Regulation of transgene expression via induced reconstitution of heterodimeric synthetic transcription factors

Abstract

The invention is about a heterodimeric synthetic transcription factor system. Importantly the domains of the synthetic transcription factor are arranged in a specific order, the system comprising a) a first exogenous nucleic acid molecule encoding a first fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promoter operably linked to a nucleic acid encoding an effector molecule, b) a second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C-terminally fused to a second binding moiety, and c) an inducer molecule, wherein the first and second binding moieties are different from each other, and Wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule.

Description

Title Regulation of transgene expression via induced reconstitution of heterodimeric synthetic transcription factors Field of the invention

[0001] The invention is related to the field of transcriptional control and split synthetic transcription factors. Key applications for these systems are in immunotherapy, producer cell lines, cell and gene therapy and basic research. Background of the invention

[0002] CAR T cells have shown tremendous clinical success in a wide range of B cell malignancies, but thus far have had limited efficacy in other forms of cancer. Some of the main challenges still associated with the technology are on-target / off-tumor toxicity, cytokine release syndrome, antigen escape, poor expansion and persistence, and generally, limited control over CAR T cell function once infused into the patient.

[0003] Lack of control over CAR T cells might lead to life-threatening side effects that would put patients in high risk. Implementation of efficient means of controlling CAR T cells in case of adverse effects are needed for a safe therapy.

[0004] In addition, outcomes of clinical trials showed that expansion of CAR T cells during the first four weeks after infusion strongly correlates with clinical response. Strategies to enhance proliferation and persistence while preventing the risk of oncogenic transformation of therapeutic cells due to uncontrolled proliferation are highly desirable.

[0005] In summary, the main technical limitations in CAR T cell therapy are: - (1) Lack of control over CAR T cells can lead to life-threatening toxicity. - (2) Poor expansion and persistence in vivo leads to inefficacy of the therapy. - (3) In absence of safeguard mechanisms, strategies to enhances proliferation can lead to oncogenic transformation.

[0006] One solution to overcome those technical limitations is the implementation of drug- inducible molecular switches that enable control of the expression of effector molecules (such as chimeric antigen receptors (CAR), proliferation factors or other molecules) in human immune cells by addition of a small molecule. Based on that, the expression of e.g. chimeric antigen receptors is coupled to the presence of an inducer molecule (e.g. a small molecule). If no inducer molecule is present, no effector molecule such as a CAR is expressed, thereby reducing side effects which may be caused by said effector molecule such as a CAR.

[0007] In addition to that, effector molecules may be factors that enhance expansion, persistence, fitness and / or overall efficacy of immune cells. By using an inducible system, the expression can be induced and a proliferation factor such as a cytokine may be released thereby affecting proliferation and persistence of e.g. immune cells expressing a CAR.

[0008] Several mechanisms for such molecular switches have been disclosed. For example the transcriptional control of CAR expression with a synthetic transcription factor fused to the murine estrogen receptor (Kotter B. et al., Cancers 2021, WO2023187031A1). In addition to that, Sakemura et. al. (Cancer Immunol Res 2016) discloses the use of synthetic transcription factors based on the bacterial TetON system. These systems can be regulated in presence of doxycycline.

[0009] Major drawbacks of these systems are e.g. that they are inherently leaky, leading to background expression of the gene of interest in absence of the inducer molecule. Moreover most systems are highly immunogenic, which may cause severe side effects when used for clinical applications. In addition to that, all the strategies described thus far require either non- FDA approved drugs, or molecules with poor pharmacological properties hampering their clinical translation.

[0010] One option to overcome the given limitations may be the use of nanobodies and in combination with FDA approved drugs. For example the use of anti-caffeine nanobodies that bind caffeine and make a homodimer has been described (Bojar D. et al., Nature Communications 2018. Caffeine-inducible gene switches controlling experimental diabetes). However, these homodimer based switches, especially when used as transcriptional switches show substantial amount of leakiness and low responsiveness, meaning that high doses of caffeine are needed for inducing a response or gene expression. All in all the homodimer-based transcriptional switch showed low functionality.

[0011] Based on that, there is a need in the art for a new or alternative system for controllable expression of effector molecules (such as CARs or proliferation factors), especially in human immune cells. Such a system should be highly controllable, leading to high expression of the transgene in the presence of the inducer molecule and reducing background expression of an effector molecule to a minimum in the absence of the inducer molecule. Brief description of the invention

[0012] The invention provides a system for inducible expression of an effector molecule in a cell, preferentially in a human cell.

[0013] Surprisingly, it was found that a specific configuration (domains of the synthetic transcription factor are arranged in a specific order) of the domains in the heterodimeric synthetic transcription factor system plays an important role in induction efficiency and expression of effector molecules encoded by endogenous or exogenous nucleic acids. It was found that a heterodimeric synthetic transcription factor system comprising a DNA binding domain N-terminally fused to a first binding moiety and a transcription regulating domain C- terminally fused to a second binding moiety leads to higher response and / or expression (in case the transcription regulating domain is a transcription activation domain) of the effector (in cells comprising the system) encoded by a nucleic acid molecule compared to other domain configurations (Figure 2a-b, 5a-d).

[0014] The system could be even more improved by using two vectors, wherein one vector comprises the expression cassette for a DNA binding domain N-terminally fused to a first binding moiety and the second vector comprises a transcription regulating domain C terminally fused to a second binding moiety. The use of the two vector system lead to reduced or complete absence background expression of the effector molecule(in cells comprising the system) in the absence of an inducer molecule (Figure 5a-d).

[0015] Even more surprisingly it was found, that this specific domain configuration is generically applicable for different heterodimeric synthetic transcription factor systems such as Caffeine Nanobody (EP23176793) and Lipocalin (as disclosed in WO2019122188A1) and is superior in regards of background expression compared to other domain configurations (Figure 5a-d, Figure 15a-d).

[0016] In a first aspect the invention provides a system comprising (a) A first exogenous nucleic acid molecule encoding a first fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety (the first exogenous nucleic acid molecule encoding said first fusion protein comprises from the N- to the C-terminus: said DNA binding domain – said first binding moiety), wherein the DNA binding domain is specific for a regulatory element in the promoter operably linked to a nucleic acid molecule encoding an effector molecule, (b) a second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a second binding moiety (the second exogenous nucleic acid molecule encoding said second fusion protein comprises from the N- terminus to the C- terminus: said second binding moiety – said transcription regulating domain) and (c) an inducer molecule, wherein the first and second binding moieties are different from each other, and wherein the first and second binding moiety are capable to dimerize in presence of the inducermolecule. This system can be used to induce the expression of an effector molecule in a cell depending on the presence and absence of the inducer molecule (Figure 1, Figure 14).

[0017] In another aspect, the invention provides an engineered cell comprising (a) A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety (the first exogenous nucleic acid molecule encoding said first fusion protein comprises from the N- to the C-terminus: said DNA binding domain – said first binding moiety), wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule, (b) a second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a binding moiety(the second exogenous nucleic acid molecule encoding said second fusion protein comprises from the N- terminus to the C-terminus: said second binding moiety – said transcription regulating domain), wherein the first and second binding moieties are different from each other, and wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and wherein said engineered cell is in combination with the inducer molecule (Figure 1).

[0018] Another aspect of the invention is an in vitro method for inducing the expression of an effector molecule comprising the steps: Step 1: Providing a composition comprising engineered cells comprising; (a) A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety (the first exogenous nucleic acid molecule encoding said first fusion protein comprises from the N- to the C-terminus: said DNA binding domain – said first binding moiety), wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule; (b) a second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C-terminally fused to a binding moiety (the second exogenous nucleic acid molecule encoding said second fusion protein comprises from the N- terminus to the C-terminus: said second binding moiety – said transcription regulating domain), and wherein the first and second binding moieties are different from each other, and wherein the first and second binding moieties are capable to dimerize in presence of the inducer molecule, and Step 2: Adding the inducer molecule to the composition comprising engineered cells.

[0019] Another aspect of the invention describes a system according to the first aspect of the invention, wherein the first and second binding moieties comprise the binding domain of a single domain antibody (VHH) or a fragment thereof, respectively, wherein the bindingdomains are specific for Xanthine or a derivate thereof and wherein the inducer molecule is xanthine or a derivate thereof such as caffeine (Figure 1).

[0020] Another aspect of the invention describes a system according to the first aspect of the invention, wherein the first binding moiety comprises a lipocalin fold molecule and second binding moiety comprises a lipocalin fold binding interaction partner and wherein the inducer molecule is a lipocalin fold ligand (Figure 14). Brief description of the drawings

[0021] Figure 1. Regulation of transgene expression via drug-induced reconstitution of heterodimeric split synthetic transcription factors. Split synthetic transcription factors consists of two independent polypeptide chains: one of them contains a DNA binding domain (e.g. a N1 zinc finger protein) fused to the first member of caffeine-responsive heterodimeric nanobodies and a nuclear localization signal (NLS). The other chain is composed of an activation domain (e.g. p65 AD) fused to the second unit of the heterodimeric nanobodies pair and an NLS for nuclear localization. Induction with caffeine brings the N1 Zinc finger protein (DNA binding domain) and the NFκB p65 activation domain in close proximity leading to the reconstitution of a fully functional synthetic transcription factor and therefore, to the expression of the GOI encoded downstream of the N1 ZF binding sites (responsive elements). NLS: Nuclear localization signal; N1 ZF: N1 zinc finger protein; VHH1: anti-caffeine nanobody 1; VHH2: anti-caffeine nanobody 2; p65: transcription factor p65; AD: activation domain; Pmin: minimal promoter; GOI: gene of interest; polyA: polyadenylation signal.

[0022] Figure 2. Domain orientation of the heterodimeric transcriptional switch components influcences the inducer dose-dependent expression of anti-CD20 CAR. a) Lentiviral vector constructs of the nanbody-based drug-inducible transcriptional switch system (Nanoswitch 1-4). Expression of both units of the split synthetic transcription factor are driven by hPGKp as independent polypeptide chains by the use of a P2A self-cleaving peptide. N1 ZF and p65 AD were cloned in different orientations (Nanoswitch1-4). Inducible gene expression cassette is encoded in the reverse strand. 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; G4S: Glycine-Glycine-Glycine- Serine (SEQ ID No: 34) linker; NLS: Nuclear localization signal; N1 ZF: N1 Zinc Finger protein; VHH1: anti-caffeine nanobody 1; P2A: Porcine teschovirus-1 self-cleaving 2A peptide; VHH2: anti-caffeine nanobody 2; p65 AD: transcription factor p65 activation domain; Pmin: minimal promoter; aCD20 CAR: anti-CD20 chimeric antigen receptor; polyA: polyadenylation signal; 3’LTR : Three Prime Long Terminal Repeat. b) Frequency of anti-CD20 CAR expressing cells in presence of different concentrations of caffeine. Human T cell line SupT1 cells were transduced with Nanoswitch1-4 and cultured in presence of caffeine at a concentration ranging between 0 μM and 500 μM. Frequency of anti-CD20 CAR was determined by flow cytometry 30 days post-induction. Bar plot depicts a representative experiment (n=3 independent experiments). c(Caffeine): concentration of caffeine; Tdx: transduction efficiency.

[0023] Figure 3. Long-term stability of the transcriptional switch and induced anti-CD20 CAR expression. a) Frequency of FLAG-tag+ cells over time. Human T cell line SupT1 cells were transduced with a Nanoswitch lentiviral vector and cultured in presence of caffeine at a concentration ranging between 0 μM and 500 μM. Tag expression was determined via flow cytometry at different timepoints post-induction. Line plot depicts a representative experiment (n=3 independent experiments). b) Frequency of anti-CD20+ cells over time. Human T cell line SupT1 cells were transduced with a Nanoswitch lentiviral vector and cultured in presence of caffeine at a concentration ranging between 0 μM and 500 μM. Anti-CD20 CAR expression was determined via flow cytometry at different timepoints post-induction. Line plot depicts a representative experiment (n=3 independent experiments).

[0024] Figure 4. ON / OFF expression kinetics of caffeine-induced anti-CD20 CAR expression. a) Timeline depicting the culturing and induction strategy followed to study the kinetics of caffeine-induced gene expression and the subsequent downregulation upon inducer withdrawal. Human T cell line SupT1 cells were transduced with Nanoswitch1-4 and cultured in presence of caffeine at a concentration ranging between 0 μM and 500 μM.. Induction was discontinued on day 15 post-induction. Induction was reactivated by addition of caffeine on day 22 after the first induction. d: day. b) Frequency of anti-CD20 CAR+ cells over time upon induction, drug discontinuation and reinduction with caffeine. Anti-CD20 CAR expression was determined by flow cytometry on the indicated timepoints. For clarity, only data regarding 500 μM Caffeine is shown. Line plot depicts a representative experiment (n=3 independent experiments). d: days. c) Expression level of anti-CD20 CAR (mean fluorescence intensity) over time upon induction, drug discontinuation and reinduction with caffeine. Anti-CD20 CAR expression was determined by flow cytometry on the indicated timepoints. For clarity, only data regarding 500 μM Caffeine is shown. Line plot depicts a representative experiment (n=3 independent experiments). MFI: mean fluorescence intensity; d: days.

[0025] Figure 5. Domain orientation of the heterodimeric transcriptional switch influcences the inducer dose-dependent expression of anti-CD20 CAR in primary humanT cells. a) Lentiviral vector constructs for expression of the first unit of the split synthetic transcription factor of the nanbody-based drug-inducible transcriptional switch system (Nanoswitch 5 & 6). LVI: Lentivirus I; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; N1 ZF: N1 Zinc Finger protein; G4S: Glycine- Glycine-Glycine-Serine (SEQ ID No: 34) linker; VHH1: anti-caffeine nanobody 1; 3xFLAG: three repeats of FLAG-tag; NLS: Nuclear localization signal; 3’LTR : Three Prime Long Terminal Repeat. b) Lentiviral vector constructs for expression of the second unit of the split synthetic transcription factor of the nanbody-based drug-inducible transcriptional switch system and the inducible gene expression casette for control of anti-CD20 CAR (Nanoswitch 7 & 8). Inducible gene expression cassette is encoded in the reverse strand. LVII: Lentivirus II; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; VHH2: anti-caffeine nanobody 2; G4S: Glycine-Glycine-Glycine-Serine (SEQ ID No: 34) linker; p65 AD: transcription factor p65 activation domain; 3xHA: 3 repeats of human influenza hemagglutinin tag; NLS: Nuclear localization signal; Pmin: minimal promoter; aCD20 CAR: anti-CD20 chimeric antigen receptor; polyA: polyadenylation signal; 3’LTR : Three Prime Long Terminal Repeat. w / o: whithout caffeine. c) Frequency of anti- CD20 CAR+ cells normalized to the frequency of transduced cells (HA-tag+ cells) upon induction in human primary T cells. Human primary T cells transduced with the indicated combinations of LVI and LVII versions were cultured either in the absence or presence of 250 µM caffeine. Frequency of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction (n=3). d) Expression level of anti-CD20 CAR (mean fluorescence intensity) in human primary T cells. Human primary T cells transduced with the indicated combinations of LVI and LVII versions were cultured either in the absence or presence of 250 µM caffeine. Frequency of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction (n=3). w / o: without caffeine; MFI: Mean Fluorescence Intensity; conv.: conventional.

[0026] Figure 6. Influence of the duration of induction and Caffeine dose-dependent expression of anti-CD20 CAR in primary human T cells double transduced with Nanoswitches 5 & 7. a) Frequency of anti-CD20 CAR-expressing human primary T cells upon induction with the indicated concentrations of caffeine (n=3). Determination via flow cytometry 24 hours post-induction. b) Expression level of anti-CD20 CAR in human primary T cells measured as mean fluorescence intensity. c) Frequency of anti-CD20 CAR+ cells upon induction for the specified duration. Human primary T cells transduced with a combination of Nanoswitch 5 and 7 were induced with 250 µM of caffeine during thespecified amount of time. Anti-CD20 CAR was detected by flow cytometry after 72 hours post- induction (n=3). d) Expression level of anti-CD20 CAR (mean fluorescence intensity) upon induction for the specified duration. Human primary T cells transduced with a combination of Nanoswitch 5 and 7 were induced with 250 µM of caffeine during the specified amount of time. Anti-CD20 CAR was detected by flow cytometry after 72 hours post-induction (n=3). c(caffeine): concentration of caffeine; MFI: mean fluorescence intensity; h: hours.

[0027] Figure 7. Cytotoxic activity of drug-inducible anti-CD20 CAR T cells in cocultures with GFP+ CD20+ Raji cells. a) Specific tumor lysis by conventional and drug-inducible anti-CD20 CAR upon induction with different doses of caffeine. Frequency of GFP+ cells was determined by flow cytometry 24 hours after the start of the coculture and normalized by the frequency of GFP+ cells in the tumor only control (Raji cells without T cells) (n=3). Caffeine at the specified concentration was added during the setup of the cocultures. GFP: green fluorescent protein; c(Caffeine): concentration of caffeine. b) Tumor GFP+ CD20+ Raji cell line concentration 24 hours post-coculture with conventional and drug-inducible anti- CD20 CAR T cells. Plots indicate the number of GFP+ cells per ml at the end of the assay. Conv.: conventional; GFP: green fluorescent protein; c(Caffeine): concentration of caffeine.

[0028] Figure 8. Modulation of the strength of induced expression of anti-CD20 CAR by the use of different activation domains. a) Lentiviral vector construct for expression of the first unit (SEQ ID NO: 27) of the split synthetic transcription factor of the nanbody-based drug-inducible transcriptional switch system (Nanoswitch 5). LVI: Lentivirus I; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; N1 ZF: N1 Zinc Finger protein; G4S: Glycine-Glycine-Glycine-Serine (SEQ ID No: 34) linker; VHH1: anti-caffeine nanobody 1; 3xFLAG: three repeats of FLAG-tag; NLS: Nuclear localization signal; 3’LTR : Three Prime Long Terminal Repeat. b) Versions of Nanoswitch 7 with different activation domains. Lentiviral vector construct for expression of the second unit of the split synthetic transcription factor of the nanbody-based drug-inducible transcriptional switch system and inducible gene expression casette for control of anti- CD20 CAR. Inducible gene expression cassette is encoded in the reverse strand. LVII: Lentivirus II; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; VHH2: anti-caffeine nanobody 2; G4S: Glycine-Glycine-Glycine-Serine (SEQ ID No: 34) linker; p65 AD: transcription factor p65 activation domain; VP64 AD: VP64 activation domain, tetrameric repeat of Human herpes simplex virus 1 Tegument protein VP16 amino acids 437-448 fragment; p65520-551AD: transcription factor p65 activation domain amino acids 520-551 fragment; CITED2 AD: Cbp / p300-interacting transactivator 2 activation domainamino acids 161-270 fragment; 3xHA: 3 repeats of human influenza hemagglutinin tag; NLS: Nuclear localization signal; Pmin: minimal promoter; aCD20 CAR: anti-CD20 chimeric antigen receptor; polyA: polyadenylation signal; 3’LTR : Three Prime Long Terminal Repeat. c) Expression level of drug-induced anti-CD20 CAR controlled by Nanobody-based Transcriptional Switches using different activation domains. Human primary T cells transduced with a combination of Nanoswitch 5 and each of the versions of Nanoswitch 7 carrying different domains were induced with 250 µM of caffeine. Expression anti-CD20 CAR (mean fluorescence intensity) was determined by flow cytometry 24 hours post-induction (n=3). w / o: without caffeine; MFI: Mean Fluorescence Intensity; conv.: conventional.

[0029] Figure 9. Evaluation of different DNA binding domains for the drug-induced expression of anti-CD20 CAR. a) Versions of lentiviral vector Nanoswitch 5 for the expression of the first unit of the split synthetic transcription factor carrying different DNA binding domains. LVI: Lentivirus I; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; N1 ZF: N1 Zinc Finger protein; GAL4 DBD: DNA binding domain of the regulatory protein GAL4; CCR5-224 ZF: synthetic zinc finger protein targeting CCR5 locus; G4S: Glycine-Glycine-Glycine-Serine (SEQ ID No: 34) linker; VHH1: anti-caffeine nanobody 1; 3xFLAG: three repeats of FLAG-tag; NLS: Nuclear localization signal; 3’LTR : Three Prime Long Terminal Repeat. b) Versions of lentiviral vector Nanoswitch 7 bearing different responsive elements with binding sites for either N1 ZF, GAL4 DBD or CCR5-224 ZF. Inducible gene expression cassette is encoded in the reverse strand. LVII: Lentivirus II; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; VHH2: anti-caffeine nanobody 2; G4S: Glycine-Glycine- Glycine-Serine (SEQ ID No: 34) linker; p65 AD: transcription factor p65 activation domain; 3xHA: 3 repeats of human influenza hemagglutinin tag; NLS: Nuclear localization signal; Pmin: minimal promoter; aCD20 CAR: anti-CD20 chimeric antigen receptor; polyA: polyadenylation signal; 3’LTR : Three Prime Long Terminal Repeat. w / o: whithout caffeine. c) Expression level of drug-induced anti-CD20 CAR controlled by Nanobody-based Transcriptional Switches carrying different DNA binding domains and their corresponding responsive elements. Human primary T cells transduced with a combination of each of the Nanoswitch 5 versions and the corresponding Nanoswitch 7 version were induced with 250 µM of caffeine. Expression anti-CD20 CAR (mean fluorescence intensity) was determined by flow cytometry 24 hours post-induction (n=3). w / o: without caffeine; MFI: Mean Fluorescence Intensity; conv.: conventional.

