Glypican-3-specific T-cell receptors and their uses for immunotherapy of hepatocellular carcinoma

Abstract

The present invention relates to glypican-3-specific T-cell receptors. The present invention further relates to soluble TCR constructs, chimeric TCRs, bi-specific antibodies, nucleic acids, expression constructs and cells comprising said TCRs or TCR constructs. The present invention further relates to the use of the TCR or the soluble TCR constructs or chimeric TCRs or bi-specific antibodies as a medicament, preferably in the detection, diagnosis, prognosis, prevention and/or treatment of liver cancer, in particular hepatocellular carcinoma, or other cancers expressing GPC3. The present invention further relates to methods of detecting, diagnosing, prognosing, preventing and/or treating liver cancer, in particular hepatocellular carcinoma, or other cancers expressing GPC3. The present invention further relates to peptides comprising glypican-3 epitope(s) and respective nucleic acids encoding them, antibodies and compositions as well as their use as (peptide) vaccines. The present invention further relates to vaccines comprising the peptide(s).

Claims

1. A T-cell receptor (TCR) comprising: (i) a T cell receptor α-chain comprising the amino acid sequence of SEQ ID NO. 1, or an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO. 1, or any variation of SEQ ID NO. 1 provided that said variation retains its functional ability to bind to the epitope with the amino acid sequence of SEQ ID NO. 14 or to its HLA-A2 bound form, (ii) a T-cell receptor β-chain comprising the amino acid sequence of SEQ ID NO. 2, or an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO. 2, or any variation of SEQ ID NO. 2 provided that said variation retains its functional ability to bind to the epitope with the amino acid sequence of SEQ ID NO. 14 or to its HLA-A2 bound form, or (iii) both (i) and (ii).

2. The T-cell receptor (TCR) of claim 1, comprising (i) a T cell receptor α-chain further comprising the amino acid sequence of SEQ ID NO. 3, or an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO. 3, (ii) a T-cell receptor β-chain further comprising the amino acid sequence of SEQ ID NO. 4, or an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO. 4, (iii) a linker or hinge region comprising the amino acid sequence of SEQ ID NO. 5, or an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO. 5, or (iv) any combination of (i), (ii), and (iii).

3. The T-cell receptor (TCR) of claim 1 comprising (i) a T cell receptor α-chain comprising the amino acid sequence of SEQ ID NO. 6, or an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO. 6, or any variation of SEQ ID NO. 6 provided that said variation retains its functional ability to bind to the epitope with the amino acid sequence of SEQ ID NO. 14 or to its HLA-A2 bound form, (ii) a T-cell receptor β-chain comprising the amino acid sequence of SEQ ID NO. 7, or an amino acid sequence that has at least 80% identity to the amino acid sequence of SEQ ID NO. 7, or any variation of SEQ ID NO. 7 provided that said variation retains its functional ability to bind to the epitope with the amino acid sequence of SEQ ID NO. 14 or to its HLA-A2 bound; form, (iii) both (i) and (ii), or (iv) the amino acid sequence of SEQ ID NO. 8.

4. A soluble T-cell receptor (sTCR) construct comprising (1) at least one of one or more T-cell receptor α-chains as defined in claim 1, and one or more T-cell receptor β-chains as defined in claim 1, (2) one or more fusion components selected from the group consisting of Fc receptors, Fc domains, cytokines, toxins, antibodies, and combinations thereof, wherein the at least one T-cell receptor chain (1) is bound to the fusion component(s) (2), and (3) a label.

5. A chimeric T-cell receptor comprising at least one of the T-cell receptor α-chain as defined in claim 1, the T-cell receptor β-chain as defined in claim 1, or the T-cell receptor of claim 1, wherein the TCR α-chain and/or the TCR β-chain is/are fused to CD3-zeta chain(s) and/or other TCR stimulation domains.

6. A bi-specific antibody comprising (a) the T-cell receptor α- and β-chain(s) of the TCR as defined in claim 1, which are linked with each other and fused to (b) an antibody or a single chain antibody fragment (scFv) which is directed against an antigen or epitope on the surface of lymphocytes.

