Chimeric antigen receptor specific for folate receptor 1

Abstract

The invention provides a protein having an antigen binding domain specific for FolRl. Specific binding of the target was shown in several different applications. Depending on the application the protein of the present invention may comprise additional structural features and / or domains. Depending on the structural features the protein having an antigen binding domain specific for FolRl may be e.g. a chimeric antigen receptor, protein drug conjugate, Bispecific T cell engager (BITE) or a detection reagent.

Description

TitleChimeric antigen receptor specific for folate receptor 1Field of the invention

[0001] The invention relates to antigen binding proteins (such as antibodies or binding fragments) specific for Folate receptor alpha (FolRl). The antigen binding proteins may be used for several applications, such as for the detection and / or treatment of cancer.Background of the invention

[0002] FolRl is a membrane protein, which binds folic acid with high affinity and mediates the cellular uptake of this vitamin via receptor-mediated endocytosis2. Folate is a basic component of cell metabolism, DNA synthesis and repair. Rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis. FolRl levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression. It has been suggested that FolRl might confer a growth advantage to the tumor by modulating folate uptake from serum or by generating regulatory signals3.

[0003] FolRl expression levels are especially high in cancer such as of triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer. Especially in ovarian cancer FolRl expression plays a critical role.

[0004] 0varian cancer is defined as a group of tumors that originate in the ovaries. Ovarian cancer is the fourth most common cancer among women and is the leading cause of gynecological cancer-related death in women. Most clinical cases are epithelial ovarian cancers, which represent approximately 90% of diagnosed patients. Epithelial ovarian cancer has four main histologic subtypes: serous, endometrioid, mucinous, and clear cell. Approximately 70% of patients present with serous histology, and clinical trials have demonstrated that a patient’s subtype has prognostic importance. Other less common types of ovarian tumors include primary peritoneal, fallopian tube, and malignant germ cell tumors1.

[0005] 0varian cancer patients have an approximate overall five-year survival rate of 49.1%. As with most cancers, the five-year survival rate can differ depending on the stage at which the cancer is diagnosed. If the cancer is caught early at a localized stage (Stage I— II), the five-year survival rate is 92.6%. Ovarian cancer that has spread to a different part of the body, also known as regional ovarian cancer (Stage III), has a five-year survival rate of 74.8%. The five-year survival rate decreases further to 30.3% in metastatic (Stage IV) ovarian cancer. Most patientspresent with advanced disease at diagnosis, and despite high responses to initial treatment, the majority will eventually relapse with incurable disease1.

[0006] Immunotherapies such as the use of protein (especially antibody) drug conjugates and chimeric antigen receptors, have been shown to be effective for cancer treatment. The chimeric antigen receptor (CAR) provides a promising approach for adoptive cell immunotherapy for cancer.

[0007] Surgery in combination with chemotherapy is still standard of care. However, the majority of patients relapse and, finally, will develop resistance to chemotherapy. Thus, ovarian cancer has a strong unmet medical need and, alternative therapeutic approaches such as immunotherapy using CAR expressing cell or protein drug conjugates are urgently needed. Especially for triple-negative breast cancer, gastric cancer, lung cancer, renal cancer and pancreatic cancers a high expression of the tumor associated FolRl was identified and it therefore represents a promising target for CAR T cell therapy. There is a need in the art for an improved or alternative immunotherapeutic approaches such as CARs specific for FolRl for use in treatment of cancer expressing FolRl.

[0008] In order to find out the stage of the disease and to prognose the effectivity of an immunotherapy also the detection of cancer cells in a patients sample plays a critical role. For this preferentially a protein comprising the antigen binding domain of the protein that is used for immunotherapy (such as CAR or protein drug conjugate) should be used. However usually proteins comprising the antigen binding domain that is used for immunotherapy show poor binding properties for other applications such as imaging or flow cytometry. In most cases such a protein does not bind to patients tissue (that has been treated with fixatives) at all. So it cannot be used for the detection and prognosis of the outcome of such an immunotherapy.

[0009] Therefore there is a need in the art, for an improved or alternative protein comprising an antigen binding domain that can be used for the detection of cancer cells expressing FolRl, disease staging and prognosis of treatment success as well as for the treatment using immunotherapy such as chimeric antigen receptors or protein drug conjugates.Brief description of the invention

[0010] The cell surface antigen FolRl is expressed on cancer cells such as triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer and can be used for targeted immunotherapy. The inventors found that a protein comprising a specific antigen binding domain specific for FolRl can be used for detecting FolRl positive cells(especially cancer cells; figure 6) and simultaneously for immunotherapy such as CART cell therapy (Examples 4-5).

[0011] The first aspect of the invention is therefore a protein comprising an antigen binding domain comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of SEQ ID No. 20(GYSFTSYW), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0012] In addition to that inventors found, that a protein comprising the antigen binding domain as disclosed herein can be used for immunotherapy such as CAR T cell therapy and protein (such as antibody) drug conjugates. They showed effective killing of cancer cells by said CAR in vivo and in vitro (figure 4 and 5). Therefore one aspect of the invention is a protein, which is a chimeric antigen receptor, comprising the antigen binding domain as defined by the first aspect of the invention, a spacer domain, a transmembrane domain and an intracellular signaling domain.

[0013] In another aspect of the invention, the protein may be a protein drug conjugate (such as an antibody drug conjugate), wherein said protein comprises an antigen binding domain as described in the first aspect of the invention and a cytotoxic payload moiety. Such a construct may be used for immunotherapeutic applications.

[0014] In yet another aspect of the invention the protein may comprise the antigen binding domain as defined in the first aspect and a detection moiety. Such a protein may be used for detection of FolRl positive cells (especially cancer cells) in a sample (preferentially a patient sample). It may be used for flow cytometry and / or imaging applications. Such a method may be used for disease staging.Brief description of the drawings

[0015] Figure 1: CAR T cell binding domain candidates. 189 human / mouse cross-reactive anti-FOLRl candidate binders were identified via phage display from a human antibody library. 19 candidates were further selected via flow cytometric screening of FOLR-variant expressing cells to select for FOLR1 -specific candidates. A CAR T cell screening approach was then used to assess functionality, specificity and cell expansion. 4 candidates were selected for further in vitro analysis, identifying one lead anti-FOLRl CAR candidate.

[0016] Figure 2: Unique binder sequences with cross-reactivity between hFOLRl and mFOLRl were identified via phage display. A) Five different panning strategies were used to identify binder candidates using hFOLRl and mFOLRl antigen for positive selection, respectively. In all strategies hFOLR2, hFOLR3 and hlgGl-Fc antigens were used for competition. Compared to strategy 1, strategy 2 used a lower amount of antigen in the third panning round. B) The used phage display antibody library revealed 2266 first hits for hFOLRl and / or mFOLRl in ELISA. C) The five different selection strategies lead to the discovery of scFvs that bind preferably to the target(s) that were used in the panning strategies, e.g. panning strategies 4 and 5 selected scFvs were binding preferably to both hFOLRl and mFOLRl antigen. D) Sequencing results confirm 349 unique sequences derived from 723 processable sequences.

[0017] Figure 3: Flow cytometric screening revealed potential CAR T cell candidates in scFv-Fc format with high specificity for FOLR1, compared to other FOLR variants. Candidates were produced in scFv-Fc format by transfection of HEK293 cells. Jurkat cells expressing the respective human or mouse FOLR variant were used to test for specificity of the candidates for FOLR1. 1x105 FOLR-expressing Jurkat cells were plated and stained with 1 pg scFv-Fc for 30 minutes at 4°C. An anti-IgG(Fc)-PE secondary antibody staining was performed for 10 minutes at 4°C. Flow cytometric measurements were performed in triplicates and are displayed as % of stained cells. Candidates staining for FOLR2 or FOLR4 and with low staining for FOLR1 were excluded. Selected candidates are marked in black, non-selected candidates in grey. A) Gating strategy to determine specificity of candidates in scFv-Fc format for hFOLRl and mFOLRl, after exclusion of debris, doublets and dead cells. B) Gating strategy to determine specificity of candidates in scFv-Fc format for hFOLR2 and mF0LR2, after exclusion of debris, doublets and dead cells. C) Gating strategy to determine specificity of candidates in scFv-Fc format for hFOLR4 and mF0LR4, after exclusion of debris, doublets and dead cells. D), E) Flow cytometric results of candidates in scFv-Fc format binding to hFOLRl overexpressing Jurkat cells. F), G) Flow cytometric results of candidates in scFv-Fc format binding to mFOLRl overexpressing Jurkat cells. H), I) Flow cytometric results ofcandidates in scFv-Fc format binding to hFOLR2 overexpressing Jurkat cells. J), K) Flow cytometric results of candidates in scFv-Fc format binding to mF0LR2 overexpressing Jurkat cells. L), M) Flow cytometric results of candidates in scFv-Fc format binding to hFOLR4 overexpressing Jurkat cells. N), O) Flow cytometric results of candidates in scFv-Fc format binding to mF0LR4 overexpressing Jurkat cells.

[0018] Figure 4: CAR T cell screening reveals functional, specific anti-FOLRl CAR T cell candidates. 2x104 GFP- and FOLR1 -expressing target cells (OV-90) were seeded and cocultured with 5x104 anti-FOLRl CAR T cells to measure antigen-dependent lysis of ovarian cancer cells. After 92h of co-culture, 2x104 ovarian cancer cells (OV-90) were added in addition, followed by 5x104 ovarian cancer cells (OV-90) after 164h. In parallel, co-culture was performed with ovarian cancer cells deficient for FOLR1 (OV-90 FOLR1 KO). Target cell lysis by the CAR T cell candidates was analyzed by decrease in GFP signal over time by measuring the Green Calibrated Units per pm2 / image for 11 days. As negative controls, target cells (OV-90) were cultured without addition of CAR T cells or co-cultured with untransduced T cells. Two positive control CAR T cell constructs known to successfully induce FOLR1 specific lysis of target cells were included. IFN-y secretion was measured after 24h of each addition of ovarian cancer cells, respectively. CAR T cell expansion was assessed by flow cytometric quantification after 92h, 164h as well as 260h of co-culture, i.e. before each addition of target cells and at end-point, respectively. A), B), C) Lysis of ovarian cancer cells (OV-90) expressing FOLR1 by 19 CAR T candidates of three different donors identified to be specific for FOLR1 in the preciously performed flow-cytometric screening. D), E), F) Lysis of ovarian cancer cells deficient for FOLR1 (OV-90 FOLR1 KO) by 19 CAR T candidates of three different donors identified to be specific for FOLR1 in the preciously performed flowcytometric screening G), H), I) CAR T cell expansion of all candidates from three different donors as CD3+ LNGFR+ cells / well after 92h, 164h as well as 260h of co-culture. J), K), L) IFN-y secretion in repeated co-cultures 24h after each addition of ovarian cancer cells expressing FOLR1 (OV-90) to candidate CAR T cells. M), N), O) IFN-y secretion in repeated co-cultures 24h after each addition of ovarian cancer cells deficient for FOLR1 (OV-90 FOLR1 KO) to candidate CAR T cells. P) Workflow scheme to identify functional and specific CAR T cells in serial in vitro co-culture assays.

