Claudin-18.2-specific immunoreceptors and T cell epitopes

Abstract

The present invention provides Claudin-18.2-specific immunoreceptors (T cell receptors and artificial T cell receptors (chimeric antigen receptors; CARs)) and T cell epitopes which are useful for immunotherapy.

Claims

1. An artificial T cell receptor which binds to claudin-18.2 (CLDN18.2) but not to CLDN18.1 and, when expressed by a T cell, mediates specific lysis of a cell expressing CLDN18.2 on its surface, the artificial T cell receptor comprising a binding domain for CLDN18.2, a transmembrane domain, an IgG1-derived spacer region which links the binding domain for CLDN18.2 to the transmembrane domain, a T cell signaling domain, and a co-stimulation domain, wherein the binding domain for CLDN18.2 comprises (i) an antibody heavy chain variable region (VH) comprising a set of VH complementarity-determining regions VHCDR1, VHCDR2 and VHCDR3: GYTFTSYW (VHCDR1), IYPSDSYT (VHCDR2), and TRSWRGNSFDY (VHCDR3) and (ii) an antibody light chain variable region (VL) comprising a set of VL complementarity-determining regions VHCDR1, VHCDR2 and VHCDR3: QSLLNSGNQKNY (VLCDR1), WAS (VLCDR2), and QNDYSYPFT (VLCDR3) and wherein the transmembrane domain and the co-stimulation domain comprise an amino acid sequence represented by SEQ ID NO: 39.

2. The artificial T cell receptor of claim 1, wherein the T cell signaling domain comprises an amino acid sequence represented by SEQ ID NO: 40.

3. The artificial T cell receptor of claim 1, wherein the IgG1-derived spacer region comprises an amino acid sequence represented by SEQ ID NO: 38.

4. The artificial T cell receptor of claim 1, further comprising a signal peptide.

5. The artificial T cell receptor of claim 4, wherein the signal peptide comprises an amino acid sequence represented by SEQ ID NO: 37.

6. The artificial T cell receptor of claim 1, wherein the VH comprises an amino acid sequence represented by SEQ ID NO: 23 and the VL comprises an amino acid sequence represented by SEQ ID NO: 30.

7. The artificial T cell receptor of claim 6, wherein the IgG1-derived spacer region comprises an amino acid sequence represented by SEQ ID NO: 38 and wherein the T cell signaling domain comprises an amino acid sequence represented by SEQ ID NO: 40.

8. The artificial T cell receptor of claim 1, wherein the artificial T cell receptor comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 41.

9. The artificial T cell receptor of claim 1, wherein the artificial T cell receptor comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 41.

10. A T cell comprising the artificial T cell receptor of claim 1.

11. A plurality of T cells comprising the artificial T cell receptor of claim 1, wherein said plurality of T cells are activated and proliferate when exposed to CLDN18.2 in vivo.

12. A method for treating a patient having cancer characterized by cancer cells expressing CLDN18.2, the method comprising administering to the patient the plurality of T cells of claim 11.

13. A method for expanding the plurality of T cells of claim 11, the method comprising contacting the plurality of T cells with CLDN18.2.

14. The method of claim 13, wherein said contacting occurs in vivo.

15. The method of claim 13, wherein the method does not require providing exogenous IL-2.

16. A T cell comprising the artificial T cell receptor of claim 8.

17. A plurality of T cells comprising the artificial T cell receptor of claim 8, wherein said plurality of T cells are activated and proliferate when exposed to CLDN18.2 in vivo.

18. A method for treating a patient having cancer characterized by cancer cells expressing CLDN18.2, the method comprising administering to the patient the plurality of T cells of claim 17.

19. A method for expanding the plurality of T cells of claim 17, the method comprising contacting the plurality of T cells with CLDN18.2.

20. The method of claim 19, wherein said contacting occurs in vivo.

21. The method of claim 19, wherein the method does not require providing exogenous IL-2.

22. An artificial T cell receptor which binds to claudin-18.2 (CLDN18.2) but not to CLDN18.1 and, when expressed by a T cell, mediates specific lysis of a cell expressing CLDN18.2 on its surface, the artificial T cell receptor comprising a binding domain for CLDN18.2, a transmembrane domain, an IgG1-derived spacer region which links the binding domain for CLDN18.2 to the transmembrane domain, a T cell signaling domain, and a co-stimulation domain, wherein the binding domain for CLDN18.2 comprises (i) an antibody heavy chain variable region (VH) comprising a set of VH complementarity-determining regions VHCDR1, VHCDR2 and VHCDR3: GYTFTSYW (VHCDR1), IYPSDSYT (VHCDR2), and TRSWRGNSFDY (VHCDR3) and (ii) an antibody light chain variable region (VL) comprising a set of VL complementarity-determining regions VHCDR1, VHCDR2 and VHCDR3: QSLLNSGNQKNY (VLCDR1), WAS (VLCDR2), and QNDYSYPFT (VLCDR3).

23. The artificial T cell receptor of claim 22, wherein the IgG1-derived spacer region comprises an amino acid sequence represented by SEQ ID NO: 38.

24. The artificial T cell receptor of claim 22, wherein the transmembrane domain and the co-stimulation domain comprise an amino acid sequence represented by SEQ ID NO: 39.