7. A nucleic acid encoding the T-cell receptor according to claim 1.

8. A nucleic acid comprising (i) the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 1 and/or SEQ ID NO. 2, or the nucleotide sequence of SEQ ID NO. 9 and/or SEQ ID NO. 10 or their complementary sequence(s), or sequence(s) that have at least 80% identity to the nucleotide sequence of SEQ ID NO. 9 or 10; (ii) the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 6 and/or SEQ ID NO. 7, or the nucleotide sequence of SEQ ID NO. 11 and/or SEQ ID NO. 12, or their complementary sequence(s), or sequence(s) that have at least 80% identity to the nucleotide sequence of SEQ ID NO. 11 or 12; or (iii) the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 8, or the nucleotide sequence of SEQ ID NO. 13, or their complementary sequence(s), or sequence(s) that have at least 80% identity to the nucleotide sequence of SEQ ID NO. 13.

9. An expression construct for expressing the T-cell receptor according to claim 1 in a cell.

10. A cell comprising the expression construct of claim 9, wherein the cell is a lymphocyte.

11. A pharmaceutical composition comprising the T-cell receptor of claim 1, and; one or more pharmaceutically acceptable excipients.

12. A method of generating genetically modified lymphocytes, comprising providing lymphocytes, providing the T-cell receptor of claim 1, introducing the T-cell receptor into the lymphocytes, thereby obtaining genetically modified lymphocytes.

13. (canceled)

14. (canceled)

15. (canceled)

16. A method of preventing and/or treating hepatocellular carcinoma, or cancers expressing GPC3, comprising the steps of (a) providing lymphocytes of a patient or a blood donor; (b) providing the T-cell receptor of claim 1; (c) ex vivo introduction of the T-cell receptor into the lymphocytes of step (a) and, thereby, obtaining genetically modified lymphocytes, (d) administering the genetically modified lymphocytes of step (c) to a subject or patient in need thereof, wherein the subject or patient is HLA-A2 positive.

17. A method of preventing and/or treating hepatocellular carcinoma, or cancers expressing GPC3, comprising the steps of (a) providing the T-cell receptor of claim 1, and (b) direct application, via injection or infusion, of the T-cell receptor of (a) to a subject or patient in need thereof, wherein the subject or patient is HLA-A2-positive.

18. A method of detecting, diagnosing, prognosing, preventing and/or treating hepatocellular carcinoma or cancers expressing GPC3, comprising the detection and/or destruction of cancer cells of a patient with the use of the soluble T-cell receptor construct of claim 4, wherein the soluble T-cell receptor is applied by intravenous, subcutaneous or intramuscular infusion or injection, or by application to the mucosa of the respiratory tract by inhalation or by spraying, wherein the patient is HLA-A2-positive.

19. A method for detecting hepatocellular carcinoma, or cancers expressing GPC3, comprising the in vitro staining of cancer cells by using the soluble T-cell receptor construct of claim 4 which is labeled.

20. A composition comprising a peptide comprising at least one GPC3 epitope comprising the amino acid sequence of SEQ ID NO. 14.

21. A nucleic acid molecule coding for at least one peptide according to claim 20 or a plasmid comprising at least one such nucleic acid molecule.

22. (canceled)

23. (canceled)

24. (canceled)

25. A vaccine composition comprising (i) at least one peptide according to claim 20, and (ii) an excipient.

26. (canceled)

27. A method of preventing and/or treating hepatocellular carcinoma or cancers expressing GPC3, comprising the step of administering a peptide of claim 20 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0351] FIG. 1

[0352] A to D) Sequence of the TCR P1-1

[0353] A) Sequence of the TCR P1-1 alpha chain.

[0354] Nucleotide and amino acid sequence of the TCR alpha chain: underlined is the variable alpha region.

[0355] B) Sequence of the TCR P1-1 beta chain.

[0356] Nucleotide and amino acid sequence of the TCR beta chain: underlined is the variable beta region.

[0357] C and D) Sequence of the TCR P1-1.

[0358] Nucleotide and amino acid sequence of the TCR: underlined is the linker region.

[0359] E to J) Sequences of the TCR P2-1

[0360] E and F) Sequence of one TCR P2-1.

[0361] Nucleotide and amino acid sequence of the TCR: underlined is the TCR alpha chain.

[0362] G and H) Sequence of another TCR P2-1.

