An Anti-her2 SCFV-secreting armoured car t cell

Abstract

The present invention provides recombinant cells, such as recombinant T cells. The recombinant cells comprise a chimeric antigen receptor (CAR), for example a CAR specifically binding to HER2, and a nucleic acid encoding a scFv specifically binding to a second tumor antigen, for example CEACAM6. Expression of the scFv is dependent on binding of the CAR to its antigen. Provided are medical uses of the cells, such as the use in the treatment of cancer.

Description

[0001] AN ANTI-HER2 SCFV-SECRETING ARMOURED CAR T CELL

[0002] Field of the invention

[0003] The present invention provides recombinant cells, such as recombinant T cells. The recombinant cells comprise a chimeric antigen receptor (CAR), for example a CAR specifically binding to HER2, and a nucleic acid encoding a scFv specifically binding to a second tumor antigen, for example CEACAM6. Expression of the scFv is dependent on binding of the CAR to its antigen. Provided are medical uses of the cells, such as the use in the treatment of cancer.

[0004] Background of the invention

[0005] Immune checkpoint inhibitors and CEACAM's

[0006] Autoreactive T cells are eliminated by apoptosis triggered by negative selection in the thymus (J Mol Med (Berl) 2014;92(7):735-41). Despite this well-regulated process, a few self-reactive T lymphocytes may still circulate in the periphery. Additional peripheral tolerance mechanisms inactivate these cells, thereby preventing auto-reactive processes (Cold Spring Harb Perspect Biol 2012;4(6): a006957). Therefore, for modulation of immune response, not only activating factors but also inactivating factors are essential (Nat Rev Immunol 2013;13(4):227-42).

[0007] Immune checkpoints ensure that the T cell response is deactivated under physiological conditions and thus prevents autoreactive processes (Nat Rev Cancer 2012;12(4):252-64). One such immune checkpoint is CTLA-4. CTLA-4 is very similar to the activating co-stimulatory molecule CD28 and binds to B7 ligands (CD80 / CD86). There are however also differences between CTLA-4 and CD28. While CD28 leads to activation, binding of CTLA-4 leads to inhibition of the T cell. If both molecules are present, they compete for binding. However, since CTLA-4 has an about 20x higher affinity for the receptor, the CTLA-4 effect outweighs that of CD28. In this way, CTLA-4 prevents the activating effect of CD28, thus leading to inactivation of the T cell, which is also reflected in a reduction of the IL-2 level (Immunol Rev 2008;224:166-82). The B7 family also includes the checkpoint molecule PD-1, which is expressed on T cells and also prevents autoimmunity and induces self-tolerance (Vet Immunol Immunopathol 2010;134(l-2):33— 8).

[0008] One difference between these two molecules is the point in time at which they intervene. While CTLA-4 can stop autoreactive cells even in the lymph node at the beginning of the activation of naive T cells, PD-1 modulates the immune response of already activated T cells in the periphery (Immunol Rev 2008;224:166-82; J Exp Med 1995;182(2):459— 65). If PD-1 binds its ligands PD-L 1 or PD-L 2, normally expressed in the peripheral tissue, the secretion of IL-2, TNFa and IFN-y, as well as the potential for proliferation are inhibited (Annu Rev Immunol2008;26:677-704). PD-L 1 is not only expressed in the peripheral tissue, tumors have also taken advantage of the ability to suppress T-cell reactivity by expressing PD-L 1, thereby avoiding destruction by the immune system (Oncol Lett 2019;18(5): 5399-407; J Clin Invest 2015;125(9):3384-91). Checkpoint inhibitors therefore became important targets for the development of therapeutics that block these mechanisms in tumors.

[0009] Another immune checkpoint that is an important target for the development of immunotherapeutic treatments is CEACAM6. CEACAM6 is a member of the CEA gene family, which is a part of the immunoglobulin superfamily (Clin Cancer Drugs 2015;2(2):100-11.9) CEA is known to be a marker for many tumor diseases (Tumour Biol 1995;16(1):32— 419). CEACAMs are membrane-bound glycoproteins, which can also be found physiologically on granulocytes and on epithelia of various organs. The genes responsible for the expression of the different CEACAMs are located on the chromosome 19ql3.1-13.2, the long arm of chromosome 19, and are independent of each other (Clin Cancer Drugs 2015;2(2):100— 11). There are at least 12 different isoforms of this family, all of whom differ in their functions (Tumour Biol 1995; 16(1):32— 41). They are all attached to the membrane either by a transmembrane domain or a glycophosphatidyl-inositol (GPI) anchor (Curr Oncol 2007;14(2):70— 3).

[0010] While CEACAM6, also known as CD66c, is one of the CEACAMs attached via a GPI anchor, CEACAM1, also known as CD66a, is attached via a transmembrane domain (Clin Cancer Drugs 2015;2(2):100— 11). The functions include physiological processes such as cell adhesion, signaling within and among cells, as well as a regulating function in T cell proliferation, thereby influencing inflammation and angiogenesis in the tumor tissue (Cancer Metastasis Rev 2013;32(3-4):643-71; Curr Opin Cell Biol 2006;18(5):565— 71). When examining adenomas and hyperplastic polyps, an increased CEACAM6 expression was found in tumor for the first time (The American Journal of Pathology 2000;156(2):595-605). It has also been found that CEACAM6 expression is also increased in a large number of tumors, including, among others, solid tumors such as pancreatic cancer, gastric cancer, cholangiocarcinoma, non-small cell lung cancer and breast cancer as well as hematological malignancies such as acute lymphoblastic leukemia and multiple myeloma (Clin Cancer Drugs 2015;2(2):100-ll; BMC Cancer 2007;7:2). Increased CEACAM6 expression promotes the progression of cancer, as well as the formation of metastases through changes in cell growth, differentiation, migration, and a reduced apoptosis effect (J Clin Oncol. 2003;21(19): 3638-3646). The anti-apoptotic effect of an increased CEACAM6 expression correlates this with an invasive behavior (Cancer Res 2005;65(19):8809-17). Furthermore, in the breast cancer cell line MCF7 it was shown that increased CEACAM6 expression is involved in the development of resistance to therapeutic agents such as tamoxifen (Clin Cancer Res 2008;14(2):405-ll). Also in pancreatic adenocarcinoma cells, resistance to chemotherapeutic gemcitabine was linked to a high CEACAM6 expression.

[0011] Silencing of the CEACAM6 gene using small interfering RNA also showed that downregulation of CEACAM6 inhibits the formation of metastases in vivo (Cancer Res 2004;64(ll):3987-93). It is therefore not surprising that a high CEACAM6 expression is a negative prognostic marker that leads to a poor prognosis (J Clin Oncol 2003;21(19):3638-46; Clin Chim Acta 2013;415:12-9). Further investigations in multiple myeloma also showed that the increased expression of CEACAM6 has a negative effect on the reactivity of cytotoxic T cells (Blood 2013;121(22):4493-503). A basic principle, important for this mechanism is the fact that CEACAMs formhomotypic bonds to other members of the CEA family, such as for example the CEACAM6-CEACAM1 interaction (Clin Cancer Drugs 2015;2(2):100- 11). CEACAM1 is also expressed by T lymphocytes and, after activation of the T cell via the TCR, is able to act as an inhibitor of the activation (Ann N Y Acad Sci 2006;1072:155-75; Nat Rev Immunol 2006;6(6):433-46). Thus, the interaction of tumor cells, expressing CEACAM6 with cytotoxic T cells leads to an inhibition of the reactivity of the T cells. If CEACAM6 is blocked via antibodies, T cell activity is increased again. In a functional T cell assay, it was shown that the IFN-y production of the T cells could be significantly increased by adding a monoclonal CEACAM6 antibody (Blood 2013;121(22):4493-503).

[0012] Analogical to PD-1 and CTLA-4, CEACAM1 can also be counted among the proliferative checkpoints (Oncoimmunology 2017;6(7):el328336.). However, not all T cells express CEACAM1; only after activation through e.g. anti-CD3 antibodies there is a CEACAM1 expression on the surface of the T cell (J Immunol 2002;168(3):1028-35). There are two different isoforms of CEACAM1 on T cells: CEACAM1-L, which has an inhibitory function in the signaling of an activated T cell, and CEACAM1-S, which can support T cell activation. However, the expression of CEACAM1-L on T cells is much more dominant than the expression of the activating S isoform (J Immunol 1999;163(3):1363-70; Eur. J. Immunol. 2000;30(9):2593-603; J Immunol 2004;172(6):3535-43; J Immunol 2002;168(10):5139-46; Immunity 2012;37(5):930- 46). In contrast to the CEACAM1-S isoform, the inhibitory CEACAM1-L isoform has two ITIM motifs expressed in the cytoplasm, ensuring the inhibitory effect (Cell Signal 2004;16(4):435-56). At the beginning of the signal chain of the T cell inactivation by CEACAM1, the ITIM motifs are phosphorylated by Src-related tyrosine kinases (J Immunol 2008;180(9):6085-93). This phosphorylation, especially by p56lck, leads to a recruitment of SHP-1 close to the TCR / CD3 complex, which in turn leads to dephosphorylation and thus inactivation of ZAP-70, which is attached to the TCR, and of TCR CD3-^ chain (J Immunol 2008;180(10):6827-35). This blocks the signal paths downstream of the TCR leading to an inactivation of the T cell via the TCR (Immunity 2006;25 (5):769— 81).

[0013] Cancer immunotherapy and CARs

[0014] Despite intensive research, the treatment of tumor diseases is still very demanding. Treatment options for cancer have long been limited to surgical removal, chemotherapy and radiation. Although these treatments have saved many people's lives, many patients remain unhealed (Front Immunol 2017;8). A therapy that is individually tailored to the patient is becoming increasingly important in order to guarantee the best treatment for each patient, ideally with fewer side effects (Cold Spring Harb Mol Case Stud 2018;4(2).

[0015] In recent years an increasing focus in the research of new cancer drugs has been on cancer immunotherapy. The focus is on boosting the immune system, so that it is able to recognize and combat the tumor disease itself (Rambam Maimonides Med J 2015;6(l):e0004). Immune checkpoint inhibitors are one way of supporting the immune system. An immune checkpoint pathway that is very popular and often used to support the anti-tumor response is the PD-1 / PD-L 1 pathway. Therefore, monoclonal antibodies are used that target either PD-1 (to influence the inhibitory receptors on immune cells) or PD-L 1 (as a ligand on the tumor cell; Curr Opin Immunol2012;24(2):207- 12). One such monoclonal antibody is Atezolizumab, which binds to PD-L1 on tumor cells and thus blocks the binding to PD-1 on immune cells. Atezolizumab is approved for the treatment of patients with advanced non-small cell lung cancer (NSCLC) after previous chemotherapy because clinical studies indicated that patients who received the therapy showed a longer overall survival (OS) (Target Oncol 2018;13(3):399-407). The blocking of immune checkpoints is therefore able to improve the response of the immune system to the tumor, thereby inhibiting tumor growth or even removing the tumor altogether (N Engl J Med 2012;366(26):2443-54; The Lancet Oncology 2015;16(5):e234-e245).

[0016] However, tumors also have mechanisms to protect themselves from being eliminated by the immune system. For example, MHC I molecules are down-regulated on tumor cells in order to remain undetected (Curr Oncol 2012;19(l):39-41; Cancer Res 2006;66(18):9281-9). To address the problem of the lack of MHC / TCR interaction, CAR T cells were developed. CAR T cells are genetically modified T cells that express a chimeric antigen receptor that can specifically recognize tumor antigens. This redirects the specificity of the cell (Nature 2017;545(7655):423-31; Curr Opin Immunol 2009;21(2):215— 23). The principle by which CAR T cells bind their antigen is based on the functionality of antibodies. The extracellular antigen binding domain is typically composed of a single chain variable fragment (scFv). scFvs consist of the variable domains of heavy (VH) and light chain (VL) of a monoclonal antibody, with a specificity for a tumor-associated antigen, typically fused together by a linker, such as a flexible glycine-serine linker (J Hematol Oncol 2018;ll(l):2). The extracellular domain and the intracellular signaling domain are typically connected via a spacer domain and a transmembrane domain for spanning the membrane (Cancers (Basel) 2017;9(9); Immunotherapy 2015;7(5):487— 97). The signaling domain, responsible for initiation of the signal within the cell, of the first generation of CAR T cells consisted only of a CD3^ signaling chain, based on the TCR receptor (Nat Rev Drug Discov 2015;14(7):499-509). In vivo, however, it was shown that this is not sufficient for an efficient activation of resting T cells (Nat Rev Cancer 2003;3(l):35— 45). The second generation CARs also comprised a costimulatory unit, such as CD28 or 4-1BB, which led to an improved anti-tumor effect through an improved activation of cytokine production (Cancers (Basel) 2017;9(9)). Third generation CARs even have two co-stimulatory domains for an even better activation of the T cell (Nat Rev Drug Discov 2015;14(7):499-509).

[0017] After activation of the CAR T cell, there is an expression of death receptors and the release of perforin and granzymes, which causes the killing of the tumor cell (J Hematol Oncol 2018;ll(l):22). Since the inhibitory tumor environment also influences the efficiency of CAR T cells and their effect is limited by the expression of immune checkpoint molecules on tumor cells, a combination of therapy with CAR T cells and immune checkpoint inhibitors is beneficial (Oncoimmunology 2016;6(2)). However, the systematic administration of immune checkpoint inhibitors also leads to side effects in some patients, which are referred to as immune-related adverse events (irAEs). This can affect many organs and, among other things, lead to hematological and neurological side effects. Moreover, the immune checkpoint inhibitors can also impair the cooperation between antigen-presenting cells and naive T cells, which affects the immune response. Therefore, it would be good to administer the inhibitors locally rather than systematically in order to minimize possible side effects (Am Soc Clin Oncol Educ Book 2015:76-83).Such local administration can be achieved with the fourth generation CARs, also known as TRUCKS. TRUCKS are CAR T cells that release a transgenic protein after activation by the target cell (Expert Opin Biol Ther 2015;15(8):1145-54). The structure and the general principle of the mode-of-action of TRUCKS is shown in Figure 1. An anti-CD19 CAR has already been modified in such a way that it secretes an anti-PD-1 scFv after activation, as a result of which the blockage of the T cell could be reversed and the anti-tumor effect was thus improved (Clin Cancer Res 2017;23(22):6982-92).

[0018] The present invention provides a novel, modified TRUCK system. A CAR with an extracellular anti-HER2 (ErbB2) domain enables the production and secretion of an anti-CEACAM6 scFv locally at the site of the tumor, thereby providing an efficient and versatile new modality for the treatment of diseases, such as cancer.

[0019] Figure legends

[0020] Figure 1 Schematic representation of a TRUCK construct and the associated signaling events

[0021] Figure 2 Dose dependent binding of scFv C6 to CEACAM6 as measured by custom made ELISA. The plates were coated with recombinant human CEACAM6 -Fc at a concentration of 5 pg / ml and the scFv C6 was titrated down from 2000 ng / ml. The scFv C6 binding was detected with anti-6xHis Tag antibody conjugated with HRP Figure 3 Binding of scFv C6 to recombinant human CEACAM6 -Fc tagged protein coated on protein A sensor chip and analysed by surface plasmon resonance.

[0022] Figure 4 Representative histograms of binding of scFv C6 to cell lines expressing CEACAM6. KS24.22 (KS) breast cancer and N87 gastric cancer cell lines were stained with 15 pg / ml scFv C6 and the bound scFv was detected with either anti-c-Myc AF488 or anti 6x His-PE or antibodies (grey histogram). The binding of anti-6xHis-PE or anti-c-Myc AF488 antibodies alone was used as a negative control (dotted histogram).

[0023] Figure 5 Representative histograms of the competitive binding of the monoclonal anti-CEACAM6 antibody (9A6) and scFv C6. KS and N87 cells were incubated with 40 pg / ml 9A6 antibody and then after washing, 15 pg / ml of the scFv C6 was added and the bound scFv was detected via anti-6xHis antibody (dark grey histogram). The signal was compared to the direct binding of scFv C6 (grey histogram) As negative control the binding of the anti-6xHis-PE antibody was used (dotted histogram).

[0024] Figure 6 Panel A: Representative histogram of expression of CEACAM1 on survivin-specific T cells as detected by FACS staining with anti-human CEACAM1 antibody (clone GM8G5) and detected by secondary BV421 conjugated anti-mouse IgG antibody. Panel B: In vitro effect of monoclonal anti-CEACAM6 Ab (9A6) on IFN-gamma secretion of survivin specific T cells. 10000 KS tumor cells were co-cultured with 10000 survivin specific T cells over 20 h in the absence or presence of different concentrations (5 to 20 pg / m) of anti-CEACAM6 (clone 9A6) monoclonal antibody. As an additional control, unrelated antibody of the same isotype was used. The concentration of IFN-gamma in the supernatant was detected by ELISA and represented in pg / ml. Statistically significant differences are indicated by asterisks and were calculated using unpaired Student's t test.Figure 7 In vitro effect of scFv C6 on IFN-gamma secretion of survivin specific T cells. 10000 KS cancer cells were co-cultured with10000 survivin T cells over 20 h in the absence or presence of scFv C6 in different concentrations (5 to 20 pg / ml in the left graph and 0,3125 to 2,5 pg / ml in the right graph). In both experiments 5 pg / ml of mAb 9A6 and an isotype matched control were used as internal negative and positive control. The concentration of IFN-gamma in the supernatant was detected by ELISA and represented in pg / ml. Statistically significant differences are indicated by asterisks and were calculated using unpaired Student's t test.