[0019] Figure 5: Advanced in vitro CAR T cell assays identify lead anti-FOLRl CAR T cell candidate. 2x104 GFP expressing cells were seeded and co-cultured with 1x104 anti- FOLRl CAR T cells to measure antigen-dependent lysis of ovarian cancer cells for five days. After 48h of co-culture, 2x104 ovarian cancer cells were added to the co-culture. Cell lysis bythe CAR T cell candidates was tracked by measurement of the green area confluency. As negative controls, ovarian cancer cells were cultured without addition of CAR T cells or with untransduced T cells. A positive control CAR T cell construct known to successfully induce FOLR1 specific lysis of target cells was included. The top four candidates identified by the CAR T cell screening were included in this assay, comprising three donors each. After 48h and 120h, flow cytometric measurement of activation marker expression CD 137 and CD69 was performed. In addition, activation markers on a fourth donor were measured upon co-culture with FOLRllow (OVCAR-3) expressing cells. A) CAR architecture of the four anti-FOLRl directed CAR T cell candidates consisting of a FOLR1 directed scFv, a CD8 hinge and transmembrane region, a 4-1BB co-stimulatory region, and a CD3(^ domain. B), C), D) Lysis of ovarian cancer cells expressing FOLR1 (OV-90 wt) of four CAR T candidates of three different donors identified to be functional in the previously performed CAR T cell screening. E), F), G) Lysis of ovarian cancer cells deficient for FOLR1 (OV-90 FOLR1 KO) of four CAR T candidates of three different donors identified to be functional in the previously performed CAR T cell screening. H), I), J) CD69 activation marker expression on CAR T cell candidates of three donors upon 48h and 120h co-culture with ovarian cancer cells. K), L), M) CD137 activation marker expression on CAR T cell candidates of three donors upon 48h and 120h coculture with ovarian cancer cells. N), O) CD69 and CD137 activation marker expression on CAR T candidates of three donors upon 48h and 120h co-culture with FOLRlhigh (OV-90 wt), FOLRllow (OVCAR-3) and FOLR1- (OV-90 FOLR1 KO) cells.

[0020] Figure 6: Immunofluorescence analysis of human cell lines and tissue revealed potential CAR T cell candidates in scFv-Fc format. Cells were seeded at a concentration of 1E6 (Jurkat) or 2E5 (OV90) cells per 24-well and fixed with 4% PFA before pre-staining with DAPI. Staining of each conjugate was automatically performed, the signal was erased and the sample was washed. A monoclonal antibody directed to the target of interest was used as a positive reference. Cyclic imaging of scFv-Fc-PE conjugates on the cells was performed in a concentration of 5 pg / mL. The scale bar indicates 200 pm. An 20x objective was used and the exposure time was set to 80ms A) Staining of target directed scFv-Fc-PE conjugates and a positive reference on target cells that do not express the antigen of interest (Jurkat wt) and B) quantification of the corresponding signal intensities and signal overlap with the positive reference C) Staining of target directed scFv-Fc-PE conjugates and a positive reference on cells with a transgenic knock-in of the antigen of interest (Jurkat KI) and D) quantification of the corresponding signal intensities and signal overlap with the positive reference E) Staining of target directed scFv-Fc-PE conjugates and a positive reference on cells with physiological targetexpression (Ov-90 wt) and F) quantification of the corresponding signal intensities and signal overlap with the positive reference G) Staining of target directed scFv-Fc-PE conjugates and a positive reference on cells with a CRISPR / Cas9-mediated knock-out of the antigen of interest (Ov-90 KO). I) Staining of target directed scFv-Fc-PE conjugates and a positive reference on target expressing diseased tissue (High-grade serous ovarian cancer) and J) quantification of the corresponding signal intensities and signal overlap with the positive reference.Detailed description of the inventionThe invention provides a protein having an antigen binding domain specific for FolRl . Specific binding of the target was shown in several different applications. Depending on the application the protein of the present invention may comprise additional structural features and / or domains. Depending on the structural features the protein having an antigen binding domain specific for FolRl may be e.g. a chimeric antigen receptor, protein drug conjugate (such as antibody drug conjugate), Bispecific T cell engager (BITE) or a detection reagent. The following paragraphs define the structural features of the protein needed for each application / construct.Protein comprising an antigen binding domain specific for FolRl

[0021] In a first aspect, the present invention provides a protein comprising an antigen binding domain (specific for FolRl) comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20) , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDSRECTIFIED SHEET (RULE 91) ISA / EP(SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0022] Said protein may comprise said heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and said light chain variable region of an antibody (VL) which may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42.

[0023] In one embodiment of the invention said protein comprising said antigen binding domain may comprise a heavy chain variable region of an antibody (VH) comprising the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No:35.

[0024] In one embodiment of the invention said protein comprising said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80

[0025] According to this aspect of the invention, the protein comprises an antigen binding domain. Any molecule that binds specifically to a target antigen (FolRl) may be used as an antigen binding domain. The antigen binding domain may be or may comprise, for example an antibody or antigen binding fragment thereof (e.g. Fab fragments or single chain Fv (scFv) fragments), VHH fragments, divalent single chain antibodies or diabodies. In a preferred embodiment of the invention, the antigen binding domain may be an antibody or antigen binding fragment thereof. In a preferred embodiment of the invention the antigen binding domain may be a scfv.

[0026] In one embodiment of the invention the protein comprising said antigen binding domain (preferentially a scfv) may comprise at least one additional domain which may be selected from the group of: cytotoxic moiety, detection moiety and Fc terminus. In one embodiment of the invention the protein comprising said antigen binding domain (preferentially an scfv) may comprise fragment crystallizable region of an antibody (Fc domain).

[0027] In another embodiment of the invention the protein comprising said antigen binding domain (preferentially an scfv) may be a chimeric antigen receptor (CAR) comprising saidantigen binding domain specific for FoLRl, a spacer domain, a transmembrane domain and an intracellular signaling domain.Chimeric antigen receptor specific for FolRl

[0028] In the second aspect, the invention provides a protein, which is a chimeric antigen receptor (CAR) comprising said antigen binding domain specific for FoLRl, a spacer domain, a transmembrane domain and an intracellular signaling domain.

[0029] Any molecule that binds specifically to the given antigen may be used as antigen binding domain. Exemplary antigen binding domains may be or may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. Preferentially the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4 / S)3-linker” or a “whitlow linker, preferentially a “(G4 / S)3-linker”.

[0030] Said CAR comprises the antigen binding domain (e.g. scfv) as defined by the first aspect of the invention. In other words the protein, of this aspect of the invention, is a chimeric antigen receptor (CAR) comprising an antigen binding domain (e.g. scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20) , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).[003 l]The protein( which is a CAR) comprises said antigen binding domain (e.g. scfv), wherein said antigen binding domain may comprise a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42.

[0032] Said protein (which is a CAR) comprises said antigen binding domain (e.g. scfv), wherein said antigen binding domain may comprise a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 35.

[0033] In one embodiment of this aspect of the invention said protein which is a CAR comprises said antigen binding domain (e.g. scfv), wherein said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80.

[0034] As defined in this aspect of the invention said protein (which is a CAR) may comprise a hinge domain as spacer, wherein the hinge domain may comprise e.g. a sequence of the hinge of CD8alpha (SEQ ID No:2) or IGG4 (SEQ ID No:72). In a preferred embodiment the CAR may comprise the hinge domain of CD8alpha, the hinge domain of CD8alpha may comprise SEQ ID NO:2.

[0035] In other words the protein, described in this aspect of the invention, is a chimeric antigen receptor (CAR) comprising an antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region (LCDR3) having the amino acid sequenceQVWDSSSDPNYV (SEQ ID No. 38) and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.

[0036] Said protein (which is a CAR) may comprise said antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42 and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.

[0037] Said protein (which is a CAR) may comprise said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No:35 and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.

[0038] In one embodiment of this aspect of the invention said protein which is a CAR and comprises said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80 and the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2.

[0039] As defined in this aspect of the invention said protein, which is a CAR may comprise a transmembrane domain comprising a sequence of the transmembrane domains from CD8alpha (SEQ ID No:4) or CD28 (SEQ ID No:76), preferentially CD8alpha (SEQ ID No:4).

[0040] In other words the protein, of this aspect of the invention, (which is a CAR) may comprise an antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domainand an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence of SEQ ID No. 37 (DDS) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38), the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2 and the transmembrane domain of CD8alpha, preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NO:4.

[0041] Said protein, (which is a CAR) may comprise said antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42, the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2 and the transmembrane domain of CD8alpha, preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NO:4.

[0042] Said protein (which is a CAR) may comprise said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variableregion of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 35, the hinge domain of CD8alpha, preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2 and the transmembrane domain of CD8alpha, preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON.

[0043] In one embodiment of the invention said protein (which is a CAR) comprises intracellular signaling domains e.g. a sequence of the intracellular signaling domains of CD3zeta (SEQ ID No:8) and / or one or more of CD28 (SEQ ID NO:78), CD137 (41BB; SEQ ID N0:6) and 0X40 (SEQ ID NO:74). In a preferred embodiment the CAR may comprise or consist of a transmembrane domain of CD8alpha, an intracellular primary (stimulatory) signaling domain of CD3 zeta and an intracellular costimulatory signaling domain of CD137 (41BB).

[0044] In other words the protein, of this aspect of the invention may comprise or consist of an antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38), the hinge domain of CD8alpha (preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2), the transmembrane domain of CD8alpha (preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON) and an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.

[0045] Said protein (which is a CAR) may comprise or consist of said antigen binding domain (preferentially an scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising anamino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42; the hinge domain of CD8alpha (preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2), the transmembrane domain of CD8alpha (preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON) and an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.