25. The artificial T cell receptor of claim 22, wherein the T cell signaling domain comprises an amino acid sequence represented by SEQ ID NO: 40.

26. The artificial T cell receptor of claim 22, wherein the transmembrane domain and the co-stimulation domain comprise an amino acid sequence represented by SEQ ID NO: 39 and wherein the T cell signaling domain comprises an amino acid sequence represented by SEQ ID NO: 40.

27. A T cell comprising the artificial T cell receptor of claim 22.

28. A plurality of T cells comprising the artificial T cell receptor of claim 22, wherein said plurality of T cells are activated and proliferate when exposed to CLDN18.2 in vivo.

Description

FIGURES

(1) FIG. 1: Representation of the TCR-CD3 complex. The intracytoplasmic CD3 immunoreceptor tyrosine-based activation motifs (ITAMs) are indicated as cylinders (adapted from “The T cell receptor facts book”, MP Lefranc, G Lefranc, 2001).

(2) FIG. 2: The design of successive generations of CARs. Schematic representation of the different generations of CARs (1G, first generation, 2G, second generation, 3G, third generation). The first generation contains extracellular scFvs and the cytoplasmic CD3 chain/ZAP70 mediating cytotoxicity, the second generation additionally CD28/PI3K promoting proliferation and the third generation furthermore 4-1BB or OX40/TRAF sustaining cell survival (Casucci, M. et al. (2011) 2: 378-382).

(3) FIG. 3: Schematic representation of the different receptor formats for the redirection of T cells against CLDN18.2. Left: a second generation CAR consisting of a CLDN18.2-specific scFv fragment, a IgG1-derived spacer domain, a CD28 costimulatory and a CD3ζ signaling domain (CAR-28ζ); middle: a novel CAR format based on the linkage of the scFv with the constant domain of the murine TCRß chain and coexpression of the constant domain of the murine TCRα chain (CAR/Cα); right: a murine TCR composed of TCR α/β chains (mu, murine TCR);

(4) FIG. 4: Profiling of CLDN18.1 and CLDN18.2 transcripts in a panel of human tissues. A, genomic structure of the CLDN18 locus (top). Hatched boxes, exons unique for CLDN18.1 (E 1.1) or CLDN18.2 (E 1.2), respectively; bottom, exon composition of CLDN18 variants; arches, the two extracellular domains; arrows, the primers used for RT-PCR. B, comparative analysis of the CLDN18 isoforms in normal human tissues (N), primary tumor specimen, and tumor cell lines by end-point RT-PCR. C, quantification in normal human tissues (N), primary tumor specimen, and tumor cell lines by real-time PCR (Sahin U et al., Clin Cancer Res 2008; 14:7624-34).

(5) FIG. 5: Ex-vivo reactivity of spleen cells from immunized HLA-A*02-transgenic mice against CLDN18.2-derived peptides analyzed by IFNy-ELISPOT assay. HLA-A*02 CLDN18.2-specific binding peptides were predicted for the first 80 amino acids of CLDN18.2 applying a specific algorithm (Rammensee H. et al. (1999) Immunogenetics 50, 213-9). Spleen cells of CLDN18.2-immunized HLA-A*02-transgenic mice were analyzed for reactivity against CLDN18.2 peptide pool or predicted HLA-A*02-binding CLDN18.2-derived peptides CLDN18.2-A2-1-6. Positive control: PMA-treated spleen cells; negative control: an irrelevant peptide pool (HIV-gag), irrelevant nonamer peptide (PLAC1-31-39).

(6) FIG. 6: Flow cytometry sorting of CLDN18.2-specific murine CD8+ T cells from HLA-A*02-transgenic mice after in-vitro restimulation. Single CD8+/CD137+ T cells were isolated after restimulation of spleen cells with CLDN18.2 overlapping peptide pool. Control: spleen cells restimulated with irrelevant peptide pool (HIV-gag).

(7) FIG. 7: Specificity testing of TCRs isolated from CD8+ T cells of CLDN18.2-immunized mice. CD8+ T cells of a HLA-A*02-positive healthy donor were transfected with TCR-α/β chain RNAs and tested for recognition of K562-A2 cells pulsed with CLDN18.2 overlapping 15 mer peptides (=CLDN18.2 pool) or CLDN18.2-derived HLA-A*02 binding peptides (CLDN18.2-A2-4, CLDN218.2-A2-5, CLDN18.2-A2-6) by IFNy-ELISPOT. Negative controls: irrelevant peptide pool (HIV-gag), irrelevant 9 mer peptide (PLAC1-31-39); Positive control: SEB;

(8) FIG. 8: Surface expression of CLDN18.2- and CLDN6-specific CARs on human preactivated CD8+ T cells. CD8+ T cells were preactivated with OKT3 and transfected with 20 μg CAR-RNA. 20 h after electroporation cells were stained with a PE-conjugated anti-CD8 antibody and idiotype-specific flurochrome-conjugated antibodies specific for the CLDN18.2-CAR and the CLDN6-CAR, respectively. Cells were gated on single CD8+T lymphocytes.