[0025] Figure 8 Survivin specific? cells transduced to express scFv C6 under the NFAT inducible cassette eradicate breast cancer cells efficiently in a real-time live cell microscopy for 90 h. Tumor cell death was evaluated using Incucyte Cytotox Red Dye. 24,000 KS breast cancer cells were cocultured either with 500 survivin T cells transduced to either express scFv under an NFAT inducible cassette, upstream of a constitutively expressed tomato fluorescent protein (Survivin iscFv C6 tomato, black circle) or with 500 survivin T cells transduced to express tomato only in the presence or absence of 5 pg / ml anti-CEACAM6 monoclonal antibody (mAB C6, black / white square or gray square, respectively). Graph shows the area of Cytotox Red positive dead cells (pm2 / image).

[0026] Figure 9 Binding of 5 pg / ml (40 nM) recombinant human CEACAM1 -Fc / His tagged protein to plate-bound recombinant human CEACAM6-His tagged protein (5 pg / ml) was blocked by scFv C6 that was added in a concentration series from 0,25-2 pg / ml (8-130 nM), as determined by competition ELISA. For detection of bound CEACAM1 -Fc / His, protein A conjugated with horseradish peroxidase (HRP) was used.

[0027] Figure 10 Schematic representation of regulatory elements and transgene of CAR and TRUCK viral vectors used for the modification of T cells from healthy donors via transduction. LTR-long terminal repeat; 6xNFAT RE -response element for NFAT repeated 6 times; IL-2 MP - minimal IL-2 promoter; PGK - PGK promoter; CMV - CMV promoter, PS - packaging signal; aCEACAM6 scFv - scFv C6; aErbB2 CD28 CD3z CAR - CAR targeting ErbB2 on the surface of tumor cells, with intracellular signaling domains derived from CD28 and CD3zeta.

[0028] Figure 11 Representative histograms of expression of CAR and CEACAM1 on CAR T cells and TRUCKS generated by viral transduction. T cells from healthy donors were transduced with either ErbB2 CAR or scFv C6 ErbB2 TRUCK constructs and stained with 1 pg / ml goat F(ab') 2 anti-human IgG PE antibody (which binds in the Fc hinge region of the CAR) to determine CAR expression, or with 2 pg / ml anti-CEACAMl (CD66a / c / e-APC) antibody to detect the expression of CEACAM1 on the transduced cells.

[0029] Figure 12 Inducible expression of scFv C6 by TRUCKS generated from a healthy donor. For this purpose 600000 T cells transduced with the TRUCK construct carrying the cassette for inducible expression of the scFv C6, the CAR construct, or untransduced (mock) cells were stimulated with 15 pg / ml plate-bound ErbB2 for 60 h. As a control the transduced and non-transduced T cells were cultured in wells coated with 15 pg / ml BSA (nonactivated). (A) The inducible expression of the scFv C6 was determined by qPCR using cDNA generated from the collected cell pellets as a template. (B) The protein secretion of scFv C6 upon activation of the TRUCKS was confirmed by custom ELISA of supernatants collected from the activated and non activated transduced T cells as described above. The supernatants were incubated on a plate coated with 5 pg / ml rh Fc-tagged CEACAM6, andthe bound scFv C6 was then detected with anti-6xHis tag antibody conjugated to HRP. As standard purified scFv C6 with known concentration was used.

[0030] Figure 13 Secretion of scFv C6 from TRUCKS upon antigen specific activation in a stimulus dose dependent manner. 260000 transduced ErbB2 specific TRUCK and CAR T cells (used as negative control) were cultured in triplicates in a 48 well plate coated with decreasing concentrations (20 - 2,5 pg / ml) of recombinant human ErbB2-Fc tagged protein for 60 h. The supernatants were then collected and subjected to custom ELISA for detection of scFv C6, where supernatants were incubated on a plate coated with 5 pg / ml rh CEACAM6-Fc tagged an the bound scFv C6 was then detected with anti-6xHis tag antibody conjugated to HRP. As standard purified scFv C6 with known concentration was used.

[0031] Figure 14 TRUCKS specific for ErbB2 outperform CART cells with the same specificity, as shown in representative luciferase cytotoxicity assays where 10000 KS or N87 cells were co-cultured for 20h with TRUCK (black bars) or CAR (gray bars) T cells in different effector to target (E: T) ratios. As a control CAR T cells were co-cultured with the target cells in the presence of soluble scFv C6 (5 pg / ml, gray dotted bars). As additional negative control, non-transduced T cells (mock) were cocultured with KS or N87 cells (light gray bars). Statistically significant differences are indicated by asterisks and were calculated using paired Student's t test. Panel A: KS cells, Panel B: N87 cells.

[0032] Figure 15 TRUCKS show an improved secretion of proinflammatory cytokine I FNy in comparison to CAR T cells alone, measured in cytokine ELISA. Supernatants of 20 h co-cultures of TRUCK (black bars) or CAR T cells (grey bars) with 10000 KS or N87 cells in different E: T ratios were collected and assessed. As a control, CAR T cells were co-cultured with the target cells in the presence of soluble scFv C6 (5 pg / ml, gray dotted bars). As additional negative control, non-transduced T cells (mock) were co-cultured with KS or N87 cells (light gray bars). Statistically significant differences are indicated by asterisks and were calculated using paired Student's t test. Panel A: KS cells, Panel B: N87 cells.

[0033] Figure 16 TRUCKS show an improvement in the secretion of the proinflammatory cytokine TNFa in comparison to CAR T cells alone, as shown in representative TNFa ELISA experiments, where the supernatants of 20 h cocultures of TRUCK (black bars) or CAR T cells (grey bars) with 10000 KS cells was measured with different E: T ratios. As a control, CAR T cells were also co-cultured with the target cells in the presence of soluble scFv C6 (5 pg / ml, grey dotted bars). As additional negative control, non-transduced T cells (mock) were co-cultured with KS cells (light gray bars). Statistically significant differences are indicated by asterisks and were calculated using paired Student's t test.

[0034] Figure 17 scFv C6 ErbB2 TRUCKS eradicate breast cancer cells more efficiently than ErbB2 CART cells as measured by real-time live cell microscopy for 100 h. The evaluation of tumor cell death was done using Incucyte Cytotox Red Dye. 24000 KS breast cancer cells were co-cultured either with 4000 TRUCKS (black circle) or CAR T cells (gray square). The graph shows the area of Cytotox Red positive dead cells (μm2 / image).

[0035] Figure 18 The presence of scFv C6 secreted by the TRUCKS increases calcium signaling. Representative calcium measurement in CAR T cells (upper 2 panels) in the absence (top panel) or presence of 5 pg / ml of soluble scFvC6 (middle panel), or TRUCKs (lower panel) after contact with target KS cells. Each line represents the signal inside a single T cell. Around 25 cells were measured per set up.

[0036] Figure 19 Summary of the data of Figure 18. The strength of the Ca2+ signaling was calculated for each imaged cell as area under the curve. Each data point represents the calculated value for each cell. The higher the signaling, the higher the calculated value. The statistical significance was denoted with asterisks and was calculated by using unpaired Student's t-test.

[0037] Figure 20 Confocal laser scanning microscopy images demonstrating the expression, secretion and binding of scFv C6-mNeonGreen (mNG) (top right) by TRUCKS upon contact with tumor cells. The images demonstrate colocalization of scFv C6-mNG with the membrane (top middle, CellBrite Steady 550) in the area of the CAR-Synapse as defined by clustering of the CAR on theT cell surface (bottom left, anti hlgG-Alexa Fluor 647). Top left shows nuclear counterstain with Hoechst33342, bottom middle shows a merge of all channels.

[0038] Figures 21 and 22 ErbB2 specific TRUCKS control tumor growth more efficiently compared to CART cells with the same specificity, as shown in a xenograft mouse model, where NSG mice were injected in both flanks with N87 tumor cell line expressing luciferase. Tumors were established for 14 days, thereafter 250000 TRUCK or CAR T cells were injected intravenously. The growth of the tumor was followed for 41 additional days and the tumor size was measured by bioluminescence. Figure 21: In vivo bioluminescence images of mice treated with CAR T cells (left) or TRUCKS (right). On the left, the days post tumor establishment are indicated. Figure 22, panel A: growth curve of the xenograft tumors of mice treated with CAR T cells (gray square) and TRUCKS (black circle). Statistically significant differences are indicated by asterisks and were calculated using Student's t test. Figure 22, panel A: survival curve of tumor bearing mice treated with CAR T cells (grey line) or TRUCKS (black line). Figure 23 CEACAM1 / CEACAM6 interaction reduces activation of CAR expressing Jurkat Lucia NFAT cells, but this effect can be ameliorated by added / secreted scFv C6. Jurkat Lucia NFAT cells transduced to express CAR or TRUCK together with either CEACAM1 or mutated delta CEACAM1 (dCEACAMl) were activated using plate bound ErbB2 (10 pg / ml) and CEACAM6 (concentrations from 0-60 pg / ml) in total concentration of 70pg / ml protein, where the differences were adjusted with IgG-Fc. 100,000 of the respective transduced Jurkat cells were added per well in 100 pl medium. The cells were cultured for 48 hours. Activation through the CAR was measured as the level of luciferase in the supernatant of the Jurkat cells. The signal produced by each of the transduced Jurkat Lucia NFAT cells in wells coated with ErbB2 only is considered as 100 % activation, and the luciferase signal in the rest of the conditions was normalized to these values. One representative experiment. Statistically significant differences are indicated by asterisks and were calculated using unpaired Student's t test.

[0039] Summary of the invention

[0040] The present disclosure relates to a recombinant cell containing a nucleic acid comprising(a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0041] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0042] (ii) a transmembrane domain; and

[0043] (iii) an intracellular signaling domain, and

[0044] (b) a second nucleic acid sequence encoding a promoter and a scFv specifically binding to a second tumor antigen,

[0045] wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

[0046] In certain embodiments, the extracellular ligand-binding domain of said chimeric antigen receptor specifically binds to Her2 (ErbB2).

[0047] In certain embodiments, the extracellular ligand-binding domain of said chimeric antigen receptor is a scFv, preferably wherein said scFv comprises a variable heavy domain comprising an HCDR1 of SEQ ID No. 21, an HCDR2 of SEQ ID No. 22, and an HCDR3 of SEQ ID No. 23, and comprising a variable light domain comprising an LCDR1 of SEQ ID No. 24, an LCDR2 of SEQ ID No. 25, and an LCDR3 of SEQ ID No. 26.

[0048] In certain embodiments, said transmembrane domain is or is derived from CD28. In certain embodiments, said transmembrane domain comprises the amino acid sequence of SEQ ID No. 30.

[0049] In certain embodiments, said intracellular costimulatory signaling domain is or is derived from CD28. In certain embodiments, said costimulatory signaling domain comprises the amino acid sequence of SEQ ID No. 31. In certain embodiments, said intracellular T cell receptor signaling domain is or is derived from CD3 zeta. In certain embodiments, said T cell receptor signaling domain comprises the amino acid sequence of SEQ ID No. 32. In certain embodiments, said chimeric antigen receptor (CAR) comprises a hinge region. In certain embodiments, wherein said hinge region is located between the extracellular ligand-binding domain and the transmembrane domain. In certain embodiments, said hinge region comprises the amino acid sequence of SEQ ID No. 33.

[0050] In certain embodiments, said chimeric antigen receptor (CAR) comprises the amino acid sequence of SEQ ID No. 34.

[0051] In certain embodiments, said the scFv specifically binding to said second tumor antigen specifically binds to CEACAM6.

[0052] In certain embodiments, said scFv specifically binding to CEACAM6 comprises a variable heavy domain comprising an HCDR1 of SEQ ID No. 7, an HCDR2 of SEQ ID No. 8, and an HCDR3 of SEQ ID No. 9, and comprisinga variable light domain comprising an LCDR1 of SEQ ID No. 10, an LCDR2 of SEQ ID No. 11, and an LCDR3 of SEQ ID No. 12.

[0053] In certain embodiments, said the promoter of said second nucleic acid comprises an inducible promoter. In certain embodiments said inducible promoter is an NFAT- inducible promoter.

[0054] In certain embodiments, said recombinant cell is a T cell or a NK cell, preferably a T cell.

[0055] In certain embodiments, the present disclosure relates to a nucleic acid comprising said first nucleic acid sequence and said second nucleic acid sequence.

[0056] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising aforementioned recombinant cells.

[0057] In certain embodiments, the present disclosure relates to aforementioned recombinant cells or aforementioned pharmaceutical composition for use in medicine. In certain embodiments, said use in medicine is the treatment of cancer or a tumor.

[0058] Definitions

[0059] The term "chimeric antigen receptor" or " CAR" refers to a recombinant polypeptide construct comprising at least an extracellular ligand-binding domain, a transmembrane domain and one or more intracellular signaling domains (also referred to herein as " cytoplasmic signaling domain") comprising a functional signaling domain derived from a stimulatory molecule.

[0060] The term "ligand-binding domain" refers to a polypeptide that is capable of binding a ligand. Prefeerably, the ligand-binding domain will be capable of interacting with a cell surface molecule. For example, the ligandbinding domain can be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease or disease state.

[0061] The term "hinge" or "spacer" as used herein refers to the hydrophilic region which is located between the ligand- binding domain and the transmembrane domain. The constructs of the present disclosure may comprise an extracellular hinge domain but is it also possible to pass such a spacer. The spacer may include Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof.

[0062] The term "transmembrane domain" refers to a portion or a region of a molecule that spans a lipid bilayer membrane.

[0063] The term "signaling domain" refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function. As such, the signaling domain recruits and interacts with specific proteins in response to an activating signal.The terms "costimulatory signaling domain" and "costimulatory domain" as used herein refer to the domain of a costimulatory molecule or costimulatory receptor responsible for mediating a costimulatory response by the T cell. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof. Commonly known costimulatory signaling domains include 4-IBB (CD137), BAFFR, 0X40, CD27, CD28, CD40, 2B4, GITR, HVEM, 0X40, RELT, TACI, TROY, TWEAK, KIR receptors, TLR1 to TLR9 receptors, IL-2, IL-7 and IL- 15 receptors.

[0064] The terms " T cell receptor signaling domain" and " TCR signaling domain" refer to cytoplasmic signaling sequences that act in a stimulatory manner to induce immune effector functions. In some embodiments, the TCR signaling domain contains a signaling motif known as Immunoreceptor Tyrosine-based Activation Motif, or ITAM. In some embodiments, the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, and DAP12. A preferred TCR signaling domain is a TCR signaling domain selected from CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon. A particularly preferred TCR signaling domain CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon.

[0065] The term "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, which interacts with an antigen. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CD Rs and four FR's arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0066] The term "antibody fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature341:544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as "single chain variable fragment", "single chain Fv" or "scFv"; see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U. S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8: 1057-1062; and U. S. Pat. No. 5,641,870).

[0067] The terms " CEACAM6", " CD66c" and " C6" refer to a surface glycoprotein that plays a role in cell adhesion and tumor progression. CEACAM6 (UniProt: P40199) has the following amino acid sequence:

[0068] MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLPQ NRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTGFY TLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYLWWV NGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVP TISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQ AHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIGVLARVALI (SEQ ID No. 1)

[0069] The term " CD28" refers to a protein essential for T-cell proliferation and survival, cytokine production and T- helper type-2 development. CD28 (UniProt: P10474) has the following amino acid sequence:

[0070] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLD SAVEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPP PYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID No. 2 )

[0071] The term " CD3" as used herein, refers to a protein complex that is composed of a CD3 gamma chain, a CD3 delta chain, and two CD3 epsilon chains. These chains associate with a TCR and a CD3 zeta chain to generate an activation signal in T lymphocytes. The TCR, CD3 zeta, and the other CD3 chains together constitute a TCR complex.

[0072] The term " CD3z" or " CD3 zeta" as used herein, refer leukocyte differentiation antigen 3 zeta. It is also known as CD247. CD3 zeta (UniProt: P20963) has the following amino acid sequence:MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No. 3 )

[0073] The terms " Her2" or " ErbB2" as used herein refers to a member of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. Her2. Amplification and / or overexpression of Her2 has been reported in numerous cancers, including breast and ovarian tumors. Her2 (UniProt: P04626) has the following amino acid sequence:

[0074] MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPAS PETHLDMLRHL YQGCQWQGNL ELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNG DPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLA LTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQC AAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSAN IQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLP DLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTV PWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQEC VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRAS PLTS IISAWG ILLVWLGWFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETEL RKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTS PKANKEILDEAYVMAGVGS P YVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVR LVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWM IDSECRPRFRELVSEFSRMARDPQRFWIQNEDLGPAS PLDSTFYRSLLEDDDMGDLVDA EEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEG AGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYV NQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGWKDVFAFGGAVENPEYLTPQ GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV (SEQ ID No. 4 )

[0075] The terms "polynucleotide", "nucleic acid sequence" and "nucleic acid" as used herein refer to a sequence of nucleoside or nucleotide monomers consisting of bases, sugars and intersugar (backbone) linkages. The term includes DNA and RNA and can be either double stranded or single stranded, and represents the sense or antisense strand. The term also includes modified or substituted sequences comprising non- naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acids of the present disclosure may be isolated from biological organisms, formed bylaboratory methods of genetic recombination or obtained by chemical synthesis or other known protocols for creating nucleic acids.

[0076] The terms "isolated polynucleotide" or "isolated nucleic acid sequence" as used herein refer to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.