[0363] Nucleotide and amino acid sequence of the TCR: underlined is the TCR alpha chain.

[0364] I and J) Sequence of a third TCR P2-1.

[0365] Nucleotide and amino acid sequence of the TCR: underlined is the TCR alpha chain.

[0366] FIG. 2. Schematic illustration of mass spectrometric analysis of MHC I bound peptides on HepG2 cells.

[0367] MHC-I peptide complexes were isolated from total cell lysate by sepharose bound anti-MHC I antibodies. After concentration, peptides were eluted from MHC I and subsequently analysed using Ultra Nano HPLC coupled on-line to the Q Exactive mass spectrometer.

[0368] FIG. 3. Time schedule for the allorestricted stimulation of T cells.

[0369] according to Wilde et al., 2012. DCs are prepared from fresh blood, maturated and electroporated with HLA-A2 RNA. GPC3 can be loaded either in form of peptide or GPC3 mRNA can be electroporated together with HLA-A2. Ten hours after electroporation DCs are co-incubated with CLTs from the same HLA-A2 negative donor. On day 14 and 21, the GPC3-specific HLA-A2-restricted T cell cultures are stained with multimer and sorted via fluorescence activated cell sorting. The sorted T cells are cloned in limiting dilution cultures or expanded as bulk lines. Functional assay for evaluation of their specific effector functions are performed.

[0370] FIG. 4.

[0371] A) Streptamer staining (P1; 367) of stimulated T cells, corresponding T cell lines, and one exemplary T cell clone. Streptamer binding T cell population was enriched from 0.6% in the primary co-culture to 57% in the T cell line. T cell clones showed strong streptamer binding

[0372] B) T cells clones specific for P1 or P2 were identified in an IFNγ secretion assay. Specific T cell clones released IFNγ on T2 cells loaded with GPC3 peptides but not on T2 cells without or with control peptides.

[0373] FIG. 5. TCR Modifications:

[0374] 1) Codon optimization for higher expression levels.

[0375] 2) Murinized constant domains to avoid miss pairing with endogenous TCR chains.

[0376] FIG. 6.

[0377] A) P1-1 transduced T cells specifically secrete IFNγ when co-incubated with T2 cells loaded with GPC3 peptide 367 but not on other target cells.

[0378] B) Killing of GPC3+ human hepatoma cells by T cells engrafted with the GPC3.sub.367 specific TCR P1-1. GPC3+ HLA-A2+ HepG2 cells and GPC3+ HLA-A2− Huh7 cells were incubated with P1-1 transduced T cells at different effector target (E:T) ratios. P1-1 enables T cells to effectively kill target cells at low E:T ratios in an HLA-A2 dependent manner.

[0379] FIG. 7.

[0380] A) Killing of GPC3+ human hepatoma cells by T cells engrafted with the GPC3 367 specific TCR P1-1. GPC3+ HLA-A2+ HepG2 cells were incubated with P1-1 transduced T cells at different effector target (E:T) ratios and target cell viabitity was measured over time. P1-1 enables T cells to rapidily kill target cells at low E:T ratios.

[0381] B) P1-1 transduced T cells specifically secrete IFNγ when co-incubated with T2 cells loaded with GPC3 peptide 367 in a peptide concentration dependent fashion.

[0382] FIG. 8.

[0383] P1-1 transduced T cells specifically secrete IFNγ, IL-2 and TNF-α and delocalize the degranulation marker LAMP-1 when co-incubated with T2 cells loaded with GPC3 peptide 367 but not on mock peptide loaded target cells.

EXAMPLES

1. Material and Methods

1.1 PBMC and Cell Lines

[0384] Peripheral blood mononuclear cells (PBMC) from healthy human donors were isolated by Ficoll density gradient centrifugation.

[0385] T2 is a HLA-A2+ TAP deficient human lymphoma cell line that was used as a target in IFNγ-release assays.

[0386] LCL-B27 is a HLA-B27+ B-lymphoblastoid cell line that was used as a feeder cell line for unspecific T cell restimulation.

[0387] Human Embryonic Kidney 293 (293T) cells were used as producer cell line of virus supernatant for T cell transduction.

[0388] HepG2 is a human hepatoma cell line expressing HLA-A2 and GPC3, which was used as a target in killing assays.