(9) FIG. 9: Specific lysis of CLDN18.2-expressing target cells mediated by the CLDN18.2-CARs. Preactivated CD8+ T cells were transfected with 20 μg CAR RNA and cocultured 20 h later together with autologous iDCs transfected with RNA encoding either with CLDN18.2-CAR using titrated E:T ratios (30:1, 10:1, 3:1). Negative controls: T cells transfected with a CLND6-specific CAR or without CAR RNA (=mock), iDCs transfected with CLDN6-RNA. Specific lysis was analyzed by luciferase-based cytotoxicity assay after 4h coculture.

(10) FIG. 10: Specific inhibition of CLDN18.2-CAR mediated lysis of CLDN18.2-expressing target cells by addition of an idiotype-specific antibody. Preactivated CD8+ T cells were transfected with 20 μg CAR RNA and cocultured 20 h later together with autologous iDCs transfected either with CLDN18.2- or with CLDN6-RNA using an E:T ratio of 30:1. Effector T cells were preincubated with or without 2 μg/ml of an idiotype-specific antibody for 1h before target cells were added. Specific lysis was analyzed by luciferase-based cytotoxicity assay after 4h coculture.

(11) FIG. 11A-11B: In vitro antigen-specific proliferation of CLDN18.2-CAR T cells. CD8.sup.+ T cells were electroporated with RNA encoding either the CLDN18.2-CAR or without RNA (mock) and labeled with carboxyfluorescein succinimidyl ester (CFSE). CAR T cells were co-cultured with iDCs transfected with 5 μg IVT-RNA encoding either CLDN18.2 or the control antigens CLDN9 or CLDN6. After 96 h co-culture cells were harvested, and the CFSE fluorescence was analyzed by flow cytometry. Cells were gated on CD8.sup.+ living single lymphocytes.

(12) FIG. 12A-12B: In vivo antigen-specific activation of CLDN18.2-CAR T cell in mice after vaccination. BALB/c-mice were i.v. engrafted with 5×10.sup.6 CLDN18-2-CAR-effLuc-GFP transduced T cells. 24 h after ACT, mice were vaccinated i.v. with RNA.sub.(F12-Lip) comprising 25 μg CLDN18.2 RNA or Ctrl RNA. FIG. 12A: Transduction efficiency of T cells was measured via flow cytometry using fluorochrome-coupled antibodies FIG. 12B: In vivo-luminescence intensities in mice were measured 48 h after vaccination. Off-color images represent light intensity (black, least intense; white up to dark-grey, most intense) which was superimposed over the greyscale reference photo.

EXAMPLES

(13) The techniques and methods used herein are described herein or carried out in a manner known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods including the use of kits and reagents are carried out according to the manufacturers' information unless specifically indicated.

Example 1: Materials and Methods

(14) Cell Lines and Reagents

(15) The human chronic myeloid leukemia cell line K562 (Lozzio, C. B. & Lozzio, B. B (1975), Blood 45, 321-334) stably transfected with HLA-A*0201 (Britten, C. M. et al. (2002), J. Immunol. Methods 259, 95-110) (referred to e.g. as K562-A2) and used for TCR validation assays was cultured under standard conditions. The primary human newborn foreskin fibroblast cell line CCD-1079Sk (ATCC No. CRL-2097) was cultured according to the manufacturers' instructions.

(16) Peripheral Blood Mononuclear Cells (PBMCs), Monocytes and Dendritic Cells (DCs)

(17) PBMCs were isolated by Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation from buffy coats. HLA allelotypes were determined by PCR standard methods. Monocytes were enriched with anti-CD14 microbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany). Immature DCs (iDCs) were obtained by differentiating monocytes for 5 days in cytokine-supplemented culture medium as described in Kreiter et al. (2007), Cancer Immunol. Immunother., CII, 56, 1577-87.

(18) Peptides and Peptide Pulsing of Stimulator Cells

(19) Pools of N- and C-terminally free 15-mer peptides with 11 amino acid overlaps corresponding to sequences of Claudin-18.2 or HIV-gag (referred to as antigen peptide pool) were synthesized by standard solid phase chemistry (JPT GmbH, Berlin, Germany) and dissolved in DMSO to a final concentration of 0.5 mg/ml. Nonamer peptides were reconstituted in PBS 10% DMSO. For pulsing stimulator cells were incubated for 1 h at 37° C. in culture medium using different peptide concentrations.

(20) Vectors for In Vitro Transcription (IVT) of RNA

(21) All constructs are variants of the previously described pST1-sec-insert-213gUTR-A(120)-Sap1 plasmid (Holtkamp, S. et al. (2006), Blood 108, 4009-4017). For generation of plasmids encoding murine TCR chains, cDNAs coding for murine TCR-α, -β.sub.1 and -β.sub.2 constant regions were ordered from a commercial provider and cloned analogously (GenBank accession numbers M14506, M64239 and X67127, respectively). Specific V(D)J PCR products were introduced into such cassettes to yield full-length TCR chains (referred to as pST1-murineTCRαß-2ßgUTR-A(120)).

(22) Full-length CLDN18.2, CLDN18.2 aa 1-80 and CLDN6 antigens were cloned linked to the MHC class I trafficking signal (MITD) in pST1 plasmids previously described (Kreiter, S. et al. (2008), J. Immunol. 180, 309-318).

