Compositions and methods for enhancing / improving CD8+ t cell function

Abstract

The present invention relates to a CD8+ T cell comprising a recombinant antigen-binding receptor and expressing BATF2. The invention also relates to a composition or kit comprising a CD8+ T cell comprising a recombinant antigen-binding receptor and expressing BATF2, and a CD4+ T cell comprising a recombinant antigen-binding receptor and overexpressing a transcription factor. The invention further relates to a method for producing such a CD8+ T cell, a pharmaceutical composition comprising the CD8+ T cell, or the composition or kit. The CD8+ T cell, composition, kit or pharmaceutical composition may be used for treating diseases in a patient.

Description

[0001] COMPOSITIONS AND METHODS FOR ENHANCING / IMPROVING CD8+T CELL FUNCTION

[0002] Technical Field

[0003] The present invention relates to a CD8+T cell comprising a recombinant antigen-binding receptor and expressing BATF2. The invention also relates to a composition or kit comprising a CD8+T cell comprising a recombinant antigen-binding receptor and expressing BATF2, and a CD4+T cell comprising a recombinant antigen-binding receptor and overexpressing a transcription factor. The invention further relates to a method for producing such a CD8+T cell, a pharmaceutical composition comprising the CD8+T cell, or the composition or kit. The CD8+T cell, composition, kit or pharmaceutical composition may be used for treating diseases in a patient.

[0004] Background

[0005] Engineered T cells have the potential to treat a wide range of diseases, including cancer and autoimmune disorders. Therapeutic T cell products are directed to specific antigens through the introduction of transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs). There are currently six FDA-approved cellular products targeting either CD19 or BCMA as cancer-associated antigens. Approved indications include hematological cancers, including leukemia, lymphoma and multiple myeloma.

[0006] The efficacy of current CAR T cell therapies is limited by non-specific homing19-17, limited persistence20-21, and the acquisition of an exhausted state upon chronic stimulation22-1-23-24, resulting in one in two patients eventually relapsing after an initial response43-25-26. The field is only beginning to understand the complex determinants that correlate with successful treatments, and limitations are mostly being addressed in an iterative, empirical process. For example, chemokine receptor overexpression27-29aims to enhance specific migration, while calibration of CAR signaling levels - either at the protein level30or through pharmacological switches such as dasatinib12- aims to prevent overstimulation. Other approaches include immune checkpoint blockade31, expression of dominant-negative receptors32or "armored" CART cells33to shield from or reengineer the immunosuppressive tumor microenvironment (TME). In addition, advances in the generation of lessdifferentiated memory subsets have led to improved expansion, engraftment and persistence11.

[0007] Recently, modulation of transcription factor levels in CAR T cells has gained interest, with the AP-1 family in the spotlight. For example, a relative imbalance of transcriptions factors BATF, IRF4 and cJUN was observed in T cells expressing a tonic signaling CAR, a model for T cell exhaustion1. This state, characterized by reduced expansion potential, terminal effector differentiation, diminished IL2 secretion, and expression of inhibitory receptors, could be reversed by AP-1 enhancement via cJUN-overexpression1. This did not only mitigate exhaustion but also enhanced the efficacy of non-tonic signaling CAR T cells1. Two other studies highlighted the role of BATF3 in memory formation, with overexpression leading to a proliferative advantage2-3. Altering the expression levels of BATF yielded context-dependent outcomes: under non-exhausting conditions proliferation and efficacy was augmented, while deepened exhaustion occurred under more challenging conditions at low effector-to-target (E: T) ratios4-5. BATF6and BATF33-6have further been confirmed as top hits in independent screening campaigns. In efforts to bring ROR1-CAR T cells to the clinic, dose-limiting toxicity was observed in an NSCLC patient with high tumor burden in the lung, marked by excessive CAR T cell activation7. Additionally, a trial using AP-1-enhanced ROR1-CAR T cells reported grade > 3 pneumonitis in a TNBC patient with metastatic lung involvement, resulting in death from respiratory failure (LYELL; https: / / firstwordpharma.com / story / 5870441)87. Nonspecific accumulation of hyperactive CAR T cells in healthy tissues such as the lung limits efficacy and poses a risk of potentially life-threatening on-target off-tumor toxicities. Reported clinical examples of lung-related toxicities include CARs targeting HER235, MSLN88, CEACAM589, and ROR17-87, with preexisting lung metastases or inflammation being potential risk factors.

[0008] Thus, available approaches are currently suffering from various limitations including nonspecific homing, limited persistence the acquisition of an exhausted state upon chronic stimulation, as well as systemic inflammatory side effects such as cytokine release syndrome (CRS) or on-target / off-tumor toxicity (OTOT).There is therefore an urgent need for CAR T cell products that balance enhanced proliferation, persistence, and exhaustion resistance while minimizing excessive activation to avoid systemic inflammatory side effects.

[0009] Brief Description of the Invention

[0010] The inventors pursued a contrasting approach by attenuating rather than enhancing AP-1 via overexpression of the transcription factor BATF2 - a dominant-negative regulator of AP-1 - in CAR T cells (CARBATF2) to introduce self-limiting features, thereby promoting longterm CAR-T cell fitness while mitigating risks for toxicity.

[0011] The inventors investigated targeted modulation of the transcription factor BATF2 as a strategy to fine-tune CAR T cell therapy and prevent toxicities. They found for instance, that BATF2 overexpression is advantageous, for example, because it shields from critical on-target off-tumor lung toxicities, e.g., due to two key characteristics: First, by detargeting CAR T cells from commonly observed nonspecific accumulation in healthy lung tissue due to attenuated basal activation and reduced expression of the key adhesion molecule LFA-1 (lymphocyte function-associated antigen-1). Second, by modulating antigen-sensitivity, enabling selective activity against high-antigen tumor cells while sparing low-antigen healthy tissue. Therefore, BATF2 overexpression allows to fine-tune CAR T cell therapy and improve safety, thereby expanding the potential for solid tumor patients.

[0012] The present invention meets or addresses at least some of the above needs and aims at solving the above problems in the art by providing CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses BATF2, as well a composition and kit comprises said CD8+T cell and a CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD4+T cell overexpresses a transcription factor, as defined by the independent claims.

[0013] The invention provides several aspects that each individually achieve an improvement over the art.In a first aspect, the invention provides a CD8+T cell comprising a recombinant antigenbinding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses (e.g., overexpresses) BATF2.

[0014] The inventors found that CD8+T cells overexpressing BATF2 exhibit a more balanced immune response by attenuating immediate effector functions and shielding from activation-induced cell death (AICD), while providing enhanced antigen-specific proliferation and a memory-like phenotype. In addition, overexpression of BATF2 in CD8+T cells comprising an antigen-binding receptor attenuates tonic signaling of the CD8+T cells in the face of continuous antigenic stimulation by overstimulation and thus reduces the CD8+T cell exhaustion. Furthermore, albeit CD8+T cells comprising an antigen-binding receptor, such as a CAR, and overexpressing BATF2 have a lower antigen sensitivity, similar maximum activation levels can be achieved at high antigen densities via the CAR. This will be particularly advantageous when targeting an antigen that is also expressed at low levels in healthy tissues. Thus, BATF2 overexpressing CD8+T cells will reduce the risk of on-target / off-tumor toxicity by being effective against tumor cells with high antigen expression while sparing healthy tissue.

[0015] Low and decreasing numbers of CAR T cells in the blood and non-specific accumulation in tissues have been reported in early clinical trials.59A case report of a patient treated with HER2-directed CAR T cells described accumulation in lung tissue with low endogenous target expression, leading to massive cytokine release, respiratory distress and eventually death.35The migratory behavior of CAR T cells has been identified as a positive predictor of survival, with high local effect-on-target (E: T) ratios and early intratumorally presence of CAR T cells being associated with improved cell product.60This highlights the importance of the migratory phenotype of a CAR T cell. The inventors showed that CD8+T cells overexpressing BATF2 expressed higher levels of βII-spectrin, thereby reducing LFA-1 surface levels compared to conventional CAR control T cells. LFA-1 has been previously described to be responsible for unspecific lung sequestration18. Thus, CD8+T cells that overexpress BATF2 have the intrinsic feature of reduced sequestration in the lung. Higherlevels of pi l-spectrin in patient CAR T products were described to be positively correlated with partial or complete responses and negatively correlated with severity of CRS.17Consequently, the intrinsic feature of CD8+T cells overexpressing BATF2 of reduced lung sequestration can be highlighted as a significant advantage over conventional CD8+T cells comprising a recombinant antigen-binding receptor, e.g. reduced unspecific tissue sequestration in the lung is expected to have a significant safety benefit for the treatment of patients with solid tumors. In addition, CD8+T cells overexpressing BATF2 show differential expression of migration-associated genes (e.g., CXCR4 and CCR7) compared to conventional CAR T cells. Less lung sequestration together with increased chemokine-specific homing into immunological niches via CXCR4 and CCR7 makes CARBATF2T cells especially attractive for the treatment of hematological malignancies like MCL, AMLor MM by potentially favoring co-localization with the tumor cells.

[0016] In a preferred embodiment of the first aspect, the invention provides a CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses (e.g., overexpresses) BATF2, wherein the antigen-binding receptor is a CAR fusion protein, comprising in an N- to C-terminal order:

[0017] a) a CAR, and

[0018] b) LCK.

[0019] The inventors have shown that CD8+T cells overexpressing BATF2 exhibit lower protein levels of the membrane-proximal kinase LCK, which may be responsible for the lower activity. The inventors have, thus, developed a receptor design comprising a direct fusion of LCK to the C-terminus of the CAR molecule. This advanced receptor design was shown to facilitate LCK availability to the CAR, enhance the control of low-antigen tumor cells, IL-2 secretion and further augmented proliferative responses in vitro of the CD8+T cell of the present invention. Therefore, such a receptor design is particularly beneficial for scenarios where high antigen-sensitivity is crucial.

[0020] In a second aspect, the invention provides a composition or kit comprising:a) a CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses (e.g., overexpresses) BATF2; and optionally

[0021] b) a CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD4+T cell expresses (e.g., overexpresses) a transcription factor.

[0022] The inventors also found that CD4+and CD8+T cell subsets have different preferences for transcription factor enhancement. For instance, BATF2 overexpression in CD4+T cells does not produce the same favorable results as observed in CD8+T cells. However, a combination of CD8+T cells overexpressing BATF2 and CD4+T cells overexpressing a transcription factor has been shown to be beneficial. Specifically, the combination of CD8+T cells overexpressing BATF2 and CD4+T cells overexpressing eJun was shown to be beneficial, and the inventors further demonstrated that CD4+T cells overexpressing eJun facilitate the proliferation of the CD8+T cells when co-cultured in vitro or co-administered in vivo. Co-administration of two heterogeneous cell products comprising CD4+T celloverexpressing eJun and CD8+T cell-overexpressing BATF2 was also shown to provide superior engraftment and tumor control in an in vivo solid tumor model.

[0023] Preferred embodiments are set out in the respective dependent claims, as well as in the embodiments described below.

[0024] 1. A CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses BATF2.

[0025] 2. The CD8+T cell according to item 1, wherein the CD8+T cell overexpresses BATF2.3. The CD8+T cell according to item 1 or 2, wherein the CD8+T cell has been modified to express BATF2 at a level that is increased compared to the level of BATF2 expressed by the CD8+T cell before it was modified to express BATF2.

[0026] 4. The CD8+T cell according to any one of items 1-3, wherein the CD8+T cell comprises an exogenous nucleic acid overexpressing BATF2, or wherein the CD8+T cell comprises an exogenous activator of transcription for an endogenous BATF2 gene.

[0027] 5. The CD8+T cell according to item 4, wherein the exogenous nucleic acid overexpressing BATF2 encodes BATF2, optionally wherein the exogenous nucleic acid comprises an expression cassette for BATF2 expression.

[0028] 6. The CD8+T cell according to item 5, wherein the exogenous nucleic acid encoding BATF2 is integrated into the genome of the CD8+T cell.

[0029] 7. The CD8+T cell according to item 6, wherein the CD8+T cell expressing BATF2 is obtainable through stable gene transfer of the exogenous nucleic acid encoding BATF2.

[0030] 8. The CD8+T cell according to any one of the preceding items, wherein the exogenous nucleic acid is a genetic expression vector encoding BATF2.

[0031] 9. The CD8+T cell according to item 8, wherein the CD8+T cell expressing BATF2 is obtainable through transient gene transfer of the exogenous nucleic acid encoding BATF2.

[0032] 10. The CD8+T cell according to any one of the preceding items, wherein the BATF2 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an aminoacid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 1.

[0033] 11. The CD8+T cell according to any one of the preceding items, wherein the CD8+T cell is a mammalian CD8+T cell.

[0034] 12. The CD8+T cell according to any one of the preceding items, wherein the CD8+T cell is a human CD8+T cell.

[0035] 13. The CD8+T cell according to any one of the preceding items, wherein the modified CD8+T cell has been obtained from an isolated CD8+T cell.

[0036] 14. The CD8+T cell according to item 13, wherein the isolated CD8+T cell is a native, naturally occurring CD8+T cell.

[0037] 15. The CD8+T cell according to any one of the preceding items, wherein the recombinant antigen-binding receptor binds to a cancer antigen.

[0038] 16. The CD8+T cell according to any one of the preceding items, wherein the recombinant antigen-binding receptor binds to an antigen selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3-Integrin, α4β1-Integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D-Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and FLT3, preferably CD19, CD20, and ROR1.

[0039] 17. The CD8+T cell according to item 16, wherein the recombinant antigen-binding receptor binds to ROR1.18. The CD8+T cell according to any one of the preceding items, wherein the recombinant antigen-binding receptor is a CAR.

[0040] 19. The CD8+T cell according to item 18, wherein the CAR comprises, in an N-to C- terminal order, at least one extracellular antigen binding domain that binds to the antigen, a spacer domain, a transmembrane domain and at least one intracellular signaling domain.

[0041] 20. The CD8+T cell according to item 19, wherein the extracellular antigen binding domain comprises an scFv that binds to the antigen, preferably wherein the antigen is a cell surface antigen.

[0042] 21. The CD8+T cell according to item 20, wherein the cell surface antigen is a cancer antigen and / or an antigen of the tumor microenvironment.

[0043] 22. The CD8+T cell according to item 20 or 21, wherein said antigen is selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3-Integrin, α4β1-Integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D-Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and FLT3, preferably ROR1.

[0044] 23. The CD8+T cell according to any one of items 21-22, wherein the antigen is ROR1, and the antigen binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 6.24. The CD8+T cell according to any one of items 21-23, wherein the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 10, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 3 or 10.

[0045] 25. The CD8+T cell according to any one of the preceding items, wherein the CD8+T cell comprises an exogenous nucleic acid encoding and expressing the recombinant antigen-binding receptor.

[0046] 26. The CD8+T cell according to item 25, wherein the CD8+T cell expressing the antigen-binding receptor is obtainable through stable gene transfer of the exogenous nucleic acid encoding and expressing the recombinant antigenbinding receptor.

[0047] 27. The CD8+T cell according to any one of items 1-25, wherein the cell expressing the antigen-binding receptor is obtainable through transient gene transfer of the exogenous nucleic acid encoding and expressing the recombinant antigenbinding receptor.

[0048] 28. The CD8+T cell according to any one of the preceding items, wherein the antigenbinding receptor and BATF2 are co-expressed from one exogenous nucleic acid.

[0049] 29. The CD8+T cell according to any one of the preceding items, wherein the antigen-binding receptor is a CAR fusion protein, comprising in an N- to C- terminal order:

[0050] a) a CAR, and

[0051] b) LCK.

[0052] 30. The CD8+T cell according to item 29, wherein the LCK is at the C-terminus of the CAR fusion protein.31. The CD8+T cell according to items 29 or 30, wherein the LCK is located the intracellular portion of the CAR fusion protein.

[0053] 32. The CD8+T cell according to any one of items 29-31, wherein the LCK is a mammalian LCK.

[0054] 33. The CD8+T cell according to any one of items 29-32, wherein the LCK is human LCK.

[0055] 34. The CD8+T cell according to any one of items 29-33, wherein the LCK comprises or consists of the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence with at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO: 7.

[0056] 35. The CD8+T cell according to any one of items 29-34, wherein the CAR fusion protein comprises a CAR comprising, in an N-to C-terminal order, at least one extracellular antigen binding domain that binds to the antigen, a spacer domain, a transmembrane domain and at least one intracellular signaling domain, and the LCK.

[0057] 36. The CD8+T cell according to any one of items 29-35, wherein the CAR is a CAR according to any one of items 15-24.

[0058] 37. The CD8+T cell according to any one of items 29-36, wherein the CAR fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence with at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO: 8.38. The CD8+T cell according to any one of the preceding items, wherein the CD8+T cell exhibits one or more improved pharmacological properties compared to a CD8+T cell that does not overexpress the BATF2.

[0059] 39. The CD8+T cell according to item 38, wherein the one or more improved pharmacological properties of the modified CD8+T cell comprise

[0060] a) enhanced proliferation;

[0061] b) decreased exhaustion;

[0062] c) reduced accumulation in non-cancerous lung tissue;

[0063] d) memory like phenotype;

[0064] e) reduced activation-induced cell death;

[0065] f) improved chemokine-dependent migration;

[0066] g) any combination of a) to e); and / or

[0067] h) all of a) to e).

[0068] 40. The CD8+T cell according to item 39, wherein the enhanced proliferation is enhanced antigen-specific proliferation.

[0069] 41. A composition comprising:

[0070] a) a CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell overexpresses BATF2.

[0071] 42. A kit comprising:

[0072] a) a CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell overexpresses BATF2.

[0073] 43. The composition or kit according to any one of items 42 or 43, preferably further comprising:b) a CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD4+T cell overexpresses a transcription factor, optionally wherein the transcription factor is selected from BATF, BATF3, TFAP4, FoxOl or eJun.

[0074] 44. The composition or kit according to item 43, wherein the transcription factor is eJun.

[0075] 45. The composition or kit according to any one of items 41-44, wherein the CD8+T cell is a CD8+T according to any one of items 1-40.

[0076] 46. The composition or kit according to item 44 or 45, wherein the CD4+T cell has been modified to express eJun at a level that is increased compared to the level of eJun expressed by the CD4+T cell before it was modified to express eJun.

[0077] 47. The composition or kit according to any one of items 44-46, wherein the CD4+T cell comprises an exogenous nucleic acid encoding and overexpressing eJun.

[0078] 48. The composition or kit according to item 47, wherein the exogenous nucleic acid encoding and overexpressing eJun is integrated into the genome of the CD4+T cell.

[0079] 49. The composition or kit according to item 48, wherein the CD4+T cell is obtainable through stable gene transfer of the exogenous nucleic acid encoding and overexpressing eJun.

[0080] 50. The composition or kit according to item 48 or 49, wherein the exogenous nucleic acid is a genetic expression vector encoding eJun.1. The composition or kit according to item 50, wherein the CD4+T cell is obtainable through transient gene transfer of the exogenous nucleic acid encoding and overexpressing eJun.

[0081] 52. The composition or kit according to any one of items 44-51, wherein the CD4+T cell comprises an exogenous activator of transcription for an endogenous eJun gene and / or activator of translation of an endogenous eJun mRNA.

[0082] 53. The composition or kit according to any one of items 44-52, wherein in the CD4+T cell the endogenous eJun gene has been genetically modified to increase eJun expression, wherein optionally the endogenous eJun gene has been genetically modified in its promoter region.

[0083] 54. The composition or kit according to any one of items 43-53, wherein the CD4+T cell is a mammalian CD4+T cell.

[0084] 55. The composition or kit according to any one of items 43-54, wherein the CD4+T cell is a human CD4+T cell.

[0085] 56. The composition or kit according to any one of items 43-55, wherein the CD4+T cell has been obtained from an isolated CD4+T cell.

[0086] 57. The composition or kit according to item 56, wherein the isolated CD4+T cell is a native, naturally occurring CD4+T cell.

[0087] 58. The composition or kit according to any one of items 43-57, wherein the recombinant antigen-binding receptor of the CD4+T cell binds to a cancer antigen.59. The composition or kit according to any one of items 43-58, wherein the recombinant antigen-binding receptor of the CD4+T cell binds to an antigen selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, av|33-lntegrin, a4 1- Integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D- Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, and FLT3, preferably CD19, CD20, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and ROR1.

[0088] 60. The composition or kit according to item 59, wherein the recombinant antigenbinding receptor of the CD4+T cell binds to ROR1.

[0089] 61. The composition or kit according to any one of items 43-60, wherein the recombinant antigen-binding receptor is a CAR.

[0090] 62. The composition or kit according to any one of items 43-61, wherein the CD4+T cell expressing the antigen-binding receptor is obtainable through stable gene transfer of a nucleic acid encoding and expressing the antigen-binding receptor.

[0091] 63. The composition or kit according to any one of items 43-62, wherein the CD4+T cell expressing the antigen-binding receptor is obtainable through transient gene transfer of a nucleic acid encoding and expressing the antigen-binding receptor.

[0092] 64. The composition or kit according to any one of items 43-63, wherein the antigenbinding receptor of the CD8+T cell and the antigen-binding receptor of the CD4+T cell target the same antigen.65. The composition or kit according to any one of items 41-64, wherein the composition or kit is substantially free of CD4+T cells overexpressing BATF2.

[0093] 66. The composition or kit according to any one of items 42-65, wherein the composition or kit comprising modified CD8+and CD4+T cells exhibits one or more enhanced properties compared to a composition or kit comprising CD8+and CD4+T cells that were not modified to have increased BATF2 and / or c-Jun expression.

[0094] 67. The composition or kit according to item 66, wherein the one or more enhanced properties of the composition and kit comprise:

[0095] a) enhanced proliferation;

[0096] b) decreased exhaustion;

[0097] c) reduced accumulation in non-cancerous lung tissue;

[0098] d) memory like phenotype;

[0099] e) reduced activation-induced cell death;

[0100] f) enhanced engraftment;

[0101] g) improved chemokine-dependent migration;

[0102] h) enhanced tumor control; and / or

[0103] i) any combination of a) to g); and / or

[0104] j) all of a) to g).

[0105] 68. A pharmaceutical composition comprising the CD8+T cell according to any one of items 1-40, or the composition according to any one of items 41, 43-67 and a pharmaceutically acceptable carrier.

[0106] 69. The CD8+T cell according to any one of items 1-40, the composition according to any one of items 41, 43-67 or the pharmaceutical composition according to item 68 for use as a medicament.70. The CD8+T cell according to any one of items 1-40, the composition according to any one of items 41, 43-67, or the pharmaceutical composition according to item 68 for use in the treatment of cancer in a patient having said cancer.

[0107] 71. Use of the CD8+T cell according to any one of items 1-40, the composition according to any one of items 41, 43-67, or the pharmaceutical composition according to item 68 for the manufacture of a medicament for the treatment of cancer.

[0108] 72. A method of treating cancer in a patient by administering the CD8+T cell according to any one of items 1-40, the composition according to any one of items 41, 43-67, or the pharmaceutical composition according to item 68.

[0109] 73. The CD8+T cell, composition, or pharmaceutical composition for use according to item 70, the use according to item 71, or the method according to item 72, wherein the cancer is a hematological cancer or solid cancer, optionally wherein the hematological cancer is leukemia, non-Hodgkin lymphoma (NHL), or multiple myeloma (MM); and / or the solid cancer is breast, ovarian, adrenocortical, or lung or thyroid cancer or a solid cancer with lung metastases which is not a lung cancer.

[0110] 74. The CD8+T cell, composition, or pharmaceutical composition for use according to item 70 or 73, the use according to item 71 or 73, or the method according to item 72 or 73, wherein the cancer expresses one or more or all of the following cancer antigens:

[0111] a) CD19;

[0112] b) BCMA;

[0113] c) ROR1;

[0114] d) FLT3;

[0115] e) CD20;f) CD22;

[0116] g) CD123;

[0117] h) ROR2;

[0118] i) Siglec-6; and

[0119] j) SLAMF7.

[0120] 75. The CD8+T cell, composition, or pharmaceutical composition for use according to item 72, wherein the recombinant antigen-binding receptor of the CD8+T cell binds to one of said cancer antigens expressed by the cancer.

[0121] 76. The CD8+T cell, composition, or pharmaceutical composition for use according to item 73, wherein the recombinant antigen-binding receptor of the CD4+T cell binds to one of said cancer antigens expressed by the cancer.

[0122] 77. The CD8+T cell, composition, or pharmaceutical composition for use according to any one of items 70, 73-76, the use according to any one of items 71, 73, or 74, or the method according to any one of items 72, 73, or 74, wherein the isolated CD8+T cell and / or CD4+T cell are allogeneic or syngeneic cells with respect to said patient.

[0123] 78. The CD8+T cell, composition, or pharmaceutical composition for use according to any one of items 70, 73-77, the use according to any one of items 71, 73, 74, 77, or the method according to any one of items 72, 73, 74, 77, wherein said treatment with a modified CD8+, or composition and kit comprising of modified CD8+and CD4+T cells exhibits one or more improved properties when administered to the patient compared to the treatment using a CD8+and a CD4+T cells that were not modified to have increased BATF2 and / or c-Jun expression, wherein said one or more improved properties comprise:

[0124] a) enhanced therapeutic efficacy;

[0125] b) enhanced the anti-tumor efficacy;

[0126] c) enhanced tumor control of the cancer;d) decreased tumor burden of the cancer in said patient;

[0127] e) decreased morbidity and mortality;

[0128] f) decreased CD8+and CD4+T cell exhaustion;

[0129] g) reduced accumulation in non-cancerous lung tissue;

[0130] h) decreased CD8+and CD4+T cell activation-induced cell death;

[0131] i) increasing CD8+and CD4+T cell engraftment and / or persistence;

[0132] j) decreased on-target / off-tu mor toxicity;

[0133] k) improved chemokine-dependent migration;

[0134] l) any combination of a) to j); and / or

[0135] m) all of a) to j).

[0136] 79. The CD8+T cell, composition, kit, or pharmaceutical composition for use according to any one of items 70, 73-78 or the use according to any one of items 71, 73, 74, 77, 78, wherein the use is also a use for eliminating the risk of pneumonitis in the treatment of the cancer.

[0137] 80. The method according to any one of items 72, 73, 74, 77, 78 wherein the method is also a method for eliminating the risk of pneumonitis in the treatment of the cancer.

[0138] 81. An in vitro method for improving CD8+T cell proliferation, reducing CD8+T cell exhaustion, reducing activation-induced cell death and / or promoting a memorylike phenotype comprising modifying the CD8+T cell to overexpress BATF2.

[0139] 82. The in vitro method according to item 81, wherein the overexpression of BATF2:

[0140] a) increases the proliferation of the modified CD8+T cell compared to proliferation of a CD8+T cell before it was modified to express the BATF2; b) decreases the exhaustion of the modified CD8+T cell compared to exhaustion of a CD8+T cell before it was modified to express the BATF2;c) decreases the activation-induced cell death of the modified CD8+T cell compared to activation-induced cell death of a CD8+T cell before it was modified to express the BATF2; and / or

[0141] d) increases the memory-like phenotype of the modified CD8+T cell compared to memory-like phenotype of a CD8+T cell before it was modified to express the BATF2.

[0142] The method according to items 81 or 82, wherein the modified CD8+T cell is a CD8+T cell according to items 1-40.

[0143] The method according to any one of items 81-83, wherein the modified CD8+T cell is co-cultured with a CD4+T cell.

[0144] The method according to item 84, wherein the CD4+T cell is a CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell overexpresses a transcription factor.

[0145] The method according to item 85, wherein the transcription factor is eJun.

[0146] The method according to any one of items 84-86, wherein the CD4+T cell is a CD4+T cell according to any one of items 48-66.

[0147] A nucleic acid or a set of nucleic acids encoding the BATF2 as defined in any one of items 1-40 and / or the CARs as defined in any one of items 15-37.

[0148] The nucleic acid or a set of nucleic acids according to item 88, wherein the BATF2 and the CAR are encoded on the same nucleic acid.90. An expression vector comprising the nucleic acid of item 89, or a set of expression vectors comprising the set of nucleic acids of item 89.

[0149] 91. The expression vector of item 90, wherein the expression vector is an expression vector for genomic integration into the host cell.

[0150] 92. Method for producing a CD8+T cell that exhibits an increased BATF2 level, wherein the method comprises introducing into the CD8+T cell an exogenous nucleic acid overexpressing BATF2 in the CD8+T cell.

[0151] 93. The method for producing the CD8+T cell according to item 90, the method comprising the steps of:

[0152] a) providing a CD8+T cell;

[0153] b) introducing into said CD8+T cell the nucleic acid or set of nucleic acids according to item 88 or 89, or the expression vector or the set of expression vectors according to item 90 or 91; and

[0154] c) co-expressing said nucleic acid or said set of nucleic acids according to item 88 or 89, or said expression vector or said set of expression vectors according to item 90 or 91;

[0155] d) thereby obtaining said CD8+T cell.

[0156] 94. The method or use according to any one of the preceding items, wherein all the steps of the method or use are carried out in vitro.

[0157] 95. The method or use according to any one of the preceding items, wherein said method or use does not comprise a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body.96. The method or use according to any one of the preceding items, wherein said method or use does not comprise a process for modifying the germ line or the genetic identity of an animal or human being and does not comprise the use of human embryos.

