Closer protein-18.2 specific immunoreceptors and t cell epitopes

Abstract

The present invention provides closed protein-18.2 specific immune receptors (T cell receptors and artificial T cell receptors (chimeric antigen receptors; CARs)) and T cell epitopes for immunotherapy.

Description

[0001] TECHNICAL FIELD

[0002] The present invention relates to the provision of closed Claudin-18.2 (Claudin-18.2) specific immune receptors (T cell receptors and artificial T cell receptors (chimeric antigen receptors; CARs)) and T cell epitopes useful for immunotherapy. BACKGROUND

[0003] Evolution of the immune system has resulted in vertebrates in a highly efficient network based on two types of defense: innate and adaptive immunity.

[0004] In contrast to the evolutionarily ancient innate immune system that relies on invariant receptors recognizing common molecular patterns associated with pathogens, adaptive immunity is based on highly specific antigen receptors on B cells (B lymphocytes) and T cells (T lymphocytes) and clonal selection.

[0005] However, B cells enhance the humoral immune response by secreting antibodies, and T cells mediate the cellular immune response leading to the destruction of the recognized cells.

[0006] In humans and animals, T cells play a central role in cell-mediated immunity. Recognition and binding of a specific antigen is mediated by the T cell receptor (TCR) expressed on the surface of T cells.

[0007] The T cell receptor (TCR) of T cells is capable of interacting with an immunogenic peptide (epitope) bound to a major histocompatibility complex (MHC) molecule and presented on the surface of a target cell. The specific binding of the TCR initiates a signal cascade inside the T cell, leading to proliferation and differentiation into mature effector T cells. In order to be able to target a wide variety of antigens, the T cell receptor needs to have a great diversity.

[0008] This diversity is achieved by gene rearrangement of different non-contiguous segments of genes that assign the genetic code to the different structural regions of the TCR. The TCR is composed of one alpha chain and one beta chain, or one gamma chain and one delta chain. The alpha / beta chains of the TCR are composed of a N-terminal highly polymorphic variable region involved in antigen recognition and an invariant constant region. At the genetic level, these chains are divided into several regions: variable (V), diversity (D) (only beta and delta chains), joining (J), and constant (C) regions. The human beta chain gene contains over 60 variable (V) segments, 2 diversity (D) segments, over 10 joining segments, and 2 constant (C) region segments. The human alpha chain gene contains over 50 V segments and over 60 J segments, but no D segments, and one C segment. The murine beta chain gene contains over 30 variable (V) segments, 2 diversity (D) segments, over 10 joining segments, and 2 constant (C) region segments. The murine alpha chain gene contains almost 100 V segments, 60 J segments, no D segments, but one C segment. During T cell differentiation, a specific T cell receptor gene is generated by rearrangement of one V, one D (only beta and delta chains), one J, and one C region gene. The diversity of the TCR is further expanded by imprecise V-(D)-J rearrangements, where random nucleotides are introduced and / or deleted at the recombination site. Since the rearrangement of the TCR loci occurs in the genome during T cell maturation, each mature T cell expresses only one specific alpha / beta TCR or gamma / delta TCR.

[0009] MHC and antigen binding are mediated by the complementarity determining regions 1, 2, and 3 (CDRl, CDR2, CDR3) of the TCR. The CDR3 of the beta chain, which is most critical for antigen recognition and binding, is encoded by the V-D-J joining of the rearranged TCR beta chain gene. The TCR is part of a complex signal transduction machinery that includes the heterodimeric complex of the TCR alpha and beta chains, the co-receptor CD4 or CD8, and the CD3 signal transduction module Figure 1 ). While the CD3 chains transmit the activation signals inside the cell, the TCR alpha / beta heterodimer is only responsible for antigen recognition. Therefore, transfer of the TCR alpha / beta chain offers the opportunity to redirect T cells to any antigen of interest.

[0010] Immunotherapy

[0011] Antigen-specific immunotherapy aims to enhance or induce a specific immune response in a patient to control infectious or malignant disease. The identification of an increasing number of pathogen and tumor-associated antigens (TAAs) has led to a broad collection of suitable targets for immunotherapy. Cells presenting immunogenic peptides (epitopes) derived from these antigens can be specifically targeted by active or passive immunization strategies.

[0012] Active immunization tends to induce and expand antigen-specific T cells in the patient, which are able to specifically recognize and kill diseased cells. In contrast, passive immunization relies on the adoptive transfer of T cells, which are expanded in vitro and optionally genetically engineered (adoptive T cell therapy).

[0013] Vaccination

[0014] Tumor vaccines aim at inducing an endogenous tumor-specific immune response by active immunization. Different antigenic forms can be used for tumor vaccination, including whole cancer cells, proteins, peptides or immunological carriers such as RNA, DNA or viral vectors, which can be applied directly in vivo by pulsing DCs or in vitro, followed by transfer into the patient.

[0015] Due to improvements in immunization strategies and methods to detect antigen-specific immune responses, the number of clinical studies to treat induced immune responses is steadily increasing (Connerotte, T. et al. (2008). Cancer Res. 68. 3931-3940; Schmitt, M. et al. (2008) Blood 111, 1357-1365; Speiser, D. E. et al. (2008) Proc. Natl. Acad. Sci. U.S.A 105, 3849-3854; Adams, S. et al. (2008) J. Immunol. 181, 776-784).

[0016] However, in most cases, the detected immune responses could not be systematically correlated with clinical outcome (Curigliano, G. et al. (2006) Ann. Oncol. 17. 750-762; Rosenberg, S. A. et al. (2004) Nat. Med. 10, 909-915).

[0017] Thus, the accurate determination of peptide epitopes derived from tumor antigens can help to improve the specificity and efficiency of vaccination strategies as well as methods for immune monitoring.

[0018] Adoptive cell transfer (ACT)

[0019] ACT-based immunotherapy can be broadly defined as a form of passive immunization with pre-sensitized T cells that have been expanded in vitro from low precursor frequencies to clinically relevant cell numbers before being transferred into a non-immune recipient or into an autologous host. Cell types that have been used in ACT experiments are lymphokine-activated killer (LAK) cells (Mule, J. J. et al. (1984) Science 225, 1487-1489; Rosenberg, S. A. et al. (1985) N. Engl. J. Med. 313, 1485-1492), tumor-infiltrating lymphocytes (TILs) (Rosenberg, S. A. et al. (1994) J. Natl. Cancer Inst. 86, 1159-1166), donor lymphocytes after hematopoietic stem cell transplantation (HSCT), and tumor-specific T cell lines or clones (Dudley, M. E. et al. (2001) J. Immunother. 24, 363-373; Yee, C, et al. (2002) Proc. Natl. Acad. Sci. U.S.A 99, 16168-16173). Adoptive T cell transfer has shown therapeutic activity against human viral infections such as CMV. However, in healthy individuals, CMV infection and reactivation of endogenous latent viruses are controlled by the immune system, which leads to significant morbidity and mortality in immunocompromised individuals such as transplant recipients or AIDS patients.

[0020] Riddell and colleagues demonstrated that adoptive T cell therapy to reconstitute viral immunity in immunosuppressed patients by transferring CD8+ CMV-specific T cell clones derived from HLA-matched CMV seropositive transplant donors (Riddell, S. R. (1992) Science 257, 238-241).

[0021] As an alternative approach, transfer of CMV- or EBV-specific T cell populations derived from polyclonal donors into transplant recipients resulted in increased persistence of the transferred T cells (Rooney, CM. et al. (1998) Blood 92, 1549-1555; Peggs, K. S. et al. (2003) Lancet 362, 1375-1377).

[0022] For adoptive immunotherapy of melanoma, Rosenberg and colleagues established an ACT approach that relies on infusion of in vitro expanded autologous tumor infiltrating lymphocytes (TILs) isolated from resected tumors in combination with non-myeloablative lymphodepleting chemotherapy and high dose IL2. Recently published clinical studies resulted in an objective response rate of ~50% of treated metastatic melanoma patients (Dudley, M.E. et al. (2005) J. Clin. Oncol. 23:2346-2357).

[0023] However, several prerequisites have to be met by patients to qualify for ACT immunotherapy. They must have a resectable tumor. The tumor must form viable TILs under cell culture conditions. The TILs must be reactive against the tumor antigen and must be expanded in vitro to sufficient numbers. Especially in cancers other than melanoma, it is difficult to obtain such tumor reactive TILs. Furthermore, repeated in vitro stimulation and clonal expansion of normal human T lymphocytes leads to a gradual decrease in telomerase activity and shortening of telomeres, resulting in replicative senescence and reduced potential persistence of transferred T cells (Shen, X. et al. (2007) J. Immunother. 30: 123-129).

[0024] ACT using genetically engineered T cells

[0025] A way to overcome the limitations of ACT is the adoptive transfer of autologous T cells that are reprogrammed to express tumor reactive immune receptors with defined specificity during short-term in vitro culture and are subsequently re-infused into the patient (Kershaw M.H. et al. (2013) Nature Reviews Cancer 13(8):525-41). This strategy makes ACT applicable to a wide variety of common malignancies even if tumor reactive T cells are not present in the patient. Since the antigen specificity of T cells is entirely dependent on the heterodimeric complex of TCR a- and β-chains, transfer of clonal TCR genes to T cells provides the possibility to redirect them to any antigen of interest. Thus, TCR gene therapy provides an attractive strategy to develop antigen specific immunotherapy with autologous lymphocytes as a therapeutic regimen. The main advantages of TCR gene transfer are the generation of therapeutic amounts of antigen specific T cells within a few days and the possibility to introduce a specificity that is not present in all components of the endogenous TCR of the patient.

[0026] Several groups have demonstrated that TCR gene transfer is an attractive strategy to redirect the antigen specificity of primary T cells (Morgan, R. A. et al. (2003) J. Immunol. 171, 3287-3295; Cooper, L. J. et al. (2000) J. Virol. 74, 8207-8212; Fujio, K. et al. (2000) J. Immunol. 165, 528-532: Kessels, H. W. et al. (2001) Nat. Immunol. 2, 957-961; Dembic, Z. et al. (1986) Nature 320, 232-238).

[0027] The feasibility of TCR gene therapy in humans was recently demonstrated by Rosenberg and his team in a clinical trial for the treatment of malignant melanoma. Adoptive transfer of autologous lymphocytes retrovirally transduced with a melanoma / melanocyte antigen-specific TCR led to cancer regression in up to 30% of treated melanoma patients (Morgan, R. A. et al. (2006) Science 314, 126-129; Johnson, L. A. et al. (2009) Blood 114, 535-546).

[0028] Chimeric antigen receptors

[0029] Chimeric antigen receptors (CARs) are engineered receptors that combine a single-chain variable fragment (scFv) of a monoclonal antibody with an intracellular part comprising one or more signal transduction domains for T cell activation. CARs recognize native antigens in a non-MHC restricted manner and can therefore be used in all individuals, regardless of their HLA type, and they are functionalized in CD4+ T cells and CD8+ T cells.

[0030] A large number of CARs have been reported in the last decade, which target a diverse set of cell surface tumor antigens. Their biological function was significantly improved by the incorporation of a costimulatory domain, resulting in a tripartite receptor (scFv, CD28, CD3ζ), called second generation CARs. Third generation CARs comprise additional domains of costimulatory molecules such as OX40 and 4-1BB to enhance the proliferative capacity and persistence of the modified T cells Figure 2 ).

[0031] Target structures for antigen-specific immunotherapy

[0032] The discovery of a variety of tumor-associated antigens (TAAs) has provided the basis for the concept of antigen-specific immunotherapy (Novellino, L. et al. (2005) Cancer Immunol. Immunother. 54, 187-207). Due to their genetic instability, TAAs are unusual proteins expressed on tumor cells that are not expressed or are expressed in limited amounts in normal cells. These TAAs can elicit specific recognition of malignant cells by the immune system.

[0033] The molecular cloning of TAAs by screening of cDNA expression libraries of tumor origin using autologous tumor-specific T cells (van der Bruggen, P. et al. (1991) Science 254, 1643-1647), or circulating antibodies (Sahin, U. et al. (1995) Proc. Natl. Acad. Sci. U.S.A 92, 11810-11813), reverse immunology approaches, biochemical methods (Hunt, D.F. et al. (1992) Science 256, 1817-1820), gene expression analysis or electronic cloning strategies (Helftenbein, G. et al. (2008) Gene 414, 76-84) has generated a considerable number of target candidates for immunotherapeutic strategies. TAAs are classified into several categories including differentiation antigens, overexpressed antigens, tumor-specific splice variants, mutated gene products, viral and cancer-testis antigens (CTAs). The cancer-testis family is a very promising class of TAAs because their expression is restricted to testis and a large number of different tumor entities (Scanlan, M.J. et al. (2002) Immunol. Rev. 188, 22-32). More than 50 CT genes have been described so far (Scanlan, M.J. et al. (2004) Cancer Immun. 4, 1) and some of them are already in clinical studies (Adams, S. et al. (2008) J. Immunol. 181, 776-784; Atanackovic, D. et al. (2004) J. Immunol. 172, 3289-3296; Chen, Q. et al. (2004) Proc. Natl. Acad. Sci. U.S.A 101, 9363-9368; Connerotte, T. et al. (2008). Cancer Res. 68, 3931-3940; Davis, I.D. et al. (2004) Proc. Natl. Acad. Sci. U.S.A 101, 10697-10702; Jager, E. (2000) Proc. Natl. Acad. Sci. U.S.A 97, 12198-12203; Marchand, M. et al. (1999) Int. J. Cancer 80, 219-230; Schuler-Thurner, B. et al. (2000) J. Immunol. 165, 3492-3496).

[0034] Despite the increasing number of attractive target structures for immunotherapeutic approaches, defined HLA-restricted specific T cell clones or lines exist only for a few of them (Chaux, P. et al. (1999) J. Immunol. 163, 2928-2936; Zhang, Y. et al. (2002) Tissue Antigens 60, 365-371; Zhao, Y. et al. (2005) J. Immunol. 174, 4415-4423).

[0035] Claudins are integral membrane proteins located within the tight junctions of epithelial and endothelial cells. Claudins are predicted to have four transmembrane segments with two extracellular loops, and the N- and C-termini are located in the cytoplasm. The claudin (CLDN) family of transmembrane proteins plays a central role in maintaining epithelial and endothelial tight junctions and can also play a role in maintaining the cytoskeleton and signaling.

[0036] CLDN18 belongs to the claudin family of cell surface molecules with four transmembrane domains involved in the formation of tight junctions (Tsukita S, Nat Rev Mol Cell Biol 2001; 2:285-93). The human CLDN18 gene has two alternative first exons, resulting in two protein isoforms differing in the N-terminal 69 amino acids (CLDN18.1 and CLDN18.2) (Niimi T, Mol Cell Biol 2001; 21 :7380-90), including the first extracellular loop Figure 4 A). A set of restriction tissue transcriptomes showed that these isoforms have different lineage orientations, with CLDN18.1 being mainly expressed in lung tissue, while CLDN18.2 showed a gastric specificity. CLDN18.2 expression is restricted to short-lived, differentiated epithelial cells of the gastric mucosa and is absent in the gastric stem cell area and any other healthy tissue. CLDN18.2 expression is associated with gastric esophageal cancer, pancreatic cancer and other cancers Figure 4 B, C; Sahin U et al., Clin Cancer Res 2008; 14:7624-34; Karanjawala ZE et al., Am J Surg Pathol 2008; 32: 188-96). Recombinant mAbs against this target are currently in clinical phase II trials, but the evaluation of CLDN18.2 as a target for T cell-based therapeutic approaches has not been addressed.

[0037] The frequent overexpression of CLDN18.2 on tumors qualifies this molecule as a very attractive target for the development of therapeutic agents against CLDN18.2, such as vaccine therapeutics and therapeutic antibodies. However, so far, no HLA-A*2 restricted CLDN18.2 T cell epitopes and T cell receptors or CARs targeting CLDN18.2 have been described and it is not known whether cancer cells expressing CLDN18.2 can be targeted in vivo by immunotherapy involving T cells using active or passive immunization approaches.

[0038] SUMMARY SUMMARY

[0039] The present invention relates to T cell receptors and artificial T cell receptors specific for the tumor-associated antigen CLDN18.2, in particular the tumor-associated antigen CLDN18.2 present on the surface of a cell, such as a diseased cell, or presented on the surface of a cell, such as a diseased cell or an antigen-presenting cell, and to peptides comprising an epitope recognized by these T cell receptors, i.e. CLDN18.2-T cell epitopes.

[0040] By adoptively transferring T cells engineered to express such T cell receptors or artificial T cell receptors, cancer cells expressing CLDN18.2 can be specifically targeted, leading to selective destruction of the cancer cells. Furthermore, the T cell epitopes provided by the present invention can be used to design vaccines against cancer expressing CLDN18.2.

[0041] In one aspect, the present invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, and 7, or a variant of said amino acid sequence. In one embodiment, the peptide has a length of 100 or less, 50 or less, 20 or less, or 10 or less amino acids. In one embodiment, the peptide can be processed to yield a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, and 7, or a variant of said amino acid sequence. In one embodiment, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, and 7, or a variant of said amino acid sequence.

[0042] In one embodiment, the peptide is an MHC class I or II presented peptide, preferably an MHC class I presented peptide, or if present within a cell, the peptide can be processed to yield a processing product which is an MHC class I or II presented peptide, preferably an MHC class I presented peptide. Preferably, the MHC class I or II presented peptide has a sequence which substantially corresponds to a given amino acid sequence, i.e. an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6 and 7 or a variant of said amino acid sequence. Preferably, the peptide of the present application is capable of stimulating a cellular response against a disease involving cells characterized by presentation of CLDN18.2 with MHC class I.

[0043] In another aspect, the present application relates to a nucleic acid comprising a nucleotide sequence encoding a peptide of the present application and a cell comprising said nucleic acid. The nucleic acid can be a recombinant nucleic acid. The nucleic acid can be present in a plasmid or expression vector and can be functionally linked to a promoter. In one embodiment, the nucleic acid is RNA. Preferably, the cell expresses the peptide. The cell can be a recombinant cell and can secrete the encoded peptide or processing product thereof, can express it on the surface, and preferably can additionally express a MHC molecule which binds to the peptide or processing product thereof, and preferably presents the peptide or processing product thereof on the cell surface. In one embodiment, the cell endogenously expresses the MHC molecule. In another embodiment, the cell expresses the MHC molecule and / or the peptide in a recombinant manner. The cell is preferably non-proliferative. In a preferred embodiment, the cell is an antigen presenting cell, in particular a dendritic cell, a monocyte or a macrophage.

[0044] In another aspect, the present application relates to a cell presenting a peptide of the present application or a processing product thereof, wherein the processing product is preferably a peptide having a given amino acid sequence, i.e. an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6 and 7 or a variant of said amino acid sequence. In one embodiment, the cell is a cell comprising a nucleic acid comprising a nucleotide sequence encoding a peptide of the present application. Preferably, the cell expresses the nucleic acid to yield the peptide. Optionally, the cell processes the peptide to yield a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6 and 7 or a variant of said amino acid sequence. The cell can present the peptide or processing product thereof via a MHC molecule on its surface. In one embodiment, the cell endogenously expresses the MHC molecule. In another embodiment, the cell expresses the MHC molecule in a recombinant manner. In one embodiment, the MHC molecule of the cell is loaded (pulsed) with the peptide by adding the peptide to the cell. The cell can express the peptide in a recombinant manner and present the peptide or processing product thereof on the cell surface. The cell is preferably non-proliferative. In a preferred embodiment, the cell is an antigen presenting cell, e.g. a dendritic cell, a monocyte or a macrophage.

[0045] In another aspect, the application relates to an immunoreactive cell that reacts with a peptide of the application, especially when presented at the surface of a cell such as a diseased cell. The immunoreactive cell can be a cell that has been sensitized in vitro to recognize the peptide. The immunoreactive cell can be a T cell, preferably a cytotoxic T cell. Preferably, the immunoreactive cell binds a sequence that substantially corresponds to a given amino acid sequence, i.e. an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7 or a variant of said amino acid, especially when bound to an MHC, such as an MHC at the surface of a cell of a diseased cell.

[0046] In another aspect, the application relates to a binding agent that binds a peptide of the application, optionally in a complex with an MHC molecule.

[0047] In another aspect, the application relates to a T cell receptor or a polypeptide chain of said T cell receptor that binds to a peptide of the application, optionally in a complex with an MHC molecule, and preferably said T cell is reactive with said peptide. In one embodiment, the polypeptide chain of said T cell receptor is a T cell receptor alpha chain or a T cell receptor beta chain.

[0048] In another aspect, the application relates to a T cell receptor alpha chain or a T cell receptor comprising said T cell receptor alpha chain,

[0049] wherein said T cell receptor alpha chain is selected from the group consisting of:

[0050] (i) a T cell receptor alpha chain comprising at least one, preferably two, more preferably all three CDR sequences selected from the group consisting of a T cell receptor alpha chain of SEQ ID NO: 8, 10, 12, 14, 16 and 18 or a variant thereof; and

[0051] (ii) a T cell receptor alpha chain comprising a T cell receptor alpha chain sequence selected from the group consisting of SEQ ID NO: 8, 10, 12, 14, 16 and 18 or a fragment thereof, or a variant of said sequence or fragment.

[0052] In one embodiment, said SEQ ID NO: is selected from the group consisting of SEQ ID NO: 8, 10, 14 and 18 and said T cell receptor is reactive with a peptide comprising an amino acid sequence of SEQ ID NO: 6 or a variant of said amino acid sequence.

[0053] In one embodiment, said SEQ ID NO: is selected from the group consisting of SEQ ID NO: 10, 12 and 14 and said T cell receptor is reactive with a peptide comprising an amino acid sequence of SEQ ID NO: 7 or a variant of said amino acid sequence.

[0054] In another aspect, the application relates to a T cell receptor beta chain or a T cell receptor comprising said T cell receptor beta chain,

[0055] wherein the T cell receptor beta chain is selected from the group consisting of:

[0056] (i) a T cell receptor beta chain comprising at least one, preferably two, more preferably all three CDR sequences of a T cell receptor beta chain selected from the group consisting of SEQ ID NO: 9, 11, 13, 15, 17 and 19, or a variant thereof; and

[0057] (ii) a T cell receptor beta chain comprising a T cell receptor beta chain sequence selected from the group consisting of SEQ ID NO: 9, 11, 13, 15, 17 and 19, or a fragment thereof, or a variant of said sequence or fragment.

[0058] In one embodiment, the SEQ ID NO: is selected from the group consisting of SEQ ID NO: 9, 11, 15 and 19, and the T cell receptor reacts with a peptide comprising the amino acid sequence of SEQ ID NO: 6, or a variant of said amino acid sequence.

[0059] In one embodiment, the SEQ ID NO: is selected from the group consisting of SEQ ID NO: 11, 13 and 15, and the T cell receptor reacts with a peptide comprising the amino acid sequence of SEQ ID NO: 7, or a variant of said amino acid sequence.

[0060] In another aspect, the present application relates to a T cell receptor selected from the group consisting of:

[0061] (I) a T cell receptor comprising:

[0062] (i) a T cell receptor alpha chain comprising at least one, preferably two, more preferably all three CDR sequences of a T cell receptor alpha chain of SEQ ID NO: x, or a variant thereof, and

[0063] (ii) a T cell receptor beta chain comprising at least one, preferably two, more preferably all three CDR sequences of a T cell receptor beta chain of SEQ ID NO: x+1, or a variant thereof;

[0064] wherein x is selected from the group consisting of 8, 10, 12, 14, 16 and 18;

[0065] and

[0066] (II) a T cell receptor comprising:

[0067] (i) a T cell receptor alpha chain comprising a T cell receptor alpha chain sequence of SEQ ID NO: x, or a fragment thereof, or a variant of said sequence or fragment, and

[0068] (ii) a T cell receptor beta chain comprising a T cell receptor beta chain sequence of SEQ ID NO: x+1 or a fragment thereof, or a variant of said sequence or fragment;

[0069] wherein x is selected from 8, 10, 12, 14, 16 and 18.

[0070] In one embodiment, said x is selected from 8, 10, 14 and 18, and said T cell receptor reacts with a peptide comprising an amino acid sequence of SEQ ID NO: 6 or a variant of said amino acid sequence.

[0071] In one embodiment, said x is selected from 10, 12 and 14, and said T cell receptor reacts with a peptide comprising an amino acid sequence of SEQ ID NO: 7 or a variant of said amino acid sequence.

[0072] In one embodiment, binding of said T cell receptor, when expressed by a T cell and / or presented on a T cell, to a CLDN18.2 -peptide epitope presented on a cell, such as a cancer cell, as described above, results in proliferation and / or activation of said T cell, wherein said activated T cell preferably releases cytotoxic factors, such as perforin and granzymes, and initiates cytolysis and / or apoptosis of the cancer cell.

[0073] In another aspect, the present application relates to an artificial T cell receptor binding claudin-18.2 (CLDN18.2). In one embodiment, the binding is specific binding.

