Combination of PRAME-specific T cell receptors with chimeric costimulatory receptors

Abstract

The present invention relates to a combination of a T cell receptor (TCR) specific for the PRAME peptide SLLQHLIGL and a chimeric co-stimulatory receptor comprising an extracellular domain derived from PD-1 (CD279) and an intracellular domain derived from 4-1BB (CD137). In particular, the present invention relates to a cell comprising said TCR and the chimeric co-stimulatory protein. Furthermore, the present invention relates to nucleic acids encoding the TCR and the co-stimulatory receptor, corresponding vectors and corresponding nucleic acid compositions. Furthermore, the present invention relates to pharmaceutical compositions accordingly. Thus, the present invention also relates to cells and nucleic acid constructs for use as medicines, in particular TCRs for use in the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to a combination of a T cell receptor (TCR) specific for the PRAME peptide SLLQHLIGL and a chimeric co-stimulatory receptor comprising an extracellular domain derived from PD-1 (CD279) and an intracellular domain derived from 4-1BB (CD137). In particular, the present invention relates to a cell comprising said TCR and the chimeric co-stimulatory protein. Furthermore, the present invention relates to nucleic acids encoding the TCR and the co-stimulatory receptor, corresponding vectors and corresponding nucleic acid compositions. Furthermore, the present invention relates to pharmaceutical compositions accordingly. Thus, the present invention also relates to cells and nucleic acid constructs for use as medicines, in particular TCRs for use in the treatment of cancer. [Background technology]

[0002] PRAME is a tumor-associated antigen expressed in various tumors, preferably melanoma. In addition, PRAME has been described as an independent biomarker for metastasis, such as uveal melanoma (Fiedl et al., Clin Cancer Res 2016 March; 22(5): 1234-1242), and as a prognostic marker for DLBCL (Mitsuhashi et al., Hematology 2014, 1 / 2014). PRAME is not expressed in normal tissues, except in the testis. This expression pattern is similar to that of other cancer-testis (CT) antigens, such as MAGE, BAGE, and GAGE. However, unlike these other CT antigens, this gene is also expressed in acute leukemia. The encoded protein acts as a repressor of the retinoic acid receptor, and this function is thought to favor the growth of cancer cells. Alternative splicing generates multiple transcript variants. Overexpression of PRAME in triple-negative breast cancer has also been found to promote cancer cell motility by inducing epithelial-mesenchymal transition (Al-Khadairi et al., Journal of Translational Medicine 2019; 17: 9). Deletion of PRAME has been reported in chronic lymphocytic leukemia, but this has no functional relevance since the gene is not expressed in B cells, and the deletion is the result of physiological immunoglobulin light chain rearrangement. Based on the described characteristics of the CT antigen PRAME, it is a suitable target for the treatment of various types of cancer by using TCR-directed cell-based immunotherapy. To do so, a TCR with high specificity for the antigen and whose cellular products can exert the effector functions required for tumor clearance, including cytokine release, cytotoxicity and proliferation, is required.

[0003] The success of immunotherapy using TCR-modified T cells depends on the choice of target antigen as well as the selection of TCR with high antigen specificity and sensitivity. An additional challenge, especially in the treatment of solid tumors, is that the immunosuppressive tumor microenvironment (TME) adversely affects the efficacy, compatibility and persistence of TCR-modified T cells. In addition to the lack of inhibitory cytokines and essential metabolic factors, T cells face the inhibitory checkpoint PD-1 / PD-L1 axis in the TME, leading to reduced infiltration of T cells and their exhaustion. As a result, new strategies are needed to equip TCR-modified T cells with traits that overcome the inhibitory immunosuppressive tumor microenvironment. More specifically, TCR-modified T cells targeting PRAME with high specificity and enhanced proliferation, cytokine release and cytotoxicity are desired. Summary of the Invention

[0004] To overcome these needs, the present invention provides a combination of a high avidity TCR and a chimeric co-stimulatory receptor of the present invention that allows the generation of highly specific T cells targeting PRAME with enhanced cytokine release, proliferation and cytotoxicity.

[0005] Thus, one object of the present invention is to (A) - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 A PRAME-specific T cell receptor (TCR) comprising: (B) - An extracellular domain containing polypeptide derived from PD-1 - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB Chimeric costimulatory receptor comprising The object of the present invention is to provide a cell comprising the

[0006] The PRAME-specific TCR used is capable of binding to the PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1) or a part thereof, or its HLA-A2 binding form. This results in high functional avidity and favorable tumor cell recognition and killing properties. In particular, the TCR of the present invention has a higher functional avidity than the TCRs disclosed in the prior art, best recognizes the tested tumor cell lines, and more efficiently lyses PRAME-positive tumor cells. The co-stimulatory receptor reverses the checkpoint inhibition axis PD-1 / PD-L1, improving the function of T cells, especially in suppressive TME. Thus, the combination of the TCR of the present invention with a chimeric co-stimulatory receptor allows for improved targeting of PRAME with high specificity and enhanced proliferation, cytokine release and cytotoxicity.

[0007] In some embodiments, the PRAME-specific TCR is an HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:04 or HLA-A * 02:09 binding form. Binding to the PRAME epitope SLLQHLIGL or a portion thereof, or its HLA-A2 binding form, induces IFN-γ secretion by TCR-transduced or transfected cells.

[0008] In some embodiments, the TCR comprises a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO: 8 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO: 9. In more specific embodiments, the TCR comprises a variable TCR alpha region having an amino acid sequence of SEQ ID NO: 8 and a variable TCR beta region having an amino acid sequence of SEQ ID NO: 9. The TCR comprises a constant TCR alpha region having an amino acid sequence identical or at least 80% identical to SEQ ID NO: 10 and a constant TCR beta region having an amino acid sequence identical or at least 80% identical to SEQ ID NO: 11.

[0009] The chimeric costimulatory receptor may comprise a transmembrane domain derived from PD-1. In a specific embodiment, the sequence of the chimeric costimulatory receptor may comprise the sequence of SEQ ID NO:26.

[0010] Thus, a further aspect is - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 A nucleic acid encoding a PRAME-specific T cell receptor (TCR) comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB A nucleic acid encoding a chimeric co-stimulatory receptor comprising The present invention relates to a composition comprising:

[0011] Further, one aspect is - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 A nucleic acid encoding a PRAME-specific T cell receptor (TCR) comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB A nucleic acid encoding a chimeric co-stimulatory receptor comprising The present invention relates to a nucleic acid comprising:

[0012] A further embodiment refers to a vector comprising a nucleic acid comprising a PRAME-specific TCR and a chimeric co-stimulatory receptor sequence. Cells comprising the nucleic acid composition and / or the vector are also included.

[0013] Typically, the cells are peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). In a specific embodiment, the cells are T cells.

[0014] Further aspects refer to pharmaceutical compositions comprising the cells, compositions, nucleic acids and vectors defined herein. Further aspects refer to the cells, compositions, nucleic acids and vectors defined herein for the treatment of cancer. [Brief description of the drawings]