[0030] Figure 10. Drug-inducible expression of factors involved in the JAK-STAT pathway enhances the persistence and expansion of CAR T cells in an inducer dose dependent manner. a) Versions of lentiviral vector I (LVI) NanoFITswitch 1 for the constitutive expression of a CAR and the first unit of the split synthetic transcription factor. LVI: Lentivirus I; 5’ LTR: Five prime Long Terminal Repeat; hEF1α: human Elongation factor 1α promoter; aCD20 CAR: anti-CD20 chimeric antigen receptor; P2A: 2A self-cleaving peptide from Porcine teschovirus; N1 ZF: N1 Zinc Finger protein; G4S: Glycine- Glycine-Glycine-Glycine-Serine (SEQ ID No: 36) linker; VHH1: anti-caffeine nanobody 1; 3xFLAG: three repeats of FLAG-tag; NLS: Nuclear localization signal; 3’LTR : Three Prime Long Terminal Repeat. b) Versions of lentiviral vector II (LVII) NanoFITswitch 2 bearing different drug-inducible proliferation factors. Inducible gene expression cassette is encoded in the reverse strand. LVII: Lentivirus II; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; VHH2: anti-caffeine nanobody 2; G4S: Glycine- Glycine-Glycine-Glycine-Serine (SEQ ID No: 36) linker; p65 AD: transcription factor p65 activation domain; 3xHA: 3 repeats of human influenza hemagglutinin tag; NLS: Nuclear localization signal; Pmin: minimal promoter; prolif. Factor: proliferation factor; GRβ: Glucocorticoid Receptor β; STAT5b N642H: signal transducer and activator of transcription 5B carrying N642H mutation; IL-15: human interleukin 15; ch. IL-15Rα: chimeric human interleukin 15 receptor alpha; polyA: polyadenylation signal; 3’LTR : Three Prime Long Terminal Repeat. c) Timeline depicting the culturing and induction strategy followed to perform the cytokine starvation assay and evaluate the persistence of T cells transduced with different inducible proliferation factors. Human T cells transduced with LVI, NanoFITswitch 1, and different versions of LVII, NanoFITSwitch 2, were expanded in media with cytokines until day 8. Then, cells were adjusted to the same count, induced with different concentrations of caffeine ranging between 0 μM and 500 μM and further cultured for 8 additional days in absence of cytokines. d) Persistence of primary human CAR T cells cultured in the absence of cytokines upon drug-induced expression of proliferation factors. Viable cell count was determined by flow cytometry upon staining with viability dye 7AAD on day 8 after cytokine withdrawal and induction. C(Caffeine): concentration of caffeine. (n = 2, error bars represent the standard error of the mean).

[0031] Figure 11. Cytotoxic activity and level of expression of drug-inducible anti-CD20 CAR T cells in cocultures with GFP+ CD20+ 526 Mel cells and different doses of caffeine. a) Specific tumor lysis by conventional and drug-inducible anti-CD20 CAR upon induction with different doses of caffeine. GFP fluorescence intensity of GFP+ CD20+ 526Mel tumor cells was monitored by live-cell imaging every 2 hours for 92 hours in cocultures with drug-inducible CAR T cells in presence of the specified doses of caffeine (n=2). Line plot represents the integrated fluorescence intensity normalized to the measurement at the start of the coculture. Doted lines represent the standard error of the mean (SEM) of two donors. b) Frequency of anti-CD20 CAR+ cells achieved upon induction with specified concentrations of caffeine for the duration of the coculture. Human primary T cells co- transduced with Nanoswitch 5 & 7 employed for the cytotoxicity assay showed a dose- dependent frequency of anti-CD20 CAR+ cells after 92 hours determined by flowcytometry. c) Level of expression of anti-CD20 CAR+ cells achieved upon induction with specified concentrations of caffeine for the duration of the coculture. Human primary T cells co- transduced with Nanoswitch 5 & 7 employed for the cytotoxicity assay showed a dose- dependent level of expression of anti-CD20 CAR+ cells after 92 hours determined by flowcytometry

[0032] Figure 12. ON / OFF expression kinetics of caffeine-induced anti-CD20 CAR expression in human primary T cells. a) Timeline depicting the culturing and induction strategy followed to study the kinetics of caffeine-induced gene expression and the subsequent downregulation upon inducer withdrawal and upregulation after re- induction. Human primary T cells transduced with Nanoswitch5+7 and cultured either in absence of presence of 250 µM of caffeine as specified in the diagram. b) Frequency of anti- CD20 CAR overtime upon induction, withdrawal and re-induction determined by flow cytometry. Error bars represent standard error (n= 3). c) Level of expression of anti-CD20 CAR overtime upon induction, withdrawal and re-induction determined by flow cytometry. Error bars represent standard error (n= 3).

[0033] Figure 13. Responsiveness of drug-inducible nanobody-based transcriptional switches by different xanthine derivatives. a) Chemical structure of caffeine. b) Chemical structure of doxofylline. c) Frequency of anti-CD20 CAR+ cells upon induction with caffeine or doxofylline. Expression of anti-CD20 CAR in primary human T cells was measured by immunostaining and flow cytometry upon induction with specified concentrations of either caffeine or doxofylline and incubation for 72 hours. c(inducer): concentration of inducer; w / o: without inducer. d) Level of expression of anti-CD20 CAR in primary human T cells upon induction with caffeine or doxofylline. Level of expression of anti-CD20 CAR in primary human T cells was measured by immunostaining and flow cytometry upon induction with specified concentrations of either caffeine or doxofylline and incubation for 72 hours. MFI: mean fluorescence intensity; c(inducer): concentration of inducer; w / o: without inducer.

[0034] Figure 14. Regulation of transgene expression via drug-induced reconstitution of heterodimeric split synthetic transcription factors. Split synthetic transcription factors consists of two independent polypeptide chains: one of them contains a DNA binding domain (e.g. a N1 zinc finger protein) fused to a variant of the human retinol binding protein 4 (RBP4) and a nuclear localization signal (NLS). The other chain is composed of an activation domain (e.g. p65 AD) fused to a specific engineered binder derived from the tenth type III domain of the human fibronectin and an NLS for nuclear localization. Induction with A1120 (PubChem CID 25138295; chemical name: 2-(4-(2-(Trifluoromethyl)phenyl)piperidine-1- carboxamido)benzoic acid) brings the N1 Zinc finger protein (DNA binding domain) and the NFκB p65 activation domain in close proximity leading to the reconstitution of a fully functional synthetic transcription factor and therefore, to the expression of the GOI encoded downstream of the N1 ZF binding sites (responsive elements). NLS: Nuclear localization signal; N1 ZF: N1 zinc finger protein; RBP4: human retinol binding protein 4 variant; RF2: engineered binder derived from the tenth type III domain of the human fibronectin; p65: transcription factor p65; AD: activation domain; Pmin: minimal promoter; GOI: gene of interest; polyA: polyadenylation signal.

[0035] Figure 15. Domain orientation of the heterodimeric transcriptional switch influcences the inducer dose-dependent expression of anti-CD20 CAR in primary human T cells. a) Lentiviral vector constructs for expression of the first unit of the split synthetic transcription factor of the lipocalin-based drug-inducible transcriptional switch system (Liposwitch 1 & 2). LVI: Lentivirus I; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; N1 ZF: N1 Zinc Finger protein; G4S: Glycine- Glycine-Glycine-Glycine-Serine (SEQ ID No: 36) linker; RBP4: retinol binding protein 4; 3xFLAG: three repeats of FLAG-tag; NLS: Nuclear localization signal; 3’LTR : Three Prime Long Terminal Repeat. b) Lentiviral vector constructs for expression of the second unit of the split synthetic transcription factor of the nanbody-based drug-inducible transcriptional switch system and the inducible gene expression casette for control of anti- CD20 CAR (Liposwitch 3 & 4). Inducible gene expression cassette is encoded in the reverse strand. LVII: Lentivirus II; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; RF2: engineered binder derived from the tenth type III domain of human fibronectin; G4S: Glycine-Glycine-Glycine-Glycine-Serine (SEQ ID No: 36) linker; p65 AD: transcription factor p65 activation domain; 3xHA: 3 repeats of human influenza hemagglutinin tag; NLS: Nuclear localization signal; Pmin: minimal promoter; aCD20 CAR: anti-CD20 chimeric antigen receptor; polyA: polyadenylation signal; 3’LTR : Three PrimeLong Terminal Repeat. w / o: whithout caffeine. c) Frequency of anti-CD20 CAR+ cells normalized to the frequency of transduced cells (HA-tag+ cells) upon induction in human primary T cells. Human primary T cells transduced with the indicated combinations of LVI and LVII versions were cultured either in the absence or presence of 50 µM A1120. Frequency of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction (n=3). d) Expression level of anti-CD20 CAR (mean fluorescence intensity) in human primary T cells. Human primary T cells transduced with the indicated combinations of LVI and LVII versions were cultured either in the absence or presence of 50 µM A1120. Mean fluorescence intensity of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction (n=3). w / o: without A1120; c(A1120): concentration of A1120; MFI: Mean Fluorescence Intensity; conv.: conventional; sTF: synthetic Transcription Factor. e) Frequency of anti-CD20 CAR+ cells normalized to the frequency of transduced cells (HA- tag+ cells) upon induction in human SupT1 T cell line. Human cell line SupT1 was transduced with the indicated combinations of LVI and LVII versions were cultured either in the absence or presence of 50 µM A1120. Frequency of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction. f) Expression level of anti-CD20 CAR (mean fluorescence intensity) in SupT1 cells. Human SupT1 cells transduced with the indicated combinations of LVI and LVII versions were cultured either in the absence or presence of 50 µM A1120. Mean fluorescence intensity of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction (n=3). w / o: without A1120; c(A1120): concentration of A1120; MFI: Mean Fluorescence Intensity; conv.: conventional; UTD: untransduced.

[0036] Figure 16. Comparison in small molecule-induced expression of anti-CD20 CAR and background expression in absence of A1120 of lipocalin-based switches with different variants of engineered binder RF2. a) Lentiviral vector constructs for expression of the first unit of the split synthetic transcription factor of the lipocalin-based drug-inducible transcriptional switch system (Liposwitch 1). LVI: Lentivirus I; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; N1 ZF: N1 Zinc Finger protein; G4S: Glycine-Glycine-Glycine-Glycine-Serine (SEQ ID No: 36) linker; RBP4: retinol binding protein 4; 3xFLAG: three repeats of FLAG-tag; NLS: Nuclear localization signal; 3’LTR : Three Prime Long Terminal Repeat. b) Lentiviral vector constructs for expression of the second unit of the split synthetic transcription factor of the nanbody-based drug- inducible transcriptional switch system and the inducible gene expression casette for control of anti-CD20 CAR (Liposwitch 3 & 5). Inducible gene expression cassette is encodedin the reverse strand. LVII: Lentivirus II; 5’ LTR: Five prime Long Terminal Repeat; hPGKp: human Phosphoglycerate Kinase promoter; RF2: engineered binder derived from the tenth type III domain of human fibronectin; RF2: alternative variant of engineered binder derived from the tenth type III domain of human fibronectin; G4S: Glycine-Glycine-Glycine-Glycine-Serine (SEQ ID No: 36) linker; p65 AD: transcription factor p65 activation domain; 3xHA: 3 repeats of human influenza hemagglutinin tag; NLS: Nuclear localization signal; Pmin: minimal promoter; aCD20 CAR: anti-CD20 chimeric antigen receptor; polyA: polyadenylation signal; 3’LTR : Three Prime Long Terminal Repeat. w / o: whithout caffeine. c) Frequency of anti- CD20 CAR+ cells upon induction in human primary T cells. Human primary T cells transduced with Liposwitch 1 and the indicated LVII versions were cultured either in the absence or presence of 100 nM of A1120. Frequency of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction (n=3). No background expression was observed in absence of A1120. Error bars indicate standard deviation. d) Expression level of anti-CD20 CAR (mean fluorescence intensity) in human primary T cells. Human primary T cells transduced with Liposwitch 1 and the indicated LVII versions were cultured either in the absence or presence of 100 nM A1120. Frequency and intensity of anti-CD20 CAR expressing cells was determined via flow cytometry on day 3 post-induction (n=3). w / o: without A1120; c(A1120): concentration of A1120; MFI: Mean Fluorescence Intensity; conv.: conventional; sTF: synthetic Transcription Factor. Error bars indicate standard deviation.

[0037] Figure 17. A1120 dose-dependent expression of anti-CD20 CAR in primary human T cells double transduced with lipocalin-based transcriptional switches (Liposwitch 1 & 3 or Liposwitch 1 & 5). a) Frequency of anti-CD20 CAR-expressing cells upon induction with the indicated concentrations of A1120 in human primary T cells transduced with Liposwitch1+3. Determination via flow cytometry 72 hours post-induction. Error bars indicate standard deviation (n=3). b) Frequency of anti-CD20 CAR-expressing cells upon induction with the indicated concentrations of A1120 in human primary T cells transduced with Liposwitch1+5. Determination via flow cytometry 72 hours post-induction. Error bars indicate standard deviation (n=3). c) Expression level of anti-CD20 CAR+ cells measured as mean fluorescence intensity in human primary T cells transduced with Liposwitch1+3. Determination via flow cytometry 72 hours post-induction with the indicated concentrations of A1120. Error bars indicate standard deviation (n=3). d) Expression level of anti-CD20 CAR+ cells measured as mean fluorescence intensity in human primary T cells transduced with Liposwitch1+5. Determination via flow cytometry 72 hours post-induction with the indicated concentrations of A1120. Error bars indicate standard deviation (n=3). e) Frequency of anti-CD20 CAR-expressing cells upon induction with the indicated concentrations of A1120 in human SupT1 cells transduced with Liposwitch1+3. Determination via flow cytometry 72 hours post-induction. f) Expression level of anti-CD20 CAR+ cells measured as mean fluorescence intensity in human SupT1 cells transduced with Liposwitch1+3. Determination via flow cytometry 72 hours post-induction with the indicated concentrations of A1120. g) Frequency of anti-CD20 CAR-expressing cells upon induction with the indicated concentrations of A1120 in human SupT1 cells transduced with Liposwitch1+5. Determination via flow cytometry 72 hours post-induction. h) Expression level of anti-CD20 CAR+ cells measured as mean fluorescence intensity in human SupT1 cells transduced with Liposwitch1+5. Determination via flow cytometry 72 hours post-induction with the indicated concentrations of A1120.

[0038] Figure 18. Cytotoxic activity of drug-inducible anti-CD20 CAR T cells in either absence or presence of 100 nM A1120 cocultured with GFP+ CD20+ 526 Mel tumor cells. a) Specific tumor lysis by conventional and drug-inducible anti-CD20 CAR upon induction with 100 nM A1120 at an efector cells to target cells (E:T) ratio of 1:1. GFP fluorescence intensity of GFP+ CD20+ 526 Mel tumor cells was monitored by live-cell imaging every 2 hours for 72 hours in cocultures with drug-inducible CAR T cells in presence of 100 nM A1120 or absence of indcuer. Line plot represents the integrated fluorescence intensity normalized to the measurement at the start of the coculture (t=0). Doted lines represent the standard error of the mean (SEM) from 4 pictures taken per timepoint of each donor (n=3). b) Specific tumor lysis by conventional and drug-inducible anti-CD20 CAR upon induction with 100 nM A1120 at an efector cells to target cells (E:T) ratio of 1:2. GFP fluorescence intensity of GFP+ CD20+ 526 Mel tumor cells was monitored by live-cell imaging every 2 hours for 72 hours in cocultures with drug-inducible CAR T cells in presence of 100 nM A1120 or absence of indcuer. Line plot represents the integrated fluorescence intensity normalized to the measurement at the start of the coculture (t=0). Doted lines represent the standard error of the mean (SEM) from 4 pictures taken per timepoint of each donor (n=3). Detailed description of the invention

[0039] The invention provides a system for inducible expression of an effector molecule in a cell, preferentially in a human cell. The expression of the effector molecule is dependent on the presence or absence of an inducer molecule.

[0040] As mentioned above the invention is about a heterodimeric synthetic transcription factor system. Importantly the domains of the synthetic transcription factor are arranged in a specific order. For this reason the first aspect of the invention provides a system comprising a. A first exogenous nucleic acid molecule encoding a first fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety (i.e. said first fusion protein comprises from the N- to the C-terminus said DNA binding domain – said first binding moiety), wherein the DNA binding domain is specific for a regulatory element in the promoter operably linked to a nucleic acid encoding an effector molecule b. A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C-terminally fused to a second binding moiety (i.e. said second fusion protein comprises from the N- terminus to the C-terminus said second binding moiety – said transcription regulating domain), c. An inducer molecule Wherein the first and second binding moieties are different from each other, and Wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule.

[0041] The system disclosed in the first aspect of the invention is comprised of three key features: - first fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety - second fusion protein comprising a transcription regulating domain C terminally fused to a second binding moiety - An inducer molecule

[0042] Importantly the first and second binding moieties are different from each other and the binding moieties are capable to dimerize in presence of the inducer molecule. In other words the first and second binding moieties form a heterodimer in the presence of the inducer molecule.

[0043] Based on the heterodimer formation of the first and second binding moieties in presence of the inducer molecule, the DNA binding domain and the transcription regulating domain are brought in close proximity to each other and a functional synthetic transcription factor is formed (Figure 1).

[0044] As the DNA binding domain of the first fusion protein is specific for a regulatory element in the promoter operably linked to a nucleic acid molecule encoding an effector molecule, the synthetic transcription factor can bind to this regulatory element and thereby regulating transcription of nucleic acid molecule encoding an effector molecule (Figure 1).Expression of the system in a cell

[0045] As disclosed herein, the invention provides a system for inducible expression of an effector molecule in a cell, preferentially in a human cell. The expression of the effector molecule is dependent on the presence or absence of an inducer molecule.

[0046] Said cells may have been engineered to express the system as disclosed herein.

[0047] Said cell may be a human cell. In one embodiment of the invention said cell may be an immune cell or immune cell subsets preferentially T cell, tumor infiltrating lymphocytes (TILs) or NK cells. In a preferred embodiment said immune cell is a T cell.

[0048] In another embodiment of the invention the cell may be a producer cell line, which can be used for vector production, such as viral (preferentially lentiviral) vector production. The producer cell lines may be selected from the group consisting of: HEK 293, HEK 293T, HEK293F and CHO. First and second binding moieties

[0049] One of the key elements of the system are the first and second binding moieties. In one embodiment of the invention the first and second binding moieties may be not naturally occurring binding moieties. In other words, the first and second binding moieties may be engineered molecules or binding moieties that are capable to heterodimerize in the presence of an inducer molecule. Several techniques for engineering of such binding moieties are known in the art (EP23176793 and WO2019122188A1).

[0050] Moreover the first and second binding moieties may be any molecule or protein capable to form heterodimeric macromolecular complexes.

[0051] In one embodiment of the invention the first and second binding moieties may be or may comprise a single domain antibody, full length heavy chain, Fab fragments, single chain Fv (scFv) fragments or VHH fragments. In a preferred embodiment of the invention the binding domain of a single domain antibody (VHH) or a fragment thereof. It is understood that both binding moiety polypeptides generate a heterodimerization complex specific for the inducer molecule (e.g. as disclosed in EP23176793). In one embodiment of the invention the moieties comprise the binding domain of a single domain antibody (VHH) or a fragment thereof, respectively, wherein the binding domains are specific for Xanthine or a derivate thereof and wherein the inducer molecule is xanthine or a derivate thereof such as doxofylline.

[0052] In another embodiment of the invention, the first binding moiety may be or may comprise a lipocalin fold molecule and the second binding moiety may be or may comprise alipocalin fold binding interaction partner, as disclosed in WO2019122188A1. In this embodiment of the invention the first binding moiety (lipocalin fold molecule) may be or may comprise lipocalin, a functional mutated lipocalin or a functional fragment thereof such as RBP4 or a mutated variant of RBP4. Moreover, the second binding moiety is a the lipocalin fold interaction partner such as an anticaline e.g. FN3 or a functional variant of FN3 or a functional fragment thereof. In this specific system the inducer molecule is a lipocalin fold ligand (Figure 14).

[0053] The first and second fusion proteins comprise two components that are fused to each other: the DNA binding domain with the first binding moiety and the transcription regulating domain with the second binding moiety. Such fusion proteins are generated by methods known in the art. The nucleic acid sequences of each fusion protein were engineered in a way that both components of the fusion protein are expressed together in one transcript, thereby linking the expression of both proteins and generating a fusion protein.

[0054] The components of each fusion protein (the DNA binding domain with the first binding moiety and the transcription regulating domain with the second binding moiety) may be linked via a linker structure. This linker structure may be a Gly-rich peptide composed by 4 to 30 amino acids, such as G4S linker. In one embodiment of the invention the linker structure may be or may comprise at least one, at least two, at least three G4S linker, preferentially one G4S linker.

[0055] In addition to that, the first and / or second fusion protein may comprise at least one nuclear localization signal (NLS). This signal may be localized on the N and / or C terminus of the fusion protein. In a preferred embodiment of the invention the NLS signal may be located on the C terminus of each fusion protein.

[0056] In one embodiment of the invention the components of each fusion protein (the DNA binding domain with the first binding moiety and the transcription regulating domain with the second binding moiety) are linked via a G4S linker structure. In addition to that, in this embodiment of the invention the first and the second fusion protein may comprise one nuclear localization signal (NLS), which is located on the C terminus of each fusion protein (Figure 2a, 5a,b, 8a,b, 9a,b). Transcription regulating domain

[0057] Another key element of the invention is the second fusion protein comprising a transcription regulating domain C-terminally fused to a second binding moiety. Thetranscription regulating domain may be a transcription activating domain (TA) or a transcription repressing domain (TR).