[0077] The terms "recombinant nucleic acid" or "engineered nucleic acid" as used herein refer to a nucleic acid or polynucleotide that is not found in a biological organism. For example, recombinant nucleic acids may be formed by laboratory methods of genetic recombination (such as molecular cloning) to create sequences that would not otherwise be found in nature. Recombinant nucleic acids may also be created by chemical synthesis or other known protocols for creating nucleic acids. Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art. The term "polypeptide" or "protein" as used herein describes a chain of amino acids. A polypeptide or protein of this disclosure can be a peptide, which usually describes a chain of amino acids of from two to about 30 amino acids. The term protein as used herein also describes a chain of amino acids having more than 30 amino acids and can be a fragment or domain of a protein or a full length protein. Furthermore, as used herein, the term protein can refer to a linear chain of amino acids or it can refer to a chain of amino acids that has been processed and folded into a functional protein. It is understood, however, that 30 is an arbitrary number with regard to distinguishing peptides and proteins and the terms can be used interchangeably for a chain of amino acids. The proteins of the present disclosure can be obtained by isolation and purification of the proteins from cells where they are produced naturally, by enzymatic (e.g., proteolytic) cleavage, and / or recombinantly by expression of nucleic acid encoding the proteins or fragments of this disclosure. The proteins and / or fragments of this disclosure can also be obtained by chemical synthesis or other known protocols for producing proteins and fragments.

[0078] The term "isolated polypeptide" refers to a polypeptide substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

[0079] The term "promoter" or "promoter region" as used herein refers to a nucleic acid sequence which controls synthesis of a transcript, e.g. a transcript comprising a coding sequence, by providing a recognition and binding site for RNA polymerase. The promoter region may include further recognition or binding sites for further factors involved in regulating transcription of said gene. A promoter may control transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible" and initiate transcription in response to an inducer, or may be "constitutive" if transcription is not controlled by an inducer. An inducible promoter is expressed only to a very small extent or not at all, if an inducer is absent. In the presence of the inducer, the gene is "switched on" or the level of transcription is increased. This is usually mediated by binding of a specific transcription factor.The term " NFAT-inducible promoter" as used herein refers to a nucleic acid sequence containing one or more NFAT responsive elements linked to a minimal promoter of any gene expressed by T-cells. Commonly used is the minimal human IL-2 promoter. The NFAT responsive elements may comprise, e.g., NFATI, NFAT2, NFAT3, and / or NFAT4 responsive elements. One exemplary promoter is the nuclear factor of activated T cell (NFAT)-derived minimal promoter (Cancer Res (2011) 71: 5697–5706; Mol Ther (2011) 19: 751–759). This promoter consists of the core promoter of IL-2 gene (from -80 to +40) (minimal IL2 promoter), downstream of regulatory sequence comprised of 6 response elements (DNA motifs) recognized by the nuclear factor of activated T cells (NFAT) transcription factors. When the CAR T cell interacts with the tumor cell, upon antigen recognition, the resulting signaling cascade leads to phosphorylation of NFAT transcription factors and their translocation into the nucleus, where the said transcription factors can bind to their response elements and drive the transcription of the gene downstream of the minimal promoter.

[0080] The term "cytokine" as used herein refers to small soluble protein substances secreted by cells, which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Cytokines can act both locally and distantly from a site of release. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin- 10; tumor necrosis factor (" TNF") -related molecules, including TNF alpha and lymphotoxin; immunoglobulin super-family members, including interleukin 1 (" IL-1"); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of other cytokines. Nonlimiting examples of cytokines include e.g., IL-1α., IL-1β., IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL- 10, IL-11, IL-12 / IL- 23 P40, IL-13, IL-15, IL-17, IL-18, IL-21, IL-23, TGF-p, IFN-gamma, GM-CSF, Gro-alpha, MCP-1 and TNF-alpha.

[0081] The term "cell" as used herein includes a single cell as well as a plurality of cells. The term "cell" includes "host cells".

[0082] The term "host cell" as used herein refers to a cell comprising a nucleic acid and / or a vector. In the context of the present disclosure, the term host cell refers to a cell comprising a nucleic acid and / or a vector as disclosed herein. Such host cell will express the CAR on the cell surface and is suitable to be used as medicine. Preferred host cells of the present invention are eukaryotic host cells, such as immune cells. Perticularly poreferred are T cells and NK cells.

[0083] The term " T cell" as used herein refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells, also referred to as T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor (TCR) on the cell surface. There are several subsets of T cells with distinct functions, including but not limited to, T helper cells, cytotoxic T cells, memory T cells, regulatory T cells and natural killer T cells. In some embodiments, theT cell is an engineered T cell.The terms " NK cell" and "natural killer cell" as used herein refer to large granular lymphocytes involved in the innate immune response. Functionally, NK cells exhibit cytolytic activity against a variety of targets via exocytosis of cytoplasmic granules containing a variety of proteins, including perforin, and granzyme proteases. Killing is triggered in a contact-dependent, non-phagocytotic process. Human NK cells are characterized by the presence of the cell-surface markers CD16 and CD56, and the absence of the T cell receptor (CD3). Human bone marrow-derived NK cells may further be characterized by the CD2+CD16+CD56+CD3- phenotype, further containing the T-cell receptor zeta-chain, and can be characterized by NKp46, NKp30 and / or NKp44 expression.

[0084] The terms "recipient", "individual", "subject", "host", and "patient", are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0085] As used herein, the terms "treatment," "treating," and the like, in some embodiments, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of affecting a partial or complete cure for a disease and / or symptoms of the disease. The terms include treatment of a disease or disorder (e.g. inflammation) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g, including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g. inflammation). The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0086] Embodiments of the invention

[0087] The present invention is based on chimeric antigen receptors of the fourth generation. Chimeric antigen receptors of the first generation consist of an extracellular ligand-binding domain, typically a scFv, and an intracellular signaling domain, typically a CD3 T cell receptor signaling domain. The ligand-binding domain and the signaling domain are connected via a transmembrane domain and, optionally a hinge region. Chimeric antigen receptors of the second generation additional contain an intracellular costimulatory signaling domain, such as the domains of CD28, 0X40 or 4-lBB(CD137). Chimeric antigen receptors of the third generation possess a second intracellular costimulatory signaling domain. Chimeric antigen receptors of the fourth contain generation typically contain only one costimulatory signaling domain, but contain a nucleic acid sequence encoding a polypeptide with an additional functionality, such as a cytokine, which is under the control of a promoter, such as a NFAT promoter. Binding of the ligand-binding domain of the CAR to its target (typically atarget associated with a disease, such as cancer) causes the promoter to produce and secrete the cytokine (Figure 1; Clin Cancer Res 2017;23(22):6982-92). Such fourth generation CARs are also referred to as TRUCKS.

[0088] The present invention makes use of this concept. The system is modified in a way that said promoter does not lead to the expression and secretion of a cytokine, but a single chain antibody. Due to the specificity of the extracellular domain of the CAR, the scFv is secreted locally at the site where it is expected to exert its anti-cancerous activity. The recombinant cell of the present invention therefore contains a chimeric antigen receptors, as well as a single chain antibody under the control of an inducible promoter, such as an NFAT-inducible promoter.

[0089] Chimeric antigen receptor

[0090] The present disclosure makes use of a chimeric antigen receptor specifically binding to a tumor antigen. In certain embodiments, said chimeric antigen receptor (CAR) comprises

[0091] (a) an extracellular ligand-binding domain comprising scFv specifically binding to a tumor antigen;

[0092] (b) a transmembrane domain; and

[0093] (c) an intracellular signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0094] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor specifically binding to a tumor antigen. In certain embodiments, said chimeric antigen receptor (CAR) comprises

[0095] (a) an extracellular ligand-binding domain comprising scFv specifically binding to a tumor antigen;

[0096] (b) a hinge region;

[0097] (c) a transmembrane domain; and

[0098] (d) an intracellular signaling domain.

[0099] In certain embodiments said tumor antigen is Her2. In certain embodiments said tumor antigen is a polypeptide comprising the amino acid sequence of SEQ ID No. 4. In certain embodiments said tumor antigen is a polypeptide consisting of the amino acid sequence of SEQ ID No. 4.

[0100] Therefore, in certain embodiments, the present disclosure makes use of a chimeric antigen receptor comprising

[0101] (a) an extracellular ligand-binding domain comprising scFv specifically binding to Her2;

[0102] (b) a transmembrane domain; and(c) an intracellular signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0103] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor comprising (a) an extracellular ligand-binding domain comprising a scFv specifically binding to a polypeptide comprising the amino acid sequence of SEQ ID No. 4;

[0104] (b) a transmembrane domain; and

[0105] (c) an intracellular signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0106] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor comprising (a) an extracellular ligand-binding domain comprising a scFv specifically binding to a polypeptide consisting of the amino acid sequence of SEQ ID No. 4;

[0107] (b) a transmembrane domain; and

[0108] (c) an intracellular signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0109] In certain embodiments, said signaling domain of the chimeric antigen receptor is a costimulatory signaling domain. In other embodiments, said signaling domain of the chimeric antigen receptor is a T cell receptor signaling domain.

[0110] In certain preferred embodiments, the chimeric antigen receptor comprises a costimulatory signaling domain and a T cell receptor signaling domain. Therefore, in certain embodiments, the present disclosure relates to a chimeric antigen receptor (CAR) comprising

[0111] (a) an extracellular ligand-binding domain comprising scFv specifically binding to Her2;

[0112] (b) a transmembrane domain;

[0113] (c) an intracellular costimulatory signaling domain, and

[0114] (d) an intracellular T cell receptor signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0115] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising (a) an extracellular ligand-binding domain comprising a scFv specifically binding to a polypeptide comprising the amino acid sequence of SEQ ID No. 4;

[0116] (b) a transmembrane domain;

[0117] (c) an intracellular costimulatory signaling domain, and(d) an intracellular T cell receptor signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0118] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising (a) an extracellular ligand-binding domain comprising a scFv specifically binding to a polypeptide consisting of the amino acid sequence of SEQ ID No. 4;

[0119] (b) a transmembrane domain;

[0120] (c) an intracellular costimulatory signaling domain, and

[0121] (d) an intracellular T cell receptor signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0122] The anti-HER2 scFv utilized in the present disclosure has the following amino acid sequence:

[0123] SEQ ID No. Description Sequence

[0124] variable heavy chain of QVQLVQS GAEVKKPGE S LKI S C KGS GYS FT S YWIAWVRQMPGKGL 19 EYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSD anti-HER2 scFv SAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSS variable light chain of QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAP 20 KLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCA anti-HER2 scFv S WD YT L S GWVF GGGT KL T VL

[0125] HCDR1 of anti-HER2 scFv

[0126] 21 SYWIA

[0127] (Ka bat)

[0128] HCDR2 of anti-HER2 scFv

[0129] 22 LIYPGDSDTKYSPSFQG

[0130] (Ka bat)

[0131] HCDR3 of anti-HER2 scFv

[0132] 23 HDVGYC S S S NC AKWPE YFQH

[0133] (Ka bat)

[0134] LCDRl of anti-HER2 scFv

[0135] 24 SGSSSNIGNNYVS

[0136] (Ka bat)

[0137] LCDR2 of anti-HER2 scFv

[0138] 25 DHTNRPA

[0139] (Ka bat)

[0140] LCDR3 of anti-HER2 scFv

[0141] 26 ASWDYTLSGWV

[0142] (Ka bat)

[0143] scFv Her2, full length incl MDFQVQI FS FLL I SAS VIMS RPAMAQVQLVQS GAEVKKPGE SLKI SCKGSGYSFTS YWIAWVRQMPGKGLEYMGLIYPGDSDTKYS PSFQ

[0144] 27 leader GQVTISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCA

[0145] KWPEYFQHWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVS

[0146]

[0147] AAPGQKVT I S C S GS S S N IGNNYVS WYQQL PGT APKLL I YDHTNRPAGVPDRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVF GGGTKLTVLG

[0148] scFv Her2, full length QVQLVQS GAEVKKPGE S LKI S C KGS GYS FT S YWIAWVRQMPGKGL EYMGLIYPGDSDTKYSPSFQGQVTISVDKSVSTAYLQWSSLKPSD

[0149] without leader S AVYFCARHDVGYC S S S NCAKWPE YFQHWGQGTLVTVS S GGGGS G 28 GGGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSW YQQLPGTAPKLLIYDHTNRPAGVPDRFSGSKSGTSASLAISGFRS EDEAD YYCASWDYTLSGWVF GGGTKLTVLG

[0150] 13 3x Gly4Ser linker GGGGS GGGGS GGGGS

[0151] 29 Igk leader sequence MDFQVQIFSFLLISASVIMSRPA

[0152]

[0153] Therefore in certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising

[0154] (a) an extracellular ligand-binding domain comprising a scFv comprising a variable heavy domain comprising an HCDR1 of SEQ ID No. 21, an HCDR2 of SEQ ID No. 22, and an HCDR3 of SEQ ID No. 23, and comprising a variable light domain comprising an LCDR1 of SEQ ID No. 24, an LCDR2 of SEQ ID No. 25, and an LCDR3 of SEQ ID No. 26;

[0155] (b) a transmembrane domain;

[0156] (c) an intracellular costimulatory signaling domain, and

[0157] (d) an intracellular T cell receptor signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region. In certain embodiments said variable heavy domain and said variable light domain are separated by a linker. In certain embodiments said linker comprises the amino acid sequence of SEQ ID No. 13.

[0158] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising (a) an extracellular ligand-binding domain comprising a scFv comprising a variable heavy domain of SEQ ID No. 19, and a variable light domain of SEQ ID No. 20;

[0159] (b) a transmembrane domain;

[0160] (c) an intracellular costimulatory signaling domain, and

[0161] (d) an intracellular T cell receptor signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region. In certain embodiments said variable heavy domain and said variable light domain are separated by a linker. In certain embodiments said linker comprises the amino acid sequence of SEQ ID No. 13.

[0162] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising (a) an extracellular ligand-binding domain comprising a scFv comprising the amino acid sequence of SEQ ID No. 28;(b) a transmembrane domain;

[0163] (c) an intracellular costimulatory signaling domain, and

[0164] (d) an intracellular T cell receptor signaling domain. Optionally, said chimeric antigen receptor additionally comprises a hinge region.

[0165] In certain embodiments, the chimeric antigen receptor of the present disclosure comprises a transmembrane domain which is or is derived from CD28. In other embodiments said transmembrane domain comprises a polypeptide of the following amino acid sequence

[0166] FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID No. 30).

[0167] Therefore, in certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising

[0168] (a) an extracellular ligand-binding domain comprising a scFv specifically binding to HER2;

[0169] (b) a transmembrane domain which is or is derived from CD28;

[0170] (c) an intracellular costimulatory signaling domain, and

[0171] (d) an intracellular T cell receptor signaling domain. Preferably, said transmembrane domain comprises the amino acid sequence of SEQ ID No. 30.

[0172] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising a) an extracellular ligand-binding domain comprising a scFv specifically binding to a polypeptide comprising the amino acid sequence of SEQ ID No. 4;

[0173] (b) a transmembrane domain which is or is derived from CD28;

[0174] (c) an intracellular costimulatory signaling domain, and

[0175] (d) an intracellular T cell receptor signaling domain. Preferably, said transmembrane domain comprises the amino acid sequence of SEQ ID No. 30.

[0176] In certain preferred embodiments, the chimeric antigen receptor of the present disclosure comprises an intracellular costimulatory signaling domain which is or is derived from CD28. In other embodiments said intracellular costimulatory signaling domain comprises a polypeptide of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID No. 31).

[0177] Therefore, in certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising

[0178] (a) an extracellular ligand-binding domain comprising a scFv specifically binding to HER2;

[0179] (b) a transmembrane domain;(c) an intracellular costimulatory signaling domain which is or is derived from CD28, and

[0180] (d) an intracellular T cell receptor signaling domain. Preferably said costimulatory signaling domain comprises the amino acid sequence of SEQ ID No. 31.

[0181] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising (a) an extracellular ligand-binding domain comprising a scFv specifically binding to a polypeptide comprising the amino acid sequence of SEQ ID No. 4;

[0182] (b) a transmembrane domain;

[0183] (c) an intracellular costimulatory signaling domain which is or is derived from CD28, and

[0184] (d) an intracellular T cell receptor signaling domain. Preferably said costimulatory signaling domain comprises the amino acid sequence of SEQ ID No. 31.

[0185] In certain preferred embodiments, the chimeric antigen receptor of the present disclosure comprises an intracellular T cell receptor signaling domain is or is derived from CD3 zeta.

[0186] In other embodiments said T cell receptor signaling domain comprises a polypeptide of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No. 32).

[0187] Therefore, in certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising

[0188] (a) an extracellular ligand-binding domain comprising a scFv specifically binding to HER2;

[0189] (b) a transmembrane domain;

[0190] (c) an intracellular costimulatory signaling domain, and

[0191] (d) an intracellular T cell receptor signaling domain which is or is derived from CD3 zeta. Preferably said T cell receptor signaling domain comprises the amino acid sequence of SEQ ID No. 32.

[0192] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising (a) an extracellular ligand-binding domain comprising a scFv specifically binding to a polypeptide comprising the amino acid sequence of SEQ ID No. 4;

[0193] (b) a transmembrane domain;

[0194] (c) an intracellular costimulatory signaling domain, and

[0195] (d) an intracellular T cell receptor signaling domain is or is derived from CD3 zeta. Preferably said T cell receptor signaling domain comprises the amino acid sequence of SEQ ID No. 32.

[0196] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprisinga) an extracellular ligand-binding domain comprising a scFv specifically binding to HER2;

[0197] (b) a transmembrane domain which is or is derived from CD28;

[0198] (c) an intracellular costimulatory signaling domain which is or is derived from CD28, and

[0199] (d) an intracellular T cell receptor signaling domain is or is derived from CD3 zeta.