[0158] Brief Description of Drawings

[0159] In order to best describe the manner in which the above-described embodiments are implemented, as well as define other advantages and features of the disclosure, a more particular description is provided below and is illustrated in the appended drawings. Understanding that these drawings depict only exemplary embodiments of the invention and are thus not to be considered to be limiting in scope, the examples will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

[0160] Figure 1. BATF2-overexpression enhances CD8+CAR T cell proliferation, memorymaintenance and exhaustion- and AICD-resistance while attenuating IL-2 secretory capacity and Aglow tumor control. (A) Schematic overview of the CAR format investigated TFs and structure of constructs. (B) Surface RORl-expression on indicated tumor cell lines via flow cytometric analysis as quantified using QuantiBrite-PE beads. (C) IL-2 and IFN-y concentrations in supernatant after 24h of co-culture with K562ROR1 tumor cells at E: T of 4:1 as measured via ELISA for 4-1BB CAR-T cells. (D) Specific lysis of Agint(JeKo-1) or Aglow(Raji) tumor cell lines in luminescence-based assay after 24h at E: T 1:1 with 4-1BB CAR-T cells. (E) Quantification of 4-1BB CAR-T cells AICD after 24h of co-culture with antigenpositive tumor cell line (JeKo-1) at E: T 4:1. (F) Proliferation of 4-1BB CAR-T cells assessed based on CFSE-dilution after 72h of stimulation with indicated antigen-positive (K562ROR1, JeKo-1) or antigen-negative control (K562) tumor cell lines at E: T 4:1. (G) Relative frequencies of stem-cell memory-like (SCM; CD45RA+CD62L+), central memory-like (CM; CD45RA" CD62L+), effector memory-like (EM; CD45RA- CD62L-) and effector-like (EF; CD45RA+CD62L ) within indicated CAR-T cell-conditions quantified via flow cytometry. (H)Differentially expressed genes comparing CD8+CAR vs CARBATF2T cells measured with the Nanostring nCounter CAR-T Characterization panel with (abs(log2FC))>1.5 and padj< 0.05. Graphs show mean ± SEM for n=3 biological replicates. Statistical significance as determined by ordinary one-way ANOVA (C, D) or two-way ANOVA (E, F, G); significances shown are referring to viable fraction in F and to SCM fraction in G) with *P< 0.05, **P< 0.01, ***p< 0.001, ****p< 0.0001

[0161] Figure 2: (A) Example for flow cytometric analysis of transgene-expression based on truncated surface markers tEGFR and HER2t for RORl-directed CAR in combination with indicated TFs. (B)-(C) Overexpression validation of indicated TFs via immunoblot. (D)E CD8+T cells were isolated, nucleofected and the fraction of the desired cell products were followed during multiple FACS-measurements based on tEGFR+and / or Her2t+of viable CD8+T cells starting on dlO for 4-lBBz (D) and CD28z (E) CAR T cells with the indicated modification. Graph shows mean ± SEM for n=3 biological replicates unless stated otherwise. Statistical significance as determined by two-way ANOVA (D) with *P< 0.05, **P< 0.01, ***p< 0.001, ****p< 0.0001.

[0162] Figure 3: (A) IL-2 and IFN-y concentrations in supernatant after 24h of co-culture with K562ROR1tumor cells at E: T 4:1 as measured via ELISA for CD28z CAR-T cells. (B) Specific lysis of Agint(JeKo-1) or Aglow(Raji) tumor cell lines in luminescence-based assay after 24h at E: T 1:1 with CD28 CAR-T cells. (C) Proliferation of CD28 CAR-T cells assessed based on CFSE-dilution after 72h of stimulation with indicated antigen-positive (K562ROR1, JeKo-1) or antigen-negative control (K562) tumor cell lines at E: T 4:1. (D) Quantification of CD28 CAR-T cells AICD after 24h of co-culture with antigen-positive tumor cell line (JeKo-1) at E: T 4:1. (E) Relative frequencies of stem-cell memory-like (SCM; CD45RA+CD62L+), central memory-like (CM; CD45RA" CD62L+), effector memory-like (EM; CD45RA" CD62L) and effector-like (EF; CD45RA+CD62L ) within indicated CD28 CAR-T cell-conditions quantified via flow cytometry. (F) Seahorse metabolic analysis of glycolysis (left) and OXPHOS (middle). (G) Seahorse metabolic analysis of OXPHOS / glycolysis ratio. Graphs show mean ± SEM for n=3 biological replicates unless stated otherwise. Statistical significance as determined by ordinary one-way ANOVA (A, B) or two-way ANOVA (C, D, E, G; significancesshown are referring to viable fraction in (D) to SCM fraction (black) or EM (grey) in E) with *P< 0.05, **P< 0.01, ***p< 0.001, ****p< 0.0001.

[0163] Figure 4: (A) Volcano Plot of Nanostring analysis of CD8+4-1BB CAR vs CARBATF2T cells for n=3 biological replicates. B-(C) Expression of the tonic signaling high-affinity GD2-directed CD28 CAR (14G2AE101K) in CD8+T cells with indicated additional TF-modification. (B) Flow cytometric quantification of activation (CD25+CD69+) on day 20 after nucleofection. (C) Quantification of the fraction (left, Ordinary one-way ANOV A) of exhaustion-marker triplepositive (PD1+LAG3+TIM3+) cells and MFI (right, two-way ANOVA) within the indicated T cell condition on day 20 after nucleofection. (D)-(F) Repetitive antigen stimulation assay. Antigen-positive tumor cells (786-0) were pre-seeded in 6-well plates and incubated until they attached to the bottom of the plate. CD8+CD28 CAR T cells with indicated modifications were added at an inverse E: T ratio of 1:4 and incubated for 48h before they were harvested and subjected to another 48h of stimulation. (D) Fold-change in cell number over 96h. (E) Flow cytometric phenotypic characterization after 96h of chronic stimulation. (F) Quantification of the fraction of exhaustion-marker triple-positive (PD1+LAG3+TIM3+) cells within the indicated T cell condition after 96h of chronic stimulation. Graphs show mean ± SEM for n=3 biological replicates unless stated otherwise. Statistical significance as determined by ordinary one-way ANOVA (B, C) left,(D, F) or two-way ANOVA (C right, E) with *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

[0164] Figure 5: Synergistic combination of CD8+CARBATF2T cells with CD4+CARdUNT cells robustly enhances proliferative potential in vitro and in vivo. (A) Specific lysis of Agint(JeKo-1) or Aglow(Raji) tumor cell lines in luminescence-based assay after 24h at E: T 4:1 with CD4+4-1BB CAR-T cells. (B) IL-2 secretion of indicated CD4+4-lBB CAR-T cell conditions upon plate-coated antigen stimulation at different concentrations normalized to the maximum of CARonlycontrol. (C) IL-2 concentrations in supernatant after 24h of co-culture with Aginttumor cells (786-0) at E: T of 4:1 as measured via ELISA for CD4+4-1BB CAR-T cells. (D) Schematic illustration of hypothesized CD8+CARBATF2T cell proliferation benefit resulting from different modifications of CD4+CAR T cells. (E) Representative histograms showingproliferation of indicated 4-1BB CAR T cell mixtures at E: T 1:1 after 72h stimulation with JeKo-1 tumor cells. (F) Quantification of CD8+4-lBBz CARBATF2T cells proliferation cocultivated with CD4+CAR T cells with indicated modification. (G) Comparison of CD4+4-1BB CAR T cells proliferation in combination with equally or different modified CD8+CAR T cells. (H) Scheme of experimental setup. NSG mice were inoculated with 1 x 106JeKo-1 ffluc tumor cells i.v. on day 0 and received the indicated population of 2.5 x 106CD4+and 2.5 x 106CD8+4-1BB T cells (5 x 106cells total) i.v. on day 7. (I) Frequencies of CD4+(top) and CD8+(bottom) CAR T cells in blood on day 14 and 21 after tumor inoculation. (n=7). (J) CD8: CD4 ratio in peripheral blood on day 14 after tumor inoculation. (n=7), (K) Kaplan-Meier survival plot. Statistical analysis by Mantel-Cox test. Graphs show mean ± SEM for n=3 biological replicates (A-G) or n=7 (H-K; only Mocks n=3). Statistical significance as determined by ordinary one-way ANOVA (A, C, F, G, I, J), two-way ANOVA (B [comparing TF-modified CAR T cells to CARonlycontrol]) or Mantel-Cox log-rank test (K) with *P< 0.05, **P< 0.01, ***p< 0.001, ****p< 0.0001

[0165] Figure 6: (A-B) Frequency of CD25+CD69+(A) and percentage of IFN-g secretion (B) normalized the maximum of conventional CAR control of indicated CD4+4-1BB CAR-T cell conditions with titrated amounts of immobilized antigen. (C) IFN-y concentrations in supernatant after 24h of co-culture with Aginttumor cells (786-0) at E: T of 4:1 as measured via ELISA for CD4+4-lBBz CAR-T cells. (D) IL-2 concentrations in supernatant after 24h coculture of indicated CD4+CD28 CAR-T cell conditions with K562ROR1tumor cells at E: T 4:1 as measured via ELISA. n=3 for all TF-modified conditions, n=2 for conventional CAR control. (E) Quantitative comparison of expansion index of different CD8+CD28 CAR T cell conditions in a 1:1 mixture with respective CD4+CD28 CAR T cells with the identical modification or co-cultivated with CD4+CARdUNT cells. (72h co-culture, K562ROR1tumor cells at E: T 4:1). (F) Long-term xCelligence cytotoxic assay with target-antigen positive adherent 786-0 cell line. An equal mixture of indicated CD4+and CD8+CAR T cells was applied at a challenging E: T 1:20. (G) Frequencies of CD4+and CD8+CAR T cells in blood on day 10 after tumor inoculation. (n=7) (H) Average tumor burden as measured via bioluminescent imaging.Figure 7: Direct fusion of LCK to the CAR molecule in BATF2-overexpressing T cells antigensensitivity, cytolytic activity and IL-2 secretion while further augmenting antigen-specific proliferative capacity in vitro. (A) Titration of plate-coated recombinant antigen. Flow cytometric analysis of the fraction of activation marker CD25+CD69+(top) CAR T cells and viable cell count (bottom) after 24h of stimulation. (B) Titration of plate-coated TCR crosslinking OKT3 antibody. Flow cytometric analysis of the fraction of activation marker CD25+CD69+(top) CAR T cells and viable cell count (bottom) after 24h of stimulation. (C) Time course of activation using a low antigen-concentration (0.2 pg / mL). Flow cytometric analysis of the fraction of activation marker CD25+CD69+double-positive (top) and exhaustion marker PD1+LAG3+TIM3+triple-positive (bottom) CAR T cells. (D) Direct flow cytometry staining of the CAR-molecule using recombinant ROR1-FC protein / a-human FC secondary antibody. Representative histograms (left) and MFI quantification (right). (E) Quantification of LCKtotallevels as measured in immunoblots (middle, n=7) and representative blot including p-actin loading control (right). (F) Schematic representation of a strategy to rescue sensitivity of CARBATF2T cells via c-terminal fusion of LCK to the CAR molecule. (G) Representative histogram for T cells from n=2 donors with indicated modification in flow cytometric CD69-expression analysis after 24h of co-culture with Aglow(Raji) and Aghigh(K562ROR1) tumor cells. (H) Proliferation of CD4+(left, n=3) and CD8+(middle, n=5) T cells with indicated modification assessed based on CFSE-dilution after 72h of stimulation with Aglow(Raji) tumor cells at an E: T of 1:1. Representative histograms showing proliferation-dependent CFSE-dilution (right). (I) Specific lysis of Aglow(Raji, top), Agint(JeKo-1, middle) and Aghigh(K562ROR1, bottom) as measured in luminescence-based cytotoxic assay at E: T 1:1 after 24h with CD4+(left) or CD8+(right) T cells with indicated modification as effector cells. Graphs show mean ± SEM for n=3 biological replicates unless stated otherwise. Statistical significance as determined by ordinary one-way ANOVA (D, H, I), two-way ANOVA (A, B, C) or unpaired t-test (E) with *P< 0.05, **P< 0.01, ***P< 0.001, ****p< 0.0001. EC50 values as determined via non-linear fit regression analysis (A).

[0166] Figure 8: (A) Representative flow cytometry plots showing gating for activation marker expression. (B) Immunoblots comparing total ERK1 / 2 and pERKl / 2 levels of CAR vs. CARBATF2T cells with 4-1BB stimulation domain subjected to different stimulationconditions (P / l - PMA / lonomycine, OKT3 - stimulation of TCR, RORllo / RORlhi- 0.5 and 2 pg / mL plate-coated recombinant RORl-protein, respectively) or left unstimulated. (C) Overexpression of BATF2 validated in immunoblots of indicated T cell conditions.

[0167] Figure 9: CD8+BATF2-ove rexpressing T cells equipped with a CAR-LCK fusion molecule and supported by CD4+cJUN-CAR T cells provide augmented solid tumor control in vivo. (A) Scheme of experimental setup. NSG mice were inoculated with 1 x 106786-0 ffluc tumor cells s.c. on day 0 and received the indicated population of 2.5 x 106CD4+and 2.5 x 106CD8+4-lBBz T cells (5 x 106cells total) i.v. on day 14. (B) Frequencies of CD8+CAR T cells in blood on day 17 and 21 after tumor inoculation (day 3 and day 7 after T cell injection). (C) CD8: CD4 ratio in peripheral blood on day 17 after tumor inoculation (day 3 after T cell injection). (D) Individual tumor burden for each treatment group as measured via bioluminescent imaging. Graphs show mean ± SEM for n=5 mice. Statistical significance as determined by ordinary one-way ANOVA (B, C) with *P< 0.05, **P< 0.01, ***P< 0.001, ****p< 0.0001

[0168] Figure 10: (A) Quantification of bioluminescence signal on day 13 after tumor inoculation with already randomized mice. (B) Frequencies of CD4+CAR T cells in blood on day 17 and 21 after tumor inoculation (day 3 and day 7 after T cell injection). (C) Activation level of respectively modified CD8+T cells before injection into mice (NSG / 786-0 model). Graphs show mean ± SEM for n=5 mice (A, B) and statistical significance was determined by ordinary one-way ANOVA with *P< 0.05, **P< 0.01, ***P< 0.001,

[0169]

[0170] 0.0001 Figure 11: BATF2-overexpression induces changes in chemokine receptor repertoire and activation state improving chemokine-dependent migration and promoting retention in circulation via increased LFA-1 recycling. (A) Nanostring analysis of CD8+4-lBBz ROR1-directed CAR T for genes related to chemokine signaling. Data duplicated and filtered from Fig. 1H. (B) Representative histograms for flow cytometric analysis of indicated chemokine receptor expression levels in CARBATF2compared to conventional CAR T cells. (C) Quantification of respective chemokine receptor MFI levels comparing CAR and CARBATF2T cells. (D) Schematic illustration of migration assay principle (left) and quantification of chemotactic index (Cl) comparing conventional CAR and CARBATF2T cells regarding potentialto migrate in response towards chemokines CCL21, CXCL12 and CCL2. (T cells n=3 biological replicates). (E) Flow cytometric quantification of CXCR4 and CCR7 expression levels (MFI) in CD4+and CD8+CAR T cells with indicated modifications. (F) Schematic illustration of experimental strategy for in vivo homing assay. Tumor-free mice (n=5) received a mixture of proliferation-dye pre-stained CAR (CTV+) or CARBATF2(CFSE+) T cells consisting of equal amounts CD4+and CD8+cells (10 x 106total T cells) via tail-vein injection. After 24h mice were sacrificed 3min after i.v. injection of an anti-human CD45 antibody to allow discrimination of blood and tissue T cells. Indicated organs were isolated, single-cell suspensions were stained for CD4+and CD8+and analyzed via flow cytometry. (G) Frequencies of indicated CD4+and CD8+CAR T cell population in respective organs of mice sacrificed after 24h. (n=5) (H) Homing index calculated as described in F. (I) Fraction of CD25+CD69+CAR vs. CARBATF2T cells to assess basal activation state (left) and quantification of activation marker MFI (right). (n=5) (J) Expression level of LFA-1 on CAR vs. CARBATF2T cells as measured via CD18 staining. (K) Histograms for MALII-lectin binding to the surface of CAR vs. CARBATF2T cells left untreated and stimulated via plate-coated OKT3 antibody for 24h. Neuraminidase A (NeuA)-treated cells were used as a control. (L) Relative expression of βII-spectrin quantified via immunoblot comparing CAR vs. CARBATF2T cells. (n=9) (M) Spontaneous migration during in vitro migration assay in absence of chemokines. Graphs show mean ± SEM for n=3 biological replicates unless stated otherwise. Statistical significance as determined by ordinary one-way ANOVA (E), two-way ANOVA (D, G, I-right) or unpaired t-test (C, I-left, J, L, M) with *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

[0171] Figure 12: (A) Exemplary gating strategy for a lung sample of an in vivo homing assay. (B) Frequencies of indicated CD4+and CD8+CAR T cell population in respective organs of mice sacrificed after 72h. (n=5 for spleen, n=4 for all other organs). (C) Homing index calculated as described in Fig 5 F. (D) Expression level of LFA-1 on CAR-L vs. CAR-LBATF2T cells as measured via CD18 staining. (n=5). Graphs show mean ± SEM for n=3 biological replicates unless stated otherwise. Statistical significance as determined by two-way ANOVA (B) or unpaired t-test (D) with *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.Figure 13: BATF2-OE opens a therapeutic window to rescue from lung toxicity induced by human / mouse cross-reactive ROR1(F) CAR T cells in tumor-free NSG mice.

[0172] A, Schematic overview of second-generation CAR constructs comprising the clone F scFv. B-C, Assessment of cross-reactivity and the effect of BATF2-overexpression on antigensensitivity against plate-coated recombinant human (top) or mouse (bottom) ROR1-protein. Quantification of the fraction of CD25+CD69+28z- (B) or BBz-CAR T cells (C).

[0173] D, Scheme of experimental setup. NSG mice received 10 x 106cells of the indicated CD8+T cell population and were closely monitored.

[0174] E, Quantification of CD18, CD25, CD69 and CXCR4 surface expression based on MFI.

[0175] F, Weight-change within indicated treatment groups over time.

[0176] G, Kaplan-Meier survival plot. Statistical analysis by Mantel-Cox test.

[0177] scFv - single-chain fragment variable, H / TM - hinge / transmembrane domain

[0178] Graphs show mean ± SEM for n=3 biological replicates. Statistical significance as determined by ordinary one-way ANOVA (E) or two-way ANOVA (B) with *P< 0.05, **P< 0.01, ***p< 0.001, ****p< 0.0001.

[0179] Figure 14:

[0180] A, Assessment of cross-reactivity against recombinant human and mouse ROR1 protein. Direct CAR staining of 28z- as well as BBz-RORl(F) CAR T cells (top) and quantification of the fraction of CD25+CD69+upon stimulation with a titration of plate-coated recombinant human or mouse ROR1 protein (bottom). Data for conventional CARs duplicated from Fig. 13B.

[0181] B, Specific lysis of K562, Raji, JeKo-1, K562ROR1and 786-0 tumor cell lines as measured in luminescence-based cytotoxic assay after 24h with T cells with indicated modification as effector cells at E: T 1:1.Graphs show mean ± SEM for n=3 biological replicates. Statistical significance as determined by unpaired t-test (A) ordinary one-way ANOVA (B) with *P< 0.05, **p< 0.01, ***P< 0.001, ****p< 0.0001.

[0182] Figure 15: BATF2-overexpressing CAR T cells enable lung tumor control without toxicity. A, Scheme of experimental setup. NSG mice received 1 x 106A549fflucNSCLC tumor cells i.v. on day 0. On day 6 mice were randomized based on BLI and weight before receiving 10 x 106of the indicated CD8+T cell population on day 7.

[0183] B, Flow cytometric characterization of A549 cell lines. (Nat - A549 native, ffluc -A549ffluc+GFP, iso - isotype control, stain - ROR1 staining.)

[0184] C, Example image showing the BLI signal located in the lungs on day 6.

[0185] D, Kaplan-Meier survival plot. Statistical analysis by Mantel-Cox test.

[0186] E, Weight change within indicated treatment groups over time.

[0187] F, Weight compared to starting weight on day 9 (day 2 after T cell infusion).

[0188] G, Quantification of bioluminescence signal at indicated time-points after tumor inoculation. Graphs show mean ± SEM for n=5 mice. Statistical significance as determined by ordinary one-way ANOVA (F, G) with *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

[0189] Description of Embodiments

[0190] All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0191] Unless context clearly indicates otherwise, each occurrence of the term "comprise", and variations such as "comprising", may optionally be substituted with the term "consist of", and variations such as "consisting of".Unless context clearly indicates otherwise, the articles "a", "an" and "the" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Unless context clearly indicates otherwise, the term "or" is used herein to mean, and is used interchangeably with, the term "and / or". The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements" connected by said term.

[0192] Unless context clearly indicates otherwise, the term "about", as used herein, refers to a deviation of ± 10 % from the recited value. When the word "about" is used herein in reference to a number, it should be understood that still another embodiment includes that number not modified by the presence of the word "about". In the absence of the term "about" and unless the context dictates otherwise, generally accepted rounding rules apply to the specified values.

[0193] Unless context clearly indicates otherwise, the term "similar", as used herein, is interchangeable for alike, analogous, comparable, corresponding, and -like, and is meant to have the same or common characteristics, and / or in a quantifiable manner to show comparable results i.e., with a variation of maximum 20 %, 10 %, more preferably 5 %, or even more preferably 1 %, or less.

[0194] Unless context clearly indicates otherwise, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0195] BATF2 (Basic Leucine Zipper ATF-Like Transcription Factor 2, or basic leucine zipper transcription factor, ATF-like 2) also known as " SARI" (suppressor of AP-1, regulated by IFN) is known in the art. It is a member of the AP-1 transcription factor family and acts via the formation of a heterodimer with JUN family proteins that recognizes and binds the DNA and regulate the expression of target genes. BATF2, as used herein, is preferablymammalian BATF2, such as human, mouse, rat or macaque BATF2. More preferably, BATF2 is human or mouse, and most preferably human. BATF2, as used herein, may comprise the amino acid sequence of an isoform of wild type BATF2 or a fragment or mutant thereof that has BATF2 activity, such as the activity of inhibiting AP-1 transcription factor activity. Preferably, BATF2 comprises or consists of an amino acid sequence represented by SEQ ID NO: 1 or comprises or consists of an amino acid sequence that has at least 90%, at least 95% or even at least 99% sequence identity to an amino acid sequence represented by SEQ ID NO: 1.

[0196] " Transcription factor", as used herein, refers to any transcription factor. Preferably, the transcription factor is a mammalian transcription factor, such as a human, mouse, rat or macaque transcription factor. More preferably, the transcription factor is human or mouse, and most preferably human. Preferably, the transcription factor is a transcription factor, which, when overexpressed in a CD4+T cell, results in the CD4+T cell exhibiting an increased level of IL-2 and IFN-y secretion. The transcription factor may be BATF, BATF3, TFAP4, FoxOl or eJun. BATF (Basic Leucine Zipper ATF-Like Transcription Factor), BATF3 (Basic Leucine Zipper ATF-Like Transcription Factor 3), TFAP4 (Transcription Factor AP-4), and FoxOl (Forkhead Box 01) are known in the art.

[0197] eJun (Proto-oncogene eJun) is also known in the art. It is a member of the AP-1 transcription factor family. eJun heterodimerizes with proteins of the FOS family to form an AP-1 transcription complex, thereby enhancing its DNA binding activity to the AP-1 consensus sequence and enhancing its transcriptional activity. eJun, as used herein, is preferably mammalian eJun, such as human, mouse, rat or macaque eJun. More preferably, eJun is human or mouse, and most preferably human. eJun, as used herein, may comprise the amino acid sequence of an isoform of wild type eJun or a fragment or mutant thereof that has eJun activity, such as AP-1 transcription factor activity. Preferably, eJun comprises or consists of an amino acid sequence represented by SEQ ID NO: 2 or comprises or consists of an amino acid sequence that has at least 90%, at least 95% or even at least 99% sequence identity to an amino acid sequence represented by SEQ ID NO: 2.LCK (Tyrosine-protein kinase Lek) is known in the art. It plays an essential role in the selection and maturation of developing T cells in the thymus and in the function of mature T cells. It plays a key role in T-cell antigen receptor (TCR)-linked signal transduction pathways. LCK, as used herein, is preferably mammalian LCK, such as human, mouse, rat or macaque LCK. More preferably, LCK is human or mouse, and most preferably human. LCK, as used herein, may comprise the amino acid sequence of an isoform of wild type LCK or a fragment or mutant thereof that has LCK activity. Preferably, LCK comprises or consists of an amino acid sequence represented by SEQ ID NO: 7 or comprises or consists of an amino acid sequence that has at least 90%, at least 95% or even at least 99% sequence identity to an amino acid sequence represented by SEQ ID NO: 7.

[0198] A "fragment", as used herein, refers to a protein in which amino acid residues are deleted as compared to the target protein itself, but where the remaining amino acid sequence is usually identical to that of the target protein. Such deletions or truncations may occur at the amino-terminus (N-terminus) or carboxy-terminus (C-terminus) of the target protein, or alternatively both. The fragment may retain one or more of the biological activities of the target protein, e.g., may comprise an enzymatic activity and / or an interaction site of the reference protein to, e.g., a cell receptor.

[0199] As used herein, "mutation" refers to a change in the amino acid sequence of a native protein. Mutations can be described by using the native sequence and then identifying the specific amino acid that has been changed. A "mutant" refers to the protein that contains the mutation. A full-length mutant sequence refers to the full amino acid sequence of the mutant protein, instead of describing the mutant as the amino acids that are different from the native protein.

[0200] The term "overexpression", as used herein, refers to the abnormal or artificial expression of a gene in increased quantity resulting in an excess production of a protein. BATF2, or a transcription factor, such as eJun, being overexpressed in a cell may lead to an increase in expression of the BATF2, or transcription factor, such as eJun of at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%,300%, 325%, 350%, 375%, 400% or more compared to the expression of the BATF2, or transcription factor, such as eJun in a normal state of the cell.

[0201] A "recombinant antigen-binding receptor" according to the invention is a transmembrane receptor, which, when expressed by an immune cell, is capable of mediating an immune response. The term "antigen-binding receptor" as used herein, is thereby understood to refer to an antigen-specific receptor. The recombinant antigen-binding receptor is a nonnatural immunoreceptor, i.e., genetically engineered. Exemplary recombinant antigenbinding receptors in accordance with the invention are T-cell receptors (TCRs), and chimeric antigen receptors (CARs). The antigen-binding receptor in its monomeric form may either consist of a single molecule comprising all its domains or consist of a heterodimer that comprises all its domains. It will be understood that a CAR and / or a TCR in accordance with the invention binds to an antigen, preferably a cancer cell antigen. While a CAR typically binds to an extracellular domain of a cancer cell antigen, i.e., a cancer cell surface antigen, a TCR typically binds to an intracellular cancer cell antigen presented by an HLA molecule.

[0202] The terms "binds", "binding" or "bind", as used herein, refer to specific binding to the antigen of interest. It is to be understood that where the terms "binds", "binding" or "bind" are mentioned, they refer to the intrinsic capability of the CAR or TCR to specifically bind to the antigen without further modification, but they do not require that the antigen must be present. The term "binding", specifically in the context of "capable of binding", as used herein, refers to the capability to form a complex with a molecule that is to be bound (e.g., CD19, FLT3, BCMA, or ROR1). Binding typically occurs non-covalently by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces and is typically reversible. Various methods and assays to determine binding capability are known in the art. Binding is usually a binding with high affinity, wherein the affinity as measured in KD values is preferably is less than 1 pM, more preferably less than 100 nM, even more preferably less than 10 nM, even more preferably less than 1 nM, even more preferably less than 100 pM, even more preferably less than 10 pM, even more preferably less than 1pM. The term, "binding", specifically in the context of "antigen-binding receptor” as used herein, refers to functional binding in the context of a CAR / TCR.

[0203] The term " T-cell receptor (TCR)", as used herein, has the meaning known in the art. Typically, a TCR is understood as a heterodimeric cell surface protein of the immunoglobulin superfamily that participate in the activation of T cells in response to the binding of an antigen. The TCR complex can consist of TCRa / R chains and CD3y / 6 / s / subunits, which can associate through hydrophobic interactions. Somatic VDJ recombination allows to generate distinct TCRa and TCRR chains, and TCRaR heterodimers are generally responsible for antigen recognition by binding to peptide-MHC complexes. CD3 can transmit the TCR-triggered signal through immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic tail, but it is generally not directly involved in antigen recognition. ITAMs are tandem duplications of a tyrosine-containing sequence (YXXL / I), and the CD3y / 6 / s chains each contain one ITAM, while the CD3 chain contains three. As a consequence of TCR engagement, ITAM phosphorylation can be induced by protein tyrosine kinases (PTKs), which allow other effector molecules to interact with the TCR complex. A TCR can be found on the surface of a cell or in soluble form. The TCR can be an intact or full-length TCR, including but not restricted to a TCR in the aR form or y6 form, as a dimeric TCR (dTCR), a single-chain TCR (scTCR). The TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigenbinding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable R chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and / or MHC-peptide complex. A TCR can contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail. Each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tailat the C-terminal end. A TCR can be associated with invariant proteins of the CD3 complex involved in mediating signal transduction.

[0204] In accordance with the invention, the term "chimeric antigen receptor (CAR)" has the meaning known in the art. A " CAR" according to the invention can be any possible form. Typically, a CAR is understood as a receptor protein that has been engineered to give T cells the new ability to target a specific antigen. The receptor is chimeric in that it combines both antigen-binding and T cell activating functions into a single receptor. CAR T cells can be derived either from T cells in a patient's own blood (autologous) or from the T cells of another, healthy, donor (allogeneic). Once isolated from a person, these T cells can be genetically engineered to express a specific CAR, which programs them to target an antigen, e.g., an antigen that is present on the cancer cell surface. The chimeric antigen receptor is expressed in T cells and allows said T cells to bind specifically to antigenexpressing cancer cells with high specificity to exert a growth inhibiting effect, preferably a cytotoxic effect, on said cancer cells.