[0074] In one embodiment, said CLDN18.2 is expressed in a cancer cell. In one embodiment, said CLDN18.2 is expressed on the surface of a cancer cell. In one embodiment, said artificial T cell receptor binds to an extracellular domain or an epitope in an extracellular domain of CLDN18.2. In one embodiment, said artificial T cell receptor binds to a natural epitope of CLDN18.2 present on the surface of a living cell. In one embodiment, said artificial T cell receptor binds to a first extracellular loop of CLDN18.2. In one embodiment, binding of said artificial T cell receptor, when expressed by a T cell and / or present on a T cell, to CLDN18.2 present on a cell, such as a cancer cell, results in proliferation and / or activation of said T cell, wherein said activated T cell preferably releases cytotoxic factors, such as perforin and granzymes, and initiates cytolysis and / or apoptosis of the cancer cell.

[0075] In one embodiment, the artificial T cell receptor of the present application comprises a binding domain for CLDN18.2. In one embodiment, the binding domain for CLDN18.2 is comprised in an exodomain of said artificial T cell receptor. In one embodiment, the binding domain for CLDN18.2 comprises a single chain variable fragment (scFv) of a CLDN18.2 antibody. In one embodiment, the binding domain for CLDN18.2 comprises a heavy chain variable region (VH) of an immunoglobulin specific for CLDN18.2 (VH(CLDN18.2)) and a light chain variable region (VL) of an immunoglobulin specific for CLDN18.2 (VL(CLDN18.2)). In one embodiment, said heavy chain variable region (VH) and the corresponding light chain variable region (VL) are connected via a peptide linker, preferably a peptide linker comprising the amino acid sequence (GGGGS)3. In one embodiment, the binding domain for CLDN18.2 comprises a VH(CLDN18.2) comprising the amino acid sequence set forth in SEQ ID NO: 23 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the binding domain for CLDN18.2 comprises a VL(CLDN18.2) comprising the amino acid sequence set forth in SEQ ID NO: 30 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the binding domain for CLDN18.2 comprises a VH(CLDN18.2) comprising the amino acid sequence set forth in SEQ ID NO: 23 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a VL(CLDN18.2) comprising the amino acid sequence set forth in SEQ ID NO: 30 or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the binding domain for CLDN18.2 comprises the amino acid sequence set forth in SEQ ID NO: 35 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0076] In one embodiment, the binding domain for CLDN18.2 recognizes the same or essentially the same epitope as a binding domain for CLDN18.2 or an antibody for CLDN18.2, wherein the binding domain for CLDN18.2 or the antibody for CLDN18.2 comprises a VH(CLDN18.2) comprising an amino acid sequence as set forth in SEQ ID NO: 23 or a fragment thereof, or a variant of said amino acid sequence or fragment, and a VL(CLDN18.2) comprising an amino acid sequence as set forth in SEQ ID NO: 30 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or the binding domain for CLDN18.2 competes with said binding domain for CLDN18.2 or antibody for CLDN18.2 for binding to CLDN18.2. In one embodiment, the binding domain for CLDN18.2 recognizes the same or essentially the same epitope as a binding domain for CLDN18.2 comprising an amino acid sequence as set forth in SEQ ID NO: 35 or a fragment thereof, or a variant of said amino acid sequence or fragment, and / or competes with said binding domain for CLDN18.2 for binding to CLDN18.2. A binding domain that competes with a second binding domain or antibody for binding to a target preferably is antagonistic with said second binding domain or antibody.

[0077] In one embodiment, the artificial T cell receptor of the application comprises a transmembrane domain. In one embodiment, the transmembrane domain is a transmembrane hydrophobic alpha helix. In one embodiment, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof.

[0078] In one embodiment, the artificial T cell receptor of the application comprises a T cell signaling domain. In one embodiment, the T cell signaling domain is intracellular. In one embodiment, the T cell signaling domain comprises CD3-zeta, preferably comprises the endodomain of CD3-zeta, optionally in combination with CD28. In one embodiment, the T cell signaling domain comprises a sequence of SEQ ID NO: 40 or a fragment thereof, or a variant of said sequence or fragment.

[0079] In one embodiment, the artificial T cell receptor of the application comprises a signal peptide that directs the nascent protein into the endoplasmic reticulum. In one embodiment, the signal peptide is before the binding domain for CLDN18.2. In one embodiment, the signal peptide comprises a sequence of SEQ ID NO: 37 or a fragment thereof, or a variant of said sequence or fragment.

[0080] In one embodiment, the artificial T cell receptor of the application comprises a spacer region linking the binding domain of CLDN18.2 to the transmembrane domain. In one embodiment, the spacer region allows the binding domain of CLDN18.2 to be oriented in different directions to facilitate recognition of CLDN18.2. In one embodiment, the spacer region comprises a hinge region from IgGl. In one embodiment, the spacer region comprises the sequence of SEQ ID NO: 38 or a fragment thereof, or a variant of said sequence or fragment.

[0081] In one embodiment, the artificial T cell receptor of the application comprises the following structure:

[0082] NH2-signal peptide-binding domain of CLDN18.2-spacer region-transmembrane domain-T cell signaling domain-COOH.

[0083] In one embodiment, the artificial T cell receptor of the application comprises the amino acid sequence of SEQ ID NO: 41 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0084] The above T cell receptors and artificial T cell receptors are preferably specific for the tumor-associated antigen CLDN18.2, especially when present on the surface of a cell, such as a diseased cell, or when presented on the surface of a cell, such as a diseased cell or an antigen-presenting cell.

[0085] The T cell receptors and artificial T cell receptors of the application can be expressed by and / or present on the surface of a cell, such as a T cell.

[0086] In another aspect, the application relates to a nucleic acid comprising a nucleotide sequence encoding a T cell receptor chain or T cell receptor of the application or encoding an artificial T cell receptor of the application. In one embodiment, the nucleic acid is a recombinant nucleic acid. In one embodiment, the nucleic acid is in the form of a vector or in the form of RNA.

[0087] In another aspect, the present application relates to a cell comprising a T cell receptor chain or a T cell receptor of the present application, or an artificial T cell receptor of the present application and / or comprising a nucleic acid comprising a nucleotide sequence encoding a T cell receptor chain or a T cell receptor of the present application or encoding an artificial T cell receptor of the present application. In one embodiment, the nucleic acid is RNA, preferably in vitro transcribed RNA. The cell can be a cell expressing a T cell receptor chain or a T cell receptor of the present application or an artificial T cell receptor of the present application and / or can have a T cell receptor chain or a T cell receptor of the present application or an artificial T cell receptor of the present application on its surface. In one embodiment, the cell is a cell useful for adoptive cell transfer. The cell can be an effector or a stem cell, preferably an immunoreactive cell. The immunoreactive cell can be a T cell, preferably a cytotoxic T cell. In one embodiment, the immunoreactive cell is reactive with the tumor associated antigen CLDN18.2. In one embodiment, the CLDN18.2 is present on the surface of a cell, e.g. a diseased cell. In one embodiment, the CLDN18.2 is presented on the surface of a cell, e.g. a diseased cell or an antigen presenting cell, and the immunoreactive cell is reactive with a peptide of the present application, especially when presented in the context of an MHC, and preferably binds a sequence substantially corresponding to a given amino acid sequence, i.e. an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 7 or a variant of said amino acid sequence. In one embodiment, the cell lacks surface expression of an endogenous TCR or is specific for an antigen unrelated to CLDN18.2.

[0088] In one embodiment, the cell of the present application is subjected to antigen specific expansion and re-challenge prior to use for adoptive cell transfer, wherein the antigen specific expansion and re-challenge can be achieved by exposing the cell to autologous antigen presenting cells, preferably presenting CLDN18.2 or a peptide fragment thereof.

[0089] In another aspect, the present application relates to a method of generating an immunoreactive cell comprising the step of transducing a T cell with a nucleic acid comprising a nucleotide sequence encoding a T cell receptor chain or a T cell receptor of the present application, or encoding an artificial T cell receptor of the present application.

[0090] Furthermore, the present application generally comprises treating a disease by targeting diseased cells, such as cancer cells, especially cancer cells expressing CLDN18.2. The method provides for selective eradication of cells expressing and / or presenting the tumor associated antigen CLDN18.2 on their surface, thereby minimizing adverse effects on normal cells not expressing and / or presenting CLDN18.2. Accordingly, the disease for which treatment is preferred is a disease in which CLDN18.2 is expressed and optionally presented, such as a cancer disease, especially a disease as described herein.

[0091] When administering the peptide of the application, the nucleic acid comprising a nucleotide sequence encoding the peptide of the application, or the cell of the application comprising said nucleic acid, the treatment preferably involves active immunization. Preferably, CLDN18.2-specific T cells are expanded in the patient, which are capable of recognizing and killing diseased cells. When administering the immunoresponsive cell of the application, the T cell receptor of the application, the artificial T cell receptor of the application, the nucleic acid of the application comprising a nucleotide sequence encoding the T cell receptor of the application or encoding the artificial T cell receptor of the application, or the cell of the application comprising the T cell receptor or artificial T cell receptor of the application and / or comprising the nucleic acid of the application comprising a nucleotide sequence encoding the T cell receptor of the application or encoding the artificial T cell receptor of the application, the treatment preferably involves passive immunization. Preferably, CLDN18.2-specific T cells, which are capable of recognizing and killing diseased cells and which are optionally genetically engineered and / or expanded in vitro, are adoptively transferred into the patient.

[0092] In one aspect, the present application relates to a pharmaceutical composition comprising one or more of:

[0093] (i) the peptide of the application;

[0094] (ii) the nucleic acid of the application;

[0095] (iii) the cell of the application;

[0096] (iv) the immunoresponsive cell of the application;

[0097] (v) the binding agent of the application;

[0098] (vi) the T cell receptor of the application; and

[0099] (vi) the artificial T cell receptor of the application.

[0100] The pharmaceutical composition of the application can comprise a pharmaceutically acceptable carrier and can optionally comprise one or more adjuvants, stabilizers, etc. The pharmaceutical composition can be in the form of a therapeutic or prophylactic vaccine. In one embodiment, the pharmaceutical composition is for use in the treatment or prevention of a cancer disease, such as those described herein.

[0101] Administering the pharmaceutical composition as described above can provide MHC class II presented epitopes, which are capable of eliciting a CD4+ helper T cell response and / or a CD8+ T cell response to CLDN18.2 (including cells expressing CLDN18.2 on the cell surface and / or presenting CLDN18.2 in the context of MHC molecules). Alternatively or in addition, administering the pharmaceutical composition as described above can provide MHC class I presented epitopes, which are capable of eliciting a CD8+ T cell response to CLDN18.2.

[0102] In another aspect, the present application relates to a method of treating or preventing a cancer disease, comprising administering to a patient a pharmaceutical composition of the present application.

[0103] In another aspect, the present application relates to a peptide of the present application, a nucleic acid of the present application, a cell of the present application, an immunoreactive cell of the present application, a binding agent of the present application, a T cell receptor of the present application or an artificial T cell receptor of the present application for use in therapy, in particular for use in treating or preventing cancer.

[0104] Another aspect relates to a method of inducing an immune response in a subject, comprising administering to the subject a pharmaceutical composition of the present application.

[0105] Another aspect relates to a method for stimulating, priming and / or expanding T cells, comprising contacting the T cells with one or more of a peptide of the present application, a nucleic acid of the present application comprising a nucleotide sequence encoding a peptide of the present application, a cell of the present application comprising said nucleic acid and / or a cell of the present application presenting a peptide of the present application or a processing product thereof. In one embodiment, the peptide of the present application is presented in the context of an MHC molecule, such as on the surface of a cell, e.g. an antigen presenting cell.

[0106] In this aspect, the present application can relate to a method of preparing CLDN18.2 specific T cells. The T cells can be stimulated, primed and / or expanded in vitro or in vivo. Preferably, the T cells are present in a sample obtained from a subject. The stimulated, primed and / or expanded T cells can be administered to a subject, and can be autologous, allogeneic, syngeneic to the subject.

[0107] In the present application in the above aspects of a method for inducing an immune response in a subject or a method for stimulating, priming and / or expanding T cells, it can relate to a method for treating a cancer disease in a subject.

[0108] Another aspect relates to a method of killing cancer cells of a subject, comprising providing to the subject a therapeutically effective amount of a peptide of the present application, a nucleic acid of the present application, a cell of the present application, an immunoreactive cell of the present application, a binding agent of the present application, a T cell receptor of the present application or an artificial T cell receptor of the present application.

[0109] The compositions and reagents described herein are preferably capable of inducing or promoting a cellular response, preferably cytotoxic T cell activity, against a disease characterized by expression of CLDN18.2 and / or presentation of CLDN18.2 with MHC class I, such as a cancer disease.

[0110] In one aspect, the present application provides the reagents and compositions described herein for use in the therapeutic methods described herein.

[0111] The treatment of a cancer disease described herein can be combined with surgical resection and / or radiation and / or conventional chemotherapy.

[0112] In another aspect, the application relates to a method for determining an immune response in a subject, comprising determining T cells that react with a peptide of the application or a cell of the application presenting a peptide of the application or a processed product thereof in a biological sample isolated from the subject. The method can comprise the following steps:

[0113] (a) incubating a sample comprising T cells isolated from a subject with one or more of:

[0114] (i) a peptide of the application;

[0115] (ii) a nucleic acid of the application comprising a nucleotide sequence encoding a peptide of the application; and

[0116] (iii) a cell of the application comprising said nucleic acid or a cell of the application presenting a peptide of the application or a processed product thereof;

[0117] and

[0118] (b) detecting specific activation of T cells, thereby determining the presence or absence of an immune response in the subject.

[0119] The application in the above aspects of a method for determining an immune response in a subject can relate to a method for diagnosing a cancer disease in a subject.

[0120] In one embodiment of the diagnostic method, the biological sample is derived from a tissue or organ, wherein cells of the tissue or organ do not express CLDN18.2 substantially when the tissue or organ is free of disease.

[0121] Generally, the level of T cells in the biological sample is compared to a reference level, wherein a deviation from the reference level is indicative of the presence and / or stage of the disease in the subject. The reference level can be a level determined in a control sample (e.g., derived from a healthy tissue or subject) or a median level derived from healthy subjects. A "deviation" from the reference level designates any significant change, e.g., an increase of at least 10%, 20% or 30%, preferably at least 40% or 50%, or even higher. Preferably, the presence of T cells in the biological sample or an increased number of T cells in the biological sample compared to the reference level is indicative of the presence of the disease.

[0122] T cells can be isolated from the peripheral blood, lymph nodes, tissue samples (such as derived from biopsies and resections or other sources) of a patient. Reactivity assays can be performed on primary T cells or other suitable derivatives. For example, T cells can be fused to produce hybridomas. Assays for measuring T cell responsiveness are known in the art and include proliferation assays and cytokine release assays.

[0123] Assays and indicators for detecting reactive T cells include, but are not limited to, the use of IFNy ELISPOT and IFNy intracellular cytokine staining. Other various methods for determining whether a T cell clone will respond to a particular peptide are known in the art. Peptides are typically added to a suspension of T cells for 1 to 3 days. T cell responses can be measured by proliferation (e.g., uptake of labeled thymidine) or release of cytokines (e.g., IL-2). Various assays can be used to detect the presence of released cytokines. T cell cytotoxicity assays can be used to detect cytotoxic T cells specific for an antigen. In one embodiment, cytotoxic T cells are tested for their ability to kill target cells that present an antigen on MHC class I molecules. Target cells that present an antigen can be labeled and added to a suspension of T cells derived from a patient sample. Cytotoxicity can be measured by quantifying the release of a marker that lyses cells. Controls for spontaneous and total release can be included in the assay.

[0124] In one embodiment of the application, the cancer described herein involves cancer cells that express CLDN18.2 and / or present CLDN18.2 in the context of MHC molecules. In one embodiment of the application, the diseased cell is a cancer cell. In one embodiment, the diseased cell (e.g., cancer cell) is a cell that expresses CLDN18.2 and / or presents CLDN18.2 in the context of MHC molecules. In one embodiment, the expression of CLDN18.2 is located on the surface of the diseased cell.

[0125] In one embodiment of the application, the cancer is an adenocarcinoma, in particular an advanced adenocarcinoma. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases of the above-mentioned cancers, Krukenberg tumors, peritoneal metastases, and / or lymph node metastases. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, in particular lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer. In one embodiment, the patient is a HER2 / neu-negative patient or a patient with a HER2 / neu-positive status who does not qualify for trastuzumab treatment.

[0126] In one embodiment of the application, the cancer cell is a cancer cell selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases of the above-mentioned cancers, Krukenberg tumors, peritoneal metastases, and / or lymph node metastases. In one embodiment, the cancer cell is a cancer cell selected from the group consisting of gastric cancer, esophageal cancer, in particular lower esophageal cancer, esophagogastric junction cancer, and gastroesophageal cancer.

[0127] According to the present application, CLDN18.2 preferably has the amino acid sequence of SEQ ID NO: 1.

[0128] Other features and advantages of the present application will be apparent from the following detailed description, and from the claims.

[0129] Detailed description of the application

[0130] Although the present application is described in detail below, it should be understood that the application is not limited to the particular methodologies, protocols and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0131] Hereinafter, elements of the present application will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to result in further embodiments. The various described embodiments and preferred embodiments should not be construed as limiting the present application to only the explicitly described embodiments. The description should be understood to support and include embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any arrangement and combination of all described elements in the present application should be considered as being disclosed by the description of the present application, unless the context indicates otherwise.

[0132] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H.

[0133] The practice of the present application will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology and recombinant DNA techniques which are explained in the literature in the field (cf., for example, Molecular Cloning: A Laboratory Manual, second edition, J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0134] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". As used herein, the terms "have", "has", "having", "include", "includes", "including", "comprise", "comprises", "comprising", or the like are used to indicate open-ended processes, machines, or apparatuses of the present application that consist of, include, and / or have, but are not limited to, the materials, actions, or steps as such terms are synonymous with the expression "including, but not limited to". Unless otherwise required by context, the terms "about", "substantially", "approximately", and the like are used on this description and in the claims to mean that the recited characteristie(s) could vary from the ideal by no more than 10%, preferably no more than 5%, and more preferably no more than 1%.

[0135] Unless otherwise indicated herein, the terms "about" and "substantially" are used in their ordinary sense to mean approximately or nearly, for example, as in "about 90 degrees" or "substantially parallel." Unless otherwise indicated herein, all methods described herein can be performed in any suitable order unless otherwise indicated herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0136] Throughout this text, a number of documents are cited. Each of the documents cited herein, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the application is not entitled to antedate such disclosure by virtue of prior application.

[0137] In the context of the present application, the term "recombinant" means "produced by genetic engineering". Preferably, in the context of the present application, a "recombinant" such as a recombinant cell is not naturally occurring.

[0138] The term "naturally occurring" as used herein refers to the fact that an item can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses) and that can be isolated from a natural source and that has not been intentionally modified by man in the laboratory is naturally occurring.

[0139] The term "immune response" refers to the comprehensive bodily reaction to an antigen, preferably to a cellular immune response or to a cellular and humoral immune response. The immune response can be protective / preventive / prophylactic and / or therapeutic.

[0140] "Inducing an immune response" can mean that there is no immune response to a particular antigen prior to induction, but can also mean that there is a certain level of immune response to a particular antigen prior to induction, and that the immune response is enhanced after induction. Thus, "inducing an immune response" also includes "enhancing an immune response". Preferably, after inducing an immune response in a subject, the subject is protected from developing a disease (such as a cancer disease) or the disease condition is improved by inducing an immune response. For example, an immune response against a tumor-associated antigen (such as CLDN18.2) can be induced in a patient having a cancer disease or in a subject at risk of developing a cancer disease. In this case, inducing an immune response can mean that the disease condition of the subject is improved, the subject does not develop metastases, or the subject at risk of developing a cancer disease does not develop a cancer disease.

[0141] "Cellular immune response", "cellular response", "cellular response against an antigen" or similar terms are intended to include a cellular response involving cells characterized by the presentation of an antigen with MHC class I or class II. The cellular response involves cells known as T cells or T lymphocytes, which act as "helpers" or "killers". Helper T cells (also known as CD4+ T cells) play a key role in modulating the immune response, and killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells, preventing the production of more diseased cells.

[0142] The term "antigen" relates to an agent comprising an epitope that will generate and / or direct an immune response. Preferably, the antigen in the context of the present application is a molecule (optionally, after processing) that induces an immune reaction, which is preferably specific for the antigen or cell expressing and / or presenting the antigen. The term "antigen" includes, inter alia, proteins and peptides. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or can be derived from tumor-associated antigens.

[0143] In particular, the antigen or peptide fragment thereof should be recognized by a T cell receptor. Preferably, the antigen or peptide is capable of inducing clonal expansion of T cells carrying a T cell receptor recognizing the antigen or peptide in the presence of an appropriate co-stimulatory signal, if recognized by a T cell receptor. In the context of embodiments of the present application, the antigen is preferably presented by a cell, preferably by an antigen-presenting cell and / or a diseased cell, in the context of an MHC molecule, which can lead to an immune reaction against the antigen (or the cell presenting the antigen).

[0144] In preferred embodiments, the antigen is a tumor-associated antigen, i.e. a component of a cancer cell that can be derived from the cytoplasm, the cell surface and the nucleus of a cancer cell, in particular those antigens that are produced (preferably in large amounts) intracellularly or as surface antigens on cancer cells.

[0145] In the context of the present application, the term "tumor-associated antigen" or "tumor antigen" relates to a protein which is expressed specifically under normal conditions in a limited number of tissues and / or organs or at a specific stage of development, for example, a tumor-associated antigen can be specifically expressed under normal conditions in gastric tissue (preferably gastric mucosa), in reproductive organs (such as testis), in trophoblast tissue (such as placenta or cells of the germ line), and in one or more tumor or cancer tissues or is aberrantly expressed. In this context, "limited number" preferably means no more than 3, more preferably no more than 2. In the context of the present application, tumor-associated antigens include, for example, differentiation antigens, preferably cell-type specific differentiation antigens, i.e. proteins which are specifically expressed under normal conditions in certain cell types at a certain stage of differentiation; cancer / testis antigens, i.e. proteins which are specifically expressed under normal conditions in testis and sometimes in placenta; and germ line specific antigens. In the context of the present application, tumor-associated antigens are preferably associated with the cell surface of cancer cells and are preferably not expressed or only expressed in small amounts in normal tissue. Preferably, the tumor-associated antigen or the aberrant expression of the tumor-associated antigen identifies a cancer cell. In the context of the present application, the tumor-associated antigen expressed by a cancer cell in a subject, for example a patient having a cancer disease, is preferably a self-protein of said subject. In a preferred embodiment, the tumor-associated antigen in the context of the present application is expressed under normal conditions, in particular in a non-essential tissue or organ, i.e. a tissue or organ whose destruction by the immune system does not lead to death of the subject, or in a body organ or structure which the immune system cannot or only with great difficulty access. Preferably, the amino acid sequence of the tumor-associated antigen is identical between the tumor-associated antigen expressed in normal tissue and the tumor-associated antigen expressed in cancer tissue. Preferably, the tumor-associated antigen is presented by the cancer cell expressing it.

[0146] Various aspects of the present application relate to the tumor-associated antigen CLDN18.2, and the present application can relate to stimulating or providing an anti-tumor CTL response against cancer cells expressing said tumor-associated antigen and preferably presenting said tumor-associated antigen with MHC class I.

[0147] Claudins are a family of proteins that are the most important components of tight junctions, where they establish a paracellular barrier that controls the flow of molecules across the intercellular space of epithelial cells. Claudins are transmembrane proteins with four transmembrane domains, with both the N- and C-termini located in the cytoplasm. The first extracellular loop or domain, termed EC1 or ECL1, consists of an average of 53 amino acids, and the second extracellular loop or domain, termed EC2 or ECL2, consists of about 24 amino acids. Cell surface proteins of the claudin family, such as CLDN18.2, are expressed in tumors of different origin, are particularly suitable as target structures in connection with antibody-mediated immunotherapy of cancer due to their selective expression (not in normal tissues associated with toxicity) and localization to the plasma membrane.

[0148] CLDN18.2 is selectively expressed in differentiated epithelial cells of the gastric mucosa of normal tissue. CLDN18.2 is expressed in differentially derived cancers such as pancreatic cancer, esophageal cancer, gastric cancer, bronchial cancer, breast cancer and ENT tumors. CLDN18.2 is a valuable target for the prevention and / or treatment of primary tumors, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and metastases of the above-mentioned cancers, in particular gastric cancer metastases, such as Krukenberg tumors, peritoneal metastases and lymph node metastases.

[0149] The term "CLDN" as used herein refers to a claudin and includes CLDN18.2. Preferably, the claudin is a human claudin.

[0150] The term "CLDN18" relates to a claudin 18 and includes any variant, which includes claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

[0151] The term "CLDN18.2" preferably relates to a human CLDN18.2, in particular to a protein comprising the amino acid sequence of SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence, preferably to a protein consisting of the amino acid sequence of SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence. The first extracellular loop or domain of CLDN18.2 preferably comprises amino acids 27 to 81 of the amino acid sequence shown in SEQ ID NO: 1, more preferably amino acids 29 to 78. The second extracellular loop or domain of CLDN18.2 preferably comprises amino acids 140 to 180 of the amino acid sequence shown in SEQ ID NO: 1. Said first and second extracellular loop or domain preferably form the extracellular part or domain of CLDN18.2.