(23) Generation of In Vitro Transcribed (IVT) RNA and Transfer into Cells

(24) Generation of IVT RNA was performed as described previously (Holtkamp, S. et al. (2006), Blood 108, 4009-4017) and added to cells suspended in X-VIVO 15 medium (Lonza, Basel, Switzerland) in a pre-cooled 4-mm gap sterile electroporation cuvette (Bio-Rad Laboratories GmbH, Munich, Germany). Electroporation was performed with a Gene-Pulser-II apparatus (Bio-Rad Laboratories GmbH, Munich, Germany) (T cells: 450 V/250 μF; K562-A2: 200 V/300 μF).

(25) In Vivo Priming of T Cells by Intranodal Immunization of HLA A2.1/DR1 Mice with IVT RNA

(26) T cells of A2/DR1 mice (Pajot A. et al. (2004), Eur. J. Immunol. 34, 3060-69) were primed in vivo against the antigen of interest by repetitive intranodal immunization using antigen-encoding IVT RNA (Kreiter S. et al. (2010), Cancer Research 70, 9031-40). For intranodal immunizations, mice were anesthetized with xylazine/ketamine. The inguinal lymph node was surgically exposed, 10 μL RNA (20 μg) diluted in Ringer's solution and Rnase-free water were injected slowly using a single-use 0.3-ml syringe with an ultrafine needle (31G, BD Biosciences), and the wound was closed. After six immunization cycles the mice were sacrificed and spleen cells were isolated.

(27) Harvest of Spleen Cells

(28) Following their dissection under sterile conditions, the spleens were transferred to PBS containing falcon tubes. The spleens were mechanically disrupted with forceps and the cell suspensions were obtained with a cell strainer (40 μm). The splenocytes were washed with PBS centrifuged and resuspended in a hypotonic buffer for lysis of the erythrocytes. After 5 min incubation at RT, the reaction was stopped by adding 20-30 ml medium or PBS. The spleen cells were centrifuged and washed twice with PBS.

(29) Single-Cell Sorting of Antigen-Specific CD8+ T Cells after CD137 Staining

(30) For antigen-specific restimulation 2.5×10{circumflex over ( )}6/well spleen cells from immunized A2/DR1 mice were seeded in a 24-well plate and pulsed with a pool of overlapping peptides encoding the antigen of interest or a control antigen. After 24 h incubation cells were harvested, stained with a FITC-conjugated anti-CD3 antibody, a PE-conjugated anti-CD4 antibody, a PerCP-Cy5.5-conjugated anti-CD8 antibody and a Dylight-649-conjugated anti-CD137 antibody. Sorting was conducted on a BD FACS Aria flow cytometer (BD Biosciences). Cells positive for CD137, CD3 and CD8 were sorted, one cell per well was harvested in a 96-well V-bottom-plate (Greiner Bio-One) containing human CCD-1079Sk cells as feeder cells, centrifuged at 4° C. and stored immediately at −80° C.

(31) RNA Extraction, SMART-Based cDNA Synthesis and Unspecific Amplification from Sorted Cells

(32) RNA from sorted T cells was extracted with the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the instructions of the supplier. A template-switch protocol was used for cDNA synthesis: Mint Reverse Transcriptase (Evrogen JSC) was combined with oligo(dT)-T-primer long for priming of the first-strand synthesis reaction and TS-short (Eurofins Genomic) introducing an oligo(riboG) sequence to allow for creation of an extended template by the terminal transferase activity of the reverse transcriptase and for template switch (Matz, M. et al. (1999) Nucleic Acids Res. 27, 1558-1560). First strand cDNA synthesized according to the manufacturer's instructions was subjected to 21 cycles of amplification with 5 U PfuUltra Hotstart High-Fidelity DNA Polymerase (Agilent Technologies) and 0.48 μM primer TS-PCR primer in the presence of 200 μM dNTP (cycling conditions: 2 min at 95° C. for, 30 s at 94° C., 30 s at 65° C., 1 min at 72° C. for, final extension of 6 min at 72° C.). Successful amplification of TCR genes was controlled with murine TCR-β constant region specific primers and consecutive clonotype-specific murine Vα-/Vβ-PCRs were only performed if strong bands were detected.

(33) Design of PCR Primers for TCR Amplification

(34) For design of murine TCR consensus primers, all functional murine TCR-Vβ and -Vα genes as listed in the ImMunoGeneTics (IMGT) database (http://www.imgt.org) together with their corresponding leader sequences were aligned with the BioEdit Sequence Alignment Editor (e.g. http://www.bio-soft.net). Forward primers of 24 to 27 bp length with a maximum of 3 degenerated bases, a GC-content between 40-60% and a G or C at the 3′end were designed to anneal to as many leader sequences as possible and equipped with a 15 bp 5′extension featuring a rare restriction enzyme site and Kozak sequence. Reverse primers were designed to anneal to the first exons of the constant region genes, with primer mTRACex1_as binding to sequences corresponding to amino acids 24 to 31 of Cα and mTRBCex1_as to amino acids (aa) 8 to 15 in Cß1 and Cß2. Both oligonucleotides were synthesized with a 5′ phosphate. Primers were bundled in pools of 2-6 forward oligos with identical annealing temperature.