[0046] Said protein (which is a CAR) may comprise or consist of said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) which may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) which may comprise the amino acid sequence SEQ ID No: 35 and the hinge domain of CD8alpha (preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2), the transmembrane domain of CD8alpha (preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON) and an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.

[0047] In one embodiment of this aspect of the invention said protein (which is a CAR) may comprise or consist of said antigen binding domain (preferentially a scfv), a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said antigen binding domain may comprise the amino acid sequence SEQ ID No: 80; the hinge domain of CD8alpha (preferentially the hinge domain of CD8alpha may comprise SEQ ID NO:2), the transmembrane domain of CD8alpha (preferentially the transmembrane domain of CD8alpha may comprise SEQ ID NON) and an intracellular signaling domain comprising the costimulatory domain of CD137 and stimulatory domain of CD3zeta.

[0048] In one embodiment of the invention said protein (which is a CAR) may comprise or consist of the amino acid sequence SEQ ID No:44.

[0049] This aspect of the invention further provides isolated nucleic acid sequences which encode the FolRl-CARs as disclosed herein (especially for this aspect of the invention).

[0050] The nucleic acid sequence encoding the CAR as disclosed herein may be contained in a vector, such as a viral vector. The vector may be a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, adenoviral vector, or a retrovirus vector, or a combination thereof. In a preferred embodiment, said nucleic acid sequence encoding the CAR as disclosed herein may be contained in a lentiviral vector.

[0051] In some embodiments of the invention, the vector further comprises a promoter wherein the promoter is an inducible promoter, a tissue specific promoter, a constitutive promoter, a suicide promoter or any combination thereof.

[0052] In another embodiment of the invention, the vector encoding the CAR can be further modified to include one or more operative elements to control the expression of CAR T cells, or to eliminate CAR-T cells by virtue of a suicide switch. The suicide switch can include, for example, an apoptosis inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the vector expressing the CAR can be further modified to express an enzyme such thymidine kinase (TK) or cytosine deaminase (CD).

[0053] This aspect of the invention further provides a composition comprising a lentiviral vector comprising a nucleic acid sequence coding for the FolRl-CAR as disclosed herein (especially for this aspect of the invention). Said composition may be used in immunotherapy. Said composition may be used for treatment of a disease such as cancer, autoimmune disease or infections disease.

[0054] This aspect of the invention further provides a composition for use in a method of treating a subject in need thereof comprising administering a lentiviral vector comprising a nucleic acid sequence coding for the FolRl-CARs as disclosed herein (especially for this aspect of the invention).

[0055] This aspect of the invention further provides an engineered cell expressing said protein which is CAR specific for FolRl as disclosed herein (especially in this aspect of the invention).

[0056] Said cell may be an immune cell. In a preferred embodiment of the invention said cell may be a T cell, tumor infiltrating lymphocytes (TILs) or NK cell. In a more preferred embodiment of the invention said cell is a T cell.

[0057] This aspect of the invention further provides an engineered cell expressing said protein which is a FolRl CAR as disclosed herein (especially in this aspect of the invention) for use in immunotherapy. The immunotherapy may be for treatment of cancer in a subject suffering from cancer, wherein the cancerous cells of said cancer express FolRl. The immunotherapy may be for treatment of cancer in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.

[0058] Said subpopulation of cancerous cells expressing FolRl may comprise at least one cell which expresses FolRl out of all cancerous cells in the subject suffering from said cancer. Preferentially said subpopulation of cancerous cells expressing FolRl may comprise at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% of all cancerous cells of a subject suffering from said cancer.

[0059] This aspect of the invention further provides a population of cells comprising cells expressing said protein which is a FolRl -CAR as disclosed herein (especially in this aspect of the invention).

[0060] Said population or isolated population of engineered cells may be expanded to therapeutically effective amount of cells before use in said immunotherapy. Said cancer may be selected from the group consisting of triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer. Said cancer may be ovarian cancer. Said cells may be immune cells or immune cell subsets, preferentially T cell, tumor infiltrating lymphocytes (TILs) or NK cell, more preferentially T-cells.

[0061] This aspect of the invention further provides a method for treating cancer comprising administering to a subject in need thereof engineered cells expressing a protein which is a FolRl -CAR as disclosed herein (especially in this aspect of the invention). The treatment of cancer may be in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.

[0062] This aspect of the invention further provides a composition comprising genetically modified cells expressing a CAR specific for the antigen FoLRl as disclosed herein (especially in this aspect of the invention) for use in treatment of disease. Such a disease may be selected from the group of cancer, autoimmune disease and infectious diseases. In one embodiment said composition may be used for the treatment of cancer (FolRl expressing cancer). In anotherembodiment said composition may be used for the treatment of cancer in a subject in need thereof.

[0063] This aspect of the invention further provides a pharmaceutical composition comprising genetically modified cells expressing a CAR specific for the antigen FoLRl as disclosed herein (especially in this aspect of the invention) and optional a pharmaceutically acceptable carrier.

[0064] Pharmaceutical acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0065] This aspect of the invention further provides a composition for use in a method of treating a subject in need thereof comprising administering genetically modified cells expressing a CAR specific for the antigen FoLRl as disclosed herein (especially in this aspect of the invention).Adapter CAR

[0066] The third aspect of the invention provides a composition comprising the protein as disclosed in the first aspect of the invention, wherein said composition comprises said protein which is a tagged protein, and an anti-Tag CAR comprising an antigen binding domain specific for said tag of said tagged protein, a transmembrane domain and an intracellular signaling domain.

[0067] In other words this aspect of the invention provides a composition comprising the protein as disclosed in the first aspect of the invention, wherein said protein is a tagged protein, and an anti-Tag CAR comprising an antigen binding domain specific for said tag of said tagged protein, a transmembrane domain and an intracellular signaling domain. Said tagged protein comprises an antigen binding domain (specific for FolRl) comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determiningregion (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38)

[0068] The antigen binding domain of said tagged protein may comprise said heavy chain variable region of an antibody (VH) which may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) may comprise a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42.

[0069] In one embodiment of this aspect of the invention said tagged protein may comprise said antigen binding domain comprising a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35.

[0070] In one embodiment of the invention said tagged protein may comprise said antigen binding domain and the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80.

[0071] The tagged protein may be any protein or protein fragment that specifically binds to a given antigen. Exemplary such proteins may be: an antibody, antigen binding fragment of an antibody, scfv, heterologous antibody (fusion scfv-IGG) VHH fragments, divalent single chain antibodies or diabodies or alike. In a preferred embodiment said tagged protein may be an antibody or an antigen binding fragment. In another embodiment antigen binding domain is fused to an Fragment crystallizable region of an antibody (Fc domain)

[0072] As described above, the tagged protein may comprise a tag. Said tag may be selected from a variety of known tags, proteins and molecules. However, it is essential that the tag canbe bound / recognized by the antigen binding domain of the anti Tag CAR. Said tag may be or may comprise parts of a protein. It may be selected from standard tags commonly known in the art. Exemplary tags are e.g. c-Myc-tag, Strep-Tag II, Flag-Tag, Polyhistidine-tag, Avi-Tag, Calmodulin binding protein-Tag, Yol-tag (derived from alpha-tubulin), E-tag, HA-Tag, S-tag, SBP-tag or V5 tag. Moreover other tags may be chosen such as biotin.

[0073] The composition of this aspect of the invention comprises two parts: the tagged protein and the anti-tag CAR. Said anti-tag CAR comprises an antigen binding domain specific for said tag of said tagged protein, a transmembrane domain and an intracellular signaling domain.

[0074] The antigen binding domain needs to be specific for the tag of the tagged protein. The antigen binding domain may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. Any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain. Often the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4 / S)3-linker” or a “whitlow linker.

[0075] The anti-tag CAR as disclosed herein may comprise a spacer domain (also known as hinge domain). Exemplary spacer domains may be selected from the group of CD8alpha and IGG4

[0076] In addition to that the anti-tag CAR may comprise a transmembrane domain, which may be selected from the group of CD28 or CD8alpha. In a preferred embodiment the transmembrane domain may be CD8alpha.

[0077] Moreover the anti-Tag CAR may comprise an intracellular signaling domain e.g. a sequence of the intracellular signaling domains of CD3zeta (SEQ ID No: 8) and / or one or more of CD28 (SEQ ID NO:78), CD137 (41BB; SEQ ID NO:6) and 0X40 (SEQ ID NO:74). In a preferred embodiment the CAR may comprise or consist of a transmembrane domain of CD8alpha, an intracellular primary (stimulatory) signaling domain of CD3 zeta and an intracellular costimulatory signaling domain of CD137 (41BB) and CD28.Protein drug conjugate

[0078] One aspect of the invention provides a protein, which is a protein drug conjugate (such as an antibody drug conjugate) comprising an antigen binding domain as described in the first aspect of the invention and a cytotoxic payload moiety.

[0079] In other words the protein, which is a protein drug conjugate comprises an antigen binding domain (specific for FolRl) and a cytotoxic payload moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38)

[0080] Said protein (protein drug conjugate) which comprises an antigen binding domain and a cytotoxic payload moiety, wherein said antigen binding domain comprises wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42.

[0081] In one embodiment of the invention said protein (which is a protein drug conjugate) may comprise said antigen binding domain and said cytotoxic payload moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35.

[0082] In one embodiment of the invention said protein (which is a protein drug conjugate) may comprise said antigen binding domain and said cytotoxic payload moiety, the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80

[0083] Any molecule that binds specifically to FolRl may be used as an antigen binding domain. The antigen may be or may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. In a preferred embodiment the antigen binding domain may be an antibody or antigen binding fragment.

[0084] In another embodiment said antigen binding domain (such as an antigen binding fragment of an antibody or an scfv) may be fused to an Fragment crystallizable region of an antibody (Fc domain). Said fusion protein may be generated by methods known in the art. Exemplary said fusion protein may be generated by enzymatic or chemical fusion. In another embodiment said fusion protein may be generated by recombinant protein expression.

[0085] In one embodiment of the invention, the cytotoxic payload moiety may be a chemotherapeutic agent (e.g. as described by Fu et al4). Said cytotoxic payload moiety (OR chemotherapeutic agent) is selected from the group of DNA-alkylating agents, Topoisomerase 1 inhibitors, Topoisomerase 2 inhibitors, Transcription inhibitors, Bcl-xL inhibitors, Tyrosine kinase inhibitors, DHFR inhibitors, Microtubule inhibitors or DNA-targeting agents. The cytotoxic domain may be fused to the protein by methods known in the art such as click chemistry. Exemplary the one of the following methods may be used / exemplary methods are disclosed in Fu et al4.