[0389] Mammalian cell culture was performed under sterile cell culture conditions and cell were incubated at 37° C. and 5% CO.sub.2.

1.2 Media

[0390] All T cell cultures were maintained in human T cell medium (hTCM) compromised of RPMI1640 medium supplemented with 10% human serum, 4 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 6 μg/ml gentamycin, 1 mM sodium pyruvate and 1× Non-Essential Amino Acids (NEAA). Human serum was isolated in our laboratory from healthy donors.

[0391] Dendritic cell cultures were maintained in DC medium consisting of very low endotoxin RPMI 1640 medium supplemented with 1.5% human serum, 100 IU/ml penicillin and 100 μg/ml streptomycin.

[0392] LCL and T2 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and 1×NEAA.

[0393] HepG2 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and 1×NEAA.

1.3 Cytokines, Peptides Multimers and Antibodies

[0394] Recombinant human IL-4 and IL-6 were obtained from GellGenix. Recombinant human GM-CSF, IL-7, IL-15, TNFα, IL-1β, PGE.sub.2 and IL-2 were purchased from Genzyme, Promokine, Peprotech, Miltenyi Biotec, R&D, Pfizer and Novartis, respectively.

[0395] The HLA-A2-restricted peptides representing defined epitopes from GPC3 (367: FIDKKVLKV; 326: TIHDSIQYV) were synthesized by iba.

[0396] HLA-A2-GPC3-multimers were generated by AG Busch, Institute for Medical Microbiology, Immunology and Hygiene, TU Munich (Knabel et al., 2002). Strap-Tactin PE for labelling of MHC-I multimers was purchased from iba.

[0397] Anti-human CD8-APC, CD8-PE, anti-CD14, anti-CD83 and anti-CD80 antibodies were obtained from BD, Immunotools, eBioscience and Immunotech, respectively.

[0398] Propidiumiodide (1 mg/ml) was obtained from Roth.

[0399] Anti-human CD3 (OKT3) was kindly provided by AG Kremmer, Institute for Molecular Immunology, Helmholtz Zentrum Munich.

1.4 Generation of Dendritic Cells (DC) for T Cell Stimulation

[0400] To generate DC PBMC from a healthy HLA-A2 negative donor were resuspended in DC medium and plated at 5×10.sup.6 cell per 75 cm.sup.2 NUNC culture flask. After incubated for 1.5 hours at 37° C. non-adherent cells were removed by extensive washing. The remaining adherent monocytes were cultured in medium containing 100 ng/ml GM-CSF and 20 ng/ml IL-4 for 24 hours. On day 2 immature DC were differentiated by addition of maturation cytokines (10 ng/ml TNFα, 10 ng/ml IL-1β, 15 ng/ml IL-6 and 1 μg/ml PGE2) directly to the culture. DC were incubated for an additional day before mature DC were harvested. Maturation was confirmed by staining with anti-CD14, anti-CD83 and anti-CD80 and subsequent flow cytometry a FACSCanto II (BD Biosciences).

[0401] Mature DC were transfected with GPC3 and HLA-A2 by RNA electroporation. 2×10.sup.6 cells were resuspended in 200 μl OptiMEM I medium and mixed with 25 μg HLA-A2 RNA and 50 GPC3 RNA. The DC-RNA mixture was placed into a 0.4 cm electroporation cuvette and incubated on ice for 3 min. Electroporation was performed with the Gene Pulser Xcell (Bio-Rad) using the exponential electroporation protocol at 300 V and 150 HLA-A2 RNA and GPC3 RNA were in vitro (ivt) transcribed using the mMES SAGE mMACHINE T7 kit and Poly(A) tailing kit (both Ambion), according to the manufacturer's instructions. HLA-A2 and GPC3 expression was assed via flow cytometry.

1.5 Allorestricted Stimulation of T Cells

[0402] CD8+ T lymphocytes were enriched from total PBMCs via negative selection using the Dynabeads® Untouched™ Human CD8 T Cells Kit from Invitrogen. Primary cultures contained 1×10.sup.6 CD8+ T cells and 1×10.sup.5 HLA-A2 and GPC3 ivt-RNA pulsed mature DC in 2 ml of hTCM medium per well of a 24-well tissue culture plate. 5 ng/ml IL-7 was added immediately to the stimulation on day 0. 50 IU/ml IL-2 were added on day 2 and thereafter every 3.sup.rd day. Primed T cells were restimulated seven days later using freshly generated ivt-RNA pulsed mature DC.

