Antigen - induced secretion of effector molecules of car t cells

Abstract

The present invention provides a combination of compositions comprising: A composition (A) comprising a population of immune effector cells comprising: A first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a tag of a tagged polypeptide and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen activated inducible promoter; and A composition (B) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said antigen-activated inducible promoter drives the expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell.

Description

Title

[0001] Antigen - induced secretion of effector molecules of CAR T cellsField of the invention

[0002] The invention relates to the field of chimeric antigen receptors (CARs) especially adapter CARs (anti tag CARs) expressed on immune cells. In addition to that, immune cells are modified to express an effector molecule dependent on CAR activity. Such an immune cell may be used for immunotherapy especially in the treatment of cancer.Background of the invention

[0003] CAR-T cell therapy is known to be a good option for the treatment of cancer, more specifically cancer of hematopoietic origin. CAR T cell therapy is based on the genetic modification of immune effector cells to express a tumor specific chimeric antigen receptor that induces T cell activation, proliferation and cytolytic activity within the patient when the tumor antigen has been recognized. However, the use of CAR immune therapy for the treatment of solid tumors had limited success. Major limitations are among others immune cell intrinsic features such as immune cell exhaustion, immunosuppressive tumor microenvironment, active cancer immunosuppressive mechanisms and insufficient CART cell activation and persistence. Moreover the lack of suited surface antigens as well as tumor evasion based on downregulation of surface antigens are one of the major challenges in immunotherapy.

[0004] For overcoming the challenge of tumor evasion based on downregulation of cell surface antigens, a flexible CAR system (known as adapter CAR or anti tag CAR system) was developed (WO2012082841A2). This is based on a tagged polypeptide, which binds to a target cell and an anti-Tag CAR which binds to the tagged polypeptide. Killing activity of the immune effector cell is triggered by binding the tagged polypeptide that is bound to the target antigen expressed on said target cell. This represents a flexible system, as the tagged polypeptides can be easily exchanged.

[0005] To overcome limitations based on immune cell intrinsic features, additional factors such as cytokines or chemokines have been administered to the patient. For example systemic administration of interleukins, such as interleukin 12 (11-12), has demonstrated anti-tumor activity in murine studies, leading to tumor regression and prolonged survival. However, the use of IL12 has also been associated with severe systemic toxicities. This is mainly due to the uncontrolled distribution of the cytokines through the whole body of the patient, thereby leading to severe side effects (1).

[0006] Therefore controlled release of effector molecules such as cytokines to the tumor site is desired. The TRUCK concept was developed to overcome the limitations (TRUCK CARs("T cell redirected for universal cytokine-mediated killing")). This concept is based on the expression of a chimeric antigen receptor directed against a tumor antigen by an immune effector cell. In addition to that, this immune effector cell comprises an expression cassette, whose expression is induced upon CAR activation by binding to a tumor antigen. Upon CAR engagement with target cells, this modified immune effector cell releases a transgenic effector molecule e g. cytokines (such as IL-18) in a restricted manner and only to the tumor site. The TRUCK strategy aims to enhance T cell activation and shape the tumor microenvironment (TME) by attracting innate immune cells, thereby facilitating a more effective attack against tumor cells.

[0007] However, the concentration of effector molecule release such as cytokine release of the TRUCK system is not controllable, as there will always be full expression of the effector molecule upon CAR binding by the immune effector cell. This may lead to undesired toxicities due to high concentrations of the effector molecule as observed for NF AT -induced IL-12 secretion in patients (1).

[0008] In addition to that, the secretion of the effector molecule cannot be induced in case of antigen loss of the tumor cells, a phenomenon that is frequently observed in tumor cells.

[0009] For this reason, there is a need in the art for a CAR concept that is flexible and enables controlled site-directed release of effector molecules such as cytokines for effective tumor treatment.Brief description of the invention

[0010] Our researchers combined the TRUCK system with the adapter CAR system to overcome the key limitations described in the prior art. It was shown that not only killing but also effector molecule production can be controlled by the concentration of the tagged polypeptide. Surprisingly, it was shown that effective killing and effector molecule production can be controlled independently from each other. Moreover the effector molecule concentration can be modulated. This enables controlled ON / OFF switching of the effector molecule production, while maintaining full effector functions of the immune effector cell.

[0011] The first aspect of the invention provides a combination of compositions comprising a. A composition (A) comprising a population of immune effector cells comprisingi. A first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a tag of a tagged polypeptide ii. A second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen activated inducible promoter and b. A composition (B) comprising a plurality of said tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell.

[0012] In addition to that, one embodiment of the invention provides a combination of compositions as defined by the first aspect of the invention. This combination of composition additionally comprises a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein the amount of tagged polypeptides in composition C is lower compared to composition B.

[0013] The second aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering to a subj ect in need thereof : a. A composition (A) comprising a population of immune effector cells comprising i. A first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a tag of a tagged polypeptide ii. A second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter and b. A composition (B) comprising an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag c. A composition (C) comprising an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag wherein said antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR tosaid tagged polypeptide that is bound to said target antigen expressed on said target cell, wherein the amount of tagged polypeptides in composition C is lower compared to composition B.Brief description of the drawings

[0014] Figure 1 : Schematic representation of an AdCAR T cell binding a tumor cell via a tagged polypeptide and secreting an effector molecule upon tumor cell binding. One or multiple polypeptides e.g., antibodies or fragments thereof (adapter) bind the target antigen of choice expressed by a cancer cell. In the next step, the antigen-adapter-complex binds a T cell that expresses a receptor specific to this adapter molecule (AdCAR) and secretes an effector molecule e g., a cytokine driven by a T cell inducible promotor. In summary, the AdCAR T cells will induce lysis of the tumor cell and secrete the effector molecule in the presence of the adapter. Both, the tumor cell lysis and the effector molecule secretion can be controlled independently of each other via the adapter.

[0015] Figure 2: CD276-specific Fab molecules tagged with LC biotin bind antigen-positive tumor cells and induced AdCAR T-cell-mediate tumor lysis and IL-18 secretion, (a) Tumor cell lysis was quantified by quantifying the impedance of attached tumor cells using the xCELLigence RTCA system. The cell index was normalized to the starting values. Each dot represents the mean obtained from n=3 healthy donors. A comparable level of tumor cell lysis was induced between 0,5 nM to 20 nM of adapter. At lower adapter concentrations the tumor lysis was decreased, (b) The supernatant of AdCAR T cells co-cultured with tumor cells and adapter was harvested after 48h. The amount of IL- 18 was quantified via ELISA. The dotted line indicates the limit of detection. The secretion of IL-18 showed a decrease between 20 nM to 0,5 nM of adapter. Below 0,5 nM no cytokine secretion above the detection limit of ELISA was found. Data shown were obtained from n=3 healthy donors and plotted with mean ± SD. (c) Normalized values of AdCAR-mediated killing and IL-18 secretion. AdCAR T cell- mediated killing and IL- 18 secretion were normalized to the control condition containing no adapter after 48h of co-culture. In contrast to the tumor cell lysis the secretion of IL-18 showed an decrease between 20nM to 0,5nM of adapter. Less than 0,5nM of adapter did not induce IL- 18 secretion. However, killing of tumor cells was induced between 20nM to 0,01nM. This demonstrates the independent regulation of tumor killing and IL-18 secretion using the AdCAR T cell system

[0016] Figure 3 : AdCAR T cells secreting IL- 18 show control of tumor growth in a lung tumor xenograft model in vivo. Female immunodeficient NOD . Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) were inoculated with 0.5xl06CD276 positive SHP-77 i.v. and 3 days later the mice were injected with IxlO7AdCAR T cells with or without CD276-specific adapter, or AdCAR T cells with inducible IL-18. A total amount of 100 pg of CD276-specific adapter was injected i.p. every day. The tumor growth was monitored via bioluminescent imaging. AdCAR T cells secreting IL-18 showed tumor control comparable to AdCAR T cells not secreting IL-18. This indicates the functionality of AdCAR T cells secreting IL- 18 in an adapter-dependent manner in vivo. At least three mice were analyzed per cohort and median values of photon flux are plotted ±SEM.

[0017] Figure 4: AdCAR T cells secreting IL-18 show enhanced expansion of CAR T cells in a lung tumor xenograft model in vivo. Female immunodeficient NOD. Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) were inoculated with 0.5xl06CD276 positive SHP-77 i.v. and 3 days later the mice were inj ected with IxlO7AdCAR T cells with or without CD276-specific adapter, or AdCAR T cells with inducible IL-18. A total amount of lOOpg of CD276-specific adapter was injected i.p. every day. The frequency of human CD45 positive cells (a) and CAR- expressing cells (b) was analyzed via flow cytometry at indicated time points. AdCAR T cells with inducible IL-18 secretion show enhanced expansion compared to control conditions. This indicates adapter-dependent secretion of the effector molecule IL-18 also in vivo. At least three mice were analyzed per cohort and median values of photon flux are plotted ±SEM.

[0018] Figure 5: Biotin-tagged CLEC12a- CD33- or CD123 specific adapters bind antigenpositive OCLAML2 tumor cells and induce AdCAR T-cell-mediated tumor lysis and IL-18 secretion, (a) Tumor cell lysis was quantified by quantifying the GFP signal of target cells using the Incucyte system. The cell index was normalized to the starting values. Each dot represents the mean obtained from n=3 healthy donors. A comparable level of tumor cell lysis was induced between 0.1 nM to 1 nM of adapter. Tumor lysis was decreased at lower adapter concentrations, (b) The supernatant of AdCAR T cells co-cultured with tumor cells and adapter was harvested after 42 h. The amount of IL-18 was quantified via ELISA. The dotted line indicates the limit of quantification. The secretion of IL-18 was adapter dose dependent between 0.01 nM or 0.02 nM to 1 nM of adapter. Without adapter, no cytokine secretion was observed. Data shown were obtained from n=3 healthy donors and plotted with mean ± SD. (c) Normalized values of AdCAR-mediated killing and IL-18 secretion. AdCAR T cell-mediated killing and IL-18 secretion were normalized to the control condition containing no adapter after 42 h of co-culture. In contrast to the tumor cell lysis, IL-18 secretion showed stronger adapter concentrationdependency at higher adapter concentrations implicating that both, the tumor cell lysis and the effector molecule secretion can be controlled independently of each other via the adapter concentration.