[0205] A CAR is designed to enhance the recognition and targeting of cancer cells or other diseased cells. A CAR according to the invention typically comprises, but is not limited to, three main components: an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain, often derived from an antibody fragment, provides specificity for the target antigen. This domain forms a single-chain variable fragment (scFv). The transmembrane domain anchors the receptor in the lymphocyte (e.g., T cell) membrane, while the intracellular signaling domain (such as CD3, CD28, IL-2 and 4-1BB) initiates activation signals upon antigen binding. When expressed in lymphocytes (e.g., T cells), the CAR can allow the modified lymphocytes (e.g., T cells) to recognize and bind to the target antigen, leading to the activation and killing of the target cells expressing the target antigen.

[0206] It will also be understood that the CAR can be any known type of CAR, e.g., a first-generation CAR, a second-generation CAR, a third-generation CAR, a fourth-generation CAR or a fifth-generation CAR. A first-generation CAR generally has an intracellular signaling domain comprising an intracellular signaling domain of CD3, FcyRI, or otherITAM-containing activating domain to provide a T cell activation signal. Second and third-generation CARs further comprise a costimulatory signaling domain (e.g., a costimulatory signaling domain from an endogenous T cell costimulatory receptor, such as CD28, 4-1BB, or ICOS) or two costimulatory signaling domains, respectively. A fourth generation CAR, instead, may express one or two costimulatory molecules together with a constitutive or inducible expression cassette containing a transgenic protein such as a cytokine or enzyme. A fifth generation CAR is known in the art and may comprise an additional intracellular domain compared to the first to fourth generation CAR. The CAR may comprise, but is not limited to, truncated intracellular domains of cytokine receptors (e.g., IL-2R chain fragment) with a motif for binding transcription factors such as STAT-3 / 5.

[0207] For CAR T cell therapy, T cells are usually manipulated and expanded ex vivo. However, in accordance with the invention, there is also the option to conduct gene transfer in vivo. One way to program immune cells such as T cells within the body is the gene transfer with DNA-carrying nanoparticles85. A second strategy is the in vivo CAR immune cell (e.g., CAR T cell) generation with viral vectors86.

[0208] In accordance with the invention, the term "cancer antigen" has the meaning known in the art. In those embodiments of the invention where the CD8+T cell or CD4+T cell contains and expresses an endogenous nucleic acid or a set of nucleic acids encoding a chimeric antigen receptor, the cancer cell antigen in accordance with the invention to which the chimeric antigen receptor binds is preferably a cancer cell surface antigen. In those embodiments of the invention where the CD8+T cell or CD4+T contains and expresses an endogenous nucleic acid or a set of nucleic acids encoding a T-cell receptor, the cancer cell antigen in accordance with the invention to which the T-cell receptor binds is preferably an intracellular cancer cell antigen presented by a HLA molecule.

[0209] The term "exogenous nucleic acid" refers to a nucleic acid that is not normally present in a cell in that form. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. For example, an exogenous nucleic acid may contain a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or it may containa sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). Thus, for example, an DNA or RNA that has been produced outside a given cell and then has been introduced into the cell is an exogenous DNA or RNA. In contrast, "endogenous nucleic acid" includes molecules or sequences that are normally present in that form in a particular cell at a particular stage of development under particular environmental conditions. Thus, for example, an endogenous gene, RNA, or DNA has not been produced outside a given cell and introduced into that cell.

[0210] The exogenous nucleic acid may comprise an expression cassette. The term "expression cassette" has the meaning known in the art. As used in accordance with the invention, an expression cassette typically comprises at least a promoter sequence, at least one open reading frame (e.g., an open reading frame encoding the at least one positive regulator of autophagy and / or an open reading frame encoding the BATF2 / transcription factor such as eJun and / or T-cell receptor / chimeric antigen receptor), and a 3' untranslated region that usually contains a polyadenylation site. An expression cassette in accordance with the invention may be monocistronic or polycistronic (e.g., bicistronic). For example, a polycistronic (e.g., bicistronic) expression cassette may comprise an open reading frame encoding the at least one positive regulator of autophagy and an open reading frame encoding the T-cell receptor or chimeric antigen receptor. Polycistronic (e.g., bicistronic) expression cassettes are generally known in the art and may comprise, for example, linkages of the open reading frames by internal ribosome entry sites (IRES). In addition, the expression cassette may appropriately comprise additional nucleotide sequences such as an adapter or a linker, an enhancer, a selectable marker (e.g., antibiotic resistance marker), a replication unit, a polyA sequence, a tag for purification (e.g., GST, poly-Arg, FLAG, histidine-tag (His-tag) or c-myc, etc. The expression cassette may comprise a selection marker. The term "selection marker" has the meaning known in the art and typically refers to a recombinant nucleic acid sequence to facilitate the identification and selection of cells or organisms that have successfully incorporated a desired genetic modification. For example, the selection marker can be one that confers a selectable phenotype, such as resistance to a specific antibiotic or the ability to grow in a selective medium. By including the selection marker in the same expression cassette, a selective pressure can be applied,such as exposure to the antibiotic or selective medium, to identify and isolate the cells or organisms that have taken up the desired genetic modification. In accordance with the invention, an expression cassette containing selection marker can be used to facilitate the identification and selection of modified lymphocytes of the invention. In an embodiment of the invention, the modified? cell of the present invention may express the EGFRt marker on the cell surface. The EGFRt marker can be used to detect, track, select and deplete the modified T cell of the present invention. Therefore, analysis of drug product persistence following administration of the modified T cell is made available. Furthermore, the EGFRt marker makes modified T cells of the invention sensitive to ADCC / CDC through the antibody Cetuximab which can therefore be used as safety switch.

[0211] The term "expression vector" or "vector" is known in the art and encompasses, for instance, a plasmid, a viral vector such as a retroviral vector, a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a transposon vector. It is understood that a vector as used in connection with the present invention is a vector which is suitable for therapeutic applications in humans.

[0212] As used in connection with the invention, the meaning of the terms "co-expressing" or "coexpress" in relation to the mammalian cells of the invention is in accordance with the common meaning of these terms and encompasses particularly the expression of two elements (e.g., proteins such as the CARs (a) and (b) as referred to herein) in the same mammalian cell.

[0213] The term "activity" or "protein activity", as used herein, means the biological activity that a given protein has in a given cell. For example, “protein activity" may refer to an activity of BATF2 (e.g., activity of inhibiting AP-1 transcription factor activity) or eJun (AP-1 transcription factor activity) in a given T cell. The BATF2 or eJun protein activity level can be determined by any known means, for example, by determining expression of BATF2 or eJun target genes and / or by measuring binding of BATF2 or eJun to target site in the genome of the T cell. This can be done, e.g., by chromatin immunoprecipitation followedby sequencing (Chip-seq) and / or quantifying RNA levels or protein levels (for example, by western blot or flow cytometry).

[0214] The term "expression level" refers to the level of gene expression from a given gene in a given cell. Gene expression in the context of protein-coding genes refers to the production of a protein via transcription of a gene encoding said protein into mRNA and translation of said mRNA into the encoded protein. Thus, the expression level can be increased by increasing transcription and / or translation from a given gene. This can be achieved e.g., by introducing activators of transcription or translation into the cell, and / or by increasing the transcription or translation of said gene in the cell by genetic modification. Conversely, the expression level can be reduced by reducing transcription and / or translation from a given gene. This can be achieved e.g., by introducing inhibitors of transcription or translation into the cell, and / or by reducing the transcription or translation of said gene in the cell by genetic modification.

[0215] The term "modified" as used herein in the context of a T cell such as a CD8+T cell or a CD4+T cell means that the T cell is different by way of physical and / or chemical manipulation that results in an increased BATF2 or other transcription factor, such as eJun, level compared to the same CD8+T cell or a CD4+T cell and the expression of a recombinant antigen-binding receptor. Thus, a "modified CD8+T cell" or "modified CD4+T cell" is different from the same CD8+T cell or a CD4+T cell that has not been modified (by way of said physical and / or chemical manipulation that results in an increased BATF2 or other transcription factor, such as eJun and the expression of a recombinant antigen-binding receptor).

[0216] The term "recombinant" as used herein in the context of a T cell such as a CD8+T cell or a CD4+T cell means that the T cell is different by way of physical and / or chemical manipulation that results in the expression of a recombinant antigen-binding receptor. Thus, a "recombinant CD8+T cell" or "recombinant CD4+T cell" is different from the same CD8+T cell or a CD4+T cell that has not been modified (by way of said physical and / or chemical manipulation that results in the expression of a recombinant antigen-binding receptor.Terms such as "treatment of cancer" or "treating cancer" according to the present invention refer to a therapeutic treatment. An assessment of whether a therapeutic treatment works can, for instance, be made by assessing whether the treatment inhibits cancer growth in the treated patient or patients. Preferably, the inhibition is statistically significant as assessed by appropriate statistical tests which are known in the art. Inhibition of cancer growth may be assessed by comparing cancer growth in a group of patients treated in accordance with the present invention to a control group of untreated patients, or by comparing a group of patients that receive a standard cancer treatment of the art plus a treatment according to the invention with a control group of patients that only receive a standard cancer treatment of the art. Such studies for assessing the inhibition of cancer growth are designed in accordance with accepted standards for clinical studies, e.g., double-blinded, randomized studies with sufficient statistical power. The term "treating cancer" includes an inhibition of cancer growth where the cancer growth is inhibited partially (i.e. where the cancer growth in the patient is delayed compared to the control group of patients), an inhibition where the cancer growth is inhibited completely (i.e. where the cancer growth in the patient is stopped), and an inhibition where cancer growth is reversed (i.e. the cancer shrinks). An assessment of whether a therapeutic treatment works can be made based on known clinical indicators of cancer progression.

[0217] The treatment of cancer according to the present invention does not exclude additional or secondary therapeutic benefits also occurring in patients. For example, an additional or secondary benefit may be an enhancement of engraftment of transplanted hematopoietic stem cells that is carried out prior to, concurrently to, or after the treatment of cancer. However, it is understood that the primary treatment for which protection is sought is for treating the cancer itself, and any secondary or additional effects only reflect optional, additional advantages of the treatment of cancer growth.

[0218] The treatment of cancer according to the invention can be a first-line therapy, a second-line therapy, a third-line therapy, or a fourth-line therapy. The treatment can also be a therapy that is beyond fourth-line therapy. The meaning of these terms is known in the artand in accordance with the terminology that is commonly used by the US National Cancer Institute.

[0219] A "subject" in the context of the invention is preferably mammalian, such as human, mouse, rat or macaque. Preferably, it is a human subject. The subject is preferably a (human) subject diagnosed with a disease to be treated using the modified CD8+T cell. " Activation Induced cell death" as used herein refers to a state of a T cell in which activation through the T-cell receptor results in apoptosis, i.e., the T cell is not able to proliferate and exhibits markers of apoptosis.

[0220] Detailed Description of Embodiments

[0221] Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0222] CD8+T cell comprising a recombinant antigen-binding receptor and overexpressing BATF2

[0223] In the first aspect, the invention provides a CD8+T cell comprising a recombinant antigenbinding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses (e.g., overexpresses) BATF2.

[0224] BATF2 according to the present invention may be mammalian BATF2, such as a human, mouse, rat, or macaque BATF2. More preferably, BATF2 is human or mouse, and most preferably human. Hence, BATF2 may comprise the amino acid sequence of a mammalian isoform of BATF2, preferably BATF2 comprises the amino acid sequence of a human isoform of BATF2.

[0225] BATF2 according to the present invention may comprise the amino acid sequence of an isoform of wild type BATF2 or a fragment or mutant thereof. Preferably, the fragment or mutant retains one or more of the biological activities of the wild-type BATF2. For instance,the fragment or mutant may have an activity of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 100% activity of the wild type BATF2. Preferably, BATF2 comprises or consists of an amino acid sequence represented by SEQ ID NO: 1 or comprises or consists of an amino acid sequence that has at least 90%, at least 95% or even at least 99% sequence identity to an amino acid sequence represented by SEQ ID NO: 1, and optionally has an AP-l-attenuating activity, or an activity of controlling the differentiation of lineage-specific cells in the immune system.

[0226] In an embodiment, the CD8+T cell of the present invention has been modified to express BATF2 at a level that is increased compared to the level of BATF2 expressed by the CD8+T cell before it was modified to express the BATF2.

[0227] The BATF2 overexpression may lead to an increase in the expression level of BATF2 of at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400% or more compared to the expression level of BATF2 expressed by the CD8+T cell before it was modified to express the BATF2. Preferably, the BATF2 level is increased compared to a control CD8+T cell, e.g., an unmodified CD8+T cell (i.e., not modified to exhibit an increased BATF2 level) of a CD8+T cell of the same species, wherein the unmodified CD8+T cell is preferably from the same subject as the modified CD8+T cell. The comparator typically is an average BATF2 level obtained from a population of such control CD8+T cell. A population of cells can be, for example, at least 10 randomly selected cells.

[0228] For example, when the CD8+T cell of the present invention is a human CD8+T cell, the BATF2 level can be increased compared to an unmodified human CD8+T cell obtained from a human subject, wherein the unmodified CD8+T cell is preferably from the same human subject as the modified CD8+T cell.

[0229] The CD8+T cell of the present invention may comprise an exogenous nucleic acid for overexpressing BATF2. Hence, preferably the exogenous nucleic acid is DNA, but can also be, for example, an mRNA encoding BATF2. The exogenous nucleic acid, when it is DNA, can be present as part of an episome or be integrated into the genome of the cell.The expression of said exogenous nucleic acid may be constitutive or inducible, preferably constitutive. In accordance with the invention, the term "constitutive" in relation to the expression of recombinant nucleic acids has the meaning known in the art. It is understood that it encompasses the continuous and uniform expression of the recombinant nucleic acid. A constitutive expression is one that is active under normal or standard conditions in the modified human lymphocytes of the invention, regardless of environmental factors or specific cellular signals.

[0230] In accordance with the invention, the term "inducible" in relation to the expression of recombinant nucleic acids refers to expression that can be regulated or controlled by specific signals or factors, e.g., by drugs which are not toxic to humans and can be administered to humans. It is well known how inducible expression can be achieved. For example, the recombinant nucleic acids may contain inducible promoters to ensure the inducible expression. It is understood that such inducible promoters may form part of the expression cassettes referred to herein.

[0231] Usually, BATF2 is not endogenously expressed in CD8+T cells. Hence, preferably the exogenous nucleic acid overexpressing BATF2 may encode the BATF2. In this instance, the exogenous nucleic acid overexpressing and encoding BATF2 may comprise expression cassette. The expression cassette may comprise a selection marker. In an embodiment of the invention, the modified T cell of the present invention may further express the EGFRt marker on the cell surface. The EGFRt marker can be used to detect, track, select and deplete the modified T cell of the present invention. Therefore, analysis of drug product persistence following administration of the modified T cell is made available. Furthermore, the EGFRt marker makes modified T cells of the invention sensitive to ADCC / CDC through the antibody Cetuximab which can therefore be used as safety switch. Suitable promoters for use in an expression cassette for overexpressing BATF2 include a MCSV, CMV or CAG promoter, preferably MCSV. For instance, the expression cassette may comprise an EF1 / HTLV hybrid promoter followed by the transgene, a 2A ribosomal skip sequence, a truncated surface marker (tEGFR / Her2t) and a Stop codon flanked by inverted terminal repeats.The exogenous nucleic acid encoding the BATF2 may be integrated into the genome of the CD8+T cell. In this instance, the CD8+T cell expressing the BATF2 may be obtainable by expressing the BATF2 through stable gene transfer. Stable gene transfer may be accomplished through viral vectors or non-viral gene transfer. Alternatively, the exogenous nucleic acid may be a genetic expression vector encoding the BATF2. In this instance, the CD8+T cell expressing the BATF2 is obtainable by expressing the BATF2 through transient gene transfer or any other means resulting in transient expression.

[0232] The CD8+T cell of the present invention may also comprise an exogenous activator of transcription for an endogenous BATF2 gene and / or activator of translation of an endogenous BATF2 mRNA. The exogenous activator of transcription for an endogenous BATF2 gene may modify the endogenous BATF2 gene genetically to induce BATF2 expression in the CD8+T cell. Preferably, the endogenous BATF2 gene may be genetically modified in its promoter region. For example, the endogenous promoter can be replaced by a promoter that provides transcription in the CD8+T cell so modified.

[0233] Expression of the endogenous BATF2 gene may also be achieved by CRISPR-mediated transcriptional activation (CRISPRa), or other CRISPR-Cas-based transcriptional engineering methods. Thus, in one embodiment, the exogenous activator of transcription for an endogenous BATF2 gene comprised by the cells (e.g. CD8+T cells) of the invention comprises or consists of Cas9 and a corresponding guide RNA, such as a nuclease defective Cas9 (dCas9), fused to activator proteins / domains (e.g. viral protein such as VP48 or VP64) and CRISPRa guide RNAs target sequences upstream of the promoter region or transcriptional start site (TSS) of BATF2 (i.e. CRISPR-mediated transcriptional activation (CRISPRa)).

[0234] An increased BATF2 level compared to the level of BATF2 expressed by the CD8+T cell before it was modified to express BATF2 can mean, for example, that the BATF2 level is increased to a detectable level in the CD8+T cell. A suitable reference may be the BATF2 level in a control CD8+T cell, e.g., an unmodified CD8+T cell (i.e., not modified to exhibitan increased BATF2 level) of the same cell type from the same species, wherein the unmodified CD8+T cell is preferably from the same subject as the modified CD8+T cell. An increased BATF2 level can also mean, for example, that the BATF2 level is the same or increased at least 2-fold, at least 3-fold, at least 5-fold, or even at least 10-fold to the BATF2 level in control immune cells of the same cell type endogenously expressing BATF2, e.g., an unmodified immune cell such as a T cell, B cell, macrophage, and dendritic cell (i.e., not modified to exhibit an increased BATF2 level) from the same species, wherein the unmodified immune cell is preferably from the same subject as the modified immune cell. The reference typically is an average BATF2 level obtained from a population of such control immune cell.

[0235] The BATF2 expression level can be determined by any known means for protein quantification, for example, flow cytometry or quantitative western blot, preferably quantitative western-blot.

[0236] It has been found by the present inventors that increased levels of BATF2 can lead to a) enhanced proliferation;

[0237] b) decreased exhaustion;

[0238] c) reduced accumulation in non-cancerous lung tissue;

[0239] d) memory like phenotype;

[0240] e) improved chemokine-dependent migration, and / or

[0241] f) reduced activation-induced cell death.

[0242] Accordingly, the invention also provides a CD8+T cell as described herein, wherein the CD8+T cell exhibits:

[0243] a) enhanced proliferation;

[0244] b) decreased exhaustion;

[0245] c) reduced accumulation in non-cancerous lung tissue;

[0246] d) memory like phenotype;

[0247] e) reduced activation-induced cell death;a) improved chemokine-dependent migration;

[0248] f) any combination of a) to e) and / or

[0249] g) all of a) to e).

[0250] Enhanced proliferation, decreased exhaustion, reduced accumulation in non-cancerous lung tissue, memory like phenotype, and / or reduced activation-induced cell death is preferably seen in comparison to a control CD8+T cell, e.g. an unmodified CD8+T cell (i.e. not modified to exhibit and increased BATF2 level) of the same cell type from the same species, wherein the unmodified CD8+T cell is preferably from the same subject as the modified CD8+T cell. The comparatortypically is an average survival of a population of such control CD8+T cell.

[0251] Thus, enhanced proliferation, decreased exhaustion, reduced accumulation in non-cancerous lung tissue, memory like phenotype, and / or reduced activation-induced cell death al can be determined, for example, by comparing the proliferation, exhaustion, accumulation in non-cancerous lung tissue, memory like phenotype, and / or activation-induced cell death in vivo of the CD8+T cell of the invention with the survival in vivo of control CD8+T cell in the same subject.

[0252] Methods to determine these properties are known to a person skilled in the art. For example, to examine proliferation, the number of CD8+T cells of the invention per ml and the number of control CD8+T cells per mL in the same (peripheral) blood sample obtained from a subject can be compared at a predetermined time point after administration, e.g. on day 0, day 1, day 3, day 7, day 14, day 21 and / or day 28 after administration. For example, the number of CD8+T cells of the invention can be at least 2 times higher, such as at least 3, 4 or 5 times higher, preferably at least 10 times higher than the number of control CD8+T cells in the same blood sample taken on day 7, day 14, day 21 and / or day 28 after administration, preferably on day 7 after administration. The number of CD8+T cells of the invention can be at least 2 times higher, such as at least 3, 4 or 5 times higher, preferably at least 10 times higher than the number of control CD8+T cells in the sameblood sample taken at even later time points, such as 2, 3, 4, 5, 6 or even 12 months after administration.

[0253] Recombinant

[0254]

[0255] In one embodiment, the antigen-binding receptor is capable of binding to an antigen, preferably to a cancer antigen, more preferably to a cancer cell surface antigen. In a preferred embodiment, the antigen-binding receptor is capable of binding to extracellular domain of a cancer antigen. In a preferred embodiment, the antigen-binding receptor is a chimeric antigen receptor. In a preferred embodiment, the antigen-binding receptor is a genetically engineered T-cell receptor.

[0256] In a preferred embodiment, the antigen-binding receptor is expressed in the CD8+T cells of the present invention. In a preferred embodiment, the antigen-binding receptor is expressed in CD8+T cell and allows said CD8+T cell to bind specifically to antigen-expressing cancer cells with high specificity to exert a growth inhibiting effect, preferably a cytotoxic effect, on said cancer cells.

[0257] For instance, the antigen-binding receptor binds to an antigen selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3-Integrin, α4β1-Integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D-Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and FLT3, preferably CD19, CD20, and ROR1, most preferably to ROR1. The invention is particularly advantageous if the antigen-binding receptor (e.g., CAR) binds to an antigen which is a cancer antigen selected from ROR1 (receptor tyrosine kinase-like orphan receptor 1), HER2 (human epidermal growth factor receptor 2), MSLN (mesothelin), CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5), and SSEA-4 (Stage-specific embryonic antigen 4). This is, for instance, because these antigens can be expressed at low, basal levels on healthy lung tissue. The invention is particularly useful to reduce toxicity of an antigen-binding receptor (e.g., CAR) that binds to such antigens. The invention is also particularly advantageous if the antigen-binding receptor (e.g., CAR) binds to an antigen selected from GD2 (disialoganglioside), CAIX (carboxy-anhydrase-IX), B7-H3 (CD276), MUC1 (mucin-1), and PSMA (prostatespecific membrane antigen). This is, for instance, because these antigens are associated with a general risk of on-target / off-tumor toxicity. The invention is also particularly useful to reduce toxicity of an antigen-binding receptor (e.g., CAR) that binds to such antigens. The CD8+T cell of the present invention may be a CD8+T cell that expresses an endogenous (physiologic) T cell receptor (TCR). Even more preferably, the CD8+T cell has been modified to express a transgenic or recombinant TCR. Even more preferably, the lymphocyte is a cell expressing a chimeric antigen receptor (CAR). A CAR is a (not naturally occurring) receptor that can be expressed on the surface of a cell and that can bind to a ligand, e.g., expressed on the surface of another cell. The receptor can thereby lead to recruitment of a cell expressing the receptor to target cells that express the ligand on their surface. Moreover, upon binding to the ligand the CAR can optionally transmit an intracellular signal within the cells on which it is expressed. Thus, for example, the CAR can be expressed on a T cell and activate the T cell upon binding to its ligand. The CD8+T cell is preferably a CD8+T cell comprising a CAR. Preferably, the CD8+T cell comprises a ROR1 specific CAR.

[0258] The CAR may comprise in an N-to C-terminal order, at least one extracellular antigen binding domain that binds to the antigen, a spacer domain, a transmembrane domain and at least one intracellular signaling domain. The extracellular antigen binding domain may comprise an scFv that binds to the antigen, preferably wherein the antigen is a cell surface antigen. The cell surface antigen may be a cancer antigen and / or an antigen of the tumor microenvironment. For instance, the antigen may be selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3-Integrin, α4β1-Integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D-Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and FLT3, preferably ROR1. The invention is particularly advantageous if the antigen-binding receptor (e.g., CAR) binds to an antigen which is a cancer antigen selected from ROR1 (receptor tyrosine kinase-like orphan receptor 1), HER2(human epidermal growth factor receptor 2), MSLN (mesothelin), CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5), and SSEA-4 (Stage-specific embryonic antigen 4). This is, for instance, because these antigens can be expressed at low, basal levels on healthy lung tissue. The invention is particularly useful to reduce toxicity of an antigen-binding receptor (e.g., CAR) that binds to such antigens. The invention is also particularly advantageous if the antigen-binding receptor (e.g., CAR) binds to an antigen selected from GD2 (disialoganglioside), CAIX (carboxy-anhydrase-IX), B7-H3 (CD276), MUC1 (mucin-1), and PSMA (prostate-specific membrane antigen). This is, for instance, because these antigens are associated with a general risk of on-target / off-tumor toxicity. The invention is also particularly useful to reduce toxicity of an antigen-binding receptor (e.g., CAR) that binds to such antigens.

[0259] The antigen binding domain may comprise an scFv that binds to the ROR1 or GD2. In this instance, the antigen binding domain may comprise or consist of SEQ ID NO: 6 or 14, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 6 or 14. Preferably, the antigen binding domain comprises or consists of SEQ ID NO: 6, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 6. In an instance, the extracellular antigen binding domain comprising an scFv that binds to the antigen and an lgG4-FC spacer domain, a transmembrane domain comprising a CD28 transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain and CD3 zeta domain. The costimulatory domain may be a CD28 cytoplasmic domain or a 4-1BB costimulatory domain. Examples of preferred CARs are shown in SEQ ID NO: 3, 10, 13. The CAR defined by SEQ ID NO: 3, for instance, comprises an scFv that binds to ROR1, an lgG4-FC spacer domain, a transmembrane domain comprising the CD28 transmembrane domain, and an intracellular signaling domain comprising the costimulatory domains 41BB / CD3 zeta co-stimulation domain. The CAR defined by SEQ ID NO: 10, for instance, comprises an scFv that binds to ROR1, a spacer domain a transmembrane domain comprising the CD28 transmembrane domain, and an intracellular signaling domain comprising the costimulatory domains CD28 / CD3 zeta co-stimulationdomain. The CAR defined by SEQ ID NO: 13, for instance, comprises an scFv that binds to GD2 (14G2a scFv with E101K mutation), an lgG4-FC spacer domain, a transmembrane domain comprising the CD28 transmembrane domain, and an intracellular signaling domain comprising the costimulatory domains CD28 / CD3 zeta co-stimulation domain. Hence, in specific embodiments, the CAR comprises or consists of SEQ ID NO: 3, 10, 13, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 3, 10, 13.

[0260] In a specific embodiment, the antigen-binding receptor is a CAR fusion protein, comprising in an N- to C-terminal order:

[0261] a) a CAR, and

[0262] b) LCK.

[0263] LCK according to the present application may be a mammalian LCK, such as human, mouse, rat or macaque LCK. More preferably, LCK is a human or mouse, and most preferably human. Hence, LCK may comprise the amino acid sequence of a mammalian isoform of LCK, preferably LCK comprises the amino acid sequence of a human isoform of LCK.

[0264] LCK may comprise the amino acid sequence of an isoform of wild type LCK or a fragment or mutant thereof. Preferably, the fragment or mutant retains one or more of the biological activities of the wild-type LCK. For instance, the fragment or mutant may have an activity of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 100% activity of wild type LCK.

[0265] Preferably, LCK comprises or consists of an amino acid sequence represented by SEQ ID NO: 7 or comprises or consists of an amino acid sequence that has at least 90%, at least 95% or even at least 99% sequence identity to an amino acid sequence represented by SEQ ID NO: 7.

[0266] The CAR fusion protein of the present invention may comprise a CAR comprising, in an N-to C-terminal order, at least one extracellular antigen binding domain that binds to the antigen, a spacer domain, a transmembrane domain and at least one intracellular signalingdomain, and LCK. In this instance, the CAR fusion protein may comprise a CAR comprising, in an N-to C-terminal order, at least one extracellular antigen binding domain that binds to the antigen, a spacer domain, a transmembrane domain and at least one intracellular signaling domain. In a specific example, the extracellular antigen ligand binding domain comprises an scFv that binds to the antigen and an lgG4-FC spacer domain, wherein the transmembrane domain comprises a CD28 transmembrane domain, and wherein the intracellular signaling domain comprises a costimulatory domain and CD3 zeta domain. For instance, the CAR fusion protein of the present invention comprises or consists of SEQ ID NO: 8, or an amino acid sequence with at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO: 8.

[0267] The CD8+T cell of the present invention may comprise an exogenous nucleic acid that encodes the antigen-binding receptor, such as the TCR, CAR or CAR fusion protein. In this instance, the exogenous nucleic acid encoding antigen-binding receptor may comprise expression cassette. The expression cassette may contain a selection marker. In an embodiment of the invention, the modified T cell of the present invention may further express the EGFRt marker on the cell surface. The EGFRt marker can be used to detect, track, select and deplete the CD8+T cell of the present invention. Therefore, analysis of drug product persistence following administration of the CD8+T cellis made available. Furthermore, the EGFRt marker makes modified CD8+T cells of the invention sensitive to ADCC / CDC through the antibody Cetuximab which can therefore be used as safety switch. Suitable promoters for use in an expression cassette for overexpressing antigen-binding receptor include a MCSV, CMV or CAG promoter, preferably MCSV. For instance, the expression cassette may comprise a EFl / HTLV hybrid promoter followed by the transgene, a 2A ribosomal skip sequence, a truncated surface marker (tEGFR / Her2t) and a Stop codon flanked by inverted terminal repeats.