[0058] In one embodiment of the invention the transcription regulating domain may be a transcription activation (or activating) domain. This transcription activation domain of a synthetic transcription factor refers to a protein or a potion thereof that autonomously facilitates the recruitment of the transcriptional machinery to initiate mRNA transcription. Examples for such transcription activating domains are fragments or tandem repeats of fragments of e.g. VP16, VP64, NFκB p65, CITED2, MYC, cmyc, HSF1, Oct4, Sox2, Klf4, Klf6, Hif1alpha, STAT3, STAT5. In a preferred embodiment of the invention the transcription activating domains are fragments or tandem repeats of fragments selected from the group consisting of NFκB p65( equivalent to the wording p65) or CITED2 (or VP16).

[0059] In one embodiment of the invention the transcription activating domains are fragments or tandem repeats of fragments of NFκB p65. Based on that, the transcription activating domain (which is NFκB p65) may comprise or consist of SEQ ID No: 12. In another embodiment of the invention the transcription activating domain may be a fragment or a tandem repeat of NFκB p65 and may comprise or consist of SEQ ID No: 13 or SEQ ID No: 14.

[0060] In one embodiment of the invention the transcription activating domains are fragments or tandem repeats of fragments of VP16. Based on that, the transcription activating domain (which is VP16) may comprise or consist of SEQ ID No: 15. In another embodiment of the invention the transcription activating domain may be a fragment or a tandem repeat of VP16 and may comprise or consist of SEQ ID No: 16 or SEQ ID No: 17 (also known as VP64).

[0061] In one embodiment of the invention the transcription activating domains are fragments or tandem repeats of fragments of CITED2. Based on that, the transcription activating domain (which is CITED2) may comprise or consist of SEQ ID No: 18. In another embodiment of the invention the transcription activating domain may be a fragment or a tandem repeat of CITED2 and may comprise or consist of SEQ ID No: 19.

[0062] In one embodiment of the invention the transcription regulating domain may be a transcription repressing domain. This domain of a synthetic transcription factor refers to a protein or a portion thereof that represses the recruitment of the transcriptional machinery to repress mRNA transcription. Common examples of such transcription repressing domains are Krüppel associated box (KRAB) repression domain, mSin Interaction Domain (SID), PIE-1 repression domain (PIE-1), QA domain within the Ubx gene, IAA28 repression domain, Tbx3 repression domain and DNA methyltransferase 3a3L (DNMT3a3L), Zim3.

[0063] In one embodiment of the invention the transcription regulating domain may be a transcription activation (or activating) domain which is C-terminally fused to a second binding moiety and may be selected from the group consisting of NFκB p65 or CITED2 (or VP16), preferentially NFκB p65 or fragments thereof or tandem repeats of these fragments. DNA binding domain

[0064] The first fusion protein comprises a DNA binding domain N-terminally fused to a first binding moiety. This DNA binding domain is specific for a DNA binding site in a regulatory element in a promoter. Said promotor is operably linked to a nucleic acid molecule encoding an effector molecule. The DNA binding domain of a synthetic transcription factor may be a protein or a fragment thereof. Such a DNA binding domain of the synthetic transcription factor may comprise at least one DNA recognition sequence, which is specific for a DNA binding site in the regulatory element of the promotor. The DNA binding domain mediates binding of the synthetic transcription factor to this specific DNA sequence.

[0065] Several proteins or fragments thereof are capable to bind such a DNA binding site comprised in a regulatory promotor such as zinc finger proteins (ZF), TALE (transcription activator-like effector) and Cas9 (Clustered Regulatory interspaced Short Palindromic Repeats – associated system) and POU homeodomain transcription factors.

[0066] In one preferred embodiment of the invention, the DNA binding domain may be a zinc finger DNA binding domain. In a preferred embodiment of the invention, the DNA binding domain may be or may comprise a zinc finger DNA binding domain or a variant thereof, which may be engineered to bind a specific DNA sequence in the regulatory element of a promotor. Said DNA binding domain (DBD) may be a polypeptide that has 3 zinc-finger domains. Each of said zinc-finger domains may comprise a recognition sequence determining the specificity of the domain to the responsive element of the DNA binding site. Such a DNA binding domain may comprise at least 3 zinc-finger domains. Each of the zinc-finger domains may comprise a recognition sequence determining the specificity of the domain to the responsive element of the DNA binding site in the promotor.

[0067] Said zinc finger DNA binding domains may be or may be derived from a protein selected from the group consisting of Sp1, N1, L8, CCR5 and Gal4 zinc finger proteins or functional fragments or mutated variants of these zinc finger proteins. In a preferred embodiment of the invention the zinc finger DNA binding domains may be or may be derived from N1 zinc finger proteins or fragments or mutated variants thereof.

[0068] The recognition sequence may be e.g. SEQ ID No: 1 (recognition sequence position -2 to 6 (relative to the start of the alpha-helix) of a finger that recognizes the target site GAA) and / or SEQ ID No: 2 (recognition sequence position -2 to 6 (relative to the start of the alpha- helix) of a finger that recognizes the target site GTA) and / or SEQ ID No: 3 (recognition sequence position -2 to 6 (relative to the start of the alpha-helix) of a finger that recognizes the target site GGG).

[0069] A zinc finger domain recognizes always 3 bp, the target site. The amino acids at position -1, 2, 3, 6 with respect to the start of the α-helix of the zinc finger domain are responsible for the DNA recognition.

[0070] A consensus zinc-finger-framework sequence derived from native and mutant versions of Sp1 zinc fingers may be SEQ ID No: 4.

[0071] The zinc finger domain (DNA binding domain) may be or may be derived from the N1 zinc-finger protein (N1 ZFP, N1 ZF or N1). The N1 may comprise the sequence of SEQ ID No:5. The corresponding responsive element for N1 may be SEQ ID No:6. The corresponding recognition motif for N1 may be SEQ ID No:7.

[0072] The DNA binding domain may be or may be derived from the GAL4 DNA binding domain (GAL4 DBD). GAL4 may comprise or consist of the sequence of SEQ ID No: 20 or a fragment thereof such as SEQ ID No: 21. The corresponding recognition motif of GAL4 DBD may be SEQ ID No: 29 and an inducible promoter for GAL4 DBD may be SEQ ID No:30.

[0073] The zinc finger domain (DNA binding domain) may be or may be derived from a synthetic zinc finger protein targeting the human CCR5 locus (CCR5 ZF). The CCR5 ZF may comprise or consist of the sequence of SEQ ID NO:22. The corresponding recognition motif for CCR5 ZF may be SEQ ID No:31 and an inducible promoter for expression induced by CCR5 ZF may be SEQ ID No:32.

[0074] The DNA binding protein binds to a region in the regulatory element in the promoter operably linked to a nucleic acid molecule encoding an effector molecule.

[0075] The promotor may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or more regulatory element(s) that may be recognized (bound) by said a DNA binding domain. In one embodiment of the invention the binding site may comprise five regulatory elements, wherein each regulatory element may comprise two direct repeats of the recognition motif SEQ ID No:7 spaced by 3 or 4 base-pairs (bps), if said zinc-finger protein is N1 ZFP (SEQ ID No: 5).

[0076] The promotor is indirectly inducible by using an inducer molecule. The inducer molecule initiates heterodimerization of the first and second binding moiety, thereby forming afunctional synthetic transcription factor. The transcription factor binds to the regulatory elements in the promotor and initiates transcription of the effector molecule.

[0077] In one embodiment of the invention the DNA binding domain which is N-terminally fused to a first binding moiety may comprise or consist of a zinc finger DNA binding domain or a variant thereof, which may be engineered to bind a specific DNA sequence in the regulatory element of a promotor. In a preferred embodiment of the invention the zinc finger DNA binding domains may be or may be derived from N1 zinc finger proteins or fragments or mutated variants thereof.

[0078] In one embodiment of the invention said transcription regulating domain may be p65 and said DNA binding domain may comprise or consist of a zinc finger DNA binding domain or a variant thereof (which may be engineered to bind a specific DNA sequence in the regulatory element of a promotor), preferentially N1 zinc finger proteins or fragments or mutated variants thereof . First and second nucleic acid and expression cassettes

[0079] According to the invention the system comprises a first and second exogenous nucleic acid molecules, wherein said exogenous nucleic acid molecules encode said first and second fusion proteins (Figure 2a, 5a-b, 8a-b, 9a-b).

[0080] Moreover the first nucleic acid molecule and the second nucleic acid molecule may be in a first and a second expression cassette, respectively. The first and second expression cassettes may be comprised in one or two different vectors, preferentially in two different vectors ( Figure 5a-b, 8a-b, 9a-b).

[0081] These expression cassettes are operably linked to a promotor, respectively, which may be inducible or constitutively active. In a preferred embodiment of the invention the first promoter operably linked to the first expression cassette and the second promoter operably linked to the second expression cassette may be constitutively active. Exemplary promotors are EF-l alpha MSCV, PGK-l, UBC, CMV, CAGG, MND, SV40 or pan-hematopoietic promoter, such as vav or functional variants thereof. In a preferred embodiment of the invention the first and second promotor is PGK-1 (SEQ ID No:33).

[0082] If the first and second expression cassette is comprised in one vector, the first and second expression cassette may be a polycistronic construct. In this embodiment of the invention the construct may be arranged in the following order: first expression cassette operably linked topromotor (preferentially constitutively active, more preferentially PGK), P2A site followed by a second expression cassette (Figure 2a).

[0083] In another embodiment of the invention, the first and second expression cassette is comprised in one vector. In this embodiment of the invention the construct may be arranged in the following order: first expression cassette operably linked to promotor (preferentially constitutively active, more preferentially PGK), followed by a second expression cassette operably linked to a promoter (preferentially constitutively active, more preferentially PGK). In this embodiment of the invention the expression cassettes are not separated by a P2A site.

[0084] In another embodiment of the invention, the first and second expression cassette is comprised in one vector. In this embodiment of the invention the construct may be arranged in the following order: first expression cassette operably linked to a bidirectional promotor (preferentially constitutively active, more preferentially PGK or EF1 alpha), followed by a second expression cassette operably linked to the same bidirectional promotor. In this embodiment of the invention the expression cassettes are not separated by a P2A site.

[0085] In yet another embodiment of the invention, the first and second expression cassette is comprised in two different vectors. In this embodiment of the invention the each construct may be arranged as follows: first vector comprises the first expression cassette operably linked to promotor (preferentially constitutively active, more preferentially PGK) and the second vector comprises the second expression cassette operably linked to a promotor (preferentially constitutively active, more preferentially PGK), or vice versa.

[0086] The use of two different vectors (or the use of a single vector without a P2A site) has the effect, that there is low or no background expression of the effector molecule in the absence of the inducer molecule (in the cell).

[0087] The vector(s), may be a retroviral vector, preferentially a lentiviral vector. Moreover the vector such as a retroviral vector or lentiviral vector may comprise said first expression cassette sequence in forward (sense) orientation, and another vector such as a retroviral vector or lentiviral vector may comprise said second expression cassette sequence in forward orientation.

[0088] In another embodiment of the invention the vector such as a retroviral vector or lentiviral vector may comprise said first and said second expression cassette in forward (sense) orientation. In this embodiment of the invention the first and said second nucleic acid sequence may be separated by a P2A site.

[0089] In one embodiment of the invention said vector such as a retroviral vector or lentiviral vector may additionally comprise a third expression cassette comprising a(n) (exogeneous) nucleic acid sequence encoding an effector molecule as disclosed herein. If this nucleic acidsequence is located on the same vector as the first and / or second expression cassette, said third expression cassette is encoded by the reverse strand of the vector (backwards orientation; (Figure 2a, 5b, 8b, 9b). This is for avoidance of readthrough and background transcription in the absence of said inducer molecule. As a result, the background expression of the effector molecule in the cell is reduced.

[0090] The one or more vectors (preferentially lentiviral vectors) may be introduced into the cell by transfection, electroporation or transduction, preferentially viral transduction, more preferentially lentiviral transduction. If the expression cassettes are comprised in different vectors (preferentially lentiviral vectors), the vectors may be introduced in a one or two step transduction procedure to the cell. In one embodiment of the invention, the first nucleic acid molecule and the second nucleic acid molecule may be in a first and a second expression cassette, wherein said first and second expression cassettes are in two different vectors ( Figure 5a,b). Said expression cassettes are operably linked to a promoter (preferentially constitutively active e.g. PGK), respectively. The expression cassettes may be comprised in separate vectors (preferentially lentiviral vectors). Preferentially, the vector such as a retroviral vector or lentiviral vector may comprise said first expression cassette sequence in forward (sense) orientation, and another vector such as a retroviral vector or lentiviral vector may comprise said second expression cassette sequence in forward (sense) orientation. In other words the first and second fusion proteins may be encoded by the forward (sense) strand of the vector (s). If the effector molecule is encoded by an exogeneous nucleic acid, one of said vectors may additionally comprise a third expression cassette comprising a(n) (exogeneous) nucleic acid sequence encoding an effector molecule as disclosed herein. Said third expression cassette is preferentially encoded in the reverse strand of the vector (Figure 5b, 8b, 9b). Effector molecule – third expression cassette

[0091] The nucleic acid sequence encoding an effector molecule may be an endogenous and / or exogenous nucleic acid molecule. In order to drive protein expression it is understood that, in any case said nucleic acid is operably linked to a promoter. Said promoter comprises the regulatory elements, needed for specific binding of the synthetic transcription factor.

[0092] In one embodiment of the invention the nucleic acid molecule may encode for one or more than one effector molecule. Said effector molecules may be same or different.

[0093] In addition to that, more than one nucleic acid may encode for more than one effector molecule, wherein said nucleic acids may be operably linked to the same promotor. In other words, more than one effector molecules may be encoded by multiple (separate) nucleic acids. Said effector molecules may be same or different.

[0094] In another embodiment of the invention, the nucleic acid sequence may encode for more than one effector molecule may be an exogenous nucleic acid molecule. In this embodiment the nucleic acid molecule may be multicistronic. Said effector molecules may be same or different.

[0095] As mentioned above, the nucleic acid (exogenous or endogenous) needs to be operably linked to a promotor. The promotor may be a naturally occurring or an engineered sequence.

[0096] Said promotor comprises a TATA box and at least one regulatory element. Said regulatory element comprises a recognition sequence, which can be specifically recognized by the DNA binding domain of the synthetic transcription factor as disclosed herein.

[0097] Said at least one regulatory element is located upstream (5’) from the TATA Box.

[0098] If the promoter is an engineered promotor, the TATA box comprised in the promotor may be selected from the group consisting of: E1b minimal promoter; MinCMV, MiniTK, Min IL2 promoter and YB TATA. In a preferred embodiment of the invention the promotor is the E1b minimal promoter (SEQ ID No:8).

[0099] If the nucleic acid is an exogenous nucleic acid it may be comprised in a third expression cassette. Said expression cassette may comprise said exogenous nucleic acid operably linked to a promoter as disclosed herein.

[0100] Said third expression cassette may be comprised in a vector, which may be a viral vector, preferentially a lentiviral vector.

[0101] Said vector may further comprise the first and / or second expression cassette, as disclosed herein. If said vector further comprises the third and first and / or second expression cassette, the first and / or second expression cassette(s) are oriented in forward direction in the sense strand, whereas the third expression cassette is encoded in the reverse strand (Figure 5a- b, 8a-b, 9a-b) of the vector. One advantage of this orientation is reduced background expression of the effector molecule.

[0102] The effector molecule encoded by the nucleic acid molecule may be a protein, (poly)peptide or polynucleotide, which may be therapeutically active. Moreover the effector molecule may be selected from the group consisting of an antibody or an antigen binding fragment thereof, a chimeric antigen receptor, a cytokine and a chemokine. In one embodimentof the invention, the effector molecule may be a regulatory RNA such as miRNA, siRNA or shRNA. In yet another embodiment of the invention, the effector molecule may be one or more proteins for virus production.

[0103] In one embodiment of the invention the effector molecule may be a chimeric antigen receptor (CAR). Said CAR may be specific for tumor associated antigens (TAAs). For example the CAR may be specific for CD19, CD20, FolR1, GD2, CD318, CD276, CD33, CD28, CD123 or CLEC12a.

[0104] In another embodiment of the invention the effector molecule may be a proliferation factor. Such a proliferation factor may be released to the extracellular space. It may affect cellular function of the releasing cell and / or surrounding cells. The Proliferation factor may affect cellular function as follows: Promote persistence, reduce exhaustion, enhance efficacy, improve migration and infiltration into solid tumors and / or resistance to the inhibitory tumor microenvironment (TME) of the immune effector cells.

[0105] In one embodiment of the invention the effector molecule (which may be a proliferation factor) may be a cytokine, cytokine receptor or a fragment thereof. Exemplary cytokines are IL2, IL7, IL12, IL15, IL18 and IL21. In addition to that exemplary cytokine receptors may be IL2RG, IL2RB, γc, IL7Ra, IL15Ra, IL21Ra and constitutively active variants thereof.

[0106] In another embodiment of the invention the effector molecule (which may be a proliferation factor) may be a protein or a variant thereof that is involved in JAK / Stat pathway such as Stat1, Stat3, Stat5a and Stat5b.

[0107] In addition to that the effector molecule may be a Glucocorticoid receptor, a Prolactin receptor or Epo Receptor. Moreover effector molecules may also include the following proteins and variants thereof: TNFRSF13B, CAML, AGTR1, ERK1, ERK2, ASS, OTC, LMO2, TAL(SCL), LYL1, LMO1, LMO2, TLX1(HOX11), TLX3(HOX11L2), NOTCH1, RhoA as well as fusion proteins (translocation products): ITK-Syk, BCR-ABL, MLL-AF4, MLL-AF9, AML1-ETO, PLZF- RARalpha, PML-RARalpha, TEL-AML1, E2A-PBX1, TFE3-ASPL, LMO2-TCRD / A, LMO2- TCRB, ETV6 / FRK, TAL1-TRB, LCK-TRB, TRB-NOTCH1, TRB-TAL2, TRB-HOX11, NRIP1-MECOM, TRD-LMO2, TRB-LMO2, TRB-CCND2, TRA / TRC-CCND2, WNT3- PRDM16, NSF-PRDM16, TRB-LYL1

[0108] In yet another embodiment of the invention, the effector molecule may be one or more proteins for virus production, preferentially lentivirus production. Based on that the effector molecule may be at least one molecule selected from the group consisting of: gag / pol, rev and a viral envelope protein .

[0109] The system as disclosed herein, comprises a first and second exogenous nucleic acid molecule, wherein said exogenous nucleic acids encode said first and second fusion protein. In this embodiment of the invention, the first nucleic acid molecule and the second nucleic acid molecule may be in a first and a second expression cassette, wherein said first and second expression cassettes are in two different vectors ( Figure 5a, 5b). Said expression cassettes are operably linked to a promotor (preferentially constitutively active e.g. PGK), respectively. The expression cassettes may be comprised in separate vectors (preferentially lentiviral vectors). Preferentially, said vector may comprise said first expression cassette in forward (sense) orientation, and another vector may comprise said second expression cassette in forward (sense) orientation. In other words the first and second fusion proteins may be encoded by the forward (sense) strand of the vector (s). If the effector molecule is encoded by an exogeneous nucleic acid, one of said vectors may additionally comprise a third expression cassette comprising a(n) (exogeneous) nucleic acid sequence encoding an effector molecule as disclosed herein. Said third expression cassette is preferentially encoded by the reverse strand of the vector (Figure 2a, 5b, 8b, 9b). In other words, if said vector further comprises the third and a first or second expression cassette, the first or second expression cassette(s) are encoded in forward strand, whereas the third expression cassette is encoded in the reverse strand (Figure 2a, 5a-b, 8a-b, 9a- b) of the vector. As mentioned above, the third nucleic acid / expression cassette is operably linked to a promotor comprising a TATA box and at least one regulatory element (comprising a recognition sequence, which can be specifically recognized by the DNA binding domain of the synthetic transcription factor as disclosed herein). Said at least one regulatory element is located upstream (5’) from the TATA Box, which is preferentially a E1b minimal promoter (SEQ ID No:8). Inducer molecule

[0110] An inducer molecule according to the current invention is capable to induce heterodimerization of the first and second binding moiety. In this invention the inducer molecule is an exogenous molecule which is not expressed by the cell. Such an inducer molecule may be a protein, a peptide or a chemical molecule / pharmaceutical molecule.

[0111] A commonly known inducer molecule is Xanthine or derivates thereof. Examples for Xanthine or derivates thereof may be caffeine, theophylline, theobromine, paraxanthine, 8- chlorotheophylline, 8-bromotheophylline, doxofylline, dyphylline, pentoxifylline, etofylline, albifylline, proxyphylline, propentofylline, pyridofylline, bamifylline, enprofylline, acefylline,xanthinol, diniprophylline, etamiphylline, lisofylline, pentifylline, arofylline, reproterol, furafylline, dasantafil, IBMX, KMUP-1, KMUP-2, KMUP-3, KMUP-4, theacrine, liberine, methylliberine or 1,3,7-trimethyluric acid.