[0015] [Figure 1] Co-expression of PD1-41BB does not change TCR expression levels. CD8+ T cells were isolated from healthy donors and activated with CD3 / CD28 antibodies in the presence of IL-7 and IL-5. Activated cells were transduced with retroviral particles containing the sequences of T23.8-2.1-027-004 (=TCR) or the combination of T23.8-2.1-027-004 and PD1-41BB (=TCR_PD1-41BB). Non-transduced (=UT) CD8+ T cells prepared in the same way were used as control. Transgene transduction efficiency and expression levels were determined by antibody staining of TCR-β chain (TRBV09) and PD-1 followed by flow cytometric analysis. [Diagram 2]The functional avidity of TCR transgenic T cells is not altered by co-expression of PD1-41BB. The functional avidity of TCR transgenic T cell populations is measured as IFN-γ release in co-culture with PD-L1 transgenic T2 cells loaded with increasing amounts of SLLQHLIGL (SLL)-peptide (10-5M to 10-10M). Half-maximal IFN-γ release is a measure of the functional avidity of TCR transgenic effector T cells. The left graph shows absolute values ​​of IFN-γ concentrations determined by ELISA after 20 hours of co-culture, while the right graph shows nonlinear regression of relative values. Co-expression of PD1-41BB increases the levels of IFN-γ released by TCR transgenic T cells in response to PD-L1 positive target cells, and this co-expression does not alter the functional avidity of the T cells. [Diagram 3] Recognition of HLA-A*02 subtypes is not altered by co-expression of PD1-41BB. In vitro co-culture of TCR-transduced T cells with selected HLA-A*02 suballele-positive lymphoblastoid cell lines (LCL; EBV-transformed B cells) at an E:T ratio of 1:1 (20.000 T cells / well). IFN-γ concentrations were determined by ELISA 20 hours after co-culture with LCL supplemented with 10-5M SLL-peptides. TCR-transgenic T cells transduced with and without PD1-41BB recognized SLL-peptides presented by MHC molecules encoded by the HLA-A*02 suballeles A*02:02, A*02:04 and A*02:09 at similar levels compared to A*02:01. [Figure 4]Successful avoidance of the risk of potential peptide off-target toxicity. To reduce the risk of potential off-target toxicity, 191 partially homologous peptides differing in up to four amino acids compared to the SLL-peptides (mismatch (MM) peptides) were selected using Expitope 2.0®. In prescreening co-cultures using PD-L1 transgenic T2 cells loaded with 10-6M MM peptides or SLL-peptides, 33 MM peptides were identified that were recognized by TCR-transduced T cells (data not shown). These 33 MM peptides were investigated for their potential to induce IFN-γ release by TCR transgenic effector T cells when the epitopes (peptides) were endogenously processed by the proteasome of the PRAME-negative target cell line SNB-19. In vitro transcribed (ivt) RNAs encoding up to five MM peptides were electroporated into SNB-19 cells. The MM peptides that induced the most IFN-γ release in TCR-transgenic T cells in prescreening cocultures (MM01, MM26, MM66) were tested individually as "midigene" constructs (~400 bp). All other MM peptides were tested as minigene constructs (~90 bp per peptide) encoding five MM peptides. A midigene construct encoding an SLL-peptide was used as a positive control. All RNA constructs contained an epitope recognized by the positive control TCR. IFN-γ concentrations were determined 20 hours after coculture of transfected SNB-19 cells with TCR-transgenic effector T cells. IFN-γ levels detected indicate that intracellularly processed MM peptides were not recognized. Thus, all MM peptides are risk-avoidant and do not result in potential off-target toxicity. Furthermore, coexpression of PD1-41BB did not alter the recognition pattern of MM peptides seen with the TCR alone. [Diagram 5]No off-target toxicity was identified using an LCL library covering frequent HLA. To evaluate potential off-target toxicity, TCR-transduced T cells transduced with and without PD1-41BB were co-cultured with a library of 36 lymphoblastoid cell line (LCL) populations covering the most frequent HLA-A, HLA-B and HLA-C alleles in Caucasian populations. These LCLs express a wide variety of endogenously expressed peptides and help identify potential cross-reactivity by recognition of endogenous peptides presented on matched HLA-A2 molecules or other most frequent HLA molecules. IFN-γ concentrations were determined by ELISA after 20 hours of co-culture with LCLs. HLA-A*02:01 positive LCLs loaded with SLL-peptides served as a positive control. TCR-transgenic T cells secreted only very low levels of IFN-γ when co-cultured with LCL number 5, and low levels of PRAME expression could be confirmed by qPCR. No other LCLs were recognized by effector T cells expressing the transgenic TCR or the transgenic TCR in combination with PD1-41BB, therefore no off-target toxicity could be identified in this safety model. [Figure 6]No off-target toxicity was identified using a panel of normal cells. Possible off-target recognition of important healthy tissues was analyzed by co-culture with normal cells originating from various tissues. As a positive control, normal cells were loaded with SLL-peptides. IFN-γ concentrations in the co-culture supernatants were determined by ELISA after 20 h of co-culture. No off-target recognition of healthy cells was observed. Only PRAME-positive mature dendritic cells (DCs) induced IFN-γ release in TCR-transgenic T cells above background of non-transduced T cells, whereas precursors of mature DCs (monocytes and immature DCs) did not result in IFN-γ release by T cells. The addition of PD1-41BB did not alter the safety profile of T cells expressing PRAME-specific TCRs. Human renal epithelial cells (HREpC), human renal cortical epithelial cells (HRCEpC), renal proximal tubular epithelial cells (RPTEC), normal human lung fibroblasts (NHLF), human osteoblasts (HOB), monocytes (Mono), immature DCs (iDCs), mature DCs (mDCs), iCell cardiomyocytes 2 (iCardio). [Figure 7-1] PD1-41BB enhances the specific release of IFN-γ in response to tumor cells expressing PD-L1. (A) PRAME-mRNA expression levels in tumor cell lines were determined by real-time quantitative PCR and normalized to the housekeeping gene GUSB. (B) TCR transgenic T cells with and without PD1-41BB were co-cultured with HLA-A*02:01-positive tumor cell lines from various indications expressing different levels of PRAME and PD-L1. To allow stable expression of PD-L1, some tumor cells were transduced with PD-L1 (TD). Furthermore, antibody staining and subsequent flow cytometry analysis determined that some cell lines showed inducible expression of PD-L1 upon IFN-γ treatment (ind), whereas others already showed some level of endogenous PD-L1 expression (end) without IFN-γ treatment. Non-transduced T cells were used as controls. IFN-γ concentrations were determined by ELISA 20 hours after coculture. Co-expression of PD1-41BB enhanced IFN-γ release in response to PD-L1-positive tumor cells. [Figure 7-2] PD1-41BB enhances the specific release of IFN-γ in response to tumor cells expressing PD-L1. (A) PRAME-mRNA expression levels in tumor cell lines were determined by real-time quantitative PCR and normalized to the housekeeping gene GUSB. (B) TCR transgenic T cells with and without PD1-41BB were co-cultured with HLA-A*02:01-positive tumor cell lines from various indications expressing different levels of PRAME and PD-L1. To allow stable expression of PD-L1, some tumor cells were transduced with PD-L1 (TD). Furthermore, antibody staining and subsequent flow cytometry analysis determined that some cell lines showed inducible expression of PD-L1 upon IFN-γ treatment (ind), whereas others already showed some level of endogenous PD-L1 expression (end) without IFN-γ treatment. Non-transduced T cells were used as controls. IFN-γ concentrations were determined by ELISA 20 hours after coculture. Co-expression of PD1-41BB enhanced IFN-γ release in response to PD-L1-positive tumor cells. [Figure 8] PD1-41BB enhances specific cytotoxic responses against three-dimensional (3D) tumor cell spheroids. TCR transgenic T cells with and without PD1-41BB were co-cultured with three-dimensional (3D) tumor cell spheroids derived from HLA-A*02:01 positive tumor cell lines expressing different levels of PRAME and PD-L1. Cytotoxicity against tumor spheroids was determined by disappearance of red fluorescence over 20 days, with images recorded every 4 hours using an Incucyte Zoom® or S3® device. Fresh tumor cell spheroids were transferred to co-culture plates on days 3, 7, 10, 13, and 16. Expression of PD1-41BB has beneficial effects on the effector function and fitness of T cells in the challenging environment of repeated exposure to tumor cells. During multiple loading of tumor cell spheroids, PD1-41BB-expressing effector T cells can better control tumor cell growth compared with effector T cells expressing only the transgenic TCR. [Figure 9] PD1-41BB increases proliferation of TCR transgenic T cells in response to tumor cells expressing PD-L1. TCR transgenic T cells with and without PD1-41BB were co-cultured with HLA-A*02:01 positive tumor cell lines expressing different levels of PRAME and PD-L1 at an effector-to-target ratio of 1:1. Untransduced T cells were used as controls. After 7 days, X-fold proliferation of T cells in the co-culture was calculated from the total cell number. Co-expression of PD1-41BB enhanced proliferation and / or survival in response to PD-L1 positive tumor cells. [Figure 10] T cells co-expressing PD1-41BB show potent antitumor reactivity in vivo. 5×106 PD-L1 transgenic MelA375 tumor cells were subcutaneously injected into 18 immunodeficient (NOD / Shi-scid / IL-2Rγnull) mice. One week later, the mice were distributed into three treatment groups, each containing six mice. Mice were injected with 10×106 TCR-positive cells (total number of cells: 16×106) with (TCR_PD1-41BB) or without (TCR)PD1-41BB or an equal amount of untransduced T cells (UT). Tumor volumes were measured two to three times a week. Effector T cells expressing PD1-41BB were able to control tumor cell growth in vivo, whereas effector T cells expressing only the transgenic TCR had little effect compared to untransduced T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention Before the present invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0017] As illustratively described below, the present invention can be suitably practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0018] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is limited not thereto but only by the claims.

[0019] When the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising of". If hereinafter a group is defined as comprising at least a certain number of embodiments, this is also understood to disclose a group that preferably consists only of these embodiments.

[0020] For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". If, below, for example, an antibody is defined as obtainable from a specific source, this is also understood to disclose the antibody obtained from this source.

[0021] When an indefinite or definite article is used with reference to a singular noun, such as "a", "an" or "the", this includes the plural of that noun unless specifically stated otherwise. In the context of the present invention, the term "about" or "approximately" describes an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the characteristic in question. The term typically indicates a deviation from the indicated numerical value of ±10%, preferably ±5%.

[0022] Technical terms are used with their ordinary meaning or meaning to one of ordinary skill in the art. Where a specific meaning is conveyed by a particular term, the definition of the term is given below in the context in which the term is used.

[0023] TCR Background TCR is composed of two distinct, separate protein chains, the TCR alpha (α) chain and the TCR beta (β) chain. The TCR α chain contains a variable (V), joining (J) and constant (C) region. The TCR β chain contains a variable (V), diversity (D), joining (J) and constant (C) region. The rearranged V(D)J regions of both the TCR α and TCR β chains contain hypervariable regions (CDRs, complementarity determining regions), among which the CDR3 region determines the specific epitope recognition. In the C-terminal region, both the TCR α and TCR β chains contain a hydrophobic transmembrane domain and terminate in a short cytoplasmic tail.

[0024] Typically, TCRs are heterodimers of one α-chain and one β-chain that can bind to peptide-presenting MHC molecules.

[0025] The term "variable TCR alpha region" or "TCR alpha variable chain" or "variable domain" in the context of the present invention refers to the TCR alpha chain variable region. The term "variable TCR beta region" or "TCR beta variable chain" in the context of the present invention refers to the TCR beta chain variable region.

[0026] TCR loci and genes are named using the International Immunogenetics and Genomics (IMGT) TCR nomenclature system (IMGT database, www.IMGT.org; Giudicelli, V., et al., IMGT / LIGM-DB, the IMGT® comprehensive database of immunoglobulin and T cell receptor nucleotide sequences, Nucl. Acids Res., 34, D781-D784 (2006). PMID: 16381979; T cell Receptor Factsbook, LeFranc and LeFranc, Academic Press ISBN 0-12- 441352-8).

[0027] target Advantageously, the TCR provided in the present invention in combination with a chimeric co-stimulatory receptor is capable of binding a peptide derived from (human) PRAME (SEQ ID NO: 1). Said TCR is therefore specific for the PRAME peptide as shown in SEQ ID NO: 1, also called PRAME-SLL. In the context of the present invention, the term "specific for" means that the TCR specifically binds to a target. PRAME (Preferentially Expressed Antigen in Melanoma, Uniprot Accession No. P78395), also called MAPE (Melanoma Antigen Preferentially Expressed in Tumors) and OIP4 (OPA-Interacting Protein 4), has been reported to be a cancer-testis antigen (CTA) of unknown function. PRAME is a protein-coding gene associated with melanoma and leukemia, and chronic myeloma. Gene Ontology (GO) annotations associated with this gene include retinoic acid receptor binding. The PRAME protein functions as a transcriptional repressor and inhibits retinoic acid signaling through the retinoic acid receptors RARA, RARB, and RARG, preventing retinoic acid-induced arrest of cell proliferation, differentiation, and apoptosis.

[0028] In particular, the present invention provides a combination of a chimeric co-stimulatory receptor and a TCR (see Table 1) capable of binding to a peptide contained within the PRAME amino acid sequence shown in SEQ ID NO: 1. The term "capable of binding" means that the TCR specifically binds to the peptide. The term "specifically binds" generally indicates that the TCR is more likely to bind to an intended antigen target through its antigen binding site than to random, unrelated, non-target antigens. In particular, the term "specifically binds" indicates that the binding specificity of the TCR is at least about 5-fold, preferably 10-fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more greater for its antigen target than for non-target antigens. The PRAME peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is also referred to herein as an "antigenic target" or "SLL-peptide". Thus, the PRAME peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is or comprises the target epitope of the TCR of the present invention.

