Immune cell expressing chimeric antigen receptor and transgenic t cell receptor

Abstract

The present invention provides T cells that express both a CAR and a tTCR (CARTCR T cells) and compositions comprising said CARTCR T cells together with T cells that express only said CAR ("CAR T cells") and / or with T cells that express only said tTCR ("tTCR T cells") for treatment of a disease. Methods for generation of these compositions are also provided.

Description

[0001] Title

[0002] Immune cell expressing chimeric antigen receptor and transgenic T cell receptor

[0003] Field of the invention

[0004] The present invention generally relates to the field of adoptive T cell therapies, e.g. use of T cell receptor and / or chimeric antigen binding receptor (CAR) expressed on the surface of immune cells for treatment of a disease such as cancer, in particular to immune cells that coexpress TCR and CAR and / or to the generation of a composition of immune cells that comprise TCR-expressing immune cells and / or, CAR-expressing T cells together with TCR and CAR co-expressing immune cells.

[0005] Background of the invention

[0006] The use of T cell receptor (TCR)- or chimeric antigen receptor (CAR)-expressing T cells redirected to specifically recognize and eliminate malignant cells, greatly increased the scope and potential of adoptive immunotherapy and is being assessed for new standard of care in certain human malignancies or other diseases. TCR-engineered T cell recognize their intracellularly processed peptide fragments in a human leukocyte antigen (HLA)-dependent manner. CARs are recombinant receptors that typically target surface molecules HLA-independently. Generally, CARs comprise an extracellular antigen recognition moiety, often a single-chain variable fragment (scFv) derived from antibodies or a Fab fragment, linked to an extracellular spacer, a transmembrane domain and intracellular co-stimulatory and signaling domains. Therapies using TCR- or CAR-modified T cells, although sometimes efficacious, have a high potential for improvements. Especially novel strategies are required to ameliorate T cell function.

[0007] In Uslu et al. 2016 the authors combined a T-cell receptor and a chimeric antigen receptor, specific for different common melanoma antigens, gplOO (PMEL) and MCSP (HMW-MAA), to generate functional CD8+ T cells expressing two additional receptors (TETARs) by electroporation of receptor-encoding mRNA, thereby generating CD8+ T cells that express transiently a transgenic TCR and a CAR.

[0008] In Simon et al. 2019 the authors generated T cells expressing two additional receptors (TETARs) by lentiviral transduction of a gplOO-specific T cell receptor (TCR) and subsequent electroporation of mRNA encoding a second-generation CSPG4-specific chimeric antigen receptor (CAR). Again, the CAR is expressed transiently only. Miyao et al. 2018 disclose a transgenic TCR together with artificial T cell-activating adapter molecules (ATAM), generated by inserting the intracellular domain (ICD) of activating T-cell signaling moieties into CD3z. ATAMs with the ICD of either CD28 or 4-1BB were generated, assembled into the TCR complex as a part of CD3z, and enhanced downstream signaling from the supramolecular activation cluster.

[0009] In Omer et al. 2018 the authors evaluated first and second generation GD2.CARs containing costimulatory endodomains derived from 4-1BB or CD28 in T cells specific for varicella zoster virus and EBV, i.e. these T cells have native TCRs.

[0010] Omer et al. 2022 discloses a T cell co-expressing a tTCR targeting an immunodominant epitope of surviving and a costimulatory CAR (CoCAR) that lacked the cytotoxic zeta chain of conventional CARs, but still provide T cell co-stimulation upon CD 19 ligation. W02019004831 discloses transgenic TCRs with specificity for ANPM1.

[0011] WO2015150526A2 discloses engineered immune cells expressing CARs with specificity for CD33.

[0012] There is a need in the art for improved or alternative adoptive immune cell such as T cell therapies including TCR and / or CAR immune cells such as T cells, especially with regard to the potential risk of tumor relapse due to HLA downregulation or surface antigen loss.

[0013] Brief description of the invention

[0014] Surprisingly, it was found that an immune cell, preferentially a T cell that expresses both a chimeric antigen receptor (CAR) specific for (targeting) a first epitope of an antigen and a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen can be used beneficially for use in treatment of a disease such as cancer in a subject suffering from said disease. It is even more surprising that a composition comprising immune cells, preferentially T cells that express both a CAR and a tTCR (“CARTCR T cells”) together with immune cells, preferentially T cells that express only said CAR (“CAR T cells”) and / or with immune cells, preferentially T cells that express only said tTCR (“tTCR T cells”) is superior for said treatment of a disease as compared to the single CARTCR T cell or a composition comprising immune cells, preferentially T cells that express only said CAR together with immune cells, preferentially T cells that express only said tTCR, i.e. a composition without said CARTCR T cell.

[0015] Combinations of specificity of CARs and tTCRs that may be usable for the immune cells and compositions as disclosed herein are, but without intention to be limited to these kind of combinations (specificity for CAR / specificity for tTCR): CD33 / ANPM1, BCMA / BOB1, CD19 / BOB1, CD20 / BOB1, CD22 / BOB1, CD123 / ANPM1, CLEC12a / ANPMl, FRa / PRAME and CSPG4 / PRAME.

[0016] Hematological malignancies are cancers that affect the blood and lymph system. The cancer may begin in blood-forming tissue, or in the cells of the immune system. Examples of hematological malignancies include myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL).

[0017] Solid tumors that may be treated with the immune cells and / or compositions as disclosed herein may be e.g. skin melanoma, synovial sarcoma, uterine cancer (uterine corpus endometrial carcinoma and uterine carcinosarcoma), ovarian cancer, uveal melanoma or lung squamous cell carcinoma.

[0018] Nucleophosmin (NPM1) is a driver gene that is frequently mutated in approximately 30% of patients with AML. Mutated NPM1 has also been observed in other types of hematological malignancies (e.g. other myeloid malignancies), although frequencies in tumors other than AML are much lower. Patients with mutated NPM1 (ANPM1, alternative notation: dNPMl) carry a characteristic 4 base pair (4-bp) frameshift insertion in exon 12 of the gene. The resulting ANPM1 protein is 4 amino acids (AA) longer than the wild type counterpart and its C-terminal 11 AA are translated in an alternative reading frame. As a result, the ANPM1 protein is dislocated from the nucleolus, where it functions as a nucleocytoplasmic shuttle protein, to the cytoplasm. The ANPM1 protein is thus localized intracellularly. However, HLA-restricted ANPM1 -derived peptides are accessible on the cell surface to T cell receptors, and thus can be recognized by T cells. One major peptide presented by HLA class I is the peptide CLAVEEVSL (SEQ ID NO: 1). SEQ ID NO: 1 is presented on the surface of primary AML cells isolated from HLA-A*02:01 positive patients with AML (W02019004831A1).

[0019] In one embodiment of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, e.g. a TAA, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen ANPM1. In a preferred embodiment of the invention said CAR has specificity for a first epitope of an antigen, e.g. a TAA, and said tTCR having specificity for an epitope of ANPM1 comprises (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO:1). In one embodiment of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen CD33, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen ANPM1. In a preferred embodiment of the invention said CAR having specificity for an epitope of CD33 comprises an antigen binding domain comprising SEQ ID NO:2, and said tTCR having specificity for an epitope of ANPM1 comprises (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0020] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen BCMA, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen BOB1, preferentially with the sequences for binding to the antigens BCMA and BOB1 as disclosed herein.

[0021] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen CD 19, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen BOB 1, preferentially with the sequences for binding to the antigens CD 19 and BOB1 as disclosed herein.

[0022] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen CD20, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen BOB 1, preferentially with the sequences for binding to the antigens CD20 and BOB1 as disclosed herein.

[0023] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen CD22, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen BOB 1, preferentially with the sequences for binding to the antigens CD22 and BOB1 as disclosed herein.

[0024] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen CD123, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen ANPM1, preferentially with the sequences for binding to the antigens CD123 and ANPM1 as disclosed herein.

[0025] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen CLEC12a, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen ANPM1, preferentially with the sequences for binding to the antigens CLEC12a and ANPM1 as disclosed herein.

[0026] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen FRa, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen PRAME, preferentially with the sequences for binding to the antigens FRa and PRAME as disclosed herein.

[0027] In other embodiments of the invention said CARTCR T cell and said composition comprising said CARTCR cells together with said CAR T cells and / or tTCR T cells comprise a CAR specific for a first epitope of an antigen, wherein said first epitope is an epitope of the antigen CSPG4, and comprise a tTCR having specificity for a second epitope of an antigen, wherein said second epitope is an epitope of the antigen PRAME, preferentially with the sequences for binding to the antigens CSPG4 and PRAME as disclosed herein.

[0028] A method for the generation of said compositions as disclosed herein is also provided.

[0029] Brief description of the drawings

[0030] Figure 1 : Schematic representation of genetically modified T cells expressing a CAR and a transgenic TCR simultaneously, which leads to increased anti-tumor cytotoxicity in cis (both TCR- and CAR-target expressed on same tumor cell) and trans (TCR- and CAR-target expressed on two different tumor cells). Figure 2: T cells were transduced with lentiviral particles encoding either ANPM1-TCR or CD33-CAR. (A) Exemplary dot plots of one representative donor displaying TCR and CAR expression of enriched ANPM1-TCR, CD33-CAR, or ANPM1-TCR and CD33-CAR dualspecific T cells, determined prior to in vitro co-culture through staining with ANPM1-HLA- A*02-specific Tetramer and CD33 -CAR-specific antibody. (B) Lysis of ANPMl-TCR-target cells or CD33-CAR (trans stimulation) after 18 hours of co-culture (E:T 1 : 1) with UTD-T cells (untransduced), ANPM1-TCR T cells, CD33-CAR T cells, or CD33-CAR / ANPM1-TCR T cells. (C) Lysis of ANPM1-TCR- / CD33 -CAR-target cells (cis stimulation) after 18 hours of coculture (E:T 1 : 1) with UTD-T cells (untransduced), ANPM1-TCR T cells, CD33-CAR T cells, or CD33-CAR / ANPM1-TCR T cells. Displayed are individual and mean values ±SD of eight different donors from two independent experiments. (D) Lysis of ANPMl-TCR-target cells or CD20-CAR-target cells (trans stimulation) after 18 hours of co-culture (E:T 1 : 1) with UTD-T cells (untransduced), ANPM1-TCR T cells, CD20-CAR T cells, or CD20-CAR / ANPM1-TCR T cells. Displayed are individual and mean values ±SD of five different donors from two independent experiments. (E) Lysis of ANPMl-TCR-target cells or CD19-CAR-target cells (trans stimulation) after 18 hours of co-culture (E:T 1 :1) with UTD-T cells (untransduced), ANPM1-TCR T cells, CD19-CAR T cells, or CD19-CAR / ANPM1-TCR T cells. Displayed are individual and mean values ±SD of two different donors. Displayed p-values were calculated using ordinary one-way analysis of variance (ANOVA) with Tukey‘s correction for multiple comparison (ns, not significant; *p < 0.05).

[0031] Figure 3: Live cell imaging via IncuCyte analysis showing cytotoxicity upon repeated target cell addition (indicated by arrow) for (A) trans and (B) cis stimulation with an E:T ratio of 1 : 1. Shown are mean values ±SEM of eight different donors from two independent experiments. The p-values were calculated using Tukey‘s multiple comparison test and the mixed-effects model with Geisser-Greenhouse correction (two-way ANOVA) (ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001).

[0032] Figure 4: Co-transduced CD33-CAR- and ANPM1-TCR- expressing T cells in vivo. (A) Schematic representation of the study design to assess the efficacy of a T cell product comprised of CAR+ / tTCR+ T cells, CAR+ T cells and tTCR+ T cells. (B) Expression and viability of T cell products prior to injection into mice. Following treatments were compared: tTCR-T cells; CAR-T cells, a 1 : 1 mixture of CAR-T cells and tTCR-T cells; and a mixture of CAR+ / tTCR+ T cells, CAR-T cells and TCR-T cells (produced through co-transduction). (C) Randomization was performed one day prior to T cell injection according to tumor burden. (D) Tumor growth of luciferase-expressing 0ci-AML3 cells was analyzed by BLI imaging quantifying the median photon flux. Eight mice were analyzed per cohort (except for Tumor only and UTD-T: five mice per cohort). Displayed are median values with error bars indicating interquartile range.

[0033] Figure 5: T cells were co-transduced with lentiviral particles encoding either ANPM1-TCR or CD123-CAR or Clecl2a-CAR. (A) Lysis of ANPM1-TCR / CD 123 -CAR-target cells (cis stimulation) after 48 hours of co-culture (E:T 0.2: 1) with UTD-T cells (untransduced), ANPM1- TCRT cells, CD123-CART cells, or CD123-CAR / ANPM1-TCRT cells. (B) Lysis of ANPM1- TCR- / Clecl2a-CAR-target cells (cis stimulation) after 48 hours of co-culture (E:T 0.1:1) with UTD-T cells (untransduced), ANPM1-TCR T cells, Clecl2a-CAR T cells, or Clecl2a- CAR / ANPM1-TCR T cells. Displayed are mean values ±SD of technical duplicates.