[0268] The exogenous nucleic acid encoding the antigen-binding receptor may be integrated into the genome of the CD8+T cell of the present invention. In this instance, the CD8+T cell expressing the antigen-binding receptor may be obtainable by expressing the antigenbinding receptor through stable gene transfer. Stable gene transfer may be accomplishedthrough viral vectors or non-viral gene transfer. Alternatively, the exogenous nucleic acid may be a genetic expression vector encoding the antigen-binding receptor. In this instance, the CD8+T cell expressing the antigen-binding receptor is obtainable by expressing the antigen-binding receptor through transient gene transfer or any other means resulting in transient expression. The expression cassette may comprise an EFl / HTLV hybrid promoter followed by a nucleic acid encoding the antigen-binding receptor and BATF2, a 2A ribosomal skip sequence, a truncated surface marker (tEGFR / Her2t) and a Stop codon flanked by inverted terminal repeats.

[0269] Hence, the amino acid sequence of the antigen-binding receptor encoded by the endogenous nucleic acid may be defined by SEQ ID NO: 3, 10, 13. Thus, the antigen-binding receptor may comprise or consist of SEQID NO: 3, 10, 13 or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 3, 10, 13.

[0270] Examples of the nucleic acid sequence of the expression cassette encoding the antigenbinding receptor, such as a CAR, comprised in the exogenous nucleic acid are shown in SEQ ID NO: 17, 22, 25. Thus, the endogenous nucleic acid encoding and expressing the recombinant antigen-binding receptor may comprise or consist of SEQ ID NO: 17, 22, 25 or an nucleic acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 17, 22, 25.

[0271] Examples of the amino acid sequence of the CAR fusion protein encoded by the endogenous nucleic acid may by defined by SEQ ID NO: 8. Thus, the CAR fusion protein may comprise or consist of SEQ ID NO: 8 or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 8.

[0272] Examples of the nucleic acid sequence of the expression cassette encoding the CAR fusion protein comprised in the exogenous nucleic acid are shown in SEQ ID NO: 20. Thus, the endogenous nucleic acid encoding and expressing the CAR fusion protein may comprise or consist of SEQ ID NO: 20 or an nucleic acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 20.The antigen-binding receptor and the BATF2 may be co-expressed from one exogenous nucleic acid. Examples of the polypeptide comprising the antigen-binding receptor and the BATF2 encoded by the endogenous nucleic acid are defined by SEQ ID NO: 4 or 11. Thus, the polypeptide comprising the antigen-binding receptor and the BATF2 may by defined by SEQ ID NO: 4 or 11 or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 4 or 11. Examples of the expression cassette encoding the antigen-binding receptor and the BATF2 comprised in the exogenous nucleic acid are shown in SEQ ID NO: 18 and 23. Thus, the endogenous nucleic acid encoding and expressing the recombinant antigen-binding receptor and BATF2 may comprise or consist of SEQ ID NO: 18 and 23 or an nucleic acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 18 and 23.

[0273] The CAR fusion protein and the BATF2 may be co-expressed from one exogenous nucleic acid. An example the polypeptide comprising the CAR fusion protein and the BATF2 encoded by the endogenous nucleic acid is defined by SEQ ID NO: 9. Thus, the polypeptide comprising the CAR fusion protein and the BATF2 encoded by the endogenous nucleic acid may comprise or consist of SEQ ID NO: 9 or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 9. An example of the expression cassette encoding the CAR fusion protein and the BATF2 comprised in the exogenous nucleic acid is shown in SEQ ID NO: 21. Thus, the endogenous nucleic acid encoding and expressing the CAR fusion protein and BATF2 may comprise or consist of SEQ ID NO: 21 or an nucleic acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 21.

[0274] Composition and kit

[0275] In a second aspect, the invention provides a composition or kit comprising:

[0276] a) a CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses (e.g., overexpresses) BATF2; and optionallyb) a CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD4+T cell expresses (e.g., overexpresses) a transcription factor.

[0277] In an embodiment, the transcription factor is selected from BATF, BATF3, TFAP4, FoxOl or eJun. In preferred embodiment, the transcription factor is eJun.

[0278] The CD8+T cell comprised in the composition or kit is preferably the modified CD8+T cell describes above.

[0279] In preferred embodiments, the composition or kit comprises CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen specific receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD4+T cell overexpresses eJun.

[0280] eJun according to the present application may be mammalian eJun, such as human, mouse, rat or macaque eJun. More preferably, eJun is human or mouse, and most preferably human. Hence, eJun may comprise the amino acid sequence of a mammalian isoform of eJun, preferably eJun comprises the amino acid sequence of a human isoform of eJun. The eJun polypeptide may comprise the amino acid sequence of an isoform of wild type eJun or a fragment or mutant thereof. Preferably, the fragment or mutant retains one or more of the biological activities of the wild type eJun. For instance, the fragment or mutant may have an activity of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 100% activity of the wild type eJun, such as AP-1 transcription factor activity.

[0281] Preferably, eJun comprises or consists of an amino acid sequence represented by SEQ ID NO: 2 or comprises or consists of an amino acid sequence that has at least 90%, at least 95% or even at least 99% sequence identity to an amino acid sequence represented by SEQ ID NO: 2.The eJun overexpression may lead to an increase in the expression level of the eJun of at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400% or more compared to the expression level of eJun expressed by the CD4+T cell before it was modified to express the eJun. In some embodiments, the CD4+T cell of the present invention has been modified to express the eJun at a level that is increased compared to the level of eJun expressed by the CD4+T cell before it was modified to express the eJun.

[0282] Preferably, the eJun level is increased compared to a control CD4+T cell, e.g., an unmodified CD4+T cell (i.e., not modified to exhibit an increased eJun level) of a CD4+T cell of the same species, wherein the unmodified CD4+T cell is preferably from the same subject as the modified CD4+T cell. The comparator typically is an average eJun level obtained from a population of such control CD4+T cell. A population of cells can be, for example, at least 10 randomly selected cells.

[0283] For example, when the CD4+T cell of the present invention is a human CD4+T cell, the eJun level can be increased compared to an unmodified human CD4+T cell obtained from a human subject, wherein the unmodified CD4+T cell is preferably from the same human subject as the modified CD4+T cell.

[0284] An increased eJun level can mean, for example, that the eJun level is increased at least 2-fold, at least 3-fold, at least 5-fold, or even at least 10-fold. A suitable reference may be the eJun level in a control CD4+T cell, e.g., an unmodified CD4+T cell (i.e., not modified to exhibit an increased eJun level) of the same cell type from the same species, wherein the unmodified CD4+T cell is preferably from the same subject as the modified CD4+T cell. The reference typically is an average eJun level obtained from a population of such control CD4+T cells.

[0285] Preferably, an increased eJun level means that the eJun protein quantity level is increased at least 2-fold, at least 3-fold, at least 5-fold, or even at least 10-fold. A suitable reference may be the eJun protein quantity level in a control CD4+T cell, e.g., an unmodified CD4+T cell (i.e., not modified to exhibit an increased eJun level) of the same cell type from thesame species, wherein the unmodified CD4+T cell is preferably from the same subject as the modified CD4+T cell. The reference typically is an average eJun protein quantity level obtained from a population of such control CD4+T cells.

[0286] The eJun level includes the eJun protein activity level and / or the protein quantity level, preferably the protein quantity level. Thus, the eJun level can be increased in various ways. The invention therefore provides a CD4+T cell as described herein, wherein the eJun level is a eJun protein activity level in the CD4+T cell. For example, the eJun level can be increased using an activator of eJun protein activity. Accordingly, in an embodiment, the CD4+T cell comprises an activator of eJun protein activity in the CD4+T cell. Thereby, the protein activity of eJun (e.g., the activity of reducing eJun expression) can be increased.

[0287] The eJun protein activity level can be determined by any known means, for example, by determining expression of eJun target genes and / or by measuring binding of eJun to target site in the genome of the CD4+T cell. This can be done, e.g., by chromatin immunoprecipitation followed by sequencing (Chip-seq) and / or quantifying RNA levels or protein levels (for example, by western blot or flow cytometry).

[0288] The CD4+T cell as described herein can also comprise an exogenous inhibitor of eJun protein degradation in the CD4+T cell. By inhibiting eJun protein degradation, the protein quantity level of eJun can be increased.

[0289] The eJun protein quantity level can be determined by any known means for protein quantification, for example, flow cytometry or quantitative western blot, preferably flow cytometry.

[0290] The eJun expression level can be determined by any known means for protein quantification, for example, flow cytometry or quantitative western blot, preferably flow cytometry.

[0291] The eJun expression level can be increased, for example, by overexpressing eJun in the CD4+T cell. Thus, the invention provides a CD4+T cell as described herein, comprising an exogenous nucleic acid encoding and overexpressing eJun. Such a nucleic acid moleculepreferably comprises an expression cassette, but can also be, for example, an mRNA encoding eJun. Suitable promoters for use in an expression cassette for overexpressing eJun include a MCSV, CMV or CAG promoter, preferably MCSV. The nucleic acid molecule, when it is DNA, can be present as part of an episome or be integrated into the genome of the cell.

[0292] The CD4+T cell may also comprise an exogenous activator of transcription from an endogenous eJun gene and / or activator of translation of an endogenous eJun mRNA. Likewise, the CD4+T cell of the present invention may comprise an endogenous eJun gene that has been genetically modified to increase eJun expression. Preferably, the endogenous eJun gene has been genetically modified in its promoter region. For example, the endogenous promoter can be replaced by a stronger promoter that provides increased transcription in the CD4+T cell so modified.

[0293] Expression of the endogenous eJun gene may also be achieved by CRISPR-mediated transcriptional activation (CRISPRa), or other CRISPR-Cas-based transcriptional engineering methods. Thus, in one embodiment, the exogenous activator of transcription for an endogenous BATF2 gene comprised by the cells (e.g. CD4+T cells) of the invention comprises or consists of Cas9 and a corresponding guide RNA, such as a nuclease defective Cas9 (dCas9), fused to activator proteins / domains (e.g. viral protein such as VP48 or VP64) and CRISPRa guide RNAs target sequences upstream of the promoter region or transcriptional start site (TSS) of eJun (i.e. CRISPR-mediated transcriptional activation (CRISPRa)).

[0294] In the context the present invention, an unmodified CD8+T cell, CD4+T cell or immune cell preferably is a (native) CD8+T cell, CD4+T cell or immune cell of the same cell type obtainable from the same subject as the modified CD8+T cell or CD4+T cell obtainable from the same subject as the CD8+T cell or CD4+T cell from which the modified CD8+T cell or CD4+T cell is produced. When the modified CD8+T cell or CD4+T cell is a human CD8+T cell or CD4+T cell, an unmodified CD8+T cell or CD4+T cell can thus be a (native) human CD8+T cell or CD4+T cell obtainable from the same human subject as the modified CD8+T cell or CD4+T cell obtainable from the same subject as the CD8+T cell or CD4+T cell from whichthe modified CD8+T cell or CD4+T cell is produced. Thus, a CD8+T cell or CD4+T cell that has been modified typically contains an exogenous nucleic acid that is not present or that is present at different levels in said CD8+T cell or CD4+T cell of the same cell type from the same species without modification. For example, the exogenous nucleic acid can be a stretch of genomic DNA with altered nucleotide sequence compared to the unmodified cell, or a protein or RNA, such as an mRNA, miRNA, siRNA or shRNA, in the modified cell that is present at lower or higher levels in the unmodified cell.

[0295] The CD8+T cell or CD4+T cell of the present invention preferably is a CD8+T cell or CD4+T cell that expresses an endogenous (physiologic) T cell receptor (TCR). Even more preferably, the CD8+T cell or CD4+T cell has been modified to express a transgenic TCR. Even more preferably, the CD8+T cell or CD4+T cell of the present invention is a cell expressing a chimeric antigen receptor (CAR). A CAR is a (not naturally occurring) receptor that can be expressed on the surface of a cell and that can bind to a ligand, e.g., expressed on the surface of another cell. The receptor can thereby lead to recruitment of a cell expressing the receptor to target cells that express the ligand on their surface. Moreover, upon binding to the ligand the CAR can optionally transmit an intracellular signal within the cells on which it is expressed. Thus, for example, the CAR can be expressed on a T cell and activate the T cell upon binding to its ligand. The CD8+T cell or CD4+T cell is preferably a CD8+T cell or a CD4+T cell expressing a CAR.

[0296] In one embodiment, the antigen-binding receptor is capable of binding to an antigen, preferably a cancer antigen, more preferably a cancer cell surface antigen. In a preferred embodiment, the antigen-binding receptor is capable of binding to extracellular domain of a cancer antigen. In a preferred embodiment, the antigen-binding receptor is a chimeric antigen receptor. In a preferred embodiment, the antigen-binding receptor is a genetically engineered T-cell receptor.

[0297] In a preferred embodiment, the immunoreceptor is expressed in CD8+T cell or CD4+T cell of the present invention. In a preferred embodiment of the invention, the immunoreceptor is expressed CD8+T cell or CD4+T cell and allows said CD8+T cell or CD4+T cell to bindspecifically to antigen-expressing cancer cells with high specificity to exert a growth inhibiting effect, preferably a cytotoxic effect, on said cancer cells.

[0298] The CAR comprised in the CD4+T cell is preferably the same as the CAR comprised in CD8+T cell. The antigen binding domain may comprise an scFv that binds to the ROR1 or GD2. In this instance, the antigen binding domain may comprise or consist of SEQ ID NO: 6 or 14, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 6 or 14. Preferably, the antigen binding domain comprises or consists of SEQ ID NO: 6, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 6.

[0299] In an instance, the extracellular antigen binding domain comprising an scFv that binds to the antigen and an lgG4-FC spacer domain, a transmembrane domain comprising a CD28 transmembrane domain, and an intracellular signaling domain comprising a costimulatory domain and CD3 zeta domain. The costimulatory domain may be a CD28 cytoplasmic domain or a 4-1BB costimulatory domain. Examples of such CARs are shown in SEQ ID NO: 3, 10, 13. The CAR defined by SEQ ID NO: 3, for instance, comprises an scFv that binds to ROR1, an lgG4-FC spacer domain, a transmembrane domain comprising the CD28 transmembrane domain, and an intracellular signaling domain comprising the costimulatory domains 41BB / CD3 zeta co-stimulation domain. The CAR defined by SEQ ID NO: 10, for instance, comprises an scFv that binds to ROR1, a spacer domain a transmembrane domain comprising the CD28 transmembrane domain, and an intracellular signaling domain comprising the costimulatory domains CD28 / CD3 zeta co-stimulation domain. The CAR defined by SEQ ID NO: 13, for instance, comprises an scFv that binds to GD2 (14G2a scFv with E101K mutation), an lgG4-FC spacer domain, a transmembrane domain comprising the CD28 transmembrane domain, and an intracellular signaling domain comprising the costimulatory domains CD28 / CD3 zeta co-stimulation domain. Hence, in specific embodiments, the CAR comprises or consists of SEQ ID NO: 3, 10, 13, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 3, 10, 13.The CD4+T cell may comprise an exogenous nucleic acid encoding the antigen-binding receptor. The exogenous nucleic acid may be the same as the exogenous nucleic acid encoding the antigen-binding receptor comprised in the CD8+T cell.

[0300] The CD4+T cell may comprise an exogenous nucleic acid that encodes the antigen-binding receptor, such as the TCR, CAR or CAR fusion protein. In this instance, the exogenous nucleic acid encoding antigen-binding receptor may comprise expression cassette. The expression cassette may contain a selection marker. Suitable promoters for use in an expression cassette for overexpressing antigen-binding receptor include a MCSV, CMV or CAG promoter, preferably MCSV. For instance, the expression cassette may comprise an EF1 / HTLV hybrid promoter followed by the transgene, a 2A ribosomal skip sequence, a truncated surface marker (tEGFR / Her2t) and a Stop codon flanked by inverted terminal repeats.

[0301] The exogenous nucleic acid encoding the antigen-binding receptor may be integrated into the genome of the CD4+T cell of the present invention. In this instance, the CD4+T cell expressing the antigen-binding receptor may be obtainable by expressing the antigenbinding receptor through stable gene transfer. Stable gene transfer may be accomplished through viral vectors or non-viral gene transfer. Alternatively, the exogenous nucleic acid may be a genetic expression vector encoding the antigen-binding receptor. In this instance, the CD4+T cell expressing the antigen-binding receptor is obtainable by expressing the antigen-binding receptor through transient gene transfer or any other means resulting in transient expression. The expression cassette may comprise an EF1 / HTLV hybrid promoter followed by a nucleic acid encoding the antigen-binding receptor and eJun, a 2A ribosomal skip sequence, a truncated surface marker (tEGFR / Her2t) and a Stop codon flanked by inverted terminal repeats.

[0302] Hence, the amino acid sequence of the antigen-binding receptor encoded by the endogenous nucleic acid may be defined by SEQ ID NO: 3, 10, 13. Thus, the antigen-binding receptor may comprise or consist of SEQ ID NO: 3, 10, 13 or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 3, 10, 13.Examples of the nucleic acid sequence of the expression cassette encoding the antigenbinding receptor, such as a CAR, comprised in the exogenous nucleic acid are shown in SEQ ID NO: 17, 22, 25. Thus, the endogenous nucleic acid encoding and expressing the recombinant antigen-binding receptor may comprise or consist of SEQ ID NO: 17, 22, 25 or an nucleic acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 17, 22, 25.

[0303] The antigen-binding receptor and the eJun may be co-expressed from one exogenous nucleic acid. Examples of the polypeptide comprising the antigen-binding receptor and the eJun encoded by the endogenous nucleic acid may by defined by SEQ ID NO: 5 or 12. Thus, the polypeptide comprising the antigen-binding receptor and the eJun may by defined by SEQ ID NO: 5 or 12 or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 5 or 12. Examples of the expression cassette encoding the antigen-binding receptor and the eJun comprised in the exogenous nucleic acid are shown in SEQ ID NO: 19 and 24. Thus, the endogenous nucleic acid encoding and expressing the recombinant antigen-binding receptor and eJun may comprise or consist of SEQ ID NO: 18 and 23 or an nucleic acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 19 and 24.

[0304] The composition or kit comprising the modified CD8+and CD4+T cells according to the present invention exhibits one or more enhanced properties compared to a composition or kit comprising CD8+and CD4+T cells that were not modified to have increased BATF2 and / or c-Jun expression, such as:

[0305] a) enhanced proliferation;

[0306] b) decreased exhaustion;

[0307] c) reduced accumulation in non-cancerous tissue, optionally reduced accumulation in non-cancerous lung tissue;

[0308] d) memory like phenotype;

[0309] e) reduced activation-induced cell death;

[0310] f) enhanced engraftment;g) improved chemokine-dependent migration;

[0311] h) enhanced tumor control; and / or

[0312] i) any combination thereof.

[0313] The above described effects are preferably seen in comparison to a control CD8+T cell, e.g., an unmodified CD8+T cell of the same cell type from the same species, wherein the unmodified CD8+T cell is preferably from the same subject as the modified CD8+T cell. The comparator typically is an average survival of a population of such control CD8+T cell. Thus, these effects can be determined, for example, by comparing the proliferation, exhaustion, accumulation in non-cancerous lung tissue, memory like phenotype, and / or activation-induced cell death in vivo of the CD8+T cell of the invention with the survival in vivo of control CD8+T cell in the same subject.

[0314] Methods to determine these properties are known to a person skilled in the art.

[0315] Therapeutic uses

[0316] Pharmaceutical compositions and formulations in accordance with the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions and formulations. For instance, the compositions and formulations are prepared in a way that they can be stored and administered appropriately, e.g., by using pharmaceutically acceptable components such as carriers, excipients or stabilizers. Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition or formulation to a patient. The pharmaceutical acceptable components added to the pharmaceutical compositions or formulations may depend on the chemical nature of the tyrosine kinase inhibitor present in the composition or formulation (depend on whether the targeting agent is e.g., an antibody or fragment thereof or a cell expressing a chimeric antigen receptor), the particular intended use of the pharmaceutical compositions and the route of administration.A pharmaceutically acceptable carrier, including any suitable diluent or, can be used herein as known in the art. As used herein, the term "pharmaceutically acceptable" means being approved by a regulatory agency of the Federal ora state government or listed in the U. S. Pharmacopeia, European Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, and more particularly in humans. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. It will be understood that the formulation will be appropriately adapted to suit the mode of administration. In one embodiment of the invention, the pharmaceutical composition may be formulated as infusion solution comprising NaCI, glucose and human serum albumin in an amount of 0.45%, 2,5% and 1%, respectively.

[0317] The present invention also relates to the CD8+T cell, composition, kit, or pharmaceutical composition or composition as described herein for use as a medicament.

[0318] The type of disease that can be treated by the CD8+T cell, composition, kit, or pharmaceutical composition of the invention is not particularly limited. This is because the present invention provides a general means of extending the in vivo activity of the modified CD8+T cell. Thus, the invention can be applied to any type of disease that is amenable to immunotherapies using CD8+T cell. Such diseases include, for example, cancer, infectious diseases and autoimmune diseases.

[0319] Accordingly, the invention provides a CD8+T cell, composition, kit, or pharmaceutical composition as described herein for use in a method of treating cancer, an infectious disease or an autoimmune disease, such as a chronic inflammatory disease or degenerative disease, preferably cancer.

[0320] The invention also provides a method of treating cancer, an infectious disease or an autoimmune disease, such as a chronic inflammatory disease or degenerative disease, using the CD8+T cell, composition, kit, or pharmaceutical composition as described herein, preferably cancer.Cancer includes all known malignancies, e.g., hematologic malignancies such as leukemia, lymphoma, multiple myeloma; solid tumors such as breast, ovarian, adrenocortical, thyroid cancer, or lung cancer, or pancreatic cancer. For instance, the cancer may be Renal cell carcinoma (ccRCC), B.cell Acute Lymphoblastic Leukaemia (B-ALL), Chronic Lymphocytic Leukaemia (CLL), Diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), Marginal Zone Lymphoma (MZL), Burkitt Lymphoma, Multiple Myeloma (MM), Acute Myeloid Leukaemia (AML), Hodgkin Lymphoma (HL), T-cell lymphoma, Hairy Cell Leukaemia (HCL), Triple-Negative Breast Cancer (TNBC), Non-small cell Lung Cancer (NSCLC), Small Cell Lung Cancer (SCLC), Ovarian Cancer, Pancreatic Cancer, Gastrointestinal Cancers (Colorectal, Gastric, Oesophageal), Prostate Cancer, Glioblastoma (GBM), Neuroblastoma, Sarcomas (Osteosarcoma, Ewing's Sarcoma, Rhabdomyosarcoma, Chondrosarcoma), or Mesothelioma.

[0321] Due to the decreased lung sequestration together with increased chemokine-specific homing into immunological niches via CXCR4 and CCR7 the CD8+T cell, composition, kit, or pharmaceutical compos are attractive forthe treatment of hematological malignancies like MCL, AML or MM. Hence, the cancer may be a hematological cancer, such as leukemia, such as acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), such as Mantle Cell lymphoma (MCL); or multiple myeloma (MM).

[0322] Hence, in specific embodiments, the cancer is a hematological cancer, lung cancer or breast cancer, optionally wherein the hematological cancer is leukemia, non-Hodgkin lymphoma (NHL), or multiple myeloma (MM), further optionally wherein the leukemia is acute myeloid leukemia (AML) and the non-Hodgkin lymphoma (NHL), is Mantle Cell lymphoma (MCL).

[0323] Infectious diseases include but are not limited to viral infections, e.g., Hepatitis B, HIV, fungal infections, e.g. aspergillosis; bacterial infections; infections with other pathogens. Autoimmune disease include, e.g., chronic inflammatory diseases or degenerative diseases. Chronic inflammatory diseases include e.g., Crohn's disease, Encephalitis disseminate. Degenerative diseases include e.g. Alzheimer's disease.For example, the (use in) the method of treatment can comprise the administration of the CD8+T cell, composition, kit, or pharmaceutical composition to a subject (in need thereof), preferably a human subject.

[0324] The CD8+T cell, composition, kit, or pharmaceutical composition of the invention may exhibit an improved therapeutic activity and / or provide an improved therapeutic outcome. For example, the improved therapeutic activity may be evidenced by faster and / or more pronounced reduction of tumor size or viral (e.g. LCMV) load.

[0325] Moreover, the modifications as described herein may lead to improved engraftment and persistence of the CD8+T cell of the present invention or improved pharmacokinetics thereof, e.g., evidenced by

[0326] greater AUC;

[0327] longer half-life (of the CD8+T cell in vivo);

[0328] superior total time period of survival in vivo;

[0329] shorter time period from adoptive transfer to peak level (of modified CD8+T cell numbers);

[0330] longer time period at or near peak level (of modified CD8+T cell numbers) longer time period of modified CD8+T cell numbers above the mean number of modified CD8+T cell during the course of treatment;

[0331] longer time period from peak level (of modified CD8+T cell numbers) to disappearance (of modified CD8+T cells).

[0332] Area-under-curve (AUC) in this context relates to integral of a curve that describes the absolute or relative number of modified CD8+T cells in peripheral blood (or another anatomical compartment, e.g., bone marrow, cerebrospinal fluid) over time.

[0333] The number of (modified) CD8+T cells are typically determined by determining the number of (modified) CD8+T cells per ml in a (peripheral) blood sample obtained from a subject at a given time point after treatment (e.g., administration of the modified lymphocytes). The peak level and disappearance of (modified) CD8+T cells can be determined by a time course, i.e., determining the number of (modified) lymphocytes at different time pointsafter treatment. The peak level corresponds to the maximal number of (modified) CD8+T cells during the course of the treatment, e.g., as determined by a time course using peripheral blood samples, in which the number of modified CD8+T cells is optionally determined once per day. Likewise, the mean number of (modified) CD8+T cells during the course of the treatment may be calculated as the mean value of the numbers determined by such a time course.

[0334] In an embodiment of the invention, the CD8+T cell, composition, kit, or pharmaceutical composition is to be administered intravenously.

[0335] The pharmaceutical composition as described above comprising the modified CD8+T cells are stored at 2-8°C. The pharmaceutical composition is stable for (at least) 48 hours after formulation and ought to be administered to the patient within this period.

[0336] Methods for obtaining a modified CD8+and CD4+cells

[0337] The methods for obtaining a (human) CD8+T cell (and CD4+T cell) according to the invention are as defined herein, including the claims. They may be performed ex vivo, e.g., using isolated human CD8+and CD4+cells as starting material. That is, preferably, the method for obtaining a modified human CD8+T cell (and CD4+T cell) according to the invention is nota method for treatment ofthe human oranimal body by surgery or therapy and is not a diagnostic method practiced on the human or animal body.

[0338] In accordance with the methods for obtaining a human CD8+(and CD4+T cell) of the invention, the step of introducing the nucleic acid or set of nucleic acids or expression vector or set of expression vectors into an mammalian cell may be performed by using any appropriate standard techniques as known in the art, for example, electroporation, electro-injection, microinjection, calcium phosphate co-precipitation, a calcium chloride / rubidium chloride method, retroviral and lentiviral infection, DEAE-dextran, a cationic liposome method, polyethylene glycol-mediated uptake, gene guns, etc., but is not limited thereto. The nucleic acid or set of recombinant nucleic acids encoding the BATF2 may be introduced prior to, simultaneously with, or after introduction of the nucleic acidor set of recombinant nucleic acids encoding and expressing the T-cell receptor or chimeric antigen receptor.

[0339] In particular, the method for producing a CD8+T cell that exhibits an increased BATF2 level may comprise introducing into the CD8+T cell an exogenous nucleic acid molecule for overexpressing BATF2 in the CD8+T cell.

[0340] In some embodiments, the method of the present invention comprises the steps of:

[0341] a) providing a CD8+T cell;

[0342] b) introducing into said CD8+T cell the nucleic acid or set of nucleic acids, or the expression vector or the set of expression vectors; and

[0343] c) co-expressing said nucleic acid or said set of nucleic acids according to claim, or an expression vector or said set of expression vectors;

[0344] d) thereby obtaining said CD8+T cell.

[0345] In an embodiment of the present invention, the method for producing a CD8+T cell or CD4+T cell comprises a step of isolating a CD8+T cell or CD4+T cell from a blood sample of a subject, before modifying the CD8+T cell or CD4+T cell. The blood sample is preferably derived from a human subject, preferably a human subject diagnosed with a disease to be treated using the modified a CD8+T cell orCD4+T cell, such as cancer, an infectious disease or an autoimmune disease, such as a chronic inflammatory disease or degenerative disease.

[0346] The method may further comprise formulating a CD8+T cell or CD4+T cell into a formulation that is suitable for administration to a human subject.

[0347] In other embodiments, the CD8+T cell or CD4+T cell is modified in vivo. Thus, the invention also provides a method for producing a CD8+T cell or CD4+T cell comprising administering a vector suitable for modifying a CD8+T cell or CD4+T cell according to the present invention to a subject (in vivo gene transfer). Preferably, the expression vector for in vivo gene transfer is a lentiviral vector pseudotyped to transduce human a CD8+T cell or CD4+T cell or a nanoparticle containing a non-viral vector suitable for delivering the non-viral vector to a CD8+T cell or CD4+T cell.The nucleic acid molecules used in the present invention, such as expression cassettes, can be in the form of expression vectors. A wide range of expression vectors for polypeptides as well as non-coding RNAs, such as siRNA or shRNAs, are known in the art and are further detailed herein. For example, in an embodiment of the invention, the expression vector is a non-viral or viral vector, and -in the context of medical purposes- preferably a non-viral vector. The expression vector can be a minimal DNA expression cassette. Moreover, an expression vector may be a DNA expression vector such as a plasmid, linear expression vector or an episome. In certain aspects, the vector comprises additional sequences, such as sequences that facilitate expression of the polypeptide, such as a promoter, enhancer, poly-A signal, and / or one or more introns. In certain aspects, the expression vector may be a transposon donor DNA molecule, preferably a minicircle DNA.