[0019] Figure 6: Biotin-tagged CLEC12a- or CD33-specific adapters bind antigen-positive THP-1 tumor cells and induce AdCAR T-cell-mediated tumor lysis and IL-18 secretion, (a) Tumor cell lysis was quantified by quantifying the GFP signal of target cells using the Incucyte system. The cell index was normalized to the starting values. Each dot represents the mean obtained from n=3 healthy donors. A comparable level of tumor cell lysis was induced between 0.1 nM to 1 nM of adapter. At lower adapter concentrations the tumor lysis was decreased, (b) The supernatant of AdCAR T cells co-cultured with tumor cells and adapter was harvested after 42 h. The amount of IL-18 was quantified via ELISA. The dotted line indicates the limit of quantification. The secretion of IL- 18 shows an adapter concentration dependent pattern, but was generally rather low and below the limit of quantification for most adapter concentrations. Data shown were obtained from n=3 healthy donors and plotted with mean ± SD.

[0020] Figure 7: Biotin-tagged CD19- or CD20-specific adapters bind antigen-positive Raji tumor cells and induce AdCAR T-cell-mediated tumor lysis and IL-18 secretion, (a) Tumor cell lysis was calculated based on flow cytometric analysis of GFP positive tumor cells after 42 h of co-culture and normalized to the control condition containing no adapter. Maximum tumor cell lysis was dependent on the adapter specificity and was reached at 1 nM for CD19 adapter and between 0.2 nM and 1 nM for CD20 adapter. Tumor lysis was decreased at lower adapter concentrations for both adapter specificities. Data shown were obtained from n=3 healthy donors and plotted with mean ± SD. (b) The supernatant of AdCAR T cells co-cultured with tumor cells and adapter was harvested after 42 h. The amount of IL-18 was quantified via ELISA. The dotted line indicates the limit of quantification. High secretion of IL- 18 was induced in co-culture with 1 nM CD19 adapter. For all other conditions, secretion of IL-18 showed an adapter concentration dependent pattern, but was below the limit of quantification for most adapter concentrations. The data demonstrates that both, tumor lysis and IL-18 secretion can be induced in an adapter dose dependent fashion. However, higher adapter concentrations are needed to induce efficient IL-18 secretion whereas tumor killing is induced already at lower adapter concentrations showing that independent regulation of both is possible by modulating the adapter concentration. Data shown were obtained from n=3 healthy donors and plotted with mean ± SD.Detailed description of the invention

[0021] The combination of compositions as disclosed herein comprises three key elements (Figure 1) a. Usage of tagged polypeptides / adapter CAR system b. Killing of a target cell by CAR T cell activation a. Controllable production of an effector molecule which is triggered by CAR T cell activation

[0022] The use of the adapter CAR system (anti tag CAR system) enables high flexibility in tumor antigen targeting. Different tagged-polypeptides or tagged-polypeptide combinations can be used for disease treatment, dependent on tumor antigen expression or disease stage for optimal tumor treatment.

[0023] The second key element of the invention is the immune effector cell expressing an anti- tag-CAR which is capable of binding a tagged polypeptide that is bound to a target molecule on the cell. This binding triggers the activation of a killing mechanism of the immune effector cell, thereby killing the target cell. Tumor cell clearance is dependent on the amount of the tagged molecule which can activate the CAR T cell, until a saturation is reached. At this point the increase in concentration of the tagged molecule has no effect on killing as the maximum is reached.

[0024] The third key element of the invention is the production of the effector molecule which is triggered by CAR T cell activation, more specifically CAR T cell activation induces the antigen activated promotor which in turn leads to the expression of the effector molecule. It was found that the expression of the effector molecule is also dependent on the concentration of the tagged polypeptides.

[0025] It was surprisingly found that even though both mechanisms are dependent on the concentration of the tagged polypeptides bound to the target antigen their dependency is different. While CAR T cell killing is triggered by a specific adapter concentration, higher concentrations are needed to trigger the expression of the effector molecule (Figure 2 a-c). For example, if the maximum of tumor cell lysis is reached at a specific concentration of adapter an increase of the adapter molecule does not affect the killing anymore (Figure 2a). However, there is still an adapter-dependent secretion of the effector molecule within the same range of adapter concentration (Figure 2b). Even more surprisingly it was shown that the killing activity of the CAR T cell can be triggered without having significant effector molecule expression (Figure 2c). In addition to that, increasing the concentration of the tagged polypeptides efficiently triggered CAR - T cell mediated killing of target cells and effector moleculeproduction. Major advantage is that the effector molecule production can be switched on and off without losing the killing activity of the CAR T cell (Figure 2c).

[0026] The described system shows unexpected possibilities and new ways of controlling CAR-T cell therapy and preventing / reducing site effects, while having full flexibility in antigen targeting. This approach makes CAR T cell controllable for effector molecule release, CAR T cell function and molecule targeting, thereby overcoming the current problems in the art. It enables to react on tumor evasion, to increase effector functions by the expression of specific molecules, with the possibility to reduce site effects by shutting off the effector molecule expression. It further provides a simple and cost effective system compared to direct CAR approach.

[0027] The first aspect of the invention provides a combination of compositions comprising a) a composition (A) comprising a population of immune effector cells comprising a. A first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a tag of a tagged polypeptide b. A second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter and b) A composition (B) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell.Composition A

[0028] The first composition (composition A) comprises a population of immune effector cells. This population of immune effector cells may be immune cells or immune cell subsets, preferentially T cells, tumor infiltrating lymphocytes (TILs) or NK cells. In a preferred embodiment said immune cells are T cells.

[0029] These immune effector cells are genetically modified to comprise a first and second exogenous nucleic acid sequence. Such modification may be done by methods known in the art. In a one embodiment of the invention, the first and second exogenous nucleic acid has been introduced into the immune effector cells by transfection, electroporation or transduction, preferentially viral transduction, more preferentially lentiviral transduction.

[0030] The first and second exogenous nucleic acid sequence may be in one single or two separate expression cassettes. In case separate expression cassettes are used, the first and second expression cassettes may be comprised in two different vectors. Moreover the nucleic acids may have been introduced in a one or two step transduction procedure.

[0031] In a preferred embodiment of the invention, the first and second nucleic acid sequence are comprised in one expression cassette, wherein said expression cassette is comprised in a single vector, which is introduced into the immune effector cell by lentiviral transduction. In this embodiment the second exogenous nucleic acid sequence may be 5 ' of the first nucleic acid sequence.

[0032] The first exogenous nucleic acid sequence codes for a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for the tag of the tagged polypeptide.

[0033] This first exogenous nucleic acid sequence may be operably linked to a promotor. This promotor may be constitutively active in order to drive permanent CAR expression in the immune effector cell. In one embodiment of the invention, said constitutively active promotor may be selected form the group of CMV (Cytomegalovirus promoter), EF-1 (Elongation factor- 1 promoter), MSCV (murine stem cell virus), hPGK (Human phosphoglycerate kinase promoter), RPBSA (Rous sarcoma virus promoter), SFFV (Spleen Focus Forming Virus promoter), SV40 (Simian Virus 40 promoter), MND (Myeloproliferative sarcoma virus promoter), tPGK (Thrombopoietin gene promoter), UBC (Ubiquitin C promoter), CAG (CMV early enhancer / chicken beta actin promoter) or fragments thereof. In a preferred embodiment said constitutively active promotor may be PGK. In a more preferred embodiment of the invention the promotor has SEQ ID No: 5. In another embodiment said constitutively active promotor may be EF-lalpha. In a more preferred embodiment of the invention the promotor has SEQ ID No: 4.

[0034] The second exogenous nucleic acid sequence encoding an effector molecule is operably linked to an antigen-activated inducible promoter. Such an antigen-activated inducible promoter drives the expression of an effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The expression of the effector molecule is therefore regulated by the activation of the CAR. It was found that the level of expression is dependent on the tagged polypeptides bound to the CAR and to the target antigen. Surprisingly the dependency is different from the dependency of killing.

[0035] The antigen-activated inducible promotor may be selected from the group consisting of: an SP1, BATF, AP-1, IRF4, RUNX, NF AT, NF-KB, STAT5 or STAT3-sensing promotor. In apreferred embodiment said promoters may be a NF AT promotor or a functional fragment thereof. In a more preferred embodiment of the invention the promotor has SEQ ID No: 1.

[0036] The CAR according to the current invention comprises an antigen binding domain specific for the tag of the tagged polypeptide.

[0037] The antigen binding domain may comprise e.g. single domain antibody, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies, each of which may be specific for said tag of the tagged polypeptide. In a preferred embodiment of the invention said antigen binding domain may be an scFv.

[0038] Said CAR comprises an antigen binding domain specific for the tag of the tagged polypeptide, a spacer, a transmembrane domain and an intracellular signaling domain.

[0039] In one embodiment the CAR may comprise a hinge domain as spacer, wherein the hinge domain may comprise e.g. a sequence of the hinge of CD8alpha or IgG4. In a preferred embodiment the CAR may comprise the hinge domain of IgG4 (SEQ ID No: 12; SEQ ID No: 13).

[0040] Moreover, the CAR comprises a transmembrane domain comprising a sequence of the transmembrane domains from CD8alpha CD28, ICOS or TNFRSF19 preferentially the transmembrane domain is CD8 (SEQ ID No: 14; SEQ ID No: 15).

[0041] The intracellular (cytoplasmic) signaling domain comprised in the CAR may comprise at least one primary cytoplasmic signaling domain comprising an immunoreceptor tyrosinebased activation motif (IT AM) and / or at least one co-stimulatory signaling domain.

[0042] The primary cytoplasmic signaling domain of said CAR may be CD3zeta (SEQ ID No:20; SEQ ID No:21)

[0043] Moreover the at least one co-stimulatory domain of said CAR, may be selected from the group consisting of ICOS, CD154, CD5, CD2, CD46, HVEM, CD8, CD97, TNFRSF18, CD30, SLAM, DAP10, CD64, CD16, CD89, MyD88, KIR-2DS, KIR-3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, CD27, 0X40, 4-1BB, and CD28. In a preferred embodiment the at least one costimulatory domain comprised in said CAR may be CD28 (SEQ ID No: 16; SEQ ID No: 17) and / or 4-1BB (SEQ ID No: 18: SEQ ID No: 19).

[0044] In one embodiment of the invention the CAR may comprise or consist of an antigen binding domain specific for the tag of the tagged polypeptide, the hinge domain of IgG4 (SEQ ID No:12; SEQ ID No: 13), a transmembrane domain of CD8 (SEQ ID No: 14: SEQ ID No: 15), a co-stimulatory of CD28 (SEQ ID No: 16; SEQ ID No: 17) and 4-1BB (SEQ ID No: 18; SEQID No: 19) and a primary cytoplasmic signaling domain of CD3zeta (SEQ ID No:20: SEQ ID No:21).