[0034] Figure 6: T cells were co-transduced with lentiviral particles encoding either PRAME-TCR or CD123-CAR or CSPGa4-CAR or FolRl-CAR. (A) Lysis of PRAME-TCR / CD 123 -CAR-target cells (cis stimulation) after 24 hours of co-culture (E:T 1:1) with UTD-T cells (untransduced), PRAME-TCR T cells, CD123-CAR T cells, or CD 123 -CAR / PRAME-TCR T cells. (B) Live cell imaging via IncuCyte analysis showing lysis of PRAME-TCR / CSPG4-CAR-target cells (cis stimulation) during 48 hours of co-culture (E:T 1:1) with UTD-T cells (untransduced), PRAME-TCR T cells, CSPG4-CAR T cells, or CSPG4-CAR / PRAME-TCR T cells. (C) Live cell imaging via IncuCyte analysis showing lysis of FolRl -CAR-target cells (trans stimulation) during 48 hours of co-culture (E:T 1 : 1) with UTD-T cells (untransduced), PRAME-TCR T cells, FolRl-CAR T cells, or FolRl -CAR / PRAME-TCR T cells. (D) Lysis of PRAME-TCR-target cells (trans stimulation) after 24h hours of co-culture (E:T 1:1) with UTD-T cells (untransduced), PRAME-TCR T cells, FolRl-CAR T cells, or FolRl -CAR / PRAME-TCR T cells. Displayed in (A) and (D) are mean values ±SD of technical duplicates.

[0035] Detailed description of the invention

[0036] In a first aspect the present invention provides an engineered immune cell expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens

[0037] Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0038] Said antigen having said first epitope may be a soluble antigen.

[0039] Said engineered immune cell, wherein said antigen having said first epitope is expressed on the surface of a target cell or is a soluble antigen, e.g. of a tumor microenvironment (TME).

[0040] Said engineered immune cell, wherein said antigen having said second epitope is a peptide of an intracellular antigen processed and presented in a major histocompatibility complex (MHC) class I or class Il-dependent manner.

[0041] Said CAR, wherein said antigen binding domain of said CAR specific for a first epitope of an antigen may be an antibody or antigen binding fragment thereof such as a scFv or a nanobody. Said CAR, wherein said intracellular signaling domain of said CAR may comprise a signaling domain such as the signaling domain of CD3zeta and / or at least one co-stimulatory domain such as the co-stimulatory domain of 4-1BB or CD28.

[0042] Alternatively, said CAR may comprise a structure as disclosed e.g. in W02020201527A1, wherein said CAR may comprise i) a polypeptide having

[0043] - an antigen binding domain;

[0044] - a TCR a-chain constant domain;

[0045] - a TCR a-chain transmembrane domain or a P-chain transmembrane domain or a 5-chain transmembrane domain or a TCR y-chain transmembrane domain; and / or ii) a polypeptide having

[0046] - an antigen binding domain;

[0047] - a TCR P-chain constant domain;

[0048] - a TCR a-chain transmembrane domain or a P-chain transmembrane domain or a y-chain transmembrane domain or a TCR 5-chain transmembrane domain.

[0049] For example, under i) a TCR 5-chain transmembrane domain may be chosen and under ii) a TCR y-chain transmembrane domain. Alternatively, under i) a TCR y-chain transmembrane domain may be chosen and under ii) a TCR 5-chain transmembrane domain. Said engineered immune cell, wherein both said antigen having said first epitope and said antigen having said second epitope are tumor antigens or tumor associated antigens of a target cell, wherein said target cell may be a cancer cell.

[0050] Said engineered immune cell, wherein both said antigen having said first epitope and said antigen having said second epitope are viral antigens of a target cell, wherein said target cell may be a cell infected by a virus.

[0051] Said engineered immune cell, wherein said antigen having said first epitope is expressed on the surface of a target cell, wherein said target cell is a non-malignant cell such as a B cell (B cell antigen), and wherein said antigen having said second epitope is presented on the surface of a virus-infected cell. Said engineered immune cell, wherein said antigen having said first epitope is expressed on the surface of a target cell, wherein said target cell is a non-malignant cell such as a B cell (B cell antigen), and wherein said antigen having said second epitope is presented on the surface of a virus-infected cell. In this embodiment of the invention, the CAR functions as booster for the immune cell expressing said CAR upon binding the first antigen such as CD 19 or CD20 that are expressed on B cells.

[0052] Said engineered immune, wherein said CAR and said tTCR are constitutively expressed in said immune cell.

[0053] Said engineered immune cell, wherein said engineered immune cell has stably integrated the nucleic acid sequence encoding said CAR and the nucleic acid sequence encoding said transgenic TCR into the genome of said immune cell.

[0054] Said engineered immune cell, wherein said immune cell is a T cell or Natural Killer (NK) cell. Said engineered immune cell, wherein said immune cell is a T cell.

[0055] Said engineered immune cell, wherein said T cell is selected from a CD4+ T cell or CD8+ T cell. Preferentially said immune cell may be a CD8+ T cell.

[0056] A population of said engineered T cell may comprise CD4+ T cells and CD8+ T cells.

[0057] Said engineered immune cell, wherein said CAR is specific for a first epitope of an antigen, e.g. a TAA, and wherein said tTCR has specificity for an epitope of the antigen ANPM1.

[0058] Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises a sequence as disclosed herein. Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0059] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0060] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0061] Said engineered immune cell, wherein said CAR is specific for a first epitope of an antigen, e.g. a TAA, and wherein said tTCR having specificity for an epitope of the antigen ANPM1 comprises:

[0062] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0063] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1), wherein the CDR3 of (a) is within a TCR a chain variable region having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 5; and optionally wherein (a) comprises a TCR alpha chain constant region, and optionally wherein the TCR alpha chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 9 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 10; and / or wherein the CDR3 of (b) is within a TCR beta chain variable region having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 8, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 6; and optionally wherein (b) comprises a TCR beta chain constant region, and optionally wherein the TCR beta chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 11 and the TCR P chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 12.

[0064] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen CD33 and wherein said tTCR has specificity for an epitope of the antigen ANPM1.

[0065] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD33 comprises SEQ ID NO:3 and SEQ ID NO:4. Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD33 comprises SEQ ID NO:2.

[0066] Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0067] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0068] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0069] Said engineered immune cell, wherein said antigen binding domain specific for CD33 comprises SEQ ID NO:2, and wherein said tTCR having specificity for ANPM1 comprises:

[0070] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0071] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0072] The peptide CLAVEEVSL (SEQ ID NO: 1) may be HLA-A*02:01 restricted.

[0073] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD33 comprises SEQ ID NO:2, and / or wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0074] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to CAVTGARLMF (SEQ ID NO: 5); and / or

[0075] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to CASSPGGLSNEQF (SEQ ID NO: 6); and / or

[0076] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO: 1, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7, and / or

[0077] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO: 1, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8, and / or

[0078] (v) the CDR3 of (a) is within a TCR a chain variable region having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 7, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 5; and optionally wherein (a) comprises a TCR alpha chain constant region, and optionally wherein the TCR alpha chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 9 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 10; and / or

[0079] (vi) the CDR3 of (b) is within a TCR beta chain variable region having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 8, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 6; and optionally wherein (b) comprises a TCR beta chain constant region, and optionally wherein the TCR beta chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 11 and the TCR P chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 12.

[0080] Said engineered immune cell may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML).

[0081] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen B cell maturation antigen (BCMA) and wherein said tTCR has specificity for an epitope of the B cell Oct binding factor- 1 (BOB1).

[0082] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen BCMA comprises SEQ ID NO: 13.

[0083] Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen BOB 1 comprises:

[0084] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0085] Said engineered immune cell, wherein said antigen binding domain specific for BCMA comprises SEQ ID NO: 13, and wherein said tTCR having specificity for BOB1 comprises:

[0086] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or

[0087] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0088] The peptide APAPTAVVL (SEQ ID NO: 14) may be HLA-B*07:02 restricted.

[0089] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen BCMA comprises SEQ ID NO: 13, and / or wherein said tTCR having specificity for the epitope of the antigen BOB1 comprises:

[0090] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to KGSSNTGKLIFGQGTTLQVKP (SEQ ID NO: 15); and / or

[0091] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASSHGPASYEQYF (SEQ ID NO: 16); and / or

[0092] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 17, and / or

[0093] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 18

[0094] Said engineered immune cell may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or diffuse large B cell lymphoma (DLBCL).

[0095] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen CD 19 and wherein said tTCR has specificity for an epitope of the B cell Oct binding factor-1 (BOB 1). Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD19 comprises SEQ ID NO: 19 or comprises SEQ ID NO:20 and SEQ ID NO:21. Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen BOB 1 comprises:

[0096] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or

[0097] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0098] Said engineered immune cell, wherein said antigen binding domain specific for CD 19 comprises SEQ ID NO: 19 (or comprises SEQ ID NO:20 and SEQ ID NO:21), and wherein said tTCR having specificity for BOB1 comprises:

[0099] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or

[0100] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0101] The peptide APAPTAVVL (SEQ ID NO: 14) may be HLA-B*07:02 restricted.

[0102] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD19 comprises SEQ ID NO: 19 (or comprises SEQ ID NO:20 and SEQ ID N0:21), and / or wherein said tTCR having specificity for the epitope of the antigen BOB1 comprises:

[0103] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to KGSSNTGKLIFGQGTTLQVKP (SEQ ID NO: 15); and / or

[0104] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASSHGPASYEQYF (SEQ ID NO: 16); and / or (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 17, and / or

[0105] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 18

[0106] Said engineered immune cell may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or diffuse large B cell lymphoma (DLBCL).

[0107] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen CD20 and wherein said tTCR has specificity for an epitope of the B cell Oct binding factor-1 (BOB 1). Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD20 comprises SEQ ID NO:22 or comprises SEQ ID NO:23 and SEQ ID NO:24. Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen BOB 1 comprises:

[0108] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or

[0109] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0110] Said engineered immune cell, wherein said antigen binding domain specific for CD20 comprises SEQ ID NO:22 (or comprises SEQ ID NO:23 and SEQ ID NO:24), and wherein said tTCR having specificity for BOB1 comprises:

[0111] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or

[0112] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0113] The peptide APAPTAVVL (SEQ ID NO: 14) may be HLA-B*07:02 restricted.

[0114] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD20 comprises SEQ ID NO:22 (or comprises SEQ ID NO:23 and SEQ ID NO:24), and / or wherein said tTCR having specificity for the epitope of the antigen BOB1 comprises:

[0115] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to KGSSNTGKLIFGQGTTLQVKP (SEQ ID NO: 15); and / or

[0116] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASSHGPASYEQYF (SEQ ID NO: 16); and / or

[0117] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 17, and / or

[0118] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 18

[0119] Said engineered immune cell may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL).

[0120] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen CD22 and wherein said tTCR has specificity for an epitope of the B cell Oct binding factor-1 (BOB 1). Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD22 comprises SEQ ID NO:25 or comprises SEQ ID NO:26 and SEQ ID NO:27. Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen BOB 1 comprises:

[0121] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or

[0122] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0123] Said engineered immune cell, wherein said antigen binding domain specific for CD22 comprises SEQ ID NO:25 (or comprises SEQ ID NO:26 and SEQ ID NO:27), and wherein said tTCR having specificity for BOB1 comprises:

[0124] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14) and / or

[0125] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide APAPTAVVL (SEQ ID NO: 14).

[0126] The peptide APAPTAVVL (SEQ ID NO: 14) may be HLA-B*07:02 restricted.

[0127] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD22 comprises SEQ ID NO:25 (or comprises SEQ ID NO:26 and SEQ ID NO:27), and / or wherein said tTCR having specificity for the epitope of the antigen BOB1 comprises:

[0128] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to KGSSNTGKLIFGQGTTLQVKP (SEQ ID NO: 15); and / or

[0129] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASSHGPASYEQYF (SEQ ID NO: 16); and / or

[0130] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 17, and / or

[0131] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO: 14, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 18

[0132] Said engineered immune cell may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or diffuse large B cell lymphoma (DLBCL).

[0133] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen CD123 and wherein said tTCR has specificity for an epitope of the antigen ANPM1.

[0134] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD123 comprises SEQ ID NO:28 or comprises SEQ ID NO:29 and SEQ ID NO:30. Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0135] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0136] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0137] Said engineered immune cell, wherein said antigen binding domain specific for CD 123 comprises SEQ ID NO:28 (or comprises SEQ ID NO:29 and SEQ ID NO:30), and wherein said tTCR having specificity for ANPM1 comprises:

[0138] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0139] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0140] The peptide CLAVEEVSL (SEQ ID NO: 1) may be HLA-A*02:01 restricted.