[0200] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising (a) an extracellular ligand-binding domain comprising a scFv comprising the amino acid sequence of SEQ ID No. 28;

[0201] (b) a transmembrane domain comprising the amino acid sequence of SEQ ID No. 30;

[0202] (c) an intracellular costimulatory signaling domain comprising the amino acid sequence of SEQ ID No. 31, and

[0203] (d) an intracellular T cell receptor signaling domain comprising the amino acid sequence of SEQ ID No. 32. The CARs exemplified in the present disclosure comprise a hinge region with the following amino acid sequence:

[0204] PKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No. 33).

[0205] Therefore, in certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising

[0206] (a) an extracellular ligand-binding domain comprising a scFv specifically binding to HER2;

[0207] (b) a hinge region;

[0208] (c) a transmembrane domain which is or is derived from CD28;

[0209] (d) an intracellular costimulatory signaling domain which is or is derived from CD28, and

[0210] (e) an intracellular T cell receptor signaling domain is or is derived from CD3 zeta. In certain embodiments, said hinge region is or is derived from a human IgGl hinge region. In certain embodiments, said human IgGl hinge region comprises amino acids 99-330 of human IgGl. In certain embodiments said human IgGl hinge region comprises amino acids 99-330 of human IgGl, wherein the cysteine at position 99 is replaced with proline. In certain embodiments said human IgGl hinge region comprises the amino acids sequence of SEQ ID No. 33.

[0211] In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising(a) an extracellular ligand-binding domain comprising a scFv comprising the amino acid sequence of SEQ ID No. 28;

[0212] (b) a hinge region comprising the amino acid sequence of SEQ ID No. 33;

[0213] (c) a transmembrane domain comprising the amino acid sequence of SEQ ID No. 30;

[0214] (d) an intracellular costimulatory signaling domain comprising the amino acid sequence of SEQ ID No. 31, and

[0215] (e) an intracellularT cell receptor signaling domain comprising the amino acid sequence of SEQ ID No. 32. In certain embodiments, the present disclosure makes use of a chimeric antigen receptor (CAR) comprising the following amino acid sequence:

[0216] QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIAWVRQMPGKGLEYMGLIYPGDSDTKYSPSFQGQV TISVDKSVSTAYLQWSSLKPSDSAVYFCARHDVGYCSSSNCAKWPEYFQHWGQGTLVTVSSGGGGSGG GGSGGGGSQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDHTNRPAGVP DRFSGSKSGTSASLAISGFRSEDEADYYCASWDYTLSGWVFGGGTKLTVLGADPAEPKSPDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGKKDPKFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No. 34).

[0217] Single chain antibody under the control of an inducible promoter

[0218] The present disclosure makes use of a single chain antibody (scFv) under the control of an inducible promoter. The inducible promoter initiates transcription upon CAR-mediated T cell activation. Any suitable promoter may be used to achieve this effect. One exemplary promoter is the nuclear factor of activated T cell (NFAT)-derived minimal promoter (Cancer Res (2011) 71: 5697–5706; Mol Ther (2011) 19: 751–759). This promoter consists of the core promoter of IL-2 gene (from -80 to +40) (minimal IL2 promoter), downstream of regulatory sequence comprised of 6 response elements (DNA motifs) recognized by the nuclear factor of activated T cells (NFAT) transcription factors. When the CAR T cell interacts with the tumor cell, upon antigen recognition, the resulting signaling cascade leads to phosphorylation of NFAT transcription factors and their translocation into the nucleus, where the said transcription factors can bind to their response elements and drive the transcription of the gene downstream of the minimal promoter.The regulatory sequence of the inducible promoter can also contain other response elements for other transcription factors, found in the promoter regions of genes, whose expression is driven through T cell activation.

[0219] Components of the inducible promoter:

[0220] SEQ ID No. Description Sequence CCCGGGACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATA

[0221] 35 IL2 minimal promoter AATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACT CAC AGT AAC C T CAAC TGC

[0222] Single NFAT response AAT TAGGAGGAAAAAC T GT T T CATACAGAAGGC GT C 36

[0223] element

[0224] AAT TAGGAGGAAAAAC T GT T T CATACAGAAGGC GT CAAT T AGGAGGAAAA ACT GT T TCATACAGAAGGC GT CAAT TAGGAGGAAAAAC T GT T T CAT ACAG

[0225] 37 Complete NFAT part AAGGCGTCAATTGGTCCCATCGAATTAGGAGGAAAAACTGTTTCATACAG AAGGCGTCAAT TAGGAGGAAAAAC T GT T T CATACAGAAGGCGT CAAT TAG GAGGAAAAAC T GT T T CATACAGAAGGCGT C AAT TAGGAGGAAAAAC T GT T T CATACAGAAGGC GT CAAT T AGGAGGAAAA ACT GT T TCATACAGAAGGC GT CAAT TAGGAGGAAAAAC T GT T T CAT ACAG

[0226] Complete NFAT region AAGGCGTCAATTGGTCCCATCGAATTAGGAGGAAAAACTGTTTCATACAG AAGGCGTCAAT TAGGAGGAAAAAC T GT T T CATACAGAAGGCGT CAAT TAG

[0227] 41 plus IL2 minimal GAGGAAAAACTGTTTCATACAGAAGGCGTCAATTGGTCCCGGGACATTTT promoter GAC AC CCCCATAATATTTTTC CAGAAT T AACAG T AT AAAT TGCATCTCTT GT T CAAGAG T T C C C T AT CAC T C T C T T T AAT CAC T AC T CAC AGT AAC C T CA ACTCCT

[0228]

[0229] Therefore, in certain embodiments, the regulatory region of the promoter comprised on the second nucleic acid sequence comprises a NFAT response element.

[0230] In certain embodiments, the promoter comprised on the second nucleic acid sequence comprises nucleic acid sequence of SEQ ID No. 36.

[0231] In certain embodiments, theregulatory region of the promoter comprised on the second nucleic acid sequence comprises one, two, three, four, five, six, seven or eight NFAT response element. Preferably, said second nucleic acid sequence comprises six NFAT elements.

[0232] In certain embodiments, the promoter comprised on the second nucleic acid sequence comprises nucleic acid sequence of SEQ ID No. 37.

[0233] In certain embodiments, the promoter comprised on the second nucleic acid sequence comprises an IL2 minimal promoter.

[0234] In certain embodiments, the promoter comprised on the second nucleic acid sequence comprises nucleic acid sequence of SEQ ID No. 35.

[0235] In certain embodiments, the promoter comprised on the second nucleic acid sequence comprises one or more NFAT elements and an IL2 minimal promoter.In certain embodiments, the promoter comprised on the second nucleic acid sequence comprises the nucleic acid sequence of SEQ ID No. 36 and SEQ ID No. 35.

[0236] In certain embodiments, the promoter comprised on the second nucleic acid sequence comprises the nucleic acid sequence of SEQ ID No. 37 and SEQ ID No. 35.

[0237] Therefore in certain embodiments, the present disclosure relates to a recombinant cell containing a nucleic acid comprising

[0238] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0239] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0240] (ii) a transmembrane domain; and

[0241] (iii) an intracellular signaling domain, and

[0242] (b) a second nucleic acid sequence comprising one or more NFAT elements and an IL2 minimal promoter and encoding a scFv specifically binding to a second tumor antigen,

[0243] wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

[0244] In certain embodiments, the present disclosure relates to a recombinant cell containing a nucleic acid comprising

[0245] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0246] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0247] (ii) a transmembrane domain; and

[0248] (iii) an intracellular signaling domain, and

[0249] (b) a second nucleic acid sequence comprising one or more NFAT elements and an IL2 minimal promoter and encoding a scFv specifically binding to a second tumor antigen.

[0250] In certain embodiments, the present disclosure relates to a recombinant cell containing a nucleic acid comprising

[0251] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0252] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0253] (ii) a transmembrane domain; and(iii) an intracellular signaling domain, and

[0254] (b) a second nucleic acid sequence comprising the nucleic acid sequence of SEQ ID No. 36 and SEQ ID No.

[0255] 35 and encoding a scFv specifically binding to a second tumor antigen,

[0256] wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

[0257] In certain embodiments, the present disclosure relates to a recombinant cell containing a nucleic acid comprising

[0258] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0259] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0260] (ii) a transmembrane domain; and

[0261] (iii) an intracellular signaling domain, and

[0262] (b) a second nucleic acid sequence comprising the nucleic acid sequence of SEQ ID No. 36 and SEQ ID No.

[0263] 35 and encoding a scFv specifically binding to a second tumor antigen.

[0264] In certain embodiments, the present disclosure relates to a recombinant cell containing a nucleic acid comprising

[0265] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0266] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0267] (ii) a transmembrane domain; and

[0268] (iii) an intracellular signaling domain, and

[0269] (b) a second nucleic acid sequence comprising the nucleic acid sequence of SEQ ID No. 37 and SEQ ID No.

[0270] 35 and encoding a scFv specifically binding to a second tumor antigen,

[0271] wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

[0272] In certain embodiments, the present disclosure relates to a recombinant cell containing a nucleic acid comprising

[0273] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0274] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;(ii) a transmembrane domain; and

[0275] (iii) an intracellular signaling domain, and

[0276] (b) a second nucleic acid sequence comprising the nucleic acid sequence of SEQ ID No. 37 and SEQ ID No.

[0277] 35 and encoding a scFv specifically binding to a second tumor antigen.

[0278] The present disclosure makes use of a scFv specifically binding to a second tumor antigen. Said scFv specifically binding to a second tumor antigen is encoded on the second nucleic acid comprised in said recombinant cell.

[0279] In certain embodiments said second tumor antigen is CEACAM6. In certain embodiments said second tumor antigen is a polypeptide comprising the amino acid sequence of SEQ ID No. 1. In certain embodiments said second tumor antigen is a polypeptide consisting of the amino acid sequence of SEQ ID No. 1.

[0280] Therefore, in certain embodiments said second nucleic acid sequence encodes a scFv specifically binding to CEACAM6.

[0281] In certain embodiments, said second nucleic acid sequence encodes a scFv specifically binding to a polypeptide comprising the amino acid sequence of SEQ ID No. 1.

[0282] In certain embodiments, said second nucleic acid sequence encodes a scFv specifically binding to a polypeptide consisting of the amino acid sequence of SEQ ID No. 1.

[0283] The amino acid sequence of the anti-CEACAM6 scFv utilized in the present disclosure is shown in Example 1. Therefore, in certain embodiments the present disclosure makes use of a second nucleic acid sequence encoding a scFv comprising a variable heavy domain comprising an HCDR1 of SEQ ID No. 7, an HCDR2 of SEQ ID No. 8, and an HCDR3 of SEQ ID No. 9, and comprising a variable light domain comprising an LCDR1 of SEQ ID No.

[0284] 10, an LCDR2 of SEQ ID No. 11, and an LCDR3 of SEQ ID No. 12.

[0285] In certain embodiments, the present disclosure makes use of a second nucleic acid sequence encoding a scFv comprising a variable heavy domain of SEQ ID No. 5, and a variable light domain of SEQ ID No. 6. In certain embodiments said variable heavy domain and said variable light domain are separated by a linker. In certain embodiments said linker comprises the amino acid sequence of SEQ ID No. 13.

[0286] In certain embodiments, the present disclosure makes use of a second nucleic acid sequence encoding a scFv comprising the amino acid sequence of SEQ ID No. 18.

[0287] Nucleic acids, vectors and host cells

[0288] The recombinant cell of the present disclosure comprises two nucleic acid sequences. The first nucleic acid sequence encodes the chimeric antigen receptor (CAR) specifically binding to a first tumor antigen, and the second nucleic acid sequence encodes a promoter and a scFv specifically binding to a second tumor antigen.In certain embodiments, said first nucleic acid sequence and said second nucleic acid sequence are contained on the same nucleic acid molecule.

[0289] In certain embodiments, said first nucleic acid sequence and said second nucleic acid sequence are contained on different nucleic acid molecules.

[0290] In certain embodiments, said first nucleic acid sequence and said second nucleic acid sequence are comprised on the same nucleic acid molecule.

[0291] In certain embodiments, said first nucleic acid sequence and said second nucleic acid sequence are comprised on different nucleic acid molecules.

[0292] Principally, any nucleic acid sequence encoding said scFv specifically binding to said first tumor antigen and any nucleic acid sequence encoding said scFv specifically binding to said second tumor antigen may be used. However, in certain embodiments said first nucleic acid sequence comprises the following nucleic acid sequence

[0293] AT GGAT T T T C AGGT GC AGAT T T T C AGC T T C C T GC TAAT C AGT GC C T C AGT C A AAT GT C T AGAC C GGC GAT GGC C C AGGT GC AGC T GGT GC AGT C T GGGGC AGAGGT GAAAAAGC C C GGGGAGT C T C T GAAGAT C T C C T GTAAGGGT T C T GGAT AC AGC T T T AC C AGC T AC T GGAT C GC C T GGGT GC GC C AGAT GC C C GGGAAA GGC C T GGAGT AC AT GGGGC T C AT C T AT C C T GGT GAC T C T GAC AC CAAAT AC AGC CCGTCCTTC C AAGG C C AGGT C AC C AT C T C AGT C GACAAGT C C GT C AGC AC T GC C T AC T T GC AAT GGAGC AGT C T GAAGC C C T C GGAC AGC GC C GT GT AT T T T T GT GC GAGACAT GAC GT GGGAT AT T GC AGT AGT T C CAAC T GC GC AAAG T GGC C T GAAT AC T T C C AGC AT T GGGGC C AGGGC AC C C T GGT C AC C GT C T C C T C AGGT GGAGGC GGT T C AGGC GGAGGT GGC T C T GGC GGT GGC GGAT CGCAGTCTGTGTT GAC GC AAC C GC C C T C AGT GT C T GC GG C C C C AGGAC AGAAGGT C AC CAT CTCCTGCTCT GGAAGC AGC T C CAAC AT T GGGAATAAT TAT GT AT C C T GGT AC C AGC AGC T C C C AGGAAC AGC C C C CAAAC T C C T CAT C T AT GAT C AC AC CAAT C GGC C C GC AGG GGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGT C C GAGGAT GAGGC T GAT TAT TACTGTGCCTCCT GGGAC T AC AC CCTCTCGGGCT GGGT GT T C GGC GGA GGAAC C AAGC T GAC C GT C C T AGGT GC GGAT C C C GC C GAGC C CAAAT C T C C T GACAAAAC T C AC AC AT G CCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG AC AC C C T C AT GAT C T C C C GGAC C C C T GAGGT C AC AT GC GT GGT GGT GGAC GT GAGC C AC GAAGAC C C T GAGGT C AAGT T CAAC T GGT AC GT GGAC GGC GT GGAGGT GC ATAAT GC CAAGAC AAAGC C GC GGGAGGA GC AGT ACAAC AGC AC GT AC C GGGT GGT C AGC GT C C T C AC C GT C C T GC AC C AGGAC T GGC T GAAT GGC A AGGAGT AC AAGT GC AAGGT C T C C AACAAAGC C C T C C C AGC C C C C AT C GAGAAAAC CAT C T C C AAAGC C AAAGGGC AGC C C C GAGAAC C AC AGGT GT AC AC CCTGCCCC CAT C C C GGGAT GAGC T GAC CAAGAAC C A GGT C AGC C T GAC C T GC C T GGT CAAAGGC T T C T AT C C C AGC GAC AT C GC C GT GGAGT GGGAGAGC AAT G GGC AGC C GGAGAAC AAC T ACAAGAC C AC GCCTCCCGTGCT GGAC T C C GAC GGCTCCTTCTTCCTCTAC AGCAAGC T C AC C GT GGACAAGAGC AGGT GGC AGC AGGGGAAC GT C T T C T CAT GC T C C GT GAT GC AT GA GGC T C T GC AC AAC C AC T AC AC GC AGAAGAGC CTCTCCCTGTCTCC GGGT AAAAAAGAT C C CAAAT T T T GGGT GC T GGT GGT GGT T GGT GGAGT CCTGGCTTGCTATAGCTTGC T AGT AAC AGT GGC C T T T AT TAT T T T C T GGGT GAGGAGTAAGAGGAGC AGGC T C C T GC AC AGT GAC T AC AT GAAC AT GAC TCCCCGCCGCCC CGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCCCCACGCGACTTCGCAGCCTATCGCTCCCTGAGAGT GAAGT T C AGC AGGAGC GC AGAC GC C C C C GC GT AC C AGC AGGGC C AGAAC C AGC T C TAT AAC GAG C T CAAT C T AGGAC GAAGAGAGGAGT AC GAT GT T T T GGACAAGAGAC GT GGC C GGGAC C C T GAGAT GGG GGGAAAGC C GAGAAGGAAGAAC C C T C AGGAAGGC C T GT AC AAT GAAC T GC AGAAAGAT AAGAT GGC GG AGGC C T AC AGT GAGAT T GGGAT GAAAGGC GAGC GC C GGAGGGGC AAGGGGC AC GAT GGC C T T T AC C AG GGT C T C AGT AC AGC C AC CAAGGAC AC C T AC GAC GC C C T T C AC AT GC AGGC CCTGCCCCCTCGC ( SEQ ID No. 38 ).