[0112] In addition to that other commonly known inducer molecules may be e.g. fenretinide, N-Ethylretinamide, all-trans retinoic acid, axerophthene, A1120 (PubChem CID 25138295; chemical name: 2-(4-(2-(Trifluoromethyl)phenyl)piperidine-1-carboxamido)benzoic acid), derivatives of A1120; 1 , 4-butanediol, sphingosine-l-phosphate, tetradecanoic acid, indicaxanthin, vulgaxanthin I, Montelukast, Cyclandelate, Oxolamine, Mazaticol, Butoctamid, Tonabersat, Novazin, Diphenidol, Neobornyval, Erlotinib, Tanespimycin, LMI070, Alloclamide, Diacetolol, Acotiamide, Acoziborole, Acumapimod, Apalutamide, ASP3026, AZD1480, BIIB021, Branaplam, Brequinar, Chlorproguanil, Clindamycin, Emricasan, Enasidenib, Enolicam, Flurazepam, ILX-295501, Indibulin, Metoclopramide, Mevastatin, MGGBYMDAPCCKCT-UHFFFAOYSA-N, MK0686, Navarixin, Nefazodone hydrochloride, Pavinetant, Proxazole, Siccanin, Sulfaguanole, Sunitinib, Suvorexant, Tiapride, Tonabersat, VNBRGSXVFBYQNN-UHFFFAOYSA-N, YUHNXU- AATAMVKD-PZJWPPBQSA-N, Ulimorelin, Xipamide, Tropesin, Triclabendazole, Triclabendazole sulfoxide, Triclabendazole sulfone and Trametinib. Caffeine switch

[0113] The invention further provides a specific variant of the system wherein the first and second binding moieties comprise the binding domain of a single domain antibody (VHH) or a fragment thereof, respectively, wherein the binding domains are specific for Xanthine or a derivate thereof and wherein the inducer molecule is xanthine or a derivate thereof.

[0114] Commonly known examples of xanthine are caffeine, theophylline, theobromine, paraxanthine, 8-chlorotheophylline, 8-bromotheophylline, doxofylline, dyphylline, pentoxifylline, etofylline, albifylline, proxyphylline, propentofylline, pyridofylline, bamifylline, enprofylline, acefylline, xanthinol, diniprophylline, etamiphylline, lisofylline, pentifylline, arofylline, reproterol, furafylline, dasantafil, IBMX, KMUP-1, KMUP-2, KMUP-3, KMUP-4, theacrine, liberine, methylliberine or 1,3,7-trimethyluric acid. In a preferred embodiment of the invention the inducer molecule may be caffeine, theophylline, theobromine or paraxanthine, preferentially caffeine.

[0115] In a preferred embodiment of the invention the system as disclosed herein, wherein the first binding moiety comprises the binding domain of a single domain antibody (VHH), wherein the VHH domain comprises (or consist of) SEQ ID No: 10 and wherein the second bindingmoiety comprises the binding domain of a single domain antibody (VHH), wherein the VHH domain comprises (or consist of) SEQ ID No: 11, and wherein the inducer molecule is caffeine.

[0116] In one embodiment of the invention the first and second binding moieties comprise the binding domain of a single domain antibody (VHH) or a fragment thereof (comprising or consisting of SEQ ID No: 10 and SEQ ID No: 11), respectively, wherein the binding domains are specific for Xanthine or a derivate thereof and wherein the inducer molecule is xanthine or a derivate thereof. Said binding moieties are linked to the DNA binding domain of the first fusion protein and the transcription regulating domain of the second fusion protein (via a G4S linker structure). In addition to that, in this embodiment of the invention the first and the second fusion protein comprises one nuclear localization signal (NLS), which is located on the C terminus of each fusion protein (Figure 2a-b, 5a-b, 8a-b, 9a-b)

[0117] In one embodiment of the invention the first and second binding moieties comprise the binding domain of a single domain antibody (VHH) or a fragment thereof (comprising or consisting of SEQ ID No: 10 and SEQ ID No: 11), respectively, wherein the binding domains are specific for Xanthine or a derivate thereof (e.g. caffeine) and wherein the inducer molecule is xanthine or a derivate thereof (e.g. caffeine). Said binding moieties are linked to the DNA binding domain of the first fusion protein and the transcription regulating domain of the second fusion protein (via a G4S linker structure). In this embodiment of the invention the transcription regulating (or activating) domain may be p65 and said DNA binding domain may comprise or consist of a zinc finger DNA binding domain or a variant thereof (which may be engineered to bind a specific DNA sequence in the regulatory element of a promoter), preferentially N1 zinc finger proteins or fragments or mutated variants thereof (Figure 1). In this embodiment of the invention the first fusion protein may comprise or consist of SEQ ID No: 25 and the second fusion protein may comprise or consist of SEQ ID No: 26.

[0118] In addition to that the first and second fusion protein as disclosed herein may comprise a nuclear localization signal (NLS). Such an NLS signal sequence may be a c-myc NLS sequence. Based on that, in one embodiment of the invention the first fusion protein may comprise or consist of SEQ ID No: 23 and the second fusion protein may comprise or consist of SEQ ID No: 24. Lipocalin system

[0119] The invention further provides a specific variant of the system wherein the first binding moiety comprises a lipocalin fold molecule and second binding moiety comprises a lipocalin fold binding interaction partner and wherein the inducer molecule is a lipocalin fold ligand. Insuch a system the lipocalin fold molecule and a lipocalin fold binding interaction partner bind to the lipocalin fold ligand, thereby forming a synthetic transcription factor. A lipocalin based heterodimerization system was disclosed in WO2019122188.

[0120] In one embodiment of the invention the lipocalin fold molecule is selected from the group of: iLBP (intracellular lipid binding protein) , a naturally occurring lipocalin or an anticalin, or is a derivative of any of these molecules. Preferentially the lipocalin fold molecule is a naturally occurring protein. This naturally occurring lipocalin fold molecule may be selected from the group of human iLBP, human RBP4, human TLC, human ApoM, human CRABPII and human FABP1 or mutated variants of these proteins. In a preferred embodiment of the invention, the lipocalin fold molecule may be RBP4 or a mutated variant of RBP4.

[0121] In one embodiment of the invention, the lipocalin fold interaction partner may not be a naturally occurring protein. In one embodiment of the invention the lipocalin fold binding interaction partner is engineered to specifically recognize the lipocalin-fold molecule with higher affinity if the lipocalin-fold molecule is bound to the lipocalin-fold ligand compared to the affinity to the lipocalin-fold molecule not bound to the lipocalin-fold ligand, and wherein the lipocalin-fold molecule is optionally engineered for binding with higher affinity to the lipocalin-fold ligand.

[0122] In one embodiment of the invention, the lipocalin fold interaction partner may comprise an antigen, a cell surface receptor, an antibody, an antibody fragment, or a non-antibody based scaffold, preferably an affibody, a lipocalin-fold molecule, preferably an iLPB or a LCN, especially an anticalin; an avimer, a DARPin, a fynomer, a Kunitz domain, a knottin, a monobody, a Sso7d-based binder, reduced charge Sso7d (rcSso7d) -based binder or Sac7d- based binder. In a preferred embodiment the lipocalin fold binding interaction partner is a an anticalin. In another preferred embodiment of the invention, said anticallin may be or may comprise FN3 (the tenth type III domain of human fibronectin) or a mutated version of FN3. In a more preferred embodiment of the invention the lipocalin fold binding interaction partner may be RF2, or RF2s11 which are a mutated variant of FN3. RF2 comprises SEQ ID NO:38 and RF2s11 comprises SEQ ID NO:39.

[0123] In one embodiment of the invention the lipocalin fold ligand may be selected from the group consisting of: fenretinide, N-Ethylretinamide, all-trans retinoic acid, axerophthene, A1120 (PubChem CID 25138295) , derivatives of A1120; 1 , 4-butanediol, sphingosine-l- phosphate, tetradecanoic acid, indicaxanthin, vulgaxanthin I, Montelukast, Cyclandelate, Oxolamine, Mazaticol, Butoctamid, Tonabersat, Novazin, Diphenidol, Neobornyval, Erlotinib,Tanespimycin, LMI070, Alloclamide, Diacetolol, Acotiamide, Acoziborole, Acumapimod, Apalutamide, ASP3026, AZD1480, BIIB021, Branaplam, Brequinar, Chlorproguanil, Clindamycin, Emricasan, Enasidenib, Enolicam, Flurazepam, ILX-295501, Indibulin, Metoclopramide, Mevastatin, MGGBYMDAPCCKCT-UHFFFAOYSA-N, MK0686, Navarixin, Nefazodone hydrochloride, Pavinetant, Proxazole, Siccanin, Sulfaguanole, Sunitinib, Suvorexant, Tiapride, Tonabersat, VNBRGSXVFBYQNN-UHFFFAOYSA-N, YUHNXU- AATAMVKD-PZJWPPBQSA-N, Ulimorelin, Xipamide, Tropesin, Triclabendazole, Triclabendazole sulfoxide, Triclabendazole sulfone and Trametinib. In a more preferred embodiment of the invention the lipocalin fold ligand may be A1120 (PubChem CID 25138295; 2-(4-(2-(Trifluoromethyl)phenyl)piperidine-1-carboxamido)benzoic acid).

[0124] In another embodiment of the invention, the first binding moiety may be or may comprise a lipocalin fold molecule such as RBP 4 (SEQ ID No: 49) or a functional mutated variant of RBP4 (SEQ ID No: 37) and the second binding moiety may be or may comprise a lipocalin fold binding interaction partners such as FN3 or a functional variant of FN3 or a functional fragment thereof (SEQ ID No: 38, 39). The inducer molecule may be a lipocalin fold ligand, which may be A1120. Said binding moieties are linked to the DNA binding domain of the first fusion protein and the transcription regulating domain of the second fusion protein (via a G4S linker structure). In addition to that, in this embodiment of the invention the first and the second fusion protein comprises one nuclear localization signal (NLS), which is located on the C terminus of each fusion protein (Figure 15a,b; Figure 16a,b).

[0125] In another embodiment of the invention, the first binding moiety may be or may comprise a lipocalin fold molecule such as RBP 4 or a functional mutated variant of RBP4 and the second binding moiety may be or may comprise a lipocalin fold binding interaction partners such as FN3 or a functional variant of FN3 or a functional fragment thereof. The inducer molecule may be a lipocalin fold ligand, which may be A1120. In this embodiment of the invention the transcription regulating (or activating) domain may be p65 and said DNA binding domain may comprise or consist of a zinc finger DNA binding domain or a variant thereof (which may be engineered to bind a specific DNA sequence in the regulatory element of a promotor), preferentially N1 zinc finger proteins or fragments or mutated variants thereof.

[0126] In one embodiment of the invention the first binding moiety comprises a lipocalin fold molecule and second binding moiety comprises a lipocalin fold binding interaction partner and wherein the inducer molecule is a lipocalin fold ligand. (comprising or consisting of SEQ ID No: 37 or SEQ ID No: 49 and SEQ ID No: 38 or SEQ ID No: 39). Said binding moieties arelinked to the DNA binding domain of the first fusion protein and the transcription regulating domain of the second fusion protein (via a G4S linker structure). In this embodiment of the invention the transcription regulating (or activating) domain may be p65 and said DNA binding domain may comprise or consist of a zinc finger DNA binding domain or a variant thereof (which may be engineered to bind a specific DNA sequence in the regulatory element of a promoter), preferentially N1 zinc finger proteins or fragments or mutated variants thereof (Figure 14). In this embodiment of the invention the first fusion protein may comprise or consist of SEQ ID No: 43 and the second fusion protein may comprise or consist of SEQ ID No: 44,45.

[0127] In addition to that the first and second fusion protein as disclosed herein may comprise a nuclear localization signal (NLS). Such an NLS signal sequence may be a c-myc NLS sequence. Based on that, in one embodiment of the invention the first fusion protein may comprise or consist of SEQ ID No: 40 and the second fusion protein may comprise or consist of SEQ ID No: 41,42.

[0128] In one embodiment of the invention the first binding moiety may be or may comprise a lipocalin fold molecule that is a modified human RBP4 (modified hRBP4), wherein said hRBP4 is stable (functional).

[0129] Such modified hRBP4 proteins usable for intracellular applications may be generated e.g. by substitutions and / or deletions of the cysteins of the wt hRBP4 (SEQ ID NO:49) resulting in disulfide free hRBP4 varaints.

[0130] For the use of a modified protein hRBP4 for an intracellularly ligand regulated protein- protein interaction system said modified protein hRBP4 may comprise e.g. at least mutations at amino acid positions C4, C70, C120, C129, C160 and C174 as compared to the wild type human RBP4 amino acid sequence comprising SEQ ID No.: 49, and wherein said mutations are the substitutions of cysteines to other naturally occurring amino acids and / or the deletion of the cysteine(s).

[0131] Alternatively, other mutations such as deletions, insertions and / or substitutions than described above in the hRBP4 may be conducted to achieve hRBP4 variants that may be stable (functional) in intracellular milieu.

[0132] In one embodiment of the invention the system is a system as disclosed herein, wherein I)i) the first binding moiety of said first fusion protein is a lipocalin fold molecule as disclosed herein, and wherein said second binding moiety of said second fusion protein is a lipocalin fold interaction partner as disclosed herein, orii) the first binding moiety of said first fusion protein is a lipocalin fold interaction partner as disclosed herein, and wherein said second binding moiety of said second fusion protein is a lipocalin fold molecule as disclosed herein; and II) the inducer molecule is a lipocalin-fold ligand as disclosed herein, wherein said lipocalin-fold ligand is a lipocalin-fold ligand with a low molecular weight of 1500 Da or below, and wherein the lipocalin-fold molecule can bind to the lipocalin-fold ligand, and wherein the lipocalin-fold molecule bound to the lipocalin-fold ligand binds to the lipocalin-fold binding interaction partner with an affinity which is at least 10-fold higher than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand, and wherein the lipocalin-fold binding interaction partner is not a naturally occurring protein which has an affinity of <10 mM to any naturally occurring lipocalin-fold molecule in the presence of any lipocalin-fold ligand.

[0133] In one embodiment of the invention the system is a system as disclosed herein, wherein I)i) the first binding moiety is a lipocalin fold molecule as disclosed herein, wherein said lipocalin fold molecule is a modified hRBP4 that is stable (functional) in intracellular milieu, and wherein said second binding moiety is a lipocalin fold interaction partner as disclosed herein, or ii) the first binding moiety is a lipocalin fold interaction partner as disclosed herein, and wherein said second binding moiety is a lipocalin fold molecule as disclosed herein, wherein said lipocalin fold molecule is a modified hRBP4 that is stable (functional) in intracellular milieu; and II) the inducer molecule is a lipocalin-fold ligand as disclosed herein, wherein said lipocalin- fold ligand is a lipocalin-fold ligand with a low molecular weight of 1500 Da or below, and wherein the lipocalin-fold molecule can bind to the lipocalin-fold ligand, and wherein the lipocalin-fold molecule bound to the lipocalin-fold ligand binds to the lipocalin-fold binding interaction partner with an affinity which is at least 10-fold higher than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand, and wherein the lipocalin-fold binding interaction partner is not a naturally occurring protein which has an affinity of <10 mM to any naturally occurring lipocalin-fold molecule in the presence of any lipocalin-fold ligand.

[0134] In one embodiment of the invention the system is a system as disclosed herein, wherein I)i) the first binding moiety is a lipocalin fold molecule as disclosed herein, wherein said lipocalin fold molecule is a modified hRBP4 that is stable (functional) in intracellular milieu, and wherein said second binding moiety is a lipocalin fold interaction partner as disclosedherein, wherein said lipocalin fold interaction partner is the polypeptide RF2 comprising SEQ ID NO:38 or is the polypeptide RF2s11 comprising SEQ ID NO: 39 or ii) the first binding moiety is a lipocalin fold interaction partner as disclosed herein, wherein said lipocalin fold interaction partner is the polypeptide RF2 comprising SEQ ID NO:38 or is the polypeptide RF2s11 comprising SEQ ID NO: 39 , and wherein said second binding moiety is a lipocalin fold molecule as disclosed herein, wherein said lipocalin fold molecule is a modified hRBP4 that is stable (functional) in intracellular milieu; and II) the inducer molecule is a lipocalin-fold ligand as disclosed herein, wherein said lipocalin- fold ligand is a lipocalin-fold ligand with a low molecular weight of 1500 Da or below, wherein said lipocalin-fold ligand is A1120 (PubChem CID 25138295), and wherein the lipocalin-fold molecule can bind to the lipocalin-fold ligand, and wherein the lipocalin-fold molecule bound to the lipocalin-fold ligand binds to the lipocalin-fold binding interaction partner with an affinity which is at least 10-fold higher than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand, and wherein the lipocalin-fold binding interaction partner is not a naturally occurring protein which has an affinity of <10 mM to any naturally occurring lipocalin-fold molecule in the presence of any lipocalin-fold ligand.

[0135] In one embodiment of the invention the system is a system as disclosed herein, wherein I) i) the first binding moiety is a lipocalin fold molecule as disclosed herein, wherein said lipocalin fold molecule is a modified hRBP4 that is stable (functional) in intracellular milieu, wherein said modified hRBP4 comprises SEQ ID NO:37 and wherein said second binding moiety is a lipocalin fold interaction partner as disclosed herein, wherein said lipocalin fold interaction partner is the polypeptide RF2 comprising SEQ ID NO:38 or is the polypeptide RF2s11 comprising SEQ ID NO:39 , or ii) the first binding moiety is a lipocalin fold interaction partner as disclosed herein, wherein said lipocalin fold interaction partner is the polypeptide RF2 comprising SEQ ID NO:38 or is the polypeptide RF2s11 comprising SEQ ID NO:39, and wherein said second binding moiety is a modified hRBP4 that is stable (functional) in intracellular milieu, wherein said modified hRBP4 comprises SEQ ID NO:37; and II) the inducer molecule is a lipocalin-fold ligand as disclosed herein, wherein said lipocalin- fold ligand is a lipocalin-fold ligand with a low molecular weight of 1500 Da or below, wherein said lipocalin-fold ligand is A1120 (PubChem CID 25138295), and wherein the lipocalin-fold molecule can bind to the lipocalin-fold ligand, andwherein the lipocalin-fold molecule bound to the lipocalin-fold ligand binds to the lipocalin- fold binding interaction partner with an affinity which is at least 10-fold higher than the affinity of the lipocalin-fold molecule not bound to the lipocalin-fold ligand, and wherein the lipocalin-fold binding interaction partner is not a naturally occurring protein which has an affinity of <10 mM to any naturally occurring lipocalin-fold molecule in the presence of any lipocalin-fold ligand. Engineered cell

[0136] In another aspect, the invention provides an engineered cell comprising (a) A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule; (b) A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a binding moiety, wherein the first and second binding moieties are different from each other, and wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and wherein said engineered cell is in combination with the inducer molecule. In other words this aspect provides a combination or a kit comprising a composition comprising an engineered cell comprising (a) A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule (b) A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a binding moiety, and wherein the first and second binding moieties are different (from each other), wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and the inducer molecule.

[0137] The engineered cell may be e.g. immune effector cell or producer cell lines.

[0138] The immune effector cell may be an immune cell or immune cell subsets preferentially T cell, tumor infiltrating lymphocytes (TILs) or NK cells. In a preferred embodiment said immune cell is a T cell.

[0139] The producer cell lines may be selected from the group consisting of: HEK 293, HEK 293T, HEK293F and CHO

[0140] In one embodiment of the invention the engineered cell is an immune cell (e.g. a T cell), the effector molecule may be a chimeric antigen receptor or a proliferation factor as disclosed herein.

[0141] In one embodiment of the invention the engineered cell is a producer cell line and the effector molecule may be gag / pol, rev and / or a viral envelope protein. Pharmaceutical composition

[0142] In another aspect, the invention provides a pharmaceutical composition comprising a population of engineered cells (e.g. immune cells such as T cells) as disclosed herein and an inducer molecule. In other words this aspect provides a combination comprising a pharmaceutical composition comprising an engineered cell comprising (a) A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N- terminally fused to a first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule (b) A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a binding moiety, and wherein the first and second binding moieties are different (from each other), wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and the inducer molecule. Optionally the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0143] Pharmaceutically 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. Disease treatment

[0144] Another aspect of the invention provides an engineered cell or a pharmaceutical composition comprising the engineered cells (e.g. immune cells such as T cells) for use in a method of treating a disease. Such a disease may be selected cancer, autoimmune disease, allergic disease or an infectious disease.

[0145] Said disease may be cancer e.g. liquid or solid cancer. In a preferred embodiment of the invention the cancer may be 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 oradults, 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 leukemia, 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 sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity or oropharyngeal cancer, osteosarcoma, 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

[0146] The disease may be an autoimmune disease. Exemplary autoimmune diseases are Bechet’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, pernicious anemia, Primary biliary cholangitis, Myasthenia gravis, Dermato polymyositis, Vasculitis, Mixed connective tissue disease, Scleroderma, Multiple sclerosis, Psoriasis, Ulcerative colitis and Uveitis.

[0147] In a preferred embodiment of the invention the engineered cell or a pharmaceutical composition comprising the engineered cells are for use in immunotherapy, preferentially CAR T cell therapy. In this preferred embodiment of the invention said engineered cell may be an immune cell or immune cell subsets preferentially T cell, tumor infiltrating lymphocytes (TILs) or NK cells. In a preferred embodiment said immune cell is a T cell.

[0148] Another aspect of the invention provides a combination of compositions as disclosed herein, for use in a method of treating a disease , the method comprising administering to a subject in need thereof a) composition comprising an engineered cell comprising A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule; and A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a binding moiety,and wherein the first and second binding moieties are different from each other, and wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and b) the inducer molecule.