[0029] The term "epitope" generally refers to a site on an antigen, typically a (poly)peptide, that is recognized by a binding domain. The term "binding domain" in its broadest sense refers to an "antigen binding site", i.e., characterized by a domain of a molecule that binds to / interacts with a specific epitope on an antigen target. An antigenic target may contain a single epitope, but typically contains at least two epitopes, and may contain any number of epitopes depending on the size, conformation, and type of antigen. The term "epitope" generally encompasses linear and conformational epitopes. A linear epitope is a continuous epitope made up of a primary sequence of amino acids, typically containing at least two or more amino acids. A conformational epitope is formed by non-contiguous amino acids juxtaposed by folding of a target antigen, particularly a target (poly)peptide.

[0030] The inventors have found that the minimal amino acid sequence recognized by the TCR of the invention corresponds to the amino acid sequence of PRAME (SEQ ID NO: 1). In particular, the TCR of the invention has been shown to (specifically) recognize an amino acid sequence comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), or the HLA-A2 binding form shown in the appended examples. This selective recognition can be obtained by a recognition motif of the TCR, which shows only some fixed positions. The amino acids LLQ and especially HLI of the sequence SLLQHLIGL (SEQ ID NO: 1) are part of this recognition motif.

[0031] One object of the present invention is to (A) - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 PRAME-specific T cell receptor (TCR), including (B) - An extracellular domain containing polypeptide derived from PD-1 - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB Chimeric costimulatory receptor comprising The object of the present invention is to provide a cell comprising the

[0032] TCR-specific sequences Thus, the TCR used in the combination of the present invention may - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 Includes.

[0033] In some embodiments, the TCR comprises a variable TCR alpha region having an amino acid sequence that is at least 80% identical to SEQ ID NO:8 and a variable TCR beta region having an amino acid sequence that is at least 80% identical to SEQ ID NO:9.

[0034] "At least 80% identical," and in particular "having an amino acid sequence that is at least 80% identical," as used herein, includes that the amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the indicated amino acid sequence.

[0035] Determination of percent identity between multiple sequences is preferably accomplished using the AlignX application of the Vector NTI Advance™ 10 program (Invitrogen Corporation, Carlsbad Calif., USA), which utilizes a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance TM 10 DNA and protein sequence analysis software. User's Manual, 2004, pp.389-662). Percent identity determination is performed using standard parameters of the AlignX application.

[0036] In a specific embodiment, the TCR comprises a variable TCR alpha region having the amino acid sequence of SEQ ID NO:8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:9.

[0037] As can be seen from the Examples, the TCR according to the present invention is specific for PRAME, particularly the PRAME epitope SLLQHLIGL (SEQ ID NO: 1), and shows very low cross-reactivity with other epitopes or antigens.

[0038] In a specific embodiment, the TCR described herein comprises a constant TCR alpha region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 and a constant TCR beta region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 11.

[0039] Thus, more particularly, in some embodiments, the TCR may comprise a variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:8, a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:9, a constant TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:10 and a constant TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:11.

[0040] In an even more specific embodiment, the TCR may comprise a variable TCR alpha region having the amino acid sequence of SEQ ID NO:8, a variable TCR beta region having the amino acid sequence of SEQ ID NO:9, a constant TCR alpha region having the amino acid sequence of SEQ ID NO:10 and a constant TCR beta region having the amino acid sequence of SEQ ID NO:11. Thus, in a specific embodiment, the TCR may comprise a TCR alpha chain having an amino acid sequence identical to or at least 80% identical to SEQ ID NO:24, and a TCR beta chain having an amino acid sequence identical to or at least 80% identical to SEQ ID NO:22.

[0041] Modifications In some embodiments, the amino acid sequence of the TCR and / or chimeric costimulatory receptor may contain one or more non-phenotype-affecting substitutions.

[0042] "Substitutions that do not affect phenotype" are also referred to as "conservative amino acid substitutions". The concept of "conservative amino acid substitutions" is understood by those skilled in the art, and preferably means that codons encoding positively charged residues (H, K, and R) are replaced with codons encoding positively charged residues, codons encoding negatively charged residues (D and E) are replaced with codons encoding negatively charged residues, codons encoding polar neutral residues (C, G, N, Q, S, T, and Y) are replaced with codons encoding polar neutral residues, and codons encoding non-polar neutral residues (A, F, I, L, M, P, V, and W) are replaced with codons encoding non-polar neutral residues. These variations may occur spontaneously, may be introduced by random mutagenesis, or may be introduced by directed mutagenesis. These changes may be made without destroying the essential properties of these polypeptides. One of skill in the art can readily and routinely screen variant amino acids and / or the nucleic acids encoding them to determine whether these variations substantially reduce or abolish ligand binding ability by methods known in the art.

[0043] Those skilled in the art will understand that the nucleic acid encoding the TCR and / or chimeric co-stimulatory receptor can also be modified. Beneficial modifications in the overall nucleic acid sequence include codon optimization of the sequence. Changes can be made that result in conservative substitutions in the expressed amino acid sequence. These variations can be made in the complementarity determining regions and non-complementarity determining regions of the amino acid sequence of the TCR chain that do not affect function. Usually, additions and deletions should not be made in the CDR3 region.

[0044] According to some embodiments of the invention, the amino acid sequence of the TCR and / or chimeric co-stimulatory receptor is modified to include a detectable label, a therapeutic agent or a pharmacokinetic-modifying moiety.

[0045] Non-limiting examples of detectable labels are radiolabels, fluorescent labels, nucleic acid probes, enzymes and imaging reagents. Therapeutic agents that can be associated with the TCR include radioactive compounds, immunomodulatory agents, enzymes or chemotherapeutic agents. Therapeutic agents can be encapsulated by liposomes linked to the TCR so that the compound can be slowly released at the target site. This avoids damage during transportation in the body and ensures that the therapeutic agent, e.g., toxins, have maximum effect after binding of the TCR to the relevant antigen-presenting cell. Other examples of therapeutic agents include:

[0046] Peptide cytotoxins, i.e. proteins or peptides capable of killing mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase and RNase. Small molecule cytotoxic agents, i.e. compounds capable of killing mammalian cells, with molecular weights of less than 700 daltons. Such compounds may contain toxic metals capable of having cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e. compounds that are broken down or transformed under physiological conditions to release the cytotoxic agent. Such agents include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, racheromycin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, mitoxantrone, auristatin E, vincristine, and doxorubicin; radionuclides, for example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225, and astatine-213. For example, radionuclides may be associated with the TCR or a derivative thereof by a chelating agent, an immunostimulant, also known as an immunostimulant, i.e., an immune effector molecule that stimulates an immune response. Exemplary immunostimulants are cytokines such as IL-2 and IFN-γ, antibodies or fragments thereof including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28 or anti-CD16); alternative protein scaffolds with antibody-like binding properties; superantigens, i.e. antigens that cause non-specific activation of T cells resulting in polyclonal T cell activation and massive cytokine release, and variants thereof; chemokines, complement activators such as IL-8, platelet factor 4, melanoma growth stimulating protein; heterologous protein domains, homologous protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0047] The therapeutic agent may preferably be selected from the group consisting of immune effector molecules, cytotoxic agents and radionuclides. Preferably, the immune effector molecule is a cytokine.

[0048] The pharmacokinetic modifying moiety may be, for example, at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialyl group, or a combination thereof. The at least one polyethylene glycol repeat unit, at least one glycol group, at least one sialyl group may be attached by several methods known to those skilled in the art. In a preferred embodiment, the unit is covalently linked to the TCR. The TCR according to the present invention may be modified by one or several pharmacokinetic modifying moieties. In particular, the soluble form of the TCR is modified by one or several pharmacokinetic modifying moieties. The pharmacokinetic modifying moiety may achieve beneficial changes to the pharmacokinetic profile of the therapeutic agent, for example, improved plasma half-life, reduced or enhanced immunogenicity, and improved solubility.

[0049] The TCR and / or chimeric co-stimulatory receptor may be modified by the addition of additional functional moieties, for example to reduce immunogenicity, increase hydrodynamic size (size in solution), solubility and / or stability (e.g., by enhancing protection against proteolytic degradation), and / or extend serum half-life.

[0050] Other useful functional moieties and modifications include "suicide" or "safety switches" that can be used to shut off effector host cells bearing the TCR of the present invention in a patient's body. One example is the inducible caspase 9 (iCasp9) "safety switch" described in Gargett and Brown Front Pharmacol. 2014; 5: 235. Briefly, effector host cells are modified by known methods to express a caspase 9 domain whose dimerization depends on a small molecule dimerizing drug such as AP1903 / CIP, resulting in rapid induction of apoptosis in modified effector cells. This system is described, for example, in EP 2173869. Examples of other "suicide" or "safety switches" are known in the art, such as herpes simplex virus thymidine kinase (HSV-TK), expression of CD20 followed by depletion using an anti-CD20 antibody, or myc tagging (Kieback et al, Proc Natl Acad Sci US A. 2008 Jan 15;105(2):623-8).

[0051] TCRs with altered glycosylation patterns are also contemplated herein. As is known in the art, glycosylation patterns may depend on the amino acid sequence (e.g., the presence or absence of specific glycosylated amino acid residues discussed below) and / or on the host cell or organism in which the protein is produced. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Addition of N-linked glycosylation sites to binding molecules is conventionally accomplished by altering the amino acid sequence to contain one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation sites may be introduced by the addition or substitution of one or more serine or threonine residues to the starting sequence.

[0052] Another means of glycosylation of TCR is by chemical or enzymatic linkage of glycosides to proteins. Depending on the linkage method used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. Similarly, deglycosylation (i.e., removal of carbohydrate moieties present on the binding molecule) can be accomplished chemically, for example, by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically, for example, by using endoglycosidases and exoglycosidases.

[0053] It is also contemplated that drugs such as small molecule compounds can be attached to the TCR, particularly to the soluble form of the TCR of the present invention. Linkage can be achieved by covalent bonds or non-covalent interactions, such as by electrostatic forces. A variety of linkers known in the art can be used to form drug conjugates.

[0054] TCRs, particularly soluble forms of the TCRs of the present invention, may be further modified to introduce additional domains that aid in the identification, tracking, purification and / or isolation of the respective molecules (tags).Thus, in some embodiments, the TCR alpha or beta chains may be modified to include an epitope tag.

[0055] Epitope tags are useful examples of tags that can be incorporated into the TCRs of the present invention.Epitope tags are short stretches of amino acids that allow specific antibody binding, and thus allow identification and tracking of the binding and movement of soluble TCRs or host cells or cultured (host) cells in the patient's body.Detection of epitope tags, and thus tagged TCRs, can be achieved using several different techniques.