[0086] Said protein drug conjugate as disclosed in this aspect of the invention may be used for immunotherapy

[0087] This aspect of the invention further provides a method for treating cancer comprising administering to a subject in need thereof said protein drug conjugate disclosed herein (in this aspect of the invention). The treatment of cancer may be in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.

[0088] This aspect of the invention further provides a pharmaceutical composition comprising a protein drug conjugate for the antigen FoLRl as disclosed herein (especially in this aspect of the invention) and optional a pharmaceutically acceptable carrier.

[0089] Pharmaceutical acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Bispecific T cell engager (BiTE)

[0090] 0ne aspect of the invention provides a protein, which is bispecific T cell engager (BITE) comprising the antigen binding domain (first antigen binding domain) as disclosed in the first aspect of the invention and at least one additional antigen binding domain (second antigen binding domain), wherein the target of said additional antigen binding domain is specific for an antigen other than FolRl .

[0091] In other words this aspect of the invention provides a protein comprising a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), wherein said first antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20) , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38) and wherein said second antigen binding domain is specific for an antigen other than FolRl.

[0092] Said protein which comprises a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), wherein said antigen binding domain comprises wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) comprises a firstframework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42 and wherein said second antigen binding domain is specific for an antigen other than FolRl.

[0093] In one embodiment of the invention said protein may comprise a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35 and wherein said second antigen binding domain is specific for an antigen other than FolRl.

[0094] In one embodiment of the invention said protein may comprise a first antigen binding domain (specific for FolRl) and at least one additional antigen binding domain (second antigen binding domain), the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80 and wherein said second antigen binding domain is specific for an antigen other than FolRl.

[0095] Any molecule or any variants thereof that bind specifically to a target antigen such as FolRl or to an antigen other than FolRl , may be used as an antigen binding domain. The antigen binding domain may be or may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. In a preferred embodiment the antigen binding domain may be an antibody or antigen binding fragment. In another embodiment said antigen binding domains (such as an antigen binding fragment of an antibody or an scfv) may be fused to an Fragment crystallizable region of an antibody (Fc domain).

[0096] Said additional antigen binding domain (second antigen binding domain) which is specific for an antigen / target other than FolRl may be CD19, CD20, CD33, CD123, FcRH5, FLT3, BCMA, GPRC5D, PSMA, EGFRvIII, DLL3, MUC17 or CLDN18.2.

[0097] Both antigen binding domains may be fused by methods known in the Art. Exemplary methods are disclosed in Kujawski et al5.

[0098] Said protein drug conjugate may be used for immunotherapy.

[0099] This aspect of the invention further provides a method for treating cancer comprising administering to a subject in need thereof said protein drug conjugate as disclosed in this aspect of the invention. The treatment of cancer may be in a subject suffering from cancer, wherein at least a subpopulation of the cancerous cells of said cancer express FolRl.

[0100] This aspect of the invention additionally provides a pharmaceutical composition comprising protein drug conjugate for the antigen FoLRl as disclosed herein and optional a pharmaceutically acceptable carrier.

[0101] Pharmaceutical acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Detection of FolRl positive cells

[0102] In another aspect of the invention the protein comprising the antigen binding domain, as described in the first aspect of the invention, may comprise a detection moiety.

[0103] In other words the protein, comprises an antigen binding domain (specific for FolRl) and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20)SEQ , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).

[0104] Said protein, which comprises an antigen binding domain and a detection moiety, may comprise an antigen binding domain comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL), wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an aminoacid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42

[0105] In one embodiment of the invention said protein, may comprise said antigen binding domain and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35.

[0106] In one embodiment of the invention said protein comprising said antigen binding domain and a detection moiety, the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80

[0107] As described above the protein comprises an antigen binding domain and a detection moiety. Said detection moiety may be a fluorescent moiety. Said detection moiety may be selected from the group of fluorescent dyes, fluorescent proteins or biotin.

[0108] In one embodiment the protein comprising the antigen binding domain may be fused to an Fc terminus by standard methods known in the art. This Fc terminus may serve as detection moiety.

[0109] Examples of fluorescent dyes are small organic molecule dyes, such as xanthene dyes, like fluorescein, or rhodamine dyes, coumarine dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyl oxazole dyes, pyromethene dyes, acridine dyes, oxadiazole dyes, carbopyronine dyes, benzpyrylium dyes, fluorene dyes, or metallo-organic complexes, such as Ru, Eu, Pt complexes. Besides single molecule entities, clusters of small organic molecule dyes, fluorescent oligomers or fluorescent polymers, such as polyfluorene.

[0110] Additionally, fluorescent moi eties might be protein-based, such as phycobiliproteins, nanoparticles, such as quantum dots, upconverting nanoparticles, gold nanoparticles, dyed polymer nanoparticles. Commonly known fluorescent proteins may be used such as GFP, RFP, YFP and variants thereof.

[0111] Said detection moiety may be directly covalently coupled or coupled via a linker unit.

[0112] Any molecule that binds specifically to a target antigen (specific for FolRl) may be used as an antigen binding domain. The antigen may be or may comprise, for example Fabfragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. In a preferred embodiment the antigen binding domain may be an antibody or antigen binding fragment. In another embodiment said antigen binding domains (such as an antigen binding fragment of an antibody or an scfv) may be fused to an Fragment crystallizable region of an antibody (Fc domain).

[0113] Said protein as disclosed in this aspect of the invention, comprising said antigen binding domain and a detection moiety, may be used in a method for the detection of cells (preferentially cancer cells) expressing FolRl (figure 6). Said method of detecting FolRl expressing cells (especially cancer cells) in a biological sample may comprise the steps:A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein according to this aspect of the invention, wherein said protein comprises a detection moietyB) Detecting said protein bound to said FolRl expressing cells

[0114] In other words this aspect of the invention provides a method for detecting FolRl expressing cells (especially cancer cells) in a biological sample, comprising the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) , wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20) , a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38).B) Detecting said protein bound to said FolRl expressing cells1

[0115] Said method for detecting FolRl expressing cells (especially cancer cells) in a biological sample may comprise the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, wherein said antigen binding domain comprises wherein said heavy chain variable region of an antibody (VH) may comprise a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No. 42 and a detection moiety; B)Detecting said protein bound to said FolRl expressing cells

[0116] In one embodiment the invention provides a method detecting FolRl expressing cells (especially cancer cells) in a biological sample, comprising the steps: a) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, said protein, comprising said antigen binding domain and a detection moiety, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) may comprise the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No: 35; b) Detecting said protein bound to said FolRl expressing cells

[0117] In one embodiment the invention provides a method for detecting FolRl expressing cells (especially cancer cells) in a biological sample, comprising the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein comprising an antigen binding domain (specific for FolRl) and a detection moiety, wherein said, the antigen binding domain may comprise the amino acid sequence SEQ ID No: 80; B) Detecting said protein bound to said FolRl expressing cells.

[0118] In a preferred embodiment said antigen binding domain is fused to an Fragment crystallizable region of an antibody (Fc domain) and said detection moiety is a fluorescent moiety or biotin, more preferentially a fluorescent moiety.

[0119] Said method may be a method for immunofluorescence or flow cytometric analysis. Specific protocols and methods are known in the art as disclosed in Kinkhabwala et al6and McKinnon7.

[0120] In the first step a sample comprising FolRl expressing cells (especially cancer cells) is contacted with said protein comprising said antigen binding domain and a detection moiety. Detection may be done directly, if the detection moiety is e.g. a fluorescent moiety or indirectly using e.g. if the detection moiety is a biotin moiety . In this case one additional protein e.g. an antibody comprising a fluorescent detection moiety and which is specific for biotin may be used.

[0121] A sample which may be used for the method disclosed in this aspect of the invention may be a tissue sample. This tissue sample may be human. The sample may be or may be derived from a cancer patient. Therefore the sample may comprise folate receptor 1 expressing cancer cells. Moreover the sample may be a sample of: of triple-negative breast cancer, gastric cancer, lung cancer, renal cancer, pancreatic cancer or ovarian cancer. Said sample may be a sample of ovarian cancer.

[0122] Depending on the detection method used the sample may be a tissue slice (e.g. for imaging applications) or the sample may be present in a dissociated form e.g. as single cell solution (e.g. for detection methods based on flow cytometry).

[0123] For optimal detection a tissue slice may be 5-20pm preferentially 8pm. The tissue may be fixed according to methods known in the art. Standard fixation methods are based on formaldehyde, formalin or acetone.

[0124] In immunofluorescent applications Step b) may be done using a standard fluorescent microscope. For detection of single cells by flow cytometry standard instruments such as the MACS Quant analyzer may be used.

[0125] After detection cells may be sorted by fluorescence activated cell sorting (FACS).

[0126] This aspect of the invention further provides a method for detecting FolRl expressing cancer cells and subsequent tumor treatment. The method may comprise the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein according to this aspect of the invention, wherein said protein comprises a detection moiety; B) detecting said protein bound to said FolRl expressing cells and D) Administering to a subject a therapeutically effective amount of a protein such as a CAR, BITE or a protein drug conjugate as disclosed herein.

[0127] This aspect of the invention further provides a method for detecting FolRl expressing cancer cells and prognosing the probability of a subject to respond to said proteins as disclosed herein. The method may comprise the steps: A) contacting a sample comprising FolRl expressing cells (especially cancer cells) with a protein according to this aspect of the invention, wherein said protein comprises a detection moiety; B) Detecting said protein bound to said FolRl expressing cells, Step C) assigning a score to the detection step (B), wherein the score is assigned based on comparison to one or more reference samples. Step D) Comparing the score in step (C) to the score of one or more reference samples wherein a score for the cancer FOLR1 level that is greater than the score for a normal or low FOLR1 expressing reference sample or a score for the cancer FOLR1 level that is equal to or greater than the score for a high FOLR1 expressing reference sample identifies the cancer as likely to respond to the immunotherapeutic treatment based on a CAR, BITE or a protein drug conjugate as disclosed herein.

[0128] Data obtained from step (b) may be used for scoring and for determining, whether an immunotherapy using this specific conjugate would be successful for treatment. Such a score may be based on the amount and frequency of cells detected as well as the expression profiles and staining intensities.