[0348] The present invention also relates to minicircle DNA comprising a polynucleotide of the present invention as defined herein. As used herein, the term "minicircle DNA" refers to vectors which are supercoiled DNA molecules that lack a bacterial origin of replication and an antibiotic resistance gene. Therefore, they are primarily composed of a eukaryotic expression cassette. In a useful embodiment the minicircle DNA of the invention is introduced into the cell in combination with mRNA encoding a transposase protein by electrotransfer, such as electroporation, nucleofection; chemotransfer with substances such as lipofectamin, fugene, calcium phosphate; nanoparticles, or any other conceivable method suitable to transfer material into a cell.

[0349] A viral vector can be, for example, a gamma retroviral vector or a lentiviral vector. Such vectors and their construction and production are commonly known in the art.

[0350] The polynucleotide or expression vector can be introduced into a CD8+T cell or CD4+T cell by any suitable means, such as by transfection or by transduction. Transfection refers to chemical or physical delivery into the cells, e.g., by electrotransfer, such as electroporation, nucleofection; chemotransfer with substances such as lipofectamin, fugene, calcium phosphate, PEI. Transduction refers to other means of (targeted) delivery into the cells including delivery by a viral vector or nanoparticles. However, the present invention is not limited to any particular method of delivery of genetic material into immune cells, suchthat also any other conceivable method suitable to transfer genetic material into a cell can be used in the context of the invention.

[0351] Typically, the polynucleotide or expression vector used in the context of the invention allows stable expression of the encoded transgene. Stable expression in this context means that the transgene and expression thereof is not lost when the cells comprising the same proliferate. Stable expression can be achieved, e.g., by expression cassettes that are integrated into the genome of the host cell, such as the CD8+T cell or CD4+T cell of the invention. Moreover, suitable promoters are known in the art that allow prolonged expression of a transgene also in vivo.

[0352] The invention also provides a CD8+T cell or CD4+T cell or formulation obtainable by the method of producing a lymphocyte as described herein.

[0353] Examples

[0354] Hereinafter, the present invention will be more specifically described based on examples. It is understood that various other aspects may be practiced, given the general and detailed descriptions provided elsewhere herein.

[0355] Human

[0356] Human peripheral blood mononuclear cells were obtained from healthy donors after written informed consent to participate in research protocols approved by the Ethics Commission of the University of Wurzburg. The material was provided by the Department for Transfusion Medicine of the University Hospital Wurzburg from leucocyte reduction chambers.

[0357] Cell lines

[0358] K562 (ATCC: CCL-243), JeKo-1 (ATCC: CRL-3006), Raji (ATCC: CCL-86), 786-0 (ATCC: CRL-1932) and DK-MG (DSMZ: ACC 277) were purchased from ATCC (VA, USA) or DSMZ (Brunswick, Germany) and cultured in RPMI-1640 supplemented with 10% FCS and 100 U / mL penicillin / streptomycin. The TM-LCL cell line was obtained from Prof. Dr. S. Riddell79. K562ROR1was generated via lentiviral transduction with a full-length ROR1 gene. All celllines were endowed with the eGFP-firefly-luciferase reporter gene to enable flow cytometric detection, bioluminescence-based cytotoxic assay, and bioluminescence imaging of xenografts in mice.

[0359] Generation of CAR T cells

[0360] CD4+or CD8+T cells were magnetically isolated (Miltenyi Biotec) from PBMC after densitygradient centrifugation (Pancoll human, Pan Biotech) and subsequently activated via anti-CD3 / anti-CD28-beads (Dynabeads, ThermoFisher). Non-viral Sleeping-Beauty-based genetransfer took place on day 2 using the P3 Primary Cell 4D-Nucleofector X Kit according to the manufacturer's protocol (Lonza). T cells were cultured in RPMI-1640 supplemented with 10% human serum, 100 U / mL penicillin / streptomycin and 50U / mL recombinant human IL-2 (Miltenyi Biotec). Transgene-positive cells were magnetically enriched using biotin-conjugated anti-tEGFR or anti-Her2t monoclonal antibodies and anti-biotin MicroBeads (Miltenyi Biotec) before entering a ten-day expansion protocol with 30ng / mL anti-CD3 mAb (OKT3 clone; Miltenyi Biotec), irradiated allogeneic PBMC and TM-LCL in medium supplemented with 50U / mL rhIL-2 (Miltenyi Biotec)79.

[0361] Cloning and constructs

[0362] All constructs generated are based on a vector initially generated by Monjezi et al.80, comprising an EFl / HTLV hybrid promoter followed by the transgene (CAR, TF or CAR-2A-TF), a 2A ribosomal skip sequence, truncated surface marker (tEGFR / Her2t) and a Stop codon flanked by inverted terminal repeats acting as binding sites for SB-mediated transposition. The RORl-directed second generation 4-lBBz CAR was previously described by Hudecek et al.81. Briefly, the CAR comprises a R12 scFv, a short lgG4-hinge region, CD28 transmembrane domain and intracellular 4-lBB / CD3z signaling domains. A variant comprising CD28 instead of 4-1BB co-stimulation was generated in this work. A tonicsignaling high-affinity GD2-directed CAR comprising the 14G2a scFvwith E101K mutation.82Genes of interested were synthesized (GeneArt, ThermoFisher) with matching restriction sites and subcloned into the donor vector. Plasmids for nucleofection were prepared from OneShot™ TOPIO E. coli cultures (Invitrogen) using column-based endotoxin-free clean-up(Macherey-Nagel). Minicircles encoding the transposase SB100X or respective CAR and transcription factors were produced by PlasmidFactory (Bielefeld, Germany).

[0363] Animal

[0364] All murine experiments were performed by certified personnel in the animal facility of the University Hospital Wurzburg, in accordance with the guidelines of and approval from the Government of Lower Franconia. Female NSG (NOD. Cg-Prkdcscid H2rgtmlWjl / SzJ) mice aged 6-10 weeks were purchased from Charles River (Sulzfeld, Germany). Mice were inoculated with 1 x 106JeKo-1 ffluc mantle cell lymphoma cells via tail vein injection on day 0. Tumor engraftment was quantified on day 7 using bioluminescence imaging, measured as average radiance (p / s / cm2 / sr) following intraperitoneal injection of 3 mg D-luciferin (Biosynth) with an IVISXRMS Series III device (Perkin Elmer). Subsequently, 2.5-5 x 106total cells of the respective T cell population were intravenously injected at a CD4: CD8-ratio of 1:1. Peripheral blood samples were collected at specified time points via tail vein puncture. At experimental endpoints, bone marrow and spleen cells were stained for flow cytometric analysis (FACSCanto II, BD). Luminescence data was analyzed using Livingimage software (Perkin Elmer). Tumor-free mice were injected i.v. with a total of 10 x 106CAR T cells per mouse. The injected T cells consisted of a 1:1 mix of CARonlyand BATF2-CAR T cells prestained with CTV and CFSE, respectively. Each modified population comprised an equal mix of CD4+and CD8+T cells. Mice were sacrificed after 24h or 72h post-injection i.v. application of an anti-human CD45 antibody to stain all vascular T cells15. Organs were minced and digested using a gentleMACS device and the Multi Tissue Dissociation Kit 1 (Miltenyi Biotec). Blood and organ-derived single-cell suspensions were stained and analyzed via flow cytometry (FACS Canto II, BD).

[0365] In vitro functional assays

[0366] Cytotoxic activity of CAR T cells was assessed in co-culture with ffluc-positive tumor cell lines at indicated effector-to-target (E: T) ratios in presence of 0.15 mg / mL D-luciferin. The luminescence signal of tumor cells was measured in a plate-reader (Tecan) at indicated time intervals for up to 24h. Specific lysis was calculated based on equally treated control T cells. The concentration of secreted cytokines IL-2 and IFN-y by T cells were determinedby ELISA (Biolegend) from the supernatants of 24h co-cultures with tumor cell lines at an E: T ratio of 4:1. Receptor-independent stimulation via PMA / lonomycin and medium only served as positive and negative controls, respectively. CD4+and CD8+T cells were individually stained with 0.1 pM CellTrace CFSE (Invitrogen) to assess proliferation via dye dilution after co-culture with irradiated tumor cells. The co-cultures were set up at E: T ratios of 4:1 or 1:1. IL-2 and medium were used as positive and negative controls, respectively. After a 72-hour incubation, the samples were stained for CD4 and CD8 and analyzed using the MACSQuant Analyzer 10 (Miltenyi Biotec). Data analysis and the calculation of the expansion index were performed using FlowJo (BD). Migratory capacity towards chemokines was evaluated in Transwell migration assays as previously described83. Briefly, T cells or JeKo-1 tumor cells were seeded in the upper chamber of the Transwell plates (5.0pm polycarbonate membrane, Costar). The lower chamber was filled with plain migration medium (RPMI1640 with 1% human serum) or supplemented with the respective chemokines CXCL12, CCL2 and CCL21 (Biotechne) at indicated concentrations. After 3h incubation the inlets were removed, cells from the lower chamber were harvested and cell counts were determined using the MACSQuant Analyzer 10 and counting beads (123counting beads, Invitrogen). The chemotactic index was calculated by dividing the number of cells in experimental wells by the number of cells in control wells. Long-term cytotoxicity analyses were conducted using the xCELLigence platform (RTCA DP analyzer, Agilent). Initially, a blank measurement with medium only was performed. Adherent 786-O target cells were then seeded at a density of 15,000 cells per well into a device-specific plate (E-Plate 16 PET, Agilent), allowing for monitoring of cell attachment and growth via impedance measurements. The cell index was recorded at 15-minute intervals for 2-4 hours until the growth curve reached a plateau. Subsequently, the respective T cells were added at the indicated E: T ratios, and measurements were continued. Repetitive antigen stimulation assays were conducted using antigen-positive tumor cells (786-0), which were pre-seeded in 6-well plates and incubated until they adhered to the bottom of the plate. CD8+T cells, either overexpressing TF or as control CAR T cells, were then added at an inverse E: T ratio of 1:4. After 48 hours of incubation, the cells underwent another 48-hour stimulation period. Following a total of 96 hours of stimulation, the differentiation status,the frequency of triple-positive exhaustion markers (PD-l+LAG-3+TIM-3+), and the foldchange in cell numbers were evaluated.

[0367] Extracellular flux analysis

[0368] Oxygen consumption rate (OCR) and glycolytic proton efflux rate (glycoPER) were analyzed using an oxygen-controlled XFe96 Extracellular Flux Analyzer (Seahorse Bioscience) and XFe96 cell culture microplates (Agilent) pre-coated with Cell-Tak (corning). T cells were seeded at 1.5 x 105cells per well in Seahorse CF RPMI medium (Agilent) supplemented with lOmM D-glucose (Sigma), 2mM L-glutamine (Gibco) and ImM sodium pyruvate (Sigma). Mitochondrial and glycolytic stress tests were performed after incubation for lh at 37°C in an incubator without CO2 control. Glycolysis was assessed by measuring basal extracellular acidification rate (ECAR) before adding 0.5mM rotenone (AdipoGen) and 0.5 mM antimycin A (Sigma) to inhibit mitochondrial complex I and III, respectively. Glycolysis was blocked completely by addition of 50mM 2-deoxy-D-glucose (Sigma). Mitochondrial respiration was analyzed by measuring basal oxygen consumption before adding 2mM of the ATP synthase inhibitor oligomycin (Cayman Chemicals), ImM of the protonophore FCCP uncoupling the mitochondria (Cayman Chemical), 0.5mM rotenone (AdipoGen) and 0.5 antimycin A (Sigma). The ratio of OCR and glycoPER was calculated to compare the metabolic phenotypes of differently modified T cells in basal and induced state.

[0369] Stimulation with plate-bound protein

[0370] RORl-specific CAR T cells were stimulated using immobilized ROR1-FC (Si noBiologica I ). Cell culture plates were coated with indicated concentrations of ROR1-FC protein in PBS for 2h at 37°C. Unbound protein was removed with two PBS washing steps. T cells were seeded in T cell medium at 0.15 x 106cells in 96-well flat bottom plates and incubated for 24 hours at 37°C. Supernatants were harvested for ELISA and expression of CD25, CD69 was analyzed. For time-course analysis with observation periods of up to 96h, individual wells were prepared for each time point and PD-1, LAG-3 and TIM-3 were included into the FACS panel. To assess phosphorylation states of signaling intermediates in Western Blots, T cells were seeded into a 48-well plate pre-coated with 0.5 or 2 pg / mL ROR1-FC at 0.75 x 106cells in lOOpL PBS per well and centrifuged at 100g for 2 minutes. After the indicatedincubation time at 37°C the supernatant was removed, and cells were resuspended in lOOpL RIPA (Sigma-Aldrich) supplemented with 1% of protease- and phosphatase inhibitors (Sigma-Aldrich) before freezing in liquid nitrogen. After centrifugation at 8000xg for 10 minutes at 4°C, the supernatant was transferred to new vials and stored at -80 °C. Western

[0371] Proteins were separated under reducing conditions using precast 4-20% polyacrylamide gels (TGX Stain-Free Protein Gels, BioRad) before transferring to 0.45pm PVDF membranes (BioRad). Blots were blocked for lh at room temperature with TBS-T supplemented with 5% Serum Albumin Fraction V (AppliChem). Primary antibodies were incubated overnight at 4°C on a shaker. Bound antibodies were labelled with HRP-conjugated secondary antibodies for lh at room temperature before being developed using Clarity Western ECL substrate (BioRad) and the ChemiDoc MP imaging system (BioRad).

[0372] Flow

[0373] Flow cytometric measurements were performed using a MACSQuant Analyzer 10 (Miltenyi Biotec) for all in vitro analyses and a FACS Canto II (BD) for samples associated with in vivo experiments. Data was analyzed with FlowJo software (BD). In general, 0.2 x 106cells were harvested, washed with FACS buffer and incubated with respective conjugated antibodies for 25 minutes at 4°C. Cells were washed twice after each incubation step. All commercial antibodies used were supplied by Biolegend or Miltenyi Biotec and were specific for human CD45, CD3, CD4, CD8, ROR1, PD-1, LAG-3, TIM-3, CD45RA, CD62L, EGFR, CD25, CD69, CD18, CXCR4, CCR7, CCR2 and HER2. EGFR (Erbitux, Cetuximab)- and HER2 (Herceptin, Trastuzumab)-specific antibodies were in-house conjugated with biotin, AF647 or PacificBlue (Thermo Fisher). Further, biotinylated Maackia Amurensis Lectin II (MALII, Vector Laboratories) and PE-conjugated streptavidin (Miltenyi Biotec) were used to detect sialic acid containing carbohydrate structures on surface proteins. Cells treated with Neuraminidase A (NEB) served as controls.

[0374]

[0375] anaRNA was isolated from 1 x 106T cells using the RNEasy Plus Mini Kit (Qiagen). Gene expression was analyzed using NanoString technology with the nCounter CAR T cell Characterization panel, enabling simultaneous analysis of 780 genes including 10 reference genes for data normalization. Briefly, RNA samples were hybridized with Reporter and Capture probes at 65°C for 16h. Subsequent sample preparation and transfer to the Cartridge was automated by the Nanostring nCounter Flex Prep Station resulting in purified and immobilized mRNA-probe complexes with respective unique fluorescent barcodes aligned to the focal plane for imaging. Quantification of the unique barcodes for each gene was done by the nCounter Analysis System. Data was analyzed by ROSALIND® (https: / / rosalind.bio / ), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA). Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step. Normalization, fold changes and p-values were calculated using criteria provided by Nanostring. Data shown are for n=3 biological replicates.

[0376] Statistical

[0377] Visualization and statistical analyses were conducted with GraphPad Prism 9 software. Data shown is mean ± SEM for experiments with n>3 biological replicates unless stated otherwise. An unpaired t-test was used to compare the means of two independent groups. One-way ANOVA was used to assess the statistical significance of data sets with more than two groups with one independent variable, while two-way ANOVA was applied for data sets involving two independent variables. Dunnett's post-hoc test was used to compare multiple conditions to a single control group, whereas Tukey's or Sidak's post-hoc test was used for all pairwise comparisons between groups. The comparison of Kaplan-Meier survival curves was conducted using the log-rank Mantel-Cox test. P-values below 0.05 were considered significant, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 Example 1: CD8+4-1BB CARBATF2T cells show attenuated IL-2 secretion and killina but enhanced proliferation, AICD-resistance and memory-maintenance

[0378] A first set of experiments aimed on characterizing the impact of TF-overexpression on the functional and phenotypic properties of CD8+T cells equipped with a second-generation 4-IBB RORl-directed CAR. The inventors generated multicistronic Sleeping-Beauty constructs encoding the respective TFs, CAR and surrogate markers under control of a human HTLV-EFla promoter (FIG. 1A). Transgene surface marker expression served for cell sorting and purity assessment (FIG. 2A). Transcription factor identity and overexpression was confirmed in immunoblots (FIG 2. B, C).

[0379] Throughout this study, effector functions were assessed using tumor cells with ROR1 expression levels ranging from antigen-negative (Agneg; K562), low (Aglow; Raji) and intermediate (Agint; JeKo-1, 786-0) to supraphysiological high (Aghigh; K562ROR1) (FIG. IB). AP-1 enhanced CARdUNT cells exhibited the highest levels of IL-2 and IFN-y secretion (Fig.

[0380] 1C) accompanied with the best Aglowtumor control compared to conventional CAR T cells (FIG. ID). In line with the hypothesis of AP-1 attenuation, CARBATF2T cells secreted less IL-2, mediated slower killing of Aginttarget cells and showed to be less susceptible to activation-induced cell death (AICD) upon antigen-encounter (FIG. 1C-E). However, the inventors also noted an inability of CARBATF2T cells to control tumor cells expressing very limited amounts of antigen (FIG. ID). The inventors found antigen-specific proliferation to be enhanced with each Batf-family member when compared to conventional CAR T cells, withCARBATF2T cells providing the highest proliferative potential (FIG. IF). Of note, CARdUNT cells did not provide a proliferation benefit (FIG. IF).

[0381] Importantly, the inventors did not observe unspecific effector functions for any of the investigated transcription factors in their dedicated in vitro assays. However, when monitoring the relative fraction of transgene-positive cells for up to 16 days after genetransfer the inventors observed an 3.6- and 3.9-fold increase for CARBATFand CARBATF3T cells, respectively (FIG. 2D). An even stronger enrichment of 8.8- and 10.8-fold was observed for CARBATF+CJUNand CARBATF3+CJUN, respectively (FIG. 2D).

[0382] Interference with transcription factor expression levels can impact the subset-composition of CAR T cell products1-2. Thus, the inventors assessed the TF-candidates' potential to promote a memory-like phenotype, that is characterized by CD62L+CD45RA+positivity and known for its enhanced persistence9 -11. Compared to conventional CAR T cells, a morepronounced CD62L+CD45RA+population was present in CARBATF2as well as CARBATF3T cells, but without any changes found for CARdUNT cells (FIG. 1G).

[0383] Transcriptional analysis of CARBATF2T cells supported our functional and phenotypic observations with downregulated effector-related genes (e.g., GZMA, GZMB, PRF1, IFNG, TBX21, ID2), upregulation of memory-related genes (LEF1, CCR7, FAS) along with downregulated exhaustion-associated genes (TOX, EOMES, HAVCR2, CTLA4, ENTPD1, FASLG) (FIG. 1H; FIG. 4A).

[0384] Taken together, the data indicates that BATF2-overexpression attenuates immediate effector functions and shields from AICD while providing enhanced antigen-specific proliferation and a memory-like phenotype.

[0385] Example 2: BATF2-overexpression in CD28-CAR T cells induces similar characteristics as in 4-1BB-CAR T cells, but without reducinq cytokine secretion and killin

[0386] All functional assays were additionally performed for otherwise identical RORl-directed CAR with CD28 co-stimulation (FIG. 3A-E). The overall impact of BATF2-overexpression was similar to the data obtained with the 4-1BB CAR (FIG. 1). In detail, CD28-CARBATF2T cells did also show enhanced proliferation, prominently reduced AICD, and improved maintenance of a memory-like phenotype (FIG. 3C-E). However, here the inventors did not observe a limitation of IL-2 secretion or reduction of specific killing (FIG. 3A, B). Analyzing the memory-composition the inventors found a more terminal differentiated phenotype for the conventional CD28-CAR compared to its respective CAR-negative T cells, while CD28-CARBATF2T cells revealed a very similar composition to Mock T cells (FIG. 3E). Due to these clear phenotypic differences the inventors were interested in the metabolic profile conveyed by TF-modified versus control 28z-CAR T cells. As memory T cells favor oxidative phosphorylation over glycolysis, the inventors asked whether the phenotypic signature resulting from BATF2-overexpression translates into the respective metabolic program. Indeed, CD28-CARBATF2T cells exhibited a significantly higher relative OCR / glycoPER-ratio compared to conventional CD28-CAR T cells, primarily due to a lowered glycolytic activity (FIG. 3F, G).To summarize, BATF2-overexpression in CD28-CAR T cells diminished AICD and terminal effector differentiation in a similar fashion as in 4-1BB-CAR T cells but without attenuating cytokine secretion and tumor cell elimination.

[0387]

[0388] 3: BATF2- alleviates tonic HA-GD2 CAR T cells activation levels and exhaustion

[0389] Next, the inventors aimed at assessing the potential of BATF2-overexpression to liberate T cells from excessive tonic signaling induced by a high-affinity GD2-directed CAR, serving as a model for exhaustion. Simple expression of this CAR under standard cultivation conditions mediated high levels of activation as measured via CD25 and CD69 expression (FIG. 4B). No other TF than BATF2 reduced the percentage of activated cells to the level of CAR-negative T cells. Analyzing exhaustion marker expression revealed a 7.8-fold reduction in PD-l+LAG-3+TIM-3+triple-positive cells for CARBATF2compared to CAR control (FIG. 4C). Interestingly, the main differences considering single exhaustion markers were observed for LAG-3 and TIM-3, with no significant differences detected for PD-1.

[0390] A similar picture was obtained for the RORl-directed CAR subjected to repetitive stimulations with tumor cells in vitro. Here, CARBATF2and CARBATF2+CJUNT cells provided the most robust expansion with simultaneously the lowest percentage of PD-l+LAG-3+TIM-3+triple-positive cells (FIG. 4D-F).

[0391] Together the data indicate that BATF2-overexpression has the potential to alleviate CAR T cells from continuous overstimulation and consequently exhaustion.

[0392] 4: CD4+CARBATF2T cells low amounts of IL-2 and fail to express activation

[0393]

[0394] markers, even at hit densities, to conventional CAR or CARdUNT cells

[0395] Next, the inventors moved on to investigate the characteristics induced by BATF2- and cJUN-overexpression in CD4+4-1BB CAR T cells. Overall, the impact of TF overexpression on tumor cell killing mediated by CD4+CAR T cells closely paralleled the findings observed in the CD8+subset (FIG. 5A). While the control of Aginttumor cells decreased for CARBATF2compared to CARonlycells, no control of Aglowtumor cells was evident. Again, CARdUNT cells provided the best tumor cell elimination at intermediate and low antigen-densities.The inventors performed in vitro dose-response experiments using a titration of plate- coated RORl-antigen using activation-marker expression and cytokine secretion as functional read-outs. BATF2-overexpression interfered with CD4+CAR T cells ability to upregulate activation-marker expression as well as IL-2 and IFN-y secretion even at high antigen-densities (FIG. 5B; FIG. 6A, B). In contrast, enhanced antigen-sensitivity and cytokine output was detected for CARdUNT cells compared to CARonlycondition in particular at low and intermediate antigen-densities (FIG. 5B FIG. 6A, B). The same pattern (CARdUN> CAR > CARBATF2) of IL-2 levels resulted from co-cultivation with antigen-positive tumor cells (FIG. 5C).

[0396] Taken together, the data indicate that CD4+CAR T cells react more sensitive towards BATF2-overexpression than CD8+CAR T cells resulting in a pronounced blockade of activation potential and effector functions.

[0397] 5: A blend of CD8+CARBATF2and CD4+CARdUNT cells enhances the

[0398]

[0399] in vitro and in vivo Above, CD8+CARBATF2T cells have shown a substantial increased proliferative capacity but intrinsically reduced IL-2 secretion (FIG. 1C, F). The inventors asked whether CD8+CARBATF2T cells proliferative phenotype could benefit from a CD4+CAR T cell population providing significant amounts of IL-2 (FIG. 5D). The inventors performed in vitro CFSE-based coculture proliferation assays and observed an increase in CD8+CARBATF2T cell proliferation corresponding to the IL-2 secretory capacity of the co-cultured CD4+CAR T cells (FIG. 5E, F). In detail, the expansion index of the CD8+subset was highest with CD4+CARdUNT cells followed by the conventional CAR and CARBATF2, respectively. A highly proliferative CD8+population could bear the risk of serving as a sink for CD4+-derived IL-2, thereby negatively impacting survival and proliferation of the CD4+subset itself. To rule this out, the inventors analyzed the expansion index of CD4+CAR T cells in the blends and observed no reduced proliferation of CD4+CAR or CARdUNT cells combined with CD8+CARBATF2T cells (FIG. 5G). The same observations were made in the context of a 28z-CAR (FIG. 6D, E)

[0400] Short-term in vitro cytotoxic assays at rather high E: T ratios do not take proliferative effects into account. Therefore, the inventors assessed long-term cytolytic effects at a challengingE: T ratio of 1:20 and observed the best control of 786-0 tumor cells provided by a blend of CD4+CARdUNand CD8+CARBATF2T cells (FIG. 6F). In line with the observed proliferative characteristics, the negative slope of the cell index increased after approximately 48 hours indicating an accelerated tumor cell elimination due to increased T cell numbers (FIG. 6F). Next, the inventors asked whether our in vitro observations would translate into improved proliferation in vivo. Mice were engrafted with the mantle cell lymphoma (MCL) cell line JeKo-1 for 7 days before being treated with a 1:1 CD4+: CD8+blend of CAR T cells (FIG. 5H). The six groups were treated with 1:1 mixture of CD4+and CD8+T cells: four groups with matching modifications in both subsets, and two blending CD8+CARBATF2T cells with either conventional CD4+CAR T cells or CD4+CARdUNT cells to account for the potentially synergistic effects observed in vitro. Frequencies of human T cells in peripheral blood were monitored over time and differences were mainly allocated to the CD8+T cell subset (FIG.

[0401] 5; FIG. 6G). Therefore, the observed inverted CD8: CD4 ratio in groups comprising CD8+CARBATF2T cells resulted from increased presence of CD8+T cells and not from decreased expansion in the CD4+subset (FIG. 5J). On day 3 p.i. CD8+CARBATF2T cells were found at higher frequencies in peripheral blood compared to conventional CAR or CARdUNT cells, without differing dependent on the co-administered CD4+CAR T cell modification (FIG. 6G). However, at day 7 p.i. the inventors observed the highest frequencies of CD8+CARBATF2T cells when combined with CD4+CARdUNT cells followed by CARonlyand CARBATF2T cells, thereby exactly mirroring our in vitro findings (FIG. 51). After substantial clearance of the primary tumor mass, T cell frequencies decreased uniformly across all groups (FIG. 51; FIG.

[0402] 6H). The lowest reduction in tumor burden was achieved in the group treated with BATF2-modified CD4+and CD8+CAR T cells (FIG. 6H). Albeit not a curative model, Kaplan-Meier survival analysis underlined that the heterogenous CD4+CARdUN: CD8+CARBATF2blend provided a longer survival compared to a homogenous CARBATF2or heterogenous CD4+CAR: CD8+CARBATF2cell product (FIG. 5K).

[0403] To summarize, the choice of individual TFs for the CD4+T cell subset can have synergistic effects onto CD8+CAR T cells modified with a different TF. The inventor's data pinpoints aheterogeneous cell product comprising CD4+CARdUNand CD8+CARBATF2T cells to provide superior engraftment in vivo.

[0404] 6: CD8+CARBATF2T cells exhibit reduced but achieve similar maximum

[0405]

[0406] activation via the CAR, while saturation at low activation levels when stimulated throuqh the TCR

[0407] As BATF2-overexpression seemed to affect tumor cell killing in an antigen-density dependent manner, the inventors next assessed antigen-sensitivity using a titration of plate-coated ROR1 or OKT3 for CAR and TCR stimulation, respectively (FIG. 7A, B). Baseline activation of CARBATF2T cells was found to be overall reduced (FIG. 7A). Increasing antigen levels led to increased activation of CARBATF2T cells, indicating that CAR molecules were not yet antigen saturated. However, at low to intermediate antigen-densities the inventors observed an overall reduced sensitivity of CARBATF2compared to conventional CAR T cells (FIG. 7A; FIG. 8A). Further, activation of CARBATF2T cells did not result in reduced viability to the extent of conventional CAR T cells at concentrations below 1 pg / mL of coated antigen (FIG. 7A). Intuitively the inventors asked whether CAR expression levels were affected by BATF2-overexpression, but direct staining of CAR molecules did not reveal any changes (FIG. 7D).

[0408] To assess whether the lower activation potential is restricted to the CAR itself, the inventors utilized titrations of OKT3 antibody for direct TCR stimulation (FIG. 7B). CARBATF2T cells activation levels reached saturation at a CD25+CD69+rate of 31% compared to 75% for conventional CAR T cells. In line with the lower activation levels of CARBATF2T cells, the inventors observed less IFN-g and IL-2 secretion for both CAR and TCR stimulation.