[0141] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CD123 comprises SEQ ID NO:28 (or comprises SEQ ID NO:29 and SEQ ID NO:30), and / or wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0142] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to CAVTGARLMF (SEQ ID NO: 5); and / or

[0143] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASSPGGLSNEQF (SEQ ID NO: 6);and / or

[0144] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO: 1, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7, and / or

[0145] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO:1, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 8, and / or

[0146] (v) the CDR3 of (a) is within a TCR a chain variable region having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 7, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 5; and optionally wherein (a) comprises a TCR alpha chain constant region, and optionally wherein the TCR alpha chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 9 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 10; and / or

[0147] (vi) the CDR3 of (b) is within a TCR beta chain variable region having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 8, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 6; and optionally wherein (b) comprises a TCR beta chain constant region, and optionally wherein the TCR beta chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 11 and the TCR P chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 12.

[0148] Said engineered immune cell may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML). Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen CLEC12a and wherein said tTCR has specificity for an epitope of the antigen ANPM1.

[0149] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CLEC12a comprises SEQ ID NO:31 or comprises SEQ ID NO:32 and SEQ ID NO:33.

[0150] Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0151] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0152] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0153] Said engineered immune cell, wherein said antigen binding domain specific for CLEC12a comprises SEQ ID NO:31, and wherein said tTCR having specificity for ANPM1 comprises:

[0154] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0155] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0156] The peptide CLAVEEVSL (SEQ ID NO: 1) may be HLA-A*02:01 restricted.

[0157] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CLEC12a comprises SEQ ID NO:31, and / or wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0158] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to CAVTGARLMF (SEQ ID NO: 5); and / or

[0159] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASSPGGLSNEQF (SEQ ID NO: 6); and / or

[0160] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO: 1, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7, and / or

[0161] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO: 1, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:8, and / or

[0162] (v) the CDR3 of (a) is within a TCR a chain variable region having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 7, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 5; and optionally wherein (a) comprises a TCR alpha chain constant region, and optionally wherein the TCR alpha chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 9 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 10; and / or

[0163] (vi) the CDR3 of (b) is within a TCR beta chain variable region having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 8, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 6; and optionally wherein (b) comprises a TCR beta chain constant region, and optionally wherein the TCR beta chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 11 and the TCR P chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 12.

[0164] Said engineered immune cell may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML).

[0165] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen Folate Receptor alpha (FRa) and wherein said tTCR has specificity for an epitope of the antigen Preferentially Expressed Antigen in Melanoma (PRAME).

[0166] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen FRa comprises SEQ ID NO:34 or comprises SEQ ID NO:35 and SEQ ID NO:36. Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen PRAME comprises:

[0167] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO: 37) and / or (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO:37).

[0168] Said engineered immune cell, wherein said antigen binding domain specific for FRa comprises SEQ ID NO:34 (or comprises SEQ ID NO:35 and SEQ ID NO:36), and wherein said tTCR having specificity for PRAME comprises:

[0169] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO: 37) and / or

[0170] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO:37).

[0171] The peptide SLLQHLIGL (SEQ ID NO:37) may be HLA-A*02:01 restricted.

[0172] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen FRa comprises SEQ ID NO:34 (or comprises SEQ ID NO:35 and SEQ ID NO:36), and / or wherein said tTCR having specificity for the epitope of the antigen PRAME comprises:

[0173] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to CAGIPRDNYGQNFVF (SEQ ID NO: 38); and / or

[0174] (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASTPWLAGGNEQFF (SEQ ID NO: 39); and / or

[0175] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO:37, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:40, and / or

[0176] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO:37, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:41. Said engineered immune cell may be for use in treatment of solid tumors such as skin melanoma, synovial sarcoma, uterine cancer (uterine corpus endometrial carcinoma and uterine carcinosarcoma), ovarian cancer, uveal melanoma or lung squamous cell carcinoma.

[0177] Said engineered immune cell, wherein said CAR is specific for an epitope of the antigen Chondroitin sulfate proteoglycan 4 (CSPG4) and wherein said tTCR has specificity for an epitope of the antigen Preferentially Expressed Antigen in Melanoma (PRAME).

[0178] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CSPG4 comprises SEQ ID NO:42 or comprises SEQ ID NO:43 and SEQ ID NO:44. Said engineered immune cell, wherein said tTCR having specificity for the epitope of the antigen PRAME comprises:

[0179] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO: 37) and / or

[0180] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO:37).

[0181] Said engineered immune cell, wherein said antigen binding domain specific for CSPG4 comprises SEQ ID NO:42 (or comprises SEQ ID NO:43 and SEQ ID NO:44), and wherein said tTCR having specificity for PRAME comprises:

[0182] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO: 37) and / or

[0183] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide SLLQHLIGL (SEQ ID NO:37).

[0184] The peptide SLLQHLIGL (SEQ ID NO:37) may be HLA-A*02:01 restricted.

[0185] Said engineered immune cell, wherein said antigen binding domain specific for the epitope of the antigen CSPG4 comprises SEQ ID NO:42 (or comprises SEQ ID NO:43 and SEQ ID NO:44), and / or wherein said tTCR having specificity for the epitope of the antigen PRAME comprises:

[0186] (i) the CDR3 of (a) has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to CAGIPRDNYGQNFVF (SEQ ID NO: 38); and / or (ii) the CDR3 of (b) has an amino acid sequence having at least 90%, at least 95% 100% sequence identity to CASTPWLAGGNEQFF (SEQ ID NO: 39); and / or

[0187] (iii) the CDR3 of (a) is within a TCR a chain variable region that specifically binds to SEQ ID NO:37, optionally wherein (a) further comprises a TCR alpha chain constant region; optionally wherein the TCR alpha chain variable region has an amino acid sequence having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:40, and / or

[0188] (iv) the CDR3 of (b) is within a TCR beta chain variable region that specifically binds to SEQ ID NO:37, optionally wherein (b) further comprises a TCR beta chain constant region, optionally wherein the TCR beta chain variable region has an amino acid sequence having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:41.

[0189] Said engineered immune cell may be for use in treatment of solid tumors such as skin melanoma, synovial sarcoma, uterine cancer (uterine corpus endometrial carcinoma and uterine carcinosarcoma), ovarian cancer, uveal melanoma or lung squamous cell carcinoma.

[0190] Said engineered immune cell, wherein said immune cell is knocked out for the endogenous TCR.

[0191] Said knocking out of said endogenous TCT may be performed by using gene editing technology such as CRIPR / CAS9 technology or TALENS.

[0192] Said engineered immune cell, wherein said T cell also expresses the endogenous TCR.

[0193] In a further aspect the present invention provides a composition comprising

[0194] A)

[0195] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0196] II) engineered immune cells expressing a) said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0197] III) engineered immune cells expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens;

[0198] Or

[0199] B)

[0200] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0201] II) engineered immune cells expressing a) said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens; or

[0202] C)

[0203] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0204] II) engineered immune cells expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens. Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0205] Said antigen having said first epitope may be a soluble antigen.

[0206] Said composition, wherein said antigen of said first epitope and said antigen of said second epitope are expressed by the same target cell.

[0207] In one embodiment of the invention, the immune cells of said composition expressing both said CAR and said tTCR may be CD8+ T cells, and said immune cells of said composition expressing solely said CAR may be CD4+ T cells or CD8+ T cells, and said immune cells of said composition expressing solely said tTCR may be CD8+ T cells.

[0208] In one embodiment of the invention, the immune cells of said composition expressing both said CAR and said tTCR may be CD4+ T cells and CD8+ T cells, and said immune cells of said composition expressing solely said CAR may be CD4+ T cells and CD8+ T cells, and said immune cells of said composition expressing solely said tTCR may be CD8+ T cells.

[0209] Said composition comprising

[0210] A)

[0211] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen that is specific for an antigen expressed on the surface of a target cell, or is a soluble antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0212] II) engineered immune cells expressing a) said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen that is specific for an antigen expressed on the surface of a target cell, or is a soluble antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0213] III) engineered immune cells expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, 1 wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens;

[0214] Or

[0215] B)

[0216] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen that is specific for an antigen expressed on the surface of a target cell, or is a soluble antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0217] II) engineered immune cells expressing a) said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen that is specific for an antigen expressed on the surface of a target cell, or is a soluble antigen ii) a transmembrane domain iii) an intracellular signaling domain, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens; or

[0218] C)

[0219] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen that is specific for an antigen expressed on the surface of a target cell, or is a soluble antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0220] II) engineered immune cells expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens. Said composition, wherein said antigen of said first epitope and said antigen of said second epitope are expressed by the same target cell.

[0221] Said composition, wherein said soluble antigen may be an antigen secreted by said same target cell.

[0222] Said composition, wherein engineered immune cells expressing said CAR does not expresses a transgenic TCR, and wherein said engineered immune cells expressing said transgenic TCR does not expresses a CAR.

[0223] Said composition, wherein said engineered immune cell expressing said CAR and said tTCR expresses said CAR and said TCR constitutively.

[0224] Said composition, wherein said tTCR is expressed constitutively and said CAR may be expressed inducibly by a synthetic drug-inducible expression system or by an antigen inducible expression system.

[0225] Said composition, wherein said immune cells may be T cells.

[0226] Said composition, wherein in A) said engineered T cells of I) may be CD4+ and / or CD8+ T cells, said engineered T cells of II) may be CD4+ and / or CD8+ T cells, and said engineered T cells of III) may be CD4+ and / or CD8+ T cells, or wherein in B) said engineered T cells of I) may be CD4+ and / or CD8+ T cells, and said engineered T cells of II) may be CD4+ and / or CD8+ T cells, or wherein in C) said engineered T cells of I) may be CD4+ and / or CD8+ T cells, and said engineered T cells of II) may be CD4+ and / or CD8+ T cells.

[0227] Said composition, wherein said antigen having said second epitope is a peptide of an intracellular antigen processed and presented in a major histocompatibility complex (MHC) class I-dependent manner.

[0228] Said composition, wherein said CAR is specific for a first epitope of an antigen, e.g. a TAA, and wherein said tTCR has specificity for an epitope of the antigen ANPM1.

[0229] Said composition, wherein said antigen binding domain is specific for a first epitope of an antigen, e.g. a TAA, and wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0230] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1).

[0231] Said composition, wherein said CAR is specific for a first epitope of an antigen, e.g. a TAA, and wherein said tTCR having specificity for an epitope of the antigen ANPM1 comprises:

[0232] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0233] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1), wherein the CDR3 of (a) is within a TCR a chain variable region having at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:7, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 5; and optionally wherein (a) comprises a TCR alpha chain constant region, and optionally wherein the TCR alpha chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 9 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 10; and / or wherein the CDR3 of (b) is within a TCR beta chain variable region having at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 8, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 6; and optionally wherein (b) comprises a TCR beta chain constant region, and optionally wherein the TCR beta chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 11 and the TCR P chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 12.

[0234] Said composition, wherein said CAR is specific for an epitope of the antigen CD33 and wherein said tTCR has specificity for an epitope of the antigen ANPM1.

[0235] Said composition, wherein said antigen binding domain specific for said epitope of the antigen CD33 comprises SEQ ID NO:2, and wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:

[0236] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0237] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1). Said composition may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML).

[0238] Said composition, wherein said CAR is specific for an epitope of the antigen BCMA and wherein said tTCR has specificity for an epitope of the antigen BOB1, wherein antigen binding domain of the CAR comprises the sequences specific for BCMA as disclosed herein, and wherein the tTRC comprises the sequences having specificity for BOB1 as disclosed herein.

[0239] Said composition may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or diffuse large B cell lymphoma (DLBCL).

[0240] Said composition, wherein said CAR is specific for an epitope of the antigen CD 19 and wherein said tTCR has specificity for an epitope of the antigen BOB1, wherein antigen binding domain of the CAR comprises the sequences specific for CD 19 as disclosed herein, and wherein the tTRC comprises the sequences having specificity for BOB 1 as disclosed herein.

[0241] Said composition may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or diffuse large B cell lymphoma (DLBCL).

[0242] Said composition, wherein said CAR is specific for an epitope of the antigen CD20 and wherein said tTCR has specificity for an epitope of the antigen BOB1, wherein antigen binding domain of the CAR comprises the sequences specific for CD20 as disclosed herein, and wherein the tTRC comprises the sequences having specificity for BOB 1 as disclosed herein.

[0243] Said composition may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or diffuse large B cell lymphoma (DLBCL).

[0244] Said composition, wherein said CAR is specific for an epitope of the antigen CD22 and wherein said tTCR has specificity for an epitope of the antigen BOB1, wherein antigen binding domain of the CAR comprises the sequences specific for CD22 as disclosed herein, and wherein the tTRC comprises the sequences having specificity for BOB 1 as disclosed herein. Said composition may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), Acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or diffuse large B cell lymphoma (DLBCL).

[0245] Said composition, wherein said CAR is specific for an epitope of the antigen CD123 and wherein said tTCR has specificity for an epitope of the antigen ANPM1, wherein antigen binding domain of the CAR comprises the sequences specific for CD123 as disclosed herein, and wherein the tTRC comprises the sequences having specificity for ANPM1 as disclosed herein.

[0246] Said composition may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML).

[0247] Said composition, wherein said CAR is specific for an epitope of the antigen CLEC12a and wherein said tTCR has specificity for an epitope of the antigen ANPM1, wherein antigen binding domain of the CAR comprises the sequences specific for CLEC12a as disclosed herein, and wherein the tTRC comprises the sequences having specificity for ANPM1 as disclosed herein.