[0152] The term "variant" of the present application relates especially to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, in particular naturally occurring variants. Allelic variants relate to changes in the normal sequence of a gene, the significance of which is usually not clear. Complete sequencing of a gene usually identifies several allelic variants of a given gene. Species homologues are nucleic acid or amino acid sequences of different species origin having a given nucleic acid or amino acid sequence. The term "variant" shall include any post-translationally modified variants and conformational variants.

[0153] According to different aspects of the present application, it is an object to induce or determine an immune response against cancer cells expressing CLDN18.2 and preferably characterized by presenting CLDN18.2, and to diagnose, treat or prevent cancer diseases involving cells expressing CLDN18.2. Preferably, the immune response involves the stimulation of an anti-CLDN18.2 CTL response against cancer cells expressing CLDN18.2 and preferably presenting CLDN18.2 with MHC class I.

[0154] According to the present application, the term "cancer expressing CLDN18.2" or "CLDN18.2 positive cancer" refers to a cancer involving cancer cells expressing CLDN18.2, preferably cancer cells expressing CLDN18.2 on their surface. Alternatively or in addition, the cancer cells expressing CLDN18.2 present CLDN18.2 in the context of MHC molecules. Cancer cells presenting CLDN18.2 in the context of MHC molecules can be targeted by immune reactive cells carrying T cell receptors, whereas cancer cells expressing CLDN18.2 on the surface can be targeted by immune reactive cells carrying artificial T cell receptors.

[0155] The use of "cell surface" is according to its normal meaning in the art, thus including the exterior of a cell accessible by binding to proteins and other molecules.

[0156] If CLDN18.2 is located at the cell surface, CLDN18.2 is expressed on the cell surface and accessible by binding with the addition of CLDN18.2 specific antibodies to the cell.

[0157] In the context of the present application, the term "extracellular part" or "extracellular domain" refers to a part of a molecule of a protein such as facing the extracellular space of a cell, and preferably accessible from the extracellular space of said cell, e.g. by an antigen binding molecule such as an antibody located extracellularly. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0158] The term "portion" refers to a fraction. For a particular structure such as an amino acid sequence or protein, the term "portion" thereof can denote a contiguous or non-contiguous fraction of said structure. Preferably, a portion of an amino acid sequence comprises at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, preferably at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90% of the amino acids of said amino acid sequence. Preferably, if the portion is a non-contiguous fraction, said non-contiguous fraction comprises 2, 3, 4, 5, 6, 7, 8 or more portions of the structure, each portion being a contiguous element of the structure. For example, a non-contiguous fraction of an amino acid sequence can comprise 2, 3, 4, 5, 6, 7, 8 or more, preferably not more than 4 portions of said amino acid sequence, wherein each portion preferably comprises at least 5 contiguous amino acids, at least 10 contiguous amino acids, preferably at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids of the amino acid sequence.

[0159] The terms "part" and "fragment" are used interchangeably herein and refer to a contiguous element. For example, a part of a structure such as an amino acid sequence or protein refers to a contiguous element of said structure. A portion, part or fragment of a structure preferably comprises one or more functional properties of said structure. For example, a portion, part or fragment of an epitope, peptide or protein is preferably immunologically equivalent to the epitope, peptide or protein from which it is derived. In the context of the present application, a "portion" of a structure such as an amino acid sequence preferably comprises, or more preferably consists of, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% of the entire structure or amino acid sequence. A portion or fragment of a protein sequence preferably comprises a sequence of at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 contiguous amino acids of the protein sequence. The term "variant" as used herein includes a portion, part or fragment as described above.

[0160] According to the present application, CLDN18.2 is essentially not expressed in the cell if the expression level is below the expression level in gastric cells or gastric tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower than the expression level in gastric cells or gastric tissue. Preferably, CLDN18.2 is essentially not expressed in the cell if the expression level does not exceed the expression level in non-cancerous tissue other than the stomach by more than 2-fold, preferably 1.5-fold, and preferably does not exceed the expression level in said non-cancerous tissue. Preferably, CLDN18.2 is essentially not expressed in the cell if the expression level is below the detection limit and / or if the expression level is too low to allow binding by a CLDN18.2 specific antibody added to the cell.

[0161] According to the present application, CLDN18.2 is expressed in the cell if the expression level exceeds the expression level in non-cancerous tissue other than the stomach preferably by more than 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10000-fold. Preferably, CLDN18.2 is expressed in the cell if the expression level is above the detection limit and / or if the expression level is high enough to allow binding by a CLDN18.2 specific antibody added to the cell. Preferably, the CLDN18.2 expressed in the cell is expressed or exposed on the surface of said cell.

[0162] A "target cell" refers to a cell that is a target of an immune response, such as a cellular immune response. Target cells include cells that present an antigen or an antigenic epitope (i.e. a peptide fragment derived from an antigen), and include any undesired cell, such as a cancer cell. In preferred embodiments, the target cell is a cell expressing CLDN18.2, which is preferably present on the cell surface and / or is presented with MHC class I.

[0163] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, i.e. a part or fragment of a molecule that is recognized by the immune system, e.g. by a T cell, especially when presented in the context of an MHC molecule. An epitope of a protein, such as a tumor-associated antigen, preferably comprises a contiguous or non-contiguous portion of said protein, and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length, e.g. an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is especially preferred that the epitope in the context of the present application is a T cell epitope.

[0164] The terms "epitope", "antigen fragment", "antigen peptide" or "immunogenic peptide" are used interchangeably herein and preferably relate to an incomplete representation of an antigen which is preferably capable of eliciting an immune response against the antigen or a cell expressing or comprising and preferably presenting the antigen. Preferably, these terms relate to an immunogenic portion of an antigen. Preferably, the portion of an antigen is recognized, i.e. specifically bound, by a T cell receptor, especially if it is presented in the context of an MHC molecule. Certain preferred immunogenic portions bind, for example, MHC class I or class II molecules on the surface of a cell and are thus MHC binding peptides. As used herein, a peptide is said to "bind" to an MHC class I or class II molecule if such binding is detectable using any assay known in the art.

[0165] Preferably, the peptides disclosed herein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7 or a variant of said amino acid sequence are capable of stimulating an immune response, preferably a cellular response against CLDN18.2 or a cell characterized by expressing CLDN18.2 and preferably by presenting CLDN18.2. Preferably, such peptides are capable of stimulating a cellular response against a cell characterized by presenting CLDN18.2 with an MHC class I and preferably are capable of stimulating a CLDN18.2 reactive CTL. Preferably, the peptides of the present application are MHC class I and / or class II presented peptides or can be processed to yield MHC class I and / or class II presented peptides. Preferably, the sequence which binds to an MHC molecule is selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7.

[0166] If the antigen peptide is directly presented, i.e. without processing, especially without cleavage, it has a length which is suitable for binding to an MHC molecule, especially a class I MHC molecule, preferably a length of 7-20 amino acids, more preferably 7-12 amino acids, more preferably 8-11 amino acids, especially 9 or 10 amino acids. Preferably, the sequence of the directly presented antigen peptide essentially corresponds to a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7 and preferably is identical to the sequence of above.

[0167] If the antigen peptide is presented after processing, in particular after cleavage, the peptide produced by processing has a length which is suitable for binding to an MHC molecule, in particular a class I MHC molecule, preferably 7-20 amino acids, more preferably 7-12 amino acids, more preferably 8-11 amino acids, in particular 9 or 10 amino acids. Preferably, the sequence of the peptide presented after processing essentially corresponds to a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7 and is preferably identical to a sequence selected from the above. Thus, in one embodiment, the antigen peptide of the application comprises a sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7 and the sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6 and 7 is produced after processing of the antigen peptide.

[0168] Peptides having an amino acid sequence which essentially corresponds to the sequence of a peptide presented by an MHC molecule can differ at one or more residues which are not necessary for TCR recognition of the peptide presented by the MHC or for binding of the peptide to the MHC. Such essentially corresponding peptides are preferably also able to stimulate an antigen-specific cellular response, such as an antigen-specific CTL. Peptides having an amino acid sequence which differs from the presented peptide at residues which do not affect TCR recognition but improve the stability of binding to the MHC can improve the immunogenicity of the antigen peptide and can be referred to herein as "optimized peptides". With the existing knowledge of which of these residues can more likely affect binding to the MHC or the TCR, a rational approach can be employed to design essentially corresponding peptides. The resulting functional peptides are considered to be antigen peptides. The term "variant" as used herein includes sequences as described above.

[0169] "Antigen processing" refers to the degradation of an antigen into processing products, which are fragments of the antigen (e.g. degradation of a protein into peptides), and the association (e.g. binding) of one or more of these fragments with an MHC molecule for presentation by a cell, preferably an antigen presenting cell, to a specific T cell.

[0170] An antigen presenting cell (APC) is a cell that displays antigens on its surface in the context of major histocompatibility complex (MHC). T cells can recognize this complex using their T cell receptors (TCRs). Antigen presenting cells process antigens and present them to T cells.

[0171] Professional antigen presenting cells very efficiently internalize antigens by phagocytosis or receptor-mediated endocytosis and then display antigen fragments bound to class II MHC molecules on their membrane. T cells recognize and interact with the antigen class II MHC molecule complex on the membrane of the antigen presenting cell. Additional co-stimulatory signals are then generated by the antigen presenting cell, leading to T cell activation. Expression of co-stimulatory molecules is a defining feature of professional antigen presenting cells. Antigen presenting cells include professional antigen presenting cells and non-professional antigen presenting cells.

[0172] The major types of professional antigen presenting cells are dendritic cells, macrophages, B cells, monocytes, and certain activated epithelial cells, with dendritic cells having the broadest range of antigen presentation and possibly being the most important antigen presenting cells.

[0173] Non-professional antigen presenting cells do not constitutively express MHC class II proteins required for initial T cell interaction; these proteins are only expressed upon stimulation of non-professional antigen presenting cells by certain cytokines such as IFNy.

[0174] Dendritic cells (DCs) are a population of leukocytes that present antigens captured in peripheral tissues to T cells via MHC class II and class I antigen presentation pathways. Dendritic cells are well known to be potent inducers of immune responses, and activation of these cells is a critical step in inducing anti-tumor immunity.

[0175] Dendritic cells and progenitor cells can be obtained from peripheral blood, bone marrow, tumor infiltrating cells, peritumoral tissue infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or fluid. For example, dendritic cells can be differentiated in vitro by adding a combination of cytokines such as GM-CSF, IL-4, IL-13, and / or TNFa to cultures of mononuclear cells harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood, or bone marrow can be differentiated into dendritic cells by adding GM-CSF, IL-3, TNFa, CD40 ligand, LPS, flt3 ligand, and / or other compounds that induce differentiation, maturation, and proliferation of dendritic cells to the culture medium.

[0176] Dendritic cells are conveniently classified as "immature" cells and "mature" cells, which can be used as a simple way to distinguish between two well-characterized phenotypes. However, this nomenclature should not be interpreted to exclude all possible intermediate stages of differentiation.

[0177] Immature dendritic cells are characterized as antigen presenting cells with high antigen uptake and processing capacity, which is associated with high expression of Fcy receptors and mannose receptors. The mature phenotype is typically characterized by lower expression of these markers, but high expression of cell surface molecules responsible for T cell activation, such as class I and II MHC, adhesion molecules (e.g. CD54 and CD11), and costimulatory molecules (e.g. CD40, CD80, CD86, and 4-1BB).

[0178] Maturation of dendritic cells is referred to as the state of dendritic cell activation, where such antigen presenting dendritic cells lead to priming of T cells, while presentation by immature dendritic cells leads to tolerance. Dendritic cell maturation is primarily induced by biological molecules with microbial signatures detected by innate receptors (bacterial DNA, viral RNA, endotoxins, etc.), proinflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 by CD40L at the surface of dendritic cells, and substances released from cells undergoing stress cell death. Dendritic cells can be obtained by in vitro culturing of bone marrow cells with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.

[0179] Cells (such as antigen presenting cells or target cells) can be loaded with MHC class I presented peptides by exposing, i.e. pulsing the cells with the peptides or transducing the cells with nucleic acids (preferably RNA) encoding peptides or proteins including the peptide to be presented, such as nucleic acids encoding an antigen.

[0180] In some embodiments, the pharmaceutical compositions of the present application comprise antigen presenting cells loaded with antigenic peptides. In this regard, protocols can rely on in vitro culturing / differentiation of dendritic cells manipulated in such a way that the dendritic cells artificially present antigenic peptides. The generation of genetically engineered dendritic cells can include the introduction of nucleic acids encoding an antigen or antigenic peptides into the dendritic cells. Transfection of dendritic cells with mRNA is a promising antigen loading technique to stimulate potent antitumor immunity. Such transfection can be performed in vitro and pharmaceutical compositions comprising such transfected cells can then be used for therapeutic purposes. Alternatively, gene delivery vectors targeting dendritic cells or other antigen presenting cells can be administered to a patient, resulting in transfection to occur in vivo. For example, in vivo and ex vivo transfection of dendritic cells can be generally performed using any method known in the art, such as the method described in WO97 / 24447 or the gene gun method described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells can be achieved by incubating dendritic cells or progenitor cells with antigens, DNA (naked or in a plasmid vector) or RNA; or with recombinant bacteria or viruses (e.g. vaccinia, fowipox, adenoviral or lentiviral vectors) expressing the antigen.

[0181] The term "immunogenicity" relates to the relative efficiency of an antigen to induce an immune response.

[0182] In the context of the present application, the term "immune effector function" comprises any function mediated by components of the immune system which result in, for example, killing of tumor cells or inhibition of tumor growth and / or inhibition of tumor progression (including inhibition of tumor dissemination and metastasis). Preferably, the immune effector function in the context of the present application is a T cell mediated effector function. Such functions include, in the case of a helper T cell (CD4 + T cell), recognition of an antigen or an antigenic peptide derived from an antigen in the context of an MHC class II molecule by a T cell receptor, release of cytokines and / or activation of CD8 + lymphocytes (CTLs) and / or B cells, and, in the case of a CTL, recognition of an antigen or an antigenic peptide derived from an antigen in the context of an MHC class I molecule by a T cell receptor, elimination of cells presented in the context of an MHC class I molecule, i.e. cells characterized by presentation of an antigen with class I MHC, e.g. by apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-g and TNF-a and specific cytolytic killing of antigen-expressing target cells.

[0183] In the context of the present application, the term "immunoreactive cell" or "immune effector cell" relates to a cell which exerts an effector function during an immune response. An "immunoreactive cell" is preferably capable of binding an antigen, e.g. an antigen expressed on the cell surface or a cell characterized by presentation of an antigen or an antigenic peptide derived from an antigen, and mediating an immune response. For example, such cells secrete cytokines and / or chemokines, kill microorganisms, secrete antibodies, recognize infected or cancerous cells and, optionally, eliminate these cells. For example, immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages and dendritic cells. Preferably, in the context of the present application, the "immunoreactive cell" is a T cell, preferably a CD4 + and / or a CD8 + T cell.

[0184] Preferably, the "immunoreactive cell" recognizes an antigen or an antigenic peptide derived from an antigen with some degree of specificity, especially if presented in the context of an MHC molecule, e.g. on the surface of an antigen presenting cell or a diseased cell, such as a cancer cell. Preferably, the recognition renders the cell responsive or reactive to the antigen or the antigenic peptide derived from the antigen. If the cell is a helper T cell (CD4 +CTL), then such responsiveness or reactivity can involve the release of cytokines and / or the activation of CD8 + lymphocytes (CTLs) and / or B cells. If the cell is a CTL, then such responsiveness or reactivity can involve the elimination of cells presenting antigens in the context of MHC class I molecules, i.e., cells characterized by the presentation of antigens with class I MHC, e.g., by apoptosis or perforin-mediated cytolysis. According to the present application, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-g and TNF-a, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen-expressing target cells. CTL responsiveness can also be determined using artificial reporters that precisely indicate CTL responsiveness. Such CTLs that recognize an antigen or an antigen peptide derived from an antigen and have responsiveness or reactivity are also referred to herein as "antigen-responsive CTLs." If the cell is a B cell, then such responsiveness can involve the release of immunoglobulins.

[0185] According to the present application, the term "immunoreactive cell" also includes cells that are capable of maturing into immune cells, e.g., T cells, especially T helper cells or cytolytic T cells, by appropriate stimulation. Immunoreactive cells include CD34 + hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. If cytolytic or T helper cells that recognize an antigen are desired, the immunoreactive cells are contacted with cells presenting an antigen or an antigen peptide under conditions that favor the production, differentiation, and / or selection of cytolytic T cells and T helper cells. Upon exposure to the antigen, T cell precursors differentiate into cytolytic T cells, analogous to clonal selection of the immune system.

[0186] "Lymphoid cells" are cells or precursor cells of such cells that are capable of producing an immune response, such as a cellular immune response, optionally after appropriate modification, e.g., after transfer of a T cell receptor, and include lymphocytes (preferably T lymphocytes), lymphoblasts, and plasma cells. Lymphoid cells can be immunoreactive cells as described herein. Preferred lymphoid cells are T cells that lack endogenous expression of a T cell receptor, which can be modified to express such a T cell receptor on the cell surface.

[0187] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) including cytolytic T cells.

[0188] T cells belong to the group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types (e.g. B cells and natural killer cells) by the presence of a specific receptor on their cell surface known as the T cell receptor (TCR). The thymus is the main organ responsible for T cell maturation of T cells. Several different T cell subsets have been discovered, each with a different function.

[0189] T helper cells assist other white blood cells in the immune process, including B cell maturation into plasma cells, activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells, as they express the CD4 protein on their surface. Helper T cells are activated when they encounter peptide antigens presented by MHC class II molecules expressed on the surface of antigen presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins known as cytokines that mediate or assist in the active immune response.

[0190] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells, as they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I present on the surface of almost every cell in the body.

[0191] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, produced from independent T cell receptor alpha and beta (TCRa and TCRp) genes, and is known as the alpha- and beta-TCR chains. Gamma delta T cells represent a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in gamma delta T cells, the TCR is composed of one gamma chain and one delta chain. This group of T cells is much less common than alpha beta T cells (2% of total T cells).

[0192] The structure of the T cell receptor is very similar to the immunoglobulin Fab fragment, which is the region of the light and heavy chains that define as a combination of arms of the antibody. Each chain of the TCR is a member of the immunoglobulin superfamily and has one N-terminal variable (V) domain, one constant (C) domain, a transmembrane / cell transmembrane region and a short cytoplasmic tail at the C-terminal end.

[0193] According to the present invention, the term "variable region of a T cell receptor" relates to the variable domain of a TCR chain.

[0194] The variable regions of the TCR alpha and beta chains have three hypervariable regions or complementarity determining regions (CDRs), while the variable region of the beta chain has an additional hypervariable region (HV4) that does not normally contact antigen and is therefore not considered a CDR. CDR3 is the major CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize the MHC. CDR4 of the beta chain is not thought to be involved in antigen recognition, but has been shown to interact with superantigens.

[0195] According to the present application, the term "at least one CDR sequence" preferably refers to at least a CDR3 sequence. The term "CDR sequence of a T cell receptor chain" preferably relates to CDR1, CDR2 and CDR3 of the alpha chain or the beta chain of a T cell receptor.

[0196] The constant domains of the TCR domains consist of short joining sequences in which the cysteine residues form disulfide bonds that link between the two chains.

[0197] All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells, which are derived from hematopoietic stem cells, reside in the thymus and expand by cell division to produce large numbers of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore classified as double negative (CD4-CD8-) cells. As they progress through their development, they become double positive thymocytes (CD4+CD8+) and finally mature into single positive (CD4+CD8- or CD4-CD8+) thymocytes, which are then released from the thymus to the peripheral tissues.

[0198] The first signal in T cell activation is provided by the binding of the T cell receptor to a short peptide presented on another cell by the major histocompatibility complex (MHC). This ensures that only T cells with that peptide-specific TCR are activated. The partner cell is usually a professional antigen presenting cell (APC), although B cells and macrophages can be important APCs, but in the case of the initial response are usually dendritic cells. Peptides presented to CD8+ T cells by MHC class I molecules are 8-10 amino acids in length; peptides presented to CD4+ T cells by MHC class II molecules are longer, as the ends of the binding groove of MHC class II molecules open up.

[0199] T cells can generally be prepared in vitro or ex vivo using standard methods. For example, using a commercially available cell separation system, T cells can be present in (or isolated from) bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a mammal (e.g., a patient). Alternatively, T cells can be derived from a relevant or irrelevant human, non-human animal, cell line, or culture. A "sample comprising T cells" can be, for example, peripheral blood mononuclear cells (PBMCs).

[0200] T cells can be stimulated with antigens, peptides, nucleic acids, and / or antigen presenting cells (APCs) expressing antigens. Such stimulation is performed under conditions and for a time sufficient to allow the generation of T cells specific for the antigen, peptide, and / or cells presenting the antigen or peptide.

[0201] Specific activation of CD4+or CD8+T cells can be detected in various ways. Methods for detecting specific T cell activation include detection of proliferation of T cells, production of cytokines (e.g., lymphokines), or production of cytolytic activity. For CD4+T cells, the preferred method for detecting specific T cell activation is detection of proliferation of T cells. For CD8+T cells, the preferred method for detecting specific T cell activation is detection of production of cytolytic activity.

[0202] For the generation of CD8+T cell lines, antigen presenting cells (preferably autologous antigen presenting cells) transfected with nucleic acids producing an antigen can be used as stimulator cells.

[0203] Nucleic acids, e.g., RNAs encoding T cell receptor (TCR) chains, can be introduced into lymphoid cells, such as T cells or other cells with lytic potential. In suitable embodiments, TCR alpha and beta chains are cloned from an antigen-specific T cell line and used for adoptive T cell therapy. In this regard, the present application provides T cell receptors specific for CLDN18.2 or CLDN18.2 peptides disclosed herein. Generally, this aspect of the present application relates to T cell receptors that recognize or bind to CLDN18.2 peptides presented in the context of MHC. The nucleic acids encoding the alpha- and beta-chains of a T cell receptor (e.g., a T cell receptor provided according to the present application) can be comprised on separate nucleic acid molecules, such as expression vectors, or on a single nucleic acid molecule. Thus, the term “nucleic acid encoding a T cell receptor” or similar terms relate to nucleic acid molecules encoding the T cell receptor chains on the same or, preferably, different nucleic acid molecules.

[0204] The term “immunoreactive cell reactive with a peptide” relates to an immunoreactive cell that, when recognizing a peptide, especially if presented in the context of an MHC molecule, e.g., on the surface of an antigen presenting cell or a diseased cell, such as a cancer cell, exerts effector functions of an immunoreactive cell as described above.

[0205] The term “T cell receptor reactive with a peptide” relates to a T cell receptor that, when present on an immunoreactive cell, recognizes a peptide, especially if presented in the context of an MHC molecule, e.g., on the surface of an antigen presenting cell or a diseased cell, such as a cancer cell, such that the immunoreactive cell exerts effector functions of an immunoreactive cell as described above.

[0206] The term "antigen-reactive T cell" or similar terms relates to a T cell which recognizes an antigen, if presented in the context of an MHC molecule, e.g. on the surface of an antigen presenting cell or a diseased cell such as a cancer cell, and which exerts effector functions of a T cell as described above.

[0207] The term "antigen-specific lymphoid cell" relates to a lymphoid cell, especially when provided with an antigen-specific T cell receptor, which recognizes an antigen, if presented in the context of an MHC molecule, e.g. on the surface of an antigen presenting cell or a diseased cell such as a cancer cell, and which preferably exerts effector functions of a T cell as described above. A T cell and other lymphoid cells are considered specific for an antigen if they kill target cells expressing and / or presenting the antigen peptide. T cell specificity can be assessed using any of a variety of standard techniques, such as chromium release assays or proliferation assays. Alternatively, synthesis of lymphokines, e.g. interferon-gamma, can be measured.

[0208] The terms "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to a complex of genes that occurs in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in an immune response, where the MHC proteins or molecules bind peptides and present them to T cell receptors for recognition. Proteins encoded by MHC are expressed on the cell surface and display both self-antigens (peptide fragments derived from the cell itself) and non-self antigens (e.g. fragments of invading microorganisms) to T cells.

[0209] The MHC region is divided into three subgroups, class I, class II, and class III. MHC class I proteins contain an alpha chain and a beta 2-microglobulin (not part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells. MHC class II proteins contain alpha- and beta-chains and they present antigen fragments to T helper cells on most immune system cells, especially antigen presenting cells. The MHC class III region encodes other immune components, such as complement components and some cytokine encoding.

[0210] In humans, the genes in the MHC region that encode antigen presenting proteins on the cell surface are known as human leukocyte antigen (HLA) genes. However, the abbreviation MHC is often used to refer to HLA gene products. The HLA genes include nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0211] In a preferred embodiment of all aspects of the application, the MHC molecule is an HLA molecule.

[0212] "Cell characterized by presentation of an antigen", "cell presenting an antigen", "antigen presented by a cell", "presented antigen" or similar expressions mean a cell such as a diseased cell (e.g. a cancer cell) or an antigen presenting cell which presents an antigen expressed by the cell or a fragment derived from the antigen (e.g. by processing the antigen) in the context of an MHC molecule, in particular an MHC class I molecule. Similarly, the term "disease characterized by presentation of an antigen" denotes a disease involving cells characterized by presentation of an antigen, in particular with class I MHC. Presentation of an antigen by a cell can be achieved by transfecting the cell with a nucleic acid such as an RNA encoding the antigen.