[0129] For scoring a value is assigned based on the comparison of reference samples.

[0130] If the scoring value is equal or bigger (preferentially bigger) compared to the score of the reference sample, then the cancer sample comprises cancer cells. The individual from which the sample was obtained is likely to respond to an immunotherapy based on a CAR, BITE or a protein drug conjugate as disclosed herein.

[0131] Reference sample may be standard tissue such as Non-neoplastic tissue, e.g. ovary, kidney, lung, cerebellum.

[0132] All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.Definitions

[0133] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0134] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0135] The term folate receptor 1, FolRl, FOLR, FOLR1, and FRl can be used interchangeably. It is understood that this also includes other state of the art synonyms such as folate receptor alpha FRalpha or FRa. Folate-binding protein, FBP or Neurodegeneration due to cerebral folate transport deficiency, NCFTD.

[0136] An antigen binding domain refers to a region of a protein (generally the variable region of an antibody or fragment thereof) that specifically binds to an antigen. It is composed of one constant and one variable domain of each of the heavy and the light chain. Key elements and most important for specific binding to an antigen are the complementarity-determining regions (CDRs) and framework region (FR) regions.

[0137] Complementarity-determining regions (CDRs) are part of the variable chains in antibodies, where these molecules bind to their specific antigen. CDRs are crucial for specific binding of the antigen binding protein.

[0138] A framework region (FR) is a subdivision of the variable region of the antibody. The framework regions are responsible for acting as a scaffold for the complementarity determining regions (CDR).

[0139] Chimeric antigen receptors (CARs) are recombinant receptors for antigens which redirect the specificity and function of T lymphocytes and / or other immune cells in a single molecule. The concept of using CARs in immunotherapy is that CARs, which are programmed targeting tumor-associated antigens or disease associated antigens, can be replicated rapidly and homogeneously.

[0140] In general, a CAR may comprise an extracellular domain (extracellular part) comprising an antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker or spacer. The extracellular domain may also comprise a signal peptide.

[0141] A "signal peptide" refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0142] Generally, an antigen binding domain of a CAR refers to the region that specifically binds to an antigen, e.g. to a tumor associated antigen (TAA), tumor specific antigen (TSA) or a disease associated antigen. The CARs of the invention may comprise one or more antigenbinding domains (e.g. a tandem CAR). Generally, the targeting regions of the CAR are localized extracellularly. The antigen binding domain may comprise an antibody, single domain antibody or an antigen binding fragment thereof. The antigen binding domain may comprise, for example Fab fragments, single chain Fv (scFv) fragments, VHH fragments, divalent single chain antibodies or diabodies. Often the antigen binding domain is a scFv. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4 / S)3 -linker” or a “whitlow linker”.

[0143] “ Spacer” or “hinge” as used herein refers to the hydrophilic region which is between the antigen binding domain of the CAR and the transmembrane domain. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge or IgG4 hinge.

[0144] The “transmembrane domain” of the CAR may be from any desired natural or synthetic source for such domain. When the source is natural the domain may be from any membranebound or transmembrane protein. The transmembrane domain may be for example from CD8alpha or CD28.

[0145] The cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR). "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions. Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and coreceptors that initiate signal transduction following antigen receptor engagement.

[0146] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co- stimulatory signalingdomain). Therefore, an intracellular signaling domain of a CAR may comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0147] Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs). Examples of ITAM containing primary cytoplasmic signaling domains often used in CARs are that those from TCRzeta (CD3zeta), FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence from CD3zeta.

[0148] The cytoplasmic domain of the CAR may be designed to comprise the CD3zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3zeta chain portion and a co-stimulatory signaling region (domain). The co-stimulatory signaling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples for a co-stimulatory molecule are CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3.

[0149] The cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo- or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet.

[0150] In some embodiments of the invention the antigen binding domain of a CAR binds a tag or hapten that is coupled to a protein or polypeptide (“haptenylated” or “tagged” polypeptide / Protein), wherein the polypeptide may bind to a disease-associated antigen such as a tumor associated antigen (TAA) that may be expressed on the surface of a cancer cell, herein FolRl.

[0151] Such a CAR may be referred to as “anti -tag” CAR or “adapterCAR” or “universal CAR” as disclosed e.g. in US9233125B2.

[0152] The haptens or tags may be coupled directly or indirectly to a polypeptide (the tagged polypeptide), wherein the polypeptide may bind to said disease associated antigen expressed on the (cell) surface of a target, e.g. FolRl. The tag may be e.g. dextran or a hapten such as biotin or fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or thiamin, but the tag may also be a peptide sequence e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide. The tag may also be streptavidin. The tag portion of the taggedpolypeptide is only constrained by being a molecule that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR. For example, when the tag is FITC (Fluorescein isothiocyanate), the tag-binding domain may constitute an anti-FITC scFv. Alternatively, when the tag is biotin or PE (phycoerythrin), the tag-binding domain may constitute an anti-biotin scFv or an anti-PE scFv, respectively.

[0153] The term “tagged protein” or “tagged polypeptide” as used herein refers to a polypeptide that has bound thereto directly or indirectly at least one additional component, i.e. the tag. The tagged polypeptide as used herein is able to bind an antigen expressed on a target cell. The polypeptide may be an antibody or antigen binding fragment thereof that binds to an antigen expressed on the surface of a target cell such as a tumor associated antigen (TAA) on a cancer cell. The polypeptide of the tagged polypeptide alternatively may be a cytokine or a growth factor or another soluble polypeptide that is capable of binding to an antigen of a target cell. The terms “adapter” or “adapter molecule” or “tagged polypeptide” as used herein may be used interchangeably. The tag may be e.g. a hapten or dextran and the hapten or dextran may be bound by the antigen binding domain a CAR comprising an antigen binding domain specific for the tag. Haptens such as e.g. FITC, biotin, PE, thiamin, streptavidin or dextran are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.

[0154] But the tag may also be a peptide sequence e.g. chemically or recombinantly coupled to the polypeptide part of the tagged polypeptide. The peptide may be selected from the group consisting of c-Myc-tag, Strep-Tag, Flag-Tag, and Polyhistidine-tag. The tag may also be streptavidin. The tag portion of the tagged polypeptide is only constrained by being a molecular that can be recognized and specifically bound by the antigen binding domain specific for the tag of the CAR. For example, when the tag is FITC (Fluorescein isothiocyanate), the tagbinding domain may constitute an anti-FITC scFv. Alternatively, when the tag is biotin or PE (phycoerythrin), the tag-binding domain may constitute an anti-biotin scFv or an anti-PE scFv.

[0155] The term “engineered” as used herein can refer to one or more human-designed alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome. An “engineered cell” or “genetically modified cell” can refer to a cell with an added, deleted and / or altered gene. Especially, the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells, preferentiallyhuman T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.

[0156] The engineered cell expressing a CAR may be further modified by genetic engineering using methods well known in the art e.g. Meganucleases, TALEN, CrisprCas, zink finger nucleases, shRNA and / or miRNA. Said cells may be modified to reduce or lack expression of a specific gene, which is normally expressed in the cell e.g. T cell receptor (TCR), MHC, co- inhibitory molecules like PD-1, CTLA-4, BTLA, TIGIT, Tim-3, CD244, LAIR, Lag-3, CD160, HVEM. Said cells may be modified to express additional transgenes such as therapeutic controls, cytokines and / or fragments, cytokine receptors and / or fragments, cytokine receptor fusion proteins, costimulatory receptors or armoring molecules.

[0157] The terms “immune cell” or “immune effector cell” may be used interchangeably and refer to a cell that may be part of the immune system and executes a particular effector function such as T cells, alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, monocytes or macrophages. Preferentially these immune cells are human immune cells. Preferred immune cells are cells with cytotoxic effector function such as T cells, alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells, macrophages or gamma-delta T cells. Most preferred immune effector cells are T cells and NK cells. "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.

[0158] The term "antibody" as used herein is used in the broadest sense to cover the various forms of antibody structures including but not being limited to monoclonal and polyclonal antibodies (including full length antibodies), single domain antibodies, multispecific antibodies (e.g. bispecific antibodies), antibody fragments, i.e. antigen binding fragments of an antibody, immunoadhesins and antibody-immunoadhesin chimeras, that specifically recognize (i.e. bind) an antigen.

[0159] "Antigen binding fragments" comprise a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof (“an antigen binding fragment of an antibody”). Examples of antigen binding fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), VHH fragments, diabodies, dsFv, Fab’, diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0160] The terms “having specificity for”, “specifically binds” or “specific for” with respect to an antigen-binding domain of an antibody, of a fragment thereof or of a CAR refer to an antigenbinding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

[0161] As used herein, the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, haptens and combinations thereof, for example a glycosylated protein or a glycolipid. The term “antigen” as used herein refers to a molecular entity that may be expressed on the surface of a target cell and that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0162] The term “epitope” means the part of an antigen, e.g. a soluble antigen, that may be recognized and specifically bound by antibodies or antigen bindings fragments thereof (antigen binding domains).

[0163] The tumor associated antigen (TAA) as used herein refers to an antigenic substance produced by tumor cells. Tumor associated antigens are useful tumor or cancer markers in identifying tumor / cancer cells with diagnostic tests and are potential candidates for use in cancer therapy. Preferentially, the TAA may be expressed on the cell surface of the tumor / cancer cell, so that it may be recognized by the antigen binding receptor as disclosed herein.

[0164] The term “target cell” as used herein refers to cell which expresses an antigen on its cell surface that should be recognized (bound) by the protein according to the current invention. Said target cell may be e.g. a cancerous cell or a cell associated with an autoimmune disease or a cell associated with an infectious disease

[0165] Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies.

[0166] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0167] The terms “therapeutically effective amount” or “therapeutically effective population” mean an amount of a cell population which provides a therapeutic benefit in a subject.

[0168] As used herein, the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the individual is a human. The subject may be a subject suffering from a disease such as cancer.

[0169] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.

[0170] The term "isolated" is used herein to indicate that the polypeptide, nucleic acid or host cell exist in a physical milieu distinct from that in which it occurs in nature. For example, the isolated polypeptide may be substantially isolated (for example enriched or purified) with respect to the complex cellular milieu in which it naturally occurs, such as in a crude extract.

[0171] The term “cancer” is known medically as a malignant neoplasm. Cancer is a broad group of diseases involving unregulated cell growth and includes all kinds of leukemia. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans.