1.6 Multimer Staining and Cloning of T Cells

[0403] After 14 days of T cell stimulation HLA-A2-restricted GPC3-specific T cells were detected by MHC-I-multimer staining. GPC3 MHC-multimers were labelled with streptactin-PE prior to staining. For each 1×10.sup.6 T cells 0.2 μg PE-labelled GPC3 MHC-multimers were used. T cells were incubated with PE-labelled GPC3 MHC-multimers for 20 min in 50 μl PBS+0.5% human serum on ice and in the dark. APC-labelled anti-human CD8 was added and incubated for an additional 20 min, before washing and analysis by flow cytometry. Dead cells were excluded by propidium iodide staining. For sorting up to 10×10.sup.6 cells were stained as described above and sorted on a FACS Aria sorter (BD Biosciences). Sorted CD8+ GPC3 MHC-multimer+ T cells were cloned by limiting dilution. Therefore 0.3 CD8+ GPC3 MHC-multimer+ T cells were seeded per well of a 96-well round-bottom plate. In Addition each well contained a feeder mixture consisting of 1×10.sup.5 allogeneic PBMC (irradiated to 35 Gy), 1×10.sup.4 LCL-B27 (irradiated to 50 Gy), 30 ng/ml OKT3 and 50 IU/ml IL-2 in 200 n1 of hTCM. After 2 weeks of incubation at 37° C. aliquots of wells containing proliferating cells were analysed with regard to their GPC3 specificity in an IFN-γ release assay and by multimer staining as described above. GPC3-specific CD8+ GPC3 MHC-multimer+ T cells were further expanded by restimulation with the feeder mixture as described.

1.7 IFN-γ Release Assay

[0404] T2 cells, which were used as targets, were loaded with 5 μM GPC3 peptide for 1.5 h prior to co-incubation with T cells. 2×10.sup.3 T cells were co-incubated with 1×10.sup.4 peptide loaded T2 cells in round-bottom 96-well plates yielding in an effector to target ratio of 0.2:1. T cells co-incubated with mock-peptide loaded T2 cells served as negative controls. After 24 h of co-culture supernatants were harvested and investigated by standard anti-human IFN-γ ELISA (BioLegend).

1.8 T Cell Receptor (TCR) Analysis and Cloning of Reactive TCR

[0405] For TCR analysis of GPC3 specific T cell clones RNA was extracted from T cell clones with Trizol and cDNA was synthesized using superscript II reverse transcriptase (Invitrogen). Variable parts of α- and β-chains were amplified with degenerated primer (Table 1) that cover 99% of the TCR variable gene segment families followed by agarose gel purification and DNA sequencing (GATC).

TABLE-US-00043 TABLE 1 FORWARD REVERSE ALPHA 5′human panVa (“VPANHUM”): 3′human CA2: 5′-TGAGTGTCCCPGAPGG2P-3′ 5′-GTGACACATTTGTTTGAGAATC-3′ (P = A or G; 2 = A, G or T) [SEQ ID NO. 43] [SEQ ID NO. 42] BETA VP1: GCIITKTIYTGGTAYMGACA CP1: [SEQ ID NO. 44] GCACCTCCTTCCCATTCAC or [SEQ ID NO. 46] VP2: CTITKTWTTGTAYCIKCAG (I = inosine, W = A/T, M = A/C,  Y = C/T, K = G/T) [SEQ ID NO. 45]

[0406] Variable parts of α and β chains of a GPC3 specific TCR were codon optimized for improved gene expression (Geneart) and cloned into the retroviral vector pMP71 via EcoRI and NotI restriction sites. Constant chains were murinized to improve pairing of the transgenic TCR and α- and β-chain were linked via a P2A element.

