5, 8, 11, 20, 21, 22, 23 and 24 hrs. Effector T-cells were co-cultured with Granta cells both with and without exogenous peptide p621 (SEQ ID NO: 55), which comprises the sequence of SEQ ID NO: 1.

[0288] Purified CD4+ T-cells and purified CD8+ T-cells transduced with Radium-1 mRNA were both found to kill both HCT116 cells and Granta cells (FIG. 9). Killing of Granta cells by both CD4+ and CD8+ T-cells was significantly higher in the presence of p621 (+TGF?RII peptide) than in its absence (no peptide). This demonstrates that Radium-1-transduced CD4+ T-cells are able to kill target cells without interaction with CD8+ T-cells.

[0289] The in vivo killing activity of T-cells transiently transduced with Radium-1 was further investigated in mice. NSG mice were injected i.p. with 10.sup.6 HCT116 cells stably transduced to express luciferase. Two days later (i.e. on day 2) the mice were injected i.p. or i.v. (intravenously) with 8-10?10.sup.6 Radium-1-transfected T-cells. Further injections of Radium-1-transfected T-cells were administered on days 5, 7, 10, 13, 15 and 21, and tumour load was evaluated by bioluminescence imaging on days 2, 7, 17, 29, 45, 53 and 60 (see FIG. 10A; final imaging not indicated).

[0290] Mice treated with Radium-1 TCR transfected T-cells showed a significantly lower tumour load than those treated with mock-transfected T-cells (FIG. 10B) (*p=0.01, Wilcoxon Mann Whitney test). Due to T-cell alloreactivity, mock-transfected T-cells had some effect on tumour growth after multiple injections, as shown. However, this effect was only temporary. As TCR expression was transient in this case, T-cells injected intravenously (i.v.) showed no effect on tumour growth.

[0291] The effectiveness of the Radium-1 TCR was compared in vitro to a known high-affinity TCR. The MART-1-specific TCR DMF5 was selected for comparison. DMF5 has been used clinically in the treatment of melanoma (Johnson, L. A. et al. (2006), J Immunol 177(9):6548-6559).

[0292] CD8? T-cells were transduced with Radium-1 and MART-1 and sorted. TCR+ T-cells were incubated with HLA-A2+ T2 cells (T2 is a human lymphoblast cell line which does not express Class II MHC molecules) loaded with the TGF?RII frameshift peptide p573 (SEQ ID NO: 1) and the MART-1 26-35 peptide analogue ELAGIGILTV (SEQ ID NO: 80) for 5 hours before staining for the degranulation marker CD107a as a marker of killing capacity followed by flow cytometry analysis. The MART-1 26-35 peptide analogue of SEQ ID NO: 80 has a single amino acid substitution relative to the wild-type peptide with SEQ ID NO: 56, i.e. the alanine at position 2 of SEQ ID NO: 56 is substituted for a leucine. The resultant analogue peptide has advantageous properties, in that it has enhanced affinity for HLA-A2, leading to enhanced presentation of the peptide by HLA-A2-containing Class I MHC molecules compared to the wild-type peptide.

[0293] The EC.sub.50 of p573 for Radium-1 was shown to be 2 nM, compared with a value of 7 nm for the MART-1 peptide of SEQ ID NO: 56 with DMF5 (FIG. 11). This indicates that Radium-1 has very high affinity for its cognate antigen/MHC complex, and CD8-independent. Furthermore, Radium-1 is shown to have a higher affinity for its cognate antigen/MHC complex than the DMF5 TCR, which is known to be clinically effective.

Example 3

[0294] The soluble, His-tagged Radium-1 TCR encoded by the scTCR of SEQ ID NO: 69 was expressed in HEK cells. The supernatant of the expressing HEK cells was isolated. SupT1 cells (an HLA-A2-negative cell line) were transduced to express HLA-A2, either fused to a non-specific, irrelevant peptide not recognised by Radium-1, or to a TGF?RII frameshift peptide. The transduced cells were incubated for 30 mins at room temperature with the soluble Radium-1 TCR; untransduced cells were also incubated with the soluble TCR as a further negative control. After incubation, the cells were washed and then stained with allophycocyanin (APC) to identify soluble TCR binding. Staining was performed using a primary mouse anti-His antibody followed by a secondary APC-conjugated anti-mouse IgG antibody. Stained cells were then analysed by flow cytometry (FIG. 12).

[0295] As shown in FIGS. 12A and 12B, essentially no staining of the negative controls was seen, demonstrating that the soluble Radium-1 TCR does not bind cells which do not express HLA-A2, or which express HLA-A2 but are not presenting the TGF?RII frameshift peptide. FIG. 12C shows that cells expressing HLA-A2 and presenting the TGF?RII frameshift peptide were recognised by the soluble Radium-1 TCR, showing it has the expected specificity.