[0056] The tag can further be used for the stimulation and proliferation of host cells bearing the TCR of the invention by culturing the cells in the presence of a binding molecule (antibody) specific for said tag.

[0057] Generally, TCR can be modified with various mutations that in some cases modify the affinity and off-rate of TCR with target antigen.In particular, the mutations can increase affinity and / or decrease off-rate.Thus, at least one CDR of TCR and its variable domain framework region can be mutated.

[0058] However, in a preferred embodiment, the CDRs of the TCR are not modified or are not subjected to in vitro affinity maturation as for the TCR in the examples. This means that the CDRs have naturally occurring sequences. This can be advantageous, since in vitro affinity maturation can lead to immunogenicity against the TCR molecule. This can lead to the production of anti-drug antibodies that reduce or inactivate therapeutic effects and treatments, and / or induce adverse effects.

[0059] The mutations may be one or more substitutions, deletions or insertions. These mutations may be introduced by any suitable method known in the art, for example, polymerase chain reaction, restriction enzyme-based cloning, ligation-independent cloning procedures, as described in Examples in Sambrook, Molecular Cloning - 4th Edition (2012) Cold Spring Harbor Laboratory Press.

[0060] Theoretically unpredictable TCR specificity with the risk of cross-reactivity may arise due to mispairing between endogenous and exogenous TCR chains. To avoid mispairing of TCR sequences, recombinant TCR sequences may be modified to contain murine or minimally murine Cα and Cβ regions, a technique that has been shown to efficiently enhance correct pairing of several different transduced TCR chains. TCR murineization (i.e., replacing human Cα and Cβ regions with their murine counterparts) is a technique that is commonly applied to improve the cell surface expression of TCRs in host cells. Without wishing to be bound by a particular theory, it is believed that murine TCRs more efficiently associate with CD3 co-receptors; and / or preferentially pair with each other, and are less likely to form mixed TCRs on human T cells that have been genetically modified ex vivo to express TCRs of desired antigen specificity, but still retain and express their "original" TCRs.

[0061] Nine amino acids have been identified that represent improved expression of murinized TCRs (Sommermeyer and Uckert, J Immunol. 2010 Jun 1; 184(11):6223-31), and it is predicted that one or all of the amino acid residues in the TCR alpha and / or beta chain constant regions will be replaced with their murine counterpart residues. This technique, also referred to as "minimal murinization", offers the advantage of enhancing cell surface expression, while at the same time reducing the number of "foreign" amino acid residues in the amino acid sequence, thereby reducing the risk of immunogenicity.

[0062] Some embodiments refer to an isolated TCR as described herein, wherein the TCR is of the single chain type, and the TCR alpha and beta chains are linked by a linker sequence.

[0063] A preferred single chain TCR format comprises a first segment composed of an amino acid sequence corresponding to the variable TCR alpha region, a second segment composed of an amino acid sequence corresponding to the variable TCR beta region fused to the N-terminus of an amino acid sequence corresponding to the TCR beta chain constant region extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may be composed of an amino acid sequence corresponding to the TCR beta chain variable region and the second segment may be composed of an amino acid sequence corresponding to the TCR alpha chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to the TCR alpha chain constant region extracellular sequence. The single chain TCR may further comprise a disulfide bond between the first and second chain, the length of the linker sequence and the position of the disulfide bond being such that the variable domain sequences of the first and second segments are oriented by mutation substantially as in the native T cell receptor. More specifically, the first segment may be constituted by an amino acid sequence corresponding to a TCR alpha chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR alpha chain constant region extracellular sequence, the second segment may be constituted by an amino acid sequence corresponding to a TCR beta chain variable region fused to the N-terminus of an amino acid sequence corresponding to a TCR beta chain constant region extracellular sequence, and a disulfide bond may be provided between the first and second chain. The linker sequence may be any sequence that does not impair the function of the TCR.

[0064] In the context of the present invention, a "functional" TCR α and / or β chain fusion protein is meant to mean a TCR or TCR variant that has been modified, for example by addition, deletion or substitution of amino acids, which maintains at least substantial biological activity. In the case of the α and / or β chain of a TCR, this is meant to mean that both chains remain capable of forming a TCR (with unmodified α and / or β chains or with another inventive fusion protein α and / or β chain) that exerts its biological function, in particular binding of said TCR to a specific peptide-MHC complex and / or functional signaling upon specific peptide:MHC interaction.

[0065] In a specific embodiment, the TCR may be modified to be a functional TCR alpha and / or beta chain fusion protein, wherein the epitope tag has a length of 6 to 15 amino acids, preferably 9 to 11 amino acids. In another embodiment, the TCR may be modified to be a functional T cell receptor (TCR) alpha and / or beta chain fusion protein, wherein the TCR alpha and / or beta chain fusion protein comprises two or more epitope tags, either spaced apart or directly in tandem. Fusion protein embodiments may contain two, three, four, five or even more epitope tags, so long as the fusion protein maintains its biological activity(ies) ("functionality").

[0066] Preferred is a functional TCR alpha and / or beta chain fusion protein according to the present invention, wherein said epitope tag is selected from, but not limited to, CD20 or Her2 / neu tag, or other conventional tags, such as myc-tag, FLAG-tag, T7-tag, HA (hemagglutinin)-tag, His-tag, S-tag, GST-tag, or GFP-tag, where myc, T7, GST, GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, for example, U.S. Pat. Nos. 4,703,004 and 4,851,341). The myc tag may be preferably used, since high quality reagents can be used for its detection. The epitope tag may of course have one or more additional functions besides recognition by an antibody. The sequences of these tags are described in the literature and are well known to those skilled in the art.

[0067] Chimeric costimulatory receptors The chimeric co-stimulatory receptor used in combination with the PRAME-specific TCR is - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB Includes.

[0068] In the present specification, the chimeric costimulatory receptor used in combination with the PRAME-specific TCR may particularly comprise an extracellular domain that contains an extracellular domain derived from PD-1 (e.g., human PD-1). In this context, the term "derived from" particularly means that the polypeptide contained in the extracellular domain comprises at least a portion of PD-1 (e.g., human PD-1), preferably the extracellular domain of PD-1, respectively. The chimeric costimulatory receptor comprising an extracellular domain derived from PD-1 has binding activity to PD-L1, PD-L2 or other inhibitory ligands of PD-1. As used herein, the term "derived from" PD-1 also allows for up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids to be substituted, deleted, and / or inserted, compared to the natural sequence of PD-1 (e.g., human PD-1) or a portion thereof (e.g., the extracellular domain).

[0069] In one embodiment, the extracellular domain containing a polypeptide derived from PD-1 comprises the sequence set forth in SEQ ID NO: 28, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 28. In a specific embodiment, the extracellular domain containing a polypeptide derived from PD-1 comprises the sequence set forth in SEQ ID NO:28.

[0070] In one embodiment of the invention, the chimeric costimulatory receptor comprises an extracellular domain containing a polypeptide derived from PD-1, and includes an amino acid sequence having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions (preferably conservative or highly conservative substitutions), deletions, and / or insertions compared to the amino acid sequence of the extracellular domain of human or mouse PD-1, e.g., human PD-1 as set forth in SEQ ID NO:28.

[0071] In the present specification, the chimeric co-stimulatory receptor used in combination with PRAME-specific TCR further comprises a transmembrane domain operably linked between the extracellular domain and the intracellular domain.In general, the transmembrane domain is not limited to a specific transmembrane domain.Preferably, the transmembrane domain allows the stable fixation of the fusion protein in the membrane of the cell (e.g., T cell) expressing the fusion protein, and further allows the extracellular domain to bind to PD-L1, respectively, and allows signal transmission to the intracellular domain containing the polypeptide derived from 4-1BB when binding to PD-L1.

[0072] In a preferred embodiment, the transmembrane domain of the chimeric costimulatory receptor is a transmembrane domain derived from PD-1. In one embodiment, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 30, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 30. In a specific embodiment, the transmembrane domain containing a PD-1 derived polypeptide comprises the sequence set forth in SEQ ID NO: 30.

[0073] In the present specification, the chimeric co-stimulatory receptor used in combination with a PRAME-specific TCR may particularly comprise an intracellular domain containing a polypeptide derived from 4-1BB (e.g., referred to as "4-1BB"), preferably an intracellular domain of 4-1BB (e.g., human 4-1BB). In this context, the term "derived from" particularly means that the polypeptide contained in the intracellular domain contains at least a portion of 4-1BB (e.g., human 4-1BB), preferably an intracellular domain of 4-1BB, respectively. A chimeric co-stimulatory receptor comprising an intracellular domain derived from 4-1BB is capable of increasing the proliferation rate of T cells expressing said chimeric co-stimulatory receptor upon stimulation with another inhibitory ligand of PD-L1, PD-L2 or PD-1, and / or increasing the effector function (such as increased IFN-γ release and / or increased cytotoxicity) of T cells expressing said chimeric co-stimulatory receptor compared to corresponding T cells not expressing said chimeric co-stimulatory receptor. As used herein, the term "derived from" 4-1BB also allows for up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids to be substituted, deleted, and / or inserted as compared to the native sequence of 4-1BB (human or mouse, preferably human 4-1BB) or a portion thereof (e.g., intracellular domain). In one embodiment, the intracellular domain containing the 4-1BB-derived polypeptide comprises the sequence set forth in SEQ ID NO:32 or an amino acid sequence at least 80% identical to SEQ ID NO:32. In a specific embodiment, the intracellular domain containing the 4-1BB-derived polypeptide comprises the sequence set forth in SEQ ID NO:32.

[0074] "At least 80% identical," and in particular "having an amino acid sequence that is at least 80% identical," as used herein, includes that the amino acid sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the indicated amino acid sequence.

[0075] Determination of percent identity between multiple sequences is preferably accomplished using the AlignX application of the Vector NTI Advance™ 10 program (Invitrogen Corporation, Carlsbad Calif., USA), which uses a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI Advance TM 10 DNA and protein sequence analysis software. User's Manual, 2004, pp.389-662). The determination of percent identity is performed using standard parameters of the AlignX application.

[0076] Nucleic acids, nucleic acid compositions and vectors Another aspect of the present invention refers to nucleic acids encoding the PRAME-specific TCRs and chimeric co-stimulatory receptors described herein.