[0172] The term Antibody-drug conjugate (ADC) is a molecule that includes an antibody (or antigen-binding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent. ADCs can be used to specifically target a drug to cancer cells through specific binding of the antibody to a tumor antigen expressed on the cell surface. Exemplary drugs for use with ADCs include of DNA-alkylating agents, Topoisomerase 1 inhibitors, Topoisomerase 2 inhibitors, Transcription inhibitors, Bcl-xL inhibitors, Tyrosine kinase inhibitors, DHFR inhibitors, Microtubule inhibitors or DNA-targeting agents.

[0173] Bispecific T cell engager or BiTE is an antigen binding molecule. It comprises a single chain polypeptide and can bind to two different antigens.

[0174] Autoimmune diseases are a condition arising from autoimmunity or disbalance in the immune homeostasis resulting in pathologies that can affect multiple different organ systems. Examples include Behcet’s disease, Juvenile idiopathic arthritis, Type 1 diabetes, Rheumatoid arthritis, Wegener Granulomatosis, Systemic lupus erythematosus, Systemic sclerosis, Crohn's disease, Graves' disease, Hashimoto thyroiditis, Goodpasture syndrome, Primary biliary cholangitis, Myasthenia gravis, Dermato polymyositis, Vasculitis, Mixed connective tissue disease, Scleroderma, Multiple sclerosis, Psoriasis, Ulcerative colitis and Uvetis.

[0175] Infection (infectious disease) is the invasion of an organism's body tissues by diseasecausing agents, their multiplication, and the reaction of host tissues to the infectious agents and the toxins they produce. Infections are caused by infectious agents (pathogens) including: viruses, bacteria, fungi and parasites. Said infection may be an acute or a chronic infection.

[0176] The term “administering” refers to local and systemic administration, e.g., including enteral, parenteral, pulmonary, and topical / transdermal administration. The administration may be directly intratumoral. Routes of administration for pharmaceutical ingredients include, e.g., oral administration, nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery, intrathecal administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, or subcutaneous administration to a subject. Administration can be by any route including parenteral and transmucosal (e.g, oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic and intracranial.Examples

[0177] The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.

[0178] The anti-FOLRl CAR T candidate selection process consisted of a an initial phage display selection, followed by a flow cytometric screening, to ensure specificity of the candidates to FOLR1 compared to other FOLR variants. Following, a CAR T screening approach and advanced in vitro co-culture assays allowed to identify a novel, functional and specific anti-FOLRl CAR candidate (Fig 1).Example 1 : Identification of antibody fragments

[0179] Two human naive scFv phage display libraries constructed at Miltenyi Biotec were used for the identification of novel binders against FOLR1. These libraries differed in the variable domains of the light chain, they contained either the K or the X isotype. For panning, mouse FOLR1 (mFOLRl), human FOLRl-hlgGlFc (hFOLRl-Fc) and human FOLR2 (hFOLR2) were purchased from Aero Biosystems. Human FOLR3 was purchased from R&D systems, human IgGl-Fc (hlgGlFc) was purchased from amsbio. Five different panning strategies were used to identify binder candidates using hFOLRl and mFOLRl antigen for positive selection, respectively. In all strategies hFOLR2, hFOLR3, and hlgGl-Fc antigens were used for competition (Fig. 2 A). For panning, each library was incubated on antigens immobilized on Maxi Sorb ELISA Plates (Nunc). After washing, scFv phage particles were eluted using trypsin. Eluted phage particles were used to infect E. coli TGI and phage production was facilitated using M13K07 helper phage. After the third panning round eluted phage particles were used to infect A. coli TGI and single cell colonies were inoculated the next day for expression of scFvs.

[0180] Expressed scFvs were analyzed for their capability to bind hFOLRl-Fc, mFOLRl, hFOLR2 or hlgGlFc in ELISA. This lead to the identification of 2266 first hits that bind either hFOLRl and / or mFOLRl and do not bind hFOLR2 or hlgGlFc (Fig. 2B). Separating the ELISA hits by panning strategy it became obvious, that binder selected in strategies without mFOLRl in the selection process indeed recognized preferably hFOLRl over mFOLRl. On the other hand panning strategy 3 with selection on mFOLRl revealed binder binding preferably to mFOLRl over hFOLRl as expected. Panning strategies using both hFOLRl and mFOLRl lead to the selection of scFvs that are cross-reactive against both targets (Fig. 2C).

[0181] 0ut of the 2266 first hits, scFv genes were sequenced for 817 clones. Alignment of the scFv genes revealed that 349 out of 817 clones contain unique scFv sequences. From these unique sequences, 189 clones were cloned as scFv-Fc fusion proteins into mammalian expression vectors (Fig. 2D). After expression and purification, 94 of these scFv-Fc proteins were used for analysis by flow cytometry.Example 2: Expression and purification of anti-FOLRl scFv-Fc candidates

[0182] Potential anti-FOLRl scFv-Fc candidates were manufactured via transient transfection of HEK293 cells. On the day of transfection, HEK293 cells were seeded at a density of 2xl06cells per well in a total volume of 800 pl expression medium in a 96 deep well plate. Next, transfection reagent was mixed with diluted plasmid DNA, encoding for the respective anti- FOLRl scFv-Fc candidate and added to the cells after an incubation period of 10 minutes. Cellswere incubated at 37°C and 8% CO2 on an orbital shaker. Supernatants were collected on day 4 after transfection and scFv-Fc candidates were purified using Protein A PhyTip® columns (Biotage) in combination with an automated workstation (Beckman Coulter). Purity was confirmed via SDS page and protein concentrations were determined using a microplate reader by measuring UV absorbance at 280 nm. Purified proteins were stored at -20°C until further processing.Example 3: Flow cytometric screening of anti-FOLRl scFv-Fc candidates

[0183] Anti-FOLRl scFv-Fc candidates were manufactured as described in example 2 and protein concentrations were normalized. For flow cytometric validation, non-target cells were labeled with CellTrace™ Violet or Cell Trace™ FarRed (ThermoFisher) and IxlO5cells per cell line were seeded into a 96-well plate. Cells were pelleted at 1200 g for 2 min at 4°C and stained with 50 pL staining mixture containing 1 pg scFv-Fc. Staining was performed for 30 min at 4°C followed by two successive washing steps with 150 pL PBS / 2 mM EDTA / 0.5% BSA (referred to as PEB, 1200 g, 2 min). Next, 50 pl of secondary staining mixture comprising the AffinitPure F(ab')2 Fragment Goat Anti-Human IgG-PE (Jackson ImmunoResearch) as well as 7-AAD as dead cell marker (Miltenyi Biotec) was added to the cells. Staining was performed for 10 min at 4°C followed by two successive washing steps with 150 pL PEB. Cells were resuspended in 50 pL PEB for subsequent flow cytometric analysis at the MACSQuant® Analyzer 10 (Miltenyi Biotec). Cells were gated on size, singularity and viability before distinguishing between the respective murine or human FOLR variant (hFOLRl SEQ ID 45, 51, 52, hFOLR2 SEQ ID 46, 53, 54, hFOLR3 SEQ ID 47, 48, 55, 56, hFOLR4 SEQ ID 49, 50, 57, 58, mFOLRl SEQ ID 59, 60, 65,66, mFOLR2 SEQ ID 61, 62, 67, 68, mFOLR4 SEQ ID 63, 64, 69, 70) expressing cell line via CellTrace (Fig. 3A-C). Staining of the respective FOLR positive target cell line with the anti-FOLRl scFv-Fc candidate was displayed as frequency (%) of stained cells. Out of 94 anti-FOLRl scFv-Fc candidates, which have been successfully produced and purified, 65 candidates showed a positive result (frequency > 15%) on hFOLRl+Jurkat cells in flow cytometric analysis (Fig. 3D,E). Out of those, 58 candidates in addition showed a positive result (frequency > 15%) on mF0LRl+Jurkat cells (Fig. 3 F,G). 19 out of those 58 candidates showed unspecific staining (frequency > 5%) on mouse FOLR2+Jurkat cells, whereas no staining was observed on hFOLR2+, mFOLR4+and hFOLR4+cells (Fig. 3H- O). The remaining 39 candidates were then selected further by excluding candidates binding to Jurkat wt cells and having a signal / noise ratio of <10 on hFOLRl+and mF0LRl+cells. Aiming for anti-FOLRl scFv-Fc candidates that show specific binding for both human and mouseFOLR1+Jurkat cells, but no signal on h / m FOLR2+as well as h / m FOLR4+Jurkat cells, in total 19 candidates (black bars) were selected for further analysis.Example 4: Identification of chimeric antigen receptors (CARs) for FOLR1 -expressing target cell recognition by CAR-transgenic T cells.

[0184] Human naive single chain variable fragment (scFv) were cloned into lentiviral vector constructs with scFv - human CD8alpha hinge (SEQ ID 1, 2) - CD8alpha transmembrane domain (SEQ ID 3, 4) - human 41BB costimulatory domain (SEQ ID 5,6) - human CD3 zeta domain configuration (SEQ ID 7, 8). The scFv contained a (648)3 linker domain (SEQ ID 9, 10). Lentiviral vectors contained a truncated human low-affinity nerve growth factor receptor (ALNGFR) expression cassette separated by a 2A element for co-expression as transduction marker. Lentiviral particles were used to transduce primary human T cells of three independent donors for CAR expression. Frequency and absolute numbers of CAR T cells were determined by quantification of CD3+LNGFR+events using flow cytometry. CAR T cells were cultured at an effector-to-target ratio of 2.5 to 1 with FOLR1 -expressing or FOLR1 KO OV-90 cells, which were transduced to express GFP. As controls, CAR T cells were cultured in absence of target cells, and OV-90 or OV-90 FOLR1 KO cells were cultured in absence of CAR T cells. Additionally, OV-90 and OV-90 FOLR1 KO cells were cultured with untransduced T cells or with T cells expressing one of two positive control CARs, respectively. After 24h cell-culture supernatants were analyzed for IFN-y secretion. After 92h CAR T cells (CD3+LNGFR+) were quantified by flow cytometry. In addition, fresh OV-90 and OV-90 FOLR1 KO cells were added according to the experimental set-up to initiate the second round of co-culture (Fig. 4P). 24h later, supernatants were again analyzed for IFN-y secretion. After 164 h, CAR T cells (CD3+LNGFR+) were quantified by flow cytometry. The third round of co-culture was started by addition of fresh OV-90 and OV-90 FOLR1 KO cells according to the experimental set-up. 24h later supernatants were again analyzed for IFN-y secretion. After 260 h, CAR T cells (CD3+LNGFR+) were quantified by flow cytometry. The total duration of three rounds of co-culture was 11 days, and cytotoxicity towards OV-90 and OV-90 FOLR1 KO cells, respectively, was quantified every 2h by acquisition of the GFP signal over time by measuring the Green Calibrated Units per pm2 / image.