1.9 Retroviral TCR Transfer into Human PBMC

[0407] 293T cells were co-transfected with 2 μg retroviral plasmid pMP71-GPC3-TCR, 1 μg pcDNA3.1-Mo-MLV and 1 μg pALF-10A1 the later representing plasmids that carry the retroviral genes for gag/pol and env, respectively. Transfection was performed with 10 lipofectamine according to the standard lipofectamin transfection protocol. Freshly isolated human PBMCs were activated on anti-CD28 and OKT3 coated 24-well plates in hTCM supplemented with 300 IU/ml IL-2 for two days. Activated PBMCs were transduced twice, on day 3 and 4. Virus containing supernatant of transfected 293T cells was harvested two days after transfection and added to RetroNectin-coated 24-well culture plates. The virus was spinoculated for 2 h at 32° C. and 2000 g. After centrifugation media was replaced with 1×10.sup.6 activated T cells/well in hTCM supplemented with 100 IU/ml IL-2. T cells were spinoculated with 1000 g for 10 min at 32° C. After 24 h of incubation medium was replaced by fresh medium containing 50 IU/ml IL2 and T cells were incubated for additional 48 h. Transduced PBMC were analysed by GPC3 MHC-multimer staining and in functional assay at multiple time points.

1.10 Functional Assays

[0408] Transduced PBMC were analysed with regard to their IFN-γ secretion capacity after co-incubation with different target cells as described above. Peptide concentrations and effector to target ratios varied. To assess killing ability of transduced PBMC the cells were co-incubated with HepG2 cells that were seeded on E-plate 96 culture plates (Roche). Target cell viability was measured on the XCelligence (Roche) over time. Alternatively cell titre blue assay was performed after 24 h of co-incubation.

2. Results

2.1 Identification of Immunodominant GPC3 Peptides Presented on MHC Class I Molecules of Human Hepatoma Cells

[0409] Immunodominant epitopes for GPC3 have not been described yet. In this study we used Ultra Nano HPLC coupled on-line to the Q Exactive mass spectrometer to obtain a comprehensive HLA class I peptidome from a GPC3 and HLA-A2 positive hepatoma cell line. The resulting data were analysed using the MaxQuant bioinformatics platform. Two HLA-A2 bound GPC3 peptides could be identified, herein referred to as [0410] GPC3 peptide 367 or GPC3.sub.367 or GPC3-P1:

TABLE-US-00044 [SEQ ID NO. 14] FIDKKVLKV
and
GPC3 peptide 326 or GPC3.sub.326 or GPC3-P2: (described in Komori et al., 2006)

TABLE-US-00045 [SEQ ID NO. 16] TIHDSIQYV.

[0411] The knowledge of these two peptides enables us to target GPC3 epitopes that are presented on GPC3 positive HCC cells.

2.2 In Vitro Production of Glypican-3 mRNA and Expression of GPC3 in DCs after mRNA Electroporation

[0412] A construct containing GPC3 has been cloned, from which GPC3 mRNA can be produced. Glypican-3 expression has been shown from the plasmid pcDNA3.1(−)GPC3 after transfection of 293T cells. Expression of GPC3 from this construct after electroporation of DCs was optimized. We were able to detect GPC3 after electroporation of DCs. GPC3 was also expressed when co-electroporated with HLA-A2. HLA-A2 expression was stable for at least 24 h. In contrast GPC3 could only be detected up to 2 h post electroporation and was rapidly degraded thereafter.

2.3 Allorestricted Stimulation of T Cells

[0413] To isolate tumour reactive high avidity T cells, an allo-restricted stimulation approach (see FIG. 3) was used, as described by Wilde et. al., 2012. For stimulation of naïve T cells, autologous dendritic cells were co-transfected with GPC3 and HLA-A2 RNA and used as antigen presenting cells. T cells from the naïve T cell repertoire of HLA-A2 negative donors were co-cultured with and expanded on these HLA-A2+ GPC3+ DCs. After two weeks, MHC streptamer-positive CD8+ T cells specific for both targeted GPC3 epitopes were detected (<1%). We were able to enrich these cell populations further to 57% GPC3-P1- and 35% GPC3-P2-MHC streptamer-positive T cell lines and grew T cell clones from them. In a co-culture with GPC3-P1/-P2 peptide loaded T2 cells we identified T cell clones displaying specific effector function by IFNγ secretion (see FIG. 4). The specific effector function could be verified in an intracellular cytokine staining for IFNγ and TNFα. Furthermore T cell clones secreted IFNγ when co-cultured with HLA-A2+ GPC3+ hepatoma cells (HepG2). Functional T cell clones showed strong GPC3 MHC streptamer binding.