[0294] In certain embodiments, said second nucleic acid sequence comprises the following nucleic acid sequence C AGGT C AC T C T GC GAGAAT CCGGCCCTGCCCTGGT GAAGC CAAC C C AGAC AC T GAC C C T GAC AT GC AC CTTCTCTGGCTTTTCCCTGTC T AC C T AC GGAAT C GGAGT GGGAT GGAT C AGGC AGC C AC C T GGC AAGG C C C T GGAGT GGC T GGC C C AC AT C T GGT GGAAC GACAAT AAGT AC TAT AGC AC AT C C C T GAAGAC AC GC C T GAC C AT C T C T AAGGAT AC C AGCAAGAAC C AGGT GGT GC T GACAAT GAC CAAT AT GGAC C C C GT GGA T AC AGC C AC C T AC T AT T GC GC C C GGAT C AGC CTGCCTTACTTC GAC T AT T GGGGC C AGGGC AC C AC AC T GAC AGT GAGC T C C GGAGGAGGAGGC T C C GGC GGC GGAGGC T C T GGC GGC GGC GGC AGC GAC AT C C AG C T GAC C C AGT C T C C T AGC TTTCTGTCCGCCTCTGTGGGC GAT AGGGT GACAAT C AC C T GTAAGGC C AG C C AGAAC GT GGGAAC AGC AGT GGC C T GGT AT C AGC AGAAGC C AGGCAAGGC C C C C AAGC T GC T GAT C T AC AGC GC C T C CAAT C GGT AT AC AGGC GT GC CAT C C AGAT T C T C T GGC AGC GGC T C C GGC AC C GAGT T T AC AC T GAC CAT C T C T AGC C T GC AGC C C GAGGAT T T C GC C AC C T AC TAT T GC C AGC AGT AT T C AAGT TA TCCTCTGACCTTCGGGGGGGGAACAAAAGTGGAAATCAAG ( SEQ ID No. 39 )

[0295] In certain embodiments, said recombinant cell contains a nucleic acid comprising the following nucleic acid sequence

[0296] GAT C TAAGC T T GAT AT C GAAT T AGGAGGAAAAAC T GT T T C AT AC AGAAGGC GT CAAT T AGGAGGAAAA AC T GT T T CAT AC AGAAGGC GT CAAT T AGGAGGAAAAAC T GT T T CAT AC AGAAGGC GT CAAT T GGT C C C AT C GAAT T AGGAGGAAAAAC T GT T T CAT AC AGAAGGC GT CAAT T AGGAGGAAAAAC T GT T T CAT AC AG AAGGC GT CAAT T AGGAGGAAAAAC T GT T T CAT AC AGAAGGC GT CAAT T GGT C C C GGGAC AT T T T GAC A C C C C CAT AAT AT T T T T C C AGAAT TAAC AGT AT AAAT T GC AT CTCTTGTT CAAGAGT T C C C TAT C AC T C T C T T TAAT C AC TACT C AC AGT AAC C T CAAC T C C T GGAT C C GC C AC CAT GGAAAC C GAC AC T C T GC T GC TGTGGGTGCTGCTGCTGTGGGTGCCAGGCT CAAC C GGAGAT GC C GC T C AGGT C AC T C T GC GAGAAT C C GGCCCTGCCCTGGTGAAGCCAACCCAGACACTGACCCTGACATGCACCTTCTCTGGCTTTTCCCTGTC T AC C T AC GGAAT C GGAGT GGGAT GGAT C AGGC AGC C AC C T GGC AAGGC C C T GGAGT GGC T GGC C C AC A T C T GGT GGAAC GAC AAT AAGT AC TAT AGC AC AT C C C T GAAGAC AC GC C T GAC C AT C T C T AAGGAT AC C AGCAAGAAC C AGGT GGT GC T GACAAT GAC CAAT AT GGAC C C C GT GGAT AC AGC C AC C T AC TAT T GC GC C C GGAT C AGC CTGCCTTACTTC GAC TAT T GGGGC C AGGGC AC C AC AC T GAC AGT GAGC T C C GGAGGAG GAGGC T C C GGC GGC GGAGGC T C T GGC GGC GGC GGC AGC GAC AT C C AGC T GAC C C AGT CTCCTAGCTTT C T GT C C GC C T C T GT GGGC GAT AGGGT GACAAT C AC C T GTAAGGC C AGC C AGAAC GT GGGAAC AGC AGT GGC C T GGT AT C AGC AGAAGC C AGGC AAGGC C C C C AAGC T GC T GAT CTACAGCGCCTC CAAT C GGT AT A C AGGC GT GC C AT C C AGAT T C T C T GGC AGC GGC T C C GGC AC C GAGT T T AC AC T GAC CAT CTCTAGCCTG C AGC C C GAGGAT T T C GC C AC C T AC T AT T GC C AGC AGT AT T CAAGT TAT C C T C T GAC C T T C GGGGGGGG AACAAAAGT GGAAAT CAAGAAGC T T GGGC C C GAACAAAAAC T C AT C T C AGAAGAGGAT C T GAAT AGC G C C GT C GAC CAT CAT CAT CAT CAT CAT T GAGAAT T CAAT T C T AC C GGGT AGGGGAGGC GCTTTTCCCAAGGC AGT C T GGAGC AT GC GC T T T AGC AGC CCCGCTGGGCACTTGGCGC T AC ACAAGT GGCCTCTGGCCT C GC AC AC AT T C C AC AT C C AC C GGT AGGC GC CAAC CGGCTCCGTTCTTTGGTGGCCCCTTCGCGC C AC C TTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAA T GGAAGT AGC AC GT C T C AC T AGT C T C GT GC AGAT GGAC AGC AC C GC T GAGC AAT GGAAGC GGGT AGGC C T T T GGGGC AGC GGC CAAT AGC AGC TTTGCTCCTTCGCTTTCT GGGC T C AGAGGC T GGGAAGGGGT GG GT C C GGGGGC GGGC T C AGGGGC GGGC T C AGGGGC GGGGC GGGC GC C C GAAGGT C C T C C GGAGGC C C GG CATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCG AC C GGT C GC C AC CAT GGAT T T T C AGGT GC AGAT T T T C AGC T T C C T GC TAAT C AGT GC C T C AGT C AT AA T GT C T AGAC C GGC GAT GGC C C AGGT GC AGC T GGT GC AGT C T GGGGC AGAGGT GAAAAAGC C C GGGGAG T C T C T GAAGAT C T C C T GTAAGGGT T C T GGAT AC AGC T T T AC C AGC T AC T GGAT C GC C T GGGT GC GC C A GAT GC C C GGGAAAGGC C T GGAGT AC AT GGGGC T C AT C T AT C C T GGT GAC T C T GAC AC C AAAT AC AGC C CGTCCTTC CAAGGC C AGGT C AC C AT C T C AGT C GACAAGT C C GT C AGC AC T GC C T AC T T GCAAT GGAGC AGT C T GAAGC C C T C GGAC AGC GC C GT GT AT T T T T GT GC GAGACAT GAC GT GGGAT AT T GC AGT AGT T C CAAC TGC GCAAAGT GGC C T GAAT AC T T C C AGC AT T GGGGC C AGGGC AC CCTGGTCACCGTCTCCTCAG GT GGAGGC GGT T C AGGC GGAGGT GGC T C T GGC GGT GGC GGAT C GC AGT C T GT GT T GAC GCAAC C GC C C TCAGTGTCTGCGGCCC C AGGAC AGAAGGT C AC CAT CTCCTGCTCT GGAAGC AGC T C CAAC AT T GGGAA TAAT TAT GT AT C C T GGT AC C AGC AGC T C C C AGGAAC AGC C C C C AAAC T C C T CAT C TAT GAT C AC AC C A ATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATC AGTGGGTTCCGGTCC GAGGAT GAGGC T GAT TAT TACTGTGCCTCCT GGGAC T AC AC CCTCTCGGGCTG GGTGTTCGGCGGAGGAACCAAGCTGACCGTCCTAGGTGCGGATCCCGCCGAGCCCAAATCTCCTGACA AAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC C C AAAAC C CAAGGAC AC C C T C AT GAT C T C C C GGAC C C C T GAGGT C AC AT GC GT GGT GGT GGAC GT GAG C C AC GAAGAC C C T GAGGT C AAGT T CAAC T GGT AC GT GGAC GGC GT GGAGGT GC AT AAT GC CAAGAC AA AGC C GC GGGAGGAGC AGT ACAAC AGC AC GT AC C GGGT GGT C AGC GT C C T C AC C GT C C T GC AC C AGGAC T GGC T GAAT GGC AAGGAGT AC AAGT GC AAGGT C T C C AACAAAGC C C T C C C AGC C C C CAT C GAGAAAAC CAT C T C CAAAGC CAAAGGGC AGC C C C GAGAAC C AC AGGT GT AC AC CCTGCCCC CAT C C C GGGAT GAGC T GAC CAAGAAC C AGGT C AGC C T GAC C T GC C T GGT CAAAGGC T T C TAT C C C AGC GAC AT C GC C GT GGAG T GGGAGAGCAAT GGGC AGC C GGAGAAC AAC T ACAAGAC C AC GCCTCCCGTGCT GGAC T C C GAC GGC T C CTTCTTCCTC T AC AGCAAGC T C AC C GT GGACAAGAGC AGGT GGC AGC AGGGGAAC GT C T T C T CAT GC T C C GT GAT GC AT GAGGC T C T GC AC AAC C AC T AC AC GC AGAAGAGC C T C T C C C T GT C T C C GGGT AAAAAA GAT C C C AAAT T T T GGGT GC T GGT GGT GGT T GGT GGAGT CCTGGCTTGCTATAGCTTGC T AGT AAC AGT GGC C T T TAT T AT T T T C T GGGT GAGGAGTAAGAGGAGC AGGC T C C T GC AC AGT GAC T AC AT GAAC AT GA CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCCCCACGCGACTTCGCAGCC TAT C GC T C C C T GAGAGT GAAGT T C AGC AGGAGC GC AGAC GC C C C C GC GT AC C AGC AGGGC C AGAAC C A GC T C TAT AAC GAGC T CAAT C T AGGAC GAAGAGAGGAGT AC GAT GT T T T GGACAAGAGAC GT GGC C GGG AC C C T GAGAT GGGGGGAAAGC C GAGAAGGAAGAAC C C T C AGGAAGGC C T GT AC AAT GAAC T GC AGAAA GATAAGAT GGC GGAGGC C T AC AGT GAGAT T GGGAT GAAAGGC GAGC GC C GGAGGGGCAAGGGGC AC GA T GGC C T T T AC C AGGGT C T C AGT AC AGC C AC CAAGGAC AC C T AC GAC GCCCTTCACAT GC AGGC C C T GC C C C C T C GC TAAT C C T C GAGGGC T GC AGC GC T GC AGAGGC C GAGT GC AGAAC T GC T C CAAAGGGAC C T C AAGGC T T T C C GAGGGAC AC T AGGC T GAC T C CAT C GAGC C AGT GT AGAGATAAGC T TAT C GAT T AGT C C AAT T T GT T AAAGAC AGGAT AT C AGT GGT C C AGGC T C T AGT T T T GAC T CAAC AAT AT C AC C AGC T GAAG C C T AT AGAGT AC GAGC CAT AGAT AAAATAAAAGAT T T T AT T T AGT C T C C AGAAAAAGGGGGGAAT GAAAGAC C C C AC C T GT AGGT T T GGCAAGC T AGC T T AAGT AAC GC CAT T T T GC AAGGC AT GGAAAAAT AC AT AAC T GAGAAT AGAGAAGT T C AGAT C AAGGT C AGGAAC AGAT GGAAC AGGGT C GAC C C T AGAGAAC CAT C AGAT GT T T C C AGGGT GC C C C AAGGAC C T GAAAT GAC CCTGTGCCTTATTT GAAC TAAC CAAT C AGT T CGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTC GGGGCGCCAGTCCTCC GAT T GAC T GAGT C GC C C GGGT AC C C GT GT AT C C AATAAAC CCTCTTGCAGTT GCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGG GGGT C T T T CAT T T GGGGGC T C GT C C GGGAT C GGGAGAC C C C T GC C C AGGGAC C AC C GAC C C AC C AC C G GGAGGT AAGC TGGCTGCCTCGCGCGTTTC GGT GAT GAC GGT GAAAAC C T C T GAC AC AT GC AGC T C C C G GAGAC GGT C AC AGC T T GT C T GTAAGC GGAT GC C GGGAGC AGAC AAGC C C GT C AGGGC GC GT C AGC GGG TGTTGGCGGGTGTC GGGGC GC AGC CAT GAC C C AGT C AC GT AGC GAT AGC GGAGT GT AGAT C C GGC T GT GGAAT GTGTGTCAGT T AGGGT GT GGAAAGT CCCCAGGCTCCC C AGC AGGC AGAAGT AT GCAAAGC AT G CAT C T CAAT T AGT C AGC AAC C AGGT GT GGAAAGT CCCCAGGCTCCC C AGC AGGC AGAAGT AT GC AAAG C AT GC AT C T CAAT T AGT C AGC AAC CAT AGT C C C GC C C C TAAC T C C GC C CAT C C C GC C C C TAAC T C C GC CCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC T C GGC C T C T GAGC TAT T C C AGAAGT AGT GAGGAGGC T T T T T T GGAGGC C T AGGC T T T T GCAAAAAGC T T AC T GGC T TAAC TAT GC GGC AT C AGAGC AGAT T GT AC T GAGAGT GC AC CAT AT GC GGT GT GAAAT AC C GC AC AGAT GC GT AAGGAGAAAAT AC C GC AT C AGGC GCTCTTCCGCTTCCTCGCTCACT GAC T C GC T GC GC T C GGT C GT T C GGC T GC GGC GAGC GGT AT C AGC T C AC T C AAAGGC GGT AAT AC GGT TAT C C AC AGAA T C AGGGGATAAC GC AGGAAAGAAC AT GT GAGC AAAAGGC C AGC AAAAGGC C AGGAAC C GTAAAAAGGC C GC GT T GC T GGC GT T T T T C C AT AGGC T C C GC C C C C C T GAC GAGC AT C AC AAAAAT C GAC GC T CAAGT C AGAGGT GGC GAAAC C C GAC AGGAC T AT AAAGAT AC CAGGCGTTTCCCCCT GGAAGC T C C C T C GT GC GC TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT T T C T CAT AGC T C AC GC T GT AGGT AT C T C AGT T C GGT GT AGGT C GT T C GC T C CAAGC T GGGC T GT GT GC ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGAC AC GAC T TAT C GC C AC T GGC AGC AGC C AC T GGT AAC AGGAT T AGC AGAGC GAGGT AT GT AGGC GG TGC T AC AGAGT T C T T GAAGT GGT GGC C TAAC T AC GGC T AC AC T AGAAGGAC AGT AT T T GGT AT C T GC G C T C T GC T GAAGC C AGT T AC C T T C GGAAAAAGAGT TGGTAGCTCTT GAT C C GGC AAACAAAC C AC C GC T GGT AGC GGTGGTTTTTTTGTTT GCAAGC AGC AGAT T AC GC GC AGAAAAAAAGGAT C T C AAGAAGAT C C T T T GAT C T T T T C T AC GGGGT C T GAC GC T C AGT GGAAC GAAAAC T C AC GT TAAGGGAT T T T GGT C AT GA GAT TAT CAAAAAGGAT C T T C AC C T AGAT C C T T T T AAAT TAAAAAT GAAGT T T T AAAT CAAT C TAAAGT AT AT AT GAGT AAAC T T GGT C T GAC AGT T AC CAAT GC T T AAT C AGT GAGGC AC C TAT C T C AGC GAT C T G T C TAT T T C GT T C AT C CAT AGT T GC C T GAC TCCCCGTCGT GT AGAT AAC T AC GAT AC GGGAGGGC T T AC CAT C T GGC C C C AGT GC T GC AAT GAT AC C GC GAGAC C C AC GCTCACCGGCTC C AGAT T T AT C AGC AAT A AAC C AGC C AGC C GGAAGGGC C GAGC GC AGAAGT GGT C C T GCAAC TTTATCCGCCTC CAT C C AGT C T AT TAAT T GT T GC C GGGAAGC T AGAGTAAGT AGT T C GC C AGT T AAT AGT T T GC GCAAC GTTGTTGCCATTG C T GC AGGC AT C GT GGT GT C AC GC T C GT C GT T T GGT AT GGCTTCATTCAGCTCCGGTTCC CAAC GAT C A AGGC GAGT T AC AT GAT CCCCCATGTTGT GCAAAAAAGC GGT T AGC TCCTTCGGTCCTCC GAT C GT T GT C AGAAGTAAGT T GGC C GC AGT GT TAT C AC T CAT GGT TAT GGC AGC AC T GC ATAAT T C T C T T AC T GT C A T GC C AT C C GT AAGAT GC T T T T C T GT GAC T GGT GAGT AC T CAAC CAAGT C AT T C T GAGAAT AGT GT AT G C GGC GAC C GAGT TGCTCTTGCCCGGCGT CAAC AC GGGAT AAT AC C GC GC C AC AT AGC AGAAC T T TAAA AGT GC T CAT C AT T GGAAAAC GT T C T T C GGGGC GAAAAC T C T CAAGGAT CTTACCGCTGTT GAGAT C C A GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT GAGCAAAAAC AGGAAGGC AAAAT GC C GC AAAAAAGGGAATAAGGGC GAC AC GGAAAT GT T GAAT AC T CAT AC TCTTCCTTTTT C AA AT T AT T GAAGC AT T TAT C AGGGT T AT T GT C T CAT GAGC GGAT AC AT AT T T GAAT GT AT T T AGAAAAATAAACAAAT AGGGGT T C C GC GC AC AT T T C C C C GAAAAGT GC C AC C T GAC GT C T AAGAAAC CAT TAT TAT CAT GAC AT T AAC C T AT AAAAAT AGGC GT AT C AC GAGGC CCTTTCGTCT T C AAGAAT T AGC T T GGC CAT T GC AT AC GT T GT AT C CAT AT CAT AAT AT GT AC AT T TAT AT T GGC T C AT GT C C AAC AT T AC C GC CAT GT T GAC AT T GAT TAT T GAC T AGT TAT TAAT AGT AAT C AAT T AC GGGGT C A T T AGT T CAT AGC C CAT AT AT GGAGT T C C GC GT T AC AT AAC T T AC GGT AAAT GGC C C GC C T GGC T GAC C GC C C AAC GAC C C C C GC C CAT T GAC GT C AAT AAT GAC GT AT GT T C C CAT AGT AAC GC CAAT AGGGAC T T T C CAT T GAC GT CAAT GGGT GGAGT AT T T AC GGTAAAC T GC C C AC T T GGC AGT AC AT CAAGT GT AT CAT AT GC CAAGT AC GC C C C C TAT T GAC GT CAAT GAC GGT AAAT GGC C C GC C T GGC AT T AT GC C C AGT AC AT GAC C T T AT GGGAC TTTCCTACTT GGC AGT AC AT C T AC GT AT T AGT CAT C GC TAT T AC CAT GGT GAT GC GGT T T T GGC AGT AC AT CAAT GGGC GT GGAT AGC GGT T T GAC T C AC GGGGAT T T C CAAGT C T C C AC C C C AT T GAC GT CAAT GGGAGT T T GT T T T GGC AC CAAAAT CAAC GGGAC T T T C CAAAAT GT C GTAACAAC T C CGCCCCATT GAC GC AAAT GGGC GGT AGGC GT GT AC GGT GGGAGGT C T AT AT AAGC AGAGC T CAAT AAA AGAGC C C ACAAC CCCTCACTCGGCGCGC C AGT C T T C C GAT AGAC T GC GT C GC C C GGGT AC C C GT AT T C CCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGA GTGATTGACTACCCACGACGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATTTGGAGACCCCTGCCCA GGGAC C AC C GAC C C AC C AC C GGGAGGT AAGC T GGC C AGCAAC TTATCTGTGTCTGTCC GAT T GT C T AG T GT C TAT GT T T GAT GT T AT GC GC C T GC GT C T GT AC T AGT T AGC T AAC T AGC T C T GT AT C T GGC GGAC C C GT GGT GGAAC T GAC GAGT T C T GAAC AC C C GGC C GC AAC C C T GGGAGAC GT C C C AGGGAC T T T GGGGG CCGTTTTTGTGGCCC GAC C T GAGGAAGGGAGT C GAT GT GGAAT C C GAC C C C GT C AGGAT AT GT GGT T C T GGT AGGAGAC GAGAAC C TAAAAC AGT T C C C GC C T C C GT C T GAAT T T T T GC T T T C GGT T T GGAAC C GA AGCCGCGCGTCTTGTCTGCTGCAGCGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCT GT AT T T GT C T GAAAAT T AGGGC C AGAC T GT T AC C AC T C C C T TAAGT T T GAC C T T AGGT C AC T GGAAAG AT GT C GAGC GGAT C GC T C ACAAC C AGT C GGT AGAT GT C AAGAAGAGAC GT T GGGT T AC CTTCTGCTCT GC AGAAT GGC CAAC C T T TAAC GT C GGAT GGC C GC GAGAC GGC AC C T T TAAC C GAGAC C T CAT C AC C C A GGT T AAGAT C AAGGT CTTTTCACCTGGCCCGCAT GGAC AC C C AGAC C AGGT C C C C T AC AT C GT GAC C T GGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCT CTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTA TCCAGCCCTCACTCCTTCTCTAGGCGCCGGAATTGAA ( SEQ ID No. 40 )