[0077] The nucleotide sequences encoding the relevant regions and domains of the PRAME-specific TCR are shown in Table 1:

[0078] [Table 1]

[0079] The nucleotide sequences encoding the relevant regions and domains of the chimeric co-stimulatory receptors are shown in Table 2:

[0080] [Table 2]

[0081] "Nucleic acid molecule" generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, may be synthesized or obtained from a natural source (e.g., isolated and / or purified), may contain natural, non-natural or modified nucleotides, and may contain natural, non-natural or modified linkages between nucleotides, such as phosphoramidate or phosphorothioate linkages instead of phosphodiesters found between nucleotides of unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule resulting from the replication of those described in (i) above. For purposes of the present invention, the replication may be in vitro or in vivo replication. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art or commercially available (e.g., from Genscript, Thermo Fisher and similar companies). See, e.g., Sambrook et al., nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (see, e.g., Sambrook et al. 2001) designed to increase the biological stability of the molecule or to enhance the physical stability of the duplexes formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Nucleic acids can include any nucleotide sequence that encodes a recombinant TCR and / or chimeric co-stimulatory receptor, polypeptide, or protein, or any functional portion or variant thereof.

[0082] For example, the disclosure also provides variants of isolated or purified nucleic acids, where the variant nucleic acids comprise a nucleotide sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence encoding a TCR described herein. Such variant nucleotide sequences encode a functional TCR that specifically recognizes PRAME, in particular the epitope SLLQHLIGL (SEQ ID NO:1) of PRAME.

[0083] For example, the disclosure also provides variants of isolated or purified nucleic acids, where the variant nucleic acid comprises a nucleotide sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a chimeric costimulatory receptor described herein, such a variant nucleotide sequence encodes a functional chimeric costimulatory receptor described herein.

[0084] As already described elsewhere herein, the nucleic acid encoding the TCR and / or chimeric co-stimulatory receptor may be modified. Useful modifications in the overall nucleic acid sequence may be codon optimization. Changes that result in conservative substitutions in the translated amino acid sequence may be made. For TCRs, these variations may be made in the complementarity determining regions and non-complementarity determining regions of the amino acid sequence of the TCR chain that do not affect function. Usually, additions and deletions should not be made in the CDR3 region.

[0085] Another embodiment refers to a vector comprising the nucleic acid encoding the TCR and chimeric co-stimulatory receptor described herein.

[0086] The vector is preferably a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector or particle and / or a vector used in gene therapy.

[0087] A "vector" is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell where synthesis of the encoded polypeptide can occur. Typically and preferably, a vector is a nucleic acid that has been engineered using recombinant DNA techniques known in the art to incorporate a desired nucleic acid sequence (e.g., a nucleic acid of the invention). A vector may comprise DNA or RNA, and / or may comprise a liposome and / or a viral particle. A vector may be a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, a lentiviral vector, an adenoviral vector or particle, and / or a vector used in gene therapy. A vector may comprise a nucleic acid sequence that allows replication in a host cell as an origin of replication. A vector may also comprise one or more selectable marker genes and other genetic elements known to those skilled in the art. A vector is preferably an expression vector that comprises a nucleic acid according to the invention operably linked to a sequence that allows expression of said nucleic acid.

[0088] Preferably, the vector is an expression vector. More preferably, the vector is a retroviral, more particularly a gamma-retroviral or lentiviral vector.

[0089] The skilled artisan will understand that the chimeric co-stimulatory receptor sequence and the sequences of the TCR chains TCR-α and TCR-β can be contained in one nucleic acid, for example in one vector, where the sequences are linked to sequences of an internal ribosome entry site (IRES) or 2A peptide sequences from the porcine virus (P2A) or from other species, such as the Thosea asigna virus 2A peptide (T2A) or the foot and mouth disease virus 2A peptide (F2A) (described in Szymczak et al.: Development of 2A peptide-based strategies in the design of multicistronic vectors), resulting in the expression in transduced cells of a single messenger RNA (mRNA) molecule under the control of a viral promoter.

[0090] In specific embodiments, the cells may comprise a nucleic acid encoding a TCR and a chimeric co-stimulatory receptor as described herein, or a vector comprising said nucleic acid.

[0091] The terms "transfection" and "transduction" are interchangeable and refer to the process by which an exogenous nucleic acid sequence is introduced into a host cell, e.g., a eukaryotic host cell. The introduction or transfer of a nucleic acid sequence can be accomplished by any number of means, including but not limited to the above-mentioned methods, electroporation, microinjection, gene gun delivery, lipofection, superfection, and infection with retroviruses or other viruses suitable for transduction or transfection. Methods for cloning and exogenous expression of TCRs are described, for example, in Engels et al. (Relapse or eradication of cancer is predicted by peptide-major histocompatibility complex affinity. Cancer Cell, 23(4), 516-26. 2013). Transduction of primary human T cells with lentiviral vectors is described, for example, in Cribbs "simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells" BMC Biotechnol. 2013; 13: 98.

[0092] The cells described and provided in connection with the present invention, comprising the nucleic acid molecules or vectors described and provided herein, are preferably capable of stably or transiently (e.g., stably) expressing (constitutively or situationally) the PRAME-specific TCR and chimeric co-stimulatory receptor of the present invention. Host cells can generally be transduced or transformed with any suitable nucleic acid molecule or vector. In one embodiment, the host cells are transduced with a retroviral or lentiviral (e.g., retroviral) vector comprising a nucleic acid molecule encoding the fusion protein of the present invention or a portion thereof (e.g., ECD, TMD, and / or ICD), as described above.

[0093] In some embodiments, the cells are peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). The cells may be natural killer cells or T cells. Preferably, the cells are T cells. The T cells may be CD4+ or CD8+ T cells. In some embodiments, the cells are stem cell-like memory T cells.

[0094] Stem cell-like memory T cells (TSCM) are a subpopulation of less differentiated CD8+ or CD4+ T cells characterized by the ability to self-renew and persist for long periods of time. When these cells encounter their antigen in vivo, they further differentiate into central memory T cells (TCM), effector memory T cells (TEM) and terminally differentiated effector memory T cells (TEMRA), with some TSCM remaining quiescent (Flynn et al., Clinical & Translational Immunology (2014)). These remaining TSCM cells show the ability to build a tolerant immune memory in vivo and are therefore considered to be an important T cell subpopulation for adoptive T cell therapy (Lugli et al., Nature Protocols 8, 33-42 (2013) Gattinoni et al., Nat. Med. 2011 Oct; 17(10): 1290-1297). Immunomagnetic selection can be used to restrict the T cell pool to the stem cell memory T cell subpopulation. See (Riddell et al. 2014, Cancer Journal 20(2): 141-44).

[0095] Pharmaceutical Compositions, Medical Treatments, and Kits Another aspect of the present invention refers to pharmaceutical compositions comprising cells comprising the PRAME-specific TCRs and chimeric costimulatory receptors described herein, or nucleic acid molecules encoding said molecules, nucleic acids encoding PRAME-specific TCRs and chimeric costimulatory receptors, compositions comprising nucleic acids encoding PRAME-specific TCRs and chimeric costimulatory receptors, and corresponding vectors described herein.

[0096] These active ingredients of the present invention are preferably used in a dosage mixed with an acceptable carrier or carrier material, so that such pharmaceutical compositions can treat or at least alleviate the disease. Such compositions can contain (in addition to the active ingredient and carrier) filler materials, salts, buffers, stabilizers, solubilizers and other materials, which are known in the art.

[0097] The term "pharmacologically acceptable" defines a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. The choice of carrier depends on the application.

[0098] The pharmaceutical composition may contain additional ingredients that enhance the activity of the active ingredient or supplement the treatment. Such additional ingredients and / or factors may be part of the pharmaceutical composition to achieve synergistic effects or minimize adverse or undesirable effects.

[0099] Techniques for formulation or preparation and application / medication of the active ingredients of the present invention are published in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. Suitable applications are parenteral applications, such as intramuscular, subcutaneous, intramedullary injection and intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injection. Intravenous injection is the preferred treatment for patients.

[0100] According to a preferred embodiment, the pharmaceutical composition is an infusion or injection.

[0101] The injectable composition is a pharma- ceutically acceptable fluid composition that contains at least one active ingredient, e.g., an expanded T cell population (e.g., autologous or allogeneic to the patient being treated) that contains a PRAME-specific TCR and a chimeric co-stimulatory receptor. The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition may further contain small amounts of one or more non-toxic auxiliary agents, such as emulsifiers, preservatives, and pH buffering agents. Such injectable compositions useful for use with the fusion proteins of the present disclosure are conventional, and suitable formulations are well known to those skilled in the art.

[0102] Typically, a pharmaceutical composition includes at least one pharma- ceutically acceptable carrier.

[0103] Accordingly, another aspect of the invention refers to a cell as described herein, a composition as described herein, a nucleic acid as described herein, and / or a vector as described herein for use as a medicament.

[0104] Some embodiments refer to a cell described herein, a composition described herein, a nucleic acid described herein, and / or a vector described herein for use in the treatment of cancer.

[0105] In one embodiment, the cancer is a hematological cancer or a solid tumor.

[0106] Blood cancers do not form solid tumors, and therefore are also referred to as blood cancers that are mainly distributed in the body. Examples of blood cancers are leukemia, lymphoma, or multiple myeloma. There are two main types of solid tumors: sarcoma and carcinoma. Sarcomas are, for example, tumors of blood vessels, bones, adipose tissue, ligaments, lymphatic vessels, muscles, or tendons. In a specific embodiment, the cancer is a solid tumor.

[0107] In one embodiment, the cancer is selected from the group consisting of prostate cancer, uterine cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-Hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, carcinoma, sarcoma or osteosarcoma.

[0108] Compositions comprising the modified T cells described herein can be utilized in methods and compositions for adoptive immunotherapy according to known techniques, or variations thereof that will be apparent to those of skill in the art based on this disclosure.

[0109] In some embodiments, the cells are formulated by first harvesting them from the culture medium, then washing the cells and concentrating them in a therapeutically effective amount in a medium and a suitable container system for administration (a "pharmaceutical acceptable" carrier). A suitable infusion medium may be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution may also be utilized. The infusion medium may be supplemented with human serum albumin.

[0110] The number of cells in the composition for effective treatment is typically greater than 10 cells, and up to 10 6 , 10 8 or 10 9 The following cells are 10 More cells may be used. The number of cells will vary depending on the final use for which the composition is intended depending on the type of cells contained therein. In the uses provided in the present invention, the cells are generally in a volume of 1 liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically 10 per ml. 6 More than 10 cells per ml 7 More than 10 per ml8 Clinically relevant numbers of immune cells exceed 10 9 , 10 10 or 10 11 The amount of the cells in the cell suspension may be distributed in multiple infusions that cumulatively equal or exceed the amount of cells in the cell suspension. The pharmaceutical compositions provided in the present invention may be in various forms, such as solid, liquid, powder, aqueous, or lyophilized forms. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and / or additives can be formulated by conventional methods and administered to subjects in suitable doses. Stabilizing agents, such as lipids, nuclease inhibitors, polymers, and chelating agents, can protect the composition from degradation in the body. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents may be included.