[0149] In a preferred embodiment of the invention said engineered cell may be an immune cell or immune cell subsets preferentially T cell, tumor infiltrating lymphocytes (TILs) or NK cells. In a preferred embodiment said immune cell is a T cell. In this embodiment of the invention the effector molecule may be a CAR or a proliferation factor.

[0150] Composition (a) and (b) may be administered sequentially or simultaneously to the subject.

[0151] In one embodiment of the invention composition (a) and (b) may be administered simultaneously to the subject.

[0152] In another embodiment of the invention, composition (b) may be administered first, followed by composition (a). This would result in a direct induction of the system, without timely delays.

[0153] In another embodiment of the invention, composition (a) may be administered to the subject first and composition (b) may be administered at time point at which effector molecule expression in the engineered cell is desired.

[0154] Dependent on the time frame of desired induction, it is understood that composition (b) may be administered to the subject repeatably. The time points, the amount of repeats and the concentration of the inducer molecule may be dependent on the system used, the effector molecule and the desired effect.

[0155] In one embodiment of the invention the inducer molecule may be administered to the subject over a time frame of at least 14 days, with at least one, at least 2 or at least 3 repeats daily.

[0156] In one embodiment of the invention, the compositions as disclosed herein comprising immune cells expressing e.g. a CAR as effector molecule 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 or an 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 system 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 pharmaceutical combination of composition as disclosed herein.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).

[0157] Populations of said genetically engineered (immune) cells may be formulated for administration to a subject using techniques known to the skilled artisan.

[0158] Formulations comprising therapeutically effective population(s) of said genetically engineered (immune) 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-injection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.

[0159] A formulation of a therapeutically effective population(s) of said engineered (immune) cells may include one population of said engineered (immune cells), or more than one population of said engineered (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.

[0160] The formulations comprising therapeutically effective population(s) of said engineered (immune) 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, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).

[0161] The formulations comprising therapeutically effective population(s) of said engineered (immune) cells that are administered to a subject comprise a number of said engineered (immune) cells that is effective for the treatment of the specific indication or disorder.

[0162] In general, formulations may be administered that comprise between about 1 x 104and about 1 x 1010said engineered (immune) cells. In most cases, the formulation may comprise between about 1 x 105and about 1 x 109said engineered (immune) cells, from about 5 x 105to about 5 x 108said engineered (immune) cells, or from about 1 x 106to about 1 x 107said engineered (immune) cells. However, the number of said engineered (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. Use of synthetic transcription factor for producer cell lines

[0163] In one aspect, the invention provides an in vitro method for inducing the expression of an effector molecule comprising the steps: (a) Providing a composition comprising engineered cells comprising A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding one or more effector molecules and A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a binding moiety, and wherein the first and second binding moieties are different from each other, and wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and (b) Adding the inducer molecule to the composition comprising engineered cells.

[0164] Said engineered cells may be cells of a producer cell line. The producer cell lines may be selected from the group consisting of: HEK 293, HEK 293T, HEK293F and CHO. In a preferred embodiment of the invention the cells of the producer cell line are HEK 293T cells

[0165] In one embodiment of the invention the effector molecule may be one or more proteins for virus production, preferentially lentivirus production. Based on that the effector molecule may be at least one molecule selected from the group consisting of: gag / pol, rev and a viral envelope protein. Said viral envelope proteins may be selected from the group consisting of: VSVG, baboon, CDV, Nipah, MV and GALV.

[0166] In another embodiment of the invention said cell line comprises at least one exogenous nucleic acid encoding at least one effector molecule.

[0167] In one embodiment of the invention the nucleic acid molecule may encode for one or more than one effector molecule. Said effector molecules may be same or different.

[0168] In addition to that, more than one nucleic acid may encode for more than one effector molecule, wherein said nucleic acids may be operably linked to the same promotor. In other words, more than one effector molecules may be encoded by separate nucleic acids. Said effector molecules may be same or different.

[0169] In a preferred embodiment of the invention the cell line comprises the exogenous nucleic acids encoding gag / pol, rev and a viral envelope protein. Said nucleic acids may be comprised in one single or separate vectors.

[0170] Optionally said producer cell line may additionally comprise a transgene encoding e.g. a CAR or cytokine.

[0171] For lentiviral production the inducer molecule is add to the cell culture and induces the expression of the viral components. This initiates the packaging of the transgene into a viral vector, thereby generating viral particles.

[0172] 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

[0173] 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.

[0174] 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.

[0175] The term “synthetic transcription factor” as used herein may comprise a DNA-binding domain fused to a first binding moiety and a transcription activating domain fused to a second binding moiety. Said binding moieties are specific for an inducer molecule. In presence of the inducer molecule the first and second binding moiety heterodimerize, thereby forming a synthetic transcription factor.

[0176] The term “exogenous” refers to a nucleic acid (sequence), an effector molecule or an exogenous protein that is not naturally expressed by a cell. In other words it is not naturally present in the genome.

[0177] The term “Endogenous” refers to a nucleic acid (sequence), an effector molecule or an exogenous protein that is naturally expressed by a cell. In other words it is naturally present in the genome.

[0178] 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.

[0179] The term “background expression of a gene or of an effector molecule” as used herein refers to the level of expression of the effector protein in the absence of the inducer molecule.

[0180] The terms “nucleic acid”, “nucleic acid sequence / molecule´” or “polynucleotide” as used interchangeably herein refer to polymers of nucleotides. Polynucleotides, which can behydrolyzed 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.

[0181] 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.

[0182] As used herein, the term “promoter” means a nucleic acid sequence which is required for transcription of a gene product (a polynucleotide and / or a polypeptide / protein) operably linked to the promoter. A promotor as used herein comprises regulatory elements and a TATA box.

[0183] 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.

[0184] 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 such as, for example, when an inducer ( e.g. an induction signal, or an induction agent such as a drug, metal ions, alcohol, oxygen, etc.) is present in the cell. A drug-inducible promoter is a promoter that may be regulatable by the presence or absence of said drug that induces the promoter.

[0185] The term “inducible system” refers to the expression of an exogenous polynucleotide encoding an effector molecule. The inducible gene expression system may be activated in a cell having said (inducible) system, when an inducer molecule, may be introduced to the cell. Said drug in the cell may bind to a synthetic transcription factor and subsequently may lead to the induction of the expression of the polypeptide encoding said effector molecule.

[0186] The terms “engineered cell” and “genetically modified cell” as used herein can be used interchangeably. The terms mean containing and / or expressing a foreign gene or nucleic acidsequence which in turn modifies the genotype and / or phenotype of the cell or its progeny. Especially, the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state.

[0187] The term "antibody" as used herein is used in the broadest sense to cover the various forms of antibody structures including but not being limited to monoclonal and polyclonal antibodies (including full length antibodies), multispecific antibodies (e.g. bispecific antibodies), antibody fragments, i.e. antigen binding fragments of an antibody, immunoadhesins and antibody-immunoadhesin chimeras, that specifically recognize (i.e. bind) an antigen. "Antigen binding fragments" comprise a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof (“an antigen binding fragment of an antibody”). Examples of antigen binding fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), single domain antibodies (nanobodies or VHHs, or single VH domains), diabodies, dsFv, Fab’, diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. The antibody or antibody fragment may be human, fully human, humanized, human engineered, non-human, and / or chimeric. The non-human antibody or antibody fragment may be humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Chimeric antibodies may refer to antibodies created through the joining of two or more antibody genes which originally encoded for separate antibodies.

[0188] A single-domain antibody (sdAb), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single- domain antibodies are much smaller than common antibodies (150–160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain). The first single- domain antibodies were engineered from heavy-chain antibodies found in camelids; these are called VHH (VHH) fragments. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called V-NAR fragments can be obtained. Alternatively, single-domain antibodies can also be based on human VH or VL domains or mutated versions thereof.

[0189] 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 antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain is specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain is specific.

[0190] The term “derivative of xanthine” refers to derivatives of the compound xanthine and are a group of alkaloids, some of which are known for their effects as mild stimulants.

[0191] 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, among many others. 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.

[0192] In general, a CAR may comprise an extracellular domain (extracellular part) comprising the 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.

[0193] The terms “immune cell” or “immune effector cell” may be used interchangeably and refer to a cell that may be part of the immune system and executes a particular effector function such as alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, regulatory T cells (Treg), monocytes or macrophages. Preferentially these immune cells are human immune cells. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells or gamma-delta T cells. Most preferred immune effector cells are T cells and NK cells. Tumor infiltrating lymphocytes (TILs) are T cells that have moved from the blood of a subject into a tumor. These TILs may be removed from a patient`s tumor by methods well known in the art, e.g. enzymatic and mechanic tumor disruption followed by density centrifugation and / or cell marker specific enrichment. TILs are genetically engineered as disclosed herein, and then given back to the patient. "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.

[0194] Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. Cancer immunotherapy as an activating immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses cell-based, preferentially T cell-based or NK cell-based cytotoxic responses to attack cancer cells. T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in-vitro and then transferred back into the cancer patient.

[0195] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0196] As used herein, the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the subject is a human. The subject may be a subject suffering from a disease such as cancer (a patient) or from an autoimmune disease or from a allergic disease or from an infectious disease or from graft rejection.

[0197] The term “producer cell line” and “packaging cell line” as used herein refers to a cell line such as HEK 293 that is able to produce retroviral vector particles or virus like particles thereof. The packaging cell line may produce said retroviral vector particles or virus like particles thereof transiently or stably, depending on the kind of preparation of the cell line.

[0198] Human embryonic kidney cells 293, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture. HEK 293 cells have been widely used in cell biology research for many years, because of their reliable growth and propensity for transfection. They are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy. Examples

[0199] 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 drug-inducible CAR T cells and evaluation of the impact of domain positioning on background expression and transcriptional output 1.1. Construct design and generation

[0200] Drug-inducible split synthetic transcription factors were cloned downstream of a constitutive human PGK promoter in a third generation lentiviral transfer vector flanked by LTR sites. The split synthetic transcription factor consists of two independent polypeptide chains separated by a furin P2A site. The first polypeptide contains a 3-finger zinc finger protein as DNA binding domain (referred to as N1 or N1 ZF) fused to the first member of the heterodimeric anti-caffein nanobodies and the nuclear localization signal (NLS) of c-Myc. The second chain is composed by a fragment of the activation domain of NFκB p65 (aa 361-551) fused to the second unit of the heterodimeric nanobodies pair and a c-Myc NLS. The two units of the synthetic transcription factor are labelled with three repeats of the DYKDDDDK-tag (SEQ ID No: 35) (commonly known as FLAG-tag) or HA-tag, respectively. Domains were separated through GGGGS (G4S) (SEQ ID No: 36) linkers. In order to study the impact of domain orientation on the transcriptional output and background activity of the system, the different domains were cloned in four possible configurations alternating the orientation of the DNA binding domain and the activation domain at N-terminal or C-terminal positions (Figure 2a, vectors NanoSwitch1-4). In addition, each of these vectors (NanoSwitch1-4) contains a second open reading frame (ORF) comprising the inducible gene expression cassette. This inducible cassette driving the expression of the gene of interest was cloned downstream of the ORF encoding for the synthetic transcription factor in the reverse strand. The inducible gene expression cassette comprises the sequence for the anti-CD20 CAR whereby the transcription of the anti-CD20 CAR is regulated by the inducible promoter. The 2nd generation anti-CD20 CAR incorporates a leader sequence from huGM-CSFR, an anti-CD20 scFv based on Leu-16, CD8 hinge, and transmembrane domain, as well as the cytoplasmic domains of 4-1BB and CD3ζ. The inducible promoter is composed of the binding sites for the N1 zinc finger (five responsive elements, with two binding sites each) linked to an E1b minimal promoter (SEQ ID NO: 9). ORFs driven by the human PGK promoter and the inducible promoter are separated by a bovine growth hormone polyA signal in reverse orientation.

[0201] Another variant of the split transcriptional switch system makes use of the human lipocalin Retinol Binding Protein 4 (RBP4) (SEQ ID NO: 37) and an anticalin (RF2) (SEQ ID NO: 38, 39) as drug-inducible heterodimer. This set of drug-inducible split synthetic transcription factors were cloned downstream of a constitutive human PGK promoter in a third generation lentiviral transfer vector flanked by LTR sites. The split synthetic transcription factor consists of two independent polypeptide chains. The first polypeptide driven by a constitutive promoter contains a 3-finger zinc finger protein as DNA binding domain (referredto as N1 or N1 ZF) fused to a variant of human Retinol Binding Protein 4 (hRBP4) and the nuclear localization signal (NLS) of c-Myc. The second chain is composed by a fragment of the activation domain of NFκB p65 (aa 361-551) fused to a modified fibronectin type III domain (FN3) that we named RF2 and a c-Myc NLS. The units of the synthetic transcription factor are labelled with three repeats of either DYKDDDDK-tag (SEQ ID NO:35) (commonly known as FLAG-tag) or HA-tag, respectively. Domains were separated through GGGGS (G4S) linkers (SEQ ID NO:36). In order to study the impact of domain orientation on the transcriptional output and background activity of the system, the different domains were cloned in four possible configurations alternating the orientation of the DNA binding domain and the activation domain at N-terminal or C-terminal positions (Figure 15a, b, vectors Liposwitch1- 4). In addition, vectors containing RF2 and p65 (Liposwitch 3 and 4) contain a second open reading frame (ORF) comprising the inducible gene expression cassette. This inducible cassette driving the expression of the gene of interest was cloned downstream of the ORF encoding for the synthetic transcription factor in the reverse strand. The inducible gene expression cassette comprises the sequence for the anti-CD20 CAR whereby the transcription of the anti-CD20 CAR is regulated by the inducible promoter. The 2nd generation anti-CD20 CAR incorporates a leader sequence from huGM-CSFR, an anti-CD20 scFv based on Leu-16, CD8 hinge, and transmembrane domain, as well as the cytoplasmic domains of 4-1BB and CD3ζ. The inducible promoter is composed of the binding sites for the N1 zinc finger (five responsive elements, with two binding sites each) linked to an E1b minimal promoter SEQ ID NO: 9. ORFs driven by the human PGK promoter and the inducible promoter are separated by a bovine growth hormone polyA signal in reverse orientation. 1.2. Generation of lentiviral particles and functional titer determination

[0202] 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 at the appropriate density in T175 culture flasks in DMEM (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10 % FCS (Biochrom) 3 days prior to transfection. At the day of transfection the culture medium was removed and replaced by DMEM (Biowest) supplemented with 2 mM L-Glutamine (Lonza). Cells were transfected with third generation lentiviral vectors encoding for VSV-G, gag+pol, rev and the psi positive transfer vector (containing either a conventional anti-CD20 CAR or the abovementioned drug- inducible synthetic transcription factor systems described in section 1.1: Nanoswitch1-4 and Liposwitch 1-4). After 48h the supernatant was collected and centrifuged for 10 min at 300g toremove cellular debris. In addition, the supernatant was filtrated trough a 0.45 µm PDVF filter and concentrated by centrifugation at 4500 rpm and 4ºC during at least 16 hours. Supernatant was discarded and the pellet was re-suspended in ice cold PBS or TexMACS medium (Miltenyi Biotec) and stored at - 80 °C.

[0203] Functional titer of VSV-G pseudotyped lentiviral vector particles was determined via titration on Sup-T1 T cells. 2E5 cells were seeded in 100 µL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) in 96 well round bottom plates. For transduction 100 µL of serially diluted lentiviral vector particles were added to the seeded cells.90 µL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10 % FCS (Biochrom) were added after 24 h. The frequency of transduced cells was quantified after 96 h by intracellular staining using either anti-DYKDDDDK-tag (SEQ ID No: 35) or anti-HA-tag antibody conjugated with APC (Miltenyi Biotec) followed by flow cytometry. Based on the frequency of tag positive cells, the number of seeded cells and the dilution of lentiviral particle used for transduction, the functional titer was determined. The titer was expressed in transducing units per mL and used to calculate the multiplicity of infection (MOI) for subsequent transductions. 1.3. Transduction, cultivation and analysis of inducible CAR T cells

[0204] Inducible CAR T cells were manufactured using primary human T cells from healthy donors. Peripheral Blood Mononuclear Cells (PMBCs) were isolated from buffy coats, whole blood or leukapheresis by density gradient centrifugation in Pancoll. T cells were isolated from PBMCs with the Pan T cell isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Prior to transduction T cells were seeded at a density of 1E6 cells / ml in TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec), IL-15 (Miltenyi Biotec) and activated with TransAct (Miltenyi Biotec). After 24 h, T cells were transduced with an MOI of 5-30 by adding the corresponding volume of lentiviral vector particles. On day 3 post- activation, TransAct was washed away and the culture medium was removed and replaced by TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec). Transduction efficiency was determined by intracellular staining of DYKDDDDK-tag (SEQ ID No: 35) and HA-tag using APC-conjugated antibodies (Miltenyi Biotec) after fixation and permeabilization with ice-cold methanol. Frequency of inducible CAR T cells was analyzed via flow cytometric determination of anti-CD20 CAR expression using anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec). Transduced T cells were usedfor functional assays after day 10 post-transduction. Cells were cultured at 37ºC and 5% CO2for the whole duration of the experiments.

[0205] Human T cell line SupT1 cells were transduced with an MOI of 10 in RPMI 1640 (Biowest) supplemented with 2 mM L-Glutamine (Lonza) at a cell density of 2E6 cells / ml. 24 hours post-transduction, cells were pelleted, media was removed and replaced by RPMI 1640 (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom). Cells were cultured at 37ºC and 5% CO2and media was exchange every 2-3 days. Transduction efficiency was determined via flow cytometry by staining with anti-DYKDDDDK-tag (SEQ ID No: 35) or anti-HA-tag antibody conjugated with APC (Miltenyi Biotec) after fixation and permeabilization with ice-cold methanol. Functional assays were performed 7 days post- transduction. 1.4. Determination of inducer dose-dependent response of drug-inducible CAR T cells upon induction with different concentrations of caffeine and evaluation of the impact of domain positioning on transcriptional output

[0206] Human T cell line SupT1 cells expressing the different nanobody-based transcriptional switches for control of anti-CD20 CAR were generated as explained in section 1.3. Transduced cells were cultured for 7 days in RPMI 1640 (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10% FCS (Biochrom). On day 8 post-transduction, cells were induced by addition of different doses of caffeine (0,1 – 500 μM) and cultured continuously in the presence of the inducer. Responsiveness of the different domain configurations was analyzed by determining the induced anti-CD20 CAR expression via flow cytometry using anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec). A clear dose-response was observed for all the constructs analyzed showing direct correlation between caffeine concentration and anti-CD20 CAR expression (Figure 2b). Detection of anti-CD20 CAR expression revealed a differential dose- response for each construct with strong differences in responsiveness among the constructs. Strikingly, the construct with N1 zinc finger in N-terminal position and p65 activation domain in C-terminal position (Nanoswitch1)showed a markedly higher anti-CD20 CAR expression demonstrating higher performance and responsiveness. By contrast, the other three constructs with different domain positioning (Nanoswitch2-4) showed a poor response, with only around 15 – 50% of transduced cells effectively expressing anti-CD20 CAR in presence of the highest concentration of caffeine. Background expression of anti-CD20 CAR in absence of the inducer also differ among the different constructs, being more prominent on those with higher responsiveness. Altogether, this demonstrates that optimal arrangement of the differentdomains comprising nanobody-based heterodimeric transcription factors (SEQ ID NO: 27, 28) is essential for maximal transcriptional output and responsiveness. Example 2: Assessment of gene expression stability of synthetic transcription factor and drug inducible anti-CD20 CAR over time

[0207] Human T cell line SupT1 cells expressing the different nanobody-based transcriptional switches for control of anti-CD20 CAR (Nanoswitch1-4) were generated and induced with different concentrations of caffeine as described in section 1.4. Stable expression of the synthetic transcription factor as well as the stable expression of the gene of interest was carefully monitored by flow cytometry upon staining with anti-DYKDDDDK-tag (SEQ ID No: 35) antibody conjugated with APC and anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec), respectively. Cells were monitored periodically for a period of 36 days. Anti- DYKDDDDK-tag (SEQ ID No: 35) staining revealed robust and stable expression of the nanobody-based synthetic transcription factor over time independently of caffeine concentration (Figure 3a). No difference was detected between induced and uninduced conditions indicating that induction does not have a negative impact on the expression of the transcription factor. Staining with anti-CD20 CAR Detection Reagent PE showed stable expression of the gene of interest with slight fluctuations over time, but no signs of epigenetic silencing during the duration of the assay (Figure 3b). Caffeine dose-dependent response was observed and maintained over time. Overall, these results demonstrate persistent expression of both, nanobody-based transcription factors and drug-induced anti-CD20 CAR and no loss of transgene expression over time. Example 3: Determination of inactivation and re-activation kinetics of drug-induced expression of anti-CD20 CAR