[0213] "Fragment of the presented antigen" or similar expressions mean that the fragment can be presented by MHC class I or class II, preferably MHC class I, e.g. when added directly to an antigen presenting cell. In one embodiment, the fragment is a fragment that is naturally presented by a cell expressing the antigen.

[0214] Some treatment methods are based on the reaction of the immune system of the patient which leads to the lysis of diseased cells presenting an antigen with class I MHC. In this respect, for example, autologous cytotoxic T lymphocytes specific for the complex of the antigen peptide and the MHC molecule can be administered to a patient suffering from the disease. The in vitro production of such cytotoxic T lymphocytes is known. An example of a method for differentiating T cells can be found in WO-A-9633265. Typically, a sample containing cells (e.g. blood cells) is taken from the patient and the cells are brought into contact with cells presenting the complex and which can cause the proliferation of cytotoxic T lymphocytes (e.g. dendritic cells). The target cells can be transfected cells such as COS cells. These transfected cells present the desired complex on their surface and stimulate the proliferation of cytotoxic T lymphocytes when brought into contact with the latter. The clonally expanded autologous cytotoxic T lymphocytes are then administered to the patient.

[0215] In another method for selecting cytotoxic T lymphocytes, a fluorescent tetramer of the MHC class I molecule / peptide complex is used to obtain specific clones of cytotoxic T lymphocytes (Altman et al. (1996), Science 274:94-96; Dunbar et al. (1998), Curr. Biol. 8:413-416, 1998).

[0216] Furthermore, cells presenting the desired complex, such as dendritic cells, can be combined with cytotoxic T lymphocytes of a healthy individual or another species, such as a mouse, which can lead to the propagation of specific cytotoxic T lymphocytes with high affinity. The high affinity T cell receptors of these propagated specific T lymphocytes can be cloned and optionally humanized to a varying degree, and the thus obtained T cell receptors can then be transduced into T cells of the patient by gene transfer, for example using retroviral vectors. These genetically altered T lymphocytes can then be used for adoptive transfer (Stanislawski et al. (2001), Nat Immunol. 2:962-70; Kessels et al. (2001), Nat Immunol. 2:957-61).

[0217] Cytotoxic T lymphocytes can also be generated in vivo in a manner known per se. One method uses non-proliferating cells expressing MHC class I / peptide complexes. The cells used here are cells that normally express the complex, for example irradiated tumor cells or cells transfected with one or both of the genes necessary for the presentation of the complex, i.e. the antigen peptide and the presented MHC molecule. Another preferred form is the introduction of the antigen in the form of a recombinant RNA, which can be introduced into the cells, for example, by liposome transfer or by electroporation. The resulting cells present the complex of interest and are recognized by autologous cytotoxic T lymphocytes, which then propagate.

[0218] A similar effect can be achieved by combining the antigen or antigen peptide with an adjuvant, to make the incorporation of antigen-presenting cells in vivo possible. The antigen or antigen peptide can be represented as protein, DNA (for example, in a vector) or RNA. The antigen can be processed to produce the peptide partner of the MHC molecule, while fragments thereof can be presented without further processing. The latter is the case, in particular, if these can bind to MHC molecules. The preferred form of administration is the complete antigen, which is processed in vivo by dendritic cells, since this also produces the T helper cell response required for an effective immune response (Ossendorp et al., Immunol Lett. (2000), 74:75-9; Ossendorp et al. (1998), J. Exp. Med. 187:693-702). In general, an effective amount of the tumor-associated antigen can be administered to the patient by, for example, intradermal injection. However, the injection can also be made into the lymph nodes (Maloy et al. (2001), Proc Natl Acad Sci USA 98:3299-303).

[0219] According to the application, the term "artificial T cell receptor" is synonymous with the terms "chimeric T cell receptor" and "chimeric antigen receptor (CAR)".

[0220] These terms relate to engineered receptors that confer arbitrary specificity to immune effector cells such as T cells, e.g. the specificity of a monoclonal antibody. In this way, large numbers of cancer-specific T cells can be generated for adoptive cell transfer. Thus, artificial T cell receptors can be present on T cells, e.g. instead of or in addition to the T cell's own T cell receptor. Such T cells do not necessarily need to be processed and presented with an antigen for recognition of a target cell but can preferentially specifically recognize any antigen presented on a target cell. Preferably, the artificial T cell receptor is expressed on the cell surface. For the purposes of the present invention, the term "T cell" as used herein includes T cells containing artificial T cell receptors.

[0221] In one embodiment, a single chain variable fragment (scFv) derived from a monoclonal antibody is fused to a CD3-zeta transmembrane and intracellular domain. Such molecules result in the transmission of a zeta signal in response to the recognition of their antigen target on a target cell and the killing of the target cell expressing the target antigen. Antigen recognition domains that can also be used include T cell receptor (TCR) alpha and beta single chains. Virtually any agent that binds a given target with high affinity can be used as an antigen recognition domain.

[0222] Upon antigen recognition, the receptors cluster and signal is transmitted to the cell. In this regard, a "T cell signaling domain" is a domain (preferably an intracellular domain) that will transmit an activation signal to a T cell upon antigen binding. The most commonly used intracellular domain composition is CD3-zeta.

[0223] Adoptive cell transfer therapy with T cells engineered to express chimeric antigen receptors (CARs) is a promising anticancer therapy because CAR-modified T cells can be engineered to target almost any tumor antigen. For example, a patient's T cells can be genetically engineered to express a CAR specific for an antigen on the patient's tumor cells and then infused back into the patient.

[0224] According to the present invention, as described above, an artificial T cell receptor can replace the function of a T cell receptor, in particular can confer a T cell reactivity (such as cytolytic activity) as described above. However, in contrast to the binding of a T cell receptor as described above to an antigen peptide-MHC complex, an artificial T cell receptor can bind to an antigen, in particular an antigen expressed on the surface of a cell.

[0225] The T cell surface glycoprotein CD3-zeta chain is a protein in humans that is encoded by the CD247 gene. CD3-zeta, together with T cell receptor alpha / beta and gamma / delta heterodimers and CD3-gamma, -delta and -epsilon, forms the T cell receptor-CD3 complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal transduction pathways. The term "CD3-zeta" preferably relates to human CD3-zeta, in particular to a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 40 of the sequence listing or a variant of said amino acid sequence.

[0226] CD28 (cluster of differentiation 28) is one of the molecules expressed on T cells that provides a costimulatory signal required for T cell activation. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2). Stimulation through CD28 in addition to the T cell receptor (TCR) can provide a potent costimulatory signal to T cells for the production of various interleukins, in particular IL-6. The term "CD28" preferably relates to human CD28, in particular to a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 39 of the sequence listing or a variant of said amino acid sequence, preferably to a protein consisting of the amino acid sequence of SEQ ID NO: 39 of the sequence listing or a variant of said amino acid sequence.

[0227] According to the present application, the CAR can typically comprise three domains.

[0228] The first domain is a binding domain that recognizes and binds to CLDN18.2.

[0229] The second domain is a costimulatory domain. The costimulatory domain serves to enhance the proliferation and survival of the cytotoxic lymphocyte when the CAR is bound to the targeting moiety. The identity of the costimulatory domain is limited only by its ability to enhance cell proliferation and survival when bound to the targeting moiety through the CAR. Suitable costimulatory domains include CD28, CD137 (4-1BB) (a member of the tumor necrosis factor (TNF) receptor family), CD134 (OX40) (a member of the receptor TNFR-superfamily), and CD278 (ICOS) (a CD28-superfamily costimulatory molecule expressed on activated T cells). Those skilled in the art will appreciate that sequence variants of these described costimulatory domains can be used without adversely affecting the present application, where the variant has the same or similar activity as the domain being emulated. Such variants have at least about 80% sequence identity to the amino acid sequence of the domain from which they are derived. In some embodiments of the present application, the CAR construct comprises 2 costimulatory domains. While specific combinations include all possible variants of the four described domains, particular examples include CD28 + CD137 (4-1BB) and CD28 + CD134 (OX40).

[0230] The third domain is an activating signaling domain (or T cell signaling domain). The activating signaling domain serves to activate the cytotoxic lymphocyte when the CAR is bound to CLDN18.2. The identity of the activating signaling domain is limited only by its ability to induce activation of the selected cytotoxic lymphocyte when bound to CLDN18.2 through the CAR. Suitable activating signaling domains include the T cell CD3 [zeta] chain and the Fc receptor [gamma]. Those skilled in the art will appreciate that sequence variants of these described activating signaling domains can be used without adversely affecting the present application, where the variant has the same or similar activity as the domain being emulated. Such variants have at least about 80% sequence identity to the amino acid sequence of the domain from which they are derived.

[0231] The CAR of the application can comprise three domains together in a fusion protein. Such fusion proteins typically comprise a binding domain, one or more costimulatory domains, and an activating signaling domain linked in N-terminal to C-terminal orientation. However, the CAR of the application is not limited to this arrangement, other arrangements are acceptable, and include a binding domain, an activating signaling domain, and one or more costimulatory domains. It will be appreciated that because the binding domain must be free to bind CLDN18.2, the placement of the binding domain in the fusion protein will generally enable the region to be displayed extracellularly. Likewise, because the costimulatory and activating signaling domains are used to induce activity and proliferation of cytotoxic lymphocytes, the fusion protein will generally display these two domains intracellularly. The CAR can include additional elements, such as a signal peptide to ensure proper export of the fusion protein to the cell surface; a transmembrane domain to ensure that the fusion protein is held as an intact membrane protein; a hinge domain (or spacer region) which imparts flexibility to the binding domain and allows for firm binding to CLDN18.2.

[0232] The cells used in conjunction with the CAR system of the application are preferably T cells, especially cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer cells (LAK). Upon activation, each of these cytotoxic lymphocytes elicits destruction of target cells. For example, cytotoxic T cells elicit destruction of target cells by one or both of the following methods. First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and elicit a caspase cascade in the cytoplasm, which leads to apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced by Fas-Fas ligand interactions between the T cell and the target tumor cell. Although heterologous or allogeneic cells can be used, the cytotoxic lymphocytes are preferably autologous cells.

[0233] According to the application, a "reference" (such as a reference sample or a reference organism) can be used to correlate and compare results obtained from a test sample or test organism in the methods of the application. Typically, the reference organism is a healthy organism, especially an organism that does not suffer from a disease such as a cancer disease. A "reference value" or "reference level" can be empirically determined from a reference by measuring a sufficiently large number of references. Preferably, the reference value is determined by measuring at least 2, preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 12, preferably at least 20, preferably at least 30, preferably at least 50, or preferably at least 100 references.

[0234] According to the present application, the term "binding agent" comprises any compound having binding capacity to a target. Preferably, such binding agent comprises at least one binding domain to the target. The term comprises molecules like antibodies and antibody fragments, bispecific or multispecific molecules, chimeric antigen receptors (CARs) and all artificial binding molecules (scaffolds) having binding capacity to a target, including but not limited to nanobodies, affibodies, anticalins, DARPins, monobodies, avimers and microbodies. In one embodiment, the binding is specific binding.

[0235] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably to antigen receptors like antibodies or B-cell receptors (BCRs). Immunoglobulins are characterized by domains having the characteristic immunoglobulin (Ig) fold, i.e. immunoglobulin domains. The term includes membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also referred to as surface immunoglobulins or membrane immunoglobulins, which are usually part of BCRs. Soluble immunoglobulins are usually referred to as antibodies. Immunoglobulins usually comprise several chains, usually 2 identical heavy chains and 2 identical light chains, which are connected by disulfide bonds. These chains are mainly composed of immunoglobulin domains, e.g. V L (variable light) domains, C L (constant light) domains and C H (constant heavy) domains C H 1, C H 2, C H 3 and C H 4. There are five types of mammalian immunoglobulin heavy chain, IgA, IgD, IgE, IgG, and IgM, which define different classes of antibodies. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy terminus. There are two types of light chain, lambda (l) and kappa (K) in mammals. Immunoglobulin chains contain variable and constant regions. The variable part is highly changeable and is responsible for antigen recognition. The constant part is less changeable and is responsible for the antibody class (IgA, IgD, IgE, IgG, and IgM).

[0236] The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0237] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. The monoclonal antibodies display single binding specificity and affinity. In one embodiment, the monoclonal antibodies are produced by a hybridoma comprising a B cell obtained from a non-human animal, such as a mouse, fused to an immortalized cell.

[0238] The term "recombinant antibody" as used herein includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to the immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

[0239] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0240] The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin of a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site can comprise an entire variable domain fused to a constant domain, or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domain. The antigen binding site can be wild-type or modified by one or more amino acid substitutions, e.g., modified to more closely resemble a human immunoglobulin. Some forms of humanized antibodies retain all of the CDR sequences (e.g., a humanized mouse antibody comprising all 6 CDRs from a mouse antibody). Other forms have one or more CDRs altered relative to the original antibody.

[0241] The term "chimeric antibody" refers to an antibody in which individual amino acid sequences of the heavy and light chains are partially homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remainder of the chains are homologous to corresponding sequences in another. Typically, the variable regions of the light and heavy chains mimic those of an antibody derived from one mammal, while the constant portions are homologous to antibody sequences derived from another. One clear advantage of this chimeric form is that the variable regions can be readily derived from currently known sources, using readily available B-cells or hybridomas from non-human host organisms combined with constant regions derived from, for example, human cell preparations. While the variable regions have the advantage of being readily prepared, and specificity is not affected by the source, the constant regions derived from humans are less likely to elicit an immune response in a human subject when the antibody is injected than constant regions from non-human sources. However, the definition is not limited to this particular example.

[0242] Antibodies can be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig, and human.

[0243] Antibodies described herein include IgA (e.g., IgAl or IgA2), IgGl, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgGl antibody, especially an IgGl, kappa or IgGl, lambda isotype (i.e., IgGl, kappa, lambda), an IgG2a antibody (e.g., IgG2a, kappa, lambda), an IgG2b antibody (e.g., IgG2b, kappa, lambda), an IgG3 antibody (e.g., IgG3, kappa, lambda), or an IgG4 antibody (e.g., IgG4, kappa, lambda).

[0244] The antibodies described herein are preferably isolated. An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 is substantially free of antibodies that specifically bind antigens other than CLDN18.2). An isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN18.2, however, can have cross-reactivity to other related antigens (e.g., derived from other species (e.g., CLDN18.2 species homologs)). Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals. In one embodiment of the application, a combination of "isolated" monoclonal antibodies involves antibodies having different specificities combined in a well-defined composition or mixture.

[0245] The term "antigen binding portion" of an antibody (or simply "binding portion") or "antigen binding fragment" of an antibody (or simply "binding fragment") or similar terms refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR) and (vii) a combination of two or more isolated CDRs, which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also included in the term "antigen binding fragment" of an antibody. Another example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those with ordinary skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0246] According to the present application, the term "binding domain of CLDN18.2" includes and preferably relates to an antigen binding portion of a CLDN18.2 antibody, i.e. an antibody directed against CLDN18.2, and preferably specific for CLDN18.2.

[0247] The term "binding domain" characterizes according to the present application a structure, e.g. of an antibody, which binds / interacts with a given target structure / antigen / epitope. Thus, a binding domain according to the present application represents an "antigen interaction site".

[0248] For the purposes of the present application, the term "antibody" includes all antibodies and derivatives of antibodies such as antibody fragments as described herein.

[0249] Antibodies can be produced by various techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although the somatic cell hybridization procedure is preferred, in principle, other techniques for producing monoclonal antibody can be used, e.g., viral or oncogenic transformation of B lymphocytes, or phage display techniques using antibody gene libraries.

[0250] A preferred animal system for preparing hybridomas secreting monoclonal antibodies is the murine system. Hybridoma production in mice is a very well established procedure. Immunization protocols and techniques for isolating immune spleen cells for fusion are known in the art. Fusion partners, such as murine myeloma cells, and fusion methods are also known.

[0251] Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0252] To generate antibodies, mice can be immunized with a peptide conjugated to a carrier derived from the sequence of the antigen (i.e., the sequence against which the antibody is directed), an enriched preparation of recombinantly expressed antigen or fragments thereof, and / or cells expressing the antigen, as described. Alternatively, mice can be immunized with DNA encoding the antigen or fragments thereof. In cases where immunization with purified or enriched antigen preparations does not result in antibodies, mice can also be immunized with cells (e.g., cell lines) expressing the antigen to promote an immune response.

[0253] The immune response can be monitored during the course of the immunization protocol, with plasma and serum samples obtained by tail vein or retro-orbital bleeds. Mice with sufficient immunoglobulin titers can be used for fusion. Mice can be boosted intraperitoneally or intravenously with cells expressing the antigen three days prior to sacrifice, and the spleen removed to increase the rate of hybridomas secreting specific antibodies.

[0254] To generate hybridomas that produce monoclonal antibodies, spleen and lymph node cells from immunized mice can be isolated and fused with an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened against the hybridomas that secrete antibodies by ELISA. Antibodies with antigen specificity can be identified by immunofluorescence and FACS analysis using cells expressing the antigen. Hybridomas that secrete antibodies can be re-inoculated, screened again, and if the monoclonal antibody is still positive, subcloned by limiting dilution. Stable subclones can then be cultured in vitro to produce antibodies in tissue culture media for characterization.

[0255] The ability of antibodies and other binding agents to bind to antigens can be determined using standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometric analysis).

[0256] Antibodies and derivatives of antibodies can be used to provide binding domains, such as antibody fragments, especially to provide VL and VH regions.

[0257] A binding domain for CLDN18.2 that can be present within an artificial T cell receptor has the ability to bind to CLDN18.2, i.e., the ability to bind to an epitope present in CLDN18.2, preferably an epitope located in the extracellular domain of CLDN18.2, especially the first extracellular loop, preferably amino acids 29 to 78 of CLDN18.2. In particular embodiments, the binding domain for CLDN18.2 binds to an epitope on CLDN18.2 that is not present on CLDN18.1. Most preferably, the binding domain for CLDN18.2 binds to an epitope on CLDN18.2 that is not present on CLDN proteins other than CLDN18.2.

[0258] A binding domain for CLDN18.2 preferably binds to CLDN18.2, but not to CLDN18.1. Preferably, the binding domain for CLDN18.2 is specific for CLDN18.2. Preferably, the binding domain for CLDN18.2 binds to CLDN18.2 expressed on the surface of a cell. In particularly preferred embodiments, the binding domain for CLDN18.2 binds to a native epitope of CLDN18.2 present on the surface of a living cell.

[0259] In a preferred embodiment, the binding domain for CLDN18.2 comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23, 24, and 25, or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0260] In a preferred embodiment, the binding domain for CLDN18.2 comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33 and 34 or a fragment thereof, or a variant of said amino acid sequence or fragment.

[0261] In certain preferred embodiments, the binding domain for CLDN18.2 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the possibilities (i) to (ix) below:

[0262] (i) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 20 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 27 or a fragment thereof,

[0263] (ii) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 21 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 26 or a fragment thereof,

[0264] (iii) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 22 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 28 or a fragment thereof,

[0265] (iv) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 24 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 31 or a fragment thereof,

[0266] (v) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 23 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 30 or a fragment thereof,

[0267] (vi) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 25 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 29 or a fragment thereof,

[0268] (vii) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 25 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 32 or a fragment thereof,

[0269] (viii) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 25 or a fragment thereof, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 33 or a fragment thereof,

[0270] (ix) the VH comprises a CDR3 comprising the sequence of SEQ ID NO: 25 or a fragment thereof, and the VL comprises a CDR3 comprising the sequence of SEQ ID NO: 34 or a fragment thereof.

[0271] In a particularly preferred embodiment, the binding domain for CLDN18.2 comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL): the VH comprises a CDR3 comprising the sequence of SEQ ID NO: 23 or a fragment thereof, and the VL comprises a CDR3 comprising the sequence of SEQ ID NO: 30 or a fragment thereof.

[0272] In another particularly preferred embodiment, the binding domain for CLDN18.2 comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL): the VH comprises a CDR3 comprising the sequence of SEQ ID NO: 21 or a fragment thereof, and the VL comprises a CDR3 comprising the sequence of SEQ ID NO: 26 or a fragment thereof.

[0273] In a preferred embodiment, the binding domain for CLDN18.2 comprises (i) a VH comprising a CDR3 comprising the sequence of SEQ ID NO: 23 or a fragment thereof, and / or (ii) a VL comprising a CDR3 comprising the sequence of SEQ ID NO: 30 or a fragment thereof.

[0274] In a preferred embodiment, the binding domain for CLDN18.2 comprises:

[0275] (i) a VH comprising the following set of complementarity determining regions CDR1, CDR2 and CDR3:

[0276] CDR1: GYTFTSYW, CDR2: IYPSDSYT, CDR3: TRSWRGNSFDY

[0277] and / or

[0278] (ii) a VL comprising the following set of complementarity determining regions CDR1, CDR2 and CDR3:

[0279] CDR1: QSLLNSGNQKNY, CDR2: WAS, CDR3: QNDYSYPFT.

[0280] In a preferred embodiment, the binding domain for CLDN18.2 comprises a combination of a VH and a VL, each comprising the following set of complementarity determining regions CDR1, CDR2 and CDR3:

[0281] VH: CDR1: GYTFTSYW, CDR2: IYPSDSYT, CDR3: TRSWRGNSFDY,

[0282] VL: CDR1 : QSLLNSGNQKNY, CDR2: WAS, CDR3: QNDYSYPFT.

[0283] Preferably, the combination of a heavy chain variable region (VH) and a light chain variable region (VL) as described herein is arranged in a single chain Fv (scFv).

[0284] The term "fragment" especially refers to one or more complementarity determining regions (CDRs), preferably at least the CDR3 variable region, in a heavy chain variable region (VH) and / or a light chain variable region (VL). In one embodiment, the one or more complementarity determining regions (CDRs) are selected from the group of complementarity determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0285] In one embodiment, the binding domain of CLDN18.2 comprising one or more CDRs, a group of CDRs or a combination of CDR groups as described herein comprises said CDRs and the framework regions intervening therebetween. Preferably, this portion also includes at least about 50% of one or both of the first and fourth framework regions, 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of binding agents by recombinant DNA techniques can result in the introduction of residues N- or C-terminal to the variable region encoded by the introduced linker to facilitate cloning or other manipulation steps, including the introduction of linkers to join the variable regions of the application to other protein sequences, including immunoglobulin heavy chains, other variable regions (for example in the production of diabodies) or protein tags.

[0286] In one embodiment, the binding domain of CLDN18.2 comprising one or more CDRs, a group of CDRs or a combination of CDR groups as described herein comprises said CDRs in a human antibody framework.

[0287] In one embodiment, the binding domain of CLDN18.2 according to the application relates to a binding domain of CLDN18.2 which recognizes (i.e. binds to) the same or essentially the same epitope as a binding domain of CLDN18.2 as described herein (e.g. an antibody comprising a combination of a heavy chain variable region (VH) and a light chain variable region (VL) as described herein) and / or which competes with said binding domain of CLDN18.2 for binding to CLDN18.2.

[0288] The term "binds" of the present application preferably relates to specific binding.

[0289] According to the present application, a reagent (such as a T cell receptor or an antibody) is capable of binding a predetermined target if it has a significant affinity for the predetermined target and binds to said predetermined target in a standard assay. "Affinity" or "binding affinity" is generally measured by the equilibrium dissociation constant (K D ) of the reagent for the target. Preferably, the term "significant affinity" means that the binding of the reagent to the predetermined target has a dissociation constant (K -5 ) of 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less. D ).

[0290] A reagent does not (essentially does not) bind a target if it has no significant affinity for the target and does not significantly bind, in particular does not detectably bind, said target in a standard assay. Preferably, the reagent does not detectably bind said target if it is present at a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg / ml or more. Preferably, a reagent has no significant affinity for a target if the K D of the reagent for the target is at least 10 times, 100 times, 10 D times, 10 3 times, 10 4 times, 10 5 times or 10 6 times higher than the K D of the reagent for the predetermined target to which the reagent is capable of binding. For example, if the K -7 of the reagent for the target to which the reagent is capable of binding is 10 D M, then the K -6 of the reagent for the target to which the reagent has no significant affinity will be at least 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 M.

[0291] An agent is specific for a predetermined target if it is able to bind to the predetermined target and is not (not substantially) able to bind to other targets, i.e. does not have a significant affinity for and does not significantly bind to other targets in standard assays. According to the present application, an agent is specific for CLDN18.2 if it is able to bind to CLDN18.2 but not (not substantially) to other targets. Preferably, an agent is specific for CLDN18.2 if the affinity for and binding to such other targets does not significantly exceed the affinity or binding to proteins not related to CLDN18.2, such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or transmembrane proteins of non-claudin proteins such as MHC molecules or transferrin receptors or any other specific polypeptide. Preferably, an agent is specific for a predetermined target if it binds to the predetermined target with a K D at least 10 times, 100 times, 10 D times, 10 3 times, 10 4 times, 10 5 times or 10 6 times lower than to a non-specific target. For example, if the K D of an agent is 10 -7 M when binding to the specific target, the K D when binding to a non-specific target will be at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 M.