[0111] The viral vector particle comprising the nucleotide sequence encoding the PRAME-specific TCR and chimeric co-stimulatory receptor provided in the present invention can be packaged as a kit.The kit can optionally include one or more components, such as instructions, devices, and additional reagents, as well as components for carrying out the method, such as tubes, containers, and syringes.The exemplary kit can include the nucleic acid encoding the recombinant TCR and chimeric co-stimulatory receptor, recombinant polypeptide, or virus provided in the present invention, and can optionally include instructions, devices for detecting the virus in a subject, devices for administering the composition to a subject, and devices for administering the composition to a subject.

[0112] Also contemplated herein is a kit comprising a polynucleotide encoding a PRAME-specific TCR and a chimeric co-stimulatory receptor.Also contemplated herein is a kit comprising a viral vector encoding a sequence of interest (e.g., a recombinant TCR) and optionally a polynucleotide sequence encoding an immune checkpoint inhibitor.

[0113] The kits contemplated herein also include kits for carrying out methods for detecting the presence of polynucleotides encoding any one or more of the TCRs and / or chimeric co-stimulatory receptors disclosed herein. In particular, such diagnostic kits may include a set of suitable amplification and detection primers and other related reagents for performing deep sequencing to detect polynucleotides encoding the TCRs and / or chimeric co-stimulatory receptors disclosed herein. In further embodiments, the kits of the present invention may include reagents for detecting the TCRs and / or chimeric co-stimulatory receptors disclosed herein, such as antibodies or other binding molecules. The diagnostic kits may also contain instructions for determining the presence of polynucleotides encoding the TCRs and / or chimeric co-stimulatory receptors disclosed herein or for determining the presence of the TCRs and / or chimeric co-stimulatory receptors disclosed herein. The kits may contain instructions. The instructions typically include tangible expressions that describe the components included in the kit, methods for administration including methods for determining the appropriate condition of the subject, appropriate dosages, and appropriate administration methods. The instructions may also include guidance for monitoring the subject over the duration of the treatment time.

[0114] The kit provided in the present invention may also include a device for administering the composition described herein to a subject.Any of the various devices known in the art for administering medicines or vaccines may be included in the kit provided in the present invention.Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as eyedroppers.Typically, the device for administering the virus of the kit corresponds to the virus of the kit, and for example, a needleless injection device, such as a high-pressure injection device, may be included in the kit with a virus that has not been damaged by high-pressure injection, but typically is not included in the kit with a virus that has been damaged by high-pressure injection.

[0115] experiment

[0116] [Example 1] Co-expression of PD1-41BB does not alter TCR expression levels. To prepare effector T cells for testing and characterization of the transgenic TCR T23.8-2.1-027-004 (=TCR) and TCR combined with PD1-41BB (=TCR_PD1-41BB), CD8+ T cells were isolated from healthy donors and activated with CD3 / CD28 antibodies in the presence of IL-7 and IL-5. Activated cells were transduced with retroviral particles containing either the sequence of the TCR or the sequence of the TCR linked to PD1-41BB. Non-transduced (=UT) CD8+ T cells prepared in the same way were used as control. Transduction efficiency and expression levels of the transgene were determined on day 14 by antibody staining for the TCR-β chain (TRBV09) and PD-1, followed by analysis by flow cytometry.

[0117] The analysis demonstrates that high transduction rates can be achieved with both constructs TCR (90.2%) and TCR_PD1-41BB (82%) (Figure 1). PD-1 expression cannot be detected in untransduced (UT) and TCR-transduced effector T cells. However, binding of PD-1 antibodies to TCR PD1-41BB-transduced T cells shows high expression levels of PD1-41BB that correlate with the expression of the transgenic TCR. Co-expression of PD1-41BB results in TCR expression levels comparable to those measured in effector T cells expressing only the transgenic TCR. This demonstrates that co-expression of the PD1-41BB switch receptor and the transgenic TCR is possible in T cells, resulting in equimolar expression at the cell surface.

[0118] [Example 2] The functional avidity of TCR transgenic T cells is not altered by co-expression of PD1-41BB. Functional avidity refers to the accumulation of the strength of multiple affinities of individual non-covalent interactions such as transgenic TCR and peptide-MHC complexes. Thus, the functional avidity of effector T cells is a measure of peptide sensitivity. TCRs that confer high peptide sensitivity can recognize smaller amounts of peptide. To investigate whether co-expression of PD1-41BB receptors affects the peptide sensitivity of TCR transgenic effector T cells, they were co-cultured with PD-L1 transgenic T2 cells (T2_PD-L1) that have the required HLA (HLA-A2:01) and overexpress PD-L1 to allow ligation with PD1-41BB.

[0119] T2_PD-L1 cells were incubated with increasing doses of SLLQHLIGL(SLL)-peptide (10 -5 M~10 -10 T2_PD-L1 cells were loaded with 1000 μg / ml of TCR-transgenic peptide (M) and co-cultured 1:1 E:T (20.000 cells) with effector T cells expressing either no transgenic TCR (UT), only transgenic TCR (TCR) or TCR in combination with PD1-41BB (TCR_PD1-41BB). IFN-γ ELISA was performed 20 h after co-culture to evaluate the reactivity of effector T cells when loaded with different concentrations of peptides presented by T2_PD-L1 cells (Figure 2). Half-maximal IFN-γ release is a measure for the functional avidity of TCR-transgenic effector T cells. In the left graph, absolute levels of IFN-γ are shown, while the right graph shows the nonlinear regression curve calculated for the relative values. As shown through the absolute IFN-γ values, co-expression of PD1-41BB increases the total amount of IFN-γ secreted by T cells compared to effectors expressing only transgenic TCR. However, the nonlinear regression curves indicate that the overall functional activity is comparable regardless of PD1-41BB expression. Thus, the peptide sensitivity of TCR-transgenic T cells is not altered by co-expression of the PD1-41BB switch receptor, even in the presence of the ligand PD-L1.

[0120] [Example 3] HLA-A * Recognition of the 02 subtype is not altered by co-expression of PD1-41BB. The HLA-A2 protein is a member of different HLA-A proteins that result in slightly different amino acid sequences. * HLA-A can be encoded by the 02 suballele. * The specific TCR that recognizes its cognate peptide in association with 02:01 is a different HLA-A * To define the genetic traits required for successful TCR-based cell therapy and to allow for the expansion of patient cohorts, we have compared TCRs with the most common HLA-A alleles. * Characterize in relation to the 02 suballele (Figure 3).

[0121] T cells expressing either no transgenic TCR (UT), only transgenic TCR (TCR), or a combination of TCR and PD1-41BB (TCR_PD1-41BB) were immunized with selected HLA-A * 02 Lower allele (HLA-A * The cells were co-cultured with lymphoblastoid cell lines (LCL; EBV-transformed B cells) bearing the transgenic TCR 02:XX at an E:T ratio of 1:1 (20,000 cells / well). To allow recognition by the transgenic TCR, 10 -5 M. IFN-γ concentrations were determined by ELISA after 20 h of coculture.

[0122] TCR transgenic effector T cells are * Similar levels of HLA-A compared to 02:01 * 02 Lower allele A * 02:02, A * 02:04 and A *The TCR transgenic effector T cells recognized SLL-peptides presented by MHC molecules encoded by 02:09. This recognition pattern was not altered by co-expression of PD1-41BB, consistent with previous results. TCR transgenic effector T cells recognized four different HLA-A peptides, regardless of co-expression of PD1-41BB. * Recognizes SLL-peptide in association with the 02 suballele.

[0123] [Example 4] Successfully avoiding the risk of potential peptide off-target toxicity. Off-target toxicity may occur when TCRs recognize not only a specific peptide (e.g., SLL-peptide) but also other peptides that share high sequence homology with the original peptide. To identify suitable peptide candidates that show high sequence similarity to a specific peptide and can be recognized by TCRs, we used Expitope 2.0 (登録商標) Computational tools such as [Jaravine V, Mosch A, Raffegerst S, et al. Expitope 2.0: a tool to assess immunotherapeutic antigens for their potential cross-reactivity against naturally expressed proteins in human tissues. BMC Cancer 2017;17(1):892.] can be used. This tool predicts peptides with the highest probability of mismatch (MM) based on genomic, transcriptomic and proteomic data. Expitope 2.0 (登録商標) By applying the search, it was possible to identify 191 MM peptides that exhibited up to four amino acid differences compared to the specific SLL-peptides. 10 -6We identified 33 MM peptides that were recognized by TCR-transduced T cells in prescreening cocultures using PD-L1 transgenic T2 cells loaded with MM peptides or SLL-peptides of M. Because exogenous loading of high concentrations of peptides does not necessarily result in physiological recognition of endogenously processed and presented peptides, the 33 MM peptides were investigated for their potential to induce IFN-γ release by TCR-transgenic effector T cells when the epitopes (peptides) were translated from in vitro transcribed RNA (ivtRNA) and endogenously processed in the PRAME-negative target cell line SNB-19. IvtRNAs encoding up to five MM peptides were electroporated into SNB-19 cells. The MM peptides that induced the most IFN-γ release in TCR-transgenic T cells in prescreening cocultures (MM01, MM26, MM66) were tested individually as "midigene" constructs (~400 bp). All other MM peptides were tested as minigene constructs (~90 bp per peptide) encoding the five MM peptides. A midigene construct encoding the SLL-peptide was used as a positive control. To confirm the success of ivtRNA transfection, all RNA constructs contained an epitope recognized by the positive control TCR. IFN-γ concentrations were determined 20 hours after co-culture of transfected SNB-19 cells with TCR transgenic effector T cells. All transfected SNB-19 cells were recognized by the positive control TCR, confirming the success of transfection (Figure 4). SNB-19 cells transfected with ivtRNA constructs encoding SLL-peptides were recognized by TCR transgenic T cells with and without PD1-41BB, whereas none of the intracellularly processed MM peptides were recognized. Thus, all MM peptides are risk-avoidant and do not result in off-target toxicity.