[0297] In certain embodiments the present disclosure relates a vector comprising aforementioned nucleic acids. In certain embodiments the present disclosure relates a vector comprising

[0298] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0299] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0300] (ii) a transmembrane domain; and

[0301] (iii) an intracellular signaling domain, and(b) a second nucleic acid sequence encoding a promoter and a scFv specifically binding to a second tumor antigen.

[0302] In certain embodiments, said first nucleic acid sequence and said second nucleic acid sequence are on the same vector. In certain embodiments, said first nucleic acid sequence and said second nucleic acid sequence are on different vectors.

[0303] In certain embodiments the present disclosure relates to a recombinant cell comprising said nucleic acid sequences or said vectors.

[0304] In certain embodiments said recombinant cell is a host cell. In certain embodiments said recombinant cell is a eukaryotic cell. In certain embodiments said recombinant cell is a eukaryotic host cell.

[0305] In certain embodiments said recombinant cell is a T cell. In certain embodiments said T cell is a CD4+ T cell. In certain embodiments said T cell is a CD8+ T cell. In certain embodiments said recombinant cell is a NK cell.

[0306] Therefore, in certain embodiments the present disclosure relates to a T cell containing a nucleic acid comprising

[0307] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0308] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0309] (ii) a transmembrane domain; and

[0310] (iii) an intracellular signaling domain, and

[0311] (b) a second nucleic acid sequence encoding a promoter and a scFv specifically binding to a second tumor antigen,

[0312] wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

[0313] In certain embodiments the present disclosure relates to a NK cell containing a nucleic acid comprising

[0314] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0315] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0316] (ii) a transmembrane domain; and

[0317] (iii) an intracellular signaling domain, and(b) a second nucleic acid sequence encoding a promoter and a scFv specifically binding to a second tumor antigen,

[0318] wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

[0319] In certain embodiments the present disclosure relates to a CAR T cell containing a nucleic acid comprising

[0320] (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,

[0321] (i) an extracellular ligand-binding domain specifically binding to a first tumor antigen;

[0322] (ii) a transmembrane domain; and

[0323] (iii) an intracellular signaling domain, and

[0324] (b) a second nucleic acid sequence encoding a promoter and a scFv specifically binding to a second tumor antigen,

[0325] wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

[0326] Pharmaceutical compositions and therapeutic use

[0327] The recombinant cells of the present disclosure can be used therapeutically for the prevention and treatment of diseases and disorders.

[0328] For such therapeutic use, said recombinant cells may be part of a pharmaceutical composition comprising said recombinant cell and a pharmaceutically acceptable carrier. Preferably, said recombinant cell is a T cell.

[0329] A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting cells of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.The administration of the recombinant cells of the present disclosure may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In an embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0330] In certain embodiments, the present disclosure provides recombinant cells or a pharmaceutical composition comprising recombinant cell, and optionally a pharmaceutically acceptable carrier, for use in the treatment of a disease or disorder, wherein said recombinant cells or said pharmaceutical composition comprising said recombinant cells comprise a chimeric antigen receptor and a scFv specifically binding to a second tumor antigen as disclosed herein. Preferably, said recombinant cell is a T cell.

[0331] In certain embodiments, the present disclosure provides recombinant T cells or a pharmaceutical composition comprising recombinant cells, and optionally a pharmaceutically acceptable carrier, for use in the prevention of a disease or disorder, wherein said recombinant cells or said pharmaceutical composition comprising said recombinant cells comprise a chimeric antigen receptor and a scFv specifically binding to a second tumor antigen as disclosed herein. Preferably, said recombinant cell is a T cell.

[0332] The most effective results in terms of efficacy of treatment or prevention in a given subject will vary depending upon a variety of factors, including but not limited to the characteristics of the recombinant T cells (including longevity, activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological cond ition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of any pharmaceutically acceptable carrier or carriers in any composition used, and the route of administration.

[0333] In certain embodiments said disease or disorder is an acute or chronic inflammatory disorder or cancer. In preferred embodiments said disease or disorder is cancer. In certain embodiments said cancer is, but not limited to, a carcinoma of the gastrointestinal tract, adrenal glands, the nervous system, the connective tissue, the liver, kidney, lung or the hematopoietic system.

[0334] In other embodiments said disease or disorder is a viral or a microbial infection, an autoimmune disease, transplant rejection, graft-versus-host disease, or a chronic inflammatory disease.Examples

[0335] Example 1: Materials and methods

[0336] Transient expression and purification of scFv

[0337] The anti-CEACAM6 scFv was produced by transient transfection of expression vector pcDNA3.1(-) myc-His\A (synthesized by Genscript). The scFv consists of the VH and VL of the mAb CEACAM6 TPP 3310, linked via a (G4S)3 linker. The scFv has an Igk leader sequence on the N-terminus to ensure secretion, and a c-Myc and a 6xHis tag on the C-terminus for detection. The amino acid sequence of the scFv is as follows:

[0338] SEQID No. Description Sequence

[0339] variable heavy chain of QVTLRESGPALVKPTQTLTLTCTFSGFSLSTYGIGVGWIRQPPGK 5 AL EWLAH IWWNDNKYYS T S L KT RL T I S KDT S KNQWL TMTNMD PV TPP 3310 DTATYYCARISLPYFDYWGQGTTLTVSS variable light chain of DIQLTQSPSFLSASVGDRVTITCKASQNVGTAVAWYQQKPGKAPK 6 LLIYSASNRYTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQ TPP 3310 YSSYPLTFGGGTKVEIK

[0340] HCDR1 of TPP 3310

[0341] 7 TYGIGVG

[0342] (Ka bat)

[0343] HCDR2 of TPP 3310

[0344] 8 HIWWNDNKYYSTSLKT

[0345] (Ka bat)

[0346] HCDR3 of TPP 3310

[0347] 9 ISLPYFDY

[0348] (Ka bat)

[0349] LCDR1 of TPP 3310

[0350] 10 KASQNVGTAVA

[0351] (Ka bat)

[0352] LCDR2 of TPP 3310

[0353] 11 SASNRYT

[0354] (Ka bat)

[0355] LCDR3 of TPP 3310

[0356] 12 QQYSSYPLT

[0357] (Ka bat)

[0358] 13 3x Gly4Ser linker GGGGS GGGGS GGGGS

[0359] Igk leader sequence

[0360] 14 METDTLLLWVLLLWVPGSTGD

[0361] (secretion signal))

[0362] 15 cMyc tag EQKLISEEDLN

[0363] 16 6xHis tag HHHHHH

[0364] scFv C6, full length incl METDTLLLWVLLLWVPGSTGDAAQVTLRESGPALVKPTQTLTLTC TFSGFSLSTYGIGVGWIRQPPGKALEWLAHIWWNDNKYYSTSLKT

[0365] 17 leader RLTISKDTSKNQWLTMTNMDPVDTATYYCARISLPYFDYWGQGT

[0366] TLTVSSGGGGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCK

[0367]

[0368] AS QNVGT AVAWYQQKPGKAPKL L I YS ASNRYT GVP S RFS GS GS GTEFTLTISSLQPEDFATYYCQQYSSYPLTFGGGTKVEIKKLGPEQK LISEEDLNSAVDHHHHHH

[0369] scFv C6, full length QVTLRESGPALVKPTQTLTLTCTFSGFSLSTYGIGVGWIRQPPGK AL EWLAH IWWNDNKYYS T S L KT RL T I S KDT S KNQWL TMTNMD PV

[0370] without leader DTATYYCARISLPYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDI 18 QLTQSPSFLSASVGDRVTITCKASQNVGTAVAWYQQKPGKAPKLL IYSASNRYTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYS SYPLTFGGGTKVEIKKLGPEQKLISEEDLNSAVDHHHHHH

[0371]

[0372] For production of the scFv, 3,5xl06HEK293T cells grown in 10 ml of DMEM Medium (Sigma), supplemented with 10% fetal calf serum (FCS) (Sigma) and 1% Pencillin / Streptomycin (Sigma) (DMEM complete) were plated in a T75 flask (TPP). After overnight incubation, cells were transfected with the help of PEIpro (Polyplus). Per flask, 10 pl PEIpro (Polyplus) were mixed with 250 pl DMEM medium without supplements and added to the diluted DNA mixture which contained 250 pl DMEM without supplements and 10 pg of the vector encoding the scFv. The mixture was incubated for 5 min at RT, and in this time the medium of the plated cells was removed and replaced by 8 ml fresh DMEM complete. After incubation, the transfection mixture was added dropwise to the plate and incubated at 37°C, 5% CO2. 18-20 h after the transfection, the medium of the plate was replaced by 14 ml DMEM supplemented with 5% FCS and 1% Pen / Strep. The flask was further incubated for 5-6 days until the supernatant was collected for the purification of the scFv. The purification of the scFv was done using 1ml HisTrap HP column (cytiva) in AktaGo chromatograpy device (cytiva). The successful expression and purification of the functional scFv was confirmed by Western Blot analysis The anti-CEACAM6 scFv on the membrane was detected with anti-H is tag antibody (clone HIS. H8, Novus Biologica Is) and secondary anti-mouse antibody conjugated with HRP (Santa Cruz).

[0373] Custom ELISA for the detection of scFv C6 or to determine blocking of CEACAM1-CEACAM6 interactions using scFv C6

[0374] In order to detect the binding of scFv C6 to CEACAM6 or to determine the presence of scFv C6 in supernatants of transduced and activated TRUCKS, a customized ELISA was developed. For this purpose, wells of a 96 well plate were coated with 100 pL coating solution (5 pg / ml rhCEACAM6-FC in in coating buffer). The plate was then covered with plastic foil and incubated overnight at 4 °C or at 37°C for one hour. After discarding the supernatant, the plate was washed twice with washing buffer, i.e. PBS-T. Then 100 pl of 4% BSA in PBS were added, and the plate was covered with plastic foil and incubated for 1 h in the dark at RT. After three more washing steps, 100 pl diluted purified scFv in concentrations between 4 and 0,06 pg / ml, or undiluted supernatants from activated TRUCKS or CAR T cells were added (260000 TRUCK or CAR T cells cultured on various concentration of ErbB2 in 200 pl T cell medium for 60 h). The ELISA plate was covered with plastic foil and incubated for 2 hours in the dark at room temperature, followed by five washing steps. Then 100 pl of 10% human AB serum (Valley Biomedical) in PBS was added, incubated for 1 hour at room temperature in the dark, again followed by five washing steps. For detection an anti-His-HRP antibody (1:1000; Miltenyi Biotec) wasdiluted in Assay Diluent (BD Biosciences), and 100 pl of this solution was added to each well. The plate was incubated for 1 hour at room temperature in the dark, followed by seven washing steps. Then lOOul Substrate Reagent (BD Bioscences) was added to each well. The plates were incubated without plastic foil in the dark until the standards turn blue, then 50 pL stop solution (H2SO4 [now it is correct]) was added in each well. The absorbance was measured at 450 nm in a Tecan Reader. The half-maximal effective concentration (EC50) values were calculated with GraphPad Prism 6.0 using a four-parameter non-linear curve fit. Concentration of the scFv C6 in the supernatant of the activated ErbB2 TRUCK T cells was extrapolated from a standard curved based on serial dilution of purified scFv C6 with known concentration.

[0375] A similar ELISA was used to detect the ability of scFv C6 to block the interaction between CEACAM6 and CEACAM1. To this end, wells in a 96 well plate were coated with 100 pl CEACAM6-H is tagged recombinant protein solution (5 pg / ml in coating buffer) and incubated overnight at 4°C. The coated plate was then washed and blocked with 4% BSA as described above. Then various concentrations of scFv C6 were added to the corresponding wells (4 - 0,25 pg / ml) and the plate was incubated for lh at room temperature. Then CEACAM1-Fc / His tagged recombinant protein was added to the corresponding wells to final concentration of 5 pg / ml and incubated for further 1,5 h. Subsequently the plate was washed and blocked again for 40 min with 4% BSA. Then the bound CEACAM-1 was detected with protein -A conjugated to HRP (1:4000 dilution, Thermo Fisher Scientific) for 1 h at room temperature. After seven washing steps, 100 ul Substrate Reagent (BD Bioscences) was added to each well. The plates were incubated without plastic foil in the dark until the standards turned blue, then 50 pL stop solution (2N H2SO4) was added in each well. The absorbance was measured at 450 nm in a Tecan Reader.

[0376] Surface plasmon resonance

[0377] Surface plasmon resonance (SPR) experiments were performed to analyze the quantitative binding of scFv C6 to its target rhCEACAM6-Fc tagged protein. The experiments were performed using Biacore X100 device (GE Healthcare Biacore, Inc., now Cytiva). Binding assays were carried out using Sensor chip protein A (Cytiva) at 25 °C in HBS-EP+ assay buffer (10 mM HEPES pH 7.4, 150 mM NaCI, 3 mM EDTA, 0.05% Surfactant P20, Cytiva). scFvC6 was used as analyte to determine its affinity. CEACAM6 was immobilized on the protein A chip via its Fc-tag at 10 pg / ml for 70 s at flow rate of 10 pl / min, prior to each analyte injection. For kinetics determination scFv C6 was injected in various concentrations (0,75 to 100 nM) at a flowrate of 30 pl / min for 80 s and dissociation was monitored for 5 min. Obtained sensograms were referenced, i.e. on-line reference cell correction. The rate constants for dissociation (kd) and association (ka) were obtained from sensograms using the global fitting with a first-order 1:1 Langmuir binding model, implemented in the Biacore Evaluation Software Package. The equilibrium dissociation constants (KD) were calculated as ratios of kaand kd.Flow cytometry

[0378] FACS-Sta in i ng was performed to analyze expression of CEACAM6 or CEACAM1 on the surface of cells, as well as for testing binding of the scFv anti-CEACAM6. All centrifugation steps in sample preparation were performed at 1400 rpm for 10 min and 4°C. For each staining 250000 to 500000 cells were used. In case of adherent cells, they were detached using PBS / EDTA (0,02%). Afterwards cells were washed with 2 ml of FACS buffer to remove remaining medium. To block unspecific binding, cells were incubated with 100 pl Kiovig (TAKEDA GmbH; 1:20 dilution in FACS buffer) for 20 min in the dark on ice. Then cells were washed with FACS buffer and live / dead staining was performed using 100 pl ZombieNir (Biolegend; 1:1000 dilution in PBS). After incubation for 20 min at RT, cells were washed again with 2 ml FACS buffer. Next primary antibody or scFv was added to the corresponding samples and incubated for 45 min on ice. The used antibodies are shown in Table 8. Afterwards cells were washed twice with 2 ml FACS buffer. Secondary antibody was added to the corresponding samples and incubated for 30 min on ice. Then, cells were washed again two times with 2 ml FACS buffer, resuspended in 150 pl FACS buffer and measured in a BD FACSLyric (BD Biosciences).