[0208] Human T cell line SupT1 cells expressing the different nanobody-based transcriptional switches for control of anti-CD20 CAR (Nanoswitch1-4) were generated and induced with different concentrations of caffeine (0, 30, 60, 222, 333 and 500 μM) as described in section 1.4. Cells were cultured continuously in the presence of the mentioned concentrations of caffeine for 36 days. Anti-CD20 CAR expression was analyzed by staining with anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec) followed by flow cytometry. Cells were monitored periodically (every 2-4 days) for a period of 36 days. On day 15 post-induction, cells were washed with fresh media and caffeine induction was discontinued. Cells were reinduced again with the same concentration of caffeine 22 days after the first induction (Figure 4a).Frequency of anti-CD20 CAR+ cells decreased over time upon caffeine discontinuation reaching either background or undetectable levels within 7 days (Figure 4b). Interestingly, expression level (measured as mean fluorescence intensity) dropped to background levels within just 4 days (Figure 4c). Upon re-induction with the specified concentration of caffeine, anti-CD20 CAR progressively increased in the subsequent days and expression level (MFI) reached a steady state within two days (Figure 4c). This demonstrates the versatility of the system to turn ON and OFF the induced expression of the gene of interest. Example 4: Evaluation of domain positioning on transcriptional output and background levels of caffeine-induced anti-CD20 CAR expression in human primary T cells using vectors carrying single units of the split synthetic transcription factor 4.1 Construct design and generation

[0209] In order to reduce background expression of the gene of interest in absence of caffeine, additional lentiviral vectors constructs carrying each of the units of the nanobody-based synthetic transcription factors individually were generated (LVI: Nanoswitch 5 & 6; LVII: Nanoswitch 7 & 8, Figure 5a,b). Lentiviral vectors carrying single units of the split synthetic transcription factor were cloned as follows: first unit of the synthetic transcription factor (LV I) comprising N1 zinc finger and first anti-caffeine nanobody fused to 3x FLAG-tag and c-Myc NLS was cloned downstream of a constitutive human PGK promoter in a third generation lentiviral transfer vector flanked by LTR sites. The DNA binding domain and the single domain antibody were cloned in two possible orientations fused by a G4S linker (Figure 5a). Second unit of the synthetic transcription factor comprised by the second anti-caffeine nanobody fused to a fragment of the NFκB p65 activation domain (aa 361-551) via G4S linker were also cloned in third generation lentiviral transfer vectors under the control of human PGK promoter (Figure 5b). This unit of the synthetic transcription factor was C-terminally fused to 3x HA-tag and the c-Myc NLS. In addition, vector containing the second unit of the synthetic transcription factor (LVII) also contains the inducible gene expression cassette cloned in the reverse strand and separated from the ORF driven by the PGK promoter via a bovine growth hormone polyA signal cloned in reverse orientation (Figure 5b). As described in section 1.4, the inducible gene expression cassette comprises the sequence for the anti-CD20 CAR whereby the transcription of the anti-CD20 CAR is regulated by the inducible promoter. The 2nd generation anti-CD20 CAR incorporates a leader sequence from huGM-CSFR, an anti-CD20 scFv based on Leu-16, CD8 hinge, and transmembrane domain, as well as the cytoplasmic domains of 4-1BB and CD3ζ. The inducible promoter is composed of the binding sites for the N1 zinc finger (fiveresponsive elements, with two binding sites each) linked to an E1b minimal promoter (SEQ ID NO:9). 4.2 Generation of lentiviral particles and functional titer determination

[0210] VSV-G psedotyped lentiviral vector particles were produced as described in section 1.2 using vectors Nanoswitch5-8 as transfer vectors. Functional titer was determined via titration on Sup-T1 T cells.2E5 cells were seeded in 100 µL RPMI (Biowest) supplemented with 2 Mm L-Glutamine (Lonza) in 96 well round bottom plates. For transduction 100 µL of serially diluted lentiviral vector particles were added to the seeded cells. 90 µL RPMI (Biowest) supplemented with 2 Mm L-Glutamine (Lonza) and 10 % FCS (Biochrom) were added after 24 h. The frequency of transduced cells was quantified after 96 h by intracellular staining using either an anti-DYKDDDDK-tag (SEQ ID No: 35) antibody (for LVI particles) or anti-HA-tag antibody (for LVII particles) conjugated with APC (Miltenyi Biotec) followed by flow cytometry. Based on the frequency of tag positive cells, the number of seeded cells and the volume of lentiviral particle used for transduction, the functional titer was determined. The titer was expressed in transducing units per Ml. 4.3 Generation of drug-inducible anti-CD20 CAR T cells in human primary T cells with lentiviral vectors carrying individual units of the split synthetic transcription factor

[0211] Inducible CAR T cells were manufactured using primary human T cells from healthy donors. Peripheral Blood Mononuclear Cells (PMBCs) were isolated from buffy coats, whole blood or leukapheresis by density gradient centrifugation in Pancoll. T cells were isolated from PBMCs with the Pan T cell isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Prior to transduction T cells were seeded at a density of 1E6 cells / ml in TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec), IL-15 (Miltenyi Biotec) and activated with TransAct (Miltenyi Biotec). Since lentiviral particles produced in section 4.2 from vectors generated in section 4.1 only contain a single unit of the synthetic transcription factors, T cells must be transduced with a combination of LVI and LVII vectors in order to deliver a fully functional transcriptional switch (Figure 5a,b). To achieve this, T cells were either co-transduced with a mixture of lentiviral vector particles (MOI 5 – 50) 24 hours post- activation or sequentially transduced by transducing with each vector on subsequent days (24 and 48 hours post-activation, respectively). On day 3 post-activation, TransAct was washed away and the culture medium was removed and replaced by TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec). Transductionefficiency was determined by intracellular staining of DYKDDDDK-tag (SEQ ID No: 35) and HA-tag using APC-conjugated antibodies (Miltenyi Biotec) after fixation and permeabilization with ice-cold methanol. Frequency of inducible CAR T cells was analyzed via flow cytometric determination of anti-CD20 CAR expression using anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec). Transduced T cells were used for functional assays after day 10 post- transduction. Cells were cultured at 37ºC and 5% CO2 for the whole duration of the experiments.

[0212] Drug-inducible CAR T cells generated with the four different possible lentiviral vector combinations that allow for theoretically functional switches (Nanoswitch5+7, 5+8, 6+7 & 6+8, Figure 5a,b) were cultured either in absence or presence of 250 μM of caffeine. Unstransduced T cells were used as negative control and T cells transduced with conventional anti-CD20 CAR as positive control. Induced anti-CD20 CAR expression was detected via flow cytometry upon staining with anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec). To exclude dead cells, Propidium iodide (PI; Miltenyi Biotec) was added to the stained cells directly before sample acquisition at the MACSQuant® Analyzer 10 (Miltenyi Biotec). Frequency of Anti-CD20 CAR+ cells upon caffeine induction strongly varied among the different constructs with distinct domain arrangements (Figure 5c). This experiment confirms once more (as in example 1.4) that the most favorable domain positioning leading to higher responsiveness is the construct with N1 zinc finger in N-terminal position and p65 activation domain in C-terminal position from their respective anti-caffeine nanobodies (LVI Nanoswitch 5 + LVII Nanoswitch7). Drug- inducible T cells generated with this particular configuration led to a positive frequency markedly higher than the rest of combinations and to a responsiveness close to 90% (understanding responsiveness as the frequency of transduced cells expressing the gene of interest). Interestingly, none of the constructs led to background expression of anti-CD20 CAR in absence of caffeine, showing that the expression of the gene of interest in our system is strictly dependent on the presence of the inducer. These results confirms our hypothesis that leaky expression in our system was a consequence of uncleaved P2A peptide generating fully functional full-length transcription factors that can trigger the expression of the inducible gene in absence of the inducer. This suggest that expressing each unit of a synthetic transcription factor from via bidirectional promoters or independent promoters (either in the same or different vectors) is preferred over the use of 2A self-cleaving peptides. The combination of constructs showing highest responsiveness (LVI Nanoswitch 5 + LVII Nanoswitch7), also led to the strongest response, achieving an anti-CD20 CAR expression almost 3 fold higher than that achieved by the EF1α-drive conventional anti-CD20 CAR control (Figure 5d). This provesthat this particular combination is optimal to trigger potent transcriptional activation of the gene of interest. 4.4. Frequency and level of expression of caffeine-induced anti-CD20 CAR show a strong dose- dependent correlation in human primary T cells

[0213] Drug-inducible CAR T cells carrying the most efficient combination of units of the split synthetic transcription factor (LVI: Nanoswitch5, LVII: Nanoswitch7) were generated as described in section 4.3. To determine the effect of inducer dose on the level of expression of inducible anti-CD20 CAR, transduced cells were cultured with different concentrations of caffeine ranging from 1 to 500 μM. Flow cytometry analysis revealed that induction of anti- CD20 CAR expression by inducible T cells is strictly dependent on the presence of caffeine with higher drug doses resulting in greater induction ratios (Figure 6a). Concurrently, surface expression (represented as MFI) steadily increased with the drug dose (Figure 6b). These results demonstrate that expression level of the gene of interest can be easily modulated by adjusting the concentration of the inducer. 4.5. Duration of induction influences the strength of the response of drug-inducible CAR T cells

[0214] Drug-inducible primary CAR T cells carrying the most efficient combination of units of the split synthetic transcription factor (LVI: Nanoswitch5, LVII: Nanoswitch7) were generated as described in section 4.3. Transduced cells were induced with 250 μM of caffeine for a period of time ranging from 0.5 to 72 hours. Upon induction for the indicated duration, cells were washed twice and resuspended in complete media (TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec) without caffeine. Level of expression of induced anti-CD20 CAR was determined 72 hours after addition of the inducer by staining with anti-CD20 detection reagent PE and analyzed by flow cytometry. Results revealed that frequency of anti-CD20 CAR+ cells as well as the expression level strongly correlate with the duration of the induction (Figure 6c, d). Example 5: Cytotoxic activity of inducible anti-CD20 CAR T cells in co-cultures with GFP+ CD20+ Raji cells.

[0215] To assess the specific tumor cell lysis mediated by inducible CAR T cells and its dependency to the caffeine dose, a killing assay using the GFP+ CD20+ tumor cell line Raji was performed. Drug-inducible primary CAR T cells carrying the most efficient combination of units of the split synthetic transcription factor (LVI: Nanoswitch5 + LVII: Nanoswitch7)were generated as described in section 4.3.1x104GFP+ CD20+ Raji cells were seeded in 50 µL RPMI medium (Biowest) supplemented with 10% FCS and 2mM L-Glutamine in a 96-well round-bottom plate. Inducible CAR T cells were subsequently added at an E:T ratio of 0.5:1 in a volume of 100 µL medium. T cell numbers were adjusted to HA-tag expression implicating equal transduced and total T cell numbers in each well. The number of control untransduced T cells was adjusted to total cell numbers. T cells transduced with a conventional anti-CD0 CAR were used as positive control. Anti-CD20 CAR expression was induced by the addition of different doses of caffeine in 50 µL of media at the start of the assay. 24 hours post-coculture, cells were analyzed by flow cytometry to determine the number and frequency of GFP+ tumor cells and calculate the specific target lysis. Tumor-specific cytotoxicity of inducible CAR T cells was strictly dependent on the presence of caffeine characterized by the absence of basal lysis in the non-induced state (Figure 7a). We observed a caffeine dose dependent cytotoxic activity of the inducible anti-CD20 CAR, effectively reducing the number of tumor cells with doses higher than 10 μM (Figure 7a,b). Conventional anti-CD20 CAR T cells showed the strongest cytotoxic activity (Figure 7a). We hypothesized that this is due to a faster killing kinetics, since conventional CARs are constitutively expressed at high level and their cytotoxic activity can be triggered shortly after coculture setup, while inducible CAR T cells need longer time to initiate, transcription, translation and transport of the CAR to the membrane upon induction leading to a delayed killing kinetics. Example 6: Cytokine secretion of inducible anti-CD20 CAR T cells in co-cultures with GFP+CD20+Raji cells.

[0216] The capacity of inducible anti-CD20 CAR T cells to secrete effector cytokines in co- cultures with tumor cells was analyzed using human MACS Plex 12 cytokine kit (Miltenyi Biotec). Cocultures of CAR T cells transduced with the most efficient combination of units of the split synthetic transcription factor (LVI: Nanoswitch5 + LVII: Nanoswitch7) and GFP+ CD20+ Raji cells were setup and analyzed as described in example 5. 24 hours after coculture setup, cells were spin down and 100μl of supernatant were collected for cytokine secretion detection. Then, 100 µl co-culture supernatant was transferred into a new 96-well plate and cytokine levels were analyzed using human MACS Plex 12 cytokine kit (Miltenyi Biotec) according to manufacturer’s instructions. Example 7: Selection of the activation domain determines the transcriptional output of the gene of interest regulated by the caffeine-inducible transcriptional switch7.1. Construct design and assembly

[0217] Vector carrying the first unit of the split synthetic transcription factor carrying the N1 ZF DNA binding domain (NanoSwitch5) remained the same for this assay (Figure 8a). Vectors for evaluation of alternative activation domains were generated by replacing the human NFκB p65 activation domain fragment (aa 361-551) from vector NanoSwitch7 by a reduced fragment of human NFκB p65 activation domain (aa 520-551), VP64 activation domain (tetrameric repeat of aa 437-448 of human herpex simplex virus 1 tegument proteinVP16) or human CITED 2 activation domain (aa 161-270) (Figure 8b). 7.2 Lentiviral vector particles production and functional titer determination

[0218] Lentiviral particles were produced and functional titer was determined as described in section 4.2 using vectors described in 7.1 as transfer vectors. 7.3 Evaluation of the fold induction of anti-CD20 CAR expression by different activation domains upon induction with caffeine

[0219] To evaluate the impact of the strength of different transcriptional activators on our transcriptional switch response, drug-inducible CAR T cells were generated as described in 4.5 by transducing with lentiviral particles carrying construct NanoSwitch5 (N1 ZF – VHH1, Figure 8a) and each of the different variants of NanoSwitch7 (VHH2 – AD) carrying the different activation domains described in section 7.1 (Figure 8b). 10 days post-transduction, cells were induced by addition of 250 μM of caffeine to the culture media. 24 hours post- induction, transduction efficiency and induced CAR expression was evaluated by immunostaining and flow cytometry as described above. Mean fluorescence intensity of anti- CD20 CAR+ cells revealed that all tested activation domain fragments are functional and suitable to induce the expression of the gene of interest at different levels (Figure 8c). In addition, anti-CD20 CAR expression by inducible T cells was strictly dependent on the presence of the inducer drug indicating that the use of different activation domains is not leading to changes in background expression. VP64 AD led to the highest fold-induction of the system as expected from a viral activation domain. Human p65 AD (aa 361-551) also showed strong transcriptional activity, 2-fold superior to the expression of a conventional CAR driven by a constitutive EF1α promoter (Figure 8c). Interestingly, a smaller fragment of human p65 (aa 520-551) showed markedly reduced transcriptional response, around 4 times lower than the CAR control. However, the used of CITED2 AD allowed to achieve expression levels comparable to a conventional anti-CD20 control. Overall, these results suggests that anyactivation domain with transcriptional activity might be suitable to function in our drug- inducible split synthetic transcription factor system. Therefore, careful selection of the activation domain can be exploited as a strategy to modulate the response of the system and achieve the optimal level of expression for the desired application. This results also identified p65 AD (aa 361-551) as the human activation domain fragment that led to highest transcriptional output. Example 8: Selection of the DNA binding domain regulating the specific recognition of the binding sites at the promoter of the gene of interest influences the transcriptional output upon drug-induction 8.1. Construct design and assembly

[0220] Vectors for evaluation of additional DNA binding domains were generated by replacing N1 zinc finger from vector NanoSwitch5 by either GAL4 DNA binding domain or a synthetic zinc finger protein targeting the CCR5 locus (referred to as CCR5-224+ ZF) (FIGURE 9a). Additionally, vector NanoSwitch7 was modified by replacing N1 ZF responsive elements by the corresponding responsive elements of the previously mentioned DNA binding domains (Figure 9b). 8.2. Lentiviral vector particles production and functional titer determination

[0221] Lentiviral particles were produced and functional titer was determined as explained in section 4.2 using the transfer vectors described in 8.1. 8.3. Evaluation of the impact of the DNA binding domain on the level of expression of caffeine- induced anti-CD20 CAR.

[0222] To evaluate the impact of different DNA binding domains on the performance of the transcriptional switch, drug-inducible CAR T cells were generated as described in section 4.5 by transduction with Nanoswitch5 lentiviral vectors carrying the different DNA binding domains mentioned above (Figure 9a) and the corresponding Nanoswitch7 version with the appropriate binding sites (Figure 9b). On day 10 post-transduction, cells were induced by addition of 250 μM of caffeine to the culture media. 24 hours post-induction, transduction efficiency and induced CAR expression was evaluated by immunostaining and flow cytometry as described before. All the different DNA binding domains tested rendered the transcriptional switch functional leading to specific anti-CD20 CAR expression in presence of caffeine (Figure 9c). Among them, N1 ZF allowed to achieve the highest fold-induction (25x over uninducedcells), leading to almost 5 times higher anti-CD20 CAR expression level than that of the EF1α- driven conventional CAR control. Construct with GAL4 DNA binding domain led to approximately 4 times expression level over the control, while CCR5 ZF allowed to achieve 2- fold higher levels. Mean fluorescence intensity of uninduced cells is comparable to that of untransduced cells, demonstrating the absence of background activity, tight control and strong dependency on caffeine for drug-induced expression of the gene of interest. Overall, these results suggest that the choice of the DNA binding domain allows to add another layer of control to fine-tune the expression level of the gene of interest. Example 9: Cytotoxic activity of inducible anti-CD20 CAR T cells in vivo.

[0223] To study the in vivo functionality of inducible CAR T cells, the immunodeficient mouse strain NOD.Cg-PrkdcscidIL2rgtm1 Wjl / SzJ (NSG™) was selected. For tumor establishment 4x105mouse-adapted RajiffLuccells were injected i.v. in 100 uL PBS into the tail vein of NSG™ mice. Tumors were engrafted for 7 days and tumor growth is monitored regularly by bioluminescent imaging and expressed in photon flux [photons / sec] over time for individual mice. Prior to T cell injection, mice groups were randomized according to the BLI signal measured on day 0 for equally distributed tumor flux among the treatment groups. Mice were treated with untransduced, inducible (Nanoswitch 5 + Nanoswitch 7) or conventional constitutively expressed (“direct”) anti-CD20 CAR T cells. T cell numbers were adjusted to ensure equal number of transduced cells and total T cell numbers per mouse. The number of untransduced T cells was adjusted to the total cell number injected in the CAR T cell groups. T cells were injected i.v. in 100 μL PBS into the tail vein of NSG™ mice. Tumor only control groups (untreated) received i.v. injections of 100 μL PBS. Caffeine was added to the drinking water at a concentration of either 0.7 g / L or 1.4 g / L and replenished on a daily basis. Control groups received water only as vehicle. Dosing with caffeine was started 5 days before T cell infusion. Example 10: Drug-induced expression of JAK-STAT pathway members enhance the persistence and expansion of CAR T cells in an inducer dose dependent manner.

[0224] To enable drug-inducible transcriptional control of immunomodulatory molecules that can enhance the expansion, persistence, infiltration and / or efficacy of CAR T cells we generated a two-vector nanobody-based switch system. The first lentiviral vector (LVI) that we termed NanoFITswitch 1, comprises a human EF1α promoter driving the expression of a CAR (anti- CD20 CAR) and the first unit of the nanobody-based switch with the optimal domainpositioning (N-terminal N1 Zinc Finger and C-terminal nanobody). Both proteins were separated by a P2A self-cleaving peptide (Figure 10a). The second lentiviral vector (LVII), NanoFITswitch 2, contains a human PGK promoter driving the expression of the second unit of the split synthetic transcription factor composed by the second anti-caffeine nanobody and a p65 activation domain (Figure 10b). In addition, this vector also contains the inducible gene expression cassette cloned in the reverse strand and separated from the ORF driven by the PGK promoter via a bovine growth hormone polyA signal cloned in reverse orientation (Figure 10b). The inducible gene expression cassette comprises the sequence of the “proliferation factor” whereby the transcription of the JAK-STAT pathway member is regulated by the inducible promoter. The inducible promoter is composed of the binding sites for the N1 zinc finger (five responsive elements, with two binding sites each) linked to an E1b minimal promoter (SEQ ID NO: 9) (Figure 10b). We generated three versions of this vector carrying different inducible genes: glucocorticoid receptor beta (GRβ, SEQ ID NO: 50), STAT5b N642H (SEQ ID NO: 51) and one containing IL-15 + IL-15Rα (Figure 10b). In order to demonstrate drug-inducible control over the expression and function of these genes, we performed a cytokine starvation assay. T cells were isolated and activated as described in 4.3 and transduced with LV1 (NanoFITswitch 1) on day 1 post-activation and with the different versions of LVII (NanoFITswitch 2 carrying either GRβ, STAT5b N642H or IL-15_P2A_chimeric IL-15Rα) on day 2 post-activation. Transduced CAR T cells were expanded in TexMACS medium supplemented with supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec). On day 8, cells were washed, induced with different doses of caffeine and, further cultured in media without cytokines in order to evaluate their survival (persistence) and expansion in absence of this essential component. Viable T cell count was determined via 7AAD staining and flow cyotmetry after 8 days in absence of recombinant cytokines (Figure 10c). Our results revealed a dose-dependent increase in persistence when inducing the expression of constitutively active STAT5b (carrying N642H mutation) or IL-15 + chimeric IL-15Rα (Figure 10d). The effect on persistence reached it maximum with 100 µM of caffeine leading to more than 5-fold increase compared to the uninduced and untransduced controls (Figure 10d). Comparable results were obtained by drug-induced expression of IL-15 and chimeric IL-15Rα with 100μM of caffeine (Figure 10d). Drug-induced expression of GRβ showed a weaker, but detectable effect on enhancing persistence of transduced cells leading to a 2-fold increase in cell counts in presence of 50 – 100 μM of caffeine (Figure 10d). Altogether, these data demonstrate the potential of our drug-inducible nanobody-based transcriptional switches to control and enhance the persistence and expansion of CAR T cells by finely tuning the concentration of the inducer andthe selected proliferation factor. Among the factors tested, controlled expression of STAT5b N642H showed the best performance.