[0248] Said composition may be for use in treatment of hematological malignancies such as myeloid malignancies such as acute myeloid leukemia (AML).

[0249] Said composition, wherein said CAR is specific for an epitope of the antigen FRa and wherein said tTCR has specificity for an epitope of the antigen PRAME, wherein antigen binding domain of the CAR comprises the sequences specific for FRa as disclosed herein, and wherein the tTRC comprises the sequences having specificity for PRAME as disclosed herein.

[0250] Said composition may be for use in treatment of solid tumors such as skin melanoma, synovial sarcoma, uterine cancer (uterine corpus endometrial carcinoma and uterine carcinosarcoma), ovarian cancer, uveal melanoma or lung squamous cell carcinoma.

[0251] Said composition, wherein said CAR is specific for an epitope of the antigen CSPG4 and wherein said tTCR has specificity for an epitope of the antigen PRAME, wherein antigen binding domain of the CAR comprises the sequences specific for CSPG4 as disclosed herein, and wherein the tTRC comprises the sequences having specificity for PRAME as disclosed herein. Said composition may be for use in treatment of solid tumors such as skin melanoma, synovial sarcoma, uterine cancer (uterine corpus endometrial carcinoma and uterine carcinosarcoma), ovarian cancer, uveal melanoma or lung squamous cell carcinoma.

[0252] Said composition, wherein said composition is composition A, and wherein said composition A is obtained by the methods as disclosed herein.

[0253] Said composition, wherein said composition is composition B, and wherein said composition B is obtained by the methods as disclosed herein.

[0254] Said composition, wherein said composition is composition C, and wherein said composition C is obtained by the methods as disclosed herein.

[0255] In a further aspect the present invention provides an engineered immune cell comprising nucleic acid sequences comprising a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, b) a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens.

[0256] Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0257] Said antigen having said first epitope may be a soluble antigen.

[0258] Said engineered immune, wherein said CAR and said tTCR are constitutively expressed in said immune cell.

[0259] Said engineered immune cell, wherein said engineered immune cell has stably integrated the nucleic acid sequence encoding said CAR and the nucleic acid sequence encoding said transgenic TCR into the genome of said immune cell.

[0260] Said immune cell, wherein said immune cell is a T cell. Said engineered immune cell, wherein said first nucleic acid sequence and said second nucleic acid sequence are on two separate nucleic acid sequences, wherein said first nucleic acid sequence comprises additionally a constitutive promotor operatively linked to said nucleic sequence encoding said CAR, and wherein said second nucleic acid sequence additionally comprises a constitutive promoter operatively linked to the nucleic acid sequence encoding said tTCR.

[0261] Said first and said second nucleic acid sequences may be viral vectors such as retroviral vectors such as lentiviral vectors. Said promoter of said first nucleic acid sequence and / or said promoter of said second nucleic acid sequence may be identical promoters or may be different promoters. Said engineered immune cell, wherein said first nucleic acid sequence and said second nucleic acid sequence are on one nucleic acid sequence, and wherein between said first nucleic acid sequence encoding said CAR and said second nucleic acid sequence encoding said tTCR is a cleavage site.

[0262] Said cleavage site may be e.g. an 2A element.

[0263] In a further aspect the present invention provides a composition comprising

[0264] A)

[0265] I) engineered immune cells comprising nucleic acid sequences comprising a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0266] II) engineered immune cells comprising said first nucleic acid sequence encoding said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0267] III) engineered immune cells comprising said second nucleic acid sequence encoding said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens;

[0268] Or B)

[0269] I) engineered immune cells comprising nucleic acid sequences comprising a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0270] II) engineered immune cells comprising said first nucleic acid sequence encoding said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens; or

[0271] C)

[0272] I) engineered immune cells comprising nucleic acid sequences comprising a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0273] II) engineered immune cells comprising said second nucleic acid sequence encoding said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens;

[0274] Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0275] Said antigen having said first epitope may be a soluble antigen. Said composition, wherein said first nucleic acid sequence and said second nucleic acid sequence of said immune cells of I) are on two separate nucleic acid sequences, wherein said first nucleic acid sequence comprises additionally a constitutive promotor operatively linked to said nucleic sequence encoding said CAR, and wherein said second nucleic acid sequence additionally comprises a constitutive promoter operatively linked to the nucleic acid sequence encoding said tTCR., and wherein said first nucleic acid sequence of said immune cell of II) comprises additionally a constitutive promotor operatively linked to said nucleic sequence encoding said CAR, and wherein said second nucleic acid sequence of said immune cell of III) comprises additionally a constitutive promotor operatively linked to said nucleic sequence encoding said TCR.

[0276] Said first and said second nucleic acid sequences may be viral vectors such as retroviral vectors such as lentiviral vectors. Said promoter of said first nucleic acid sequence and / or said promoter of said second nucleic acid sequence may be identical promoters or may be different promoters.

[0277] In a further aspect the present invention provides an in-vitro method for the generation of a composition, wherein the composition comprises

[0278] A) engineered immune cells co-expressing a CAR and a transgenic TCR,

[0279] B) engineered immune cells expressing said CAR, and

[0280] C) engineered immune cells expressing said transgenic TCR, the method comprising the step: transducing simultaneously immune cells such as T cells a) with a first viral vector comprising a first nucleic acid sequence comprising encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) with a second viral vector comprising a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens, thereby obtaining said composition.

[0281] Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells. Said antigen having said first epitope may be a soluble antigen.

[0282] Said in-vitro method, wherein said immune cells are T cells.

[0283] Said in-vitro method, wherein composition obtained by said method also comprises nontransduced immune cells such as non-transduced T cells.

[0284] Said in-vitro method, wherein the multiplicity of infection MOI ratio of said first viral vector and said second viral vector used for the transduction step is between 1 : 10 and 10: 1.

[0285] Said method, wherein said MOI ratio is between 1 :5 and 5: 1, between 1 :2 and 2: 1, or is about 1 : 1.

[0286] Said method, wherein said MOI ratio is 1 : 10, 1 :9, 1 :8, 1 :7: 1 :6, 1 :5; 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

[0287] Said method, wherein said method is an automated method.

[0288] Said method, wherein said method is performed in a closed system

[0289] Said method, wherein said method is an automated method performed in a closed system.

[0290] In one embodiment of the invention the invention provides an in-vitro method for the generation of a composition, wherein the composition comprises

[0291] A) engineered immune cells co-expressing a CAR and a transgenic TCR,

[0292] B) engineered immune cells expressing said CAR, and

[0293] C) engineered immune cells expressing said transgenic TCR, the method comprising the steps: a) providing a sample comprising T cells b) enrichment of said T cells from said sample comprising T cells c) activation of the enriched T cells d) genetic modification of the activated T cells by transducing simultaneously said activated T cells

[0294] A) with a first viral vector comprising a first nucleic acid sequence comprising encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and B) with a second viral vector comprising a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens, and e) optionally expanding said genetically modified T cells, thereby obtaining said composition.

[0295] Said sample comprising T cells may comprise CD4+ T cells and CD8+ T cells.

[0296] Said enrichment of T cells may be the enrichment of CD4+ T cells.

[0297] Said enrichment of T cells may be the enrichment of CD8+ T cells.

[0298] Said enrichment of T cells may be the enrichment of CD4+ T cells and CD8+ T cells.

[0299] Activation of the enriched T cells may be with modulatory agents.

[0300] In another aspect the present invention provides an in-vitro method for the generation of a composition of engineered immune cells co-expressing a CAR and a tTCR, the method comprising

[0301] A) transducing or electroporating immune cells of a sample comprising said immune cells with a viral vector comprising a nucleic acid sequence comprising a) first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, b) a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein between said first nucleic acid and said second nucleic acid is a cleavage site, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens; or

[0302] B) a) transducing or electroporating immune cells of a sample comprising immune cells with

[0303] I) a first viral vector comprising a nucleic acid sequence comprising a) first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0304] II) a second viral vector comprising a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens, and optionally b) enriching the engineered immune cells that co-express said CAR and said tTCR.

[0305] Said enrichment of engineered immune cells that co-express said CAR and said tTCR may be performed e.g. by fluorescence activated cell sorting (FACS).

[0306] Said method, wherein between said first nucleic acid and said second nucleic acid of said viral vector of A) is a cleavage site such as a 2A element.

[0307] Said method, wherein said nucleic acid sequence(s) is is / are lentiviral vector(s).

[0308] In another aspect the present invention provides an in-vitro method for the generation of a composition, wherein the composition comprises

[0309] I) immune cells co-expressing a CAR and a transgenic TCR,

[0310] II) immune cells expressing said CAR, the method comprising the step:

[0311] A) transducing or electroporating immune cells of a sample comprising said immune cells with a viral vector comprising a nucleic acid sequence comprising a) first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, b) a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein between said first nucleic acid and said second nucleic acid is a cleavage site, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens;

[0312] B) transducing or electroporating immune cells of a sample comprising immune cells with a viral vector comprising a nucleic acid sequence comprising a) said first nucleic acid sequence encoding said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0313] C) combining the engineered immune cells of steps A) and step B) in a ratio of between 10:1 to 1:10.

[0314] Said method, wherein said ratio is between 1:5 and 5:1, between 1:2 and 2:1, or is about 1:1. Said method, wherein said ratio is 1:10, 1:9, 1:8, 1:7: 1:6, 1:5; 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0315] In a further aspect the present invention provides an in-vitro method for the generation of a composition, wherein the composition comprises

[0316] A) immune cells co-expressing a CAR and a transgenic TCR,

[0317] B) immune cells expressing said transgenic TCR, the method comprising the step:

[0318] A) transducing or electroporating immune cells of a sample comprising said immune cells with a viral vector comprising a nucleic acid sequence comprising a) first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, b) a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein between said first nucleic acid and said second nucleic acid is a cleavage site, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens;

[0319] B) transducing or electroporating immune cells of a sample comprising immune cells with a viral vector comprising a nucleic acid sequence comprising said second nucleic acid sequence encoding said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, and

[0320] C) combining the engineered immune cells of steps A) and step B) in a ratio of between 10:1 to 1:10.

[0321] Said method, wherein said ratio is between 1:5 and 5:1, between 1:2 and 2:1, or is about 1:1. Said method, wherein said ratio is 1:10, 1:9, 1:8, 1:7: 1:6, 1:5; 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0322] Said antigen having said first epitope may be a soluble antigen.

[0323] In another aspect the present invention provides a pharmaceutical composition comprising engineered T cells expressing the CAR and the TCR as disclosed herein, and optionally a pharmaceutical acceptable carrier.

[0324] In another aspect the present invention provides a pharmaceutical composition comprising engineered T cells comprising nucleic acid sequences encoding a CAR and a TCR as disclosed herein, and optionally a pharmaceutical acceptable carrier.

[0325] In a further aspect the present invention provides a pharmaceutical composition comprising a composition as disclosed herein, and optionally a pharmaceutical acceptable carrier.

[0326] In one aspect the present invention provides an engineered immune cell for use in immune cell therapy, wherein said engineered immune cell expresses a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens.

[0327] Said immune cell therapy may be a treatment of a disease such as cancer, an autoimmune disease or an infectious disease.

[0328] Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0329] Said antigen having said first epitope may be a soluble antigen.

[0330] In one aspect the present invention provides a composition for use in immune therapy, wherein said composition comprises A)

[0331] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0332] II) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0333] III) engineered immune cells expressing a transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens;

[0334] Or

[0335] B)

[0336] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and

[0337] II) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens; or c)

[0338] I) engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, and II) engineered immune cells expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens.

[0339] In another aspect the present invention provides an in-vivo method for treating a disease in a subject in need thereof comprising

[0340] I) administering to said subject an engineered immune cell expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens.

[0341] Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0342] Said antigen having said first epitope may be a soluble antigen.

[0343] In a further aspect the present invention provides an in-vivo method for treating a disease in a subject in need thereof comprising

[0344] A)

[0345] I) administering to said subject engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens,

[0346] II) administering an engineered immune cell expressing a) said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0347] III) administering an engineered immune cell expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen,

[0348] Or

[0349] B)

[0350] I) administering to said subject engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens,

[0351] II) administering engineered immune cells expressing a) said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain, or

[0352] C)

[0353] I) administering to said subject engineered immune cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) said transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens, II) administering engineered immune cell expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen.

[0354] Said antigen having said first epitope and said second antigen having said second epitope may be expressed or presented on the surface of the same target cell or on the surfaces of different target cells.

[0355] Said antigen having said first epitope may be a soluble antigen.

[0356] Said in-vivo method, wherein said administration of the immune cells of A) I), II) and III) is simultaneously.

[0357] Said in-vivo method, wherein said administration of the immune cells of B) I) and II) is simultaneously.

[0358] Said in-vivo method, wherein said administration of the immune cells of C) I) and II) is simultaneously.

[0359] Said in-vivo method, wherein said administration of the immune cells of A) I), II) and III) is subsequently in the order: I), II), III) or I), III), II) or II), I), III) or II), III), I) or III), II), I) or

[0360] III), II), I).