2.4 T Cells Specific for Peptide GPC3.SUB.367

[0414] We have identified and cloned the T cell receptor (TCR) specific for GPC3 peptide 367 from our T cell clones. This TCR is also referred to as P1-1. The T cell receptor sequence coding for the variable parts of alpha and beta chain of P1-1 were codon optimized to increase the level of expression after viral transfer into T cells. Furthermore the constant domains were exchanged with murine constant domains to avoid miss pairing with endogenous TCR chains and hence ensure optimal expression (see FIG. 5).

[0415] T cells engrafted with this optimized GPC3 specific TCR showed strong GPC3 MHC streptamer binding. When co-cultured with GPC3 peptide loaded target cells or a GPC3 expressing hepatoma cell line (HepG2), GPC3 TCR transduced T cells secreted IFNγ, TNFα, IL2 and the degranulation marker Lamp-1 (see FIG. 6A and data not shown). Furthermore cytotoxicity was observed by killing of up to 60% of HepG2 cells (see FIG. 6B).

[0416] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

[0417] Baumhoer D, Tornillo L, Stadlmann S, Roncalli M, Diamantis E K, Terracciano L M. Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am J Clin Pathol. 2008 June; 129(6):899-906. [0418] Boulter J M and Jakobsen B K. Stable, soluble, high-affinity, engineered T cell receptors: novel antibody-like proteins for specific targeting of peptide antigens. Clin Exp Immunol. 2005; 142(3): 454-460. [0419] Csoka M, Strauss G, Debatin K M, Moldenhauer G. Activation of T cell cytotoxicity against autologous common acute lymphoblastic leukemia (CALL) blasts by CD3×CD19 bispecific antibody. Leukemia 1996 November; 10(11): 1765-72. [0420] Engels B, Cam H, Schüler T, Indraccolo S, Gladow M, Baum C, Blankenstein T, Uckert W. Retroviral vectors for high-level transgene expression in T lymphocytes. Hum Gene Ther. 2003 Aug. 10; 14(12):1155-68. [0421] Hamid O et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med. 2013 Jul. 11; 369(2):134-44. [0422] Han E Q, Li X L, Wang C R, Li T F, Han S Y. Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges. J Hematol Oncol. 2013 Jul. 8; 6:47. [0423] Knabel, M., Franz, T. J., Schiemann, M., Wulf, A., Villmow, B., Schmidt., B., Bernhard, H., Wagner, H. and Busch, D. Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nature Medicine 2002, 8 (6), 631-637. [0424] Komori H, Nakatsura T, Senju S, Yoshitake Y, Motomura Y, Ikuta Y, Fukuma D, Yokomine K, Harao M, Beppu T, Matsui M, Torigoe T, Sato N, Baba H, Nishimura Y. Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma. Clin Cancer Res. 2006 May 1; 12(9):2689-97. [0425] Lunde E, Løset GÅ, Bogen B, Sandlie I. Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins. BMC Biotechnology 2010, 10:61 [0426] Maiti S N, Huls H, Singh H, Dawson M, Figliola M, Olivares S, Rao P, Zhao Y J, Multani A, Yang G, Zhang L, Crossland D, Ang S, Torikai H, Rabinovich B, Lee D A, Kebriaei P, Hackett P, Champlin R E, Cooper L J. Sleeping beauty system to redirect T-cell specificity for human applications. J Immunother. 2013 February; 36(2): 112-23. [0427] Miyoshi H, Blömer U, Takahashi M, Gage F H, Verma I M. Development of a self-inactivating lentivirus vector. J Virol. 1998 October; 72(10):8150-7. [0428] Yu S F, von Rüden T, Kantoff P W, Garber C, Seiberg M, Rüther U, Anderson W F, Wagner E F, Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Prot Natl Acad Sci USA 1986 May; 83(10):3194-8. [0429] Wilde S, Geiger C, Milosevic S, Mosetter B, Eichenlaub S, Schendel D J. Generation of allo-restricted peptide-specific T cells using RNA-pulsed dendritic cells: A three phase experimental procedure. Oncoimmunology. 2012 Mar. 1; 1 (2): 129-140.