[0225] Example 11: Switchability and ON / OFF kinetics of drug-induced gene expression in human primary T cells

[0226] To evaluate the drug-induced switchability of the system and determine the ON / OFF / ON kinetics of the transcriptional switch, drug-inducible CAR T cells were generated as described in section 4.3 by transduction with the most efficient combination of units of the split synthetic transcription factor (LVI: Nanoswitch5, LVII: Nanoswitch7). Transduced cells were cultured in TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec) on different caffeine regimes at a concentration of 250 µM (Figure 12a). First group was continuously cultured in absence of caffeine (- / - / -), while a second group was continuously cultured in presence of 250 µM of caffeine (+ / + / +). Two additional groups were used to evaluate the switchability and ON / OFF / ON kinetics. Both groups were cultured for 4 days in presence of caffeine and then in absence of caffeine either until the end of the experiment (+ / - / -) or for 6 days and then reinduced with caffeine (+ / - / +) (Figure 12a). Level of expression of induced anti-CD20 CAR was determined 24 hours after addition of the inducer by staining with anti-CD20 detection reagent PE and analyzed by flow cytometry. Additional measurements were performed 4, 6, 7, 10 and 11 days post-induction. Results revealed that uninduced cells (- / - / -) did not present any leaky expression of the inducible gene of interest, while cells continuously cultured in presence of caffeine (+ / + / +) showed progressively increasing frequencies of anti-CD20 CAR+ cells (Figure 12b). Upon drug withdrawal, anti-CD20 CAR+ cells were detected for up to 6 days longer. No positive cells were detected on day 7 post-withdrawal (groups + / - / - and + / - / +). Interestingly, expression level of anti-CD20 CAR+ (MFI) was downregulated to background levels in just 3 days post- withdrawal (Figure 12c) Reinduced primary T cells (+ / - / +) showed high expression levels of anti-CD20 CAR+ cells in just 24 hours post-reinduction. These data indicate that the switch provides fast ON-kinetics and slower OFF-kinetics. Similar kinetics were observed when analyzing expression levels of the induced gene (Figure 12c).

[0227] Example 12: Inducer dose-response curves and functionality of nanobody-based drug- inducible transcriptional switch to different Xanthine-derived molecules

[0228] To evaluate the responsiveness of the described drug-inducible transcriptional switch to different Xanthine-derived molecules, drug-inducible CAR T cells were generated as describedin section 4.3 by transduction with the most efficient combination of units of the split synthetic transcription factor (LVI: Nanoswitch5, LVII: Nanoswitch7). Transduced cells were cultured in TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec) and induced with different concentrations ranging from 0 to 500 µM of either caffeine (Figure 13a) or doxofylline (Figure 13b). Induced expression of anti_CD20 CAR was determined by flow cytometry 3 days post-induction. These data demonstrates that this nanobody-based molecular switch is functional and leads to a comparable inducer dose- response curve with both xanthine derivatives inducers (caffeine and doxofylline) (Figure 13c, d).

[0229] Example 13: In vitro cytotoxic activity of inducible anti-CD20 CAR T cells in co- cultures with GFP+ CD20+ 526-Mel tumor cells upon induction with different doses of caffeine.

[0230] To assess the specific tumor cell lysis mediated by inducible CAR T cells and its dependency to the caffeine dose, a killing assay using the GFP+ CD20+ 526-Mel tumor cell line was performed. Drug-inducible primary CAR T cells carrying the most efficient combination of units of the split synthetic transcription factor (LVI: Nanoswitch5 + LVII: Nanoswitch7) were generated as described in section 4.3. 1x104GFP+ CD20+ 526-Mel cells were seeded in 250 µL RPMI medium (Biowest) supplemented with 10% FCS and 2mM L- Glutamine in a 96-well flat-bottom plate 24 hours before co-culture setup. Inducible CAR T cells were counted and HA-tag frequency was determined by flow cytometry on the day of co- culture. Subsequently, 1x104HA-tag+ cells were added to each well of the plate already containing attached GFP+ CD20+ 526-Mel cells. T cell numbers were adjusted to HA-tag expression implicating equal transduced and total T cell numbers in each well. In order to adjust total T cell numbers, untransduced cells were spiked-in when necessary. In the case of the group transduced with conventional anti-CD20 CAR, cells were adjusted based on the frequency of anti-CD20 CAR+ cells. The number of control untransduced T cells was adjusted to total cell number. T cells transduced with a conventional anti-CD0 CAR were used as positive control. Inducible anti-CD20 CAR expression was induced by the addition of different doses of caffeine in 50 µL of media at the start of the assay. Co-cultures were monitored by live-cell imaging using the Incucyte S3 Live-Cell Analysis system (Sartorius) for 96 hours. Analysis of green fluorescence intensity revealed that tumor-specific cytotoxicity of inducible CAR T cells was strictly dependent on the presence of caffeine characterized by the absence of basal lysis in the non-induced state (Figure 11a). Transduced cells cultured with the different caffeine concentrations were also analyzed by flow cytometry by detecting anti-CD20 CAR expression. We observed a caffeine dose dependent cytotoxic activity of the inducible anti-CD20 CAR, effectively reducing the number of tumor cells with doses higher than 10 μM (Figure 11a). Conventional anti-CD20 CAR T cells showed the strongest cytotoxic activity (Figure 11a) and are comparable to the induction with 500 µM of caffeine. Analysis of anti- CD20 CAR expression upon induction with different doses of caffeine revealed a direct correlation between the concentration of caffeine and frequency and expression level (Figure 11b, c). These variation in frequency of CAR+ cells depending on the inducer dose explains the difference in cytotoxic activity among groups induced with different concentrations of caffeine. Example 14: Evaluation of domain positioning on transcriptional output and background levels of A1120-induced anti-CD20 CAR expression in human primary T cells co-transduced with vectors carrying single units of lipocalin-based split synthetic transcription factor 14.1 Construct design and generation

[0231] Lentiviral vector constructs carrying each of the units of the lipocalin-based synthetic transcription factors individually were generated (LVI: Liposwitch 1 & 2; LVII: Liposwitch 3 & 4, Figure 15a,b). Lentiviral vectors carrying single units of the split synthetic transcription factor were cloned as follows: first unit of the synthetic transcription factor (LV I) comprising N1 zinc finger and first protein of the of lipocalin heterodimer pair (e.g. RBP4) to 3x FLAG- tag and c-Myc NLS was cloned downstream of a constitutive human PGK promoter in a third generation lentiviral transfer vector flanked by LTR sites. The DNA binding domain and the lipocalin RBP4 were cloned in two possible orientations fused by a G4S linker (Figure 15a). Second unit of the synthetic transcription factor comprised by the second protein of the heterodimer pair (e.g. RF2) fused to a fragment of the NFκB p65 activation domain (aa 361- 551) via G4S linker were also cloned in third generation lentiviral transfer vectors under the control of human PGK promoter (Figure 15b). This unit of the synthetic transcription factor was C-terminally fused to 3x HA-tag and the c-Myc NLS. In addition, the vector containing the second unit of the synthetic transcription factor (LVII) also contains the inducible gene expression cassette cloned in the reverse strand and separated from the ORF driven by the PGK promoter via a bovine growth hormone polyA signal cloned in reverse orientation (Figure 15b). As described in section 1.4, the inducible gene expression cassette comprises the sequence for the anti-CD20 CAR whereby the transcription of the anti-CD20 CAR is regulated by the inducible promoter. The 2nd generation anti-CD20 CAR incorporates a leader sequence from huGM-CSFR, an anti-CD20 scFv based on Leu-16, CD8 hinge, and transmembrane domain, as well as the cytoplasmic domains of 4-1BB and CD3ζ. The inducible promoter is composed ofthe binding sites for the N1 zinc finger (five responsive elements, with two binding sites each) linked to an E1b minimal promoter (SEQ ID NO: 9). 14.2 Generation of lentiviral particles and functional titer determination

[0232] VSV-G psedotyped lentiviral vector particles were produced as described in section 1.2 using vectors Liposwitch 1-4 as transfer vectors. Functional titer was determined via titration on Sup-T1 T cells.2x105cells were seeded in 100 µL RPMI 1640 (Biowest) supplemented with 2 mM L-Glutamine (Lonza) in 96 well round bottom plates. For transduction 100 µL of serially diluted lentiviral vector particles were added to the seeded cells. 90 µL RPMI (Biowest) supplemented with 2 mM L-Glutamine (Lonza) and 10 % FCS (Biochrom) and 2 µM retinol (Sigma Aldrich) were added after 24 h. The frequency of transduced cells was quantified after 96 h by intracellular staining using either an anti-DYKDDDDK-tag (SEQ ID NO:35) antibody (for LVI particles) or anti-HA-tag antibody (for LVII particles) conjugated with APC (Miltenyi Biotec) followed by flow cytometry. Based on the frequency of tag positive cells, the number of seeded cells and the volume of lentiviral particle used for transduction, the functional titer was determined. The titer was expressed in transducing units per mililiter (TU / ml). 14.3 Generation of drug-inducible anti-CD20 CAR T cells in human primary T cells with lentiviral vectors carrying individual units of the lipocalin-based split synthetic transcription factor

[0233] Inducible CAR T cells were manufactured using primary human T cells from healthy donors. Peripheral Blood Mononuclear Cells (PMBCs) were isolated from buffy coats, whole blood or leukapheresis by density gradient centrifugation in Pancoll. T cells were isolated from PBMCs with the Pan T cell isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Prior to transduction T cells were seeded at a density of 1x106cells / ml in TexMACS medium (Miltenyi Biotec) supplemented with 12.5 ng / ml of IL-7 (Miltenyi Biotec), 12.5 ng / ml of IL-15 (Miltenyi Biotec) and activated with 1:100 TransAct (Miltenyi Biotec). Since lentiviral particles produced in section 13.2 from vectors generated in section 13.1 only contain a single unit of the synthetic transcription factors, T cells must be transduced with a combination of LVI and LVII vectors in order to deliver a fully functional transcriptional switch (Figure 15a,b). To achieve this, T cells were either co-transduced with a mixture of lentiviral vector particles (MOI 5 – 50) 24 hours post-activation or sequentially transduced by transducing with each vector on subsequent days (24 and 48 hours post-activation, respectively). On day 3 post-activation, TransAct was washed away and the culture medium was removed and replaced by RPMI 1640medium (Biowest) supplemented with 2 mM L-Glutamine (Lonza), 10 % FCS (Biochrom), IL- 7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec). Transduction efficiency was determined by intracellular staining of DYKDDDDK-tag (SEQ ID NO:35) and HA-tag using APC-conjugated antibodies (Miltenyi Biotec) after fixation and permeabilization with ice-cold methanol. Frequency of inducible CAR T cells was analyzed via flow cytometric determination of anti- CD20 CAR expression using anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec). Transduced T cells were used for functional assays after day 10 post-transduction. Cells were cultured at 37ºC and 5% CO2 for the whole duration of the experiments.

[0234] Drug-inducible CAR T cells generated with the four different possible lentiviral vector combinations that allow for theoretically functional switches (Liposwitch1+3, 1+4, 2+3 & 2+4, Figure 15a, b) were cultured either in absence or presence of 500 nM of A1120. Unstransduced T cells were used as negative control and T cells transduced with conventional anti-CD20 CAR as positive control. Induced anti-CD20 CAR expression was detected via flow cytometry upon staining with anti-CD20 CAR Detection Reagent PE (Miltenyi Biotec). To exclude dead cells, Propidium iodide (PI; Miltenyi Biotec) was added to the stained cells directly before sample acquisition at the MACSQuant® Analyzer 10 (Miltenyi Biotec). Frequency of anti-CD20 CAR+ cells upon A1120 induction strongly varied among the different constructs with distinct domain arrangements (Figure 15c, d). This experiment confirms once more (as in example 1.4 and 4.3) that the most favorable domain positioning leading to higher responsiveness of heterodimeric switches is the construct with N1 zinc finger in N-terminal position and p65 activation domain in C-terminal position from their respective anti-caffeine nanobodies (LVI Liposwitch1 + LVII Liposwitch3). Drug-inducible T cells generated with this particular configuration led to a positive frequency markedly higher than the rest of combinations and to a responsiveness close to 100% (understanding responsiveness as the frequency of transduced cells expressing the gene of interest). Interestingly, none of the constructs led to background expression of anti-CD20 CAR in absence of A1120, showing that the expression of the gene of interest in our system is strictly dependent on the presence of the inducer. The combination of constructs showing highest responsiveness (LVI Liposwitch1 + LVII Liposwitch3), also led to the strongest response, achieving a 2-fold higher anti-CD20 CAR expression than with the rest of the configurations (Figure 15d). This proves that this particular combination is optimal to trigger potent transcriptional activation of the gene of interest. Comparable results were obtained in SupT1 cell line and same configuration showed the highest responsiveness and transcriptional output in absence of background (Figure 15e,f). Interestingly, the optimaldomain positioning of the lipocalin-based synthetic transcription factor is equivalent to the most efficient orientation of the nanobody-based split synthetic transcription factor (Figure 5a-d, Figure 15a-d).

[0235] Example 15: Lipocalin-based split synthetic transcription factors are functional with different RF2 variants and show a strong inducer dose – response correlation. 15.1. Functionality of lipocalin-based drug-inducible switches carrying different variants of engineered binder RF2 Since we determined that Liposwitch1 contains the most efficient domain orientation for the split sTF1 (SEQ ID NO: 46) we proceeded with this construct for further experiments. An alternative version of the most efficient orientation of LVII (Liposwitch 3, split sTF2 (SEQ ID NO: 47) was generated by replacing RF2 sequence (SEQ ID NO: 38) by variant RF2s11 (SEQ ID NO: 39) (Liposwitch5, split sTF2 with RF2s11 (SEQ ID NO: 48) (Figure 16b) via restriction – ligation method. Human primary T cells were co-transduced with either a combination of Liposwitch1+3 or Liposwitch1+5 lentiviruses (Figure 16 a, b) and cultured either in presence of 100 nM A1120 or absence of inducer. Detection of anti-CD20 CAR via flow cytometry revealed that both versions of the lipocalin-based switch carrying different variants of RF2 are functional and do not lead to background expression in absence of the inducer (Figure 16c). Moreover, level of expression of anti-CD20 CAR measured as mean fluorescence intensity (MFI) is comparable for both liposwitch versions (Figure 16d). 15.2. Frequency and level of expression of A1120-induced anti-CD20 CAR show a strong dose- dependent correlation in human primary T cells independently of the RF2 variant used

[0236] Drug-inducible CAR T cells carrying the most efficient orientation domain of the split synthetic transcription factor (LVI: Liposwitch 1, LVII: Liposwitch 3(RF2) or Liposwitch 5(RF2s11)) were generated as described in section 13.2. To determine the effect of inducer dose on the level of expression of inducible anti-CD20 CAR, transduced human primary T cells were cultured with different concentrations of A1120 ranging from 1 to 50000 nM. Flow cytometry analysis revealed that frequency of anti-CD20 CAR+ cells is detectable with doses of A1120 as low as 1 nM and increases with higher concentrations of inducer up to 100 nM when using variant RF2 (Figure 17a) and 50 nM when using RF2s11 (Figure 17b). In both cases, the frequency of CAR+ T cells is strictly dependent on the presence of A1120 with higher drug doses resulting in greater induction ratios (Figure 17a, b). Concurrently, surface expression (represented as MFI) steadily increased with the drug dose and plateaued with concentrationsabove 100 nM A1120 (Figure 17 c, d). These results demonstrate that expression level of the gene of interest can be easily modulated by adjusting the concentration of the inducer in primary T cells transduced with both functional units of the lipocalin-based switch. Comparable results were obtained in SupT1 cells transduced with the abovementioned combination of lipocalin- based switches LVI and LVII (Figure 17 e-h).

[0237] Example 16: Cytotoxic activity of human primary T cells transduced with lipocalin- based transcriptional switches controlling the expression of anti-CD20 CAR in co-cultures with CD20+ GFP+ 526-Mel tumor cells. Drug-inducible CAR T cells were generated by cotransduction of human primary T cells with both functional units of the optimal configuration of the lipocalin-based switches, including both RF2 versions (either Liposwitch 1 + Liposwitch 3 or Liposwitch 1 + Liposwitch 5). See section 13.3. Positive control was generated by transduction of human primary T cell with a lentiviral vector carrying a conventional anti-CD20 CAR driven by EF1alpha promoter. Cells were cultured with RPMI 1640 + 10% FCS + 2 mM L-Glutamine + 12.5 ng / ml IL7 + 12.5 ng / ml IL15 for 10 days. One day before the start of the assay, 1x104and 2x104CD20+ GFP+ 526-Mel tumor cells per well were seeded on a 96-well plate and cultured overnight with full RPMI without cytokines. On the day of co-culture setup, frequency of transduced T cells was determined by flow cytometry via detection of HA-tag or anti-CD20 CAR. Transduction efficiency was adjusted by spiking-in untransduced T cells to the respective groups. 1x104effector T cells were seeded in each well containing tumor cells. Co-cultures were incubated at 37ºC and 5% CO2 either in presence of 100 nM A1120 or in absence of inducer and monitored by live-cell imaging using the Incucyte S3 Live-Cell Analysis system (Sartorius) for 72 hours. Analysis of green fluorescence intensity revealed that tumor-specific cytotoxicity of inducible CAR T cells was strictly dependent on the presence of A1120 characterized by the absence of basal lysis in the non-induced state (Figure 18a). Moreover, both lipocalin-based switches led to comparable efficacy to the conventional anti-CD20 CAR, although with slower kinetics. Differences in killing kinetics can be explained by the fact that conventional CAR is constitutively expressed and therefore, active from the beginning of the co-culture while inducible CARs require several hours before CAR molecules are displayed on the cell surface. Comparable results were obtained when increasing the starting number of tumor cells (E:T 1:2) (Figure 18b). These results demonstrate that, in presence of inducer, our lipocalin-based switches can achieve comparable efficacy to a conventional anti-CD20 CAR.Sequences

[0238] SEQ ID NO:1 (recognition sequence; amino acid sequence): SQSSNLVR

[0239] SEQ ID NO:2 (recognition sequence; amino acid sequence): SQSSSLVR

[0240] SEQ ID NO:3 (recognition sequence; amino acid sequence): SRSDKLVR

[0241] SEQ ID NO:4 (consensus zinc-finger-framework sequence derived from native and mutant versions of Sp1 zinc fingers; X= any natural amino acid): MAQAALEPKEKPYACPECGKSFXXXXXXXXHQRTHTGEKPYKCPECGKSFXXXXXXXXHQ RTHTGEKPYKCPECGKSFXXXXXXXXHQRTHTGKKTSGQAG

[0242] SEQ ID NO:5 (N1 zinc-finger protein; amino acid sequence): MAQAALEPKEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSSLVRHQRT HTGEKPYKCPECGKSFSRSDKLVRHQRTHTGKKTSGQAG

[0243] SEQ ID NO:6 (responsive element for N1; DNA sequence): ggggtagaaaaaggggtagaa

[0244] SEQ ID NO:7 (Recognition motif for N1; DNA sequence, n= a, c, g or t): ggggtagaan

[0245] SEQ ID NO:8 (E1b minimal promoter; DNA sequence): agggtatataatg

[0246] SEQ ID NO:9 (E1b minimal promoter with 5 responsive elements for N1; DNAsequence): ggggtagaaaaaggggtagaaccgagcggggtagaaaaaggggtagaactcttaggggtagaaaaaggggtagaagttaatggggtagaaaa aggggtagaataacagggggtagaaaaaggggtagaaagtcgactctagagggtatataatg

[0247] SEQ ID NO:10 (VHH1, acVHH dfM1-1; amino acid sequence): QVQLVESGGGLVQAGGSLRLSATAYGKTGTIYSMAWFRQAPGKEREFLAPVALGLQSTYYM DSVKGRFTISRDKGKNTVYLQMDSLKPEDTAVYYAAATRAYSVGYDYWGQGTQVTVTS

[0248] SEQ ID NO: 11 (VHH2, acVHH-V104D); amino acid sequence): QVQLVESGGGLVQAGGSLRLSCTASGRTGTIYSMAWFRQAPGKEREFLATVGWSSGITYYM DSVKGRFTISRDKGKNTVYLQMDSLKPEDTAVYYCTATRAYSDGYDYWGQGTQVTVSS

[0249] SEQ ID NO:12 (full length NFκB p65; amino acid sequence): MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKING YTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQ AISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTA ELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTP PYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPF SGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPP QVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQR PPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS

[0250] SEQ ID NO:13 (NFκB p65 amino acids 331 – 551 fragment; amino acid sequence): DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAP PAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAP HTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQIS S

[0251] SEQ ID NO:14 (NFκB p65 amino acids 521 – 551 fragment; amino acid sequence): APGLPNGLLSGDEDFSSIADMDFSALLSQISS