[0124] [Example 5] No off-target toxicities were identified using an LCL library covering common HLA domains. To obtain information on the possible cross-reactivity of TCR transgenic T cells with other HLA allotypes, a library of lymphoblastoid cell lines (LCLs) covering the most frequent HLA-A, -B and -C alleles in the Caucasian population was used as target cells. These LCLs express a wide variety of endogenously expressed peptides, helping to identify potential cross-reactivity by recognition of endogenous peptides presented on matched HLA-A2 molecules or other most frequent HLA molecules. If cross-recognition of a particular HLA allotype is detected, patients carrying the respective HLA allele will be excluded from the clinical study. T cells expressing either no transgenic TCR (UT), only transgenic TCR (TCR) or a combination of TCR and PD1-41BB (TCR_PD1-41BB) were co-cultured with 36 different LCLs at an E:T ratio of 1:1. IFN-γ concentrations were determined by ELISA 20 h after co-culture. TCR transgenic T cells with and without PD1-41BB were transfected with HLA-A loaded SLL-peptide, which served as a positive control. * 02:01 positive LCL (Figure 5). One HLA-A without any exogenous peptides * IFN-γ was released only in coculture with 02:01 LCL. Low levels of PRAME-RNA were detected by quantitative real-time polymerase chain reaction (qPCR) in this cell line, so the slight recognition was likely on-target recognition of SLL-peptides presented by HLA-A2. None of the other LCLs were recognized by effector T cells expressing the transgenic TCR or the transgenic TCR combined with PD1-41BB. Therefore, off-target toxicity due to recognition of endogenous peptides presented on matched HLA-A2 molecules or most frequently other HLA molecules could not be detected.

[0125] [Example 6] No off-target toxicity was identified using a panel of normal cells. The aim of this experiment is to evaluate the possible on-target / off-tumor and off-target toxicities caused by PRAME-specific TCR transgenic T cells with and without PD1-41BB. * 02:01 positive primary normal cells and induced pluripotent stem cell (iPS) derived cell lines representing essential tissues or organs were tested for recognition by TCR-transduced T cells. Matched to the characteristics of the individual targets, cells were seeded 1–7 days prior to the start of co-culture at cell densities according to the manufacturer's instructions and grown in monolayer in flat-bottom wells. To distinguish between on-target / off-tumor and potential off-target toxicity, PRAME mRNA expression in all tested normal cells was analyzed by quantitative real-time polymerase chain reaction (qPCR). 10 -5 Target cells loaded with M peptide served as an internal positive control (SLL-peptide). IFN-γ concentrations were determined by ELISA after 20 h of coculture. All normal cells loaded with specific SLL-peptides were recognized by TCR transgenic T cells with and without PD1-41BB (Figure 6). Without peptide loading, only mature dendritic cells (mDCs) yielded IFN-γ levels above background in non-transduced cells. As mDCs express PRAME, this recognition is due to on-target recognition of SLL-peptides presented by HLA-A2. None of the other target cells were recognized by effector T cells expressing transgenic TCR or transgenic TCR in combination with PD1-41BB. Therefore, no off-target toxicity due to recognition of endogenous peptides was observed.

[0126] [Example 7] PD1-41BB enhances the specific release of IFN-γ in response to tumor cells expressing PD-L1. The interaction of PD-L1 on tumor cells with PD-1 on T cells normally results in an inhibitory signal that reduces T cell activity. PD1-41BB reverses this signal and increases T cell reactivity when the transgenic TCR binds its cognate peptide-MHC complex. To test the effect of PD1-41BB co-expression on TCR transgenic T cell reactivity, effector T cells with or without PD1-41BB were co-cultured with tumor cells expressing the ligand PD-L1. For this co-culture, tumor cells from various indications that express the specific antigen PRAME at different levels were selected (Figure 7A). PRAME-RNA expression levels in tumor cell lines were determined by real-time quantitative PCR and normalized to the housekeeping gene GUSB. Ten tumor cell lines showed PRAME expression, whereas PRAME mRNA expression could not be detected in four tumor cell lines. However, these four PRAME-negative tumor cell lines expressed the PD1-41BB ligand PD-L1, serving as negative controls to confirm that PD1-41BB did not adversely affect the specificity of transgenic TCR-T cells. PD-L1 expression levels were determined by antibody staining followed by flow cytometry analysis. Some tumor cells were transduced with PD-L1 to allow stable expression (TD). Some tumor cell lines showed endogenous (end) expression of PD-L1, whereas in others, expression of PD-L1 could be induced by treatment with IFN-γ (ind). The IFN-γ levels used to induce expression were comparable to those produced in co-culture experiments with specific T cells recognizing the antigen. Endogenous PD-L1 expression levels in tumor cells could generally be further elevated by IFN-γ treatment (end, ind).

[0127] To determine the effect of PD1-41BB co-expression on cytokine release, TCR transgenic T cells with and without PD1-41BB were co-transfected with selected HLA-A antigens expressing different levels of PRAME and PD-L1. *02:01 positive tumor cell line (Figure 7B). Untransduced (UT) T cells were used as a control. TCR transgenic T cells and tumor cells were co-cultured at an E:T ratio of 1:1 (20.000 cells), and IFN-γ concentrations were measured by ELISA 20 hours after co-culture. Co-expression of PD1-41BB enhanced the release of IFN-γ in response to PD-L1 positive tumor cells, indicating that co-expression of PD1-41BB improved T cell reactivity in response to PD-L1 positive tumor cells. At the same time, PRAME negative tumor cells showing PD-L1 expression were not recognized, demonstrating that co-expression of PD1-41BB does not affect the specificity of TCR transgenic T cells. Increased cytokine release is observed only when transgenic TCR T cells recognize specific PRAME antigens.

[0128] [Example 8] PD1-41BB enhances specific cytotoxic responses against 3D tumor cell spheroids. To determine whether simultaneous co-expression of PD1-41BB has a beneficial effect on cytotoxicity, TCR transgenic T cells were co-cultured with PD-L1 positive 3D tumor cell spheroids (Figure 8). This 3D tumor cell spheroid should serve as an in vitro model of solid tumors. From the tumor cell panel introduced in Example 7, three HLA-A spheroids that showed different levels of PRAME expression and expressed red fluorescent protein (NucLight-Red) were identified. *02:01 positive tumor cell lines were selected. Cytotoxicity against tumor spheroids was determined by disappearance of red fluorescence over 20 days, with images recorded every 4 hours using an Incucyte Zoom® or S3® device. To investigate the effect of simultaneous co-expression of PD1-41BB on T cell fitness and resilience, fresh tumor cell spheroids were transferred to co-culture plates on days 3, 7, 10, 13 and 16. In this harsh environment of repeated exposure to tumor cells, expression of PD1-41BB has a beneficial effect on the effector function and fitness of T cells. In the process of multiple loading of tumor cell spheroids, PD1-41BB expressing effector T cells can better control tumor cell growth compared with effector T cells expressing only transgenic TCR. In addition, PRAME negative PD-L1 positive tumor cells were not targeted by transgenic TCR T cells independent of expression of PD1-41BB. Thus, although T cells co-expressing PD1-41BB remain fully antigen-dependent, the specific cytotoxic response against PD-L1-positive 3D tumor cell spheroids is enhanced.

[0129] [Example 9] PD1-41BB increases the proliferation of TCR transgenic T cells in response to tumor cells expressing PD-L1. The increased effector function and resilience of TCR transgenic T cells co-expressing PD1-41BB was determined by enhanced cytokine release and cytotoxicity in response to PD-L1-positive tumor cells (Examples 7, 8). In particular, better control of tumor cells, even after multiple loading of tumor cell spheroids, indicates increased T cell compatibility in a suppressive tumor cell environment and may also be associated with better survival or proliferation of T cells. The 4-1BB signaling domain contained in the PD1-41BB switch receptor is known to provide costimulation that enhances the proliferation rate of T cells (Choi et al., 4-1BB signaling activates glucose and fatty acid metabolism to enhance CD8+T cell proliferation; 2017). To investigate whether this increase in T cell proliferation can also be observed when PD1-41BB interacts with its ligand PD-L1, TCR transgenic T cells were co-cultured with PD-L1-positive tumor cells expressing different levels of the PRAME antigen (Figure 9). TCR transgenic T cells and HLA-A *02:01 positive tumor cell lines were co-cultured 1:1 E:T, and untransduced T cells (UT) were used as a control. To measure the X-fold expansion of T cells in co-culture with PD-L1 positive tumor cells, cells were harvested on day 7 and total cell numbers were determined using a MACSQuant® X Analyzer. Flow cytometry-based cell counting allowed easy discrimination between T cells and tumor cells that may still be present in the co-culture. As expected, untransduced T cells did not proliferate in response to PRAME positive tumor cells due to the absence of specific TCR stimulation required for proliferation. Transgenic TCR-T cells proliferate in response to PD-L1 positive tumor cells in a manner that appears to depend on the level of the specific antigen PRAME. Co-expression of PD1-41BB enhanced proliferation and survival in response to PD-L1 positive tumor cells in an antigen level-dependent manner compared to T cells expressing only transgenic TCR. Thus, expression of PD1-41BB in TCR-transgenic T cells improves proliferation rates and contributes to increased survival of the cells in the challenging tumor cell environment containing inhibitory PD-L1 receptors.

[0130] [Example 10] T cells co-expressing PD1-41BB exhibit potent anti-tumor reactivity in vivo. Co-expression of PD1-41BB increased the anti-tumor effector function of TCR transgenic T cells in in vitro assays. To confirm this positive effect of PD1-41BB on TCR transgenic T cells in vivo, we used immunodeficient (NOD / Shi-scid / IL-2Rγnull) mice and PRAME / HLA-A * We developed a mouse model using the 02:01 positive melanoma cell line MelA375. To mimic the immunosuppressive environment of solid tumors, MelA375 cells were transduced with PD-L1. 5×10 6One week after subcutaneous injection of PD-L1 transgenic MelA375, mice developed palpable tumors. At this time, mice were distributed into treatment groups containing 6 mice each. Mice were injected with 10×10 6 TCR positive cells (total number of cells was 16 × 10 6 Each mouse was injected with either 1000 mm2 or an equal amount of untransduced T cells (UT). Tumor volumes were measured 2–3 times a week. 3 Mice that exceeded 100% were sacrificed. Tumors in mice treated with non-transduced T cells grew rapidly, reaching maximum tumor volume within 2–4 weeks after T cell injection (Figure 10). Effector T cells expressing only the transgenic TCR had little effect on tumor growth. Only T cells co-expressing PD1-41BB were able to reject tumors, resulting in tumor disappearance 3.5 weeks after treatment. These data indicate that combining a PRAME-specific TCR that shows potent anti-tumor reactivity in vitro with PD1-41BB results in highly efficient T cells that can eliminate invasively growing tumor cells in an in vivo model.