[0292] Binding of an agent to a target can be determined experimentally by any suitable method; see, e.g., Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, NY (1992), and methods described herein. Affinities can be readily determined using conventional techniques, e.g., by equilibrium dialysis; by using the general protocols outlined by the manufacturer using a BIAcore 2000 instrument; by radioimmunoassay using radiolabeled target antigen; or by other methods known to the skilled artisan. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51 :660 (1949). The affinity of a particular antibody-antigen interaction can be variable if measured under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other antigen binding parameters (e.g., K D , IC 50 ) are preferably made with standardized solutions of antibody and antigen and standardized buffers.

[0293] It will be appreciated that the peptide and protein agents described herein can be provided in vitro or in vivo, which can be in the form of nucleic acids (e.g., RNA encoding the agent) and / or host cells comprising nucleic acids (e.g., RNA encoding the agent). In particular, a variety of methods can be used to introduce the CAR construct into T cells, including non-viral based DNA transfection, transposon-based systems, and viral-based systems. Non-viral based DNA transfection has a low risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids without integration elements. Viral-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to produce, can efficiently and permanently transduce T cells, and have been preliminarily proven safe from an integration standpoint in primary human T cells. Lentiviral vectors can also efficiently and permanently transduce T cells, but are more costly to produce. They can also be safer than retrovirus-based systems.

[0294] The peptide and protein agents described herein can be delivered to a patient by administration of a nucleic acid, such as an RNA encoding the agent, and / or by administration of a host cell comprising a nucleic acid, such as an RNA encoding the agent. When administered to a patient, the nucleic acid can be present in naked form or in a suitable delivery vehicle, for example in the form of a liposome or viral particle, or within a host cell. The provided nucleic acid can produce the agent in a sustained manner over an extended period of time to mitigate at least some of the observed instability of the therapeutic protein. If the nucleic acid is administered to a patient in a manner that is not present within a host cell, it is preferred that it is taken up by a cell of the patient to express the agent encoded by the nucleic acid. If the nucleic acid is administered to a patient in a manner that is present within a host cell, it is preferred that it is expressed by the host cell within the patient in order to produce the agent encoded by the nucleic acid.

[0295] The term "nucleic acid" as used herein is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. The nucleic acid can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present application, the nucleic acid is preferably an isolated nucleic acid.

[0296] The nucleic acid can be comprised in a vector. The term "vector" as used herein includes any vector known to the skilled person, including a plasmid vector, a cosmid vector, a phage vector such as a lambda phage, a viral vector such as an adenoviral or baculoviral vector, or an artificial chromosome vector such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). The vector includes expression vectors as well as cloning vectors. Expression vectors include plasmids as well as viral vectors and typically contain the desired coding sequence and suitable DNA sequences necessary for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in an in vitro expression system. Cloning vectors are typically used for engineering and amplification of certain desired DNA fragments and can lack functional sequences required for expression of the desired DNA fragments.

[0297] In the context of the present application, the term "RNA" relates to a molecule comprising ribonucleotide residues and preferably consisting entirely or essentially of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of a beta-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA which differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, for example to the end(s) of the RNA or for example internally to one or more nucleotides of the RNA. The nucleotides in the RNA molecule can also comprise non-standard nucleotides, for example non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA.

[0298] According to the present application, the term "RNA" includes and preferably relates to "mRNA", which means "messenger RNA" and relates to a "transcript" which can be produced using DNA as a template and which encodes a peptide or protein. mRNA typically comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). The half-life of mRNA is limited within cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the application, the RNA is obtained by in vitro transcription or chemical synthesis. Methods of in vitro transcription are known to the person skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0299] In one embodiment of the application, the RNA is a self-replicating RNA, for example a single-stranded self-replicating RNA. In one embodiment, the self-replicating RNA is a positive-sense single-stranded RNA. In one embodiment, the self-replicating RNA is a viral RNA or a RNA derived from a viral RNA. In one embodiment, the self-replicating RNA is an alphavirus genomic RNA or derived from an alphavirus genomic RNA. In one embodiment, the self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is a Semliki forest virus. In one embodiment, the self-replicating RNA comprises one or more transgenes, wherein at least one of the transgenes encodes an agent described herein. In one embodiment, if the RNA is a viral RNA or derived from a viral RNA, the transgene(s) can partially or completely replace viral sequences, for example viral sequences encoding structural proteins. In one embodiment, the self-replicating RNA is an in vitro transcribed RNA.

[0300] To increase the expression and / or stability of the RNA used according to the present application, it can be modified, preferably without changing the sequence of the expressed peptide or protein.

[0301] In the context of the RNA used according to the present application, the term "modification" comprises any modification of the RNA which does not naturally occur in said RNA.

[0302] In one embodiment of the present application, the RNA used according to the present application does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

[0303] The RNA according to the present application can have modified naturally occurring or synthetic ribonucleotides to increase its stability and / or to reduce cellular toxicity. For example, in one embodiment, in the RNA used according to the present application, 5-methylcytidine partially or completely, preferably completely, replaces cytidine. Alternatively or in addition, in one embodiment, in the RNA used according to the present application, pseudouridine partially or completely, preferably completely, replaces uridine.

[0304] In one embodiment, the term "modification" relates to providing the RNA with a 5'-cap or a 5'-cap analog. The term "5'-cap" refers to the cap structure found on the 5'-end of an mRNA molecule, typically consisting of a guanosine nucleotide linked to the mRNA by an unusual 5' to 5' triphosphate bond. In one embodiment, the guanosine is methylated at the 7-position. The term "conventional 5'-cap" refers to the naturally occurring RNA 5' cap, preferably a 7-methylguanosine cap (m7G). In the context of the present application, the term "5'-cap" includes 5'-cap analogs which resemble the RNA cap structure and are modified to have the ability to stabilize the RNA, if attached thereto, preferably in vivo and / or in a cell.

[0305] Providing the RNA with a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analog, wherein said 5'-cap is co-transcriptionally incorporated into the RNA strand produced, or the RNA can be produced, e.g., by in vitro transcription, and the 5'-cap can be attached to the RNA using a capping enzyme (e.g., a capping enzyme of a vaccinia virus) after transcription.

[0306] The RNA can comprise further modifications. For example, a further modification of the RNA used in the present application can be an elongation or truncation of the naturally occurring poly(A) tail, or a change in the 5' or 3' untranslated region (UTR), e.g. the introduction of a UTR which is not related to the coding region of the RNA, e.g. the insertion of one or more, preferably two, copies of a 3'-UTR derived from a globin gene (e.g. alpha2-globin, alpha 1 -globin, beta-globin, preferably beta-globin, more preferably human beta-globin).

[0307] Thus, in order to improve the stability and / or expression of the RNA used in the present application, it can be modified to be present in form of a linkage to a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, in particular 100 to 150 adenosine residues. In a particularly preferred embodiment, the poly-A sequence has a length of about 120 adenosine residues. Furthermore, the incorporation of two or more 3'-untranslated regions (UTRs) into the 3'-untranslated region of the RNA molecule can lead to an increase in the translation efficiency. In a particular embodiment, the 3'-UTR is derived from the human beta-globin gene.

[0308] The term "stability" of an RNA relates to the "half-life" of the RNA. The "half-life" relates to the time required for the activity, amount or number of the molecules to be halved. In the context of the present application, the half-life of the RNA is indicative of the stability of the RNA. The half-life of the RNA can influence the "duration of expression" of the RNA. It can be expected that an RNA having a long half-life will be expressed for an extended period of time.

[0309] In the context of the present application, the term "transcription" relates to the process of transcribing the genetic code in a DNA sequence into RNA. Subsequently, the RNA can be translated into a protein. According to the present application, the term "transcription" includes "in vitro transcription", wherein the term "in vitro transcription" relates to the process of the synthesis of RNA, in particular mRNA, in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, cloning vectors are used for the production of the transcripts. These cloning vectors are often referred to as transcription vectors and are included in the term "vector" according to the present application.

[0310] The term "translation" according to the present application relates to the process in which a messenger RNA chain directs the assembly of an amino acid sequence to produce a peptide or protein in the ribosomes of a cell.

[0311] According to the present application, the nucleic acid can be present alone or in combination with other nucleic acids, which can be homologous or heterologous. In a preferred embodiment, the nucleic acid is functionally linked to an expression control sequence, which can be homologous or heterologous to the nucleic acid. The term "homologous" means that the nucleic acid is also naturally functionally linked, the term "heterologous" means that the nucleic acid is not naturally functionally linked.

[0312] The nucleic acid and the expression control sequence are "functionally" linked to each other if they are covalently linked to each other in such a way that the expression or transcription of the nucleic acid is controlled or influenced by the expression control sequence. If the nucleic acid is to be translated into a functional protein, the induction of the expression control sequence leads to the transcription of the nucleic acid as the expression control sequence is functionally linked to the coding sequence, without causing a shift in the reading frame in the coding sequence or the coding sequence cannot be translated into the desired protein or peptide.

[0313] The term "expression control sequence" or "expression control element" includes promoters, ribosomal binding sites, enhancers and other control elements which regulate transcription or translation of mRNA according to the present application. In a particular embodiment of the present application, the expression control sequence can be regulated. The precise structure of the expression control sequence can vary as a function of the species or cell type, but generally includes 5'-nontranscribed and 5'- and 3'-nontranslated sequences which play an important role in initiating transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. More specifically, the 5'-nontranscribed expression control sequence includes a promoter region, which comprises a promoter sequence for the transcription control of the nucleic acid functionally linked thereto. The expression control sequence can also comprise an enhancer sequence or an upstream activator sequence.

[0314] According to the present application, the term "expression" is used in its broadest meaning and includes, for example, the production of RNA and / or peptides or proteins by transcription and / or translation. With respect to RNA, the term "expression" or "translation" relates in particular to the production of peptides or proteins. The term "expression" also includes partial expression of a nucleic acid. Furthermore, expression can be transient or stable. According to the present application, the term "expression" also includes "aberrant expression" or "abnormal expression".

[0315] According to the present application, "aberrant expression" or "abnormal expression" means that the expression is altered, preferably increased, compared to a reference, e.g. a state of a subject not having a disease associated with aberrant expression or abnormal expression of certain proteins, such as tumor antigens. An increase in expression means an increase of at least 10%, in particular at least 20%, at least 50% or at least 100% or more. In one embodiment, expression is only found in diseased tissue, whereas expression in healthy tissue is suppressed.

[0316] The term "specifically expressed" means that a protein is expressed substantially only in a particular tissue or organ. For example, a tumor antigen that is specifically expressed in the gastric mucosa means that the protein is expressed primarily in the gastric mucosa and not expressed in other tissues, or not expressed to a significant degree in other tissue or organ types. Thus, a protein that is expressed only in cells of the gastric mucosa and expressed to a significantly lower degree in any other tissue, such as the testes, is specifically expressed in cells of the gastric mucosa. In some embodiments, a tumor antigen can also be specifically expressed under normal conditions in more than one tissue type or organ, for example in 2 or 3 tissue types or organs, but preferably in no more than 3 different tissue or organ types. In this case, the tumor antigen is specifically expressed in these organs. For example, if a tumor antigen is expressed under normal conditions, preferably in lung and stomach to approximately equal degrees, then the tumor antigen is specifically expressed in lung and stomach.

[0317] According to the present application, the term "nucleic acid encoding" means that the nucleic acid, if present in the appropriate environment, preferably within a cell, can be expressed to produce the protein or peptide it encodes.

[0318] Some aspects of the present application rely on the adoptive transfer of host cells, which are transfected in vitro with nucleic acids, such as RNAs encoding reagents as described herein, and preferably transferred to a recipient, such as a patient, after expansion from low precursor frequencies to clinically relevant cell numbers in vitro. According to the present application, the host cells used for therapy can be autologous, allogeneic or syngeneic to the recipient of the therapy.

[0319] The term "autologous" is used to describe anything that is derived from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or organs derived from the same subject. Such methods are advantageous because they overcome the immunological barrier that would otherwise lead to rejection.

[0320] The term "allogeneic" is used to describe anything that is derived from a different individual of the same species. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are not identical.

[0321] The term "syngeneic" is used to describe anything that is derived from an individual or tissue having an identical genotype, i.e. identical twins or the same inbred animal or tissue thereof.

[0322] The term "heterologous" is used to describe something that is composed of multiple different elements. For example, the transfer of bone marrow from one individual to a different individual constitutes a heterologous transplantation. A heterologous gene is a gene derived from a source other than the subject.

[0323] The term "transfection" relates to the introduction of nucleic acids, especially RNA, into a cell. For the purposes of the present application, the term "transfection" also includes the introduction of nucleic acids into a cell or the uptake of nucleic acids by such a cell, wherein the cell can be present in a subject, such as a patient. Thus, according to the present application, the cells for transfection of nucleic acids described herein can be present in vitro or in vivo, e.g. the cells can form part of an organ, tissue and / or organism of a patient. According to the present application, the transfection can be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is only expressed transiently. Since the nucleic acids introduced in the process of transfection are usually not integrated into the nuclear genome of the cell, the foreign nucleic acid will be diluted by mitosis or degradation. Cells that allow for episomal amplification of the nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection has to be carried out. RNA can be transfected into a cell for the transient expression of the protein it encodes.

[0324] According to the present application, any technique for introducing, i.e. transferring or transfecting, nucleic acids into a cell can be used. Preferably, the RNA is transfected into the cell by standard techniques. These techniques include electroporation, lipofection and microinjection. In a particularly preferred embodiment of the present application, the RNA is introduced into the cell by electroporation.

[0325] Electroporation or electropermeabilization involves a significant increase in the conductivity and permeability of the cytoplasmic membrane caused by an externally applied electric field. It is commonly used in molecular biology as a way to introduce some substances into cells.

[0326] According to the present application, it is preferred that the introduction of the nucleic acid encoding a protein or a peptide into the cell leads to the expression of said protein or peptide.

[0327] According to the present application, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising 2 or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, especially 100 amino acids, which are covalently linked by peptide bonds. The term "protein" refers to large peptides, preferably peptides having more than 100 amino acid residues, but often the terms "peptide" and "protein" are synonymous and used interchangeably herein.

[0328] According to the present application, the peptides can comprise natural amino acids and non-natural amino acids. In one embodiment, the peptides comprise only natural amino acids.

[0329] According to the present application, the term "non-natural amino acid" refers to an amino acid having a structure different from the 20 natural amino acids. Since the non-natural amino acid has a structure similar to the natural amino acid, the non-natural amino acid can be classified as a derivative or an analogue of a given natural amino acid.

[0330] Preferably, the proteins and peptides of the present application are isolated. The term "isolated protein" or "isolated peptide" means that the protein or peptide has been separated from its natural environment. The isolated protein or peptide can be in a substantially purified state. The term "substantially purified" means that the protein or peptide is essentially free of other components with which it associates in nature or in vivo.

[0331] The teachings given herein with respect to a particular amino acid sequence, such as those shown in the sequence listing, are to be interpreted to also relate to variants of the particular sequence which result in a sequence which is functionally equivalent to the particular sequence, e.g., an amino acid sequence which shows the same or similar properties as the particular amino acid sequence. One important property is to retain the binding of the peptide to an MHC molecule and / or a T cell receptor, or the binding of a T cell receptor to its target, or to maintain the effector function of a T cell. Preferably, a sequence which is modified relative to a particular sequence, when it replaces the particular sequence in a T cell receptor, retains the binding of the T cell receptor to a target, and preferably retains the function of the T cell receptor or a T cell carrying the T cell receptor described herein.

[0332] For example, the sequences shown in the sequence listing can be modified to remove one or more, preferably all, free cysteine residues, in particular by replacing the cysteine residues with an amino acid other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, leucine or methionine, most preferably alanine or serine.

[0333] The skilled person will appreciate that, in particular, the sequences of the CDR sequences, hypervariable regions and variable regions can be modified without losing the ability to bind to a target. For example, the CDR regions will be identical or highly homologous to the antibody regions specified herein. By "highly homologous" it is intended that between 1 and 5, preferably 1 to 4, for example 1 to 3 or 1 or 2 substitutions can be made in the CDRs. Furthermore, the hypervariable regions and variable regions can be modified such that they show a substantial homology to the regions specifically disclosed herein.

[0334] A peptide "variant" can retain the immunogenicity of a given peptide (e.g., the ability of the variant to react with a T cell line or clone is not substantially diminished relative to the given peptide). In other words, the ability of the variant to react with a T cell line or clone can be enhanced or unchanged relative to the given peptide, or can be diminished by less than 50%, preferably less than 20%, relative to the given peptide.

[0335] Variants can be identified by evaluating the ability of the variant to bind to an MHC molecule. In a preferred embodiment, the variant peptide has a modification such that the ability of the variant peptide to bind to an MHC molecule is increased relative to a given peptide. The ability of the variant peptide to bind to an MHC molecule can be increased at least 2-fold, preferably at least 3-fold, 4-fold or 5-fold relative to a given peptide. Thus, in certain preferred embodiments, the peptide comprises a variant in which 1 to 3 amino acid residues within the immunogenic portion are substituted such that the ability to react with a T cell line or clone is statistically greater than the ability of the unmodified peptide. Such substitutions are preferably located within the MHC binding site of the peptide. Preferred substitutions allow for increased binding to MHC class I or class II molecules. Certain variants contain conservative substitutions.

[0336] The term "variant" in the context of the present application also includes mutants, splice variants, conformers, isoforms, allelic variants, species variants and species homologues, especially naturally occurring variants. Allelic variants relate to changes in the normal sequence of a gene, the significance of which is often unclear. Complete sequencing of a gene often identifies multiple allelic variants of a given gene. Species homologues are nucleic acid or amino acid sequences of different species origin having a given nucleic acid or amino acid sequence. The term "variant" shall include any post-translationally modified variants and conformers.

[0337] For the purposes of the present application, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. Amino acid deletion variants comprising deletions of amino acids at the N- and / or C-terminus of a protein are also referred to as N-terminal and / or C-terminal truncation variants.

[0338] Amino acid insertion variants comprise the insertion of a single or two or more amino acids into a particular amino acid sequence. In the case of an amino acid sequence variant with insertions, one or more amino acid residues are inserted into the sequence at a particular point, although random insertions into the resulting product can also be suitably screened.

[0339] Amino acid addition variants comprise amino- and / or carboxyl-terminal fusions of one or more amino acids, such as 1, 2, 3, 4, 5, 10, 20, 30, 50 or more amino acids.

[0340] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example by removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein.

[0341] Amino acid substitution variants are characterized by the replacement of at least one residue in the sequence with another residue. Preferably, the modification is considered in the context of the position in the amino acid sequence that is not conserved between homologous proteins or peptides, and / or the amino acid is replaced with another amino acid having similar properties. Preferably, the amino acid change in a protein variant is a conservative amino acid change, i.e. substitution by an amino acid of similar charge or no charge. Conservative amino acid changes involve substitution of one of the amino acid families with which its side chain is associated. Naturally occurring amino acids are generally divided into four families: (1) acidic (aspartic acid, glutamic acid), (2) basic (lysine, arginine, histidine), (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0342] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Preferably, the degree of similarity or identity is given for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, preferably the degree of similarity or identity is given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200, preferably contiguous, amino acids. In a preferred embodiment, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed with tools known in the art, preferably using optimal sequence alignment, e.g. using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0343] “Sequence similarity” means the percentage of amino acids that are identical or represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences means the percentage of amino acids that are identical between the sequences.

[0344] The term "percent identity" is intended to mean the percentage of amino acid residues that are identical between two sequences being compared, after optimal alignment, which is purely statistical, the differences between the two sequences being randomly distributed and spanning their entire length. Sequence comparisons between two amino acid sequences are typically performed by comparing the two amino acid sequences after optimal alignment of the two amino acid sequences, the comparison being performed by segments or by "comparison windows", in order to identify and compare local regions of sequence similarity. In addition to manual, optimal alignment, the optimal alignment of sequences for comparison can be produced, e.g., by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482; by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444 or by computer programs using algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0345] The percent identity is calculated by determining the number of positions at which the two sequences be ing compared are identical, dividing this number by the number of positions compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0346] According to the present application, homologous amino acid sequences show at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0347] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, e.g., by recombinant DNA manipulation. Sambrook et al. (1989) describe in detail DNA sequence manipulations for making proteins and peptides with substitutions, additions, insertions or deletions of amino acids. Furthermore, the peptides and amino acid variants described herein can be readily prepared by means of known peptide synthesis techniques, such as by solid phase synthesis and similar methods.

[0348] The present application includes derivatives of the peptides or proteins as described herein comprising the terms "peptide" and "protein". According to the present application, a "derivative" of a protein and a peptide is a modified form of the protein and the peptide. Such modifications include any chemical modification and include single or multiple substitutions, deletions and / or additions of any molecule associated with the protein or peptide (e.g. carbohydrates, lipids and / or proteins or peptides). In one embodiment, a "derivative" of a protein or peptide includes modified analogs resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective / blocking groups, proteolytic cleavage, or binding to an antibody or additional cellular ligand. The term "derivative" also extends to all functionally chemically equivalent of the proteins and peptides. Preferably, the modified peptides have increased stability and / or increased immunogenicity.

[0349] Also included are mimetics of the peptides. Such mimetics can comprise amino acids linked to one or more amino acid mimetics (i.e., one or more amino acids within the peptide can be replaced by an amino acid mimetic) or can be entirely non-peptide mimetics. An amino acid mimetic is a compound that resembles the conformation of an amino acid, e.g., such that it can be substituted for an amino acid without substantially decreasing the ability to react with a T cell line or clone. A non-peptide mimetic is a compound that does not contain amino acids and that has an overall conformation similar to a peptide, e.g., such that the ability of the mimetic to react with a T cell line or clone is not substantially decreased relative to the given peptide.

[0350] According to the present application, a variant, derivative, modified form, fragment, part or portion of an amino acid sequence, a peptide or a protein preferably has the functional properties of the amino acid sequence, the peptide or the protein, respectively, from which it is derived, i.e. it is functionally equivalent. In one embodiment, a variant, derivative, modified form, fragment, part or portion of an amino acid sequence, a peptide or a protein is immunologically equivalent to the amino acid sequence, the peptide or the protein, respectively, from which it is derived. In one embodiment, the functional properties are immunological properties.

[0351] A particular property is the ability to form a complex with an MHC molecule and, where appropriate, to stimulate an immune response, preferably by stimulating the production of cytotoxic or T helper cells.

[0352] The term "immunologically equivalent" means that immunologically equivalent molecules, e.g. immunologically equivalent amino acid sequences, display the same or essentially the same immunological properties and / or exert the same or essentially the same immunological effects, e.g. with respect to the type of immunological effect like induction of a humoral and / or cellular immune response, the strength and / or duration of the induced immune response, or the specificity of the induced immune response. In the context of the present application, the term "immunologically equivalent" is preferably used for the immunological effects or properties of a peptide or peptide variant for use in immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if the immune response induced by the amino acid sequence has the specificity of the response of the reference amino acid sequence when exposed to the immune system of a subject.

[0353] According to the present application, the term "derived from" means that a particular entity, in particular a particular sequence, is present in the subject, in particular an organism or molecule, from which it is derived. In the case of an amino acid sequence, in particular a particular sequence region, "derived from" in particular means that the relevant amino acid sequence is derived from the amino acid sequence in which it is present.

[0354] The term "cell" or "host cell" preferably relates to an intact cell, i.e. a cell with an intact membrane, which has not released its normal intracellular components such as enzymes, organelles, or genetic material. The intact cell is preferably a viable cell, i.e. a living cell, which is capable of performing its normal metabolic functions. Preferably, according to the present application, the term relates to any cell which can be transfected with an exogenous nucleic acid. Preferably, the cell can express the nucleic acid in a recipient when transfected with the exogenous nucleic acid and transferred to the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include cells from strains of gram-negative bacteria, such as Escherichia coli, Proteus, and Pseudomonas, as well as gram-positive bacteria, such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of the genera Saccharomyces (e.g. Saccharomyces cerevisiae), Schizo saccharomyces (e.g. Schizo saccharomyces pombe), Pichia (e.g. Pichia pastoris and Pichia methanolicd), and Hansenula. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins can also be used. Mammalian cells are particularly preferred for adoptive transfer, e.g. cells derived from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a large number of tissue types and include primary cells and cell lines, e.g. cells of the immune system, especially antigen presenting cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells, and other cell types. Antigen presenting cells are cells which display an antigen on the cell surface in the context of the major histocompatibility complex. T cells can recognize this complex using their T cell receptor (TCR).

[0355] The cell comprising the nucleic acid molecule preferably expresses the peptide or protein encoded by the nucleic acid.

[0356] The cell can be a recombinant cell and can secrete the encoded peptide or protein, can express the peptide or protein on the surface and preferably can additionally express MHC molecules which bind to the peptide or protein or processing products thereof. In one embodiment, the cell endogenously expresses MHC molecules. In another embodiment, the cell expresses MHC molecules and / or the peptide or protein or processing products thereof in a recombinant manner. The cell is preferably non-proliferating. In a preferred embodiment, the cell is an antigen presenting cell, especially a dendritic cell, a monocyte, or a macrophage.

[0357] The term "clonal expansion" refers to a process in which a particular entity is multiplied. In the context of the present application, the term is preferably used in the context of an immune response, wherein lymphocytes are stimulated by an antigen, proliferate, and specific lymphocytes recognizing said antigen are expanded. Preferably, clonal expansion leads to differentiation of the lymphocytes.