[0045] In one embodiment of the invention the tag of the tagged protein may be or may comprise biotin or a derivate thereof. Based on that, the antigen binding domain of the CAR (expressed by the immune effector cell) comprised in composition A may be specific for said biotin or a derivate thereof, preferentially LC biotin or a derivative thereof. Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially LC biotin or a derivative thereof) and comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No:6 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No:8. The CAR may additionally comprise or consist of the hinge domain of IgG4 (SEQ ID No: 12; SEQ ID No: 13), a transmembrane domain of CD8 (SEQ ID No: 14; SEQ ID No: 15), a co-stimulatory of CD28 (SEQ ID No: 16; SEQ ID No:17) and 4-1BB (SEQ ID No:18; SEQ ID No: 19) a primary cytoplasmic signaling domain of CD3zeta (SEQ ID No:20; SEQ ID No:21).

[0046] Said CAR may comprise or consist of an antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain is specific for biotin or a derivate thereof (especially for LC biotin or a derivative thereof) and may comprise the amino acid sequence SEQ ID No: 10. The CAR may additionally comprise or consist of the hinge domain of IgG4(SEQ ID No: 12; SEQ ID No: 13), a transmembrane domain of CD8 (SEQ ID No: 14; SEQ ID No: 15), a co-stimulatory of 4-1BB (SEQ ID No: 18; SEQ ID No: 19) and CD28 (SEQ ID No: 16; SEQ ID No: 17), a primary cytoplasmic signaling domain of CD3zeta (SEQ ID No:20; SEQ ID No:21). In one embodiment the CAR binding biotin or a derivative thereof (especially LC biotin or a derivative thereof) may comprise or consist of the amino acid sequence SEQ ID No:22.Composition B and C

[0047] According the invention the combination of compositions comprises composition B. In a specific embodiment the combination of compositions also comprises composition C. Composition B and C comprise a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag.

[0048] The antigen binding domain of said tagged polypeptides may be, may comprise or may consist of e.g. single domain antibody, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies, diabodies or other alternative antigen binding fragments thereof that bind to a target antigen of expressed on the surface of the target cell.

[0049] The tagged polypeptides (each of the tagged polypeptides) comprise a tag. Said tag may be a hapten tag e.g. FITC, biotin, PE, streptavidin or dextran. In another embodiment the tag may be a peptide tag such as c-Myc-tag, Strep-Tag, Flag-Tag, and Polyhistidine-tag. In a preferred embodiment the tag may be biotin or a derivate thereof. In another preferred embodiment, the tag may be LC biotin or a derivative thereof. LC biotin is a biotin derivate which additionally comprises a C6 chain for example as disclosed in EP23192028.1 and EP23192030.7.

[0050] As mentioned above, binding of the antigen binding domain of said CAR to the tagged polypeptide that is bound to said target antigen expressed on said target cell leads to effective killing of the target cell Depending on the concentration of the tagged polypeptide that is bound to said target antigen expressed on said target cell level of activation of the CAR can be modulated. The activation threshold that needs to be achieved for effective killing is lower than the activation threshold that is needed for expressing the effector molecule. In other words, the level of expression of the effector molecule by the immune effector cell is dependent on the concentration of the tagged polypeptide bound to the CAR and the target antigen.

[0051] In one embodiment of the invention, the combination of compositions may additionally comprise a third composition (C), comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention, the amount of tagged polypeptides in composition C is lower compared to composition B or vice versa. As a consequence there is decreased activation of the inducible promotor and decreased expression of the effector molecule, while effective killing is maintained. In a preferred embodiment of the invention, the lower amount of the tagged polypeptides leads to effective killing of the target cell without the expression of the effector molecule.

[0052] The amount of said tagged polypeptides in composition C e.g. is capable to induce the expression of the effector molecule in the immune effector cell at lower levels compared to composition B. In a preferred embodiment, the amount of said tagged polypeptides in composition C is not capable to induce the expression of the effector molecule in the immuneeffector cell. Importantly (in both scenarios) killing and elimination of the target cells is maintained.

[0053] In other words the combination of compositions as disclosed herein, additionally comprising a third composition (C), comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein the amount of tagged polypeptides in composition C is lower compared to composition B, wherein the amount of tagged polypeptides of composition B is sufficient to induce the expression of the effector molecule in the immune effector cells and to lead to saturation of activation of said immune effector cells resulting in (maximum of) tumor cell lysis, and wherein the amount of tagged polypeptides of composition C is lower compared to composition B but is still sufficient to lead to activation of said immune effector cells resulting in tumor cell lysis, but is too low to induce the expression of the effector molecule in the immune effector cell.

[0054] According to the present invention the antigen binding domain comprised in the tagged polypeptides (in composition B and C) bind to a target antigen which is expressed on a target cell, more specifically on the surface of a target cell. Such a target cell expressing the target antigen may be a cancer cell, an immune cell, a cell associated with an autoimmune disease, a cell associated with an allergic disease, a cell associated with an infectious disease (e g. and infected cell) or a cell associated with graft rejection in a subject. In one embodiment of the invention, the target antigen is expressed on the surface of a target cell. In a preferred embodiment of the invention, the target antigen is expressed on the surface of a target cell such as a cancer cell comprised in solid cancer.

[0055] The target antigen which is expressed on the cell surface of the target cells may be a tumor associated antigen (TAA). Based on that the target antigen (e.g. TAA) may be selected from the group of: B7H3, ROR1, CD56, GD2, FOLR1, CD318, CLDN3, CLEC12A CD33 CD123, CD19 CD20, Her2, BCMA, FAP, EGFRvIII, IL13Ra2, mesothelin, PSMA, MUC1, CLDN18.2, CEA, EpCAM, GPC3 and CD22. In a preferred embodiment the target antigen is B7H3.

[0056] According to the current invention compositions B and C comprise a plurality of tagged polypeptides whose antigen binding domain is specific for a target antigen expressed on the surface of the target cell.

[0057] For high flexibility and to overcome problems of limited availability of cell surface antigens and tumor evasion, based on downregulation of surface antigens, each composition may comprise tagged polypeptides specific for same or different target antigens. The target antigen of composition B and C may be same (identical) or different. In other words in one embodiment the invention provides a combination of compositions as disclosed herein, wherein said tagged polypeptides of composition B and composition C are identical (the same), or wherein said tagged polypeptides of composition B are different from said tagged polypeptides of composition C.

[0058] In one embodiment of the invention the tagged polypeptides comprising an antigen binding domain specific for a target antigen are comprised in the plurality of the tagged polypeptides are same in compositions B and C. Such antigens may be selected from the group of: B7H3, R0R1, CD56, GD2, FOLR1, CD318, CLDN3, CLEC12A CD33 CD123, CD19 CD20, Her2, BCMA, FAP, EGFRvIII, IL13Ra2, mesothelin, PSMA, MUC1, CLDN18.2, CEA, EpCAM, GPC3 and CD22. In a preferred embodiment of the invention, the tagged polypeptides comprising an antigen binding domain specific for a target antigen comprised in the plurality of the tagged polypeptides in compositions B and C is specific for B7H3.

[0059] In one embodiment of the invention, the tagged polypeptides comprising an antigen binding domain specific for a target antigen are comprised in the plurality of the tagged polypeptides are different in compositions B and C. Such antigens may be selected from the group of : B7H3, ROR1, CD56, GD2, FOLR1, CD318, CLDN3, CLEC12A CD33 CD123, CD19 CD20, Her2, BCMA, FAP, EGFRvIII, IL13Ra2, mesothelin, PSMA, MUC1, CLDN18.2, CEA, EpCAM, GPC3 and CD22. In a preferred embodiment of the invention the tagged polypeptides comprising an antigen binding domain specific for a target antigen comprised in the plurality of the tagged polypeptides in composition B is specific for CD276 (B7H3) and antigen binding domains comprised in the plurality of the tagged polypeptides in composition C is specific for GD2, FolRl, ROR1 or CD56 or vice versa.

[0060] In another embodiment of the invention, composition B and / or C may comprise tagged polypeptides comprising an antigen binding domain specific for different target antigens expressed on the surface of the target cell. In other words composition B and / or composition C may be a mixture of tagged polypeptides comprising antigen binding domains specific for different antigens. For example such a composition may comprise a mixture of tagged polypeptides comprising antigen binding domains specific for at least 2, at least 3 or at least 4 different antigens. Such antigens may be selected from the group of : B7H3, ROR1, CD56, GD2, FOLR1, CD318, CLDN3, CLEC12A CD33 CD123, CD19 CD20, Her2, BCMA, FAP,EGFRvIII, IL13Ra2, mesothelin, PSMA, MUC1, CLDN18.2, CEA, EpCAM, GPC3 and CD22. In a preferred embodiment a mixture of tagged polypeptides comprising antigen binding domains specific for at least 2 different antigens expressed on the surface of said target cells, wherein said antigens are GD2, FolRl, ROR1 or CD56 .Effector molecule

[0061] According the current invention an effector molecule is expressed upon activation of the CAR. This effector molecule may be released in a restricted manner and only to the tumor site. This aims to enhance T cell activation and shape the tumor microenvironment (TME) by attracting innate immune cells, thereby facilitating a more effective attack against tumor cells.

[0062] Said effector molecule may be an antibody or antigen binding fragment thereof, a therapeutic peptide or protein, an immune modulatory protein (such as a cytokine, a chemokine), a receptor, a transcription factor, a siRNA, or shRNA. Said effector molecule may be secreted by the immune effector cell.

[0063] In one embodiment of the invention, the effector molecule may be an immune modulatory protein, preferentially a cytokine. The cytokine may be secreted by the immune effector cell. The cytokine may allow local and dose-controlled shaping of the microenvironment that activated the CAR of the immune cell. The cytokine may be a Pro- inflammatory Cytokine such as - Interleukin-1 (IL-1), Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-a); an anti-inflammatory Cytokine such as Interleukin- 10 (IL-10), Transforming Growth Factor-beta (TGF-[3 ); a T Helper Cell Differentiation Cytokine such as IL21; Interleukin-2 (IL-2), Interferon -gamma (IFN-y), Interleukin-4 (IL-4), Interleukin-5 (IL- 5), Interleukin- 13 (IL-13); Chemotactic Cytokines (Chemokines) such as CCL2 (MCP-1), CXCL8 (IL-8)); Growth Factors such as Epidermal Growth Factor (EGF), Platelet-derived Growth Factor (PDGF), IL-7, IL.15; Regulatory Cytokines such as (Interleukin-12 (IL-12), Interleukin- 18 (IL- 18)).