[0185] Three consecutive rounds of co-culture of CAR T cells with OV-90 target cells allowed to identify CAR T candidates, which were able to mediate killing of FOLR1 -expressing cells (Fig. 4A). Candidates FOLR1 CAR 8 and 19 (SEQ ID NOs: 88 and 98) failed to be expressed and were excluded from further analysis. FOLR1 CAR 5, 7, 12, 16, 18 (SEQ ID NOs: 85, 87,80, 95 and 97) efficiently lysed tumor cells over three rounds of co-culture. These functional CAR T candidates were confirmed in co-cultures of OV-90 cells with CAR T cells of two additional donors (Fig. 4B, C). In contrast, candidates FOLR1 CAR 6, 9, 10, 11, 13, 15, 17, 20, 21 (SEQ ID NOs: 86, 89, 90, 91, 92, 94, 96, 99 and 100) were either least or not functional after three rounds of co-culture from at least one donor. Co-cultures of CAR T cells with OV-90 FOLR1 KO cells allowed to identify CAR T candidates, which mediated cytotoxicity in the first and up to the third round of co-culture (Fig. 4D-F), i.e. antigen-independent cytolysis. Candidates FOLR1 CAR 16 lysed tumor cells unspecifically in the absence of FOLR1.

[0186] CAR T cell expansion was observed for four CAR constructs, i.e. FOLR1 CAR 5, 7, 12, 18, for all three donors in co-cultures with OV-90 cells at the end of the first and the third round, i.e. numbers were exceeding the initial amount ofCD3+LNGFR+T cells plated at day 0 (Fig. 4 G-I). This was also true for T cells expressing the CAR, i.e. FOLR1 CAR 16„ which mediated cytotoxicity towards OV-90 FOLR1 KO cells. Proliferation was not measurable in at least one donor for candidates FOLR1 CAR 1, 2, 3, 4, 6, 9, 10, 11, 13, 14, 15, 17, 20, and 21 (SEQ ID NOs: 81, 82, 83, 84, 86, 89, 90, 91, 92, 93, 94, 96, 99 and 100).Expression of these four functional CARs by T cells, i.e. FOLR1 CAR 5, 7, 12, and 18 was associated with secretion of IFN-y in each round ofc o-culture with OV-90 cells (Fig. 4 J-L). In contrast, IFN-y was not measurable in at least one donor for candidates FOLR1 CAR 1, 2, 3, 4, 6, 9, 10, 11, 13, 14, 15, 17, 20, and 21. The CAR candidate, which induced T cell-mediated cytotoxicity in OV-90 FOLR1 KO co-cultures, also induced IFN-y secretion in each round of co-culture with OV-90 and OV-90 FOLR1 KO cells, respectively (Fig. 4 M-O). In sum, in vitro co-cultures allowed to select for CARs that equipped T cells with the ability to mediate repeated killing, cytokine production as well as expansion upon target cell encounter while sparing target antigen KO cells, namely FOLR1 CAR 5, 7, 12, 18.Example 5: Generation and identification of a lead chimeric antigen receptor (CAR) for FOLR1 -expressing target cell recognition.

[0187] The four lead CAR T candidates (FOLR1 CAR5, FOLR1 CAR7, FOLR1 CAR12 and FOLR1 CAR18) that were identified in the previous CAR T screening approach were further tested in more advanced in vitro assays. CAR constructs were generated lacking the LNGFR reporter by cloning human naive FOLR1 directed scFvs into lentiviral vector constructs together with a CD8alpha hinge (SEQ ID 1,2), a CD8alpha transmembrane domain (SEQ ID 3,4), a human 41BB costimulatory domain (SEQ ID 5,6) and a human CD3 zeta domain linker(SEQ SEQ ID 7,8) (Fig. 5A). Heavy and light chain of the scFv were linked via a (G4S)s linker (SEQ ID 9,10). Lentiviral particles were produced via transfection of HEK293T cells and were harvested from cell culture supernatants. To determine the functional titer of the different lentiviral particles, SUP-T1 cells were transduced with varying amounts of lentiviral vector and transduction efficiency was determined via flow cytometry by detection of the CAR using biotinylated FOLRl-Fc protein. All four candidate CARs were expressed at high and comparable levels.

[0188] To validate functionality and specificity of the CAR T candidates, CAR T cells of three different donors were co-cultured with repeatedly added GFP-expressing target cells (OV-90 wt) or non-target cells (OV-90 FOLR1 KO) at an initial effector-to-target ratio of 0.5 to 1. As controls, OV-90 cells were cultured without T cells as well as with untransduced T cells. In addition, positive control CAR T cells were co-cultured with OV-90 cells, known to specifically and efficiently induce lysis of FOLR1 -expressing cells. After 48h, fresh OV-90 cells were added to the co-culture. OV-90 cell lysis causes a decrease in GFP signal, whereas inefficient or no lysis increases GFP signal over time. OV-90 cell lysis was measured for 120h and is displayed as green area confluency [%]. All CAR T candidates from all three donors efficiently eradicate OV-90 cells expressing FOLR1 after two additions of OV-90 cells, slightly outperforming the positive control (Fig. 5B-D). Additionally, all CAR T candidates from all three donors do not eradicate OV-90 FOLR1 KO cells (Fig. 5E-G).

[0189] 48h and 120h after co-culture set-up, the expression of activation markers CD137 and CD69 was measured on the CAR T candidates via flow cytometry. CAR T cells of all three donors were efficiently activated as shown in increased CD69 and CD 137 expression compared to untransduced T cells. While activation profiles between candidates are similar after 48 of coculture, 120h co-culture results show stronger differences between candidates. FOLR1 CAR 18 and FOLR1 CAR 7 show lower activation marker expression compared to FOLR1 CAR 5 and FOLR1 CAR 12 after co-culture with OV-90 cells expressing FOLR1. CD137 activation marker expression of all candidates was comparable to untransduced T cells after co-culture with OV-90 FOLR1 KO cells (Fig. 5K-M). However, CD69 expression of FOLR1 CAR 18 and FOLR1 CAR 7 is elevated compared to FOLR1 CAR 5, FOLR1 CAR 12 and the positive control CAR T cells after co-culture with OV-90 FOLR1 KO cells (Fig. 5 H-J).

[0190] In another co-culture experiment, anti-FOLRl CAR T candidates were co-cultured with FOLRllow(OVCAR-3) expressing cells and compared to OV-90 wt (FOLRlhlgh) and OV-90 FOLR1 KO cells. CD69 and CD137 activation marker expression of CAR T candidates was increased after 48h of co-culture with OV-90 cells expressing FOLR1, while minimal activationof all CAR T candidates was observed after co-culture with OV-90 FOLR1 KO cells. Although OVCAR-3 cells only displayed 15% FOLR1 expression, the positive control CAR T cells and FOLR1 CAR 12 show higher expression of the activation markers CD69 and CD137, compared to untransduced T cells and other candidates (Fig. 5N, O).

[0191] Taken together, all four tested CAR T candidates are functional and specific in vitro. As FOLR1 CAR 12 was activated by FOLRllowexpressing target cells and showed superior activation after co-culture with FOLRlhlghexpressing cells compared to other candidates, FOLR1 CAR 12 was nominated as lead candidate (SEQ ID 11-44).Example 6: Immunofluorescence detection of FOLR1 expression in human cell lines and tissue using anti-FOLRl scFv-Fc candidates.

[0192] The four lead CAR T candidates (FOLR1 CAR5, FOLR1 CAR7, FOLR1 CAR12 and FOLR1 CAR18) that were identified in the previous CAR T screening approach were further characterized for their capability to detect FOLR1 expressing cells via immunofluorescence analysis. The scFv-Fc fusion proteins as described in example 3 were used. Moreover, the candidates were directly labeled with a fluorescent dye namely PE.

[0193] In a first step Jurkat cells which do not express FOLR1 were analyzed with the PE- labeled scFv-Fc fusion proteins (Fig. 6A). Here, specificity of the anti-FOLRl scFv-Fc fusion proteins was assessed and detect no significant signal of the candidates relative to the positive and negative control antibodies (Fig. 6B).

[0194] Secondly, Jurkat cells were transduced to express FOLR1 and were analyzed with the PE-labeled scFv-Fc fusion proteins (Fig. 6C). Here, specificity of the anti-FOLRl scFv-Fc fusion proteins was assessed and detect increased signals of the candidates relative to the negative control antibody (Fig. 6D) and relative to Jurkat cells not expressing FOLR1 (Fig. 6B). Moreover, the signal of the scFv-Fc candidates co-localizes with the positive control.

[0195] Thirdly, OV-90 cells expressing FOLR1 were analyzed with the PE-labeled scFv-Fc fusion proteins (Fig. 6E). Here, specificity of the anti-FOLRl scFv-Fc fusion proteins was assessed and detect elevated signals of the candidates relative to the negative control antibody (Fig. 6F).

[0196] Next, OV-90 cells were modified via CRISPR / CAS9 to ablate endogenous FOLR1 expression. Subsequently, these OV-90 FOLR1 KO cells were analyzed with the PE-labeled scFv-Fc fusion proteins (Fig. 6G). Here, specificity of the anti-FOLRl scFv-Fc fusion proteins was assessed and detect no significant signal of the candidates relative to the positive andnegative control antibodies. Moreover, the signal of the scFv-Fc candidates co-localizes with the positive control.