[0409] Interestingly, upon CAR-antigenic stimulation ERK1 / 2 phosphorylation (pERKl / 2) was below detection limit in CARBATF2T cells whereas pERKl / 2 was detectable in conventional CAR T cells (FIG. 8B). However, stimulation via TCR or receptor-independently via PMA / lonomycin (P / l) led to similar pERKl / 2 in both conditions.

[0410] The inventors asked whether the lower rate of activation-marker positive cells at a low antigen-density is a matter of a slower activation kinetics, thus longer stimulation wouldyield similar overall activation levels. Cells were stimulated with a low concentration of plate-coated ROR1 and analyzed regarding activation and exhaustion-marker expression for upto 96 h (FIG. 7C). CARBATF2T cells pattern of activation was similar to the CART control but at an overall lower level. No notable fraction of exhaustion-marker triple positive cells was observed for CARBATF2T cells even after 96 hours of continuous stimulation.

[0411] Together, the data indicate that CD8+CARBATF2T cells exhibit reduced sensitivity but achieve similar maximum activation via the CAR, while showing early saturation at low activation levels when stimulated through the TCR. This could indicate a limitation in membrane-proximal signaling intermediates.

[0412] Example 7: Direct fusion of LCK to the CAR molecule rescues Aq / Owtumor cell control of BATF2-overexpressinq T cells in vitro

[0413] As antigen-sensitivity and CAR T cell activity is greatly dependent on CAR-proximal kinases12 14, the inventors started correlating sensitivity and LCK-levels. The inventors found total LCK levels to be reduced in CARBATF2compared to conventional CAR T cells (FIG.

[0414] 7E).

[0415] To assess the role of diminished LCK expression, the inventors introduced a novel CAR-LCK (CAR-L) fusion molecule (FIG. 7F). The inventors observed restored activation levels for CAR-LBATF2compared to CARBATF2T cells upon encounter of antigen-positive tumor cells (FIG. 7G). Further, CAR-L augmented the proliferative capacity of CD4+and CD8+T cells with CAR-LBATF2conditions providing the most significantly enhanced expansion index (FIG. 7H). Finally, control of Aglow, Agintand Aghightumor cells was fully rescued by the LCK-fusion with no significant difference comparing CAR-LBATF2T cells to conventional CAR or CAR-Lonlyfor both CD4+and CD8+subsets (FIG. 71). Supporting the inventors' hypothesis, and the postulated mechanism of action, the impact of the CAR-LCK fusion was highest at low-antigen densities and faded with increasing target molecules (FIG. 71).

[0416] Taken together the data show that an advanced receptor design facilitating LCK availability to the CAR molecule can rescue the control of tumor cells with limited antigen-expression thereby enhancing the efficacy of CAR-LBATF2compared to CARBATF2T cells.8: CD8+BATF2-< T cells equipped with a CAR-LCK fusion molecule

[0417]

[0418] CD4+CARdUNT cells id tumor control in vivo The inventors have shown that a special CAR design incorporating a LCK fusion molecule was able to rescue the tumor control while enhancing the proliferative potential of CARBATF2T cells in vitro (FIG. 7). However, BATF2-overexpression did not even lead to diminished effector functions in the context of a 28z-CAR (FIG. 3A, B). After our initial characterization shown in Figure 1, the inventors moved on with the BBz-CAR due to the observed pronounced terminal differentiation induced by the 28z-CAR (FIG. 3E). To finally identify the best CAR design for BATF2-overexpressing CD8+T cells blended with CD4+CARdUNT cells, the inventors aimed to compare three receptor variants in vivo: (i) BBz (ii) BBz-CAR-L or (iii) 28z-CAR. Here the inventors chose a subcutaneous clear cell renal cell cancer (ccRCC) xenograft model in NSG mice (FIG. 9A).

[0419] The inventors were in particular interested whether their in vitro findings regarding the CAR-LCK fusion molecule would translate to better solid tumor control in vivo. Tumor cells were engrafted for 14 days and mice were randomized before T cell transfer (FIG. 10A). Three days after treatment, the inventors analyzed T cell abundance in peripheral blood and found the highest frequencies for CARBATF2T cells followed by CAR-LBATF2and 28z- CARBATF2T cells (FIG. 9B).

[0420] Interestingly, this distribution in abundance inversely correlated with pre-infusion activation level as determined via CD69 expression (FIG. IOC). The higher frequency of BATF2-modified CD8+T cells was reflected in higher CD8: CD4 ratios compared to the CARonlycontrol, as no significant changes could be detected in frequencies within the CD4+subset (FIG. 9C). Overall T cell frequencies contracted between day 17 and day 21 (FIG. 9B, FIG. 10B).

[0421] Further follow-up showed a contraction to baseline by day 28 (data not shown). All treatment conditions conferred a rapid tumor regression within 4 days after T cell transfer (FIG. 9D). Among the lead-candidates, the most homogenous tumor clearance was provided by CD8+CAR-LBATF2T cells co-administered with CD4+CARdUNT cells (cJ: CAR-LB2) with 5 out of 5 mice reaching BLI intensities in the magnitude of background levels (FIG.9D). Interestingly, 2 out of 5 mice treated with a blend of CD8+CARBATF2T cells and CD4+CARdUNT cells (CARd: CARB2), that did not initially reach a tumor reduction to baseline were able to control the tumor from day 35 on.

[0422] Within the cJUN-overexpressing control group 4 out of 5 mice reached the same tumor reduction while a major part of mice (3 / 5) treated with the conventional CARonlycontrol conferred suboptimal tumor clearance (FIG. 9D).

[0423] To conclude, the inventors identified two cell product blends comprising CD4+CARdUNT cells either with CD8+CARBATF2or with CD8+CAR-LBATF2as synergistic cell product compositions achieving superior tumor control in a solid tumor ccRCC model.

[0424] 9: BATF2-> induces in chemokine activation state h chemokine-i and retention in

[0425]

[0426] circulation via increased LFA-1 Migration-associated genes (e.g. CCR7, CXCR4, CCR2) were among the most significantly differential expressed genes comparing CD8+CARBATF2to conventional CAR T cells on transcriptional level (Fig. 1H; FIG. 11A). These changes were particularly interesting as chemokine receptors can enhance the specific homing ability of CAR T cells. Therefore, transcriptional differences were confirmed on translational level for the two upregulated receptors CCR7 and CXCR4 as well as CCR2 as a downregulated one (FIG. 11B, C).

[0427] The inventors performed Transwell migration assays to assess whether these findings would translate to a functional difference (FIG. 11D). Indeed, the inventors observed augmented specific migration of CARBATF2T cells compared to conventional CAR T cells based on CCR7 and CXCR4 towards CCL21 and CXCL12, respectively (FIG. 11D).

[0428] Next, the inventors asked whether the migratory phenotype observed in vitro would translate to differences in vivo. Before deciding on a suitable experimental design, the inventors analyzed relative expression levels of CCR7 and CXCR4 in CD4+and CD8+cJUN-, BATF2- or non-TF-modified CAR T cells. The inventors did not observe significant differences for CARdUNcompared to conventional CAR T cells in both subsets, but the increased CXCR4 expression resulting from BATF2-overexpression was not only apparentin CD8+but also in CD4+CART cells (FIG. HE). Therefore, the inventors decided to compare the homing of CARBATF2T cells to conventional CAR T cells. Tumor-free mice were injected with a single mixture of proliferation-dye pre-stained T cells comprising equal amounts of the four populations of interest to enable a direct comparison regarding their body distribution (FIG. 11F). Mice were sacrificed 24 hours later after injecting a human CD45-specific antibody to stain all vascular T cells15, thereby allowing to discriminate intravascular and tissue T. Blood, lung, bone marrow and spleen were analyzed and a specific homing index was calculated for each organ (FIG. 11G, H; FIG. 12A). The inventors observed a higher proportion of CARBATF2T cells in the blood and reduced accumulation in the lung tissue (FIG. 11G). Instead, the inventors detected more CD8+CARBATF2T cells in the spleen while this was not the case for CD4+CARBATF2T cells. This observation is in line with CCR7 expression levels as this receptor is the main GPRC known for its critical role for T cell entry into the spleen16(FIG. 11E).

[0429] The inventors were interested whether the reason behind the reduced unspecific tissue sequestration of CARBATF2T cells in the lung is related to LFA-1 recycling via the previously described ST3GALl-|3ll-spectrin axis17-18. Upon in vitro activation ST3GAL1 is upregulated and stabilizes LFA-1 (CDlla / CD18) by adding sialic acid residues17. On the other hand, pi I-spectrin acts as a cytoskeletal force increasing endocytic recycling of LFA-1 and is downregulated upon activation17. Indeed, CARBATF2T cells showed drastically lower baseline activation (FIG. Ill) and significantly lower cell surface CD18 levels (FIG. 11J; FIG.

[0430] 11D). However, staining of surface sialic acid residues using MALII-lectin did not reveal glycosylation differences for resting or re-stimulated cells (FIG. 11K). Instead, the inventors detected enhanced pi l-spectrin levels in CARBATF2T cells compared to conventional CAR T cells (FIG. 11L). Enhanced LFA-1 recycling was described to rescue from unspecific migration while maintaining a specific migrational behaviour17that is in line with characteristics of CARBATF2T cells in our hands (FIG. 11D, M).

[0431] Taken together, BATF2-overexpression induced preferential characteristics enhancing specific migration and minimizing lung sequestration compared to conventional CAR T cells due to increased LFA-1 recycling via pi l-spectri n.Discussion of results shown in Examples 1-9

[0432] Our a

[0433]

[0434] and

[0435] In this study the inventors performed a phenotypic and functional side-by-side comparison of CD4+and CD8+CAR T cells overexpressing a variety of AP-1 transcription factors. The inventors identified BATF2 as a lead-candidate for CD8+CAR T cell modification enhancing the proliferative capacity, exhaustion- and AICD-resistance while providing a memorysignature on phenotypic, transcriptional, and metabolic level. An overall reduced basal activation state of CARBATF2T cells contributed to reduced unspecific tissue sequestration via LFA-1 commonly observed in vivo for in vitro activated CAR T cells17. Further, the inventors observed reduced unspecific but enhanced CCR7- and CXCR4-dependent migration towards spleen and bone marrow in-line with the respective chemokine-receptor expression levels.

[0436]

[0437] nic risk mediated byTF-i

[0438] Modulation of transcription factor levels in T cells bears the risk of promoting oncogenesis in this cell type of highly proliferative nature. Therefore, any risk for malignant transformation must be carefully assessed. cJUN, BATF and BATF3 are naturally expressed in T cells, take part in multiple cellular processes, and have also been involved in T cell malignant transformation.

[0439] BATF expression was found to be increased in NSCLC tissues and knockdown via shRNA inhibited proliferation and enhanced apoptosis rate of the NSCLC cell line A54946. In a mouse model with T cells overexpressing human BATF, over 90% of mice developed a lymphoproliferative disorder within one year.47

[0440] BATF3, IRF4 and IZKF1 were defined as preferentially essential genes in ALIC anaplastic large cell lymphoma (ALCL) cell lines in a CRISPR screening campaign (depmap.org)48and part of a core regulatory circuit driving pro-survival genes like MYC in leukemia and lymphoma49-51. BATF3 can also drive proliferation of malignant T cells via binding to IL2R regulatory regions sustaining high IL2-Ra levels that are correlated with a lower event-free survival (EFS)52. More recently, bi-allelic loss of TET2 resulted in antigen-independenthyperproliferation of CAR T cells with sustained expression of BATF3 driving a MYC-dependent proliferative program38.

[0441] BATF2 is mainly expressed in monocytes / macrophages53and non-hematopoietic tissues8but not in T cells - in contrast to BATF, BATF3 and cJUN. Screening the literature for oncogenic association of BATF2 revealed a tumor suppressive role with expression level as well as subcellular localization acting as prognostic markers of survival. BATF2 was shown to be constitutively expressed in multiple lineage-specific normal cells (e.g., melanocytes, astrocytes, pancreatic mesothelial cells, breast, prostate epithelial cells) but not in tumorigenic counterparts8. Further, artificial restoration of BATF2-expression in diverse cancer cells inhibited proliferation and enhanced apoptosis without affecting "normal" immortalized cell lines8. In CIVIL cells BATF2 was transcriptionally suppressed by BCR-ABL54and low BATF2 levels have been associated with a poor prognosis for glioma55, gastric cancer56and NSCLC57.

[0442] Proliferation and synergistic effects

[0443] Here, the inventors observed a proliferation benefit of CARBATFand CARBATF3BBz-CAR T cells after initial bead-stimulation and gene-transfer in culture with exogenous IL-2 that was similarly observed by Ataide et al.2. Interestingly, this effect was not present within 28z-CAR T cells and based on our AICD-assessment unlikely due to a higher resistance as neither BATF- nor BATF3-overexpression showed higher viabilities compared to the conventional CAR control upon stimulation via the CAR. Importantly, the inventors did not observe any autonomous proliferation, unspecific killing, or cytokine secretion for CARBATF2T cells with any CAR construct tested. In vivo, CD8+CARBATF2T cells combined with conventional CD4+CAR or CARdUNT cells provided the best antigen-specific expansion and but also robust contraction after clearance of the major tumor mass. CD4+CARdUNT cells were more effective than conventional CD4+CAR in boosting the proliferation of CD8+CARBATF2T cells most likely due to their higher IL-2 secretory potential. In-line with the severely diminished effector functions observed for CD4+CARBATF2T cells in vitro, a cell product consisting ofBATF2-overexpressing CD4+and CD8+CAR T cells yielded no beneficial proliferative effects, and the survival was significantly reduced compared to thecombination with a CD4+CARdUNT cell. Enhanced CD8+T cell proliferation and effector functions in presence of IL-2 producing tumor-specific CD4+T cells has previously been described58. The inventors' findings highlight the need to consider transcriptional engineering for different subsets of therapeutically utilized cells individually to provide synergistic effects and maximize curative potential.

[0444] Rescue from u

[0445]

[0446] n

[0447] Low and decreasing CAR T cell numbers in the blood and unspecific accumulation in tissues have been reported already in early clinical trials59. A case report of a patient treated with HER2-directed CAR T cells described accumulation in lung tissue with low endogenous target expression resulting in massive cytokine release, respiratory distress and finally death35. Further, tumor-specific migration favors high local E: T ratios and early intertumoral presence of CAR T cells was identified as a positive predictor of survival60, underlining the importance of the migratory phenotype of a CART cell product. Therefore, the CARBATF2T cell intrinsic feature of reduced lung sequestration the inventors observed here can be highlighted as a significant advantage over conventional CAR T cells. However, this feature was unlikely to be solely dependent on the detected different chemokine repertoire pattern. It has been described that activated CD8+T cells can be retained in noninflamed pulmonary interstitium in an LFAl-dependent manner.18Recent work by Hong and Walling et al.17identified LFA-1, that is regulated by the ST3GALl-|3ll-spectrin axis, as responsible for unspecific tissue sequestration of CAR T cel Isl7. Upon activation ST3GAL1 was found to be upregulated, stabilizing LFA-1 via addition of sialic acid residues while pi I-spectrin, that promotes endocytic recycling of LFA-1, was downregulatedl7. Indeed, quantities of LFA-1 were significantly reduced on the surface of CARBATF2T cells compared to conventional CAR control T cells. While the inventors did not observe changed quantities of cell surface sialic acid via MALII-binding as a surrogate for ST3GAL1 activity, the inventors indeed detected enhanced βII-spectrin expression correlating with lower basal activation levels of CARBATF2T cells. Interestingly, higher levels of βII-spectrin in patient CART products were described to be positively correlated with partial or complete responses and negatively correlated with severity of CRS17. Taken together, less lung sequestrationtogether with increased chemokine-specific homing into immunological niches via CXCR4 and CCR7 makes CARBATF2T cells especially attractive for the treatment of hematological malignancies like MCL, AML or MM by potentially favoring co-localization with the tumor cells.

[0448] Potential mechanistic

[0449]

[0450] The inventors found a reduced phosphorylation state of ERK in CARBATF2T cells compared to the conventional CAR control. BATF2 was previously described to bind to p53, enhancing protein stability and thereby inhibiting phosphorylation of ERK in gastric cancer cells56. Another potential mechanism for lower pERK is based on AP-1 suppressive actions mediated byBATF2. NFAT+AP1 induces CD25 expression61that the inventors found to be significantly reduced in CARBATF2T cells. The IL-2 receptor signals via RasGTPase interacting with the serin / threonine kinase RAF-1 that in turn redirects activity of the MAP kinases MEK1 and MEK2 to phosphorylate ERK1 / 262'63. Thus, the lower quantity of available CD25 could result in less pERKl / 2. Further, less IL-2 signaling would diminish expression of STAT5A / B target genes FoxP364'65, Tbx2166, Gata367and IL2R-a68that is supported by our transcriptional data. IL-2 signaling was described to sustain glycolytic metabolism via mTOR and loss of mTORCl led to reduced glycolytic activity while oxidative phosphorylation was maintained69'70(as reviewed in62), that is in line with the inventor's observations. Further, mTORCl is involved in induction of effector molecules perforin, granzyme and IFN y69expression but also repression of adhesion molecules and chemokine receptors CD62L, CCR7 and CXCR4 via HIFla69-71. Inhibition of mTOR during ex vivo manufacturing of CAR-T cells under IL-2 regimen previously led to upregulation of CXCR4 and enhanced migration towards BM and improved AML elimination72. Together the summarized findings perfectly align with the phenotype induced by BATF2-overexpression. Therefore, the inventors hypothesize that one part of the BATF2 mode-of-action is based on the AP-1 inhibitory effects, leading to down-tuning of IL2-induced STAT5A / B and mTORCl signaling thereby promoting favorable characteristics with manageable drawbacks.

[0451] Novel CAR-LCK fusion molecule identified as best receptor design for BATF2- T cellsCARBATF2T cells have shown desirable characteristics, albeit with the drawback of reduced anti-tumor activity on a per cell basis. The inventors found lower levels of LCK in CARBATF2T cells compared to conventional CAR control potentially contributing to the observed lower sensitivity. Membrane-proximal CD8-bound LCK has been previously shown to be important to enhance TCR-signaling for low-affinity antigens73. Therefore, the inventors directly fused LCK c-terminally to our second generation 4-1BB CAR resulting in restored control of low-antigen tumor cells, IL-2 secretion and further augmented proliferative responses in vitro. The inventors conducted another experiment to validate our findings in a solid tumor ccRCC model in vivo. The inventors included a treatment-group with BATF2- overexpression in CD8+28z-CAR T cells, as CD28-costimulation was previously described to enable LCK-independent CAR signaling via FYN74 that might have been responsible for a compensatory effect in the inventor's initial in vitro experiments with BATF2- overexpressing 28z-CAR T cells. A blend of CD4+CARdUNand CD8+CAR-LBATF2T cells proved superior, providing augmented tumor clearance.

[0452] 10: BATF2-OE opens a ic window to rescue

[0453]

[0454] cross-reactive CAR T cells in tumor-free NSG mice The inventors hypothesized, that alleviating CAR T cells from nonspecific accumulation in non-target tissue could improve safety. We utilized a human / mouse cross-reactive ROR1- CAR (clone F), previously reported to induce pulmonary toxicity in NSG mice90-14-91to assess the potential of BATF2 to overcome pulmonary toxicities. Two CAR variants were cloned: one with CD28 H / TM and CD28 costimulatory domain (RORl(F)-28z) and one with CD8a H / TM and 4-1BB costimulatory domain (RORl(F)-BBz) - each with or without BATF2 transgene (Fig.l3A). Comparable binding to and stimulation by human and mouse ROR1 was confirmed (Fig.l4A). Plate-bound antigen titration revealed a shift toward higher activation thresholds in the context of both 4-1BB and CD28 costimulation, suggesting the potential to spare healthy tissues with low antigen expression ( Fig.13 B, C). Consistent with the data derived from plate-coated antigen titration, B2-RORl(F)-28z lysed AglowRaji cells while B2-RORl(F)-BBz failed to achieve control ( Fig.14B). However, B2-RORl(F)-BBz CAR T cells controlled K562ROR1and 786-0 cell lines, albeit less effective than all other conditions(Fig.l4B). Taken together, the RORl(F)-constructs proved to be cross-reactive, and BATF2-overexpression lowered antigen-sensitivity, thereby opening a therapeutic window that spares healthy tissues with low, basal antigen expression levels while retaining control of tumor cells.

[0455] This encouraged us to move on with the evaluation of lung toxicity in tumor-free NSG mice (Fig.l3D). On the day of injection, BATF2-overexpressing CAR T cells showed their characteristic features of lower activation, reduced LFA-1 and enhanced CXCR4 expression (Fig.l3E). Control groups treated with either cJUN-overexpressing or conventional ROR1(F)-CAR T cells showed progressive signs of toxicity and weight loss, ultimately reaching humane endpoints within 3 days (Fig.l3F, G). Strikingly, the B2-RORl(F)-28z and B2-RORl(F)-BBz groups were largely unaffected, with the only symptom being a transient weight-loss on day 2 for B2-RORl(F)-28z, which was eventually resolved by day 3 (Fig.l3F, G). Thus, BATF2-overexpression completely protected mice from lethal toxicity induced by the ROR1(F)-CAR, resulting in 100% survival without any detectable difference compared to Mock-treated mice.

[0456]

[0457] 11: BATF2-overexpressinq CAR T cells enable lunq tumor control without toxicity To date, we have shown that BATF2 de-targets CAR T cells from nonspecific lung retention and associated critical pulmonary toxicity. This encouraged us to evaluate, whether BATF2-overexpression could enable effective lung tumor control without inducing toxicity using the same ROR1(F) CAR-model. NSG mice were injected i.v. with ROR1+A549 non-small cell lung cancer (NSCLC) and lung engraftment was confirmed by BLI before administering the respective T cell products on day 7 (Fig.l5A-C).

[0458] As before, conventional 28z- and BBz-CAR T cells triggered toxicity manifesting in rapid weight-loss and death (Fig.l5D-F). Specifically, all mice receiving conventional 28z-CAR T cells reached the endpoint two days after infusion, whereas only 2 of 5 BBz-CAR T-treated mice did so on day 2, with the remaining three reaching endpoint one day later (Fig.15 D, E). In contrast, all mice receiving BATF2-overexpressing CAR T cells survived the initial toxicity phase (Fig.lSD). The most pronounced weight-loss occurred in conventional 28z- and BBz-CAR T groups, followed by B2-28z CAR T cells, while B2-BBz CAR T cell-treated micemaintained stable weights comparable to Mock controls (Fig.15 E, F). Although B2-28z CAR T cell recipients experienced transient weight-loss until day 3 after infusion, they subsequently recovered to baseline weight over the next five days (Fig.15E). Tumor control was evident as early as one day after T cell transfer for all groups except conventional 28z-CAR T cells relative to Mock-control ( Fig.15G ). By day 4 after T cell transfer, B2-28z and B2-BBz CAR T cells reduced tumor burden by approximately 13-fold and 4-fold compared to Mock control, respectively, but did not induce complete remission (Fig.15G).

[0459] Overall, BATF2-overexpression effectively prevented on-target / off-tumor toxicity in the ROR1(F)-CAR model while retaining anti-tumor activity, thereby prolonging the observed tumor-related survival. However, the extent of protection from toxicity appears to depend on the costimulatory domain, with CD28-based constructs remaining more susceptible to toxicity-associated weight-loss than 4-lBB-based constructs.

[0460] Discussion

[0461] Collectively, three critical roadblocks in the field emerge from available data: First, there is a lack of true tumor-specific antigens for solid tumors. Second, expression of target antigens might be upregulated in non-tumor tissues under specific pathophysiological conditions. Third, the need to balance antigen sensitivity to achieve effective tumor recognition while avoiding on-target off-tumor toxicity. The inventors present BATF2-overexpression in CAR T cells as a strategy supporting a favorable migratory profile to secondary lymphoid organs accompanied with reduced nonspecific lung accumulation and attenuated overactivation, while opening a therapeutic window sparing healthy tissues. Hybrid toxicity models with lung-localized tumors to integrate efficacy and safety in vivo proved tumor control while mitigating toxicities using BATF2 to fine-tune CAR T cell sensitivity and biodistribution. In synthesis, BATF2-overexpression is expected to have a strong clinical impact across multiple target entities by overcoming a critical limitation, currently hindering cancer patients with lung cancer or metastases to receive a promising treatment modality.Industrial Applicability

[0462] The present invention is useful in a wide range of fields, including the methods and uses as described (e.g., for treating cancer or for in vitro uses). The CD8+T cell, composition, kit or pharmaceutical composition may be industrially manufactured in accordance with known standards for the manufacture of pharmaceutical products.

[0463] Thus, the present invention is industrially applicable.

[0464] Further applications and practical exploitation in industry may be derived from the present description by the skilled person's general knowledge.

[0465] Sequences

[0466] SEQ ID NO: 1 (BATF2) MHLCGGNGLLTQTDPKEQQRQLKKQKNRAAAQRSRQKHTDKADALHQQHESLEKDNLALRKEIQS LQAELAWWSRTLHVHERLCPMDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQTPGSC YPAQPLSPGPQPHDSPSLLQCPLPSLSLGPAVVAEPPVQLSPSPLLFASHTGSSLQGSSSKLSALQPSLT AQTAPPQPLELEHPTRGKLGSSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPHPLLAFPLLS SAQVHF SEQ ID NO: 2 (eJun) MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGL LKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVN GAGMVAPAVASVAGGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQ QQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKC RKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF SEQ ID NO: 3 (CAR of ROR-1 CAR 4-lBBz construct: R12sh4-lBBz EGFRt_SB, scFV12- inker- Cp28-41SS- CD3z-T2A- EGFR) MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLE WIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQG EAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYUIPSVQADDEADYYCGADYIGGYVFGGGT QLTVTGESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWV / ('RGR / (' / ('££Y / F / ('QPFMRP\ / Q7T j9^ GC5CRFP GGC RyKfSR_S DAPAY^

[0467]

[0468] CLKpjlRKNP& EjaLYNELClKp^ RLEGGGEGRGSLL TCGD VEEN PG PR M LLLVTS LLLC ELPHPAFLLI PRKVCNGIGIGEFKDSLSINATNI KHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEII RGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRG ENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPE CLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNC TYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 4 (BATF2+CAR of ROR-1 CAR 4-lBBz construct, hBatf2 T2A R12sh4-lBBz T2A tEGFR) MHLCGGNGLLTQ. TDPKEQQRQLKKQKNRAAAQRSRQKHTDKADALHQQHESLEKDNLALRKEIQS LQAELAWWSRTLHVHERLCPMDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQ. TPGSC YPAQPLSPGPQPHDSPSLLQCPLPSLSLGPAVVAEPPVQLSPSPLLFASHTGSSLQGSSSKLSALQPSLT AQTAPPQPLELEHPTRGKLGSSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPHPLLAFPLLS SAQVHFLEGGGEGRGSLLTCGDVEENPGPRASEFLEATMLLLVTSLLLCELPHPAFLLIPQEQLVESGG RLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQ NTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQ SPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGA DRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGESKYGPPCPPCPMFWVLVVVGGVLACYS LLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLC ELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDIL KTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNK NLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMG ENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 5 (cJun+CAR of ROR-1 CAR 4-lBBz construct, hcJun T2A R12sh4-lBBz T2A tEGFR) MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGL LKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVN GAGMVAPAVASVAGGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQ QQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKC RKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTFLEGG GEGRGSLLTCGDVEENPGPRASEFLEATMLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTL SCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSL TAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSP AKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQAD DEADYYCGADYIGGYVFGGGTQLTVTGESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWV KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPR KVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLI QAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTIN WKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWK YADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 6 (scFV of the non-humanized R12 antibody) QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGR FTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGG GGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPD RFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG SEQ ID NO: 7 (partial human LCK)IRNGSEVRDPLVTYEGSNPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTT GQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQN QGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEV PRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYA VVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTL SCKIADFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNP EVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP SEQ ID NO: 8 (CAR of ROR-1 CAR-L 4-lBBz construct, R12sh4-lBBz-LCKfusion-tEGFR) MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLE WIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWG PGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAP RYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVT GESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGIR NGSEVRDPLVTYEGSNPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTG QEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQ GEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVP RETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYAV VTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTLS CKIADFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPE VIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP££GGG£ G / ? GS££7’CGDV'££ / VPGP / ? M£££\ / 7’S£££C££PHPAF££ / P / ? KVCNGIGIGEFKDSLSINATNIKHFKNCTSI SGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVC HALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTG RGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPT NGPKIPSIATGMVGALLLLLVVALGIGLFMSEQ ID NO: 9 (BATF2+CAR-L of ROR-1 CAR-L 4-lBBz construct, BATF2+CAR-L: B2 R12sh4-lBBz-LCKfusion tEGFR SB) MHLCGGNGLLTQTDPKEQQRQLKKQKNRAAAQRSRQKHTDKADALHQQHESLEKDNLALRKEIQS LQAELAWWSRTLHVHERLCPMDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQTPGSC YPAQPLSPGPQPHDSPSLLQCPLPSLSLGPAVVAEPPVQLSPSPLLFASHTGSSLQGSSSKLSALQPSLT AQTAPPQPLELEHPTRGKLGSSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPHPLLAFPLLS SAQVHFLEGGGEGRGSLLTCGDVEENPGPRASEFLEATMLLLVTSLLLCELPHPAFLLIPQEQLVESGG RLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQ NTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQ SPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGA DRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGESKYGPPCPPCPMFWVLVVVGGVLACYS LLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGIRNGSEVRDPLVTYEGSNPPASPLQDNLVIAL HSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSLEPEPWFFKNLSRKD AERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHELV RHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKV AVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTIN KLLDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGAKFPIKWTAP EAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCW KERPEDRPTFDYLRSVLEDFFTATEGQYQPQPLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCEL PHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILK TVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNK NLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRE CVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMG ENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 10 (CAR of ROR-1 CAR CD28z construct, EFl R12shCD28z T2A tEGFR SB) MLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLE WIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAP RYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVT GESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE GGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFK NCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTK QHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKA TGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAM NITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPG LEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0469] SEQ ID NO: 11 (BATF2+CAR of ROR-1 CAR CD28z construct, hBatf2 T2A R12shCD28z T2A tEGFR SB) MHLCGGNGLLTQ. TDPKEQQRQLKKQKNRAAAQRSRQKHTDKADALHQQHESLEKDNLALRKEIQS LQAELAWWSRTLHVHERLCPMDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQ. TPGSC YPAQPLSPGPQPHDSPSLLQCPLPSLSLGPAVVAEPPVQLSPSPLLFASHTGSSLQGSSSKLSALQPSLT AQTAPPQPLELEHPTRGKLGSSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPHPLLAFPLLS SAQVHFLEGGGEGRGSLLTCGDVEENPGPRASEFLEATMLLLVTSLLLCELPHPAFLLIPQEQLVESGG RLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQ NTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQ SPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGA DRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTGESKYGPPCPPCPMFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLL CELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELD ILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISG NKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRG RECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGL FM SEQ ID NO: 12 (cJun+CAR of ROR-1 CAR CD28z construct, eJun T2A R12shCD28z T2A tEGFR SB) MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGL LKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVN GAGMVAPAVASVAGGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQ QQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKC RKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTFLEGG GEGRGSLLTCGDVEENPGPRASEFLEATMLLLVTSLLLCELPHPAFLLIPQEQLVESGGRLVTPGGSLTL SCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSL TAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSP AKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQAD DEADYYCGADYIGGYVFGGGTQLTVTGESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNEL NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIP RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFL LIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTIN WKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWK YADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0470] SEQ ID NO: 13 (CAR of HA-GD2-CAR CD28z, HA-GD2shortCD28z SB) MLLLVTSLLLCELPHPAFLLIPVQEQLVESGGALVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLE WIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMKYWGQGTSVTVSS GGGGSGGGGSGGGGSDVVMTQ. TPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKL LIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKESKYGPPC PPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLL TCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSP EGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDN CIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIP SIATGMVGALLLLLVVALGIGLFM