(35) PCR Amplification and Cloning of V(D)J Sequences

(36) 6 μl of preamplified cDNA from isolated T cells was subjected to 40 cycles of PCR in the presence of 0.6 μM mVα-/mVβ-specific oligo pool, 0.6 μM mCα- or mCβ-oligo, 200 μM dNTP and 5 U PfuUltra II Fusion HS DNA Polymerase (Agilent; cycling conditions: 1 min at 95° C., 30 s at 94° C., 30 s annealing temperature, 30 s at 72° C., final extension time of 3 min at 72° C.). PCR products were analyzed using Qiagen's capillary electrophoresis system. Samples with bands at 470-550 bp were size fractioned on agarose gels, the bands excised and purified using a Gel Extraction Kit (Qiagen, Hilden, Germany). Sequence analysis was performed to reveal the sequence of both the V(D)J domain and 13 constant region, as mTRBCex1_as and mTRBCex1_as primer, respectively, match to both TCR constant region genes Cβ1 and Cβ2 in mouse. DNA was digested and cloned into the IVT vectors containing the appropriate backbone for a complete murine TCR-α/β chain.

(37) Flow Cytometric Analyses

(38) Cell surface expression of transfected TCR genes was analyzed by flow cytometry using fluorochrome-conjugated anti-TCR antibody against the appropriate variable region family or the constant region of the TCR β chain (Beckman Coulter Inc., Fullerton, USA) in combination with antibodies directed against CD3, CD8 or CD4 (BD Biosciences). Cell surface expression of transfected CARs was analyzed using a fluorochrome-conjugated idiotype-specific antibodies (Ganymed Pharmaceuticals) recognizing the scFv fragments contained in the respective CAR construct. Flow cytometric analysis was performed on a FACS CANTO II flow cytometer using the FACS Diva software (BD Biosciences).

(39) Luciferase Cytotoxicity Assay

(40) For assessment of cell-mediated cytotoxicity a bioluminescence-based assay was used as an alternative and optimization to .sup.51Cr release. In contrast to the standard chromium release assay, this assay measures lytic activity of effector cells by calculating the number of viable luciferase expressing target cells following coincubation. The target cells were stably or transiently transfected with the luciferase gene coding for the firefly luciferase from firefly Photinus pyralis (EC 1.13.12.7). Luciferase is an enzyme catalyzing the oxidation of luciferin. The reaction is ATP-dependent and takes place in two steps:
luciferin+ATP.fwdarw.luciferyl adenylate+PP.sub.i
luciferyl adenylate+O.sub.2.fwdarw.oxyluciferin+AMP+light

(41) Target cells were plated at a concentration of 10.sup.4 cells per well in white 96-well plates (Nunc, Wiesbaden, Germany) and were cocultivated with varying numbers of TCR-transfected T cells in a final volume of 100 μl. 3 h later 50 μl of a D-Luciferin (BD Biosciences) containing reaction mix (Luciferin (1 μg/μl), HEPES-buffer (50 mM, pH), Adenosine 5′-triphosphatase (ATPase, 0.4 mU/μl, Sigma-Aldrich, St. Louis, USA) was added to the cells. By addition of ATPase to the reaction mix luminescence resulting from luciferase released from dead cells was diminished.

(42) After a total incubation time of 4 h bioluminescence emitted by viable cells was measured using the Tecan Infinite 200 reader (Tecan, Crailsheim, Germany). Cell-killing activity was calculated in regard to luminescence values obtained after complete cell lysis induced by the addition of 2% Triton-X 100 and in relationship to luminescence emitted by target cells alone. Data output was in counts per second (CPS) and percent specific lysis was calculated as follows:
(1−(CPS.sub.exp−CPS.sub.min)/(CPS.sub.max−CPS.sub.min)))*100.

(43) Maximum luminescence (maximum counts per second, CPSmax) was assessed after incubating target cells without effectors and minimal luminescences (CPSmin) was assessed after treatment of targets with detergent Triton-X-100 for complete lysis.

(44) ELISPOT (Enzyme-Linked ImmunoSPOT Assay)

(45) Microtiter plates (Millipore, Bedford, Mass., USA) were coated overnight at room temperature either with an anti-human IFNγ antibody 1-D1k (Mabtech, Stockholm, Sweden) or overnight at 4° C. with an anti-murine IFNy antibody AN18 (Mabtech) and blocked with 2% human albumin (CSL Behring, Marburg, Germany) or with murine culture medium. In the murine setting 5×10.sup.5 spleen cells were distributed per well, while in the human setting 2-5×10.sup.4/well antigen presenting stimulator cells were plated in triplicates together with 0.3-3×10.sup.5/well TCR-transfected CD4+ or CD8+ effector cells 24 h after electroporation. The plates were incubated overnight (37° C., 5% CO.sub.2), washed with PBS 0.05% Tween 20, and incubated for 2 hours with the anti-human IFNγ biotinylated mAB 7-B6-1 (Mabtech) or anti-murine IFNγ biotinylated mAb R4-6A2 (Mabtech) at a final concentration of 1 μg/ml at 37° C. Avidin-bound horseradish peroxidase H (Vectastain Elite Kit; Vector Laboratories, Burlingame, USA) was added to the wells, incubated for 1 hour at room temperature and developed with 3-amino-9-ethyl carbazole (Sigma, Deisenhofen, Germany).