[0064] In a preferred embodiment the effector molecule may be a regulatory cytokine, preferentially IL-18. In one embodiment of the invention the regulatory cytokine is IL-18 and has SEQ ID No: 2; SEQ ID No:3.Specific embodiments - aspect 1

[0065] In one embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of T cells immune effector cells. These immune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigenreceptor (CAR) comprising an antigen binding domain specific for the tag of the tagged polypeptide and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen activated inducible promoter. In addition to that the combination of composition comprises a composition (B) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. The antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said effector molecule is secreted by the T cell. Optionally, the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) and wherein the amount of said tagged polypeptides in composition C is lower compared to composition B, hereby altering the level of expression of the effector molecule.

[0066] In one embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells immune effector cells. These immune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a tag of the tagged polypeptide and a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL- 18) operably linked to an antigen activated inducible promoter. In addition to that the combination of composition comprises composition (B). This composition (B) comprises a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. The antigen-activated inducible promoter drives expression of said immunomodulatory molecule such as a cytokine upon binding of said antigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said immunomodulatory molecule such as a cytokine (e.g. IL-18) is secreted by the population of immune effector cells, preferentially T cells. (Optionally) the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) and wherein the amount of taggedpolypeptides in composition C is lower compared to composition B, hereby altering the level of expression of the effector molecule.

[0067] In one embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells immune effector cells. These immune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for the tag of the tagged polypeptide and a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL- 18) operably linked to an antigen activated inducible promoter. The antigen activated inducible promoter is an NF AT promotor. Moreover the combination of composition comprises composition B. Composition (B) comprises a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. The antigen-activated inducible promoter drives the expression of said immunomodulatory molecule such as a cytokine (e.g. IL- 18) upon binding of said antigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said immunomodulatory molecule such as a cytokine (e g. IL- 18) is secreted by the population of immune effector cells, preferentially T cells. (Optionally) the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) and wherein the amount of tagged polypeptides in composition C is lower compared to composition B, hereby altering the level of expression of the effector molecule.

[0068] In another embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells. The immune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof (preferentially LC biotin or a derivate thereof) and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen activated inducible promoter. Moreover the combination of compositions comprises composition B. Composition (B) comprising a plurality of said tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tag is biotin or a derivative thereof (preferentially LC-biotin or a derivate thereof). The antigen- activated inducible promoter drives expression of said effector molecule upon binding of saidantigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said effector molecule is secreted by the population of immune effector cells preferentially T cells. (Optionally) the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tag is biotin or a derivative thereof (preferentially LC-biotin or a derivate thereof), wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) and wherein the amount of tagged polypeptides in composition C is lower compared to composition B, thereby altering the level of expression of the effector molecule.

[0069] In another embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells. The immune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof (preferentially LC biotin or a derivate thereof). In addition to that the immune effector cells comprise a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen activated inducible promoter. This antigen activated inducible promoter is an NF AT promotor. Moreover the combination of composition comprises composition B. Composition (B) comprises a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment said tag comprises or consist of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof). The antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said effector molecule is secreted by the population of immune effector cells preferentially T cells. (Optionally) the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) and wherein the amount of tagged polypeptides in composition C is lower compared to composition B, hereby altering the level of expression of the effector molecule.

[0070] In another embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells. Theimmune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof (preferentially LC -biotin or a derivate thereof). In addition to that the immune effector cells comprise a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL-18) operably linked to an antigen activated inducible promoter. Moreover the combination of composition comprises a composition B. Composition (B) comprises a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tag is biotin or a derivative thereof (preferentially LC-biotin or a derivate thereof). The antigen- activated inducible promoter drives expression of said encoding an immunomodulatory molecule such as a cytokine (e.g. IL-18) upon binding of said antigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said encoding an immunomodulatory molecule such as a cytokine (e.g. IL- 18) is secreted by the population of immune effector cells preferentially T cells. (Optionally) the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tag is biotin or a derivative thereof (preferentially LC-biotin or a derivate thereof), wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) and wherein the amount of tagged polypeptides in composition C is lower compared to composition B, hereby altering the level of expression of the effector molecule.

[0071] In another embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells. The immune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof. Moreover the immune effector cell may comprise a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL-18) operably linked to an antigen activated inducible promoter. In this embodiment of the invention the antigen activated inducible promotor is an NF AT promotor. The combination of compositions further comprises composition B. Composition (B) comprises a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag is biotin or a derivative thereof. The antigen-activated inducible promoter drives expression of said encoding an immunomodulatory molecule such as a cytokine (e.g. IL-18) upon binding of saidantigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL- 18) is secreted by the population of immune effector cells preferentially T cells. (Optionally) the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) and wherein the amount of tagged polypeptides in composition C is lower compared to composition B, hereby altering the level of expression of the effector molecule.

[0072] In another embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells. The immune effector cells further comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain. In this embodiment of the invention the antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 6 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 8. In addition to that the immune effector cell further comprises second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen activated inducible promoter. Moreover, the combination of composition further comprises a composition (B) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag comprises or consists of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof). The tag can be bound by the antigen binding domain of the CAR expressed by the immune effector cells in composition A. The antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said effector molecule is secreted by the population of immune effector cells preferentially T cells. (Optionally) the combination of compositions may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag, wherein said tag comprises or consists of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof), wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical) andwherein the amount of tagged polypeptides in composition C is lower compared to composition B thereby altering the level of expression of the effector molecule.

[0073] In another embodiment of the invention, the combination of compositions may comprise a composition (A) comprising a population of immune effector cells, preferentially T cells. The immune effector cells comprise a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain. The antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 6 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 8. The immune effector cell additionally comprise a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL- 18) operably linked to an antigen activated inducible promoter. The combination of composition also comprises a composition B. Composition (B) comprises a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention the tag comprises or consists of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof) The antigen-activated inducible promoter drives expression of said immunomodulatory molecule such as a cytokine (e.g. IL- 18) upon binding of said antigen binding domain of said CAR said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially said immunomodulatory molecule such as a cytokine (e.g. IL-18) is secreted by the population of immune effector cells preferentially T cells. Preferentially the target antigen expressed on said target cell is B7H3. (Optionally) the combination of composition may comprise a composition (C) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell (preferentially the target antigen expressed on said target cell is B7H3) and said tag, wherein said tag comprises or consists of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof), wherein said tagged polypeptides of compositions B and C may be identical or different, (preferentially they may be identical). In addition to that, the amount of tagged polypeptides in composition C is lower compared to composition B thereby altering the level of expression of the effector molecule.

[0074] In one embodiment of the invention the first and second exogenous nucleic acid sequence may be comprised on one single expression cassette, wherein said expression cassette is comprised in a single vector, which is introduced into the immune effector cell by lentiviral transduction. The first nucleic acid sequence codes for a chimeric antigen receptor (CAR), wherein the CAR binds biotin or a derivative thereof (especially LC biotin or a derivativethereof) and may comprise or consist of the amino acid sequence SEQ ID No:22. This first exogenous nucleic acid sequence may be operably linked to a constitutively active promotor, preferentially EF-lalpha (SEQ ID No: 4) or PGK (SEQ ID No: 5). The second exogenous nucleic acid sequence encoding an effector molecule is operably linked to an antigen-activated inducible promoter. In this embodiment of the invention the effector molecule may be a regulatory cytokine preferentially IL-18 (SEQ ID No: 2; SEQ ID No:3). The antigen activated inducible promotor may be a NF AT promotor or a fragment thereof, preferentially the promotor has SEQ ID No: 1. In this embodiment of the invention the second exogenous nucleic acid sequence may be 5' of the first nucleic acid sequence.

[0075] In another aspect the invention provides a pharmaceutical composition as disclosed herein. The aspect the invention provides a combination of pharmaceutical compositions comprising. a. A composition (A) comprising a population of immune effector cells comprising i. A first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a tag of a tagged polypeptide ii. A second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter and b. A composition (B) comprising a plurality of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag wherein said antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell.

[0076] In this aspect of the invention the combination of compositions may optionally comprise a pharmaceutical acceptable carrier.

[0077] Pharmaceutical acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0078] Another aspect of the invention provides a combination of compositions for use in the treatment of a disease according to the features and embodiments described in the first aspect of the invention. In addition to that this aspect of the invention provides a combination of compositions for use in the treatment of a cancer or an autoimmune disease or an allergic disease or an infectious disease or a graft rejection. This aspect of the invention provides a combination of compositions for treating a disease in a subject suffering from said disease.Combination of compositions for use in a method of treating a disease

[0079] All features and embodiments described in the previous aspects of the invention also apply for this aspect of the invention.

[0080] Another aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for said tag of the tagged polypeptide and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. The antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lower compared to composition B.

[0081] Based on that the expression of the effector molecule by the immune effector cell is at a lower level by administration of composition C as compared to the administration of composition B.

[0082] In other words, the amount of tagged polypeptides in composition B lead to a lower expression of said effector molecule compared to amount of tagged polypeptides in composition C. In a preferred embodiment the amount of tagged polypeptides in composition B leads to no expression of the effector molecule by the immune effector cell. More importantly the immune cell retains killing activity and still induces tumor cell lysis.

[0083] In order to reach the desired effect, it is understood that during this method of treatment the composition B and C are administered to the patient separately from each other. In addition to that, the effect is coupled to the presence of composition A (immune effector cells expressing the CAR) in the patient.

[0084] Several treatment strategies may be beneficial for the patient.

[0085] One embodiment of the invention is a combination of compositions for use in a method of treating a disease and each composition may be administered separately to the patient. In one embodiment of the invention composition A may be administered first to the patient. Composition B and C may be administered separately in specific time intervals.

[0086] Independently from the order of administration of the compositions, each composition may optionally administered repeatedly.

[0087] In another embodiment composition A and B or A and C may be administered at the same time, followed by the administration of composition C or B (respectively) in a specific time interval.

[0088] In yet another embodiment of the invention, composition B or C may be administered first to the patient, followed by the administration of composition A (immune effector cells), which is then followed by the administration of composition C or B (respectively) in specific time intervals. Time intervals between the administrations are dependent on the half life time of the tagged polypeptides. Different tagged polypeptides may have different half-life times, which is e.g. dependent on the structure of the tagged polypeptide and the tag.

[0089] The initial administration of the tagged polypeptides (composition B or C) to the patient, may have the advantage that the tagged polypeptides may have bound the target already before administration of the CART cell expressing immune effector cell.