[0358] Diseases associated with expression of an antigen can be detected based on the presence of T cells specifically reacting with the peptide in a biological sample. In certain methods, a biological sample comprising CD4+ and / or CD8+ T cells isolated from a patient is incubated with a peptide of the application, a nucleic acid encoding such a peptide, and / or an antigen-presenting cell expressing and / or presenting at least an immunogenic portion of such a peptide, and it is detected whether specific activation of T cells occurs. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from a patient by conventional techniques, e.g., by Ficoll / Hypaque density gradient centrifugation of peripheral blood lymphocytes. For CD4+ T cells, activation is preferably detected by assessing proliferation of the T cells. For CD8+ T cells, activation is preferably detected by assessing cytolytic activity. A level of proliferation that is at least 2-fold higher and / or a level of cytolytic activity that is at least 20% higher compared to in a disease-free subject indicates the presence of a disease associated with expression of the antigen in the subject.

[0359] "Decrease" or "inhibit" as used herein means the ability to cause an overall decrease in a level, preferably a decrease of 5% or more, 10% or more, 20% or more, more preferably 50% or more, most preferably 75% or more. The term "inhibit" or similar phrases includes complete or substantially complete inhibition, i.e., a decrease to zero or substantially zero.

[0360] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, most preferably at least 100%.

[0361] The agents, compositions, and methods described herein can be used to treat a subject suffering from a disease, e.g., a disease characterized by the presence of CLDN18.2-expressing and preferably CLDN18.2-presenting in the context of MHC molecules diseased cells. Examples of diseases that can be treated and / or prevented include all diseases expressing CLDN18.2. Particularly preferred diseases are cancer diseases.

[0362] The agents, compositions, and methods described herein can also be used for immunization or vaccination to prevent a disease described herein.

[0363] The term "normal tissue" or "normal condition" refers to a healthy tissue or condition of a healthy subject, i.e. a non-pathological condition, wherein "healthy" preferably means non-cancerous.

[0364] The term "disease" refers to an abnormal condition that affects the body of an individual. A disease is generally interpreted as a medical condition associated with specific symptoms and signs. A disease can be caused by factors originally from outside the body, such as infectious diseases, or it can be caused by internal dysfunction, like autoimmune diseases. In humans, "disease" is often used more generally to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In a more general sense, disease sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts, especially in

[0365] According to the present application, a "disease involving cells expressing CLDN18.2" or similar expressions means that CLDN18.2 is expressed in cells of a diseased tissue or organ. In one embodiment, the expression of CLDN18.2 in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. Increased means an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or more. In one embodiment, the expression is only found in diseased tissue, whereas the expression in healthy tissue is suppressed. According to the present application, a disease involving cells expressing CLDN18.2 comprises a cancer disease. Furthermore, according to the present application, the cancer disease is preferably a cancer disease wherein the cancer cells express CLDN18.2.

[0366] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningoma, and pituitary adenoma. According to the present application, the term "cancer" also includes metastasis of a cancer. Preferably, the "cancer disease" is characterized by cells expressing CLDN18.2, and the cancer cells express CLDN18.2.

[0367] The diseased cell is preferably a cell expressing CLDN18.2, which is preferably present on the surface of the cell as a transmembrane protein and / or is presented by the cell in the context of MHC, such as MHC I. The cell expressing CLDN18.2 is preferably a cancer cell, preferably a cancer cell of a cancer as described herein.

[0368] In one embodiment, the cancer disease is a malignant disease characterized by anaplasia, invasiveness and metastasis. Malignant tumors can be contrasted with non-cancerous benign tumors, because malignant tumors are not self-limiting in their growth, are able to invade into adjacent tissues, and are able to spread to distant tissues (metastasis), while benign tumors do not have these properties.

[0369] In one embodiment, the cancer of the present application involves cancer cells expressing CLDN18.2. In one embodiment, the cancer is CLDN18.2 positive. In one embodiment, the expression of CLDN18.2 is located at the cell surface. In one embodiment, at least 50%, preferably 60%, 70%, 80% or 90% of the cancer cells are CLDN18.2 positive, and / or at least 40%, preferably at least 50% of the cancer cells are positive for surface expression of CLDN18.2. In one embodiment, at least 95% or at least 98% of the cancer cells are CLDN18.2 positive. In one embodiment, at least 60%, at least 70%, at least 80% or at least 90% of the cancer cells are positive for surface expression of CLDN18.2.

[0370] In one embodiment, the CLDN18.2-expressing cancer, the cancer involving CLDN18.2-expressing cancer cells or the CLDN18.2-positive cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and metastases thereof, in particular gastric cancer metastases such as Krukenberg tumors, peritoneal metastases and lymph node metastases. In one embodiment, the cancer is an adenocarcinoma, in particular an advanced adenocarcinoma. Particularly preferred cancer diseases are adenocarcinomas of the stomach, esophagus, pancreatic duct, bile duct, lung and ovary. In one embodiment, the cancer is selected from the group consisting of gastric cancer, esophageal cancer, in particular lower esophageal cancer, esophagogastric junction cancer and gastroesophageal cancer. In a particularly preferred embodiment, the cancer is a gastroesophageal cancer, for example a metastatic, refractory or relapsed advanced gastroesophageal cancer.

[0371] According to the present application, the term "tumor" or "tumorous disease" refers to a swelling or lesion formed by the abnormal growth of cells, known as neoplastic cells or tumor cells. A "tumor cell" refers to an abnormal cell that grows by rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissues, and usually form distinct mass of tissue, which can be benign, pre-malignant, or malignant.

[0372] According to the present application, a "carcinoma" is a malignant tumor derived from epithelial cells. This group represents the most common cancers, including the common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0373] An "adenocarcinoma" is a cancer that originates in glandular tissue. This tissue is also part of a larger category of tissue known as epithelial tissue. Epithelial tissue includes the skin, glands, and many other tissues that line the body's cavities and organs. Epithelial cells are embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as an adenocarcinoma, the cells do not necessarily have to be part of a gland, only that they have secretory properties. This form of cancer can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originated, while less-differentiated ones can not. Through staining of cells from a biopsy, a pathologist will determine whether a tumor is an adenocarcinoma or some other type of cancer. Because glands are ubiquitous in the body, adenocarcinomas can occur in many tissues of the body. While each gland can not secrete the same substance, as long as the cells have an exocrine function, they are considered a gland, and thus their malignant form is called an adenocarcinoma. Malignant adenocarcinomas invade other tissues, and often have enough time to metastasize. Ovarian adenocarcinoma is the most common type of ovarian cancer. It includes serous and mucinous adenocarcinomas, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0374] Lymphomas and leukemias are malignancies that arise from hematopoietic (blood-forming) cells.

[0375] Blastic tumors or blastomas are tumors (usually malignant) that resemble immature or embryonic tissue. Many of these tumors are most common in children.

[0376] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane into body cavities and blood vessels, and then, after being transported by the blood, infiltration of the target organ. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, since tumor cells or components can remain and develop metastatic potential. In one embodiment, according to the present application, the term "metastasis" relates to "distant metastasis", which relates to metastasis away from the primary tumor and the regional lymph node system. In one embodiment, the term "metastasis" according to the present application relates to lymph node metastasis.

[0377] The cells of a secondary or metastatic tumor resemble those in the original tumor. This means, for example, that if an ovarian cancer metastasizes to the liver, the secondary tumor is composed of abnormal ovarian cells, not abnormal liver cells. The tumor in the liver is then called metastatic ovarian cancer, not liver cancer.

[0378] A relapse or recurrence occurs when a person is again affected by a condition that affected them in the past. For example, if a patient has a tumor disease, has received a successful treatment of said disease, and develops said disease again, the newly developed disease can be considered a relapse or recurrence. However, according to the present application, a relapse or recurrence of a tumor disease can but does not necessarily occur at the site of the original tumor disease. Thus, for example, if a patient has an ovarian tumor and has received a successful treatment, the relapse or recurrence can be the occurrence of an ovarian tumor or the occurrence of a tumor at a site different from the ovary. A relapse or recurrence of a tumor also includes the case where a tumor occurs at a site different from the site of the original tumor as well as at the site of the original tumor. Preferably, the original tumor for which the patient receives a treatment is a primary tumor and the tumor at a site different from the site of the original tumor is a secondary or metastatic tumor.

[0379] The term "treatment" or "therapeutic treatment" relates to any treatment which improves the health state of an individual and / or prolongs (increases) the life of an individual. The treatment can eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the likelihood of recurrence in an individual who currently has or who previously has had a disease.

[0380] The term "prophylactic treatment" or "preventive treatment" relates to any treatment which aims at preventing an individual from developing a disease. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

[0381] The terms "individual" and "subject" are used interchangeably herein. They refer to a human, a non-human primate or other mammal (such as a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse or primate) who can be afflicted with or susceptible to a disease or disorder, such as cancer, but who can or can not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not denote a particular age and thus include adults, elderly, children and neonates. In preferred embodiments of the present application, the "individual" or "subject" is a "patient". According to the present application, the term "patient" means a subject for treatment, in particular a diseased subject.

[0382] "Being at risk" means that a subject, i.e. a patient, is identified to have a higher than normal likelihood of developing a disease, in particular cancer, compared to the general population. Furthermore, a subject who has had or who currently has a disease, in particular cancer, is a subject at increased risk of developing a disease, as such a subject can continue to develop the disease. A subject who currently has or who has had cancer is also at increased risk of metastasis of the cancer.

[0383] The term "immunotherapy" refers to a treatment which involves a specific immune response.

[0384] In the context of the present application, terms such as "protect", "prevent", "prophylactic", "preventive" or "protective" relate to the prevention or treatment or both of the occurrence and / or spread of a disease in a subject, in particular to minimize or delay the development of a disease in a subject. For example, as mentioned above, a person at risk of developing a tumor would be a candidate for a treatment to prevent the tumor.

[0385] Preventive administration of immunotherapy, such as the preventive administration of a reagent or composition of the application, preferably protects the recipient from the development of a disease. Therapeutic administration of immunotherapy, such as the therapeutic administration of a reagent or composition of the application, can lead to the inhibition of the progression / growth of a disease. This includes the slowing of the progression / growth of a disease, in particular the disruption of the progression of a disease, which preferably leads to the elimination of the disease.

[0386] Immunotherapy can be performed using any of a variety of techniques, wherein the reagents provided herein are preferably used to remove cells expressing CLDN18.2 from a patient. Such removal can occur as a result of enhancing or inducing an immune response in the patient against CLDN18.2 or cells expressing CLDN18.2 and / or the presentation of CLDN18.2 specificity in the context of MHC molecules.

[0387] In certain embodiments, immunotherapy can be active immunotherapy, wherein the treatment relies on the administration of immune response-modifying agents, such as the peptides and nucleic acids provided herein, to stimulate the endogenous host immune system in vivo, thereby reacting against diseased cells.

[0388] In other embodiments, immunotherapy can be passive immunotherapy, wherein the treatment involves the delivery of reagents, such as effector cells, with defined tumor-immune reactivity, which can directly or indirectly mediate an anti-tumor effect, and do not necessarily rely on an intact host immune system. Examples of effector cells include T lymphocytes, such as CD8+ cytotoxic T lymphocytes and CD4+ T helper lymphocytes, and antigen-presenting cells, such as dendritic cells and macrophages. T cell receptors specific for the CLDN18.2 peptides described herein and artificial T cell receptors specific for CLDN18.2 can be transferred into effector cells for adoptive immunotherapy.

[0389] As mentioned above, the immunoreactive peptides provided herein can be used to rapidly expand antigen-specific T cell cultures to produce sufficient numbers of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic cells, macrophages, monocytes, fibroblasts, and / or B cells, can be pulsed with the immunoreactive peptides or transfected with one or more nucleic acids using standard techniques well known in the art. The cultured effector cells used for therapy must be able to grow and distribute widely, and survive long-term in the body. Studies have shown that cultured effector cells can be induced to grow and survive long-term in the body in large numbers by repeated stimulation with antigen supplemented with IL-2 (see, e.g., Cheever et al. (1997), Immunological Reviews 157, 177.

[0390] Alternatively, nucleic acids expressing the peptides described herein can be introduced into antigen-presenting cells taken from a patient and clonally propagated in vitro for implantation back into the same patient.

[0391] The transfected cells can be reintroduced into the patient using any method known in the art, preferably in sterile form, by intravenous, intracavitary, intraperitoneal or intratumoral administration.

[0392] The methods disclosed herein can involve administering autologous T cells that are activated in response to the peptide or the peptide-expressing antigen presenting cells. Such T cells can be CD4+ and / or CD8+ and can be propagated as described above. The T cells can be administered to the subject in an effective amount to inhibit the progression of the disease.

[0393] The term "immunization" or "vaccination" describes the process of treating a subject with the aim to induce an immune response for therapeutic or prophylactic reasons.

[0394] The term "in vivo" relates to the situation in a subject.

[0395] According to the present application, a "sample" can be any useful sample according to the present application, especially a biological sample, e.g. a tissue sample, which includes a body fluid and / or a cell sample, and can be obtained in a conventional manner, e.g. by tissue biopsy (including punch biopsy) and by taking blood, bronchial aspirate, sputum, urine, feces or other body fluids. According to the present application, the term "sample" also includes processed samples, e.g. fractions or isolates of a biological sample, such as nucleic acid and peptide / protein isolates.

[0396] The compounds and agents described herein can be administered in any suitable pharmaceutical composition.

[0397] The pharmaceutical compositions of the present application are preferably sterile and contain an effective amount of the agents described herein and optionally additional agents discussed herein to produce the desired reaction or desired effect.

[0398] The pharmaceutical compositions are generally provided in unit dosage form and can be prepared in a manner known per se. The pharmaceutical compositions can be in the form of, for example, a solution or a suspension.

[0399] The pharmaceutical compositions can comprise salts, buffer substances, preservatives, carriers, diluents and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" means a nontoxic material that does not interact with the active ingredients of the pharmaceutical compositions in a way that would interfere with their function.

[0400] Pharmaceutically unacceptable salts can be used for the preparation of pharmaceutically acceptable salts and are included in the present application. Such pharmaceutically acceptable salts include, in a non-limiting manner, salts prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid and the like. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0401] Suitable buffering agents for use in pharmaceutical compositions include acetates, citrates, borates and phosphates.

[0402] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorbutanol, parabens and thimerosal.

[0403] Injectable formulations can include pharmaceutically acceptable excipients, such as Ringer Lactate.

[0404] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, in which the active ingredient is combined to facilitate, enhance or enable administration. According to the present application, the term "carrier" also includes one or more compatible solid or liquid filler diluents or encapsulating substances suitable for administration to a patient.

[0405] Possible carrier substances for parenteral administration are, for example, sterile water, Ringer's solution, Ringer-Lactate solution, sterile sodium chloride solution, polyalkylene glycols, naphthalenes, and especially biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.

[0406] The term "excipient" as used herein is intended to mean all substances which can be present in a pharmaceutical composition and are not active ingredients, such as carriers, binders, lubricants, thickening agents, surfactants, preservatives, emulsifiers, buffers, flavorings or colorings.

[0407] The agents and compositions described herein can be administered by any conventional route, for example, by parenteral administration (including by injection or infusion). Parenteral administration, for example intravenous, intraarterial, subcutaneous, intradermal or intramuscular administration, is preferred.

[0408] Compositions suitable for parenteral administration typically comprise sterile aqueous or nonaqueous formulations of the active compound, which are preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspension medium.

[0409] The agents and compositions described herein are administered in effective amounts. An "effective amount" means the amount which achieves the desired response or the desired effect, alone or in combination with other doses. In the case of treating a particular disease or particular condition, the desired response preferably relates to the inhibition of the disease process. This includes slowing down the progression of the disease, in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease or condition can also be the delay of the onset of said disease or said condition or the prevention of the onset of said disease or said condition.

[0410] An effective amount of an agent or composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient including age, physiological condition, size and weight, the duration of treatment, the type of accompanying therapy, if any, the specific route of administration and similar factors. Thus, the dosage of agents described herein can vary depending on different such parameters. When the response in a patient is insufficient using an initial dosage, higher doses (or effective higher doses achieved by different, more localized routes of administration) can be used.

[0411] Agents and compositions described herein can be administered, for example, in vivo to a patient, to treat or prevent various disorders such as those described herein. Preferred patients include human patients having a disorder that can be corrected or ameliorated by administration of agents and compositions described herein. This includes disorders involving cells characterized by expression of CLDN18.2.

[0412] For example, in one embodiment, agents described herein can be used to treat a patient having a cancer disease, such as a cancer disease described herein, characterized by the presence of cancer cells expressing CLDN18.2.

[0413] The pharmaceutical compositions and methods of treatment described herein can also be used for immunization or vaccination to prevent diseases described herein.

[0414] The pharmaceutical compositions of the present application can be administered together with supplementary immunopotentiating substances, such as one or more adjuvants, and can comprise one or more immunopotentiating substances to further increase their effectiveness, preferably to achieve a synergistic effect of the immunostimulation. The term "adjuvant" relates to a compound that prolongs or enhances or accelerates the immune response. Depending on the type of adjuvant various mechanisms are possible in this respect. For example, compounds that allow the maturation of DCs, such as lipopolysaccharides or CD40 ligands, form a first class of suitable adjuvants. In general, any agent that influences the "danger signals" type of the immune system (LPS, GP96, dsRNA, etc.) or any agent that influences the cytokine type of the immune system, such as GM-CSF, can be used as an adjuvant that is able to enhance the immune response and / or to influence the immune response in a controlled manner. Optionally, CpG oligodeoxynucleotides can also be used herein, although as mentioned above, their side effects in certain cases have to be taken into account. Particularly preferred adjuvants are cytokines, such as monokines, lymphokines, interleukins or chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN alpha, IFN gamma, GM-CSF, LT-alpha or growth factors such as hGH. Other known adjuvants are aluminum hydroxide, Freund's adjuvant or oils such as Most preferred are ISA 51. Lipopeptides such as Pam3Cys are also suitable as adjuvants in the pharmaceutical compositions of the present application.

[0415] The pharmaceutical composition can be administered topically or systemically, preferably systemically.

[0416] The term "systemic administration" refers to administration of an agent such that the agent is distributed throughout the body of an individual in an effective amount and produces the desired effect. For example, the agent can produce its desired effect in the blood and / or reach its desired site of action via the vasculature. Typical systemic routes of administration include administration by introducing the agent directly into the vasculature or oral, pulmonary, or intramuscular administration, wherein the agent is absorbed, enters the vasculature, and is carried via the blood to one or more desired sites of action.

[0417] According to the present application, systemic administration is preferably performed by parenteral administration. The term "parenteral administration" refers to administration of an agent such that the agent does not pass through the intestinal tract. The term "parenteral administration" includes intravenous administration, subcutaneous administration, intradermal administration, or intra-arterial administration, but is not limited to these.

[0418] Oral, intraperitoneal, or intramuscular administration can also be performed, for example.

[0419] The agents and compositions provided herein can be used alone or in combination with conventional treatment regimens, such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

[0420] The present application is described in detail by way of the following drawings and examples, which are for illustrative purposes only and not intended to limit the application. Other embodiments, which are equally effective as described and exemplified, are available to those skilled in the art from the description and examples. BRIEF DESCRIPTION OF DRAWINGS

[0421] Figure 1 : Representative of the TCR-CD3 complex. Intracytoplasmic CD3 immunoreceptor tyrosine activation motif (ITAM) is represented as a cylinder (adapted from "The T cell receptor factbook", MP Lefranc, G Lefranc, 2001).

[0422] Figure 2 : Design of successive generations of CARs. Schematic representation of different generations of CARs (1G, first generation, 2G, second generation, 3G, third generation). The first generation comprises an extracellular scFv and a cytoplasmic CD3 zeta chain / ZAP70 mediating cytotoxicity, the second generation additionally has CD28 / PI3K promoting proliferation, the third generation has also 4-1BB or OX40 / TRAF maintaining cell survival (Casucci, M. et al. (2011) 2: 378-382).

[0423] Figure 3 : Schematic of different receptor formats for T cell redirection against CLDN18.2. Left: second generation CAR consisting of a CLDN18.2-specific scFv fragment, an IgGl-derived spacer domain, a CD28 costimulatory and a CD3 zeta signaling domain (CAR-28z); middle: novel CAR format based on the linkage of the scFv to the constant domain of a murine TCR beta chain and the co-expression of the constant domain of a murine TCR alpha chain (CAR / Ca); right: murine TCR comprising TCR alpha / beta chains (mu, murine TCR);

[0424] Figure 4 : Analysis of CLDN18.1 and CLDN18.2 transcripts in a panel of human tissues. A, genomic structure of the CLDN18 locus (top). Shaded boxes, exons unique to CLDN18.1 (E1.1) or CLDN18.2 (E 1.2), respectively; bottom, exon composition of CLDN18 variants; arches, 2 extracellular domains; arrows, primers used for RT-PCR. B, comparative analysis of CLDN18 isoforms by endpoint RT-PCR in normal human tissues (N), primary tumor samples, and tumor cell lines. C, quantification by real-time PCR in normal human tissues (N), primary tumor samples, and tumor cell lines (Sahin U et al., Clin Cancer Res 2008; 14:7624-34).

[0425] Figure 5 : Analysis of the ex vivo reactivity of splenocytes from immunized HLA-A*02-transgenic mice against CLDN18.2-derived peptides by IFNy-ELISPOT assay. The first 80 amino acids of CLDN18.2 were predicted for HLA-A*02 CLDN18.2-specific binding peptides using a specific algorithm (Rammensee H. et al. (1999) Immunogenetics 50, 213-9). Splenocytes of CLDN18.2-immunized HLA-A*02 transgenic mice were analyzed for reactivity against CLDN18.2 peptide pools or predicted HLA-A*02-binding CLDN18.2-derived peptides CLDN18.2-A2-1-6. Positive control: PMA-treated splenocytes; negative controls: irrelevant peptide pool (HIV-gag), irrelevant nonamer peptide (PLAC1-31-39).

[0426] Figure 6Flow cytometric sorting of CLDN18.2-specific murine CD8+ T cells from HLA-A*02-transgenic mice after in vitro re-stimulation. Single CD8+ / CD137+ T cells were isolated after re-stimulation of splenocytes with the CLDN18.2 overlapping peptide pool. Control: re-stimulation of splenocytes with an unrelated peptide pool (HIV-gag).

[0427] Figure 7 Specificity test of TCR isolated from CD8+ T cells of CLDN18.2 immunized mice. CD8+ T cells of HLA-A*02 positive healthy donors were transfected with TCR-alpha / beta chain RNA and tested for recognition of K562-A2 cells pulsed with CLDN18.2 overlapping 15mer peptides (= CLDN18.2 pool) or CLDN18.2 derived HLA-A*02 binding peptides (CLDN18.2-A2-4, CLDN18.2-A2-5, CLDN18.2-A2-6) by IFN gamma-ELISPOT. Negative controls: unrelated peptide pool (HIV-gag), unrelated 9mer peptide (PLAC1-31-39); positive control: SEB;

[0428] Figure 8 Surface expression of CLDN18.2 and CLDN6 specific CARs on human pre-activated CD8+ T cells. CD8+ T cells were pre-activated with OKT3 and transfected with 20 pg of CAR-RNA. 20 h after electroporation, cells were stained with PE-conjugated anti-CD8 antibodies and idiotype-specific fluorescent dye-conjugated antibodies specific for CLDN18.2-CAR and CLDN6-CAR, respectively. Cells were gated on single CD8+ T lymphocytes.

[0429] Figure 9 Specific lysis of CLDN18.2 expressing target cells mediated by CLDN18.2-CAR. Pre-activated CD8+ T cells were transfected with 20 pg CAR RNA and subsequently co-cultured with autologous iDCs transfected with RNA encoding for CLDN18.2-CAR using titrated E:T ratios (30:1, 10:1, 3:1) for 20 h. Negative controls: T cells transfected with CLND6 specific CAR or without CAR RNA (= mock), iDCs transfected with CLDN6-RNA. Specific lysis was analyzed by luciferase-based cytotoxicity assay 4 h after co-culture.

[0430] Figure 10The study aimed to specifically inhibit CLDN18.2-CAR-mediated lysis of target cells expressing CLDN18.2 by adding a idiotype-specific antibody. Pre-activated CD8+ T cells were transfected with 20 μg CAR RNA and then co-cultured for 20 h with autologous iDCs transfected with CLDN18.2- or CLDN6- RNA at an E:T ratio of 30:1. Effector T cells were pre-incubated for 1 h with or without 2 μg / ml idiotype-specific antibody before adding target cells. Specific lysis was analyzed by a luciferase-based cytotoxicity assay after 4 h of co-culture.

[0431] Figure 11 In vitro antigen-specific proliferation of CLDN18.2-CAR T cells. CD8+ T cells were electroporated with RNA encoding CLDN18.2-CAR or without RNA (simulated) and labeled with carboxyfluorescein succinimide (CFSE). CAR T cells were co-cultured with iDCs transfected with 5 μg of IVT-RNA encoding CLDN18.2 or control antigens CLDN9 or CLDN6. Co-cultured cells were harvested after 96 hours, and CFSE fluorescence was analyzed by flow cytometry. Cells were then propagated using live, single-CD8+ T cells. + Lymphocytes are phylogenetic.