[0361] Said in-vivo method, wherein said administration of the immune cells of B) I) and II) is subsequently in the order: I), II), or II), I).

[0362] Said in-vivo method, wherein said administration of the immune cells of C) I) and II) is subsequently in the order: I), II), or II), I).

[0363] All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.

[0364] Definitions

[0365] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0366] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0367] A T cell receptor (TCR) is a molecule found on the surface of T cells (T lymphocytes) that is responsible for recognizing a peptide that is bound to (presented by) a major histocompatibility complex (MHC) molecule on a target cell. HLA-A*02:01 is a globally common human leukocyte antigen serotype within the HLA-A serotype group. Peptides that are presented by HLA-A*02:01 to TCRs are described as being “HLA-A*02:01 restricted”.

[0368] HLA-B*07:02 is a globally common human leukocyte antigen serotype within the HLA-B serotype group. Peptides that are presented by HLA-B*07:02 to TCRs are described as being “HLA-B*07:02” restricted”.

[0369] The TCR is composed of two different polypeptide chains. In humans, 95% of TCRs consist of an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB respectively). When the TCR engages with peptide in the context of HLA (e.g. in the context of HLA-A*02:01), the T cell is activated through signal transduction.

[0370] The alpha and beta chains of the TCR are highly variable in sequence. Each chain is composed of two extracellular domains, a variable region (V) and a constant region (C). The constant region is proximal to the T cell membrane followed by a transmembrane region and a short cytoplasmic tail while the variable region binds to the peptide / HLA-A complex.

[0371] The variable region of each chain has three hypervariable regions (also called complementarity determining regions (CDRs)). Accordingly, the TCR alpha chain comprises a CDR1, a CDR2 and a CDR3 and the TCR beta chain also comprises a (different) CDR1, CDR2, and a CDR3. In each of the alpha and beta chains, it is CDR3 that is mainly responsible for recognizing the peptide being presented by HLA-A.

[0372] The peptide CLAVEEVSL (SEQ ID NO: 1) may be presented by HLA-A*02:01 (i.e. may be HLA-A*02:01 restricted).

[0373] The peptide APAPTAVVL (SEQ ID NO: 14) may be presented by HLA-B*07:02 (i.e. may be HLA-B*07:02 restricted).

[0374] The peptide SLLQHLIGL (SEQ ID NO:37) may be presented by HLA-A*02:01 (i.e. may be HLA-A*02:01 restricted).

[0375] Nucleophosmin (NPM1) is a driver gene that is frequently mutated in approximately 30% of patients with AML. Mutated NPM1 has also been observed in other types of hematological malignancies (e.g. other myeloid malignancies), although frequencies in tumors other than AML are much lower.

[0376] CD33 or Siglec-3 is a transmembrane receptor expressed on cells of myeloid lineage. It is usually considered myeloid-specific, but it can also be found on some lymphoid cells. CD33 is the target of gemtuzumab ozogamicin an antibody-drug conjugate (ADC) for the treatment of patients with acute myeloid leukemia. The intracellular transcription factor B cell Oct binding protein 1 (Bobl) encoded by gene POU2AF1 was identified as a suitable target for TCR-based immunotherapies of B cell malignancies and multiple myeloma. The Bobl polypeptides may be used as immunogens, or targets for immunotherapy.

[0377] B-cell maturation antigen (BCMA or BCM), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a protein that in humans is encoded by the TNFRSF17 gene. This receptor is preferentially expressed in mature B lymphocytes, and may be important for B cell development and autoimmune response. This receptor has been shown to specifically bind to the tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B / TALL- 1 / BAFF). TNFRSF17 is implicated in leukemia, lymphomas, and multiple myeloma.

[0378] CD 19 is widely expressed during all phases of B cell development until terminal differentiation into plasma cells. During B cell lymphopoiesis, CD 19 surface expression starts during immunoglobulin (Ig) gene rearrangement, which coincides during B lineage commitment from hematopoietic stem cell. Throughout development, the surface density of CD19 is highly regulated. CD 19 expression in mature B cells is threefold higher than that in immature B cells. CD 19 is expressed on all normal, mitogen-stimulated, and malignant B cells, excluding plasma cells. CD 19 expression is even maintained in B lineage cells that undergo neoplastic transformation. Because of its ubiquity on all B cells, it can function as a B cell marker and a target for immunotherapies targeting neoplastic lymphocytes.

[0379] CD20 is expressed on the surface of all B-cells beginning at the pro-B phase (CD45R+, CD117+) and progressively increasing in concentration until maturity.

[0380] CD20 is the target of the monoclonal antibodies rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, and ublituximab, which are all active agents in the treatment of all B cell lymphomas, leukemias, and B cell-mediated autoimmune diseases. CD22 is a molecule belonging to the SIGLEC family of lectins- It is found on the surface of mature B cells and to a lesser extent on some immature B cells. Because CD22 is restricted to B cells, it is an excellent target for immunotherapy of B cell malignancies.

[0381] The interleukin-3 receptor (CD123) is a molecule found on cells which helps transmit the signal of interleukin-3, a soluble cytokine important in the immune system. CD123 is expressed across acute myeloid leukemia (AML) subtypes, including leukemic stem cells.

[0382] CLEC12a gene encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. In the immunotherapy of acute myeloid leukemia (AML), CLEC12a becomes one of the target due to its high expression in AML cells while being absent in normal hematopoietic stem cells. CLEC12a is also expressed on the surface of leukemic stem cells (LSC), which possesses the ability to indefinitely self-renew, produce plenty of leukemic cells and are associated with leukemia relapses.

[0383] Folate Receptor alpha (FRa) is responsible for binding to folic acid and its derivatives, which becomes crucial during fetal development. By adding folate supplementation during pregnancy, neural tube defects in the fetus are prevented. FRa, due to its high expression in some tumors, is an attractive therapeutic target for the development of novel anti-cancer agents in order to limit toxic side-effects on off-target tissues. FRa can be overexpressed by a number of epithelial-derived tumors including ovarian, breast, renal, lung, colorectal, and brain.

[0384] CSPG4 plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. It represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells.

[0385] The Preferentially Expressed Antigen of Melanoma (PRAME) gene is expressed at a high level in a large proportion of tumors, including melanomas, non-small-cell lung carcinomas, renal cell carcinoma (RCC), breast carcinoma, cervix carcinoma, colon carcinoma, sarcoma, neuroblastoma, as well as several types of leukemia.

[0386] In general, a CAR may comprise an extracellular domain (extracellular part) comprising the antigen binding domain (that may be specific for an epitope of the antigen), a transmembrane domain and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker or spacer. The extracellular domain may also comprise a signal peptide.

[0387] A "signal peptide" refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0388] Generally, an “antigen binding domain” refers to the region of the CAR that specifically binds to an antigen (to an epitope of the antigen), e.g. to a tumor associated antigen (TAA) or tumor specific antigen (TSA). The CARs of the invention may comprise one or more antigen binding domains (e.g. a tandem CAR). Generally, the targeting regions on the CAR are extracellular. The antigen binding domain may comprise an antibody or an antigen binding fragment thereof. The antigen binding domain may comprise, for example, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, divalent single chain antibodies or diabodies. Any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain. Often the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4 / S)3 -linker”.

[0389] In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will be used in. For example, when it is planned to use it therapeutically in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human or humanized antibody or antigen binding fragment thereof. Human or humanized antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art.

[0390] “Spacer” or “hinge” as used herein refers to the hydrophilic region which is between the antigen binding domain and the transmembrane domain. The CARs of the invention may comprise an extracellular spacer domain but is it also possible to leave out such a spacer. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge. The transmembrane domain of the CAR may be derived from any desired natural or synthetic source for such domain. When the source is natural the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived for example from CD8alpha or CD28. When the key signaling and antigen recognition modules (domains) are on two (or even more) polypeptides then the CAR may have two (or more) transmembrane domains. The splitting key signaling and antigen recognition modules enable for a small molecule-dependent, titratable and reversible control over CAR cell expression (e.g. WO2014127261A1) due to small molecule-dependent heterodimerizing domains in each polypeptide of the CAR.

[0391] The cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR, otherwise it is indicated explicitly as an inhibitory CAR (iCAR)). "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions.

[0392] Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement.

[0393] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co-stimulatory signaling domain). Therefore, an intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0394] Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs).

[0395] Examples of IT AM containing primary cytoplasmic signaling domains often used in CARs are that those derived from TCR^ (CD3Q, FcRgamma, FcRbeta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence derived from CD3^.

[0396] The cytoplasmic domain of the CAR may be designed to comprise the CD3^ signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3^ chain portion and a co-stimulatory signaling region (domain). The co-stimulatory signaling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples for a co-stimulatory molecule are CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3.

[0397] The cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo- or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet. As an example, the cytoplasmic domain may comprise the signaling domain of CD3^ and the signaling domain of CD28. In another example the cytoplasmic domain may comprise the signaling domain of CD3^ and the signaling domain of CD137. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3^, the signaling domain of CD28, and the signaling domain of CD137.

[0398] As aforementioned either the extracellular part or the transmembrane domain or the cytoplasmic domain of a CAR may also comprise a heterodimerizing domain for the aim of splitting key signaling and antigen recognition modules of the CAR.

[0399] The CAR may be further modified to include on the level of the nucleic acid encoding the CAR one or more operative elements to eliminate CAR expressing immune cells by virtue of a suicide switch. The suicide switch can include, for example, an apoptosis inducing signaling cascade or a drug that induces cell death. In one embodiment, the nucleic acid expressing and encoding the CAR can be further modified to express an enzyme such thymidine kinase (TK) or cytosine deaminase (CD). The CAR may also be part of a gene expression system that allows controlled expression of the CAR in the immune cell. Such a gene expression system may be an inducible gene expression system and wherein when an induction agent is administered to a cell being transduced with said inducible gene expression system, the gene expression system is induced and said CAR is expressed on the surface of said transduced cell.

[0400] In some embodiments, the endodomain may contain a primary cytoplasmic signaling domains or a co-stimulatory region, but not both.

[0401] In some embodiment of the invention the CAR may be a “SUPRA” (split, universal, and programmable) CAR, where a “zipCAR” domain may link an intra-cellular costimulatory domain and an extracellular leucine zipper (WO2017 / 091546). This zipper may be targeted with a complementary zipper fused e.g. to an scFv region to render the SUPRA CAR T cell tumor specific. This approach would be particularly useful for generating universal CAR T cells for various tumors; adapter molecules could be designed for tumor specificity and would provide options for altering specificity post-adoptive transfer, key for situations of selection pressure and antigen escape.

[0402] The CARs of the present invention may be designed to comprise any portion or part of the above-mentioned domains as described herein in any order and / or combination resulting in a functional CAR, i.e. a CAR that mediated an immune effector response of the immune effector cell that expresses the CAR as disclosed herein. The term "antibody" as used herein is used in the broadest sense to cover the various forms of antibody structures including but not being limited to monoclonal and polyclonal antibodies (including full length antibodies), multispecific antibodies (e.g. bispecific antibodies), antibody fragments, i.e. antigen binding fragments of an antibody, immunoadhesins and antibody - immunoadhesin chimeras, that specifically recognize (i.e. bind) an antigen. "Antigen binding fragments" comprise a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof (“an antigen binding fragment of an antibody”). Examples of antigen binding fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), single domain antibodies (nanobodies), diabodies, dsFv, Fab’, diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. The antibody or antibody fragment may be human, fully human, humanized, human engineered, non-human, and / or chimeric. The non-human antibody or antibody fragment may be humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Chimeric antibodies may refer to antibodies created through the joining of two or more antibody genes which originally encoded for separate antibodies.

[0403] The terms “having specificity for”, “specifically binds” or “specific for” e.g. with respect to an antigen-binding domain of an antibody, of a fragment thereof or of a CAR refer to an antigenbinding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain is specific.

[0404] As used herein, the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, and combinations thereof, for example a glycosylated protein or a glycolipid. The term “antigen” as used herein refers to a molecular entity that may be expressed e.g. on the surface of a target cell and that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0405] The term “epitope” means the part of an antigen, that may be recognized and specifically bound e.g. by antibodies or antigen bindings fragments thereof (antigen binding domains).

[0406] The terms “immune cell” or “immune effector cell” may be used interchangeably and refer to a cell that may be part of the immune system and executes a particular effector function such as alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, regulatory T cells (Treg), monocytes or macrophages. Preferentially these immune cells are human immune cells. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells or gamma-delta T cells. Most preferred immune effector cells are T cells and NK cells. Tumor infiltrating lymphocytes (TILs) are T cells that have moved from the blood of a subject into a tumor. These TILs may be removed from a patient's tumor by methods well known in the art, e.g. enzymatic and mechanic tumor disruption followed by density centrifugation and / or cell marker specific enrichment. TILs are genetically engineered as disclosed herein, and then given back to the patient. "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.