[0090] One embodiment of the invention is a combination of compositions for use in a method of treating a disease, wherein each composition is administered in the following order: A + B +C; A +C +B; B +A +C, B+ C + A, C + A + B, C + B + A, A / B +C, C+ A / B. A / C + B, B+A / C, B / C + A, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B, B+ A+ C, or C+A+B. In a preferred embodiment each composition is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, A / B +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B, or C+A+B

[0091] In one embodiment of the invention, the compositions as disclosed herein comprising immune cells expressing the CAR (anti tag CAR) as disclosed herein may be for use e.g. in the treatment of a subject suffering from cancer or an autoimmune disease or an allergic disease oran infectious disease or a graft rejection as disclosed herein. The subject may suffer from said cancer (a patient) or said autoimmune disease or may be a healthy subject. These immune cells are genetically modified (in vitro) to express said CAR as disclosed herein. These engineered cells may be activated and expanded in vitro to a therapeutically effective population of expressing cells. In cellular therapy these engineered cells may be infused to a recipient in need thereof as a combination of pharmaceutical compositions as disclosed herein. The infused cells in the recipient may be e.g. able to kill (or at least stop growth of) cancerous cells expressing the antigen which is recognized by the CAR system as disclosed herein or may reduce the effect of the autoimmune disease. The recipient may be the same subject from which the cells were obtained (autologous cell therapy) or may be from another subject of the same species (allogeneic cell therapy).

[0092] Populations of said CAR expressing (immune) cells may be formulated for administration to a subject using techniques known to the skilled artisan.

[0093] Formulations comprising therapeutically effective population(s) of said CAR expressing cells may include pharmaceutically acceptable excipient(s) (carrier or diluents). Excipients included in the formulations will have different purposes depending, for example, on the nature of the tag-binding domain of the anti-tag-CAR, the (sub)population of immune cells used, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for-inj ection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.

[0094] A formulation of a therapeutically effective population(s) of said CAR expressing cells may include one population of said CAR expressing (immune) cells, or more than one population of said CAR expressing (immune) cells. The different populations of said CAR expressing (immune) cells may vary based on the identity of the activation domain, the identity of the (sub)population of immune cells, or a combination thereof.

[0095] The formulations comprising therapeutically effective population(s) of said CAR expressing cells may be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous injection. Other modes include, without limitation, intratumoral, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m ), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).

[0096] The formulations comprising therapeutically effective population(s) of said CAR expressing cells that are administered to a subject comprise a number of said CAR expressing cells such immune cells that is effective for the treatment of the specific indication or disorder.

[0097] In general, formulations may be administered that comprise between about 1 x 104and about 1 x 1010said CAR expressing cells such as immune cells. In most cases, the formulation may comprise between about 1 x 105and about 1 x 109said CAR expressing cells such as immune cells, from about 5 x 105to about 5 x 108said CAR expressing cells such as immune cells, or from about 1 x 106to about 1 x 107said CAR expressing cells such as immune cells. However, the number of said CAR expressing cells such as immune cells administered to a subject may vary between wide limits, depending upon the location, source, identity, extent and severity of the disorder, the age and condition of the individual to be treated, etc. A physician may ultimately determine appropriate dosage.

[0098] The tagged polypeptide(s) as disclosed herein may be formulated for administered to a subject using techniques known to the skilled artisan. Formulations of the tagged polypeptide(s) may include pharmaceutically acceptable excipient(s) (carriers or diluents). Excipients included in the formulations will have different purposes depending, for example, on the nature of the tag, the antigen binding domain of the tagged polypeptide, and the mode of administration. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for-inj ection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.

[0099] The tagged polypeptide(s) may be administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intravenous, intraperitoneal, and intratumoral injection. Other modes include, without limitation, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m.), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). ges to be used.

[0100] Formulations comprising the polypeptide(s) are administered to a subject in an amount which is effective for treating the specific indication or disorder. In general, formulations comprising at least about 1 pg / kg to about 100 mg / kg body weight of the tagged polypeptide(s) may be administered to a subject in need of treatment. In most cases, the dosage may be from about 100 pg / kg to about 10 mg / kg body weight of the tagged polypeptide(s) daily, taking into account the routes of administration, symptoms, etc. The amount of tagged polypeptide(s) in formulations administered to a subject may vary between wide limits, depending upon thelocation, source, identity, extent and severity of the disorder, the age and condition of the individual to be treated, etc. A physician may ultimately determine appropriate dosages to be used.

[0101] The timing between the administration of the CARs expressing cell formulation and the tag polypeptide-formulation may range widely depending on factors that include the type of (immune) cells being used, the binding specificity of the CARs, the identity of the tag, the antigen binding moiety of the tagged polypeptide, the identity of said soluble antigen, the identity of the target cell, e.g. cancer cell to be treated, the location of the target cell in the subject, the means used to administer the formulations to the subject, and the health, age and weight of the subject being treated. Indeed, the tagged polypeptide formulation may be administered prior to, simultaneous with, or after the genetically engineered (immune) cell formulation.

[0102] Depending on the disorder being treated the step of administering the CARs expressing cell formulation, or the step of administering the tagged polypeptide formulation, or both, can be repeated one or more times. When two or more formulations of engineered cells such as immune cells expressing the CARs of the invention are applied to a subject, the engineered cells may be of the same cell type or of different cell types, e.g. T cells and / or NK cells. A formulation of cells such as immune cells may also comprise more than one cell type, each expressing the CARs of the invention.

[0103] The disease may be cancer, more specifically solid cancer. For example the cancer types may be adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain / CNS tumors in children or adults, breast cancer, cervical cancer, colon / rectum cancer, endometrial cancer, esophagus cancer, ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST), gestation trophoblastic disease, hodgkin disease, kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leuckemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinum cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, oral cavity or oropharyngeal cancer, osteosarcoa, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, sarcoma, basal skin cancer, squamous cell skin cancer, melanoma,merkel cell skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, or wilms tumor

[0104] The disease may be an autoimmune disease. Exemplary autoimmune diseases are Behcet’s disease, Juvenile idiopathic arthritis, Type 1 diabetes, Rheumatoid arthritis, Wegener Granulomatosis, Systemic lupus erythematosus, Systemic sclerosis, Crohn's disease, Graves' disease, Hashimoto thyreoiditis, Goodpasture syndrome, pernicieuse anemia, Primary biliary cholangitis, Myasthenia gravis, Dermato polymyositis, Vasculitis, Mixed connective tissue disease, Scleroderma, Multiple sclerosis, Psoriasis, Ulcerative colitis and Uvetis.Specific Embodiments

[0105] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of T cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for said tag of the tagged polypeptide and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. The antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lower compared to composition B. Preferentially said effector molecule is secreted by the population of immune effector cells. Preferentially the combination of compositions for use in a method of treating a disease is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, A / B +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B, or C+A+B..

[0106] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells, preferentially T cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific forsaid tag of the tagged polypeptide and a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL- 18). operably linked to an antigen- activated inducible promoter. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. The antigen-activated inducible promoter drives expression of said immunomodulatory molecule such as a cytokine (e.g. IL- 18) upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lower compared to composition B. Preferentially said immunomodulatory molecule such as a cytokine (e.g. IL-18) is secreted by the population of immune effector cells, preferentially T cells. Preferentially the combination of compositions for use in a method of treating a disease is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, A / B +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B, or C+A+B..

[0107] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells, preferentially T cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof. Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof (preferentially LC-biotin or a derivative thereof). The antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lower compared to composition B. Preferentially said effector molecule is secreted by the population of immune effector cells, preferentially T cells. Preferentially the combination of compositions for use in a method of treating a diseaseis administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, A / B +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B,or C+A+B.

[0108] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells, preferentially T cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof (preferentially LC-biotin or a derivative thereof) and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen- activated inducible promoter, wherein said antigen activated inducible promotor is an NF AT promotor. Composition (B) comprises an amount of said tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof (preferentially LC-biotin or a derivative thereof). Composition (C) comprises an amount of said tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof. The antigen-activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lower compared to composition B. Preferentially said effector molecule is secreted by the population of immune effector cells, preferentially T cells. Preferentially the combination of compositions for use in a method of treating a disease is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, A / B +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B, or C+A+B.

[0109] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells, preferentially T cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof (preferentially LC-biotin or a derivative thereof) and a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e g. IL- 18) operably linked to an antigen-activated inducible promoter. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specificfor a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof (preferentially LC-biotin or a derivative thereof). Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof (preferentially LC-biotin or a derivative thereof). The antigen-activated inducible promoter drives expression of said immunomodulatory molecule such as a cytokine (e.g. IL- 18) upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lower compared to composition B. Preferentially said effector molecule is secreted by the population of immune effector cells, preferentially T cells. Preferentially the combination of compositions for use in a method of treating a disease is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, A / B +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B, or C+A+B.

[0110] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells, preferentially T cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for biotin or a derivative thereof (preferentially LC-biotin or a derivative thereof) and a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e g. IL- 18) operably linked to an antigen-activated inducible promoter, wherein said antigen activated inducible promotor is an NF AT promotor. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof. Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention said tag of the tagged polypeptides is biotin or a derivative thereof (preferentially LC- biotin or a derivative thereof). The antigen-activated inducible promoter drives expression of said immunomodulatory molecule such as a cytokine (e.g. IL- 18) upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lowercompared to composition B. Preferentially said effector molecule is secreted by the population of immune effector cells, preferentially T cells. Preferentially the combination of compositions for use in a method of treating a disease is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, AZB +C, A / C + B, A + B / C, A + B + C; A+C+B, AZB +C, A / C +B, or C+A+B.

[0111] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells, preferentially T cells comprising: a first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain comprising an antigen binding domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 6 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 8 and a second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention the tag comprises or consists of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof) Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention the tag comprises or consists of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof). The antigen- activated inducible promoter drives expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. The amount of tagged polypeptides in composition C is lower compared to composition B. Preferentially said effector molecule is secreted by the population of immune effector cells, preferentially T cells. Preferentially the combination of compositions for use in a method of treating a disease is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, AZB +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B, or C+A+B

[0112] This aspect of the invention provides a combination of compositions for use in a method of treating a disease, said method comprising administering the compositions as disclosed herein to a subject in need thereof. The composition (A) comprises a population of immune effector cells, preferentially T cells comprising: a first exogenous nucleic acid sequenceencoding a chimeric antigen receptor (CAR) comprising an antigen binding domain comprising an antigen binding domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 6 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 8 and a second exogenous nucleic acid sequence encoding an immunomodulatory molecule such as a cytokine (e.g. IL-18) operably linked to an antigen- activated inducible promoter. Composition (B) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention the tag comprises or consists of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof). Composition (C) comprises an amount of tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag. In this embodiment of the invention the tag comprises or consist of biotin or a derivative thereof (preferentially LC biotin or a derivative thereof). The antigen-activated inducible promoter drives expression of said immunomodulatory molecule such as a cytokine (e.g. IL- 18) upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell. Preferentially the target antigen expressed on said target cell is B7H3. The amount of tagged polypeptides in composition C is lower compared to composition B. Preferentially said effector molecule is secreted by the population of immune effector cells, preferentially T cells. Preferentially the combination of compositions for use in a method of treating a disease is administered in the following order: A + B +C; A +C +B; B +A +C, C + A + B, A / B +C, A / C + B, A + B / C, A + B + C; A+C+B, A / B +C, A / C +B,or C+A+B.All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.Definitions

[0113] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0114] The term B7H3 or CD276 can be used interchangeably. It is understood that this also includes other state of the art synonyms.