[0432] Figure 12 : In vivo antigen-specific activation of CLDN18.2-CAR T cells in mice after vaccination. Using 5 × 10 6 CLDN18-2-CAR-effLuc-GFP transduced T cells were intravenously (iv) implanted into BALB / c mice. Twenty-four hours after ACT, the cells were administered RNA containing 25 μg of CLDN18.2 RNA. (F12-Lip) Alternatively, control RNA was intravenously injected into mice. (A) T cell transduction efficiency was measured by flow cytometry using a fluorescent dye-conjugated antibody. (B) In vivo luminescence intensity was measured in mice 48 hours post-inoculation. Color-changing images represent light intensity superimposed on a grayscale reference photograph (black, least intense; white to dark gray, most intense). Example

[0433] The techniques and methods used herein are described or practiced in a manner known to them, as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Unless otherwise expressly stated, all methods, including those used in kits and reagents, were performed based on the manufacturer’s information.

[0434] Example 1 : Materials and methods

[0435] Cell lines and reagents

[0436] The human chronic myelogenous leukemia cell line K562 stably transfected with HLA-A*0201 and used for TCR validation assays (Lozzio, C. B. & Lozzio, B. B (1975), Blood 45, 321-334) (Britten, C. M. et al. (2002), J. Immunol. Methods 259, 95-110) (referred to as e.g. K562-A2) were cultured under standard conditions. The primary human neonatal foreskin fibroblast cell line CCD-1079Sk (ATCC No. CRL-2097) was cultured according to the manufacturer's instructions.

[0437] Peripheral blood mononuclear cells (PBMC), monocytes and dendritic cells (DC)

[0438] PBMC were isolated from buffy coats by Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. HLA alleles were determined by standard PCR methods. Monocytes were enriched with anti-CD14 microbeads (Miltenyi Biotech. Bergisch-Gladbach, Germany). Immature DC (iDC) were obtained by differentiating monocytes for 5 days in cytokine-supplemented medium as described in Kreiter et al. (2007), Cancer Immunol. Immunother., CII, 56, 1577-87.

[0439] Peptides and peptide pulsing of stimulator cells

[0440] N- and C-terminally free 15-mer peptide pools (referred to as antigen peptide pools) corresponding to the sequences of closed protein-18.2 or HIV-gag with 11 amino acid overlaps 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. Stimulator cells were incubated with different peptide concentrations in culture medium for 1 h at 37°C for pulsing.

[0441] Vectors for RNA in vitro transcription (IVT)

[0442] All constructs are variants of the previously described pST1-sec-insert-2βgUTR-A(120)-Sap1 plasmid (Holtkamp, S. et al. (2006), Blood 108, 4009-4017). To generate plasmids encoding murine TCR chains, cDNAs encoding murine TCR-alpha, -beta1 and -beta2 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 generate full-length TCR chains (designated pST1-murine TCR alpha beta-2βgUTR-A(120)).

[0443] Cloning of full-length CLDN18.2, CLDN18.2 aa 1-80 and CLDN6 antigens into MHC class I transport signals (MITD) in the previously described pST1 plasmid (Kreiter, S. et al. (2008), J. Immunol. 180, 309-318).

[0444] Generation and transfer of in vitro transcribed (IVT) RNA into cells

[0445] Generation of IVT RNA was performed as previously described (Holtkamp, S. et al. (2006). Blood 108, 4009-4017) and added to cells suspended in X-VIVO 15 medium (Lonza, Basel, Switzerland) in pre-chilled 4 mm gap sterile electroporation cuvettes (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).

[0446] In vivo priming of T cells by intranodal immunization of HLA A2.1 / DR1 mice with IVT RNA

[0447] T cells of A2 / DR1 mice against an antigen of interest were elicited in vivo by repeated intranodal immunization with IVT RNA encoding the antigen (Pajot A. et al. (2004), Eur. J. Immunol. 34, 3060-69) (Kreiter S. et al. (2010), Cancer Research 70, 9031-40). For intranodal immunization, mice were anesthetized with xylazine / ketamine. The inguinal lymph nodes were surgically exposed, and 10 pl RNA (20 pg) diluted in Ringer’s solution and RNase-free water were slowly injected using a disposable 0.3-ml syringe with an ultrafine needle (31G, BD Biosciences), and the wound was closed. After 6 cycles of immunization, mice were sacrificed and splenocytes were isolated.

[0448] Harvesting of splenocytes

[0449] After dissection, the spleen was transferred into a centrifuge tube containing PBS under sterile conditions. The spleen was mechanically disrupted with forceps and a cell strainer (40 pm) was used to obtain the cell suspension. Splenocytes were washed with PBS, centrifuged and resuspended in a hypotonic buffer for lysis of red blood cells. After incubation for 5 min at room temperature, the reaction was stopped by adding 20-30 ml of culture medium or PBS. Splenocytes were centrifuged and washed twice with PBS.

[0450] Single cell sorting of antigen-specific CD8+ T cells after CD137 staining

[0451] For antigen-specific restimulation, 2.5 x 10^6 / well of splenocytes from immunized A2 / DR1 mice were seeded into 24-well plates and pulsed with overlapping peptide pools encoding the antigen of interest or a control antigen. After 24 h incubation, cells were harvested, stained with FITC-conjugated anti-CD3 antibody, PE-conjugated anti-CD4 antibody, PerCP-Cy5.5-conjugated anti-CD8 antibody and Dylight-649-conjugated anti-CD137 antibody. Sorting was performed 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 96-well V-bottom plates containing human CCD-1079Sk cells as feeder cells, centrifuged at 4°C and immediately stored at -80°C.

[0452] RNA extraction from sorted cells, SMART-based cDNA synthesis and unspecific amplification

[0453] RNA was extracted from sorted T cells using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the supplier's instructions. cDNA synthesis was performed using a template-transformation protocol: Mint reverse transcriptase (Evrogen JSC) was combined with a long oligo(dT)-T primer for initiating the first-strand synthesis reaction and a short TS primer (Eurofins Genomic) for introducing the oligo(riboG) sequence to allow the generation of an extended template via the terminal transferase activity of the reverse transcriptase for template transformation (Matz, M. et al. (1999) Nucleic Acids Res. 27, 1558-1560). The first-strand cDNA synthesized according to the manufacturer's instructions was amplified in the presence of 200 μM dNTPs using 5 U Pfu Ultra Hotstart High-Fidelity DNA polymerase (Agilent Technologies) and 0.48 μM TS-PCR primers for 21 cycles (cycle conditions: 95°C for 2 min, 94°C for 30 s, 65°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 6 min). Successful amplification of the TCR gene was controlled using mouse TCR-β constant region-specific primers, and only sequential clone-specific mouse Vα- / Vβ-PCR was performed if a strong band was detected.

[0454] PCR primers designed for TCR amplification

[0455] To design mouse TCR common primers, the ImMunoGeneTics (IMGT) database ( http: / / www.imgt.org All functional mouse TCR-Vβ and TCR-Vα genes listed in [reference needed] and their corresponding leader sequences were aligned using a BioEdit sequence alignment editor (e.g., http: / / www.bio-soft.net). Forward primers of 24-27 bp length with up to 3 degenerate bases, 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 were equipped with a 15 bp 5' extension characterized by rare restriction enzyme sites and Kozak sequences. Reverse primers were designed to anneal to the first exon of the constant region genes; primer mTRACex1_as binds to amino acid sequences 24-31 corresponding to Cα, and mTRBCex1_as binds to amino acid sequences 8-15 corresponding to Cβ1 and Cβ2. Both oligonucleotides were synthesized using 5' phosphate. The primers were pooled in pools of 2-6 forward oligonucleotides with the same annealing temperature.

[0456] PCR amplification and cloning of the V(D)J sequence

[0457] In the presence of 0.6 μΜ mVα- / mVβ-specific oligonucleotide pool, 0.6 μΜ mCα- or mCβ-oligonucleotide, 200 μΜ dNTPs and 5 U Pfu Ultra II Fusion HS DNA polymerase, 6 μl of pre-amplified cDNA from isolated T cells were subjected to 40 PCR cycles (Agilent; cycle conditions: 1 min at 95 °C, 30 s at 94 °C, annealing temperature for 30 s, 30 s at 72 °C, final 72 °C for 3 min elongation time). PCR products were analyzed using the Qiagen capillary electrophoresis system. Samples with bands at 470-550 bp were size fractionated on agarose gels, excised and purified from bands using the gel recovery kit (Qiagen, Hilden, Germany). Sequence analysis was performed to show the sequence of both V(D)J domains and β constant region, as mTRBCex1_as and mTRBCex1_as primers match the TCR constant region genes Cβ1 and Cβ2 in mice, respectively. DNA was digested and cloned into IVT vectors containing the appropriate scaffolds for the complete murine TCR-α / β chains.

[0458] Flow cytometry analysis

[0459] Cell surface expression of transfected TCR genes was analyzed by flow cytometry using a combination of fluorescent dye-conjugated anti-TCR antibodies directed against the appropriate variable region family or constant region of the TCR β chain (Beckman Coulter Inc., Fullerton. USA) together with antibodies against CD3, CD8 or CD4 (BD Biosciences). Cell surface expression of transfected CARs was analyzed using fluorescent dye-conjugated idiotype specific antibodies (Ganymed Pharmaceuticals) recognizing the scFv fragment contained in the respective CAR constructs. Flow cytometry analysis was performed on a FACSCANTO II flow cytometer using FACS Diva software (BD Biosciences).

[0460] Luciferase cytotoxicity assay

[0461] To assess cell-mediated cytotoxicity, an assay based on bioluminescence was used as readout against 51Alternative and optimal choice for Cr release. In contrast to the standard chromium release assay, this assay measures the cytolytic activity of effector cells by calculating the number of luciferase-expressing target cells that are alive after co-incubation. Target cells are stably or transiently transfected with a luciferase gene encoding luciferase from Photinus pyralis (EC 1.13.12.7). Luciferase is an enzyme that catalyzes the oxidation of luciferin. The reaction is ATP-dependent and occurs in two steps:

[0462] Luciferin + ATP + O2 ATP → Luciferinyl adenylate + PPi PP i

[0463] Luciferinyl adenylate + O2 O2 → Oxyluciferin + AMP AMP + light

[0464] Target cells are plated in white 96-well plates (Nunc, Wiesbaden, Germany) at a concentration of 10 4 cells per well and co-cultured with different numbers of TCR-transfected T cells in a final volume of 100 μl. After 3 h, 50 μl of D-luciferin (BD Biosciences) containing the reaction mixture (luciferin (1 μg / μl), HEPES buffer (50 mM, pH), adenylate 5'-triphosphatase (ATPase, 0.4 mU / μl, Sigma-Aldrich, St. Louis, USA)) are added to the cells. By adding ATPase to the reaction mixture, the fluorescence generated from luciferase released from dead cells is attenuated.

[0465] After an incubation time of 4 h in total, the bioluminescence emitted by the living cells is measured using a Tecan Infinite 200 reader (Tecan, Crailsheim, Germany). The cell-killing activity is calculated against the fluorescence obtained after induction of complete cell lysis by addition of 2% Triton-X 100 and correlated to the fluorescence emitted by the target cells alone. The data output is counts per second (CPS), and the percentage of specific lysis is calculated as follows:

[0466] (l-(CPS exp -CPS min ) / (CPS max –CPS min )))*100.

[0467] After incubation of the target cells without effectors, the maximum fluorescence (maximum counts per second, CPS max), and the minimal fluorescence (CPS min ) was assessed after complete solubilization of the targets by treatment with the detergent Triton-X-100.

[0468] ELISPOT (enzyme-linked immunospot assay)

[0469] Microtiter plates (Millipore, Bedford, MA, USA) were coated overnight at room temperature with anti-human IFNy antibody 1-D1 k (Mabtech, Stockholm, Sweden) or at 4°C with anti-mouse IFNy antibody AN18 (Mabtech) and blocked with 2% human serum albumin (CSL Behring, Marburg, Germany) or with mouse culture medium. In a murine background, 5 x 10 5 splenocytes per well were distributed, whereas in a human background, 2-5 x 10 4 antigen-presenting stimulator cells were seeded in triplicates together with 0.3-3 x 10 5 TCR-transfected CD4+ or CD8+ effector cells per well 24 h after electroporation. Plates were incubated overnight (37°C, 5% C02), washed with PBS 0.05% Tween 20, and incubated with 1 pg / ml final concentration of anti-human IFNy biotinylated mAB 7-B6-1 (Mabtech) or anti-mouse IFNy biotinylated mAb R4-6A2 (Mabtech) for 2 h at 37°C. Avidin-conjugated horseradish peroxidase H (Vectastain Elite Kit; Vector Laboratories, Burlingame, USA) was added to the wells for 1 h at room temperature and developed with 3-amino-9-ethylcarbazole (Sigma, Deisenhofen, Germany).

[0470] CFSE (carboxyfluorescein succinimidyl ester) proliferation assay

[0471] CAR RNA-transfected CD8+ T cells were labeled with 0.8 mM CFSE about 20 h after transfection. Labeled T cells were washed and co-cultured with RNA-transfected autologous iDCs (ratio E:T = 10:1). After 4 days of co-culture, cells were harvested and proliferation was analyzed by flow cytometry based on the progressive halving of CFSE fluorescence in daughter cells after cell division.

[0472] Animals

[0473] BALB / c mice were purchased from Javier Labs. Age (8 weeks old) and gender (female) matched animals were used throughout the experiment. Congenic BALB / c-Thyl.1 mice were housed in the animal facility of BioNTech AG, Germany.

[0474] Retroviral gene manipulation and preparation of CAR T cells for adoptive T cell transfer

[0475] Splenocytes of BALB / c-Thyl.1 mice were isolated and pre-activated by 2 pg / mL soluble anti-CD3 (eBioscience) and 1 pg / mL soluble anti-CD28 (Novus Biologicals) in the presence of 5 ng / mL rh IL-7 and 10 ng / mL rh IL-15 (both from Miltenyi). Twenty-four and 48 hours after pre-activation, T cells were transduced (retroviruses (MLV-E)) with a tri-cistronic vector encoding CLDN18.2-CAR-effLuc-GFP using the RetroNectin technology (Takara). Transduced T cells were then expanded for 3 days in the presence of 5 ng / mL rh IL-7 and 10 ng / mL rh IL-15, followed by a ficoll wash with Ficoll-Paque PREMIUM (1.084) and adoptive transfer into mice.

[0476] Mouse experiments

[0477] 5xl06 6 CLDN18.2 CAR transduced BALB / c-Thyl.1 + T cells were intravenously (i.v.) transferred into BALB / c mice. Subsequently, 24 hours after adoptive T cell transfer (ACT), mice were intravenously (i.v.) inoculated with RNA (Lip) at a ratio of F12:RNA of 1.3:2. Whole body bioluminescence imaging was performed.

[0478] In vivo bioluminescence imaging (BLI)

[0479] In vivo bioluminescence imaging was used to assess the expansion of CLDN18.2-CAR-effLuc-GFP transduced T cells using an IVIS Lumina imaging system (Caliper Life Sciences). Briefly, 5 minutes after injection of a D-luciferin aqueous solution (80 mg / kg body weight; Perkin Elmer), the emitted photons were quantified (1 min integration time). The intensity of transmitted light originating from luciferase-expressing cells in the animals was represented as a gray scale image, where black is the least intense bioluminescence signal and white to dark gray is the most intense bioluminescence signal. A gray scale reference image of the mice was obtained under LED low light. The images were overlaid using Living Image 4.0 software.

[0480] Example 2: Isolation of high affinity HLA-A*02 restricted murine TCR specific for claudin 18.2

[0481] We verified the immunogenic potential of CLDN18.2 in A2 / DR1 mice by repeated intranodal immunization with IVT-RNA encoding amino acids 1-80 of CLDN18.2. The human CLDN18 gene has 2 alternative first exons, resulting in 2 protein isoforms that differ in 69 amino acids at the N-terminus (CLDN18.1 and CLDN18.2) Figure 4 A). Since CLDN18.1 is also expressed in normal tissue, especially in the lung, we used only the N-terminal part of CLDN18.2 in order to specifically induce CLDN18.2 specific T cell reactivity. We used splenocytes of these mice for isolation of CLDN18.2 specific T cells and subsequent cloning of the corresponding TCR genes. Splenocytes of immunized mice with successful induction of CLDN18.2 specific T cells were analyzed and their reactivity to predicted HLA-A*02 binding CLDN18.2 peptides was analyzed ex vivo by IFNy-ELISPOT Figure 5 ).

[0482] By RNA immunization, a significant frequency of CLDN18.2 specific T cells could be induced in all 3 mice, while T cell reactivity was focused on 2 CLDN18.2 peptides predicted to bind to HLA-A*0201 (CLDN18.2-A2-5 and CLDN18.2-A2-6).

[0483] For isolation of CLDN18.2 specific T cells, splenocytes of immunized mice were restimulated in vitro and single cells were isolated by flow cytometry based on CD137 upregulation induced by activation Figure 6 ).

[0484] CLDN18.2-specific CD8+ T cells could be retrieved from all 3 immunized A2 / DR1 mice and a total of 6 CLDN18.2-specific TCRs were cloned from single sorted murine T cells.

[0485] The TCRs were subjected to immunological validation assays which showed that all 6 CLDN18.2-TCRs recognized one or both of the 2 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 Figure 7 ).

[0486] Example 3: Generation and in vitro validation of closed protein-18.2 specific CARs

[0487] We produced second generation CARs targeting CLDN18.2 which comprise the signaling part of CD3 zeta and CD28 and the costimulatory part, respectively. The deletion of the lck binding part in the CD28 intracellular structure abrogates IL2 secretion once the CAR is engaged to prevent the induction of regulatory T cells (Kofler D.M. et al., (2011) Molecular Therapy 19(4), 760-767). The modification of the IgGl Fc "spacer region" domain in the extracellular part of the CAR avoids "off-target" activation and unintended initiation of innate immune responses (Hombach A. et al., (2010) Gene Therapy 17, 1206-1213).

[0488] 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 pre-activated and transfected with IVT-RNA encoding for CLDN18.2-CAR or a CLDN6 specific CAR as control. Surface expression of the CARs was confirmed by flow cytometry after staining with a fluorescent dye-conjugated antibody Figure 8 ). Both CARs were well expressed on the surface of CD8+ T cells. CAR transfected T cells were incubated with autologous iDCs transfected with CLDN18.2- or CLDN6-RNA using different effector-target ratios and specific lysis was calculated after 4 hours of co-culture Figure 9 ). Both CLDN18.2-CAR and CLDN6-CAR mediated specific lysis of iDCs expressing CLDN18.2 and CLDN6, respectively. No lysis was observed when iDCs expressed the respective control antigens.

[0489] For analysis, if CLDN18.2-CAR-mediated lysis of target cells expressing CLDN18.2 can be inhibited by the addition of the idiotype-specific antibody, CAR T cells can be pre-incubated with or without an antibody specifically binding to the scFv fragment comprised in the CLDN18.2-CAR prior to the start of co-culture with target cells and analyzed for lysis using a luciferase-based cytotoxicity assay Figure 10 ).

[0490] CLDN18.2-CAR-mediated lysis of target cells expressing CLDN18.2 can be efficiently inhibited by blocking the binding of the CLDN18.2-CAR to its target antigen, even at high E:T ratios of 30:1. No inhibition of CLDN6-CAR-mediated lysis was observed, confirming the selective binding of the antibody to the CLDN18.2-CAR. This experiment confirms on the one hand that CLDN18.2-CAR-mediated lysis is completely dependent on the CLDN18.2 specificity of the CAR and on the other hand that the idiotype-specific antibody used for detection of the CLDN18.2-CAR can in principle also be applied for the inhibition of CLDN18.2-CAR T cells in vivo in case of severe adverse events.

[0491] A fundamental prerequisite for the anti-tumor efficacy of CAR-engineered T cells is their ability to proliferate and persist in the patient’s body. For analysis, if CLDN18.2-CAR T cells efficiently proliferate in response to ectopically expressed CLDN18.2 in iDC, a carboxyfluorescein succinimidyl ester (CFSE)-based in vitro co-culture assay was performed. CD8 + T cells transfected with IVT-RNA encoding for the CLDN18.2-CAR were labeled with CFSE and co-cultured with autologous iDC transfected with IVT-RNA encoding for CLDN18.2 or control antigens CLDN9 or CLDN6. Surface expression of the CAR was analyzed by flow cytometry using a fluorescent dye-coupled anti-idiotype specific antibody Figure 11 A). After 4 days of co-culture, antigen-specific proliferation of CFSE-labeled CAR-transfected CD8 + T cells was analyzed by flow cytometry. In response to CLDN18.2, a CLDN18.2-CAR-mediated proliferation of about 86% of CD8 + T cells was observed, whereas in response to control antigen-transfected iDC (CLDN9, CLDN6) only background proliferation of CAR T cells could be observed Figure 11 B). 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 iDC.

[0492] The efficacy of CLDN18.2-CAR in mediating antigen-specific activation and amplification of CAR-carrying T cells was examined in vivo in a homologous mouse model. To track the in vivo fate of adoptive-transferred CAR-T cells, a tricistronic retroviral vector encoding a luciferase (effLuc) and eGFP reporter gene downstream of the CLDN18.2-CAR sequence separated by the viral T2A sequence was used.

[0493] CLDN18.2-CAR-transduced mouse T cells were transplanted into naïve BALB / c mice. On the day of transfer, CLDN18.2-CAR expression on the transduced T cells was assessed by flow cytometry using a fluorescent dye-conjugated anti-idiotype-specific antibody in combination with eGFP reporter gene expression. CLDN18.2-CAR expression was found in approximately 36% of CD8+ cells. + and about 45% of CD4 + Highly expressed in T cells ( Figure 12 A). The transplanted mice were then treated with IVT-RNA encoding either the CLDN18.2 or control (Ctrl) antigen. Two days after mRNA inoculation, a strong increase in luminescence was observed in the CLDN18.2 RNA compared to mice treated with control RNA, indicating that CLDN18.2-CAR T cells were significantly activated and proliferated in response to the homologous antigen. Figure 12 B). These data clearly demonstrate the function and antigen specificity of CLDN18.2 CAR in T cells in vivo.