(46) CFSE (Carboxyfluorescein Succinimidyl Ester) Proliferation Assay

(47) CD8.sup.+ T cells were transfected with CAR RNA and about 20h later labeled with 0.8 μM CFSE. Labeled T cells were washed and cocultured with RNA-transfected autologous iDCs (E:T ratio=10:1). After 4 days of coculture cells were harvested and proliferation was analyzed by flow cytometry based on the progressive halving of CFSE fluorescence within daughter cells following cell divisions.

(48) Animals

(49) BALB/c mice were purchased from Javier Labs. Age (8 weeks old) and sex (female) matched animals were used throughout the experiments. Congenic BALB/c-Thy1.1 mice were bred in the animal facility of the BioNTech AG, Germany

(50) Retroviral Gene Manipulation and Preparation of CAR T Cells for Adoptive T Cell Transfer

(51) Splenocytes of BALB/c-Thy1.1 mice were isolated and pre-activated by 2 μg/mL soluble anti-CD3 (eBioscience) and 1 μg/mL soluble anti-CD28 (Novus Biologicals) in the presence of 5 ng/mL rh IL-7 and 10 ng/mL rh IL-15 (both Miltenyi). 24 h and 48 h after pre-activation, T cells were retrovirally (MLV-E) transduced with tricistronic vector encoding CLDN18.2-CAR-effLuc-GFP using RetroNectin-technique (Takara). Transduced T cells were then 3 days expanded in the presence of 5 ng/mL rh IL-7 and 10 ng/mL rh IL-15 and were subsequently ficoll cleaned with Ficoll-Paque PREMIUM (1.084) prior adoptive transfer into mice.

(52) Mouse Experiments

(53) 5×10.sup.6 CLDN18.2 CAR transduced BALB/c-Thy1.1.sup.+ T cells were intravenously (i.v.) transferred into BALB/c mice. Subsequently, mice were i.v. vaccinated with an F12:RNA ratio of 1.3:2 of RNA.sub.(Lip) 24 hours after adoptive T cells transfer (ACT). Whole body bioluminescence imaging was performed.

(54) In Vivo Bioluminescence Imaging (BLI)

(55) Expansion of CLDN18.2-CAR-effLuc-GFP transduced T cells was evaluated by in vivo bioluminescence imaging using the IVIS Lumina imaging system (Caliper Life Sciences). Briefly, 5 min after injection of an aqueous solution of D-luciferin (80 mg/kg body weight; Perkin Elmer), emitted photons were quantified (integration time of 1 min). The intensity of transmitted light originating from luciferase expressing cells within the animal was represented as a greyscale image, where black is the least intense and white to dark-grey the most intense bioluminescence signal. Greyscale reference images of mice were obtained under LED low light illumination. The images were superimposed using the Living Image 4.0 software.

Example 2: Isolation of High-Affinity HLA-A*02-Restricted Murine TCRs Specific for Claudin-18.2

(56) We validated the immunogenic potential of CLDN18.2 in A2/DR1 mice by repetitive intranodal immunization with IVT-RNA encoding aa 1-80 of CLDN18.2. The human CLDN18 gene has two alternative first exons, giving rise to two protein isoforms (CLDN18.1 and CLDN18.2) differing in the N-terminal 69 amino acids (FIG. 4A). As CLDN18.1 is also expressed in normal tissues, especially in the lung, we only used the N-terminal part of CLDN18.2 in order to exclusively induce CLDN18.2-specific T cell reactivities. We used spleen cells of these mice for isolation of CLDN18.2-specific T cells and subsequent cloning of the corresponding TCR genes. Spleen cells of immunized mice were analyzed for the successful induction of CLDN18.2-specific T cells and their reactivity against predicted HLA-A*02 binding CLDN18.2 peptides ex-vivo by IFNγ-ELISPOT assay (FIG. 5).

(57) Significant frequencies of CLDN18.2-specific T cells could be induced In all three mice by RNA immunization, whereas T cell reactivity was focused on two CLDN18.2 peptides predicted to bind to HLA-A*0201(CLDN18.2-A2-5 and -CLDN18.2-A2-6).

(58) For isolation of CLDN18.2-specific T cells, spleen cells of immunized mice were restimulated in-vitro and single cells were isolated by flowcytometry based on the activation-induced upregulation of CD137 (FIG. 6).

(59) CLDN18.2-specific CD8+ T cells could be retrieved from all three immunized A2/DR1 mice and a total of 6 CLDN18.2-specific TCRs were cloned from single-sorted murine T cells. TCRs were subjected to immunological validation assays, which revealed that all six CLDN18.2-TCRs recognized one or both of the two HLA-A*0201-restricted epitopes CLDN18.2 aa 7-15 (CLDN18.2-A2-5) and CLDN18.2 aa 8-16 (CLDN18.2-A2-6), which were previously identified by ex-vivo ELISPOT analysis (FIG. 7).

Example 3: Generation and In-Vitro Validation of a Claudin-18.2-Specific CAR

(60) We generated a second generation CAR targeting CLDN18.2 that contains the signaling and costimulatory moieties of CD3 and CD28, respectively. A deletion of the lck binding moiety in the CD28 endodomain abrogates IL2 secretion upon CAR engagement to prevent induction of regulatory T cells (Kofler D. M. et al., (2011) Molecular Therapy 19 (4), 760-767). A modification of the IgG1 Fc ‘spacer’ domain in the extracellular moiety of the CAR avoids ‘off-target’ activation and unintended initiation of an innate immune response (Hombach A. et al., (2010) Gene Therapy 17, 1206-1213).