[0379] The detection of CAR and CEACAM1 expression on the surface of transduced T cells was done in a similar manner. T cells were collected and washed with FACS buffer and after blocking and life / dead staining was performed, the T cells were stained with anti-CD3 antibody conjugated with AlexaFluor 700 and anti-CD66a / c / e APC conjugated antibody. After 30 min on ice incubation, the cells were washed and subsequently stained with Goat F(ab')2 anti-Human IgG PE conjugated antibody for another 30 min on ice. After that the T cells were washed two times with FACS buffer, resuspended in 150 pl FACS buffer and acquired on a BD FACSLyric device (BD Biosciences).

[0380] Table 8:

[0381] Antibodies for FACS-Staining Manufacturer

[0382] Goat F(ab')2 Anti-Human IgG, Mouse ads-PE SouthernBiotech

[0383] CEACAM-6 / CD66c Antibody (9A6), isotype mouse IgGl Enzo Life Sciences CEACAM-1 Antibody (GM8G5), isotype mouse IgGl Enzo Life Sciences

[0384] Anti-mouse IgGl BV421 BioLegend

[0385] PE anti-H is Tag Antibody, isotype mouse IgGl BioLegend

[0386] AF488 anti c-Myc Antibody, isotpe mouse IgGl BioRad

[0387] Alexa Fluor® 700 anti-human CD3 Antibody BioLegend

[0388] APC anti-human CD66a / c / e Antibody BioLegend

[0389]

[0390] Transduction of T cells

[0391] Peripheral blood mononuclear cells (PBMCs) derived from platelet cone (Department of Clinical Chemistry and Laboratory Medicine, Transfusion medicine-Blood donation) via gradient centrifugation were frozen and stored in liquid nitrogen. 2 days prior to transduction they were thawed and transferred in 10 mLT cell medium supplemented with 50 U / mL benzonase. After centrifugation, the supernatant was discarded, the pellet was resuspended in fresh T cell medium, and the cells were counted. The cell number was adjusted to 0.,6x106cells / mL and CD3 at 200 ng / mL, CD28 at 50 ng / mL and IL-2 and 1000 lU / mL were added. Cells were incubated at 37 °C and 5% CO2 for 2 days.

[0392] On the same day, HEK 293T cells were washed with PBS and detached with trypsin. The cells were collected and centrifuged for 5 min at 1400 rpm. The supernatant was discarded, and the pellet was resuspended in DMEM complete (Sigma). Cells were counted and 3 x 106 cells in 10 mL DMEM complete were plated in a 10 cm dish. The plates were incubated overnight at 37°C and 5% CO2.

[0393] HEK 293T cells were transfected for the generation of viral supernatant. Therefore, a DNA mix containing 250 pL DMEM without supplements, 10 pg encoding vector (pSIN), 5 pg helper vector (pHIT60) and 5 pg envelope vector (pCOLT-GALV) was prepared for each 10 cm plate. Also, for each 10 cm plate a transfection reagent mix containing 250 pL DMEM (Sigma) and 20 pL PEIpro transfection reagent ( Pol pl us) was prepared. DNA mix and transfection reagent were mixed and incubated for 5 min at room temperature (RT). Meanwhile the medium of the 10 cm plates was exchanged with 10 mLT cell medium and 500 pL of DNA -transfection reagent mix were added dropwise to each plate. The plates were incubated overnight at 37°C and 5% CO2. Additionally, PDL coated T25 culture flasks were prepared forT cell spinfection by adding 2 mL PDL solution (10 pg / mL diluted in ddH2O) to a T25 culture flask, followed by overnight incubation at 4 °C.

[0394] For the transduction of T cells, 10 mL of the virus particle containing supernatant was collected from the HEK293 T cells in a 15 mL tube. The removed supernatant was replaced with 10 mL T cell medium and the HEK293T cells were kept at 37°C and 5% CO2. Meanwhile the activated PBMCs were counted and 5x106cells were placed in a 15 mL falcon tube and centrifuged for 5 min at 300 g. After removing the supernatant, the pellet was resuspended with the supernatant containing the virus particle and 200 lU / mL IL-2 was added. The T cell / virus supernatant mixture was added to the flasks, which were then centrifuged for 60 min at 1600g at 32 °C and reduced break. Finally, the flasks were incubated overnight at 37 °C and 5% CO2 (in lying position). New T25 culture flasks were coated with PDL (see above) for the repetition of the transduction procedure on the next day.

[0395] On the next day, the T cells from the T25 flasks were collected, centrifuged and the pellet was resuspended in 5 mL of T cell medium. After counting the cells were subjected to magnetic activated cell sorting as described below.Magnetic activated cell sorting (MACS)

[0396] The following protocol was applied for cell counts of 10x106cells or less. If more cells were used, all volumes were adjusted appropriately. After transduction, cells were counted and centrifuged at 1000 rpm for 5 min. After removing the supernatant, the cell pellet was resuspended in Buffer / antibody (47,5 pL MACS buffer and 2,5 pL biotinylated goat a-human antibody (SouthernBiothec)). The cell suspension was incubated for 5 min at 4 °C and washed by adding 2 mL MACS buffer. The centrifugation step was repeated and the cell pellet was resuspended in 80 pL MACS buffer. 20 pL anti-Biotin MicroBeads (Miltenyi Biotec) were added, mixed by pipetting and incubated for 15 min at 4°C. Cells were washed by adding 2 mL MACS buffer and centrifuged for 5 min at 1000 rpm. After removing the supernatant completely, the cells were resuspended with 500 pL MACS buffer. For preparing the MS column (Miltenyi Biotec), 500 pL MACS buffer was pipetted into the filter for column equilibration. Then samples were added onto the pre-separation filter, which was washed three times with 500 pL MACS buffer. Between each step, complete flow through was ensured. The column was removed and placed onto a fresh 15 mL falcon. To elute the CART cells, 1 mL MACS buiffer was added to the column and pressed through with a plunger. Cells were counted, adjusted to 0,6x106cells per mL in T cell medium supplemented with IL-2 (1000 U / mL), IL-7 (600 U / mLl) (Miltenyi Biotec) and IL-15 (300 U / mL) (Miltenyi Biotec) and incubated at 37 °C and 5% CO2.

[0397] Luciferase kill assay

[0398] Tumor cells were detached from their flasks, centrifuged, resuspended in T cell medium and adjusted to 2x105cells per mL. 50 pL cell suspension (1x104cells) were added to each well of a white 96 well plate. 100 pL enriched ErbB2 CAR T cells and scFv C6 ErbB2 TRUCKS were added in different E: T ratios to tumor cells on the plate. The plates were incubated overnight (17-20h) at 37 °C and 5% CO2. After centrifugation of the plate, the supernatant was discarded and 40 pL lysis buffer were added to each well. Plates were incubated for 15 min at RT, then 60 pL luc buffer were added and the luminescence immediately measured in a Tecan reader.

[0399] Table 9:

[0400] Buffer Component

[0401] ddH2O

[0402] 415 mM DTT

[0403] B2 buffer

[0404] 35 mM ATP

[0405] 1 mM AMP

[0406] ddH2O

[0407] 50 mM HEPES

[0408] 0,5 mM EDTA

[0409] BL buffer

[0410] 0,33 mM Phenylacetic acid

[0411] 0,07 mM Oxalic acid

[0412] pH Adjustment to 7.6

[0413] PBS FACS Buffer

[0414]

[0415] 1% FCSLuciferin solution ddH2O

[0416] 0,45 mM D_luciferin

[0417] Addition of NaOH until color change Luciferase buffer 86,25 % BL buffer

[0418] 10 % B2 buffer

[0419] 1,25 % Luciferin solution

[0420] 2,5 % MgSO4 (IM stock)

[0421] Luciferase assay lysis buffer BL buffer

[0422] 10% TritonX-100

[0423] MACS Buffer PBS

[0424] 2m M EDTA

[0425] 0,5% Filtered AB serum

[0426] ELISA coating buffer ddH20

[0427] 0,1 M NaHCO3

[0428] 33,6 mM Na2CO3

[0429] pH Adjustment to 9,5

[0430] PBS-T PBS

[0431] 0,05% Tween 20

[0432] ddH20

[0433] 0,65 % NaCI

[0434] RINGER's solution 0,042 % KCL

[0435] 0,025 % CaCI2

[0436]

[0437] 0,02 % Sodium bicarbonate

[0438] Activation of Jurkat-Lucia™ NFAT Cells -newly added

[0439] Jurkat-Lucia™ NFAT Cells (Invivogen) were transduced with viral particles encoding for CAR or TRUCK construct as well as viral particles encoding for CEACAM1 or delta CEACAM1, using the protocol described above for T cells. The resulting Jurkat-Lucia cell lines CAR CEACAM1, CAR dCEACAMl, TRUCK CEACAM1 and TRUCK dCEACAMl were then activated with a plate bound ErbB2 (10 pg / ml) in the absence or the presence of different concentration of CEACAM6 (20-60 pg / ml). All cell lines were diluted to 106 cells / ml and 100 pl of the cell suspension was pipetted in the respective wells of the 96 well plate coated with the proteins of interest. Cells were cultured for 48 hours. To determine the effect of CEACAM6 / CEACAM1 interaction on the activation through the CAR, luciferase was measured. Jurkat-Lucia™ NFAT Cells carry a luciferase expression cassette downstream of inducible NFAT regulated promoter, so when the cells are activated through the CAR, the cells secrete luciferase in the supernatant. The amount of produced luciferase correlates to the level of activation. To measure the released luciferase, 20 pl of the supernatant of the activated Jurkat -Lucia cells was transferred in a new 96 well plate and 50 pl QUANTI-Luc™ 4 Reagent (Invivogen) was added, then luminescence was immediately measured atTecan reader.Real-time live-cell imaging assay

[0440] The eradication of KS cancer cells by transduced T cells was determined by real-time live-cell imaging using Incucyte SX5 live cell imager (Sartorius). 24000 KS cells were co-cultured in different E: T ratios of transduced T cells. Afterwards, Incucyte Cytotox Red Dye was added to cells (1:4000, Sartorius, # 4632). Cells were imaged for the indicated time points at a lOx magnification. Tumor cell apoptosis was quantified with the Incucyte 2021C software.

[0441] Interferon-y (IFN-y) and tumor necrosis factor-a (TNFa) ELISA

[0442] All IFN-y and TNFa ELISA experiments were performed using BD OptEIA™Human IFN-y or Human TNF ELISA Sets (BD Biosceinces), according to manufacturers instructions. In short 96 well plates were coated with 100 pL coating solution (for one plate: 44 pL capture antibody and 11 mL coating buffer (see table 9)). Plates were covered with plastic foil and incubated overnight at 4°C. After discarding the supernatant from the coated plates, the plates were washed with washing buffer, i.e. PBS-T. 100 pL BD OptEIA Assay Diluent (BD Biosciences) were added per well and the plates were incubated for 1 h in the dark at RT. As samples, supernatants from the plates used for either XTT based or luciferased based kill assays were collected and centrifuged to remove any remaining cells or cell debris. Standards were prepared according to manufacturer instruction and samples for IFN-y were diluted 1:8 with BD OptEIA Assay Diluent (BD Biosceices). Samples for TNFa ELISA were not diluted. The plates were then washed three times and 100 pL of diluted sample and standards were added in each well. The plates were covered with plastic foil and incubated for 2 h in the dark at RT. After plates were washed five times, 100 pL working solution (BD Bioscences) was added to each well and the plates were incubated for 1 h in the dark at RT. Washing was repeated seven times and 100 pL per well substrate reagent (BD Bioscences) was added. The plates were incubated without plastic foil in the dark until the standards turned blue, then 50 pL stop solution (2N H2SO4) was added in each well. The absorbance was measured at 450 nm in a Tecan Reader.

[0443] Calcium imaging

[0444] One day prior to the imaging 150 000 breast cancer cells -(KS cells) have been plated on ibi Treat p-dishes (ibidi), that were coated for 1 h at 37°C with 100 pg / ml poly D-lysine. As a control, in one dish we added 5 pg / ml soluble scFv C6 for 30 minutes. TRUCK or CAR T cells were preactivated to ensure the secretion of the scFv C6 at the time of the co-culture. To this end transduced T cells were cultured overnight on plates coated with 2 pg / ml of CD3 antibody (clone OKT3, e Bioscience) with the addition of 0,5 pg / ml CD28 antibody (clone CD28.2, Biolegend). The activated CAR T cells and TRUCKS were collected in serum free OptiMEM medium (Gibco) and were stained with lOpM Fura-2-AM dye (Life Technologies). At the time of measurement, medium was replaced with RINGER's solution. After 10 min of initial imaging of the KS cells the stained transduced TRUCK and CAR T cells were added to the target cells in effector to target ratio of 1:1. Calcium signals in the T cells were detectedby measuring the 340 / 380 nm wavelength ratio. Imaging was performed by the group of Prof. Christian Wetzel (Molecular Neuroscience, University of Regensburg).

[0445] Confocal imaging

[0446] TRUCKS expressing scFv C6 were pre-activated with a CD3 coated plate (2pg / m I), 0,5pg / ml CD28 and 1000U / ml IL-2 for 24h. Then they were collected and co-cultured with tumor cells (KS cells) at 1:1 E: T ratio for 30 min in presence of Hoechst 33342 (nuclear stain), CellBrite Steady 550 (membrane stain) and the Goat F(ab')2 Anti-Human lgG-AF647 Antibody (staining of the CAR) before image acquisition.

[0447] Images were acquired using a Leica Stellaris 8 confocal microscope equipped with HC PL APO CS2 63x / 1,20 WATER objective lens (NA 1,2). A zoom factor of 3,24 was applied and line average set to 2 to enhance signal-to- noise ratio. Hoechst was excited with a 405 nm laser at 0,67% intensity, emission was detected from 430-509 nm. In the same setting, CellBrite Steady 550 was excited by a white light laser split into a 561 nm beam at 1,19% intensity with emission being detected at 568-663 nm.ln another setting, mNG-tagged scFv C6 was excited at 504 nm with 1,42% intensity. Emission was detected at 509-561 nm. Simultaneously, the AF647-labelled antibody was excited at 653 nm and 1,45% intensity. Here the detector was set to 663-775 nm. Images were cropped and deconvoluted using the Leica Las X software. Z-stacks were processed using maximum projection to reduce dimensionality. Scale bar represents 5 pm.

[0448] Image acquisition of 310 frames took a total 629,257 seconds. It was a unidirectional scan with a scan speed of 400 Hz and a pixel dwell time of 1,575 ps.

[0449] The complete image spans has dimensions of 56,9 pm x 56,9 pm x 21,96 pm. Pixel / Voxel size in x and y is 0,056pm, in z it is 0,36pm.

[0450] Xenograft Tumor and in vivo CAR T cell treatment

[0451] For this purpose NSG (NOD. Cg-Prkdcscid / ll2rgtmlWjl / SzJ) mice were purchased from The Jackson Laboratory and housed in a specific-pathogen-free environment. For testing the in vivo effector functions of scFv C6 ErbB2 TRUCKS and ErbB2 CAR T cells, seven-week-old female NSG mice were administered a subcutaneous (s.c.) injection in both flanks, each containing 3x106N87.ffLuc cells in 100 pL of PBS mixed with an equal volume of Matrigel (BD Biosciences, San Jose, CA, USA). Tumor growth was monitored with an IVIS Spectrum CT instrument (Perkin Elmer, Waltham, MA, USA). Before measurement, isoflurane-anesthetized animals were injected IP with D-luciferin (150 mg / kg). A bioluminescence image was obtained and analysed after 10 min using Living Image software Version 4.0 (Caliper Life Sciences, Waltham, MA, USA). A region of interest of the same size was drawn over the tumor region, and the intensity of the signal measured as total photons per second per square centimeter per steradian (p / s / cm2 / sr) was obtained. Mice received a single dose of 0.25x106scFv C6 ErbB2 TRUCKS or ErbB2 CAR T cells i.v. on day 14 post-tumor cell inoculation.Example 2: Titration of the purified scFv

[0452] Functionality and specificity of the produced anti-CEACAM6 scFv was tested in a CEACAM6-specific ELISA assay (see Example 1). scFv C6 was added to a plate coated with rhCEACAM6-Fc tagged in concentrations between 2000 to 0,13 ng / ml (corresponding to 70 to 0.005 nM). The binding of the scFv C6 to the CEACAM6 was detected with anti-His-HRP conjugated antibody. Results are shown in Figure 2. The anti-CEACAM6 scFv binds to the target antigen in a dose-dependent manner. An EC50 of 5 ng / ml (0,18 nM) was determined.

[0453] Example 3: Affinity determination by surface plasmon resonance

[0454] In this experiment, the affinity of the purified scFv was determined by surface plasmon resonance. To this end rhCEACAM6 was used, which was immobilized via its Fc - tag on a protein A chip. For kinetics determination scFv C6 was used as analyte in concentrations between 0,75 to 100 nM.

[0455] Results are shown in Figure 3. The affinity (KD) was calculated as 2,5 nM. This is the same affinity range as the monovalent dissociation constant KD = 13 nM, calculated for the parental monoclonal antibody (Oncoimmunology 2021;11(1):2008110). Hence, no loss of affinity was observed due to the isolation of the variable fragment.

[0456] Example 4: The purified scFv binds to CEACAM6-positive cells, but not to CEACAM6-negative cells

[0457] Next it was tested if the produced scFv C6 also binds to CEACAM6 expressed on living cells. Therefore flow cytometry staining with scFv C6 was performed, followed by anti-His-PE antibody as a secondary antibody of CEACAM6-positive KS22.24 (KS) (Cancer Immunol Immunother. 2005; 54:129-140. ) and NCI-N87 (N87)-fLuc (Cancer Lett. 2020; 484:1-8) cells, and CEACAM6-negative T cells isolated from healthy donors as described in Example 1.