[0115] The term “antigen binding domain refers” to a region of a protein or a polypeptide (normally the variable region of an antibody or fragment thereof) that specifically binds to an antigen. It may be composed of one constant and one variable domain of each of the heavy and the light chain.

[0116] Chimeric antigen receptors (CARs) are recombinant receptors for antigens which redirect the specificity and function of T lymphocytes and / or other immune cells in a single molecule. The concept of using CARs in immunotherapy is that CARs, which are programmed targeting tumor-associated antigens or disease associated antigens, can be replicated rapidly and homogeneously.

[0117] In general, a CAR may comprise an extracellular domain (extracellular part) comprising an antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker or spacer. The extracellular domain may also comprise a signal peptide.

[0118] The CAR as disclosed herein may be referred to as “anti-tag” CAR or “adapterCAR” or “universal CAR” or “adCAR” as disclosed e.g. in US9233125B2. Such a CAR has a similar domain structure compared to the standard direct CAR, besides that the adapter CAR binds a target antigen indirectly by binding an adapter molecule ( tagged polypeptide) which binds to the target antigen.

[0119] A "signal peptide" refers to a peptide sequence that directs the transport and localization of the protein within a cell, e g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0120] Generally, an antigen binding domain of a CAR refers to the region that specifically binds to an antigen. In the present invention the CAR binds the adapter / tagged polypeptide. The CARs of the invention may comprise one or more antigen binding domains (e.g. a tandem CAR). Generally, the targeting regions of the CAR are localized extracellularly. The antigen binding domain may comprise an antibody, single domain antibody or an antigen binding fragment thereof. The antigen binding domain may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. Often the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4 / S)3 -linker” or a “whitlow linker”.

[0121] “Spacer” or “hinge” as used herein refers to the hydrophilic region which is between the antigen binding domain of the CAR and the transmembrane domain. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge or IgG4 hinge.

[0122] The “transmembrane domain” of the CAR may be from any desired natural or synthetic source for such domain. When the source is natural the domain may be from any membranebound or transmembrane protein. The transmembrane domain may be for example from CD8alpha or CD28.

[0123] The cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR).

[0124] "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions. Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement.

[0125] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co-stimulatory signaling domain). Therefore, an intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0126] Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs). Examples of ITAM containing primary cytoplasmic signaling domains often used in CARs are that those from TCRzeta(CD3zeta), FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence from CD3zeta.

[0127] The cytoplasmic domain of the CAR may be designed to comprise the CD3zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3zeta chain portion and a co-stimulatory signaling region (domain). The co-stimulatory signaling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples for a co-stimulatory molecule are CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3.

[0128] The cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo- or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet.

[0129] As mentioned above, according to the present invention, the antigen binding domain of a CAR binds a tag or hapten that is coupled to a protein or polypeptide (“haptenylated” or “tagged” polypeptide / Protein), wherein the polypeptide may bind to a disease-associated antigen such as a tumor associated antigen (TAA)

[0130] The haptens or tags may be coupled directly or indirectly to a polypeptide (the tagged polypeptide), wherein the polypeptide may bind to said disease associated antigen expressed on the (cell) surface of a target. The tag may be e.g. dextran or a hapten such as biotin or fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or thiamin, but the tag may also be a peptide sequence (a peptide) e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide. The tag may also be streptavidin. The tag portion of the tagged polypeptide is only constrained by being a molecule that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR. For example, when the tag is FITC (Fluorescein isothiocyanate), the tag-binding domain may constitute an anti-FITC scFv. Alternatively, when the tag is biotin or PE (phycoerythrin), the tag-binding domain may constitute an anti-biotin scFv or an anti-PE scFv, respectively.

[0131] In a preferred embodiment of the invention the tag may be biotin and the tag binding domain may be an anti-biotin scfv.

[0132] In a more preferred embodiment of the invention the tag may be or may comprise LC biotin and the tag binding domain may be an anti LC-biotin binding domain / scfv. Such an adapter CAR system is disclosed in EP23192028.1 and EP23192030.7.

[0133] LC biotin is a biotin derivate which additionally comprises a CL chain for example as disclosed in EP23192028.1 and EP23192030.7. This LC biotin can be coupled as a tag to a polypeptide having an antigen binding domain, thereby generating a tagged polypeptide as disclosed herein. The structure of the LC biotin may make up a specific recognition motif, which can be specifically recognized by the anti-tag binding domain of the CAR.

[0134] The term “tagged protein” or “tagged polypeptide” as used herein refers to a polypeptide that has bound thereto directly or indirectly at least one additional component, i.e. the tag. The tagged polypeptide as used herein is able to bind an antigen expressed on a target cell. The polypeptide may be an antibody or antigen binding fragment thereof that binds to an antigen expressed on the surface of a target cell such as a tumor associated antigen (TAA) on a cancer cell.

[0135] The terms “adapter” or “adapter molecule” or “tagged polypeptide” as used herein may be used interchangeably. The tag may be e g a hapten or dextran and the hapten or dextran may be bound by the antigen binding domain a CAR comprising an antigen binding domain specific for the tag. Haptens such as e.g. FITC, biotin, PE, thiamin, streptavidin or dextran are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.

[0136] But the tag may also be a peptide sequence e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide. The peptide may be selected from the group consisting of c-Myc-tag, Strep-Tag, Flag-Tag, and Polyhistidine-tag. The tag may also be streptavidin. The tag portion of the tagged polypeptide is only constrained by being a molecular that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR.

[0137] The term “engineered” as used herein can refer to one or more human-designed alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome. An “engineered cell” or “genetically modified cell” can refer to a cell with an added, deleted and / or altered gene. Especially, the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigenreceptor on their cell surface. As disclosed herein, cells are also engineered to comprise an inducible nucleic acid construct.

[0138] The engineered cell expressing a CAR may be further modified by genetic engineering using methods well known in the art e.g. Meganucleases, TALEN, CrisprCas, zink finger nucleases, shRNA and / or miRNA. Said cells may be modified to reduce or lack expression of a specific gene, which is normally expressed in the cell e.g. T cell receptor (TCR), MHC, co- inhibitory molecules likePD-1, CTLA-4, BTLA, TIGIT, Tim-3, CD244, LAIR, Lag-3, CD160, HVEM.

[0139] The terms “immune cell” or “immune effector cell” may be used interchangeably and refer to a cell that may be part of the immune system and executes a particular effector function such as T cells, alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, monocytes or macrophages. Preferentially these immune cells are human immune cells. Preferred immune effector cells are T cells and NK cells. "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.

[0140] The terms “having specificity for”, “specifically binds” or “specific for” with respect to an antigen-binding domain of an antibody, of a fragment thereof or of a CAR refer to an antigenbinding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

[0141] As used herein, the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, haptens and combinations thereof, for example a glycosylated protein or a glycolipid. The term “antigen” as used herein refers to a molecular entity that may be expressed on the surface of a target cell and that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof,antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0142] The tumor associated antigen (TAA) as used herein refers to an antigenic substance produced by tumor cells. Tumor associated antigens are useful tumor or cancer markers in identifying tumor / cancer cells with diagnostic tests and are potential candidates for use in cancer therapy. Preferentially, the TAA may be expressed on the cell surface of the tumor / cancer cell, so that it may be recognized by the antigen binding receptor as disclosed herein.

[0143] The term “target cell” as used herein refers to cell which expresses an antigen on its cell surface that should be recognized (bound) by the protein according to the current invention. Said target cell may be e.g. a cancerous cell or a cell associated with an autoimmune disease or a cell associated with an infectious disease

[0144] Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies.

[0145] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0146] The terms “therapeutically effective amount” or “therapeutically effective population” mean an amount of a cell population which provides a therapeutic benefit in a subject.

[0147] As used herein, the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the individual is a human. The subject may be a subject suffering from a disease such as cancer.

[0148] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.

[0149] The term “cancer” is known medically as a malignant neoplasm. Cancer is a broad group of diseases involving unregulated cell growth and includes all kinds of leukemia. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans.

[0150] Autoimmune diseases are a condition arising from autoimmunity or disbalance in the immune homeostasis resulting in pathologies that can affect multiple different organ systems.Examples include Behcet’s disease, Juvenile idiopathic arthritis, Type 1 diabetes, Rheumatoid arthritis, Wegener Granulomatosis, Systemic lupus erythematosus, Systemic sclerosis, Crohn's disease, Graves' disease, Hashimoto thyroiditis, Goodpasture syndrome, Primary biliary cholangitis, Myasthenia gravis, Dermato polymyositis, Vasculitis, Mixed connective tissue disease, Scleroderma, Multiple sclerosis, Psoriasis, Ulcerative colitis and Uvetis.

[0151] Infection (infectious disease) is the invasion of an organism's body tissues by diseasecausing agents, their multiplication, and the reaction of host tissues to the infectious agents and the toxins they produce. Infections are caused by infectious agents (pathogens) including: viruses, bacteria, fungi and parasites. Said infection may be an acute or a chronic infection.

[0152] The term “administering” refers to local and systemic administration, e.g., including enteral, parenteral, pulmonary, and topical / transdermal administration. The administration may be directly intratumoral. Routes of administration for pharmaceutical ingredients include, e.g., oral administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery, intrathecal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, or subcutaneous administration to a subject. Administration can be by any route including parenteral and transmucosal (e.g, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic and intracranial.

[0153] The term “effector molecule” as used herein refers to a molecule expressed or synthesized by the immune cell as disclosed herein triggered by an activated adapterCAR (antitag CAR) as disclosed herein that has bound to the cell surface antigen antigen as disclosed herein. The effector molecule has an effect on the effector expressing immune cell itself or on the environment of the effector expressing immune cell when expressed and secreted, wherein said effect is not present when the effector molecule is not expressed and / or synthesized in said immune cell. If the effector molecule has an effect on the immune cell expressing said effector, then the effector may exert its effect intracellularly or alternatively, the effector may be secreted by said immune cell and exerts its effect on said immune cell extracellularly.

[0154] The effector molecule may be an antibody or antigen binding fragment thereof, a therapeutic peptide or protein, a cytokine, a chemokine, a receptor, a transcription factor, a siRNA, or shRNA.