(61) To analyze the specific lysis of CLDN18.2-expressing target cells by CLDN18.2-CAR T cells a luciferase-based cytotoxicity assay was performed. CD8+ T cells were preactivated and transfected with IVT-RNA encoding either the CLDN18.2-CAR or the CLDN6-specific CAR as a control. CAR surface expression was confirmed after staining with fluorochrome-conjugated antibodies by flowcytometry (FIG. 8). Both CARs were well expressed on the surface of CD8+ T cells. CAR-transfected T cells were cultured with autologous iDCs transfected either with CLDN18.2- or CLDN6-RNA using different effector-to-target ratios and the specific lysis was calculated after 4h of coculture (FIG. 9). Both, the CLDN18.2-CAR and the CLDN6-CAR, mediated specific lysis of iDCs expressing CLDN18.2 and CLDN6, respectively. No lysis could be observed, when iDCs expressed the respective control antigen.

(62) In order to analyze, if the CLDN18.2-CAR-mediated lysis of CLDN18.2-expressing target cells may be inhibited by addition of an idiotype-specific antibody, CAR T cells were preincubated with or without the antibody that specifically binds to the scFv fragment contained in the CLDN18.2-CAR before coculture with target cells was initiated and lysis was analyzed using a luciferase-based cytotoxicity assay (FIG. 10).

(63) The CLDN18.2-CAR-mediated lysis of CLDN18.2-expressing target cells could be efficiently inhibited even with a high E:T ratio of 30:1 by blocking the binding of the CLDN18.2-CAR to its target antigen. No inhibition of the CLDN6-CAR mediated lysis could be observed confirming the selective binding of the antibody to the CLDN18.2-CAR. This experiment confirmed on the one hand that the CLDN18.2-CAR-mediated lysis is exclusively dependent on the CLDN18.2 specificity of the CAR and on the other hand that the idiotype-specific antibody that is used for CLDN18.2-CAR detection could in principle also be applied for inhibition of CLDN18.2-CAR T cells in-vivo in case of a severe adverse event.

(64) An essential prerequisite for the anti-tumoral efficacy of CAR-engineered T cells is their ability to proliferate and persist in the patient. In order to analyze, if CLDN18.2-CAR T cells efficiently proliferate in response to CLDN18.2 ectopically expressed in iDCs, a carboxyfluorescein succinimidyl ester (CFSE) based in vitro co-culture assay was performed. CD8.sup.+ T cells transfected with IVT-RNA encoding the CLDN18.2-CAR were labeled with CFSE and co-cultured with autologous iDCs transfected with IVT-RNA encoding either CLDN18.2 or the control antigens CLDN9 or CLDN6. CAR surface expression was analyzed by flow cytometry using a fluorochrome-coupled anti-idiotype-specific antibody (FIG. 11A). After four days of co-culture, the antigen-specific proliferation of CFSE-labeled CAR-transfected CD8.sup.+ T cells was analyzed by flow cytometry. The CLDN18.2-CAR mediated proliferation of about 86% of CD8.sup.+ T cells in response to CLDN18.2, while only background proliferation of CAR T cells in response to control antigen transfected iDCs (CLDN9, CLDN6) could be observed (FIG. 11B). These data confirm that efficient antigen-specific activation and expansion of CLDN18.2-CAR T cells can be achieved by ectopic CLDN18.2 expression in human iDCs.

(65) The potency of the CLDN18.2-CAR to mediate antigen-specific activation and expansion of CAR bearing T cells in vivo was examined in a syngenic mouse model. To follow the fate of adoptively transferred CAR-T cells in vivo, a tricistronic retroviral vector was used which encodes luciferase (effLuc) and eGFP reporter genes downstream of CLDN18.2-CAR separated by viral T2A sequences.

(66) Naïve BALB/c mice were engrafted with CLDN18.2-CAR transduced murine T cells. At the day of transfer, CLDN18.2-CAR expression on transduced T cells was assessed by flow cytometry using fluorochrome-coupled anti-idiotype-specific antibody in combination with eGFP reporter expression. The CLDN18.2-CAR was highly expressed in about 36% of CD8.sup.+ and in about 45% of CD4.sup.+ T cells (FIG. 12A). Engrafted mice were then treated with IVT-RNA encoding either CLDN18.2 or a control (Ctrl) antigen. A strong increase of light emission could be observed in CLDN18.2 RNA compared to control RNA treated mice two days after mRNA vaccination indicating significant activation and proliferation of CLDN18.2-CAR T cells in response to the cognate antigen (FIG. 12B). These data clearly showed the functionality and antigen specificity of the CLDN18.2 CAR in T cells in vivo.