[0407] Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. Cancer immunotherapy as an activating immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses cell-based, preferentially T cell-based or NK cell-based cytotoxic responses to attack cancer cells. T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in-vitro and then transferred back into the cancer patient. Then the immunotherapy is referred to as “CAR cell immunotherapy” or in case of use of T cells only as “CAR T cell therapy” or “CAR T cell immunotherapy”.

[0408] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0409] The term “autologous” as used herein refers to any material derived from the same subject to who it is later re-introduced.

[0410] The term “allogeneic” as used herein refers to any material derived from a different subject of the same species as the subject to who the material is re-introduced. The terms “therapeutically effective amount” or “therapeutically effective population” mean an amount of a cell population which provides a therapeutic benefit in a subject.

[0411] As used herein, the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the subject is a human. The subject may be a subject suffering from a disease such as cancer (a patient) or from an autoimmune disease or from a allergic disease or from an infectious disease or from graft rejection.

[0412] The term "expression" as used herein is defined as the transcription of a particular nucleotide sequence into RNA and optionally subsequent translation of said RNA into a polypeptide sequence or a protein.

[0413] The term “transduction” means the transfer of genetic material from a viral agent such as a lentiviral vector particle into a eukaryotic cell such as a T cell.

[0414] By way of example only, the viral agent may be a viral vector, such as a retroviral vector or a lentiviral vector. Adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus, herpes virus, minicircle vectors may also be used.

[0415] The term “electroporation” is a technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell.

[0416] A transgene may be a gene that has been transferred by genetic engineering techniques into a host that normally does nor bear this gene. The gene may be a naturally gene that occurs in other cells or may be a recombinant gene. Most prominent transgenes used in the present invention may be the T cell receptor and the chimeric antigen receptor.

[0417] The terms “engineered cell” and “genetically modified cell” as used herein can be used interchangeably. The terms mean containing and / or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.

[0418] The term “cancer” is known medically as a malignant neoplasm. Cancer is a broad group of diseases involving unregulated cell growth and includes all kinds of leukemia. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans.

[0419] The terms “nucleic acid”, “nucleic acid sequence” or “polynucleotide” as used interchangeably herein refer to polymers of nucleotides. Polynucleotides, which can be hydrolyzed into monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, the term “polynucleotides” encompasses, but is not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0420] A constitutive promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product such as a CAR and / or a tTCR, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. Then the cell expresses said transgene(s) constitutively.

[0421] The term “closed system” as used herein refers to any closed system which reduces the risk of cell culture contamination while performing culturing processes such as the introduction of new material, e.g. by transduction, and performing cell culturing steps such as proliferation, differentiation, activation, separation of cells, and / or electroporation if an in-line electroporation unit is connected. Such a system allows to operate under GMP or GMP-like conditions (“sterile”) resulting in cell compositions which are clinically applicable. Herein exemplarily the CliniMACS Prodigy® (Miltenyi Biotec B.V. & Co. KG, Germany) connected to the CliniMACS® Electroporator (Miltenyi Biotec B.V. & Co. KG, Germany) is used as a closed system. The CliniMACS Prodigy® is disclosed in W02009 / 072003. But it is not intended to restrict the use of the method of the present invention to the CliniMACS Prodigy®. The process of the invention may be performed in a closed system, comprising a centrifugation chamber comprising a base plate and cover plate connected by a cylinder, pumps, valves, a magnetic cell separation column and a tubing set. The blood samples or other sources comprising T cells may be transferred to and from the tubing set by sterile docking or sterile welding. A suitable system is disclosed in W02009 / 072003.

[0422] The closed system may comprise a plurality of tubing sets (TS) where cells are transferred between TS by sterile docking or sterile welding.

[0423] Different modules of the process may be performed in different functionally closed TS with transfer of the product (cells) of one module generated in the one tubing set to another tubing set by sterile means. For example, T cells can be magnetically enriched in a first tubing set (TS) TS100 by Miltenyi Biotec and the positive fraction containing enriched T cells is welded off the TS100 and welded onto a second tubing set TS730 by Miltenyi Biotec for further activation, modification, cultivation and washing.

[0424] The terms “automated method” or “automated process” as used herein refer to any process being automated through the use of devices and / or computers and computer software. Methods (processes) that have been automated require less human intervention and less human time. In some instances the method of the present invention is automated if at least one step of the present method is performed without any human support or intervention. Preferentially the method of the present invention is automated if all steps of the method as disclosed herein are performed without human support or intervention other than connecting fresh reagents to the system. Preferentially the automated process is implemented on a closed system such as CliniMACS Prodigy® as disclosed herein.

[0425] The closed system may comprise a) a sample processing unit comprising an input port and an output port coupled to a rotating container (or centrifugation chamber) having at least one sample chamber, wherein the sample processing unit is configured to provide a first processing step to a sample or to rotate the container so as to apply a centrifugal force to a sample deposited in the chamber and separate at least a first component and a second component of the deposited sample; and b) a sample separation unit coupled to the output port of the sample processing unit, the sample separation unit comprising a separation column holder, a pump, and a plurality of valves configured to at least partially control fluid flow through a fluid circuitry and a separation column positioned in the holder, wherein the separation column is configured to separate labeled and unlabeled components of sample flown through the column.

[0426] Said rotating container may also be used as a temperature controlled cell incubation and cultivation chamber (CentriCult Unit = CCU). This chamber may be flooded with defined gas mixes, provided by an attached gas mix unit (e.g. use of pressurized air / N2 / CO2 or N2 / CO2 / O2).

[0427] All agents may be connected to the closed system before process initiation. This comprises all buffers, solutions, cultivation media and supplements, MicroBeads, used for washing, transferring, suspending, cultivating, harvesting cells or immunomagnetic cell sorting within the closed system. Alternatively, such agents might by welded or connected by sterile means at any time during the process.

[0428] The cell sample comprising T cells may be provided in transfer bags or other suited containers which can be connected to the closed system by sterile means.

[0429] The term “providing a (cell) sample comprising T cells” means the provision of a cell sample, preferentially of a human cell sample of hematologic origin. Normally, the cell sample may be composed of hematologic cells from a donor or a patient. Such blood product can be in the form of whole blood, buffy coat, leukapheresis, PBMCs or any clinical sampling of blood product. It may be from fresh or frozen origin.

[0430] The modulatory agents may be selected from the group consisting of agonistic antibodies or antigen binding fragment thereof, cytokines, recombinant costimulatory molecules and small drug inhibitors. Said modulatory agents may be anti-CD3 and anti-CD28 antibodies or antigenbinding fragments thereof coupled to beads or nanostructures. The modulatory agents may be a nanomatrix, the nanomatrix comprising a) a matrix of mobile polymer chains, and b) attached to said matrix of mobile polymer chains anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof, wherein the nanomatrix is 1 to 500 nm in size. The anti-CD3 and anti-CD28 antibodies or antigen-binding fragments thereof may be attached to the same or to separate matrices of mobile polymer chains. If the anti-CD3 and anti-CD28 antibodies or antigenbinding fragments thereof may be attached to separate matrices of mobile polymer chains, finetuning of nanomatrices for the stimulation of the T cells may be possible. The nanomatrix may be biodegradable. The nanomatrix may be of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. A polysaccharide may include for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum or alginate.

[0431] The term “particle” as used herein refers to a solid phase such as colloidal particles, microspheres, nanoparticles, or beads. Methods for generation of such particles are well known in the field of the art. The particles may be magnetic particles. The particles may be in a solution or suspension or they may be in a lyophilised state prior to use in the present invention. The lyophilized particle is then reconstituted in convenient buffer before contacting the sample to be processed regarding the present invention.

[0432] The term “magnetic” in “magnetic particle” as used herein refers to all subtypes of magnetic particles which can be prepared with methods well known to the skilled person in the art, especially ferromagnetic particles, superparamagnetic particles and paramagnetic particles. "Ferromagnetic" materials are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is removed. "Paramagnetic" materials have only a weak magnetic susceptibility and when the field is removed quickly lose their weak magnetism. "Superparamagnetic" materials are highly magnetically susceptible, i.e. they become strongly magnetic when placed in a magnetic field, but, like paramagnetic materials, rapidly lose their magnetism. For enrichment, isolation or selection in principle any sorting technology can be used. This includes for example affinity chromatography or any other antibody-dependent separation technique known in the art. Any ligand-dependent separation technique known in the art may be used in conjunction with both positive and negative separation techniques that rely on the physical properties of the cells. An especially potent sorting technology is magnetic cell sorting. Methods to separate cells magnetically are commercially available e.g. from Invitrogen, Stem cell Technologies, in Cellpro, Seattle or Advanced Magnetics, Boston. For example, monoclonal antibodies can be directly coupled to magnetic polystyrene particles like Dynal M 450 or similar magnetic particles and used e.g. for cell separation. The Dynabeads technology is not column based, instead these magnetic beads with attached cells enjoy liquid phase kinetics in a sample tube, and the cells are isolated by placing the tube on a magnetic rack. However, in a preferred embodiment for enriching CD4+ and / or CD8+ T cells from a sample comprising T cells according the present invention monoclonal antibodies or antigen binding fragments thereof are used in conjunction with colloidal superparamagnetic microparticles having an organic coating by e.g. polysaccharides (Magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec B.V. & Co. KG, Germany)). These particles (nanobeads or MicroBeads) can be either directly conjugated to monoclonal antibodies or used in combination with anti-immunoglobulin, avidin or anti-hapten-specific MicroBeads.

[0433] The MACS technology allows cells to be separated by incubating them with magnetic nanoparticles coated with antibodies directed against a particular surface antigen. This causes the cells expressing this antigen to attach to the magnetic nanoparticles. Afterwards the cell solution is transferred on a column placed in a strong magnetic field. In this step, the cells attach to the nanoparticles (expressing the antigen) and stay on the column, while other cells (not expressing the antigen) flow through. With this method, the cells can be separated positively or negatively with respect to the particular antigen(s) / marker(s).

[0434] In case of a positive selection the cells expressing the antigen(s) of interest, which attached to the magnetic column, are washed out to a separate vessel, after removing the column from the magnetic field.

[0435] In case of a negative selection the antibody used is directed against surface antigen(s) which are known to be present on cells that are not of interest. After application of the cells / magnetic nanoparticles solution onto the column the cells expressing these antigens bind to the column and the fraction that goes through is collected, as it contains the cells of interest. As these cells are non-labelled by an antibody coupled to nanoparticels, they are “untouched”. The procedure can be performed using direct magnetic labelling or indirect magnetic labelling. For direct labelling the specific antibody is directly coupled to the magnetic particle. Indirect labelling is a convenient alternative when direct magnetic labelling is not possible or not desired. A primary antibody, a specific monoclonal or polyclonal antibody, a combination of primary antibodies, directed against any cell surface marker can be used for this labelling strategy. The primary antibody can either be unconjugated, biotinylated, or fluorophore- conjugated. The magnetic labelling is then achieved with anti-immunoglobulin MicroBeads, anti-biotin MicroBeads, or anti -fluor ophore MicroBeads.

[0436] Embodiments

[0437] In one embodiment of the invention the composition comprises

[0438] A)

[0439] I) engineered T cells expressing a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen, wherein said antigen of said first epitope is CD33 and wherein said antigen binding domain comprises SEQ ID NO:2 (scFv), ii) a transmembrane domain such as the transmembrane domain of CD8a iii) an intracellular signaling domain comprising the co-stimulatory domain of 4- IBB and the stimulatory domain of CD3zeta, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said second epitope is ANPM1, and wherein said tTCR comprises:

[0440] (a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and / or

[0441] (b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1),

[0442] II) engineered T cells expressing a) said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and III) engineered T cells expressing said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are different antigens. Said engineered T cells may be for use in treatment of AML.

[0443] In one embodiment of the invention the invention provides an in-vitro method for the generation of a composition, wherein the composition comprises

[0444] A) engineered immune cells co-expressing a CAR and a transgenic TCR,

[0445] B) engineered immune cells expressing said CAR, and

[0446] C) engineered immune cells expressing said transgenic TCR, the method comprising the steps: a) providing a sample comprising T cells b) enrichment of said T cells from said sample comprising T cells c) activation of the enriched T cells d) genetic modification of the activated T cells by transducing simultaneously said activated T cells

[0447] A) with a first viral vector comprising a first nucleic acid sequence comprising encoding a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and

[0448] B) with a second viral vector comprising a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens, and e) optionally expanding said genetically modified T cells, thereby obtaining said composition.

[0449] Said enriched T cells may be CD8+ T cells, or CD4+ and CD8+ T cells.

[0450] In another embodiment of the invention the invention said method for the generation of said composition is an automated method performed in a closed system. Examples

[0451] The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.

[0452] Example 1: Genetic modification of T cells with lentiviral particles encoding a CAR or a transgenic TCR construct.