[0471] SEQ ID NO: 14 (scFv GD2) FV'QZ QSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGR ATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMKYWGQGTSVTVSSGGGGSGGGGSGGGGSDVV MTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSG SGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

[0472] SEQ ID NO: 17 (CAR of ROR-1 CAR 4-lBBz construct: R12sh4-lBBz EGFRt_SB) ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCC CAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACACCTGGCGGCAGCCTGACCCTGAGC TGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTCCGCCAGGCCCCTGGCA AG G G ACTG G AATG G ATCG CCACC ATCTACCCCAG CAG CGG CAAG ACCTACTACG CC ACCTG GGT GAACGGACGGTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACAGC CTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCGACGACGGCGCCC TGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTAGCGGCGGAGGCGGATCTGGTGG CGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCC CTG G G AAG CCCTGCC AAG ATC ACCTGTACCCTG AG C AG CGCCCAC AAG ACCG AC ACC ATCG ACT GGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCCGACCGCTACCT GATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTACTGTGGCGCCGACTACATCGGC GGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGCGAATCTAAGTACGGACCGCCCT GCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTG CTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTG CCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACG ACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACC CCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCG GCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCG CCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGCTCGAGGGCGGCGG AGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCT TCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAA AGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTA AACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGT GACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGA AATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGA ACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTG AACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGG AAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGA AAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGC CTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTC AGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTG GAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCAC AGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAG ACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGC CATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTG TCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGC TGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0473] SEQ ID NO: 18 (BATF2+CAR of ROR-1 CAR 4-lBBz construct, hBatf2 T2A R12sh4-lBBz T2A tEGFR) ATGCACCTGTGCGGCGGCAACGGCCTGCTGACCCAGACCGACCCCAAGGAGCAGCAGAGGCAG CTGAAGAAGCAGAAGAACAGGGCCGCCGCCCAGAGGAGCAGGCAGAAGCACACCGACAAGGCCGACGCCCTGCACCAGCAGCACGAGAGCCTGGAGAAGGACAACCTGGCCCTGAGGAAGGAGAT CCAGAGCCTGCAGGCCGAGCTGGCCTGGTGGAGCAGGACCCTGCACGTGCACGAGAGGCTGTG CCCCATGGACTGCGCCAGCTGCAGCGCCCCCGGCCTGCTGGGCTGCTGGGACCAGGCCGAGGGC CTGCTGGGCCCCGGCCCCCAGGGCCAGCACGGCTGCAGGGAGCAGCTGGAGCTGTTCCAGACCC CCGGCAGCTGCTACCCCGCCCAGCCCCTGAGCCCCGGCCCCCAGCCCCACGACAGCCCCAGCCTG CTGCAGTGCCCCCTGCCCAGCCTGAGCCTGGGCCCCGCCGTGGTGGCCGAGCCCCCCGTGCAGC TGAGCCCCAGCCCCCTGCTGTTCGCCAGCCACACCGGCAGCAGCCTGCAGGGCAGCAGCAGCAA GCTGAGCGCCCTGCAGCCCAGCCTGACCGCCCAGACCGCCCCCCCCCAGCCCCTGGAGCTGGAG CACCCCACCAGGGGCAAGCTGGGCAGCAGCCCCGACAACCCCAGCAGCGCCCTGGGCCTGGCCA GGCTGCAGAGCAGGGAGCACAAGCCCGCCCTGAGCGCCGCCACCTGGCAGGGCCTGGTGGTGG ACCCCAGCCCCCACCCCCTGCTGGCCTTCCCCCTGCTGAGCAGCGCCCAGGTGCACTTCCTCGAG GGCGGAGGCGAAGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCC CAGAGCTAGCGAATTCCTCGAGGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAG CTGCCCCACCCCGCCTTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGT GACACCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTAC ATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCCACCATCTACCCCAGCA GCGGCAAGACCTACTACGCCACCTGGGTGAACGGACGGTTCACCATCTCCAGCGACAACGCCCA GAACACCGTGGACCTGCAGATGAACAGCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCC AGAGACAGCTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAA TCTCTAGCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTG CTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATCACCTGTACCCTGAG CAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATAC CTGATGCAGGTGCAGAGCGACGGCAGCTACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGC GGATCTAGCTCTGGCGCCGACCGCTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCG ATTACTACTGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGT GACCGGCGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGG TCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAA CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCA AGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGT GAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAG ATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGAC AAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCA CGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAG GCCCTGCCCCCAAGGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGAC GTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACC ACACCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAG ACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATC TCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGG AACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAA AACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATG GTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGA TAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGG AAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCT GCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCC CAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTT CTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCC TGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCA CTAC ATTG ACGG CCCCCACTG CGTC AAG ACCTGCCCG G CAG G AGTC ATG GG AG AAAAC AAC ACC CTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGG ATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTG GGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0474] SEQ ID NO: 19 (cJun+CAR of ROR-1 CAR 4-lBBz construct, hcJun T2A R12sh4-lBBz T2A tEGFR) ATGACCGCCAAGATGGAGACCACCTTCTACGACGACGCCCTGAACGCCAGCTTCCTGCCCAGCGA GAGCGGCCCCTACGGCTACAGCAACCCCAAGATCCTGAAGCAGAGCATGACCCTGAACCTGGCC GACCCCGTGGGCAGCCTGAAGCCCCACCTGAGGGCCAAGAACAGCGACCTGCTGACCAGCCCCG ACGTGGGCCTGCTGAAGCTGGCCAGCCCCGAGCTGGAGAGGCTGATCATCCAGAGCAGCAACG GCCACATCACCACCACCCCCACCCCCACCCAGTTCCTGTGCCCCAAGAACGTGACCGACGAGCAGGAGGGCTTCGCCGAGGGCTTCGTGAGGGCCCTGGCCGAGCTGCACAGCCAGAACACCCTGCCCA GCGTGACCAGCGCCGCCCAGCCCGTGAACGGCGCCGGCATGGTGGCCCCCGCCGTGGCCAGCGT GGCCGGCGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGCACAGCGAGCCCCCCGTGTACGC CAACCTGAGCAACTTCAACCCCGGCGCCCTGAGCAGCGGCGGCGGCGCCCCCAGCTACGGCGCC GCCGGCCTGGCCTTCCCCGCCCAGCCCCAGCAGCAGCAGCAGCCCCCCCACCACCTGCCCCAGCA GATGCCCGTGCAGCACCCCAGGCTGCAGGCCCTGAAGGAGGAGCCCCAGACCGTGCCCGAGAT GCCCGGCGAGACCCCCCCCCTGAGCCCCATCGACATGGAGAGCCAGGAGAGGATCAAGGCCGA GAGGAAGAGGATGAGGAACAGGATCGCCGCCAGCAAGTGCAGGAAGAGGAAGCTGGAGAGGA TCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCG CCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACGTGAACAGCG GCTGCCAGCTGATGCTGACCCAGCAGCTGCAGACCTTCCTCGAGGGCGGAGGCGAAGGCAGAG GCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCCCAGAGCTAGCGAATTCCTCGA GGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGC TGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACACCTGGCGGCAGCCTGAC CCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTCCGCCAGGCCC CTGGCAAGGGACTGGAATGGATCGCCACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCAC CTG G GTG AACGG ACG GTTC ACC ATCTCC AG CG AC AACG CCC AG AAC ACCGTG G ACCTGC AG ATG AACAGCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCGACGACG GCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTAGCGGCGGAGGCGGATC TGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTGCTGACCCAGAGCCCCTCTGTGTC TGCTGCCCTGGGAAGCCCTGCCAAGATCACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCA TCGACTGGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACG GCAGCTACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCCGACCG CTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTACTGTGGCGCCGACTACA TCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGCGAATCTAAGTACGGACC GCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACA GCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTAT ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCG ATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGC CCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAA GAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGA GATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGT CCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGCTCGAGGG CGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAG GATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCC ACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGA AT ATTA A AC ACTTC A A A A ACTG C ACCTCC ATC AGTG G CG ATCTCC AC ATCCTG CCG GTG G C ATTTA GGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTA AAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTT TGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCA GCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATT TCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGG TCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGC CATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGA ATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGT TTGTG GAG AACTCTG AGTG CATAC AGTGCC ACCC AG AGTG CCTG CCTC AG GCCATG AAC ATC ACC TGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGT CAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGC CGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAG GCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTG CTG CTG GTG GTGG CCCTG GG G ATCG G CCTCTTCATGTG A

[0475] SEQ ID NO: 20 (CAR of ROR-1 CAR-L 4-lBBz construct, R12sh4-lBBz-LCKfusion-tEGFR) ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCC CAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACACCTGGCGGCAGCCTGACCCTGAGC TGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTCCGCCAGGCCCCTGGCA AG G G ACTG GAATG G ATCG CCACCATCTACCCCAG CAG CGG CAAG ACCTACTACG CC ACCTG GGT GAACGGACGGTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACAGC CTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTAGCGGCGGAGGCGGATCTGGTGG CGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCC CTG G G AAG CCCTGCC AAG ATC ACCTGTACCCTG AG C AG CGCCCAC AAG ACCG AC ACC ATCG ACT GGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCT ACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCCGACCGCTACCT GATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTACTGTGGCGCCGACTACATCGGC GGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGCGAATCTAAGTACGGACCGCCCT GCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTG CTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATT CAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTC CAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTG CCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACG ACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACC CCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCG GCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCG CCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGGGCTCCGGAATCAG AAACGGCAGCGAAGTGCGCGATCCCCTCGTGACATACGAGGGCTCTAATCCTCCTGCAAGCCCTC TGCAGGACAACCTCGTGATTGCCCTGCACAGCTACGAGCCTAGCCACGATGGCGATCTGGGCTTT GAGAAGGGCGAGCAGCTGAGAATCCTGGAACAGTCTGGCGAGTGGTGGAAGGCCCAGTCTCTG ACAACAGGCCAAGAGGGCTTCATCCCCTTCAACTTCGTGGCCAAGGCCAACAGCCTGGAACCTGA GCCATGGTTCTTCAAGAATCTGAGCCGGAAGGACGCCGAGAGACAGCTGCTTGCCCCTGGAAAT ACCCACGGCAGCTTTCTGATCCGCGAGAGCGAATCTACAGCCGGCTCCTTTTCTCTGTCCGTGCG GGACTTCGACCAGAACCAGGGCGAAGTGGTCAAGCACTACAAGATCCGGAACCTGGACAACGG CG GCTTCTACATC AG CCCCAG AATC ACATTCCCCG G CCTG CATG AACTCGTGCGG C ACTAC ACCA ATGCCAGCGACGGCCTGTGTACCAGACTGAGCAGACCTTGCCAGACACAGAAGCCTCAGAAACC TTGGTGGGAAGATGAGTGGGAAGTGCCCCGGGAAACCCTGAAGCTGGTGGAAAGACTTGGCGC CGGACAGTTCGGCGAAGTGTGGATGGGCTACTACAACGGCCACACCAAGGTGGCCGTGAAGTCT CTGAAGCAGGGCTCTATGAGCCCCGATGCCTTTCTGGCCGAGGCCAATCTGATGAAGCAGCTGC AGCACCAGAGACTTGTGCGGCTGTATGCCGTGGTCACACAAGAGCCCATCTACATCATCACCGAG TACATGGAAAACGGCTCCCTGGTGGACTTCCTGAAAACCCCTAGCGGCATCAAGCTGACCATCAACAAGCTGCTGGACATGGCCGCTCAGATCGCCGAAGGCATGGCCTTCATCGAGGAACGGAACTAC ATCCACCGGGACCTGAGAGCCGCCAACATCCTGGTGTCTGATACCCTGAGCTGCAAGATTGCCGA CTTCGGCCTGGCCAGACTGATCGAGGACAACGAGTACACAGCCAGAGAGGGCGCTAAGTTCCCC ATCAAGTGGACAGCCCCTGAGGCCATCAACTACGGCACCTTCACCATCAAGTCCGATGTGTGGTC CTTCGGCATCCTGCTGACCGAGATCGTGACACACGGCAGAATCCCTTATCCTGGCATGACAAACC CCGAAGTGATCCAGAATCTGGAACGGGGCTACAGAATGGTCCGACCTGACAACTGCCCCGAGGA ACTGTACCAGCTGATGCGGCTGTGCTGGAAAGAGAGGCCCGAGGACAGACCCACCTTCGACTAC CTGAGAAGCGTGCTGGAAGATTTCTTCACCGCCACCGAGGGCCAGTACCAGCCTCAACCTCTCGA GGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCC TAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGAT CCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTA CGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCAT TTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCG TAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCC TTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGT CAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAA TTTCAG G AAAC AAAAATTTGTG CTATG CAAATACAATAAACTG G AAAAAACTGTTTGG G ACCTCC GGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCT GCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCG GAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGA GTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCA CCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGC GTC AAG ACCTG CCCGG CAG G AGTC ATG GG AG AAAAC AACACCCTGGTCTG G AAGTACG C AG AC GCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGA AGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCT TGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0476] SEQ ID NO: 21 (BATF2+CAR-L of ROR-1 CAR-L 4-lBBz construct, BATF2+CAR-L: B2 R12sh4-lBBz-LCKfusion tEGFR SB)ATGCACCTGTGCGGCGGCAACGGCCTGCTGACCCAGACCGACCCCAAGGAGCAGCAGAGGCAG CTGAAGAAGCAGAAGAACAGGGCCGCCGCCCAGAGGAGCAGGCAGAAGCACACCGACAAGGC CGACGCCCTGCACCAGCAGCACGAGAGCCTGGAGAAGGACAACCTGGCCCTGAGGAAGGAGAT CCAGAGCCTGCAGGCCGAGCTGGCCTGGTGGAGCAGGACCCTGCACGTGCACGAGAGGCTGTG CCCCATGGACTGCGCCAGCTGCAGCGCCCCCGGCCTGCTGGGCTGCTGGGACCAGGCCGAGGGC CTGCTGGGCCCCGGCCCCCAGGGCCAGCACGGCTGCAGGGAGCAGCTGGAGCTGTTCCAGACCC CCGGCAGCTGCTACCCCGCCCAGCCCCTGAGCCCCGGCCCCCAGCCCCACGACAGCCCCAGCCTG CTGCAGTGCCCCCTGCCCAGCCTGAGCCTGGGCCCCGCCGTGGTGGCCGAGCCCCCCGTGCAGC TGAGCCCCAGCCCCCTGCTGTTCGCCAGCCACACCGGCAGCAGCCTGCAGGGCAGCAGCAGCAA GCTGAGCGCCCTGCAGCCCAGCCTGACCGCCCAGACCGCCCCCCCCCAGCCCCTGGAGCTGGAG CACCCCACCAGGGGCAAGCTGGGCAGCAGCCCCGACAACCCCAGCAGCGCCCTGGGCCTGGCCA GGCTGCAGAGCAGGGAGCACAAGCCCGCCCTGAGCGCCGCCACCTGGCAGGGCCTGGTGGTGG ACCCCAGCCCCCACCCCCTGCTGGCCTTCCCCCTGCTGAGCAGCGCCCAGGTGCACTTCCTCGAG GGCGGAGGCGAAGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCC CAGAGCTAGCGAATTCCTCGAGGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAG CTGCCCCACCCCGCCTTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGT GACACCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTAC ATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCCACCATCTACCCCAGCA GCGGCAAGACCTACTACGCCACCTGGGTGAACGGACGGTTCACCATCTCCAGCGACAACGCCCA GAACACCGTGGACCTGCAGATGAACAGCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCC AGAGACAGCTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAA TCTCTAGCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTG CTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATCACCTGTACCCTGAG CAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATAC CTGATGCAGGTGCAGAGCGACGGCAGCTACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGC GGATCTAGCTCTGGCGCCGACCGCTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCG ATTACTACTGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGT GACCGGCGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGG TCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTGAAA CGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGT GAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAG CTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAG ATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGAC AAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCA CGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAG GCCCTGCCCCCAAGGGGCTCCGGAATCAGAAACGGCAGCGAAGTGCGCGATCCCCTCGTGACAT ACGAGGGCTCTAATCCTCCTGCAAGCCCTCTGCAGGACAACCTCGTGATTGCCCTGCACAGCTAC GAGCCTAGCCACGATGGCGATCTGGGCTTTGAGAAGGGCGAGCAGCTGAGAATCCTGGAACAG TCTGGCGAGTGGTGGAAGGCCCAGTCTCTGACAACAGGCCAAGAGGGCTTCATCCCCTTCAACTT CGTGGCCAAGGCCAACAGCCTGGAACCTGAGCCATGGTTCTTCAAGAATCTGAGCCGGAAGGAC GCCGAGAGACAGCTGCTTGCCCCTGGAAATACCCACGGCAGCTTTCTGATCCGCGAGAGCGAAT CTACAGCCGGCTCCTTTTCTCTGTCCGTGCGGGACTTCGACCAGAACCAGGGCGAAGTGGTCAAG CACTACAAGATCCGGAACCTGGACAACGGCGGCTTCTACATCAGCCCCAGAATCACATTCCCCGG CCTGCATGAACTCGTGCGGCACTACACCAATGCCAGCGACGGCCTGTGTACCAGACTGAGCAGA CCTTGCCAGACACAGAAGCCTCAGAAACCTTGGTGGGAAGATGAGTGGGAAGTGCCCCGGGAA ACCCTGAAGCTGGTGGAAAGACTTGGCGCCGGACAGTTCGGCGAAGTGTGGATGGGCTACTACA ACGGCCACACCAAGGTGGCCGTGAAGTCTCTGAAGCAGGGCTCTATGAGCCCCGATGCCTTTCTG GCCGAGGCCAATCTGATGAAGCAGCTGCAGCACCAGAGACTTGTGCGGCTGTATGCCGTGGTCA CACAAGAGCCCATCTACATCATCACCGAGTACATGGAAAACGGCTCCCTGGTGGACTTCCTGAAA ACCCCTAGCGGCATCAAGCTGACCATCAACAAGCTGCTGGACATGGCCGCTCAGATCGCCGAAG GCATGGCCTTCATCGAGGAACGGAACTACATCCACCGGGACCTGAGAGCCGCCAACATCCTGGT GTCTGATACCCTGAGCTGCAAGATTGCCGACTTCGGCCTGGCCAGACTGATCGAGGACAACGAG TACACAGCCAGAGAGGGCGCTAAGTTCCCCATCAAGTGGACAGCCCCTGAGGCCATCAACTACG GCACCTTCACCATCAAGTCCGATGTGTGGTCCTTCGGCATCCTGCTGACCGAGATCGTGACACAC GGCAGAATCCCTTATCCTGGCATGACAAACCCCGAAGTGATCCAGAATCTGGAACGGGGCTACA GAATGGTCCGACCTGACAACTGCCCCGAGGAACTGTACCAGCTGATGCGGCTGTGCTGGAAAGA GAGGCCCGAGGACAGACCCACCTTCGACTACCTGAGAAGCGTGCTGGAAGATTTCTTCACCGCC ACCGAGGGCCAGTACCAGCCTCAACCTCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTA ACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTIll

[0477] CTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTG GTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCA TCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTC TGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAG GCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGA CCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCT CCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAAT ACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAG GTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTG GGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGA CAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGC CACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTA TCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGA GAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAA ACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCC GTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGC CTCTTCATGTGA

[0478] SEQ ID NO: 22 (CAR of ROR-1 CAR CD28z construct, EFl R12shCD28z T2A tEGFR SB) ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCC CAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACACCTGGCGGCAGCCTGACCCTGAGC TGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTCCGCCAGGCCCCTGGCA AG G G ACTG G AATG G ATCG CCACC ATCTACCCCAG CAG CGG CAAG ACCTACTACG CC ACCTG GGT GAACGGACGGTTCACCATCTCCAGCGACAACGCCCAGAACACCGTGGACCTGCAGATGAACAGC CTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCGACGACGGCGCCC TGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTAGCGGCGGAGGCGGATCTGGTGG CGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTGCTGACCCAGAGCCCCTCTGTGTCTGCTGCC CTG G G AAG CCCTGCC AAG ATC ACCTGTACCCTG AG C AG CGCCCAC AAG ACCG AC ACC ATCG ACT GGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACGGCAGCTACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCCGACCGCTACCT GATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTACTGTGGCGCCGACTACATCGGC GGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGCGAATCTAAGTACGGACCGCCCT GCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTG CTGGTCACCGTGGCCTTCATCATCTTTTGGGTCCGCAGCAAGCGGAGCAGAGGCGGCCACAGCG ACTACATGAACATGACCCCTAGACGGCCTGGCCCCACCAGAAAGCACTACCAGCCCTACGCCCCT CCCCGGGACTTTGCCGCCTACAGAAGCCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCT ACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACG TCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCC AGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCA TGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCA CCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGCTCGAGGGCGGCGGAGA GGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTC CTG GTG AC AAG CCTTCTGCTCTGTG AGTTACC AC ACCCAG C ATTCCTCCTG ATCCC ACG CAAAGTG TGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACA CTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACT CCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCA CAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTA GAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACAT AACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACA AAAATTTGTG CTATG CAAATAC AATAAACTG G AAAAAACTGTTTGG G ACCTCCG GTCAG AAAACC AAAATTATAAG CAAC AG AG GTG AAAAC AGCTG CAAG GCC ACAG GCC AG GTCTGCC ATG CCTTGT GCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCG AGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAA CTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGAC GGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGC CCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTG TGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAAC GAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTG GTGGCCCTGGGGATCGGCCTCTTCATGTGASEQ ID NO: 23 (BATF2+CAR of ROR-1 CAR CD28z construct, hBatf2 T2A R12shCD28z T2A tEGFR SB) ATGCACCTGTGCGGCGGCAACGGCCTGCTGACCCAGACCGACCCCAAGGAGCAGCAGAGGCAG CTGAAGAAGCAGAAGAACAGGGCCGCCGCCCAGAGGAGCAGGCAGAAGCACACCGACAAGGC CGACGCCCTGCACCAGCAGCACGAGAGCCTGGAGAAGGACAACCTGGCCCTGAGGAAGGAGAT CCAGAGCCTGCAGGCCGAGCTGGCCTGGTGGAGCAGGACCCTGCACGTGCACGAGAGGCTGTG CCCCATGGACTGCGCCAGCTGCAGCGCCCCCGGCCTGCTGGGCTGCTGGGACCAGGCCGAGGGC CTGCTGGGCCCCGGCCCCCAGGGCCAGCACGGCTGCAGGGAGCAGCTGGAGCTGTTCCAGACCC CCGGCAGCTGCTACCCCGCCCAGCCCCTGAGCCCCGGCCCCCAGCCCCACGACAGCCCCAGCCTG CTGCAGTGCCCCCTGCCCAGCCTGAGCCTGGGCCCCGCCGTGGTGGCCGAGCCCCCCGTGCAGC TGAGCCCCAGCCCCCTGCTGTTCGCCAGCCACACCGGCAGCAGCCTGCAGGGCAGCAGCAGCAA GCTGAGCGCCCTGCAGCCCAGCCTGACCGCCCAGACCGCCCCCCCCCAGCCCCTGGAGCTGGAG CACCCCACCAGGGGCAAGCTGGGCAGCAGCCCCGACAACCCCAGCAGCGCCCTGGGCCTGGCCA GGCTGCAGAGCAGGGAGCACAAGCCCGCCCTGAGCGCCGCCACCTGGCAGGGCCTGGTGGTGG ACCCCAGCCCCCACCCCCTGCTGGCCTTCCCCCTGCTGAGCAGCGCCCAGGTGCACTTCCTCGAG GGCGGAGGCGAAGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCC CAGAGCTAGCGAATTCCTCGAGGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAG CTGCCCCACCCCGCCTTTCTGCTGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGT GACACCTGGCGGCAGCCTGACCCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTAC ATGAGCTGGGTCCGCCAGGCCCCTGGCAAGGGACTGGAATGGATCGCCACCATCTACCCCAGCA GCGGCAAGACCTACTACGCCACCTGGGTGAACGGACGGTTCACCATCTCCAGCGACAACGCCCA GAACACCGTGGACCTGCAGATGAACAGCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCC AGAGACAGCTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAA TCTCTAGCGGCGGAGGCGGATCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTG CTGACCCAGAGCCCCTCTGTGTCTGCTGCCCTGGGAAGCCCTGCCAAGATCACCTGTACCCTGAG CAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATAC CTGATGCAGGTGCAGAGCGACGGCAGCTACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGC GGATCTAGCTCTGGCGCCGACCGCTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCG ATTACTACTGTGGCGCCGACTACATCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGCGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGG TCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTCCGC AGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCTAGACGGCCTGGCCCC ACCAGAAAGCACTACCAGCCCTACGCCCCTCCCCGGGACTTTGCCGCCTACAGAAGCCGGGTGAA GTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTG AACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGAT GGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAA GATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACG ACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCC CTGCCCCCAAGGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTG GAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACA CCCAGCATTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACT CACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCC ACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAAC TGG ATATTCTG AAAACCGTAAAG G AAATCAC AGG GTTTTTG CTG ATTC AG G CTTGG CCTG AAAAC AGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTC AGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAA GTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAA AAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCA AGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAG GGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTG GAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGC CTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTAC ATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGG TCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGC ACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGA TGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0479] SEQ ID NO: 24 (cJun+CAR of ROR-1 CAR CD28z construct, eJun T2A R12shCD28z T2A tEGFR SB)ATGACCGCCAAGATGGAGACCACCTTCTACGACGACGCCCTGAACGCCAGCTTCCTGCCCAGCGA GAGCGGCCCCTACGGCTACAGCAACCCCAAGATCCTGAAGCAGAGCATGACCCTGAACCTGGCC GACCCCGTGGGCAGCCTGAAGCCCCACCTGAGGGCCAAGAACAGCGACCTGCTGACCAGCCCCG ACGTGGGCCTGCTGAAGCTGGCCAGCCCCGAGCTGGAGAGGCTGATCATCCAGAGCAGCAACG GCCACATCACCACCACCCCCACCCCCACCCAGTTCCTGTGCCCCAAGAACGTGACCGACGAGCAG GAGGGCTTCGCCGAGGGCTTCGTGAGGGCCCTGGCCGAGCTGCACAGCCAGAACACCCTGCCCA GCGTGACCAGCGCCGCCCAGCCCGTGAACGGCGCCGGCATGGTGGCCCCCGCCGTGGCCAGCGT GGCCGGCGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGCACAGCGAGCCCCCCGTGTACGC CAACCTGAGCAACTTCAACCCCGGCGCCCTGAGCAGCGGCGGCGGCGCCCCCAGCTACGGCGCC GCCGGCCTGGCCTTCCCCGCCCAGCCCCAGCAGCAGCAGCAGCCCCCCCACCACCTGCCCCAGCA GATGCCCGTGCAGCACCCCAGGCTGCAGGCCCTGAAGGAGGAGCCCCAGACCGTGCCCGAGAT GCCCGGCGAGACCCCCCCCCTGAGCCCCATCGACATGGAGAGCCAGGAGAGGATCAAGGCCGA GAGGAAGAGGATGAGGAACAGGATCGCCGCCAGCAAGTGCAGGAAGAGGAAGCTGGAGAGGA TCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCG CCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACGTGAACAGCG GCTGCCAGCTGATGCTGACCCAGCAGCTGCAGACCTTCCTCGAGGGCGGAGGCGAAGGCAGAG GCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCCCAGAGCTAGCGAATTCCTCGA GGCCACCATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGC TGATCCCCCAGGAACAGCTCGTCGAAAGCGGCGGCAGACTGGTGACACCTGGCGGCAGCCTGAC CCTGAGCTGCAAGGCCAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTCCGCCAGGCCC CTGGCAAGGGACTGGAATGGATCGCCACCATCTACCCCAGCAGCGGCAAGACCTACTACGCCAC CTG G GTG AACGG ACG GTTC ACC ATCTCC AG CG AC AACG CCC AG AAC ACCGTG G ACCTGC AG ATG AACAGCCTGACAGCCGCCGACCGGGCCACCTACTTTTGCGCCAGAGACAGCTACGCCGACGACG GCGCCCTGTTCAACATCTGGGGCCCTGGCACCCTGGTGACAATCTCTAGCGGCGGAGGCGGATC TGGTGGCGGAGGAAGTGGCGGCGGAGGATCTGAGCTGGTGCTGACCCAGAGCCCCTCTGTGTC TGCTGCCCTGGGAAGCCCTGCCAAGATCACCTGTACCCTGAGCAGCGCCCACAAGACCGACACCA TCGACTGGTATCAGCAGCTGCAGGGCGAGGCCCCCAGATACCTGATGCAGGTGCAGAGCGACG GCAGCTACACCAAGAGGCCAGGCGTGCCCGACCGGTTCAGCGGATCTAGCTCTGGCGCCGACCG CTACCTGATCATCCCCAGCGTGCAGGCCGATGACGAGGCCGATTACTACTGTGGCGCCGACTACA TCGGCGGCTACGTGTTCGGCGGAGGCACCCAGCTGACCGTGACCGGCGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACA GCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTCCGCAGCAAGCGGAGCAGAGGCGGCCA CAGCGACTACATGAACATGACCCCTAGACGGCCTGGCCCCACCAGAAAGCACTACCAGCCCTACG CCCCTCCCCGGGACTTTGCCGCCTACAGAAGCCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCC TGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTA CGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGA ACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGA TCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCA CCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGCTCGAGGGCGG CGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGAT GCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACG CAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATA TTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGG GTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGG AAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAG AACCTAG AAATCATACG CG G CAG G ACC AAG C AAC ATG GTC AGTTTTCTCTTGC AGTCGTC AGCCT GAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAG GAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAG AAAACC AAAATTATAAG CAAC AG AG GTG AAAAC AGCTG C AAG G CC ACAG G CC AGGTCTG CC ATG CCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGT CAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGT GGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCA CAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAA GACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGG CCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCT GTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTG CTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGTGA

[0480] SEQ ID NO: 25 (CAR of HA-GD2-CAR CD28z, HA-GD2shortCD28z SB)ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATTCCT GAGGTGCAGCTGGTGCAGTCTGGCGCCGAGGTGGAAAAACCTGGCGCCTCCGTGAAGATCAGC TGCAAGGCCAGCGGCAGCAGCTTCACCGGCTACAACATGAACTGGGTGCGCCAGAACATCGGCA AGAGCCTGGAATGGATCGGCGCCATCGACCCCTACTACGGCGGCACCAGCTACAACCAGAAGTT CAAGGGCAGAGCCACCCTGACCGTGGACAAGAGCACCAGCACCGCCTACATGCACCTGAAGTCC CTGCGGAGCGAGGACACCGCCGTGTACTACTGTGTGTCCGGCATGAAATACTGGGGCCAGGGCA CAAGCGTGACCGTGTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGA TCTGATGTCGTGATGACCCAGACCCCCCTGAGCCTGCCTGTGACACCTGGCGAACCTGCCAGCAT CAGCTGTCGGAGCAGCCAGAGCCTGGTGCACAGAAACGGCAACACCTACCTGCACTGGTATCTG CAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCCACAAGGTGTCCAACCGGTTCAGCGGCGTGC CCGACAGATTTTCCGGCAGCGGCTCTGGCACCGACTTCACCCTGAAGATCTCCCGGGTGGAAGCC GAGGACCTGGGCGTGTACTTCTGCAGCCAGTCCACCCACGTGCCCCCCCTGACATTTGGCGCCGG AACAAAGCTGGAACTGAAAGAGAGCAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTCTGG GTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCAT CTTTTGGGTCCGCAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCTAGA CGGCCTGGCCCCACCAGAAAGCACTACCAGCCCTACGCCCCTCCCCGGGACTTTGCCGCCTACAG AAGCCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTG TACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGG GACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTG CAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGG CAAG GG CC ACG ACGG CCTGTATCAG GG CCTGTCC ACCG CC ACCAAG G ATACCTACG ACGCCCTG CACATGCAGGCCCTGCCCCCAAGGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACA TGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTG TG AGTTACC ACACCCAG CATTCCTCCTG ATCCCACGC AAAGTGTGTAACG G AATAG GTATTG GTG AATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCA GTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGG ATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCT TGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCA AGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCC TCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGT GAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGG GCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACA AGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCA CCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATC CAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAG AAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAAC TGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGT CCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCT CTTCATGTGA

[0481] SEQ ID NO: 15 (cJUNonly) ATGACCGCCAAGATGGAGACCACCTTCTACGACGACGCCCTGAACGCCAGCTTCCTGCCCAGCGA GAGCGGCCCCTACGGCTACAGCAACCCCAAGATCCTGAAGCAGAGCATGACCCTGAACCTGGCC GACCCCGTGGGCAGCCTGAAGCCCCACCTGAGGGCCAAGAACAGCGACCTGCTGACCAGCCCCG ACGTGGGCCTGCTGAAGCTGGCCAGCCCCGAGCTGGAGAGGCTGATCATCCAGAGCAGCAACG GCCACATCACCACCACCCCCACCCCCACCCAGTTCCTGTGCCCCAAGAACGTGACCGACGAGCAG GAGGGCTTCGCCGAGGGCTTCGTGAGGGCCCTGGCCGAGCTGCACAGCCAGAACACCCTGCCCA GCGTGACCAGCGCCGCCCAGCCCGTGAACGGCGCCGGCATGGTGGCCCCCGCCGTGGCCAGCGT GGCCGGCGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGCACAGCGAGCCCCCCGTGTACGC CAACCTGAGCAACTTCAACCCCGGCGCCCTGAGCAGCGGCGGCGGCGCCCCCAGCTACGGCGCC GCCGGCCTGGCCTTCCCCGCCCAGCCCCAGCAGCAGCAGCAGCCCCCCCACCACCTGCCCCAGCA GATGCCCGTGCAGCACCCCAGGCTGCAGGCCCTGAAGGAGGAGCCCCAGACCGTGCCCGAGAT GCCCGGCGAGACCCCCCCCCTGAGCCCCATCGACATGGAGAGCCAGGAGAGGATCAAGGCCGA GAGGAAGAGGATGAGGAACAGGATCGCCGCCAGCAAGTGCAGGAAGAGGAAGCTGGAGAGGA TCGCCAGGCTGGAGGAGAAGGTGAAGACCCTGAAGGCCCAGAACAGCGAGCTGGCCAGCACCG CCAACATGCTGAGGGAGCAGGTGGCCCAGCTGAAGCAGAAGGTGATGAACCACGTGAACAGCG GCTGCCAGCTGATGCTGACCCAGCAGCTGCAGACCTTCCTCGAGGGCGGAGGCGAAGGCAGAG GCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCCCAGAATGCTGCTGCTCGTGAC CAGCCTGCTGCTGTGTGAACTGCCTCATCCTGCTTTTCTGCTGATTCCTTGTCACCCCGAGTGCCAGCCTCAGAATGGCAGCGTGACCTGTTTTGGCCCTGAGGCCGATCAGTGTGTGGCCTGCGCTCACT AC AAG G ATCCTCCCTTTTG CGTG G CCCG GTGCCCTTCTG GCGTG AAG CCTG ACCTGAG CTACATG CCCATCTGGAAGTTCCCCGACGAGGAAGGCGCCTGCCAGCCCTGCCCAATCAATTGCACACACAG CTGCGTGGACCTGGACGACAAGGGCTGTCCTGCCGAGCAGAGAGCCTCTCCACTGACCGGCGGA GGAAGTGGCGGCGGATCTATCATCTCTGCCGTCGTGGGCATCCTGCTGGTGGTGGTGCTGGGCG TGGTGTTCGGCATCCTGATCTGA

[0482] SEQ ID NO: 16 (BATF2only) ATGCACCTGTGCGGCGGCAACGGCCTGCTGACCCAGACCGACCCCAAGGAGCAGCAGAGGCAG CTGAAGAAGCAGAAGAACAGGGCCGCCGCCCAGAGGAGCAGGCAGAAGCACACCGACAAGGC CGACGCCCTGCACCAGCAGCACGAGAGCCTGGAGAAGGACAACCTGGCCCTGAGGAAGGAGAT CCAGAGCCTGCAGGCCGAGCTGGCCTGGTGGAGCAGGACCCTGCACGTGCACGAGAGGCTGTG CCCCATGGACTGCGCCAGCTGCAGCGCCCCCGGCCTGCTGGGCTGCTGGGACCAGGCCGAGGGC CTGCTGGGCCCCGGCCCCCAGGGCCAGCACGGCTGCAGGGAGCAGCTGGAGCTGTTCCAGACCC CCGGCAGCTGCTACCCCGCCCAGCCCCTGAGCCCCGGCCCCCAGCCCCACGACAGCCCCAGCCTG CTGCAGTGCCCCCTGCCCAGCCTGAGCCTGGGCCCCGCCGTGGTGGCCGAGCCCCCCGTGCAGC TGAGCCCCAGCCCCCTGCTGTTCGCCAGCCACACCGGCAGCAGCCTGCAGGGCAGCAGCAGCAA GCTGAGCGCCCTGCAGCCCAGCCTGACCGCCCAGACCGCCCCCCCCCAGCCCCTGGAGCTGGAG CACCCCACCAGGGGCAAGCTGGGCAGCAGCCCCGACAACCCCAGCAGCGCCCTGGGCCTGGCCA GGCTGCAGAGCAGGGAGCACAAGCCCGCCCTGAGCGCCGCCACCTGGCAGGGCCTGGTGGTGG ACCCCAGCCCCCACCCCCTGCTGGCCTTCCCCCTGCTGAGCAGCGCCCAGGTGCACTTCCTCGAG GGCGGAGGCGAAGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCAGGCCC CAGAATGCTGCTGCTCGTGACCAGCCTGCTGCTGTGTGAACTGCCTCATCCTGCTTTTCTGCTGAT TCCTTGTCACCCCG AGTG CCAG CCTC AG AATGG CAG CGTG ACCTGTTTTGG CCCTG AG G CCG ATC AGTGTGTGGCCTGCGCTCACTACAAGGATCCTCCCTTTTGCGTGGCCCGGTGCCCTTCTGGCGTG AAGCCTGACCTGAGCTACATGCCCATCTGGAAGTTCCCCGACGAGGAAGGCGCCTGCCAGCCCT GCCCAATCAATTGCACACACAGCTGCGTGGACCTGGACGACAAGGGCTGTCCTGCCGAGCAGAG AGCCTCTCCACTGACCGGCGGAGGAAGTGGCGGCGGATCTATCATCTCTGCCGTCGTGGGCATC CTG CTG GTG GTGGTG CTGG G CGTG GTGTTCG G CATCCTG ATCTG AReference List

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[0504] 26 Spiegel, J. Y. et al. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial.

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[0507] 28 Sun, R. et al. CXCR4-modified CAR-T cells suppresses MDSCs recruitment via STAT3 / NF-KB / SDF-la axis to enhance efficacy against pancreatic cancer.Molecular Therapy 31, 3193-3209 (2023). https: / / doi.org:https: / / doi.org / 10.1016 / j.ymthe.2023.09.010

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Claims

Claims1. A CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell expresses BATF2.

2. The CD8+T cell according to claim 1, wherein the CD8+T cell overexpresses BATF2.

3. The CD8+T cell according to claim 1 or 2, wherein the CD8+T cell has been modified to express BATF2 at a level that is increased compared to the level of BATF2 expressed by the CD8+T cell before it was modified to express BATF2.

4. The CD8+T cell according to any one of claims 1-3, wherein the CD8+T cell comprises an exogenous nucleic acid overexpressing BATF2, or wherein the CD8+T cell comprises an exogenous activator of transcription for an endogenous BATF2 gene.

5. The CD8+T cell according to claim 4, wherein the exogenous nucleic acid overexpressing BATF2 encodes BATF2, optionally wherein the exogenous nucleic acid comprises an expression cassette for BATF2 expression.

6. The CD8+T cell according to claim 5, wherein the exogenous nucleic acid encoding BATF2 is integrated into the genome of the CD8+T cell.

7. The CD8+T cell according to claim 6, wherein the CD8+T cell expressing BATF2 is obtainable through stable gene transfer of the exogenous nucleic acid encoding BATF2.

8. The CD8+T cell according to any one of the preceding claims, wherein the exogenous nucleic acid is a genetic expression vector encoding BATF2.

9. The CD8+T cell according to claim 8, wherein the CD8+T cell expressing BATF2 is obtainable through transient gene transfer of the exogenous nucleic acid encoding BATF2.

10. The CD8+T cell according to any one of the preceding claims, wherein the BATF2 comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 1.

11. The CD8+T cell according to any one of the preceding claims, wherein the CD8+T cell is a mammalian CD8+T cell.

12. The CD8+T cell according to any one of the preceding claims, wherein the CD8+T cell is a human CD8+T cell.

13. The CD8+T cell according to any one of the preceding claims, wherein the modified CD8+T cell has been obtained from an isolated CD8+T cell.

14. The CD8+T cell according to claim 13, wherein the isolated CD8+T cell is a native, naturally occurring CD8+T cell.

15. The CD8+T cell according to any one of the preceding claims, wherein the recombinant antigen-binding receptor binds to a cancer antigen.

16. The CD8+T cell according to any one of the preceding claims, wherein the recombinant antigen-binding receptor binds to an antigen selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3-Integrin, α4β1-Integrin, LILRB4, EpCAM-1,MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D-Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and FLT3, preferably CD19, CD20, and ROR1.

17. The CD8+T cell according to claim 16, wherein the recombinant antigen-binding receptor binds to ROR1.

18. The CD8+T cell according to any one of the preceding claims, wherein the recombinant antigen-binding receptor is a CAR.

19. The CD8+T cell according to claim 18, wherein the CAR comprises, in an N-to C- terminal order, at least one extracellular antigen binding domain that binds to the antigen, a spacer domain, a transmembrane domain and at least one intracellular signaling domain.

20. The CD8+T cell according to claim 19, wherein the extracellular antigen binding domain comprises an scFv that binds to the antigen, preferably wherein the antigen is a cell surface antigen.

21. The CD8+T cell according to claim 20, wherein the cell surface antigen is a cancer antigen and / or an antigen of the tumor microenvironment.

22. The CD8+T cell according to claim 20 or 21, wherein said antigen is selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3-Integrin, α4β1-Integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D-Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2,GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and FLT3, preferably ROR1.

23. The CD8+T cell according to any one of claims 21-22, wherein the antigen is ROR1, and the antigen binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 6.

24. The CD8+T cell according to any one of claims 21-23, wherein the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 10, or an amino acid sequence with at least 90%, such as at least 95%, or at least 97%, or at least 99% sequence identity with SEQ ID NO: 3 or 10.

25. The CD8+T cell according to any one of the preceding claims, wherein the CD8+T cell comprises an exogenous nucleic acid encoding and expressing the recombinant antigen-binding receptor.

26. The CD8+T cell according to claim 25, wherein the CD8+T cell expressing the antigen-binding receptor is obtainable through stable gene transfer of the exogenous nucleic acid encoding and expressing the recombinant antigenbinding receptor.

27. The CD8+T cell according to any one of claims 1-25, wherein the cell expressing the antigen-binding receptor is obtainable through transient gene transfer of the exogenous nucleic acid encoding and expressing the recombinant antigenbinding receptor.

28. The CD8+T cell according to any one of the preceding claims, wherein the antigen-binding receptor and BATF2 are co-expressed from one exogenous nucleic acid.

29. The CD8+T cell according to any one of the preceding claims, wherein the antigen-binding receptor is a CAR fusion protein, comprising in an N- to C- terminal order:a) a CAR, andb) LCK.

30. The CD8+T cell according to claim 29, wherein the LCK is at the C-terminus of the CAR fusion protein.

31. The CD8+T cell according to claims 29 or 30, wherein the LCK is located the intracellular portion of the CAR fusion protein.

32. The CD8+T cell according to any one of claims 29-31, wherein the LCK is a mammalian LCK.

33. The CD8+T cell according to any one of claims 29-32, wherein the LCK is human LCK.

34. The CD8+T cell according to any one of claims 29-33, wherein the LCK comprises or consists of the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence with at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO: 7.

35. The CD8+T cell according to any one of claims 29-34, wherein the CAR fusion protein comprises a CAR comprising, in an N-to C-terminal order, at least one extracellular antigen binding domain that binds to the antigen, a spacer domain, a transmembrane domain and at least one intracellular signaling domain, and the LCK.

36. The CD8+T cell according to any one of claims 29-35, wherein the CAR is a CAR according to any one of claims 15-24.

37. The CD8+T cell according to any one of claims 29-36, wherein the CAR fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence with at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with SEQ ID NO: 8.

38. The CD8+T cell according to any one of the preceding claims, wherein the CD8+T cell exhibits one or more improved pharmacological properties compared to a CD8+T cell that does not overexpress the BATF2.

39. The CD8+T cell according to claim 38, wherein the one or more improved pharmacological properties of the modified CD8+T cell comprisea) enhanced proliferation;b) decreased exhaustion;c) reduced accumulation in non-cancerous lung tissue;d) memory like phenotype;e) reduced activation-induced cell death;f) improved chemokine-dependent migration;g) any combination of a) to e); and / orh) all of a) to e).

40. The CD8+T cell according to claim 39, wherein the enhanced proliferation is enhanced antigen-specific proliferation.

41. A composition comprising:a) a CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell overexpresses BATF2.

42. A kit comprising:a) a CD8+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell overexpresses BATF2.

43. The composition or kit according to any one of claims 42 or 43, preferably further comprising:b) a CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD4+T cell overexpresses a transcription factor, optionally wherein the transcription factor is selected from BATF, BATF3, TFAP4, FoxOl or eJun.

44. The composition or kit according to claim 43, wherein the transcription factor is eJun.

45. The composition or kit according to any one of claims 41-44, wherein the CD8+T cell is a CD8+T according to any one of claims 1-40.

46. The composition or kit according to claim 44 or 45, wherein the CD4+T cell has been modified to express eJun at a level that is increased compared to the level of eJun expressed by the CD4+T cell before it was modified to express eJun.

47. The composition or kit according to any one of claims 44-46, wherein the CD4+T cell comprises an exogenous nucleic acid encoding and overexpressing eJun.

48. The composition or kit according to claim 47, wherein the exogenous nucleic acid encoding and overexpressing eJun is integrated into the genome of the CD4+T cell.

49. The composition or kit according to claim 48, wherein the CD4+T cell is obtainable through stable gene transfer of the exogenous nucleic acid encoding and overexpressing eJun.

50. The composition or kit according to claim 48 or 49, wherein the exogenous nucleic acid is a genetic expression vector encoding eJun.

51. The composition or kit according to claim 50, wherein the CD4+T cell is obtainable through transient gene transfer of the exogenous nucleic acid encoding and overexpressing eJun.

52. The composition or kit according to any one of claims 44-51, wherein the CD4+T cell comprises an exogenous activator of transcription for an endogenous eJun gene and / or activator of translation of an endogenous eJun mRNA.

53. The composition or kit according to any one of claims 44-52, wherein in the CD4+T cell the endogenous eJun gene has been genetically modified to increase eJun expression, wherein optionally the endogenous eJun gene has been genetically modified in its promoter region.

54. The composition or kit according to any one of claims 43-53, wherein the CD4+T cell is a mammalian CD4+T cell.

55. The composition or kit according to any one of claims 43-54, wherein the CD4+T cell is a human CD4+T cell.

56. The composition or kit according to any one of claims 43-55, wherein the CD4+T cell has been obtained from an isolated CD4+T cell.

57. The composition or kit according to claim 56, wherein the isolated CD4+T cell is a native, naturally occurring CD4+T cell.

58. The composition or kit according to any one of claims 43-57, wherein the recombinant antigen-binding receptor of the CD4+T cell binds to a cancer antigen.

59. The composition or kit according to any one of claims 43-58, wherein the recombinant antigen-binding receptor of the CD4+T cell binds to an antigen selected from the group consisting of CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, av|33-lntegrin, a4 1- Integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, Ll-CAM, c-kit, NKG2D, NKG2D- Ligand, PD-L1, PD-L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvlll, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesothelin, PSMA, PSCA, VEGFR, and FLT3, preferably CD19, CD20, Siglec-6, Podoplanin, CEACAM5, SSEA-4, B7-H3, and ROR1.

60. The composition or kit according to claim 59, wherein the recombinant antigenbinding receptor of the CD4+T cell binds to ROR1.

61. The composition or kit according to any one of claims 43-60, wherein the recombinant antigen-binding receptor is a CAR.

62. The composition or kit according to any one of claims 43-61, wherein the CD4+T cell expressing the antigen-binding receptor is obtainable through stable gene transfer of a nucleic acid encoding and expressing the antigen-binding receptor.

63. The composition or kit according to any one of claims 43-62, wherein the CD4+T cell expressing the antigen-binding receptor is obtainable through transient gene transfer of a nucleic acid encoding and expressing the antigen-binding receptor.

64. The composition or kit according to any one of claims 43-63, wherein the antigen-binding receptor of the CD8+T cell and the antigen-binding receptor of the CD4+T cell target the same antigen.

65. The composition or kit according to any one of claims 41-64, wherein the composition or kit is substantially free of CD4+T cells overexpressing BATF2.

66. The composition or kit according to any one of claims 42-65, wherein the composition or kit comprising modified CD8+and CD4+T cells exhibits one or more enhanced properties compared to a composition or kit comprising CD8+and CD4+T cells that were not modified to have increased BATF2 and / or c-Jun expression.

67. The composition or kit according to claim 66, wherein the one or more enhanced properties of the composition and kit comprise:a) enhanced proliferation;b) decreased exhaustion;c) reduced accumulation in non-cancerous lung tissue;d) memory like phenotype;e) reduced activation-induced cell death;f) enhanced engraftment;g) improved chemokine-dependent migration;h) enhanced tumor control; and / ori) any combination of a) to g); and / orj) all of a) to g).

68. A pharmaceutical composition comprising the CD8+T cell according to any one of claims 1-40, or the composition according to any one of claims 41, 43-67 and a pharmaceutically acceptable carrier.

69. The CD8+T cell according to any one of claims 1-40, the composition according to any one of claims 41, 43-67 or the pharmaceutical composition according to claim 68 for use as a medicament.

70. The CD8+T cell according to any one of claims 1-40, the composition according to any one of claims 41, 43-67, or the pharmaceutical composition according to claim 68 for use in the treatment of cancer in a patient having said cancer.

71. Use of the CD8+T cell according to any one of claims 1-40, the composition according to any one of claims 41, 43-67, or the pharmaceutical composition according to claim 68 for the manufacture of a medicament for the treatment of cancer.

72. A method of treating cancer in a patient by administering the CD8+T cell according to any one of claims 1-40, the composition according to any one of claims 41, 43-67, or the pharmaceutical composition according to claim 68.

73. The CD8+T cell, composition, or pharmaceutical composition for use according to claim 70, the use according to claim 71, or the method according to claim 72, wherein the cancer is a hematological cancer or solid cancer, optionally wherein the hematological cancer is leukemia, non-Hodgkin lymphoma (NHL), or multiple myeloma (MM); and / or the solid cancer is breast, ovarian, adrenocortical, lung or thyroid cancer or a solid cancer with lung metastases which is not a lung cancer.

74. The CD8+T cell, composition, or pharmaceutical composition for use according to claim 70 or 73, the use according to claim 71 or 73, or the method according toclaim 72 or 73, wherein the cancer expresses one or more or all of the following cancer antigens:a) CD19;b) BCMA;c) ROR1;d) FLT3;e) CD20;f) CD22;g) CD123;h) ROR2;i) Siglec-6; andj) SLAMF7.

75. The CD8+T cell, composition, or pharmaceutical composition for use according to claim 72, wherein the recombinant antigen-binding receptor of the CD8+T cell binds to one of said cancer antigens expressed by the cancer.

76. The CD8+T cell, composition, or pharmaceutical composition for use according to claim 73, wherein the recombinant antigen-binding receptor of the CD4+T cell binds to one of said cancer antigens expressed by the cancer.

77. The CD8+T cell, composition, or pharmaceutical composition for use according to any one of claims 70, 73-76, the use according to any one of claims 71, 73, or 74, or the method according to any one of claims 72, 73, or 74, wherein the isolated CD8+T cell and / or CD4+T cell are allogeneic or syngeneic cells with respect to said patient.

78. The CD8+T cell, composition, or pharmaceutical composition for use according to any one of claims 70, 73-77, the use according to any one of claims 71, 73, 74, 77, or the method according to any one of claims 72, 73, 74, 77, wherein said treatment with a modified CD8+, or composition and kit comprising of modifiedCD8+and CD4+T cells exhibits one or more improved properties when administered to the patient compared to the treatment using a CD8+and a CD4+T cells that were not modified to have increased BATF2 and / or c-Jun expression, wherein said one or more improved properties comprise:a) enhanced therapeutic efficacy;b) enhanced the anti-tumor efficacy;c) enhanced tumor control of the cancer;d) decreased tumor burden of the cancer in said patient;e) decreased morbidity and mortality;f) decreased CD8+and CD4+T cell exhaustion;g) reduced accumulation in non-cancerous lung tissue;h) decreased CD8+and CD4+T cell activation-induced cell death;i) increasing CD8+and CD4+T cell engraftment and / or persistence;j) decreased on-target / off-tu mor toxicity;k) improved chemokine-dependent migration;l) any combination of a) to j); and / orm) all of a) to j).

79. The CD8+T cell, composition, kit, or pharmaceutical composition for use according to any one of claims 70, 73-78 or the use according to any one of claims 71, 73, 74, 77, 78, wherein the use is also a use for eliminating the risk of pneumonitis in the treatment of the cancer.

80. The method according to any one of claims 72, 73, 74, 77, 78 wherein the method is also a method for eliminating the risk of pneumonitis in the treatment of the cancer.

81. An in vitro method for improving CD8+T cell proliferation, reducing CD8+T cell exhaustion, reducing activation-induced cell death and / or promoting a memorylike phenotype comprising modifying the CD8+T cell to overexpress BATF2.

82. The in vitro method according to claim 81, wherein the overexpression of BATF2: a) increases the proliferation of the modified CD8+T cell compared to proliferation of a CD8+T cell before it was modified to express the BATF2; b) decreases the exhaustion of the modified CD8+T cell compared to exhaustion of a CD8+T cell before it was modified to express the BATF2; c) decreases the activation-induced cell death of the modified CD8+T cell compared to activation-induced cell death of a CD8+T cell before it was modified to express the BATF2; and / ord) increases the memory-like phenotype of the modified CD8+T cell compared to memory-like phenotype of a CD8+T cell before it was modified to express the BATF2.

83. The method according to claims 81 or 82, wherein the modified CD8+T cell is a CD8+T cell according to claims 1-40.

84. The method according to any one of claims 81-83, wherein the modified CD8+T cell is co-cultured with a CD4+T cell.

85. The method according to claim 84, wherein the CD4+T cell is a CD4+T cell comprising a recombinant antigen-binding receptor, wherein the antigen-binding receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and wherein the CD8+T cell overexpresses a transcription factor.

86. The method according to claim 85, wherein the transcription factor is eJun.

87. The method according to any one of claims 84-86, wherein the CD4+T cell is a CD4+T cell according to any one of claims 48-66.

88. A nucleic acid or a set of nucleic acids encoding the BATF2 as defined in any one of claims 1-40 and / or the CARs as defined in any one of claims 15-37.

89. The nucleic acid or a set of nucleic acids according to claim 88, wherein the BATF2 and the CAR are encoded on the same nucleic acid.

90. An expression vector comprising the nucleic acid of claim 89, or a set of expression vectors comprising the set of nucleic acids of claim 89.

91. The expression vector of claim 90, wherein the expression vector is an expression vector for genomic integration into the host cell.

92. Method for producing a CD8+T cell that exhibits an increased BATF2 level, wherein the method comprises introducing into the CD8+T cell an exogenous nucleic acid overexpressing BATF2 in the CD8+T cell.

93. The method for producing the CD8+T cell according to claim 90, the method comprising the steps of:a) providing a CD8+T cell;b) introducing into said CD8+T cell the nucleic acid or set of nucleic acids according to claim 88 or 89, or the expression vector or the set of expression vectors according to claim 90 or 91; andc) co-expressing said nucleic acid or said set of nucleic acids according to claim 88 or 89, or said expression vector or said set of expression vectors according to claim 90 or 91;d) thereby obtaining said CD8+T cell.

94. The method or use according to any one of the preceding claims, wherein all the steps of the method or use are carried out in vitro.

95. The method or use according to any one of the preceding claims, wherein said method or use does not comprise a method for treatment of the human or animal body by surgery or therapy or a diagnostic method practiced on the human or animal body.

96. The method or use according to any one of the preceding claims, wherein said method or use does not comprise a process for modifying the germ line or the genetic identity of an animal or human being and does not comprise the use of human embryos.

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