(67) TABLE-US-00004 CLDN18.2-specific T cell epitopes A2-1 (aa 68-76) (SEQ ID NO: 2) TLLGLPAML A2-2 (aa 71-79) (SEQ ID NO: 3) GLPAMLQAV A2-3 (14-22) (SEQ ID NO: 4) SLIGIAGII A2-4 (17-25) (SEQ ID NO: 5) GIAGIIAAT A2-5 (7-15) (SEQ ID NO: 6) QGLGFVVSL A2-6 (8-16) (SEQ ID NO: 7) GLGFVVSLI CLDN18.2-specific T cell receptors mTCR.sub.CD8-CL18#2 >Alpha V12D.1 J33 C (MN lacks N-terminal; V .fwdarw. G und S .fwdarw. C) (SEQ ID NO: 8) MRPGTCSVLVLLLMLRRSNGDSVTQTEGLVTVTEGLPVKLNCTYQTTYLTIAFFWYVQ YLNEAPQVLLKSSTDNKRTEHQGFHATLHKSSSSFHLQKSSAQLSDSALYYCALMDSNY QLIWGSGTKLIIKPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDK TVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNL NFQNLSVMGLRILLLKVAGFNLLMTLRLWSS* >Beta V13.3 D1 J1.4*02 C1 (SEQ ID NO: 9) MGSRLFFVVLILLCAKHMEAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDT GHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSINERLF FGHGTKLSVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNG KEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPE GSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMA MVKRKNS* mTCR.sub.CD8-CL18#4 >Alpha V6D.7 *04 J26 C (S .fwdarw. F) (SEQ ID NO: 10) MNSITGFMTVMLLIFTRAHGDSVTQTEGQVALSEEDFLTIHCNYSASGYPALFWYVQYP GEGPQFLFRASRDKEKGSSRGFEATYDKGTTSFHLRKASVQESDSAVYYCALGDYAQG LTFGLGTRVSVFPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKT VLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNL NFQNLSVMGLRILLLKVAGFNLLMTLRLWSS* >Beta V2 D2 J2.7 C2 (CASSQEWGGYEQYF) (SEQ ID NO: 11) MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAK KPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQEWGG YEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSW WVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEED KWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNS* mTCR.sub.CD8-CL18#5 >Alpha V6D.7*04 J47 C (N .fwdarw. D und S .fwdarw. F) (SEQ ID NO: 12) MDSITGFMTVMLLIFTRAHGDSVTQTEGQVALSEEDFLTIHCNYSASGYPTLFWYVQYP GEGPQLLFRASRDKEKGSSRGFEATYDKGTTSFHLRKASVQESDSAVYYCALSVDYAN KMIFGLGTILRVRPHIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDK TVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNL NFQNLSVMGLRILLLKVAGFNLLMTLRLWSS* >Beta V1 D2 J2.7 C2 (SEQ ID NO: 13) MWQFCILCLCVLMASVATDPTVTLLEQNPRWRLVPRGQAVNLRCILKNSQYPWMSWY QQDLQKQLQWLFTLRSPGDKEVKSLPGADYLATRVTDTELRLQVANMSQGRTLYCTCS PLTGSYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHV ELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHG LSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVL VSGLVLMAMVKKKNS* mTCR.sub.CD8-CL18#8 >Alpha V8D.2*02 or V8N.2 J31 C (SEQ ID NO: 14) MNRFLGISLVTLWFQVAWAKSQWGEENLQALSIQEGEDVTMNCSYKTYTTVVQWYRQ KSGKGPAQLILIRSNEREKRSGRLRATLDTSSQSSSLSITGTLATDTAVYFCATDNRIFFG DGTQLVVKPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLD MKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQN LSVMGLRILLLKVAGFNLLMTLRLWSS* >Beta V23 D2 J2.7 C2 (SEQ ID NO: 15) MGARLICYVALCLLGAGSFDAAVTQKPRYLIKMKGQEAEMKCIPEKGHTAVFWYQQK QSKELKFLIYFQNQQPLDQIDMVKERFSAVCPSSSLCSLGIRTCEAEDSALYLCSSSQSGG YEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSW WVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEED KWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNS* mTCR.sub.CD8-CL18#9 >Alpha V9N.3 J21 C (P .fwdarw. S) (SEQ ID NO: 16) MLLALLSVLGIHFLLRDAQAQSVTQPDARVTVSEGASLQLRCKYSYFGTPYLFWYVQY PRQGLQLLLKYYPGDPVVQGVNGFEAEFSKSNSSFHLRKASVHWSDWAVYFCAVSKY YNVLYFGSGTKLTVEPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFIT DKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETD MNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS* >Beta V2 D2 J2.7 C2 (CASSQDQGGQGQYF) (SEQ ID NO: 17) MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAK KPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSQDQGG QGQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSW WVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEED KWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL VLMAMVKKKNS* mTCR.sub.CD8-CL18#12 >Alpha V6.3*02 or V6D.3 J26 C (N .fwdarw. T) (SEQ ID NO: 18) MNTSPALVTVMLFILGRTHGDSVIQMQGQVTLSENDFLFINCTYSTTGYPTLFWYVQYS GEGPQLLLQVTTANNKGSSRGFEATYDKGTTSFHLQKTSVQEIDSAVYYCAIGNYAQGL TFGLGTRVSVFPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTV LDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNF QNLSVMGLRILLLKVAGFNLLMTLRLWSS* >Beta V2 D2 J2.7 C2 (CASSPDWGAEYEQYF) (SEQ ID NO: 19) MGSIFLSCLAVCLLVAGPVDPKIIQKPKYLVAVTGSEKILICEQYLGHNAMYWYRQSAK KPLEFMFSYSYQKLMDNQTASSRFQPQSSKKNHLDLQITALKPDDSATYFCASSPDWGA EYEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELS WWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSE EDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVS GLVLMAMVKKKNS*