[0155] The effector molecule may be an antibody or antigen binding fragment thereof. Said antibody or antigen binding fragment thereof may be secreted after triggering the adCAR. Saidantibody or antigen binding fragment may be a therapeutic or nontherapeutic antibody or antigen binding fragment thereof.

[0156] The effector molecule may be a cytokine and may be secreted. Said cytokine may allow local and dose-controlled shaping of the microenvironment that activated the CAR of the immune cell.

[0157] The terms “nucleic acid (sequences)” or “polynucleotide” can be as used interchangeably herein refer to polymers of nucleotides. Polynucleotides, which can be hydrolyzed into monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, the term “polynucleotides” encompasses, but is not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0158] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0159] The term “promoter” as used herein refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the transcription of a specific polynucleotide sequence.

[0160] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0161] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only in the presence or absence of certain conditions. As disclosed herein the inducer may be the activation of the intracellular signaling domain of a CAR. Such a promotor is herein also referred to as “antigen activated inducible promoter” and may also be referred to as “Immune cell inducible promotor”Examples

[0162] The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.Example 1 Generation of Anti-Tag CAR T CellsConstruct Design:

[0163] The expression vector is composed of two expression cassettes.

[0164] The AdCAR system as disclosed in EP23192028.1 and EP23192030.7 was used. This is a variant of an adapter CAR system. The tags and the binding moieties of the adCAR are identical to the ones disclosed in the cited patent applications. For sake of simplicity the binding moieties are referred to as biotin binding moiety and the tag moiety is referred to a biotin tag.

[0165] AdCAR T cells contained an anti-biotin scFv as a binding moiety (SEQ ID No. 10; SEQ ID No. 11). The scFv was linked to the human CD8 (SEQ ID No. 14; SEQ ID No. 15) transmembrane domain via hIgG4 hinge domain (SEQ ID No. 12; SEQ ID No. 13). The signaling domain was composed of CD28 (SEQ ID No. 16; SEQ ID No. 17), 4-1BB (SEQ ID No. 18; SEQ ID No. 19) and 3 (SEQ ID No. 20; SEQ ID No. 21) . Upstream of the CAR, the inducible IL-18 (SEQ ID No. 2; SEQ ID No 3) expression cassette was located. The cassette contained an NF AT (SEQ ID No. 1) promoter upstream of human IL18.Generation of LV Particles and Titration:

[0166] Lentiviral vector particles were manufactured via transient transfection of HEK-293T cells. The lentiviral vector particles were pseudotyped with VSV-G. For transfection, HEK- 293T cells were seeded in T175 culture flasks in DMEM (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom) 3 days before transfection. On the day of transfection, the culture medium was removed and replaced by DMEM (Biowest) supplemented with 2 mM L-Glutamine (Lonza). The cells were transfected with a three-plasmid system encoding for VSV-G, gag / pol / rev and the psi positive transfer vector. After 48 h the supernatant was collected and centrifuged for 10 min at 1000 rpm to remove cellular debris. In addition, the supernatant was filtrated through a 0.45 pm filter. The pellet was re-suspended in ice-cold PBS and stored at -80° C.

[0167] A functional titer of VSV-G pseudotyped lentiviral vector particles was determined via titration on Sup-Tl cells. 2xl05cells were seeded in 150 pL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) in a 96 well-round bottom plates. For transduction 50 pL of serial diluted lentiviral vector particles were added to the seeded cells. 90 pL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom) was added after 24h. The frequency of transduced cells was quantified after 96 h by flow cytometry using a biotin CAR detection reagent (Miltenyi Biotec). Based on the frequency of positive cells, the number of seeded cells and the volume of lentiviral particles used for transduction, the titer was calculated. The titer was expressed in transducing units per mL.Manufacturing of AdCAR T cells:

[0168] Initially, 2xl06T cells were plated in a 24-well plate with 2 mL of TexMACS™ medium, enriched with 12.5 ng / mL of both recombinant human IL-7 and IL-15. The cells were then activated using a 1 :100 dilution of TransAct™. After 24 hours, lentiviral vectors (LVVs) were introduced to achieve a multiplicity of integration (MOI) of 10, followed by a resuspension of the cells. On the third day, the old medium was completely replaced with fresh medium. Subsequently, the T cells were split at a 1 :2 ratio every other day starting from day three. This expansion process continued for a minimum of ten days before the T cells were utilized in any functional assays. Cell concentration was analyzed by flow cytometry using propidium iodide to discriminate live and dead cells.Example 2

[0169] Induction of effector molecule secretion and tumor cell lysis via tagged polypeptides

[0170] LC biotin-specific AdCARs expressed by T cells can engage atumor cell via a LC biotin tagged polypeptide. The tagged polypeptide e.g., a CD276-specific Fab will bind to CD276 positive tumor cells and induce clustering of CAR molecules on the T cell surface. The AdCAR will trigger the intrinsic signaling pathways such as NFAT / Ap-1 or NF-KB. In addition to the AdCAR, the T cells can be modified to contain an NF AT inducible expression cassette that induces e.g. the secretion of IL-18.

[0171] Therefore, the binding of e.g., the CD276-specific Fab adapter (SEQ ID NO: 24 and SEQ ID NO:25) will induce the killing of tumor cells and activation-induced secretion of IL- 18 (Figure 1). IL-18 plays a multifaceted role in the immune response, particularly in the function of T cells. IL- 18 does increase the secretion of other pro-inflammatory cytokines such as IFN-y. In the presence of IL-18 better persistence and long-term functionality of CAR T cells can be observed. However, like other cytokines, IL- 18 affects a multitude of immune cells such as NK cells and NKT cells, which can influence T cell responses indirectly. Therefore, control of the secreted effector molecule is needed for clinical application in case of side effects induced by the effector molecule. The AdCAR system in combination with the NFTA-inducible IL-18 cassette allows independent control for both, the adapter-mediated lysis of tumor cells and theadapter-mediated secretion of the effector molecule. At low adapter concentrations, tumor cell lysis can be induced but no secretion of the effector molecule is induced. To reach the activation threshold that is needed to induce the secretion of the effector molecule the adapter concentration must be increased. Therefore, the system described here provides a high degree of control of the distinct modes of action of a CAR T cell equipped with an activation-induced effector molecule.Example 3

[0172] Independent modulation of activation-induced IL- 18 secretion and tumor cell killing.

[0173] CD276 expressing H446-cells were cultured in the presence or absence of 20nM to OnM CD276-specific Fabs (SEQ ID NO: 24 and SEQ ID NO:25) conjugated with the biotin-tag and AdCAR T cells specific for the biotin-tag. The AdCAR expression cassette contained IL-18 regulated via the NF AT promotor. AdCAR T cells and target cells were co-cultured at a ratio of 2: 1 in 200 pL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom) at 37°C and 5% CO2. The lysis of tumor cells was quantified by the impedance of attached tumor cells using the xCELLigence RTCA system at time intervals of 15 min. The values were normalized to the start values. The lysis of tumor cells was dependent on the CD276 adapter concentration. The maximum tumor cell lysis was reached between 0,5 nM to 20 nM of adapter. At lower concentrations of adapter the tumor cell lysis was decreasing (Figure 2a).

[0174] After 48h supernatant was collected to analyze the amount of secreted IL- 18 via the Human Interleukin 18 (IL 18) ELISA Kit (abbexa). The assay was done according to the manufacturer's instructions. IL- 18 secretion was detected between 0,5nM to 20nM of adapter. In contrast to the tumor cell lysis ofH446-cells, the level of IL-18 showed an adapter-dependent decrease and did not plateau. Most strikingly, no IL-18 was detected at 0,05nM to 0,01nM of the adapter. This contrasts with the tumor cell lysis which was still induced at this adapter contractions (Figure 2b-c). This indicates that the AdCAR system in combination with the NFTA inducible IL-18 can either induce only tumor cell lysis or tumor cell lysis and IL-18 secretion. Furthermore, the amount of the secreted effector molecule can be fine-tuned via the adapter concentration.Example 4

[0175] Application of AdCAR T cells secreting NF AT -induced IL-18 in vivo.

[0176] First, female immunodeficient NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) were inoculated with 0.5xl06CD276 positive SHP-77 i.v. and 3 days later the mice were randomizedaccording to their tumor burden. Before the AdCAR T cells were injected the mice were preconditioned with 100pg CD276-specificFab adapter tagged with biotin. Next lx 107AdCAR T cells or AdCAR T cells with NFTA-inducible IL- 18 were administered i.v. Control cohorts were injected with PBS instead of an adapter. The mice were injected with lOOpg adapter every day. At regular time intervals, the tumor growth was monitored via bioluminescent imaging.

[0177] Therefore, D-Luciferin potassium was dissolved in PBS to achieve a final concentration of 30 mg / mL. Subsequently, mice were injected withlOO pL of D-Luciferin potassium salt. Imaging was performed using the IVIS Lumina III imaging system, with the device automatically adjusting the aperture and exposure time. The bioluminescent signal was quantified within a manually defined region of interest that encompassed the entire body of the animal, and this measurement was recorded.

[0178] Both, AdCAR T cells and AdCAR T cells with NF AT -inducible IL-18 showed improved tumor control compared to the controls in the absence of an adapter. AdCAR T cells and AdCAR T cells with NFAT-inducible IL-18 showed comparable tumor lysis in vivo. This indicates the adapter-dependent anti-tumor efficacy of AdCAR T cells with NFAT-inducible IL-18 in vivo (Figure 3). The system is as functional as its counterpart without inducible IL-18Example 5

[0179] Improving the persistence of AdCAR T cells in vivo via NFAT-inducible IL-18 secretion

[0180] Following the procedures detailed in example 4, mice were subjected to treatment. At specified time intervals, blood samples were obtained through a vena facialis puncture. These blood samples were promptly collected in capillary tubes coated with heparin. Subsequently, the blood was transferred into microvette tubes containing EDTA. To separate plasma from cells, the samples underwent centrifugation at 13,000 rpm for 15 min at 4 °C. The remaining cell pellet was resuspended in 1 mL of red blood cell lysis buffer, following the manufacturer's instructions. After a 5-minute incubation period, the cells were centrifuged at 300 g and then resuspended in 100 pL of PEB. The cellular analysis was carried out using flow cytometry.

[0181] As described in example 2 one effect of IL-18 is the enhanced expansion and persistence of T cells. As expected AdCAR T cells with the capability to secrete IL- 18 in an adapterdependent context showed up to 40% of human CD45-positive cells circulating in the blood. In all other cohorts without IL- 18 or adapter lower frequencies of approx. 10% of CD45-positive cells were detected (Figure 4a). A similar trend was observed for AdCAR-expressing cells. In the presence of IL-18 the highest proportion of CAR positive cells was detected with up to 60%.In the control cohorts, the frequency of CAR positive cells ranged between 10% to 40% (Figure 4b).