[0494] CLDN18.2 - Specific T cell epitope

[0495] A2-1(aa 68-76)

[0496] TLLGLPAML

[0497] A2-2(aa 71-79)

[0498] GLPAMLQAV

[0499] A2-3(14-22)

[0500] SLIGIAGII

[0501] A2-4(17-25)

[0502] GIAGIIAAT

[0503] A2-5(7-15)

[0504] QGLGFVVSL

[0505] A2-6(8-16)

[0506] GLGFVVSLI

[0507] CLDN18.2-specific T cell receptor

[0508] mTCR CD8 - CL18#2

[0509] > alpha V12 D.1 J33 C (MN missing N-terminus; V→G und S→C)

[0510]

[0511]

[0512] > beta V13.3 D1 J1.4 *02 C1

[0513]

[0514] mTCR CD8 - CL18#4

[0515] > alpha V6 D.7 *04 J26 C (S→F)

[0516]

[0517] > beta V2 D2 J2.7 C2 (CASSQEWGGYEQYF)

[0518]

[0519]

[0520] mTCR CD8 - CL18#5

[0521] > alpha V6 D.7 *04 J47 C (N→D und S→F)

[0522]

[0523] > beta V1 D2 J2.7 C2

[0524]

[0525] mTCR CD8 - CL18#8

[0526] > alpha V8 D.2 *02 or V8 N.2 J31 C

[0527]

[0528] > beta V23 D2 J2.7 C2

[0529]

[0530] mTCR CD8 - CL18 #9

[0531] >αV9N.3 J21 C(P→S)

[0532]

[0533] >βV2 D2 J2.7 C2(CASSQDQGGQGQYF)

[0534]

[0535] mTCR CD8 - CL18#12

[0536] >αa V6.3*02 or V6D.3 J26 C(N→T)

[0537]

[0538] >βV2 D2 J2.7 C2(CASSPDWGAEYEQYF)

[0539] SEQUENCE LISTING <110> BioNTech Cell & Gene Therapies GmbH et al. <120> Immune receptors and T cell epitopes specific for Clostridial protein-18.2 <130> 674-152 PCT <150> PCT / EP2015 / 060357 <151> 11 May 2015 <160> 47 <170> SIPO SequenceListing 1.0 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <400> 1 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly lie Ala Gly lie lie Ala Ala Thr Cys Met Asp Gin Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gin Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gin Ala Val Arg 65 70 75 80 Ala Leu Met lie Val Gly lie Val Leu Gly Ala lie Gly Leu Leu Val 85 90 95 Ser lie Phe Ala Leu Lys Cys lie Arg lie Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly lie Met Phe lie Val Ser 115 120 125 Gly Leu Cys Ala lie Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gin Thr Val Gin Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> T cell epitope <400> 2 Thr Leu Leu Gly Leu Pro Ala Met Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> T cell epitope <400> 3 Gly Leu Pro Ala Met Leu Gln Ala Val 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> T cell epitope <400> 4 Ser Leu Ile Gly Ile Ala Gly Ile Ile 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> T cell epitope <400> 5 Gly Ile Ala Gly Ile Ile Ala Ala Thr 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> T cell epitope <400> 6 Gln Gly Leu Gly Phe Val Val Ser Leu 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> T cell epitope <400> 7 Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 <210> 8 <211> 268 <212> PRT <213> Homo sapiens <400> 8 Met Arg Pro Gly Thr Cys Ser Val Leu Val Leu Leu Leu Met Leu Arg 1 5 10 15 Arg Ser Asn Gly Asp Ser Val Thr Gln Thr Glu Gly Leu Val Thr Val 20 25 30 Thr Glu Gly Leu Pro Val Lys Leu Asn Cys Thr Tyr Gln Thr Thr Tyr 35 40 45 Leu Thr Ile Ala Phe Phe Trp Tyr Val Gln Tyr Leu Asn Glu Ala Pro 50 55 60 Gln Val Leu Leu Lys Ser Ser Thr Asp Asn Lys Arg Thr Glu His Gln 65 70 75 80 Gly Phe His Ala Thr Leu His Lys Ser Ser Ser Ser Phe His Leu Gln 85 90 95 Lys Ser Ser Ala Gln Leu Ser Asp Ser Ala Leu Tyr Tyr Cys Ala Leu 100 105 110 Met Asp Ser Asn Tyr Gln Leu Ile Trp Gly Ser Gly Thr Lys Leu Ile 115 120 125 Ile Lys Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys 130 135 140 Asp Pro Arg Ser Gin Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gin He Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe He Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly 180 185 190 Ala He Ala Trp Ser Asn Gin Thr Ser Phe Thr Cys Gin Asp He Phe 195 200 205 Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala 210 215 220 Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gin 225 230 235 240 Asn Leu Ser Val Met Gly Leu Arg He Leu Leu Leu Lys Val Ala Gly 245 250 255 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 9 <211> 301 <212> PRT <213> Homo sapiens <400> 9 Met Gly Ser Arg Leu Phe Phe Val Val Leu He Leu Leu Cys Ala Lys 1 5 10 15 His Met Glu Ala Ala Val Thr Gin Ser Pro Arg Ser Lys Val Ala Val 20 25 30 Thr Gly Gly Lys Val Thr Leu Ser Cys His Gin Thr Asn Asn His Asp 35 40 45 Tyr Met Tyr Trp Tyr Arg Gin Asp Thr Gly His Gly Leu Arg Leu Ile 50 55 60 His Tyr Ser Tyr Val Ala Asp Ser Thr Glu Lys Gly Asp Ile Pro Asp 65 70 75 80 Gly Tyr Lys Ala Ser Arg Pro Ser Gin Glu Asn Phe Ser Leu Ile Leu 85 90 95 Glu Leu Ala Ser Leu Ser Gin Thr Ala Val Tyr Phe Cys Ala Ser Ser 100 105 110 Ile Asn Glu Arg Leu Phe Phe Gly His Gly Thr Lys Leu Ser Val Leu 115 120 125 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 130 135 140 Ser Lys Ala Glu Ile Ala Asn Lys Gin Lys Ala Thr Leu Val Cys Leu 145 150 155 160 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 165 170 175 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gin Ala Tyr Lys 180 185 190 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 195 200 205 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gin Val Gin Phe 210 215 220 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 225 230 235 240 Val Thr Gin Asn He Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 245 250 255 He Thr Ser Ala Ser Tyr Gin Gin Gly Val Leu Ser Ala Thr He Leu 260 265 270 Tyr Gin He Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 275 280 285 Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 290 295 300 <210> 10 <211> 267 <212> PRT <213> Homo sapiens <400> 10 Met Asn Ser Phe Pro Gly Phe Met Thr Val Met Leu Leu He Phe Thr 1 5 10 15 Arg Ala His Gly Asp Ser Val Thr Gin Thr Glu Gly Gin Val Ala Leu 20 25 30 Ser Glu Glu Asp Phe Leu Thr Ile His Cys Asn Tyr Ser Ala Ser Gly 35 40 45 Tyr Pro Ala Leu Phe Trp Tyr Val Gin Tyr Pro Gly Glu Gly Pro Gin 50 55 60 Phe Leu Phe Arg Ala Ser Arg Asp Lys Glu Lys Gly Ser Ser Arg Gly 65 70 75 80 Phe Glu Ala Thr Tyr Asp Lys Gly Thr Thr Ser Phe His Leu Arg Lys 85 90 95 Ala Ser Val Gin Glu Ser Asp Ser Ala Val Tyr Tyr Cys Ala Leu Gly 100 105 110 Asp Tyr Ala Gin Gly Leu Thr Phe Gly Leu Gly Thr Arg Val Ser Val 115 120 125 Phe Pro Tyr Ile Gin Asn Pro Glu Pro Ala Val Tyr Gin Leu Lys Asp 130 135 140 Pro Arg Ser Gin Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser 145 150 155 160 Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp 165 170 175 Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala 180 185 190 Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys 195 200 205 Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr 210 215 220 Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gin Asn 225 230 235 240 Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe 245 250 255 Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 11 <211> 305 <212> PRT <213> Homo sapiens <400> 11 Met Gly Ser Ile Phe Leu Ser Cys Leu Ala Val Cys Leu Leu Val Ala 1 5 10 15 Gly Pro Val Asp Pro Lys Ile Ile Gln Lys Pro Lys Tyr Leu Val Ala 20 25 30 Val Thr Gly Ser Glu Lys Ile Leu Ile Cys Glu Gin Tyr Leu Gly His 35 40 45 Asn Ala Met Tyr Trp Tyr Arg Gin Ser Ala Lys Lys Pro Leu Gin Phe 50 55 60 Met Phe Ser Tyr Ser Tyr Gin Lys Leu Met Asp Asn Gin Thr Ala Ser 65 70 75 80 Ser Arg Phe Gin Pro Gin Ser Ser Lys Lys Asn His Leu Asp Leu Gin 85 90 95 Ile Thr Ala Leu Lys Pro Asp Asp Ser Ala Thr Tyr Phe Cys Ala Ser 100 105 110 Ser Gin Gin Trp Gly Gly Tyr Gin Gin Tyr Phe Gin Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu Gin Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser 130 135 140 Leu Phe Gin Pro Ser Lys Ala Gin Ile Ala Asn Lys Gin Lys Ala Thr 145 150 155 160 Leu Val Gin Leu Ala Arg Gly Phe Phe Pro Gin His Val Gin Leu Ser 165 170 175 Trp Trp Val Asn Gin Lys Gin Val His Ser Gin Val Ser Thr Gin Pro 180 185 190 Gln Gin Tyr Gin Gin Gin Ser Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 195 200 205 Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 210 215 220 Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly 225 230 235 240 Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg 245 250 255 Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser 260 265 270 Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 275 280 285 Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn 290 295 300 Ser 305 <210> 12 <211> 268 <212> PRT <213> Homo sapiens <400> 12 Met Asp Ser Phe Pro Gly Phe Met Thr Val Met Leu Leu Ile Phe Thr 1 5 10 15 Arg Ala His Gly Asp Ser Val Thr Gln Thr Glu Gly Gln Val Ala Leu 20 25 30 Ser Glu Glu Asp Phe Leu Thr Ile His Cys Asn Tyr Ser Ala Ser Gly 35 40 45 Tyr Pro Thr Leu Phe Trp Tyr Val Gln Tyr Pro Gly Glu Gly Pro Gln 50 55 60 Leu Leu Phe Arg Ala Ser Arg Asp Lys Glu Lys Gly Ser Ser Arg Gly 65 70 75 80 Phe Glu Ala Thr Tyr Asp Lys Gly Thr Thr Ser Phe His Leu Arg Lys 85 90 95 Ala Ser Val Gln Glu Ser Asp Ser Ala Val Tyr Tyr Cys Ala Leu Ser 100 105 110 Val Asp Tyr Ala Asn Lys Met Ile Phe Gly Leu Gly Thr Ile Leu Arg 115 120 125 Val Arg Pro His Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys 130 135 140 Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly 180 185 190 Ala lie Ala Trp Ser Asn Gin Thr Ser Phe Thr Cys Gin Asp He Phe 195 200 205 Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala 210 215 220 Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gin 225 230 235 240 Asn Leu Ser Val Met Gly Leu Arg He Leu Leu Leu Lys Val Ala Gly 245 250 255 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 13 <211> 307 <212> PRT <213> Homo sapiens <400> 13 Met Trp Gin Phe Cys He Leu Cys Leu Cys Val Leu Met Ala Ser Val 1 5 10 15 Ala Thr Asp Pro Thr Val Thr Leu Leu Glu Gin Asn Pro Arg Trp Arg 20 25 30 Leu Val Pro Arg Gly Gin Ala Val Asn Leu Arg Cys He Leu Lys Asn 35 40 45 Ser Gin Tyr Pro Trp Met Ser Trp Tyr Gin Gin Asp Leu Gin Lys Gin 50 55 60 Leu Gin Trp Leu Phe Thr Leu Arg Ser Pro Gly Asp Lys Glu Val Lys 65 70 75 80 Ser Leu Pro Gly Ala Asp Tyr Leu Ala Thr Arg Val Thr Asp Thr Glu 85 90 95 Leu Arg Leu Gin Val Ala Asn Met Ser Gin Gly Arg Thr Leu Tyr Cys 100 105 110 Thr Cys Ser Pro Leu Thr Gly Ser Tyr Gin Gin Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Leu Gin Asp Leu Arg Asn Val Thr Pro Pro Lys 130 135 140 Val Ser Leu Phe Gin Pro Ser Lys Ala Glu lie Ala Asn Lys Gin Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Gin 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gin Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser 195 200 205 Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe 210 215 220 Arg Cys Gin Val Gin Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro 225 230 235 240 Glu Gly Ser Pro Lys Pro Val Thr Gin Asn He Ser Ala Glu Ala Trp 245 250 255 Gly Arg Ala Asp Cys Gly He Thr Ser Ala Ser Tyr His Gin Gly Val 260 265 270 Leu Ser Ala Thr He Leu Tyr Glu He Leu Leu Gly Lys Ala Thr Leu 275 280 285 Tyr Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys 290 295 300 Lys Asn Ser 305 <210> 14 <211> 264 <212> PRT <213> Homo sapiens <400> 14 Met Asn Arg Phe Leu Gly He Ser Leu Val Thr Leu Trp Phe Gin Val 1 5 10 15 Ala Trp Ala Lys Ser Gin Trp Gly Glu Glu Asn Leu Gin Ala Leu Ser 20 25 30 He Gin Glu Gly Glu Asp Val Thr Met Asn Cys Ser Tyr Lys Thr Tyr 35 40 45 Thr Thr Val Val Gin Trp Tyr Arg Gin Lys Ser Gly Lys Gly Pro Ala 50 55 60 Gln Leu He Leu He Arg Ser Asn Glu Arg Glu Lys Arg Ser Gly Arg 65 70 75 80 Leu Arg Ala Thr Leu Asp Thr Ser Ser Gin Ser Ser Ser Leu Ser He 85 90 95 Thr Gly Thr Leu Ala Thr Asp Thr Ala Val Tyr Phe Cys Ala Thr Asp 100 105 110 Asn Arg He Phe Phe Gly Asp Gly Thr Gin Leu Val Val Lys Pro Asn 115 120 125 He Gin Asn Pro Glu Pro Ala Val Tyr Gin Leu Lys Asp Pro Arg Ser 130 135 140 Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gin He Asn 145 150 155 160 Val Pro Lys Thr Met Glu Ser Gly Thr Phe He Thr Asp Lys Thr Val 165 170 175 Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala He Ala Trp 180 185 190 Ser Asn Gin Thr Ser Phe Thr Cys Gin Asp He Phe Lys Glu Thr Asn 195 200 205 Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu 210 215 220 Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val 225 230 235 240 Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu 245 250 255 Met Thr Leu Arg Leu Trp Ser Ser 260 <210> 15 <211> 305 <212> PRT <213> Homo sapiens <400> 15 Met Gly Ala Arg Leu Ile Cys Tyr Val Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Ser Phe Asp Ala Ala Val Thr Gln Lys Pro Arg Tyr Leu Ile Lys 20 25 30 Met Lys Gly Gln Glu Ala Glu Met Lys Cys Ile Pro Glu Lys Gly His 35 40 45 Thr Ala Val Phe Trp Tyr Gln Gln Lys Gln Ser Lys Glu Leu Lys Phe 50 55 60 Leu Ile Tyr Phe Gln Asn Gln Gln Pro Leu Asp Gln Ile Asp Met Val 65 70 75 80 Lys Glu Arg Phe Ser Ala Val Cys Pro Ser Ser Ser Leu Cys Ser Leu 85 90 95 Gly Ile Arg Thr Cys Glu Ala Glu Asp Ser Ala Leu Tyr Leu Cys Ser 100 105 110 Ser Ser Gln Ser Gly Gly Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser 130 135 140 Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu 195 200 205 Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 210 215 220 Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly 225 230 235 240 Ser Pro Lys Pro Val Thr Gin Asn He Ser Ala Glu Ala Trp Gly Arg 245 250 255 Ala Asp Cys Gly He Thr Ser Ala Ser Tyr His Gin Gly Val Leu Ser 260 265 270 Ala Thr He Leu Tyr Glu He Leu Leu Gly Lys Ala Thr Leu Tyr Ala 275 280 285 Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn 290 295 300 Ser 305 <210> 16 <211> 268 <212> PRT <213> Homo sapiens <400> 16 Met Leu Leu Ala Leu Leu Ser Val Leu Gly He His Phe Leu Leu Arg 1 5 10 15 Asp Ala Gin Ala Gin Ser Val Thr Gin Pro Asp Ala Arg Val Thr Val 20 25 30 Ser Glu Gly Ala Ser Leu Gin Leu Arg Cys Lys Tyr Ser Tyr Phe Gly 35 40 45 Thr Pro Tyr Leu Phe Trp Tyr Val Gin Tyr Pro Arg Gin Gly Leu Gin 50 55 60 Leu Leu Leu Lys Tyr Tyr Pro Gly Asp Pro Val Val Gin Gly Val Asn 65 70 75 80 Gly Phe Glu Ala Glu Phe Ser Lys Ser Asn Ser Ser Phe His Leu Arg 85 90 95 Lys Ala Ser Val His Trp Ser Asp Trp Ala Val Tyr Phe Cys Ala Val 100 105 110 Ser Lys Tyr Tyr Asn Val Leu Tyr Phe Gly Ser Gly Thr Lys Leu Thr 115 120 125 Val Glu Pro Asn Ile Gin Asn Pro Glu Pro Ala Val Tyr Gin Leu Lys 130 135 140 Asp Pro Arg Ser Gin Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gin Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly 180 185 190 Ala Ile Ala Trp Ser Asn Gin Thr Ser Phe Thr Cys Gin Asp Ile Phe 195 200 205 Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala 210 215 220 Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln 225 230 235 240 Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly 245 250 255 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 17 <211> 305 <212> PRT <​​​​​​​​​​​​​​​​​​​​​​​​​​​Ile Thr Ala Leu Lys Pro Asp Asp Ser Ala Thr Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Asp Gln Gly Gly Gln Gly Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser 130 135 140 Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu 195 200 205 Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 210 215 220 Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly 225 230 235 240 Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg 245 250 255 Ala Asp Cys Gly lie Thr Ser Ala Ser Tyr His Gin Gly Val Leu Ser 260 265 270 Ala Thr lie Leu Tyr Glu lie Leu Leu Gly Lys Ala Thr Leu Tyr Ala 275 280 285 Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn 290 295 300 Ser 305 <210> 18 <211> 267 <212> PRT <213> Homo sapiens <400> 18 Met Asn Thr Ser Pro Ala Leu Val Thr Val Met Leu Phe lie Leu Gly 1 5 10 15 Arg Thr His Gly Asp Ser Val lie Gin Met Gin Gly Gin Val Thr Leu 20 25 30 Ser Glu Asn Asp Phe Leu Phe lie Asn Cys Thr Tyr Ser Thr Thr Gly 35 40 45 Tyr Pro Thr Leu Phe Trp Tyr Val Gin Tyr Ser Gly Glu Gly Pro Gin 50 55 60 Leu Leu Leu Gin Val Thr Thr Ala Asn Asn Lys Gly Ser Ser Arg Gly 65 70 75 80 Phe Glu Ala Thr Tyr Asp Lys Gly Thr Thr Ser Phe His Leu Gin Lys 85 90 95 Thr Ser Val Gin Glu lie Asp Ser Ala Val Tyr Tyr Cys Ala lie Gly 100 105 110 Asn Tyr Ala Gin Gly Leu Thr Phe Gly Leu Gly Thr Arg Val Ser Val 115 120 125 Phe Pro Tyr lie Gin Asn Pro Gin Pro Ala Val Tyr Gin Leu Lys Asp 130 135 140 Pro Arg Ser Gin Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser 145 150 155 160 Gln lie Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe lie Thr Asp 165 170 175 Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala 180 185 190 lie Ala Trp Ser Asn Gin Thr Ser Phe Thr Cys Gin Asp lie Phe Lys 195 200 205 Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr 210 215 220 Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gin Asn 225 230 235 240 Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe 245 250 255 Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 19 <211> 306 <212> PRT <213> Homo sapiens <400> 19 Met Gly Ser Ile Phe Leu Ser Cys Leu Ala Val Cys Leu Leu Val Ala 1 5 10 15 Gly Pro Val Asp Pro Lys Ile Ile Gln Lys Pro Lys Tyr Leu Val Ala 20 25 30 Val Thr Gly Ser Glu Lys Ile Leu Ile Cys Glu Gln Tyr Leu Gly His 35 40 45 Asn Ala Met Tyr Trp Tyr Arg Gln Ser Ala Lys Lys Pro Leu Glu Phe 50 55 60 Met Phe Ser Tyr Ser Tyr Gln Lys Leu Met Asp Asn Gln Thr Ala Ser 65 70 75 80 Ser Arg Phe Gln Pro Gln Ser Ser Lys Lys Asn His Leu Asp Leu Gln 85 90 95 Ile Thr Ala Leu Lys Pro Asp Asp Ser Ala Thr Tyr Phe Cys Ala Ser 100 105 110 Ser Pro Asp Trp Gly Ala Glu Tyr Glu Gin Tyr Phe Gly Pro Gly Thr 115 120 125 Arg Leu Thr Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val 130 135 140 Ser Leu Phe Glu Pro Ser Lys Ala Glu lie Ala Asn Lys Gin Lys Ala 145 150 155 160 Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu 165 170 175 Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp 180 185 190 Pro Gin Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg 195 200 205 Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg 210 215 220 Cys Gin Val Gin Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu 225 230 235 240 Gly Ser Pro Lys Pro Val Thr Gin Asn lie Ser Ala Glu Ala Trp Gly 245 250 255 Arg Ala Asp Cys Gly lie Thr Ser Ala Ser Tyr His Gin Gly Val Leu 260 265 270 Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr 275 280 285 Ala Val Leu Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys 290 295 300 Asn Ser 305 <210> 20 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 20 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 21 <211> 118 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Antibody fragment <400> 21 Gln Ile Gln Leu Val Gin Ser Gly Pro Gin Leu Lys Lys Pro Gly Gin 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Gin Pro Thr Tyr Ala Gin Gin Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Gin Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gin Ile Asn Asn Leu Lys Asn Gin Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 22 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 22 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 23 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 23 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 24 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> antibody fragment <400> 24 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 25 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> antibody fragment <400> 25 Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Phe 20 25 30 Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp 35 40 45 Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr Gly Glu Lys 50 55 60 Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala 65 70 75 80 Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr 85 90 95 Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 26 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 26 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 27 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 27 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gin Gin Lys Pro Gly Thr Ser Pro Lys Leu Trp lie Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Arg Ser Ser Tyr Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 100 105 <210> 28 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 28 Asp lie Val Met Thr Gin Ser Gin Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser lie Thr Cys Lys Ala Ser Gin Asn Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Ala Leu lie 35 40 45 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 29 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 29 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 30 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 30 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 31 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 31 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 32 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 32 Asp Ile Val Met Ser Gin Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gin Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gin 85 90 95 Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 33 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 33 Asp Ile Val Met Ser Gin Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gin Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Gin Asp Leu Ala Val Tyr Tyr Cys Gin Gin 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Gin Leu 100 105 110 Lys <210> 34 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Antibody fragment <400> 34 Asn Ile Val Met Thr Gin Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 35 <211> 251 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> mouseIgG Vh‑Vl (κ) <400> 35 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp lie Asn Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp lie 35 40 45 Gly Asn lie Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gin Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gin Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp lie Val Met Thr Gin Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gin 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gin Lys Asn Tyr Leu Thr Trp Tyr Gin 165 170 175 Gln Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Ser Asp Pro Ala 245 250 <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 36 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 37 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> signal peptide <400> 37 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser <210> 38 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> Human IgGl Fc fragment <400> 38 Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140 Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 39 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Human CD28 (TM + ic) <400> 39 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gin Ala Tyr Ala Ala Ala Arg Asp Phe Ala 50 55 60 Ala Tyr Arg Ser 65 <210> 40 <211> 113 <212> PRT <213> Artificial Sequence (Artificial Sequence) <220> <223> Human CD3-zeta (ic) <400> 40 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gin Gin 1 5 10 15 Gly Gin Asn Gin Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 20 25 30 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 35 40 45 Lys Pro Arg Arg Lys Asn Pro Gin Glu Gly Leu Tyr Asn Glu Leu Gin 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu lie Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gin Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gin Ala Leu Pro Pro 100 105 110 Arg <210> 41 <211> 686 <212> PRT <213> Artificial Sequence <220> <223> Artificial T cell receptor <400> 41 Met Asp Trp He Trp Arg He Leu Phe Leu Val Gly Ala Ala Thr Gly 1 5 10 15 Ala His Ser Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Val Arg 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Trp He Asn Trp Val Lys Gin Arg Pro Gly Gin Gly Leu 50 55 60 Glu Trp He Gly Asn He Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 65 70 75 80 Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gin Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly 115 120 125 Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys 165 170 175 Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr 180 185 190 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp 195 200 205 Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 210 215 220 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp 225 230 235 240 Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe 245 250 255 Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ser Asp Pro Ala Glu Pro 260 265 270 Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro 275 280 285 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 290 295 300 Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 305 310 315 320 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 325 330 335 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 340 345 350 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 355 360 365 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 370 375 380 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 385 390 395 400 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 405 410 415 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 420 425 430 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 435 440 445 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 450 455 460 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 465 470 475 480 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 485 490 495 Leu Ser Pro Gly Lys Lys Asp Pro Lys Phe Trp Val Leu Val Val Val 500 505 510 Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile 515 520 525 Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr 530 535 540 Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln 545 550 555 560 Ala Tyr Ala Ala Ala Arg Asp Phe Ala Ala Tyr Arg Ser Leu Arg Val 565 570 575 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 580 585 590 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 595 600 605 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 610 615 620 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 625 630 635 640 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 645 650 655 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 660 665 670 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 675 680 685 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 42 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 43 Ile Tyr Pro Ser Asp Ser Tyr Thr 1 5 <210> 44 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 44 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr 1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 45 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr 1 5 10 <210> 46 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 46 Trp Ala Ser 1 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR sequence <400> 47 Gln Asn Asp Tyr Ser Tyr Pro Phe Thr 1 5

Claims

1. A chimeric antigen receptor (CAR) comprising a binding domain, a spacer region, a transmembrane domain, a costimulatory domain, and a signal transduction domain of closure protein-18.2 (CLDN18.2), wherein the binding domain, spacer region, costimulatory domain, and signal transduction domain are connected in order from the N-terminus to the C-terminus. The binding domain of closure protein-18.2 (CLDN18.2) includes: (a) An antibody heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 23 or a VH comprising CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NO: 42, 43, and 44; and (b) An antibody light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 30 or a VL comprising CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 consist of the amino acid sequences shown in SEQ ID NO: 45, 46, and 47. The VH and VL are arranged in a single chain Fv. The spacer subregion contains the amino acid sequence shown in SEQ ID NO:

38. The co-stimulatory domain mentioned above is the co-stimulatory domain of CD28, wherein the lck-binding portion of the intracellular domain of CD28 is missing, and The signal conduction structure domain mentioned therein is the CD3-ζ signal conduction structure domain.

2. The chimeric antigen receptor of claim 1, wherein the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

40.

3. The chimeric antigen receptor of claim 1, comprising the amino acid sequence shown in SEQ ID NO:

39.

4. The chimeric antigen receptor of claim 1, wherein it comprises a signal peptide.

5. The chimeric antigen receptor of claim 4, comprising the amino acid sequence shown in SEQ ID NO:

37.

6. The chimeric antigen receptor of claim 1, wherein the VH and VL are linked via a peptide linker comprising the amino acid sequence (GGGGS)3.

7. A nucleic acid comprising a nucleic acid encoding a chimeric antigen receptor as claimed in any one of claims 1-6, wherein the nucleic acid is mRNA or self-replicating RNA.

8. A vector comprising the nucleic acid of claim 7.

9. The vector as described in claim 8, wherein it is a viral vector or a virus-based vector.

10. The vector of claim 9, wherein the virus-based vector is a gamma-retrovirus, a lentiviral vector, or an adenovirus vector.

11. A cell comprising the nucleic acid of claim 7 or expressing the chimeric antigen receptor of any one of claims 1-6.

12. Use of the chimeric antigen receptor of any one of claims 1 to 6, the nucleic acid of claim 7, the vector of any one of claims 8 to 10, or the cell of claim 11 in the preparation of a medicament for the treatment or prevention of gastric cancer, esophageal cancer, or pancreatic cancer in an individual, wherein the cancer comprises cancer cells expressing CLDN18.2.

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