[0453] 1.1. Construct design and production of lentiviral particles

[0454] All second-generation CAR constructs used contained a CD8 hinge and transmembrane domain, a 4-1BB co-stimulatory domain and a CD3(^ activation domain. The CD33-CAR (My96 scFv, SEQ ID NO:2), CD123-CAR (scFv: SEQ ID NO:28), Clecl2a (scFv: SEQ ID NO:31), and FolRl-CAR (MORAb-003, SEQ ID NO:34) were subcloned under control of EFla promotor, CD20-CAR (Leul6 scFv, SEQ ID NO:22) and CD19-CAR (FMC63 scFv, SEQ ID NO: 19) were subcloned under control of PGK promoter. The alpha- and beta-chains of ANPM1 and PRAME-TCR (SEQ ID NO:7 and 8, SEQ ID NO:40 and 41) were separated by T2A element and subcloned under control of EFla promoter. Vesicular stomatitis virus glycoprotein (VSV- G)-pseudotyped lentiviral vectors were produced using HEK 293-T cells. For production of CD33 / CD 19 / CD20 / CD123 / Clecl2a / FolRl -CAR- or ANPMl / PRAME-TCR-encodig lentiviral particles, 4E7 cells were seeded in 500 ml medium in a 5-layer Cellstack chamber 72 hours prior to transfection. 109.93 pg VSV-G encoding plasmid, 164,27 pg gag / pol encoding plasmid, 82.13 pg rev encoding plasmid and 98 pg transfer vector plasmid were diluted in 62 ml DMEM without additives and mixed with 62 ml DMEM supplemented with 2.5 ml PEI (1 mg / ml). The transfection mixture was incubated for 20 min at RT. HEK 293-T medium was completely removed from the cells and replaced with 324 ml DMEM without additives. Subsequently, the transfection mixture was carefully added to the cells. After 4 - 6 h 50 ml FCS were added and cultured for 24 h before 10 ml sodium butyrate (500 mM) was additionally added. 48 h after transfection, supernatant was collected, sterile filtrated and concentrated at 4°C for 24 h at 4700 x g. Pelleted lentiviral particles were diluted in PBS and freshly used or stored at -70°C.

[0455] 1.2. Isolation, expansion and modification of T cells

[0456] Unless mentioned to the contrary, kits and reagents were used according to the manufacturer's protocol. All kits and reagents, unless mentioned otherwise, were from Miltenyi Biotec.

[0457] T cells were isolated from freshly isolated PBMC using the human CD8 T cell isolation Kit and cultured in TexMACS supplemented with 12.5 ng / ml recombinant human IL-7 and 12.5 ng / ml recombinant human IL-15. T cells were activated MACS GMP T Cell TransAct with a titer of 1 : 17.5 or T cell TransAct, human with a titer of 1 : 100. For this purpose, 1E6 CD8 T cells per 1 ml were cultured in a 24-well plate for 72 h at 37°C and 5% CO2 atmosphere before the stimulation reagent was removed. T cells were transduced 24 h after activation with frozen, carefully resuspended VSV-G pseudotyped lentiviral particles. In case of co-transduction, lentiviral particles encoding CAR or TCR were added to the T cells simultaneously.

[0458] Example 2: Functionality of genetically modified T cells

[0459] 2.1. Combination of CD33-CAR and ANPM1-TCR

[0460] For all in vitro co-culture experiments, genetically modified T cells were enriched using MACSQuant Tyto. CD33-CAR and ANPM1-TCR were stained and sorted using CD33-CAR Detection Reagent or ANPMiMHC-Tetramer, respectively. After sorting, T cells were expanded for at least five more days (Figure 2 A). For trans stimulation of CD33-CAR+ / ANPM1-TCR+ T cells, 1E4 T cells were co-cultured with 5E4 CAR-target expressing cells (CD33+ wtNPMl 0ci-AML2, CellTrace-labeled / GFP+) and 5E4 TCR-target expressing cells (CD33-ko ANPM1 0ci-AML3, GFP+), while for cis stimulation 1E4 T cells were co-cultured with 1E4 CAR- and TCR-target-expressing cells (CD33+ ANPM1+ 0ci-AML3, GFP+). After 18 hours of coculture in TexMACS without supplements, killing was analyzed using flow cytometry (Figure 2 B and C). Using trans stimulation CD33-CAR+ / dNPMl-TCR + T cells showed superior cytolytic activity towards the TCR-target expressing cells compared to dNPMl-TCR+ T cells Lysis of cells expressing both CAR- and TCR-target (cis stimulation) by CD33-CAR+ / dNPMl- TCR + T cells revealed significant improvement over lysis by dNPMl-TCR+ or CD33-CAR+ T cells.

[0461] In parallel, the same experimental set-up was used for long-term co-culture experiments with repetitive stimulation (Figure 3 A and B). In total, three rounds of target cell addition were performed by removing supernatant from the co-culture and adding 1E4 fresh target cells. Killing of GFP+ target cells was analyzed via live cell imaging using IncuCyte S3 device. After 2ndand 3rdround of restimulation CD33-CAR+ / dNPMl-TCR+ T cells were able to lyse their target cells significantly more efficient than CD33-CAR+ T cells or dNPMl-TCR+ T cells. This suggests not only a higher cytolytic potency of CD33-CAR+ / dNPMl-TCR+ T cells but also a diminished exhaustion profile. 2.2. Combination of CD20-CAR / CD19-CAR and ANPM1-TCR

[0462] CD20-CAR / CD19-CAR and ANPM1-TCR were stained and sorted using anti-ALNGFR antibody (via transduction marker) or ANPMiMHC-Tetramer, respectively. After sorting, T cells were expanded for at least five more days. For trans stimulation of CD20-CAR+ / ANPM1- TCR+ T cells with an E:T ratio of 2: 1, 2E4 T cells were co-cultured with 5E4 CAR-target expressing cells (GFP+ / CellTrace-labeled CD20+ or CD19+ Raji cells) and 5E4 TCR-target expressing cells (ANPM1 0ci-AML3, GFP+). After 18 hours of co-culture in TexMACS without supplements, killing was analyzed using flow cytometry (2 Figure D and E). Lysis of TCR-target cells by CD20-CAR+ / ANPM1-TCR+ T cells and CD19-CAR+ / ANPM1-TCR+ T was significantly improved.

[0463] 2.3. Combination of CD123-CAR / Clecl2a-CAR and ANPM1-TCR

[0464] For cis stimulation of CD123-CAR+ / ANPM1-TCR+ T cells 1E4 T cells were co-cultured with an E:T ratio of 0.2: 1 with CAR- and TCR-target expressing 0ci-AML3 cells. For cis stimulation of Clecl2a-CAR+ / ANPM1-TCR+ T cells 1E4 T cells were co-cultured with an E:T ratio of 0.1 : 1 with CAR- and TCR-target expressing 0ci-AML3 cells. After 48 hour of co-culture in TexMACS without supplements, killing was analyzed using flow cytometry (Figure 5 A and B). Cytoltytic activity of both CD123-CAR+ / ANPM1-TCR+ T cells as well as Clecl2a- CAR+ / ANPM1-TCR+ T cells was clearly enhanced compared to CD123 / Clecl2a-CAR+ cells or ANPM1-TCR+ T cells.

[0465] 2.4. Combination of CD123-CAR / CSPG4 / FolRl CAR and PRAME-TCR

[0466] For cis stimulation of CD123-CAR+ / PRAME-TCR+ T cells 1E4 T cells were co-cultured with an E:T ratio of 1 : 1 with CAR- and TCR-target expressing 0ci-AML2 cells. After 24 hour of co-culture, killing was analyzed using flow cytometry (Figure 6 A). For cis stimulation of CSPG4-CAR+ / PRAME -TCR+ T cells 1E4 T cells were co-cultured with an E:T ratio of 1 : 1 with CAR- and TCR-target expressing Mel256 cells. During 48 hour of co-culture, killing was analyzed via live cell imaging using IncuCyte S3 device (Figure 6 B). CD123 / CSPG4- CAR+ / PRAME-TCR+ T cells showed superior cytolytic activity. For trans stimulation of FolRl-CAR+ / PRAME-TCR+ T cells 1E4 T cells were co-cultured with 0.5E4 CAR-target expressing Ov90 cells and 0.5E4 TCR-target expressing U266 cells with an E:T ratio of 1 : 1. Killing of Ov90 cells was monitored over time via live cell imaging using IncuCyte S3 device (Figure 6 C). After 24 hour of co-culture, killing of U266 cells was analyzed using flow cytometry (Figure 6 D). Lysis of CAR- and TCR-target expressing cells was significantly improved by FolRl-CAR+ / PRAME-TCR+ T cells.

[0467] Example 3: Combination of CAR and TCR in vivo

[0468] Genetically modified T cells were manufactured in large-scale using the automated T Cell Transduction Process on the CliniMACS Prodigy (Figure 4 A, B). I x lO6Oci-AML3 cells expressing luciferase were injected intravenously in six- to eight- week-old female immunodeficient NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) mice. One day prior to T cell injection, mice were randomized based on tumor burden (Figure 4 C). Whole body luminescence (p / s) was measured twice a week via an IVIS Lumina III instrument in vivo imaging system upon injection of 100 pl (30 mg / ml) D-Luciferin (Gold Biotechnology) (Figure 4 D). Following treatments were compared in vivo: untransduced cells (UTD-T); CD33-CAR- T cells; ANPM1-TCR-T cells; a 1 : 1 mixture of CD33-CAR-T cells and ANPM1-TCR-T cells (Double-T); and a mixture of co-transduced cells, CD33-CAR-T cells and ANPM1-TCR-T cells (Triple-T). The latter is automatically occurring in case of co-transduction of lentiviral particles encoding either CD33-CAR or ANPM1-TCR. In all treatment groups, 5 x 106modified T cells (7.9 x 106total T cells) were injected intravenously on day 4. The health status of the animals was monitored daily. Euthanasia was applied when study endpoint criteria were reached as defined by institutional regulations. Following some primary delay the Triple-T condition developed a powerful tumor control of a very challenging tumor modell in vivo, further supporting the benefit of combining CAR-and TCR signaling in a novel therapeutic approach.

[0469] References

[0470] Uslu U, Schuler G, Dome J, Schaft N. Combining a chimeric antigen receptor and a conventional T- cell receptor to generate T cells expressing two additional receptors (TETARs) for a multi-hit immunotherapy of melanoma. Exp Dermatol. 2016 Nov;25(l 1): 872-879. doi: 10. 1111 / exd. 13095. Epub 2016 Aug 3. PMID: 27246630.

[0471] Simon B, Harrer DC, Schuler-Thumer B, Schuler G, Uslu U. Arming T Cells with a gplOO-Specific TCR and a CSPG4-Specific CAR Using Combined DNA- and RNA-Based Receptor Transfer. Cancers (Basel). 2019 May 20; 11(5):696. doi: 10.3390 / cancersl 1050696. PMID: 31137488; PMCID: PMC6562862.

[0472] Miyao K, Terakura S, Okuno S, Julamanee J, Watanabe K, Hamana H, Kishi H, Sakemura R Koyama D, Goto T, Nishida T, Murata M, Kiyoi H. Introduction of Genetically Modified CD3^ Improves Proliferation and Persistence of Antigen-Specific CTLs. Cancer Immunol Res. 2018 Jun;6(6):733-744. doi: 10.1158 / 2326-6066. CIR-17-0538. Epub 2018 Apr 13. PMID: 29653982. Omer B, Castillo PA, Tashiro H, Shum T, Huynh MTA, Cardenas M, Tanaka M, Lewis A, Sauer T, Parihar R, Lapteva N, Schmueck-Henneresse M, Mukherjee M, Gottschalk S, Rooney CM. Chimeric Antigen Receptor Signaling Domains Differentially Regulate Proliferation and Native T Cell Receptor Function in Virus-Specific T Cells. Front Med (Lausanne). 2018 Dec 11;5:343. doi: 10.3389 / fmed.2018.00343. PMID: 30619856; PMCID: PMC6297364.

[0473] Omer B, Cardenas MG, Pfeiffer T, Daum R, Huynh M, Sharma S, Nouraee N, Xie C, Tat C, Perconti S, Van Pelt S, Scherer L, DeRenzo C, Shum T, Gottschalk S, Arber C, Rooney CM. A Costimulatory CAR Improves TCR-based Cancer Immunotherapy. Cancer Immunol Res. 2022 Apr 1 ; 10(4):512-524. doi: 10.1158 / 2326-6066. CIR-21-0307. PMID: 35176142; PMCID: PMC8978620.