[0131] The present application further includes the following items:

[0132] Item 1: (A) - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 PRAME-specific T cell receptor (TCR), including (B) - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB Chimeric costimulatory receptor comprising Cells containing

[0133] Item 2: The cell of item 1, wherein the TCR is capable of binding to a PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1) or a portion thereof, or an HLA-A2 binding form thereof.

[0134] Item 3: HLA-A2 is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:04 or HLA-A * The cell according to item 2, wherein the molecule is encoded by 02:09.

[0135] Item 4: The cell according to any one of items 1 to 3, wherein binding to the sequence SLLQHLIGL (SEQ ID NO: 1) or a part thereof, or an HLA-A2 binding form thereof induces IFN-γ secretion by the TCR-transduced or transfected cell.

[0136] Item 5: The cell of any one of the preceding items, wherein the TCR comprises a variable TCR alpha region having an amino acid sequence that is at least 80% identical to SEQ ID NO:8 and a variable TCR beta region having an amino acid sequence that is at least 80% identical to SEQ ID NO:9.

[0137] Item 6: The cell of any one of the preceding items, wherein the TCR comprises a variable TCR alpha region having the amino acid sequence of SEQ ID NO:8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:9.

[0138] Item 7: TCR is 10. The cell of any one of the preceding items, comprising a constant TCR alpha region having an amino acid sequence that is at least 80% identical to SEQ ID NO:10 and a constant TCR beta region having an amino acid sequence that is at least 80% identical to SEQ ID NO:11.

[0139] Item 8: TCR is 10. The cell of any one of the preceding items, comprising a constant TCR alpha region having the amino acid sequence of SEQ ID NO:10 and a constant TCR beta region having the amino acid sequence of SEQ ID NO:11.

[0140] Item 9: The cell of any one of the preceding items, wherein the extracellular domain containing the PD-1-derived polypeptide comprises the sequence of SEQ ID NO: 28.

[0141] Item 10: The cell of any one of the preceding items, wherein the intracellular domain containing the 4-1BB-derived polypeptide comprises the sequence of SEQ ID NO: 32.

[0142] Item 11: The cell of any one of the preceding items, wherein the transmembrane domain is derived from PD-1.

[0143] Item 12: The cell of any one of the preceding items, wherein the transmembrane domain-containing polypeptide from PD-1 comprises the sequence of SEQ ID NO: 30.

[0144] Item 13: The cell of any one of the preceding items, wherein the chimeric co-stimulatory receptor comprises the sequence of SEQ ID NO: 26.

[0145] Item 14: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, and - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 A nucleic acid encoding a PRAME-specific TCR comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB A nucleic acid encoding a chimeric co-stimulatory receptor comprising A composition comprising:

[0146] Item 15: - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3 and a CDR3 having the amino acid sequence of SEQ ID NO: 4, and - a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7 A nucleic acid encoding a PRAME-specific TCR comprising: - an extracellular domain containing polypeptide derived from PD-1; - a transmembrane domain, and - an intracellular domain containing a polypeptide derived from 4-1BB A nucleic acid encoding a chimeric co-stimulatory receptor comprising A nucleic acid comprising:

[0147] Item 16: The composition of item 14 or the nucleic acid of item 15, wherein the TCR is capable of binding to a PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1) or a portion thereof, or an HLA-A2 binding form thereof.

[0148] Item 17: HLA-A2 is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:04 or HLA-A * 17. The composition or nucleic acid according to item 16, which is a molecule encoded by 02:09.

[0149] Item 18: The composition according to items 14 and 16 to 17 or the nucleic acid according to items 15 to 17, wherein binding to the sequence SLLQHLIGL (SEQ ID NO: 1) or a part thereof, or an HLA-A2 binding form thereof induces IFN-γ secretion by TCR-transduced or transfected cells.

[0150] Item 19: The composition according to items 14 and 16 to 18 or the nucleic acid according to items 15 to 18, wherein the TCR comprises a variable TCR alpha region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 8 and a variable TCR beta region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 9.

[0151] Item 20: The composition according to items 14 and 15 to 19 or the nucleic acid according to items 15 to 19, wherein the TCR comprises a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO: 9.

[0152] Item 21: The composition according to items 14 and 15 to 20 or the nucleic acid according to items 15 to 20, wherein the TCR comprises a constant TCR alpha region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 10 and a constant TCR beta region having an amino acid sequence that is at least 80% identical to SEQ ID NO: 11.

[0153] Item 22: The composition according to items 14 and 16 to 21 or the nucleic acid according to items 15 to 21, wherein the TCR comprises a constant TCR alpha region having the amino acid sequence of SEQ ID NO: 10 and a constant TCR beta region having the amino acid sequence of SEQ ID NO: 11.

[0154] Item 23: The composition according to items 14 and 16 to 22 or the nucleic acid according to items 15 to 22, wherein the extracellular domain containing the Pd-1-derived polypeptide comprises the sequence of SEQ ID NO: 28.

[0155] Item 24: The composition according to items 14 and 16 to 23 or the nucleic acid according to items 15 to 23, wherein the intracellular domain containing the 4-1BB-derived polypeptide comprises the sequence of SEQ ID NO: 32.

[0156] Item 25: The composition according to items 14 and 16 to 24 or the nucleic acid according to items 15 to 24, wherein the transmembrane domain is derived from PD-1.

[0157] Item 26: The composition according to items 14 and 16 to 25, or the nucleic acid according to items 15 to 25, wherein the transmembrane domain containing a polypeptide derived from PD-1 comprises the sequence of SEQ ID NO: 30.

[0158] Item 27: The composition according to items 14 and 16 to 26 or the nucleic acid according to items 15 to 26, wherein the chimeric co-stimulatory receptor comprises the sequence of SEQ ID NO: 26.

[0159] Item 28: A vector comprising the nucleic acid according to Items 15 to 27.

[0160] Item 29: A cell comprising the composition according to items 14 and 16 to 27, the nucleic acid according to items 15 to 27, or the vector according to item 28.

[0161] Item 30: The cell according to items 1 to 13 and 29, which is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).

[0162] Item 31: The cell according to any one of items 1 to 13 and items 29 to 30, which is a T cell.

[0163] Item 32: A pharmaceutical composition comprising the cell according to items 1 to 13, the cell according to items 29 to 31, the composition according to items 14 and 16 to 27, the nucleic acid according to items 15 to 27 and / or the vector according to item 28.

[0164] Item 33: The pharmaceutical composition according to Item 20, comprising at least one pharma- ceutically acceptable carrier.

[0165] Item 34: The cell according to items 1 to 13, the cell according to items 29 to 31, the composition according to items 14 and 16 to 27, the nucleic acid according to items 15 to 27 and / or the vector according to item 28 for use as a medicament.

[0166] Item 35: The cell according to items 1 to 13, the cell according to items 29 to 31, the composition according to items 14 and 16 to 27, the nucleic acid according to items 15 to 27 and / or the vector according to item 28 for use in the treatment of cancer.

[0167] Item 36: The cancer is preferably melanoma, bladder cancer, colon cancer, and breast cancer, sarcoma, prostate cancer, uterine cancer, uveal cancer, uveal melanoma, head and neck squamous cell carcinoma, synovial cancer, Ewing's sarcoma, triple-negative breast cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, The cell according to items 1 to 13, the cell according to items 29 to 31, the composition according to items 14 and 16 to 27, the nucleic acid according to items 15 to 27 and / or the vector according to item 28 for use in the treatment of cancer selected from the group consisting of breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, preferably the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer or colorectal cancer, sarcoma or osteosarcoma.

Claims

1. (A) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:2, a CDR2 having the amino acid sequence of SEQ ID NO:3 and a CDR3 having the amino acid sequence of SEQ ID NO:4, and a TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3 having the amino acid sequence of SEQ ID NO:7; A PRAME-specific T cell receptor (TCR) comprising: (B) - an extracellular domain comprising the extracellular domain from PD-1, - a transmembrane domain, and - an intracellular domain containing an intracellular domain derived from 4-1BB Chimeric costimulatory receptor comprising Cells containing

2. The cell of claim 1, wherein the TCR is capable of binding to a PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO:1), or a portion thereof, or an HLA-A2 binding form thereof.

3. The HLA-A2 is HLA-A ＊ 02:01, HLA-A ＊ 02:02, HLA-A ＊ 02:04 or HLA-A ＊ The cell of claim 2, wherein the molecule is encoded by 02:

09.

4. The cell of claim 1, wherein the TCR comprises a variable TCR alpha region having an amino acid sequence identical or at least 80% identical to SEQ ID NO:8 and a variable TCR beta region having an amino acid sequence identical or at least 80% identical to SEQ ID NO:

9.

5. The TCR is The cell of claim 1 , comprising a constant TCR alpha region having the amino acid sequence of SEQ ID NO: 10 and a constant TCR beta region having the amino acid sequence of SEQ ID NO:

11.

6. the extracellular domain comprising the extracellular domain derived from PD-1 comprises the sequence of SEQ ID NO:28; The cell of claim 1, wherein the intracellular domain containing the 4-1BB-derived intracellular domain comprises the sequence of SEQ ID NO:

32.

7. 2. The cell of claim 1, wherein the transmembrane domain is derived from PD-1, preferably the transmembrane domain containing the PD-1 derived transmembrane domain comprises the sequence of SEQ ID NO: 30, and preferably the chimeric co-stimulatory receptor comprises the sequence of SEQ ID NO:

26.

8. - a nucleic acid encoding a T cell receptor (TCR) as defined in claim 1, and - a nucleic acid encoding a chimeric co-stimulatory receptor as defined in claim 1 A composition comprising:

9. - a nucleic acid encoding a T cell receptor (TCR) as defined in claim 1, and - a nucleic acid encoding a chimeric co-stimulatory receptor as defined in claim 1 A nucleic acid comprising:

10. A vector comprising the nucleic acid of claim 9.

11. A cell comprising the composition of claim 8, the nucleic acid of claim 9 or the vector of claim 10.

12. The cell of claim 1, which is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC), preferably a T cell.

13. A pharmaceutical composition comprising a cell according to claim 1, a composition according to claim 8, a nucleic acid according to claim 9 and / or a vector according to claim 10.

14. A cell according to claim 1, a composition according to claim 8, a nucleic acid according to claim 9 and / or a vector according to claim 10 for use as a medicament.

15. A cell according to claim 1, a composition according to claim 8, a nucleic acid according to claim 9 and / or a vector according to claim 10 for use in the treatment of cancer.

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