[0458] Results are shown in Figure 4. It could be confirmed that the scFv C6 binds to CEACAM6-positive cells (KS; N87), but not to CEACAM6-negative T cells (grey histograms).

[0459] Example 5: The purified scFv competes for binding with the CEACAM6-specific antibody 9A6

[0460] In this experiment it was tested if the purified scFv C6 competes for binding to CEACAM6 with another anti-CEACAM6 antibody, 9A6 (WO2016150899). For this purpose, KS or N87 cells were incubated with 40 pg / mL ofmAB 9A6 for one hour at 4°C. After washing, 15 pg / mL scFv C6 was added and incubated for 1 h at 4°C. The bound scFv C6 was detected using anti-His-PE conjugated antibody as described in Example 1. As a control, the cells were stained with the scFv C6 only without pre incubation of the mAb 9A6.

[0461] Results are shown in Figure 5. It could be confirmed that the purified scFv cross competes with antibody 9A6 as shown by the 70% reduction of MFI for KS cells and 49 % reduction of MFI for N87 cells.

[0462] Example 6: Anti-CEACAM6 antibodies trigger an increased INF-y release

[0463] The expression of CEACAM1 on Survivin-specific T cells (generated from healthy donors and expanded as described in Blood Cancer J. 2011;l(3):ell) was confirmed by staining of these cells with anti-human CEACAM1 monoclonal antibody (clone GM8G5, Enzo Life Sciences), which was subsequently detected by anti-mouse IgG-BV421 conjugated secondary antibody (Figure 6A, grey histogram). As a control staining only with the secondary antibody was used (dotted histogram). Next, 10000 Survivin-specific T cells were co-cultured with CEACAM6-expressing 10000 KS cells at effector to target (E: T) ratio of 1:1 with and without the presence of monoclonal anti-human CEACAM6 antibody (clone 9A6, Enzo Life sciences) at various concentrations (5-20 pg / ml) and isotype matched control antibody at the highest concentration (20 pg / ml)(Figure 6B), or in the presence or absence of scFv C6 in concentrations between 20 pg / ml to 0,3125 pg / ml. As a control full length antibody 9A6 and isotype matched antibody were added in concentration of 5 pg / ml. IFN-y secretion in the supernatant of the co-culture after 17 h was measured. As shown in figure 6B and 7, both the full length 9A6 antibody and scFv C6 improved significantly the IFN-g secretion in a concentration dependent manner.

[0464] Example 7: The inducible expression of scFv C6 by survivin specific T cells improves the eradication of tumor cells in real- timelive-cell imaging assay

[0465] To determine if the inducible expression of scFv C6 in T cells improves their cytotoxicity, transduced survivin specific T cells were used. These T cells carried constructs comprising either the inducible cassette of scFv C6 upstream of a constitutive expression cassette encoding tomato fluorescent protein (survivin iscFv C6 tomato), or only the cassette for tomato fluorescent protein alone (survivin tomato). Cells were sorted using fluorescence assisted cell sorting. The sorted survivin iscFv C6 tomato and survivin tomato cells were used in real-time livecell imaging assay, where the transduced survivin specific T cells were co-cultured with 24000 KS cells in an E: T ratio of 1:48 in the presence of Incucyte Cytotox Red Dye (which binds to apoptotic cells). Cells were co-cultured for 90 h and imaged every hour. The cytotoxicity was measured as the red area in the wells (μm2 / image). As a control KS and survivin tomato cells were co-cultured in the presence of 5 pg / ml of the monoclonal CEACAM6 antibody (clone9A6, mAb C6).It could be shown that the survivin scFv C6 tomato T cells eradicate the KS tumor cells more efficiently than survivin tomato T cells. See Figure 8. The presence of the mAB C6 improved cytotoxicity, but not as efficiently as the secretion of the scFv C6 directly into the immunological synapse between the T cells and tumor cells.

[0466] Example 8: The interaction of CEACAM1 and CEACAM6 is blocked by anti-CEACAM6 antibodies

[0467] To determine the ability of the scFv C6 to block the interaction between CEACAM6 and CEACAM1, an ELISA experiment was performed. Plates were coated with recombinant human His-tagged CEACAM6 and recombinant His-tagged human CEACAMl-Fc was added at a concentration of 5 pg / ml (40 nM) in the absence or presence of the scFv C6 at different concentrations (between 4 and 0,25pg / ml (130-8 nM)). Binding was detected with HRP-conjugated protein A. As depicted in Figure 9, the binding of CEACAM1 to CEACAM6 was inhibited by 80% in the presence of the scFv C6.

[0468] Example 9: Generation of CARs and TRUCKS

[0469] Two different CARs were generated, a classical CAR and a TRUCK. The molecular architecture of the constructs is shown in Figure 10.

[0470] Both constructs were transduced into T cells and the expression of the CAR and CEACAM1 was confirmed after cell enrichment via MACS using FACS as described in Example 1 and as shown on Figure 11. The CAR is expressed to similar levels (~95%) on both, TRUCKS and CAR T cells. The expression of CEACAM1 is in the range of 50%.

[0471] Example 10: CEACAM1 / CEACAM6 interaction inhibits CAR driven activation in transduced Jurkat Lucia NFAT cells and can be rescued by scFv C6

[0472] The same constructs were used to generate Jurkat -Lucia NFAT cells expressing either the CAR or the TRUCK. To confirm the relevance of CEACAM1 / CEACAM6 interaction in dampening CAR mediated activation, Jurkat cells were additionally transduced to express either CEACAM1 or deltaCEACAMl (dCEACAMl). Delta CEACAM1 contains the extracellular part of CEACAM1 but lacks the intracellular domain containing the inhibitory ITIM motifs. This construct was used as a control to confirm that the inhibitory signal through CEACAM1 ITIM is responsible for reduced signalling through the CAR. As shown on Figure 23, increasing concentrations of CEACAM6 added to ErbB2 reduced the activation of Jurkat cells through the CAR in the CEACAM1 expressing CAR Jurkat cells (as can be seen by the reduced reporter expression). The signal was reduced by half in the case when 20 pg / ml of CEACAM6 was used for coating, and nearly 80 % reduction of the signal was observed at 60 pg / ml of CEACAM6 (white bars). CAR and TRUCK Jurkat cells expressing dCEACAMl were largely resistant toCEACAM6 / CEACAM1 mediated inhibition (light grey bars and dark grey bars), with exception of the highest concentration of CEACAM6, which is likely a consequence of steric hindrance of the activation through ErbB2 due to the very high concentration of CEACAM6. However, the addition of scFv C6 (dotted bars) or expression by the TRUCK Jurkat cells (grey bars) could partially rescue the phenotype and reduce the inhibition mediated by the CEACAM6 / CEACAM1 interaction. Noteworthy, the secretion of the scFv C6 by the TRUCK Jurkat cells still reduced the negative effect of CEACAM6 at very high concentrations, whereas the addition of scFv C6 had no further effect, pointing to a beneficial effect of local secretion directly at the synapse side.

[0473] Example 11: Inducible expression of scFv C6 in activated TRUCKS

[0474] To determine the inducibility of the expression and secretion of the scFv C6 by the TRUCKS and CAR T cells, transduced cells were cultured either on plates coated with 15 pg / ml of ErbB2 (activated) or 15 pg / ml BSA (non activated). Non-transduced T cells were used as control. After 60h activation, the supernatant was collected, cells were lysed, mRNA was isolated and cDNA was generated. cDNA was then used as template for a qPCR with primers specific for the scFv C6.

[0475] As shown in Figure 12A, significant amplification was only observed with cDNAfrom activated TRUCKS. mRNA transcription of the scFv C6 can occur to a small degree prior to activation due to tonic signaling mediated by the CAR, there is however a 10 fold increase of the mRNA transcription with activated, transduced T cells. The same increase of expression could be observed for the scFv C6 protein in the supernatant of the activated transduced T cells (Figure 12B), confirming on protein level the results observed on mRNA level.

[0476] It was further confirmed that the TRUCKS are able to secrete scFv C6 in a dose dependent, antigen specific manner. For this purpose, both TRUCKS and CAR T cells were activated with plate-bound recombinant human ErbB2-Fc protein in a concentration range of 20 to 2,5 pg / ml for 60 h. After that the supernatant was collected and used to detect scFv C6 in a custom scFv C6 ELISA (as described in Example 1). The concentration of the scFv C6 was determined in comparison to a standard curve generated by dilution of purified scFv C6 with known concentration. As can be seen in Figure 13, scFv C6 is detected only in the supernatant of the activated TRUCKS, and the concentration of the secreted protein is dependent on the stimulus concentration.

[0477] Example 12: TRUCKS have a superior cytotoxic capacity and improved secretion of proinflammatory cytokines compared to CAR T cells

[0478] To determine if the secretion of the scFv C6 TRUCKS improve their cytotoxicity in comparison to T cells that only express the CAR, a cytotoxicity assay was performed using a co-culture of the TRUCKS or CAR T cells in range of effector to target ratios of 5:1 to 1:24 with 10000 ErbB2 expressing cells of cell lines KS and N87. After 17-20 h supernatants were collected to determine the presence of IFN-y and TN Fa (for KS cells), and the amount ofsurviving target cells (using a luciferase assay). Based hereon the level of T cell mediated cytotoxicity was calculated.

[0479] In Figure 14 (Panel A: KS cells, Panel B: N87 cells) a representative experiment from 5 independent experiments is depicted. It could observed that at high E: T ratios, there is not a major difference in the functionality of the TRUCKS and the CART cells, but at lower E: T ratios TRUCKS have a significantly higher capacity to kill target cells. The presence of soluble scFv C6 has little to no effect on the cytotoxicity of CART cells, showing that the secretion of the scFv C6 in the synapse and the local blockade of CEACAM1, as done by the TRUCKS, is superior in improving their functionality. In comparison to the CAR T cells, the TRUCKS also showed a significant increase in the secretion of IFN-y when cultured with KS and N87 cells (Figure 15; Panel A: KS cells, Panel B: N87 cells), and significant effect on the secretion of TNFa when TRUCKS were co-cultured with KS cells (Figure 16), thus confirming their improved functionality.

[0480] Example 13: The inducible expression of scFv C6 by TRUCKS improves the eradication of tumor cells in a real-time live-cell imaging assay

[0481] To confirm the superior functionality of the TRUCKS in eradicating tumor cells, real-time live-cell imaging was used. TRUCKS and CAR T cells were co-cultured with 24000 KS breast cancer cells in an effector to target ratio of 1:6 over 100 h in the presence of Incucyte Cytotox Red Dye (which binds to apoptotic cells) in an Incucyte livecell imager. Cells were imaged every hour and cytotoxicity was measured as the red area in the wells (μm2 / image). The scFv C6 secreting TRUCKS (black circles) eradicated the KS tumor cells more efficiently than the CAR T cells (grey squares). See Figure 17. As a control non-transduced T cells were used (grey diamonds).

[0482] Example 14 Calcium Imaging

[0483] Calcium is a secondary messenger in T cell signaling and propagates signal in waves. Due to the wave nature of the signal, it is important to measure the amplitude, frequency and intercellular propagation of calcium signals on a single-cell resolution. The fluorescent dye Fura-2-AM was used to measure intracellular calcium concentrations in transduced CART cells and TRUCKS. The ratiometric nature of this dye prevents imaging-based side-effects (e.g. photobleaching). CAR T cells and TRUCKS were labelled with Fura-2-AM and were added to KS cells in a 1:1 E: T ratio. The interaction of TRUCKS and CAR T cells with the target cells induced distinct calcium signaling peaks. The presence of scFv C6, secreted by the TRUCKS (lower graph, black lines), enhanced the calcium signal in respect to the frequency and amplitude of the peaks (Figure 18) in comparison to CAR T cells (upper graph, grey lines). Preincubating the KS cells with soluble scFv C6 prior to imaging (Figure 18, middle panel, dotted grey lines) had no effect on the calcium signaling compared to CAR T cells alone. Data are summarized in Figure 19, where the area under the curve was calculated for every imaged cell (TRUCKS co-cultured with KS cells (black circles), CAR T cells co-cultured with KS cells (grey squares), CAR T cells co-cultured with KS cells and pre-incubated with soluble scFv C6 (white / black squares)). It could be shown that calcium signaling is significantly enhanced when scFv C6 is secreted by the TRUCKS.

[0484] Example 15: Visualization of the expression of scFv C6 by TRUCKS in contact with tumor cell

[0485] To visualize the expression and the localization of scFv C6 upon T cell-tumor cell contact, TRUCKS were generated that express a fusion protein consisting of scFv C6 and mNeonGreen fluorescent protein in an inducible manner. Confocal laser scanning microscopy was used (Figure 20) to demonstrate that scFv C6-mNeonGreen (mNG) (top right) is expressed by TRUCKS intracellularly upon contact with tumor cells (membrane stain -Cell Brite Steady 555, top middle panel, upper cell = tumor cell, lower cell = TRUCK). The CAR staining (anti-hIgG -AF647, bottom left), shows the expression of the CAR and provides context for the formation of the Immunological synapse. The arrows point to the scFv C6-mNG in the TRUCKS present intracellularly, directed towards the tumor cells. Notably, scFv C6-mNG is also localized at the point of T cell-tumor cells contact within the synapse. The top left panel shows a nuclear counterstain with Hoechst33342; the bottom middle panel a merge of all channels.

[0486] Example 16: Xenograft models

[0487] To investigate the effect of the CAR and TRUCK T cells in solid tumors an in vivo xenograft mouse model was used. 2x106N87 -ffLuc expressing tumor cells were subcutaneously injected in each of the flanks of NSG mice. Tumors were allowed to grow for 14 days, then 250000 TRUCK or CAR T cells were injected intravenously into the mice. The tumor growth was followed and measured by bioluminescence for 40 additional days.

[0488] Figures 21 and 22 show representative results of two independent experiments. In Figure 21 the bioluminescence imaging of the mice over the course of the experiment is shown. Figure 22 depicts the growth curves (panel A) and survival curves (panel B). It could be observed that TRUCKS are superior to CAR T cells in controlling tumor growth, and mice treated with the TRUCK survived significantly longer.

Claims

Claims1. A recombinant cell containing a nucleic acid comprising(a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein said chimeric antigen receptor comprises,(i) an extracellular ligand-binding domain specifically binding to HER2;(ii) a transmembrane domain; and(iii) an intracellular signaling domain, and(b) a second nucleic acid sequence encoding a promoter and a scFv specifically binding to a second tumor antigen,wherein said recombinant cell expresses and secretes said second scFv when the extracellular ligandbinding domain of the chimeric antigen receptor binds to said first tumor antigen.

2. The recombinant cell according to claim 1, wherein the extracellular ligand-binding domain of said chimeric antigen receptor that specifically binding to HER2 is a scFv.

3. The recombinant cell according to claim 2, wherein said scFv that specifically binding to HER2 comprises a variable heavy domain comprising an HCDR1 of SEQ ID No. 21, an HCDR2 of SEQ ID No. 22, and an HCDR3 of SEQ ID No. 23, and comprising a variable light domain comprising an LCDR1 of SEQ ID No. 24, an LCDR2 of SEQ ID No. 25, and an LCDR3 of SEQ ID No. 26.

4. The recombinant cell according to any one of the preceding claims, wherein said transmembrane domain is or is derived from CD28, preferably wherein said transmembrane domain comprises the amino acid sequence of SEQ ID No. 30.

5. The recombinant cell according to any one of the preceding claims, wherein said intracellular costimulatory signaling domain is or is derived from CD28, preferably wherein said costimulatory signaling domain comprises the amino acid sequence of SEQ ID No. 31.

6. The recombinant cell according to any one of the preceding claims, wherein said intracellular T cell receptor signaling domain is or is derived from CD3 zeta, preferably wherein said T cell receptor signaling domain comprises the amino acid sequence of SEQ ID No. 32.

7. The recombinant cell according to any one of the preceding claims, wherein said chimeric antigen receptor (CAR) comprises a hinge region, preferably wherein said hinge region is located between the extracellular ligand-binding domain and the transmembrane domain, more preferably wherein said hinge region comprises the amino acid sequence of SEQ ID No. 33.

8. The recombinant cell according to any one of the preceding claims, wherein said chimeric antigen receptor (CAR) comprises the amino acid sequence of SEQ ID No. 34.

9. The recombinant cell according to any one of the preceding claims, wherein the scFv specifically binding to said second tumor antigen specifically binds to CEACAM6.

10. The recombinant cell according to claim 9, wherein said scFv specifically binding to CEACAM6 comprises a variable heavy domain comprising an HCDR1 of SEQ ID No. 7, an HCDR2 of SEQ ID No. 8, and an HCDR3 of SEQ ID No. 9, and comprising a variable light domain comprising an LCDR1 of SEQ ID No. 10, an LCDR2 of SEQ ID No. 11, and an LCDR3 of SEQ ID No. 12.

11. The recombinant cell according to any one of the preceding claims, wherein the promoter of said second nucleic acid is an inducible promoter, preferably an NFAT-inducible promoter.

12. The recombinant cell according to any one of the preceding claims, wherein said recombinant cell is a T cell or a NK cell, preferably a T cell.

13. A nucleic acid comprising said first nucleic acid sequence and said second nucleic acid sequence contained in the recombinant cell according to any one of the preceding claims.

14. A pharmaceutical composition comprising a recombinant cell according to claim 1-13.

15. A recombinant cell according to claim 1-12 or a pharmaceutical composition according to claim 14 for use in medicine, preferably wherein said use in medicine is the treatment of cancer or a tumor.

Images