[0474] Sequence listing (in standard one letter code for amino acids)

[0475] SEQ ID NO: 1 (dNPMl tTCR-directed target)

[0476] CLAVEEVSL

[0477] SEQ ID NO:2 (CD33 -specific scFv)

[0478] EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWAST RESGVPDRFTGSGSGTDFTLTIS S VQPEDL AIYYCHQYLS SRTFGQGTKLEIKRGGGGS GGGGSSGGGSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLE WVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRL RYFD VWGQGTT VT VS S

[0479] SEQ ID NO: 3 (VL of CD33 -specific scFv)

[0480] EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWAST RESGVPDRFTGSGSGTDFTLTIS S VQPEDL AIYYCHQYLS SRTFGQGTKLEIKR

[0481] SEQ ID NO:4 (VH of CD33 -specific scFv)

[0482] QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGND DISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGT TVTVS S

[0483] SEQ ID NO: 5 (dNPMl CDR3 of a TCR alpha chain polypeptide) CAVTGARLMF

[0484] SEQ ID NO: 6 (dNPMl CDR3 of a TCR beta chain polypeptide)

[0485] CASSPGGLSNEQF

[0486] SEQ ID NO:7 (dNPMl TCR alpha chain variable region)

[0487] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWY RQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVTGA RLMFGDGTQLVVKP

[0488] SEQ ID NO:8 (dNPMl TCR beta chain variable region)

[0489] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQT PGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPG GLSNEQFFGPGTRLTVL SEQ ID NO:9 (dNPMl TCR alpha chain variable region CDR1) SDRGSQS

[0490] SEQ ID NO: 10 (dNPMl TCR a chain variable region CDR2)

[0491] FIYSNGD

[0492] SEQ ID NO: 11 (dNPMl TCR beta chain variable region CDR1)

[0493] SGHRS

[0494] SEQ ID NO: 12 (dNPMl TCR 0 chain variable region CDR2)

[0495] EYFSETQRNKGNF

[0496] SEQ ID NO: 13 (BCMA-specific VHHs (single-domain antibody, clones VHH1 and VHH2)), EVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYY AESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQ VTVSSTSGGGGSGGGGSGGGGSAVQLVESGGGLVQAGDSLRLTCTASGRAFSTYFM AWFRQAPGKEREFVAGIAWSGGSTAYADSVKGRFTISRDNAKNTVYLQMNSLKSED TAVYYC ASRGIEVEEFGAWGQGTQ VT VS S

[0497] SEQ ID NO: 14 (BOB1 tTCR-directed target)

[0498] AP APT AWL

[0499] SEQ ID NO: 15 (BOB1 CDR3 of a TCR alpha chain polypeptide) KGSSNTGKLIFGQGTTLQVKP

[0500] SEQ ID NO: 16 (BOB1 CDR3 of a TCR beta chain polypeptide)

[0501] CASSHGPASYEQYF

[0502] SEQ ID NO: 17 (BOB1 TCR alpha chain variable region)

[0503] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQ ELGKGPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASKGSS NTGKLIFGQGTTLQVKP

[0504] SEQ ID NO: 18 (BOB1 TCR beta chain variable region)

[0505] MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWY KQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASS

[0506] HGPASYEQYFGPGTRLTVT

[0507] SEQ ID NO: 19 (CD19-specific scFv)

[0508] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGS GGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIW GSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY WGQGTSVTVSS

[0509] SEQ ID NO:20 (VL of CD19-specific scFv)

[0510] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT SEQ ID NO:21 (VH of CD19-specific scFv)

[0511] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT

[0512] YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQG TSVTVSS

[0513] SEQ ID NO:22 (CD20-specific scFv)

[0514] MLLLVTSLLLCELPHPAFLLIPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMH

[0515] WVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDS

[0516] ADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAI LS ASPGEKVTMTCRAS S S VNYMDWYQKKPGS SPKPWIYATSNL ASGVPARF SGSGSG TSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0517] SEQ ID NO:23 (VL of CD20-specific scFv)

[0518] DIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGV

[0519] PARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIK

[0520] SEQ ID NO:24 (VH of CD20-specific scFv)

[0521] EVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGN GDTS YNQKFKGK ATLTADKS S ST AYMQL S SLT SED S AD YYC ARSNYYGS S YWFFD V

[0522] WGAGTTVTVSS

[0523] SEQ ID NO:25 (CD22-specific scFv)

[0524] QVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS

[0525] KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDAFDIWGQ

[0526] GTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVYASVGDKVTITCRASQDVSGWL

[0527] AWYQQKPGLAPQLLISGASTLQGEVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA KYFPYTFGQGTKLEIK

[0528] SEQ ID NO:26 (VL of CD22-specific scFv)

[0529] DIQMTQSPSSVYASVGDKVTITCRASQDVSGWLAWYQQKPGLAPQLLISGASTLQGE

[0530] VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKYFPYTFGQGTKLEIK

[0531] SEQ ID NO:27 (VH of CD22-specific scFv)

[0532] QVQLQQSGPGLVKHSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS

[0533] KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAQEVEPHDAFDIWGQ GTMVTVSS

[0534] SEQ ID NO:28 (CD 123 -specific scFv)

[0535] MLLLVTSLLLCELPHPAFLLIPEVQLLESGGGLVKPGGSLRLSCAASGFTFSNAWMSW

[0536] VRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTED

[0537] TAVYYCTTGLLWFGTRNYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQSAL TQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLVIYDVSNRPSGLSN RFSGSKSGNTASLTISGLQAEDEADYYCNSYAGSGSWVFGGGTKLTVL

[0538] SEQ ID NO:29 (VL of CD 123 -specific scFv)

[0539] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLVIYDVSNRPS

[0540] GLSNRFSGSKSGNTASLTISGLQAEDEADYYCNSYAGSGSWVFGGGTKLTVL SEQ ID NO:30 (VH of CD 123 -specific scFv)

[0541] MLLLVTSLLLCELPHPAFLLIPEVQLLESGGGLVKPGGSLRLSCAASGFTFSNAWMSW VRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTED TAVYYCTTGLLWFGTRNYYYGMDVWGQGTTVTVSS

[0542] SEQ ID NO:31 (CLEC12a-specific scFv)

[0543] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGS

[0544] TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGLDLDAFDIWGQGT

[0545] MVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQPITISCTGSYSDVGYYDYVS

[0546] WYQQHPGKAPKLIIYDVTKRPSGASNRFSGSKSGNTASLTISGLQAEDEADYYCNSYT ARDTWVFGGGTKLTVL

[0547] SEQ ID NO:32 (VL of CLEC12a-specific scFv)

[0548] QSALTQPASVSGSPGQPITISCTGSYSDVGYYDYVSWYQQHPGKAPKLIIYDVTKRPS

[0549] GASNRFSGSKSGNTASLTISGLQAEDEADYYCNSYT ARDTWVFGGGTKLTVL

[0550] SEQ ID NO:33 (VH of CLEC12a-specific scFv)

[0551] EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGS

[0552] TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGLDLDAFDIWGQGT MVTVSS

[0553] SEQ ID NO:34 (FRalpha-specific scFv)

[0554] MEVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGG

[0555] SYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWG

[0556] QGTPVTVSSASTKGGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCSVSSSIS

[0557] SNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYY

[0558] CQQWS S YPYMYTFGQGTKVEIKRT

[0559] SEQ ID NO:35 (VL of FRalpha-specific scFv)

[0560] DIQLTQ SP S SLS AS VGDRVTITC S VS S SIS SNNLHW YQQKPGK APKPWIYGTSNL ASGV

[0561] PSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIKRT

[0562] SEQ ID NO:36 (VH of FRalpha-specific scFv)

[0563] MEVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGG

[0564] SYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWG QGTPVTVSSASTKG

[0565] SEQ ID NO:37 (PRAME tTCR-directed target)

[0566] SLLQHLIGL

[0567] SEQ ID NO:38 (PRAME CDR3 of a TCR alpha chain polypeptide)

[0568] CAGIPRDNYGQNFVF

[0569] SEQ ID NO:39 (PRAME CDR3 of a TCR beta chain polypeptide)

[0570] CASTPWLAGGNEQFF

[0571] SEQ ID NO:40 (PRAME TCR alpha chain variable region)

[0572] MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDP

[0573] GEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGIPRDNYGQ NFVFGPGTRLSVLP SEQ ID NO:41 (PRAME TCR beta chain variable region)

[0574] MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYR QDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCAST PWLAGGNEQFFGPGTRLTVL

[0575] SEQ ID NO:42 (CSPG4-specific scFv)

[0576] DIELTQSPKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPEPLLFSASYRYTG

[0577] VPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGGGTKLEIKRAAAEGGG

[0578] GSGGGGSGGGGSAMAQVKLQQSGGGLVQPGGSMKLSCVVSGFTFSNYWMNWVRQ SPEKGLEWIAEIRLKSNNFGRYYAESVKGRFTISRDDSKSSAYLQMINLRAEDTGIYY CTS YGNYVGHYFDHWGQGTT VT VS S

[0579] SEQ ID NO:43 (VL of CSPG4-specific scFv)

[0580] DIELTQSPKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPEPLLFSASYRYTG

[0581] VPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGGGTKLEIKRAAAE

[0582] SEQ ID NO:44 (VH of CSPG4-specific scFv)

[0583] AMAQVKLQQSGGGLVQPGGSMKLSCVVSGFTFSNYWMNWVRQSPEKGLEWIAEIR

[0584] LKSNNFGRYYAESVKGRFTISRDDSKSSAYLQMINLRAEDTGIYYCTSYGNYVGHYF DHWGQGTTVTVSS

Claims

Claims1) An in-vitro method for the generation of a composition, wherein the composition comprisesA) engineered immune cells co-expressing a CAR and a transgenic TCR,B) engineered immune cells expressing said CAR, andC) engineered immune cells expressing said transgenic TCR, the method comprising the step: transducing simultaneously immune cells a) with a first viral vector comprising a first nucleic acid sequence comprising encoding a chimeric antigen receptor (CAR) comprising1) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) with a second viral vector comprising a second nucleic acid sequence encoding a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens, thereby obtaining said composition.2) The in-vitro method of claim 1, wherein said immune cells are T cells.3) The in-vitro method of claim 1 or 2, wherein the multiplicity of infection (MOI) ratio of said first viral vector and said second viral vector used for the transduction step is between 1 : 10 and 10:1.4) The in-vitro method of claim 3, wherein said MOI ratio is about 1 : 10.5) A composition comprisingA)I) engineered immune cells comprising a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, andb) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, andII) engineered immune cells comprising said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, andIII) engineered immune cells comprising said transgenic T cell receptor (tTCR) having specificity for said second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens; orB)I) engineered immune cells comprising a) a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, andII) engineered immune cells comprising said chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for said first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens; orC)I) engineered immune cells comprising a)a chimeric antigen receptor (CAR) comprising i) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, and b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, andII) engineered immune cells comprising said transgenic T cell receptor (TCR) having specificity for said second epitope of an antigen,wherein said antigen of said first epitope and said antigen of said second epitope are the same antigen or are different antigens.6) The composition of claim 5, wherein said engineered immune cells are T cells.7) The composition of claim 5 or 6, wherein said CAR is specific for an epitope of the antigen CD33 and wherein said tTCR has specificity for an epitope of the antigen ANPM1.8) The composition of claim 7, wherein said antigen binding domain specific for said epitope of the antigen CD33 comprises SEQ ID NO:2, and wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:(a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and(b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1), wherein the CDR3 of (a) is within a TCR a chain variable region having at least 90% sequence identity to SEQ ID NO:7, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 5; and wherein (a) comprises a TCR alpha chain constant region, wherein the TCR alpha chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 9 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 10; and wherein the CDR3 of (b) is within a TCR beta chain variable region having at least 90% sequence identity to SEQ ID NO: 8, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 6; and wherein (b) comprises a TCR beta chain constant region, and wherein the TCR beta chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 11 and the TCR P chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 12.9) The composition of claim 5 to 8, wherein said composition is composition A, and wherein said composition A is obtained by the method of any one of claims 1 to 4.10) A pharmaceutical composition comprising the composition of claims 5 to 9, and optionally a pharmaceutical acceptable carrier.11) An engineered immune cell comprising a) a chimeric antigen receptor (CAR) comprisingi) an antigen binding domain specific for a first epitope of an antigen ii) a transmembrane domain iii) an intracellular signaling domain, b) a transgenic T cell receptor (tTCR) having specificity for a second epitope of an antigen, wherein said antigen of said first epitope and said antigen of said second epitope are different antigens, and wherein said CAR and said tTCR are expressed constitutively in said immune cell, wherein said CAR is specific for an epitope of the antigen CD33 and wherein said tTCR has specificity for an epitope of the antigen ANPM1.12) The engineered immune cell of claim 11, wherein said antigen binding domain specific for said epitope of the antigen CD33 comprises SEQ ID NO:2, and wherein said tTCR having specificity for the epitope of the antigen ANPM1 comprises:(a) a polypeptide comprising a CDR3 of a TCR alpha chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1) and(b) a polypeptide comprising a CDR3 of a TCR beta chain polypeptide that specifically binds to the peptide CLAVEEVSL (SEQ ID NO: 1), wherein the CDR3 of (a) is within a TCR a chain variable region having at least 90% sequence identity to SEQ ID NO:7, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 5; and wherein (a) comprises a TCR alpha chain constant region, wherein the TCR alpha chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 9 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 10; and wherein the CDR3 of (b) is within a TCR beta chain variable region having at least 90% sequence identity to SEQ ID NO: 8, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 6; and wherein (b) comprises a TCR beta chain constant region, and wherein the TCR beta chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 11 and the TCR P chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 12.13) The engineered immune cell of claim 11 or 12, wherein said immune cell is a T cell.14) A pharmaceutical composition comprising the engineered immune cell of any one of claims 11 to 13, and optionally a pharmaceutical acceptable carrier.

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