[0182] These data indicate that AdCAR T cells do secrete IL-18 in the presence of an adapter driven by the NF AT promotor. The IL-18 is functional and improves the persistence and expansion of both CAR T cells and T cells.Example 6

[0183] Adapter concentration dependent modulation of activation-induced IL-18 secretion and tumor cell killing.

[0184] To evaluate the system not only for solid tumor models (Figure 2-4), but also hematologic malignancies, biotin-tag specific AdCAR T cells containing the IL-18 expression cassette were tested on two acute myeloid leukemia (AML) models in combination with different biotin-tagged Fab molecules. As first AML model, CLEC12a, CD33, and CD123 expressing OCI-AML2 cells were cultured in the presence or absence of 1 nM to 0 nM CLEC12a, CD33 (SEQ ID NO: 26 and SEQ ID NO:27) or CD 123 -specific (SEQ ID NO: 28 and SEQ ID NO 29) Fabs conjugated with the biotin-tag and AdCAR T cells specific for the biotin-tag. As second AML model CLEC12a and CD33 expressing THP-1 tumor cells were cultured in the presence or absence of InM to OnM CLEC12a or CD33-specific Fabs conjugated with the biotin-tag and AdCAR T cells specific for the biotin-tag. The AdCAR expression cassette contained IL-18 regulated via the NF AT promotor. OCI-AML2 cells and AdCAR T cells were co-cultured at a ratio of 1 :1 in 200 pL Alpha MEM Eagle medium (PAN-Biotech) supplemented with 20% FCS (Biochrom) at 37°C and 5% CO2. THP-1 tumor cells and AdCAR T cells were co-cultured at a ratio of 1 : 1 in 200 nL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom) at 37°C and 5% CO2. The lysis of tumor cells was quantified by the GFP signal of the tumor cells using the Incucyte system at time intervals of 3 h. The values were normalized to the start values. The lysis of tumor cells was dependent on the target-specific adapter concentration. The maximum tumor cell lysis was reached between 0.1 nM to 1 nM of adapter. At lower adapter concentrations, the tumor cell lysis was decreasing (Figure 5a, Figure 6a).

[0185] After 42h, supernatant was collected to analyze the amount of secreted IL-18 via the Human Interleukin 18 (IL 18) ELISA Kit (abbexa). The assay was done according to the manufacturer's instructions. For co-cultures using OCI-AML2 target cells, IL-18 secretion was dependent on the adapter specificity as well as adapter concentration used and detected between 0.01 nM or 0.02 nM to 1 nM of adapter (Figure 5b). In coculture with THP-1 target cells,generally less IL- 18 was produced. Due to the quantification limit of the IL- 18 ELISA, the amount of produced IL-18 could not be quantified reliably. However, the data shows an adapter concentration dependent pattern of IL- 18 production for the THP-1 tumor model, as well (Figure 6b). Interestingly, similar to the solid tumor model in example 3, a plateau of tumor cell lysis was observed at higher adapter concentrations whereas the IL- 18 secretion pattern was more Adapter concentration dependent. Especially at lower adapter concentrations, stronger tumor cell lysis than IL-18 secretion was induced. In general, tumor cell lysis as well as the amount of secreted effector molecule can be fine-tuned via the adapter concentration (Figure 5a-c, Figure 6a-b).Example 7

[0186] Adapter concentration dependent modulation of activation-induced IL-18 secretion and tumor cell killing.

[0187] To prove the broad range of application of the system, its functionality was tested on a further tumor model, namely the Burkitt’s lymphoma cell line Raji. For this, Raji tumor cells were co-cultured with various concentrations of CD19- (SEQ ID NO: 32 and SEQ ID NO:33) and CD20-specific (SEQ ID NO: 30 and SEQ ID NO:31) Fabs conjugated with the biotin-tag and AdCAR T cells specific for the biotin-tag. The AdCAR expression cassette contained IL- 18 regulated via the NF AT promotor. Raji cells and AdCAR T cells were co-cultured at a ratio of 1: 1 in 200 pL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom) at 37°C and 5% CO2. Lysis of tumor cells was analyzed 42 h post co-culture set-up via Flow cytometry using 7-AAD as live / dead marker and GFP expression to discriminate target cells from T cells. Lysis of target cells was normalized to the control condition containing no adapter. The lysis of tumor cells was adapter concentration dependent showing its maximum between 0.2 nM to 1 nM of adapter for CD20 Adapter and 1 nM of adapter for CD19 adapter. At lower adapter concentrations, the tumor cell lysis was decreasing (Figure 7a).

[0188] After 42h, supernatant was collected to analyze the amount of secreted IL-18 via the Human Interleukin 18 (IL 18) ELISA Kit (abbexa). The assay was done according to the manufacturer's instructions. For most adapter concentrations tested, the amount of secreted IL- 18 was below the quantification limit of the assay and IL 18 concentrations could not be quantified reliably. However, 1 nM of CD19-specific Adapter led to very strong secretion of IL-18 with a mean concentration of 44.8 pg / ml. All other adapter concentrations induced only IL- 18 levels below the quantification limit, but still in an adapter dose dependent pattern. Incontrast, tumor cell lysis induced by the adapters showed a rather mild adapter concentration dependency and no drastic increase at 1 nM Adapter demonstrating that induction of strong IL- 18 production can be regulated independently of the tumor lysis by selection of an appropriate adapter and adapter concentration (Figure 7 a-b). In general, higher adapter concentrations are required to induce efficient 11-18 release whereas tumor lysis is induced already at lower Adapter concentrations.

[0189] Description of the sequences of the sequence protocolSEQ ID NO: 1: NF ATSEQ ID NO 2: IL-18 (aa)SEQ ID NO:3: IL18 (nt)SEQ ID NO:4: EFlaSEQ ID NO: 5: PGKSEQ ID NO:6: VH biotin (aa)SEQ ID NO:7: VH biotin (nt)SEQ ID NO:8: VL biotin (aa)SEQ ID NOV: VL biotin (nt)SEQ ID NO: 10: full binder biotin (aa)SEQ ID NO: 11 : full binder biotin (nt)SEQ ID NO: 12: hIgG4 hinge (aa)SEQ ID NO: 13: hIgG4 hinge (nt)SEQ ID NO: 14: CD8 TM (aa)SEQ ID NO: 15: CD8 TM (nt)SEQ ID NO: 16: CD28 (aa)SEQ ID NO: 17: CD28 (nt)SEQ ID NO: 18: 4-1BB (aa)SEQ ID NO: 19: 4-1BB (nt)SEQ ID NO:20: CD3 zeta (aa)SEQ ID NO:21: CD3 zeta (nt)SEQ ID NO: 22: Adapter CAR biotin (aa)SEQ ID NO:23: Adapter CAR biotin (nt)SEQ ID NO:24: anti-CD276 Adapter (VL)SEQ ID NO:25: anti-CD276 Adapter (VH)SEQ ID NO:26: anti-CD33 Adapter (VL)SEQ ID NO:27: anti-CD33 Adapter (VH)SEQ ID NO:28: anti-CD123 Adapter (VL)SEQ ID NO:29: anti-CD123 Adapter (VH)SEQ ID NO:30: anti-CD20 Adapter (VL) SEQ ID N0:31 : anti-CD20 Adapter (VH)SEQ ID NO:32: anti-CD19 Adapter (VL)SEQ ID NO 33: anti-CD19 Adapter (VH) References

[0190] 1 : Zhang L, Morgan RA, Beane ID, Zheng Z, Dudley ME, Kassim SH, Nahvi AV, Ngo LT, Sherry RM, Phan GQ, Hughes MS, Kammula US, Feldman SA, Toomey MA, Kerkar SP, Restifo NP, Yang JC, Rosenberg SA. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin- 12 for the immunotherapy of metastatic melanoma. Clin Cancer Res. 2015 May 15;21(10):2278-88. doi: 10.1158 / 1078- 0432.CCR-14-2085. Epub 2015 Feb 18. PMID: 25695689; PMCID: PMC4433819

Claims

Claims1. A combination of compositions for use in a method of treating a disease, wherein the disease is cancer or an autoimmune disease, the method comprising administering to a subj ect in need thereof : a. A composition (A) comprising a population of immune effector cells comprising i. A first exogenous nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain specific for a tag of a polypeptide ii. A second exogenous nucleic acid sequence encoding an effector molecule operably linked to an antigen-activated inducible promoter and b. A composition (B) comprising tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag c. A composition (C) comprising tagged polypeptides comprising an antigen binding domain specific for a target antigen expressed on the surface of a target cell and said tag wherein said antigen-activated inducible promoter drives the expression of said effector molecule upon binding of said antigen binding domain of said CAR to said tagged polypeptide that is bound to said target antigen expressed on said target cell wherein the amount of tagged polypeptides in composition C is lower compared to composition B, wherein composition B and composition C are administered to the subject separately from each other, wherein said tagged polypeptides of composition B and composition C are identical, wherein the effector molecule is an immune modulatory protein.

2. The combination of compositions for use in a method of treating a disease according to claim 1, wherein the level of expression of said effector molecule is dependent on the amount of the tagged polypeptides3. The combination of compositions for use in a method of treating a disease according to claim 1 or 2 , wherein the level of expression of said effector molecule is dependent on the amount of the tagged polypeptides and therefore varies between the presence of composition B and composition C in the presence of composition A in the subject, respectively.

4. The combination of compositions for use in a method of treating a disease according to any one of claims 1 to 3, wherein the amount of tagged polypeptide in composition C is in such a way that it activates the CAR immune effector cells thereby leading to killing of the target cell, but does not trigger the secretion of the effector molecule from the effector immune cell, and wherein the amount of tagged polypeptide in composition B is in such a way that it triggers the secretion of the effector molecule from the effector immune cell.

5. The combination of compositions for use in a method of treating a disease according to any one of claims 1 to 4, wherein said antigen-activated inducible promoter is selected from the group of: NF AT, NF-KB and AP-1.

6. The combination of compositions for use in a method of treating a disease according to any one of claims 1 to 5, wherein the effector molecule is IL-187. The combination of compositions for use in a method of treating a disease according to any one of claims 1 to 6, wherein the antigen binding domain of the CAR is specific for a hapten or a peptide sequence and wherein the tag of said tagged polypeptides is said hapten or said peptide sequence.

8. The combination of compositions for use in a method of treating a disease according to any one of claims 1 to 7, wherein said effector immune cells are T cells.

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