[0252] SEQ ID NO:15 (Full length Human herpes simplex virus 1 Tegument protein VP16, amino acid sequence): MDLLVDELFADMNADGASPPPPRPAGGPKNTPAAPPLYATGRLSQAQLMPSPPMPVPPAALF NRLLDDLGFSAGPALCTMLDTWNEDLFSALPTNADLYRECKFLSTLPSDVVEWGDAYVPERT QIDIRAHGDVAFPTLPATRDGLGLYYEALSRFFHAELRAREESYRTVLANFCSALYRYLRASV RQLHRQAHMRGRDRDLGEMLRATIADRYYRETARLARVLFLHLYLFLTREILWAAYAEQM MRPDLFDCLCCDLESWRQLAGLFQPFMFVNGALTVRGVPIEARRLRELNHIREHLNLPLVRSA ATEEPGAPLTTPPTLHGNQARASGYFMVLIRAKLDSYSSFTTSPSEAVMREHAYSRARTKNNY GSTIEGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG

[0253] SEQ ID NO:16 (Human herpes simplex virus 1 Tegument protein VP16 amino acids 437 – 448 fragment, amino acid sequence): DALDDFDLDML

[0254] SEQ ID NO:17 (VP64 activation domain, tetrameric repeat of Human herpes simplex virus 1 Tegument protein VP16 amino acids 437 – 448 fragment, amino acid sequence): DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML

[0255] SEQ ID NO:18 (Full length Cbp / p300-interacting transactivator 2 CITED2, amino acid sequence): MADHMMAMNHGRFPDGTNGLHHHPAHRMGMGQFPSPHHHQQQQPQHAFNALMGEHIHY GAGNMNATSGIRHAMGPGTVNGGHPPSALAPAARFNNSQFMGPPVASQGGSLPASMQLQKL NNQYFNHHPYPHNHYMPDLHPAAGHQMNGTNQHFRDCNPKHSGGSSTPGGSGGSSTPGGSG SSSGGGAGSSNSGGGSGSGNMPASVAHVPAAMLPPNVIDTDFIDEEVLMSLVIEMGLDRIKEL PELWLGQNEFDFMTDFVCKQQPSRVSC

[0256] SEQ ID NO: 19 (CITED 2 amino acids 161 – 270 fragment, amino acid sequence): SGGSSTPGGSGGSSTPGGSGSSSGGGAGSSNSGGGSGSGNMPASVAHVPAAMLPPNVIDTDFI DEEVLMSLVIEMGLDRIKELPELWLGQNEFDFMTDFVCKQQPSRVSC

[0257] SEQ ID NO: 20 (Saccharomyces cerevisiae full length regulatory protein GAL4, amino acid sequence):MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLER LEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHR ISATSSSEESSNKGQRQLTVSIDSAAHHDNSTIPLDFMPRDALHGFDWSEEDDMSDGLPFLKTD PNNNGFFGDGSLLCILRSIGFKPENYTNSNVNRLPTMITDRYTLASRSTTSRLLQSYLNNFHPY CPIVHSPTLMMLYNNQIEIASKDQWQILFNCILAIGAWCIEGESTDIDVFYYQNAKSHLTSKVF ESGSIILVTALHLLSRYTQWRQKTNTSYNFHSFSIRMAISLGLNRDLPSSFSDSSILEQRRRIWW SVYSWEIQLSLLYGRSIQLSQNTISFPSSVDDVQRTTTGPTIYHGIIETARLLQVFTKIYELDKTV TAEKSPICAKKCLMICNEIEEVSRQAPKFLQMDISTTALTNLLKEHPWLSFTRFELKWKQLSLII YVLRDFFTNFTQKKSQLEQDQNDHQSYEVKRCSIMLSDAAQRTVMSVSSYMDNHNVTPYFA WNCSYYLFNAVLVPIKTLLSNSKSNAENNETAQLLQQINTVLMLLKKLATFKIQTCEKYIQVL EEVCAPFLLSQCAIPLPHISYNNSNGSAIKNIVGSATIAQYPTLPEENVNNISVKYVSPGSVGPSP VPLKSGASFSDLVKLLSNRPPSRNSPVTIPRSTPSHRSVTPFLGQQQQLQSLVPLTPSALFGGAN FNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSNVHDNFMNNEITASKIDDGNNSK PLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDDEDTPPNPKKE

[0258] SEQ ID NO: 21 (Saccharomyces cerevisiae regulatory protein GAL4 amino acids 1 – 93 fragment, amino acid sequence): MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLER LEQLFLLIFPREDLDMILKMDSLQDIKALL

[0259] SEQ ID NO: 22 (Synthetic zinc finger protein targeting human CCR5 locus, CCR5-224+ ZF, amino acid sequence): FQCRICMRNFSDRSNLSRHIRTHTGEKPFACDICGRKFAISSNLNSHTKIHTGSQKPFQCRICMR NFSRSDNLARHIRTHTGEKPFACDICGRKFATSGNLTRHTKIHLRGSQL

[0260] SEQ ID NO: 23 (N1 Zinc Finger – G4S linker - acVHH_dfM1-1 (VHH1) – G4S linker – cmyc NLS, amino acid sequence): MAQAALEPKEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSSLVRH QRTHTGEKPYKCPECGKSFSRSDKLVRHQRTHTGKKTSGQAGGGGGSNIQVQLVESGGG LVQAGGSLRLSATAYGKTGTIYSMAWFRQAPGKEREFLAPVALGLQSTYYMDSVKGRFTISR DKGKNTVYLQMDSLKPEDTAVYYAAATRAYSVGYDYWGQGTQVTVTSGGGGSTGPAAKR VKLD

[0261] SEQ ID NO: 24 (acVHH_V106D (VHH2) – G4S linker – p65 AD – G4S linker – cmyc NLS, amino acid sequence): MQVQLVESGGGLVQAGGSLRLSCTASGRTGTIYSMAWFRQAPGKEREFLATVGWSSGI TYYMDSVKGRFTISRDKGKNTVYLQMDSLKPEDTAVYYCTATRAYSDGYDYWGQGTQ VTVSSGGGGSVYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVP VLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEF QQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIA DMDFSALLSQISSTSYGGGGSRTPAAKRVKLD

[0262] SEQ ID NO: 25 (N1 Zinc Finger – G4S linker - acVHH_dfM1-1 (VHH1), amino acid sequence): MAQAALEPKEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSSLVRH QRTHTGEKPYKCPECGKSFSRSDKLVRHQRTHTGKKTSGQAGGGGGSNIQVQLVESGGG LVQAGGSLRLSATAYGKTGTIYSMAWFRQAPGKEREFLAPVALGLQSTYYMDSVKGRFTISR DKGKNTVYLQMDSLKPEDTAVYYAAATRAYSVGYDYWGQGTQVTVTS

[0263] SEQ ID NO: 26 (acVHH_V106D (VHH2) – G4S linker – p65 AD, amino acid sequence): MQVQLVESGGGLVQAGGSLRLSCTASGRTGTIYSMAWFRQAPGKEREFLATVGWSSGI TYYMDSVKGRFTISRDKGKNTVYLQMDSLKPEDTAVYYCTATRAYSDGYDYWGQGTQ VTVSSGGGGSVYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVP VLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEF QQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIA DMDFSALLSQISSTSY

[0264] SEQ ID NO: 27 (N1 Zinc Finger – G4S linker - acVHH_dfM1-1 (VHH1) – G4S linker – 3xFLAG tag - cmyc NLS, amino acid sequence): MAQAALEPKEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSSLVRH QRTHTGEKPYKCPECGKSFSRSDKLVRHQRTHTGKKTSGQAGGGGGSNIQVQLVESGGG LVQAGGSLRLSATAYGKTGTIYSMAWFRQAPGKEREFLAPVALGLQSTYYMDSVKGRFTISR DKGKNTVYLQMDSLKPEDTAVYYAAATRAYSVGYDYWGQGTQVTVTSGGGGSTG DYKDDDDKDYKDDDDKDYKDDDDKPAAKRVKLD

[0265] SEQ ID NO: 28 (acVHH_V106D (VHH2) – G4S linker – p65 AD – G4S linker – 3x HA tag – cmyc NLS, amino acid sequence): MQVQLVESGGGLVQAGGSLRLSCTASGRTGTIYSMAWFRQAPGKEREFLATVGWSSGI TYYMDSVKGRFTISRDKGKNTVYLQMDSLKPEDTAVYYCTATRAYSDGYDYWGQGTQ VTVSSGGGGSVYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVP VLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEF QQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIA DMDFSALLSQISSTSYGGGGSRT YPYDVPDYAYPYDVPDYAYPYDVPDYAPAAKRVKLD

[0266] SEQ ID NO:29 (Recognition motif for GAL4 DBD; DNA sequence): CGGGTTACAGCAAGCCG

[0267] SEQ ID NO:30 (E1b minimal promoter with 7 binding sites for GAL4 DBD; DNA sequence): CGGGCTATATGCAACCGAGCATCGGAAGGGATGCCCGAGCGGAAGAGAGGCTTCCGCAT GGCGGGTTACAGCAAGCCGATTGACGGAGGGTAAGTCGCCGAGTGGAGTACTGTCCTCC GAGCGGAGTACTGTCCTCCGGTCGACTCTAGAGGGTATATAATG

[0268] SEQ ID NO:31 (Recognition motif for CCR5-224+ ZF; DNA sequence): GATGAGGATGAC

[0269] SEQ ID NO:32 (E1b minimal promoter with 8 binding sites for CCR5-224+ ZF; DNA sequence): ATGAGGATGACCACGAGATGAGGATGACTTCAATTCAAGATGAGGATGACTTAGAGATG AGGATGACATCAGGATGAGGATGACTAAGAGATGAGGATGACAACGAGATGAGGATGA CTTACAGATGAGGATGACAGTCGACTCTAGAGGGTATATAATG

[0270] SEQ ID NO:33 (human Phosphoglycerate kinase 1 promoter; DNA sequence): GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTC TGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTC CGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCT GCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACG GAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGC GCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCGGGGCGCGCCGAGAGCA GCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTC CTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCC CTCGTTGACCGAATCACCGACCTCTCTCCCCG

[0271] SEQ ID NO:34 (G4S linker): GGGS

[0272] SEQ ID NO:35 (DYKDDDDK-tag): DYKDDDDK

[0273] SEQ ID NO:36 (G4S): GGGGS

[0274] SEQ ID NO: 37 (human Retinol Binding Protein 4 (hRBP4) variant, amino acid sequence): ERDYRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRV RLLNNWDVMADMVGTFTDTEDPAKFKMKYWGVASFLQNGNDDFWIVDTDYDTYAVQYSP RLLNLDGTAADSYSFVFSRDPNGLPPEAQKIVRQRQEELGLAGQYRLIVHNGL

[0275] SEQ ID NO: 38 (RF2 variant of human fibronectin type III domain (FN3), amino acid sequence): VSDVPRDLEVVAATPTSLLISWYYPNASHAGYYRITYGETGGNSPVQEFTVPFSIRYTIATISGL KPGVDYTITVYAVTDYAYYYRLSEPISINYRTEIDKPSQ

[0276] SEQ ID NO: 39 (RF2s11 variant of human fibronectin type III domain (FN3), amino acid sequence): VSDGPRDLEVVAATPTSLLISWYYPNASHAGYYRITYGETGGNSPVQEFTVPFSIRYTIATISGL KPGVDYTITVYAVTDYDYYYRMSEPISINYRTEIDKPSQ

[0277] SEQ ID NO: 40 (N1 Zinc Finger – G4S linker – hRBP4 – G4S linker – cmyc NLS, amino acid sequence): MAQAALEPKEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSSLVRH QRTHTGEKPYKCPECGKSFSRSDKLVRHQRTHTGKKTSGQAGGGGGSNIERDYRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSTSY

[0282] SEQ ID NO: 45 (RF2s11 – G4S linker – p65 AD, amino acid sequence): VSDGPRDLEVVAATPTSLLISWYYPNASHAGYYRITYGETGGNSPVQEFTVPFSIRYTIAT ISGLKPGVDYTITVYAVTDYDYYYRMSEPISINYRTEIDKPSQGGGGSVYDEFPTMVFPSGQ ISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPE AITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSTSY

[0283] SEQ ID NO: 46 (N1 Zinc Finger – G4S linker – hRBP4 – G4S linker – 3xFLAG tag - cmyc NLS, amino acid sequence): MAQAALEPKEKPYACPECGKSFSQSSNLVRHQRTHTGEKPYKCPECGKSFSQSSSLVRH QRTHTGEKPYKCPECGKSFSRSDKLVRHQRTHTGKKTSGQAGGGGGSNI ERDYRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRV RLLNNWDVMADMVGTFTDTEDPAKFKMKYWGVASFLQNGNDDFWIVDTDYDTYAVQYSP RLLNLDGTAADSYSFVFSRDPNGLPPEAQKIVRQRQEELGLAGQYRLIVHNGLGGGGSTG DYKDDDDKDYKDDDDKDYKDDDDKPAAKRVKLD

[0284] SEQ ID NO: 47 (RF2 – G4S linker – p65 AD – G4S linker – 3x HA tag – cmyc NLS, amino acid sequence): VSDVPRDLEVVAATPTSLLISWYYPNASHAGYYRITYGETGGNSPVQEFTVPFSIRYTIAT ISGLKPGVDYTITVYAVTDYAYYYRLSEPISINYRTEIDKPSQGGGSVYDEFPTMVFPSGQIS QASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTL SEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEA ITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSTSYGGGGSRTYPY DVPDYAYPYDVPDYAYPYDVPDYAPAAKRVKLD

[0285] SEQ ID NO: 48 (RF2s11 – G4S linker – p65 AD – G4S linker – 3x HA tag – cmyc NLS, amino acid sequence): VSDGPRDLEVVAATPTSLLISWYYPNASHAGYYRITYGETGGNSPVQEFTVPFSIRYTIAT ISGLKPGVDYTITVYAVTDYDYYYRMSEPISINYRTEIDKPSQGGGSVYDEFPTMVFPSGQI SQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGT LSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPE AITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSTSYGGGGSRTYP YDVPDYAYPYDVPDYAYPYDVPDYAPAAKRVKLD

[0286] SEQ ID NO:49 (wt human RBP4): ERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRV RLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSC RLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRSERNLL

[0287] SEQ ID NO: 50 (Glucocorticoid Receptor isoform β): MDSKESLTPGREENPSSVLAQERGDVMDFYKTLRGGATVKVSASSPSLAVASQSDSKQRRLL VDFPKGSVSNAQQPDLSKAVSLSMGLYMGETETKVMGNDLGFPQQGQISLSSGETDLKLLEE SIANLNRSTSVPENPKSSASTAVSAAPTEKEFPKTHSDVSSEQQHLKGQTGTNGGNVKLYTTD QSTFDILQDLEFSSGSPGKETNESPWRSDLLIDENCLLSPLAGEDDSFLLEGNSNEDCKPLILPD TKPKIKDNGDLVLSSPSNVTLPQVKTEKEDFIELCTPGVIKQEKLGTVYCQASFPGANIIGNKM SAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSLGTLNFPGRTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASGCHYGVLTCGSCK VFFKRAVEGQHNYLCAGRNDCIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQAT TGVSQETSENPGNKTIVPATLPQLTPTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLGG RQVIAAVKWAKAIPGFRNLHLDDQMTLLQYSWMFLMAFALGWRSYRQSSANLLCFAPDLII NEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSVPKDGLKSQELFDEIR MTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHENVMWLKPESTSHTLI

[0288] SEQ ID NO 51 (STAT5b N642H): MAVWIQAQQLQGEALHQMQALYGQHFPIEVRHYLSQWIESQAWDSVDLDNPQENIKATQLL EGLVQELQKKAEHQVGEDGFLLKIKLGHYATQLQNTYDRCPMELVRCIRHILYNEQRLVREA NNGSSPAGSLADAMSQKHLQINQTFEELRLVTQDTENELKKLQQTQEYFIIQYQESLRIQAQF GPLAQLSPQERLSRETALQQKQVSLEAWLQREAQTLQQYRVELAEKHQKTLQLLRKQQTIIL DDELIQWKRRQQLAGNGGPPEGSLDVLQSWCEKLAEIIWQNRQQIRRAEHLCQQLPIPGPVEE MLAEVNATITDIISALVTSTFIIEKQPPQVLKTQTKFAATVRLLVGGKLNVHMNPPQVKATIISE QQAKSLLKNENTRNDYSGEILNNCCVMEYHQATGTLSAHFRNMSLKRIKRSDRRGAESVTEE KFTILFESQFSVGGNELVFQVKTLSLPVVVIVHGSQDNNATATVLWDNAFAEPGRVPFAVPDK VLWPQLCEALNMKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGLSVSWSQFNREN LPGRNYTFWQWFDGVMEVLKKHLKPHWNDGAILGFVNKQQAHDLLINKPDGTFLLRFSDSE IGGITIAWKFDSQERMFWHLMPFTTRDFSIRSLADRLGDLNYLIYVFPDRPKDEVYSKYYTPVP CESATAKAVDGYVKPQIKQVVPEFVNASADAGGGSATYMDQAPSPAVCPQAHYNMYPQNP DSVLDTDGDFDLEDTMDVARRVEELLGRPMDSQWIPHAQS References

[0289] Kotter B, Engert F, Krueger W, Roy A, Rawashdeh WA, Cordes N, Drees B, Webster B, Werchau N, Lock D, Dapa S, Schneider D, Ludwig S, Rossig C, Assenmacher M, Mittelstaet J, Kaiser AD. Titratable Pharmacological Regulation of CAR T Cells Using Zinc Finger-Based Transcription Factors. Cancers (Basel). 2021 Sep 22;13(19):4741. doi:

[0290] Sakemura R, Terakura S, Watanabe K, Julamanee J, Takagi E, Miyao K, Koyama D, Goto T, Hanajiri R, Nishida T, Murata M, Kiyoi H. A Tet-On Inducible System for Controlling CD19-Chimeric Antigen Receptor Expression upon Drug Administration. Cancer Immunol Res. 2016 Aug;4(8):658-68. doi: 10.1158 / 2326-6066.CIR-16-0043. Epub 2016 Jun 21. PMID: 27329987.

[0291] Bojar D, Scheller L, Hamri GC, Xie M, Fussenegger M. Caffeine-inducible gene switches controlling experimental diabetes. Nat Commun. 2018 Jun 19;9(1):2318. doi: 10.1038 / s41467-018-04744-1. PMID: 29921872; PMCID: PMC6008335.

Claims

Claims 1. A system comprising a. A first exogenous nucleic acid molecule encoding a first fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety, i.e. said first fusion protein comprises from the N- to the C-terminus said DNA binding domain – said first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promoter operably linked to a nucleic acid molecule encoding an effector molecule b. A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a second binding moiety, i.e. said second fusion protein comprises from the N- terminus to the C-terminus said second binding moiety – said transcription regulating domain, c. An Inducer molecule, Wherein the first and second binding moieties are different from each other, and Wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule.

2. A system according to claim 1, wherein the first nucleic acid molecule and the second nucleic acid molecule are in a first and a second expression cassette, respectively, wherein the first and second expression cassettes are comprised in two different vectors.

3. A system according to any of the claims 1-2, wherein the nucleic acid molecule encoding an effector molecule is an endogenous or exogenous nucleic acid molecule.

4. A system according to any of the claims 1-3, wherein the effector molecule is selected from the group consisting of an antibody or an antigen binding fragment thereof, a chimeric antigen receptor, a cytokine and a chemokine.

5. A system according to any of the claims 1-4, wherein the transcription regulating domain is a transcription activation domain.

6. A system according to claim 5, wherein the DNA binding domain is a zinc finger domain.

7. A system according to any of the claims 1-6 wherein the first and second binding moieties comprise the binding domain of a single domain antibody (VHH) or a fragment thereof, respectively and, wherein the binding domains are specific for Xanthine or a derivate thereof and wherein the inducer molecule is xanthine or a derivate thereof 8. A system according to claim 7, wherein the first binding moiety comprises the binding domain of a single domain antibody (VHH), wherein the VHH domain comprises SEQ ID No: 10 and wherein the second binding moiety comprises the binding domain of a single domain antibody (VHH), wherein the VHH domain comprises SEQ ID No: 11, and wherein the inducer molecule is caffeine.

9. An engineered cell comprising a. A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety, i.e. said first fusion protein comprises from the N- to the C-terminus said DNA binding domain – said first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule b. A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a second binding moiety, i.e. said second fusion protein comprises from the N- terminus to the C-terminus said second binding moiety – said transcription regulating domain, and wherein the first and second binding moieties are different from each other, and Wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and wherein said engineered cell is in combination with the inducer molecule.

10. A pharmaceutical composition comprising a population of engineered cells according to claim 9 and an inducer molecule.

11. An engineered cell according to the claim 9 for use in a method of treating a disease.

12. An engineered cell according to claim 9 for use in immunotherapy.

13. An in vitro method for inducing the expression of an effector molecule comprising the steps: a) Providing a composition comprising engineered cells comprising i) A first exogenous nucleic acid molecule encoding a fusion protein comprising a DNA binding domain N-terminally fused to a first binding moiety i.e. said first fusion protein comprises from the N- to the C-terminus said DNA binding domain – said first binding moiety, wherein the DNA binding domain is specific for a regulatory element in the promotor operably linked to a nucleic acid molecule encoding an effector molecule, ii) A second exogenous nucleic acid molecule encoding a second fusion protein comprising a transcription regulating domain C terminally fused to a binding moiety, i.e. said second fusion protein comprises from the N- terminus to the C-terminus said second binding moiety – said transcription regulating domain, and wherein the first and second binding moieties are different from each other, and Wherein the first and second binding moiety are capable to dimerize in presence of the inducer molecule, and b) Adding the inducer molecule to the composition comprising engineered cells

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