[0197] Lastly, the PE-labeled anti-FOLRl scFv-Fc fusion proteins were used to analyze primary human ovarian cancer (Fig. 61). Here, specificity of the anti-FOLRl scFv-Fc fusion proteins was assessed and detect significant signal of the candidates relative to the positive and negative control antibodies (Fig. 6J).[001981Description of the sequences of the sequence protocolSEQ ID NO 1 : Human CD8 alpha hinge nucleotide sequence SEQ ID NO 2: Human CD8 alpha hinge amino acid sequence SEQ ID NO 3 : Human CD8alpha transmembrane domain nucleotide sequence SEQ ID NO 4: Human CD8alpha transmembrane domain amino acid sequence SEQ ID NO 5: Human 4 IBB costimulatory domain nucleotide sequence SEQ ID NO 6: Human 41BB costimulatory domain amino acid sequence SEQ ID NO 7: Human CD3 zeta domain nucleotide sequence SEQ ID NO 8: Human CD3 zeta domain amino acid sequence SEQ ID NO 9: (G4S)3 linker nucleotide sequence SEQ ID NO 10: (G4S)3 linker amino acid sequence SEQ ID NO 11 : FOLR1 CAR 12 scFv Vh nucleotide sequence SEQ ID NO 12: FOLR1 CAR 12 scFv Vh CDR1 nucleotide sequence SEQ ID NO 13: FOLR1 CAR 12 scFv Vh CDR2 nucleotide sequence SEQ ID NO 14: FOLR1 CAR 12 scFv Vh CDR3 nucleotide sequence SEQ ID NO 15: FOLR1 CAR 12 scFv Vh FR1 nucleotide sequence SEQ ID NO 16: FOLR1 CAR 12 scFv Vh FR2 nucleotide sequence SEQ ID NO 17: FOLR1 CAR 12 scFv Vh FR3 nucleotide sequence SEQ ID NO 18: FOLR1 CAR 12 scFv Vh FR4 nucleotide sequence SEQ ID NO 19: FOLR1 CAR 12 scFv Vh amino acid sequence SEQ ID NO 20: FOLR1 CAR 12 scFv Vh CDR1 amino acid sequence SEQ ID NO 21 : FOLR1 CAR 12 scFv Vh CDR2 amino acid sequence SEQ ID NO 22: FOLR1 CAR 12 scFv Vh CDR3 amino acid sequence SEQ ID NO 23: FOLR1 CAR 12 scFv Vh FR1 amino acid sequence SEQ ID NO 24: FOLR1 CAR 12 scFv Vh FR2 amino acid sequence SEQ ID NO 25: FOLR1 CAR 12 scFv Vh FR3 amino acid sequence SEQ ID NO 26: FOLR1 CAR 12 scFv Vh FR4 amino acid sequence SEQ ID NO 27: FOLR1 CAR 12 scFv VI nucleotide sequence SEQ ID NO 28: FOLR1 CAR 12 scFv VI CDR1 nucleotide sequence SEQ ID NO 29: FOLR1 CAR 12 scFv VI CDR2 nucleotide sequence SEQ ID NO 30: FOLR1 CAR 12 scFv VI CDR3 nucleotide sequence SEQ ID NO 31 : FOLR1 CAR 12 scFv VI FR1 nucleotide sequence SEQ ID NO 32: FOLR1 CAR 12 scFv VI FR2 nucleotide sequence SEQ ID NO 33: FOLR1 CAR 12 scFv VI FR3 nucleotide sequence SEQ ID NO 34: FOLR1 CAR 12 scFv VI FR4 nucleotide sequence SEQ ID NO 35: FOLR1 CAR 12 scFv VI amino acid sequence SEQ ID NO 36: FOLR1 CAR 12 scFv VI CDR1 amino acid sequence SEQ ID NO 37: FOLR1 CAR 12 scFv VI CDR2 amino acid sequence SEQ ID NO 38: FOLR1 CAR 12 scFv VI CDR3 amino acid sequence SEQ ID NO 39: FOLR1 CAR 12 scFv VI FR1 amino acid sequenceRECTIFIED SHEET (RULE 91) ISA / EPSEQ ID NO 40: FOLR1 CAR 12 scFv VI FR2 amino acid sequence SEQ ID NO 41 : FOLR1 CAR 12 scFv VI FR3 amino acid sequence SEQ ID NO 42: FOLR1 CAR 12 scFv VI FR4 amino acid sequence SEQ ID NO 43: FOLR1 CAR 12 nucleotide sequence SEQ ID NO 44: FOLR1 CAR 12 amino acid sequence SEQ ID NO 45: Human FOLR1, Pl 5328, aa25-235 SEQ ID NO 46: Human FOLR2, P14207, aal7-230 SEQ ID NO 47: Human FOLR3 Isoform 1, P41439, aa23-245 SEQ ID NO 48: Human FOLR3 Isoform2, P41439-4, aa23-172 SEQ ID NO 49: Human FOLR4 Isoform 1, A6ND01, aa20-228 SEQ ID NO 50: Human FOLR4 Isoform2, A6ND01-2, aa20-243 SEQ ID NO 51 : recombinant hFOLRl nucleotide sequence including 3x HA tag SEQ ID NO 52: recombinant hFOLRl amino acid sequence including 3x HA tag SEQ ID NO 53: recombinant hFOLR2 nucleotide sequence including 3x HA tag SEQ ID NO 54: recombinant hFOLR2 amino acid sequence including 3x HA tag SEQ ID NO 55: recombinant hFOLR3 nucleotide sequence including 3x HA tag and hFOLRl GPI-anchor domainSEQ ID NO: 56: recombinant hFOLR3 amino acid sequence including 3x HA tag and hFOLRl GPI-anchor domainSEQ ID NO 57 recombinant hFOLR4 nucleotide sequence including 3x HA tag SEQ ID NO 58 recombinant hFOLR4 amino acid sequence including 3x HA tag SEQ ID NO 59 recombinant mFOLRl nucleotide sequence including 3x HA tag SEQ ID NO 60 recombinant mFOLRl amino acid sequence including 3x HA tag SEQ ID NO 61 recombinant mFOLR2 nucleotide sequence including 3x HA tag SEQ ID NO 62 recombinant mFOLR2 amino acid sequence including 3x HA tag SEQ ID NO 63 recombinant mFOLR4 nucleotide sequence including 3x HA tag SEQ ID NO 64 recombinant mFOLR4 amino acid sequence including 3x HA tag SEQ ID NO 65 mFOLRl nucleotide sequence, 14275 SEQ ID NO 66 mFOLRl amino acid sequence, P35846 SEQ ID NO 67 mFOLR2 nucleotide sequence, 14276 SEQ ID NO 68 mFOLR2 amino acid sequence, Q05685 SEQ ID NO 69 mFOLR4 nucleotide sequence, 64931 SEQ ID NO 70 mFOLR4 amino acid sequence, Q9EQF4 SEQ ID NO 71 Human IgG4 hinge nucleotide sequence SEQ ID NO 72 Human IgG4 hinge amino acid sequence SEQ ID NO 73 0X40 intracellular domain nucleotide sequence SEQ ID NO 74 0X40 intracellular domain amino acid sequence SEQ ID NO 75 CD28 transmembrane nucleotide sequence SEQ ID NO 76 CD28 transmembrane amino acid sequence SEQ ID NO 77 CD28 intracellular domain nucleotide sequence SEQ ID NO 78 CD28 intracellular domain amino acid sequence SEQ ID NO 79 FOLR1 CAR 12 scFv nucleotide sequence SEQ ID NO 80 FOLR1 CAR 12 scFv amino acid sequence SEQ ID NO 81 FOLR1 CAR 1 scFv sequence SEQ ID NO 82 FOLR1 CAR 2 scFv sequence SEQ ID NO 83 FOLR1 CAR 3 scFv sequence SEQ ID NO 84 FOLR1 CAR 4 scFv sequence SEQ ID NO 85 FOLR1 CAR 5 scFv sequence SEQ ID NO 86 FOLR1 CAR 6 scFv sequence SEQ ID NO 87 FOLR1 CAR 7 scFv sequence SEQ ID NO 88 FOLR1 CAR 8 scFv sequenceSEQ ID NO: 89: FOLR1 CAR 9 scFv sequence SEQ ID NO: 90: FOLR1 CAR 10 scFv sequence SEQ ID NO: 91 : FOLR1 CAR 11 scFv sequence SEQ ID NO: 92: FOLR1 CAR 13 scFv sequence SEQ ID NO: 93: FOLR1 CAR 14 scFv sequence SEQ ID NO: 94: FOLR1 CAR 15 scFv sequence SEQ ID NO: 95: FOLR1 CAR 16 scFv sequence SEQ ID NO: 96: FOLR1 CAR 17 scFv sequence SEQ ID NO: 97: FOLR1 CAR 18 scFv sequence SEQ ID NO: 98: FOLR1 CAR 19 scFv sequence SEQ ID NO: 99: FOLR1 CAR 20 scFv sequence SEQ ID NO: 100: FOLR1 CAR 21 scFv sequence

[0199] References1. Hansen T., Datamonitor Healthcare, Informa Pharma Intelligence, Ovarian cancer disease analysis, Ref Code: DMKC0215129, 20212. Leamon CP, Low PS, 1991, PNAS; Kandalaft et al., 2012, J Transl Med3. Kelemen, 2006, Int. J. Cancer4. Fu, Z., Li, S., Han, S. et al. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Sig Transduct Target Ther 7, 93 (2022). https : / / doi .org / 10.1038 / s41392-022-00947-75. Kujawski M, Li L, Bhattacharya S, Wong P, Lee WH, Williams L, Li H, Chea J, Poku K, Bowles N, Vaidehi N, Yazaki P, Shively JE. Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy. BMC Cancer. 2019 Sep 5;19(I):882. doi: 10.1186 / sl2885-019-6056-8. PMID: 31488104; PMCID: PMC6727398.6. Kinkhabwala A, Herbel C, Pankratz J, Yushchenko DA, Riiberg S, Praveen P, ReiB S, Rodriguez FC, Schafer D, Kollet J, Dittmer V, Martinez-Osuna M, Minnerup L, Reinhard C, Dzionek A, Rockel TD, Borbe S, Buscher M, Krieg J, Nederlof M, Jungblut M, Eckardt D, Hardt O, Dose C, Schumann E, Peters RP, Miltenyi S, Schmitz J, Muller W, Bosio A. MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors. Sci Rep. 2022 Feb 3; 12(1): 1911. doi: 10.1038 / s41598-022-05841-4. PMID: 35115587; PMCID: PMC8813936.7. McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018 Feb 21;120:5.1.1-5.1.11. doi: 10.1002 / cpim.40. PMID: 29512141; PMCID: PMC5939936.

Claims

Claims1. A protein comprising an antigen binding domain specific for FolRl comprising a heavy chain variable region of an antibody (VH) and a light chain variable region of an antibody (VL) ,- wherein said heavy chain variable region of an antibody (VH) comprises a first complementarity determining region (HCDR1) comprising an amino acid sequence of GYSFTSYW (SEQ ID No. 20), a second complementarity determining region (HCDR2) comprising the amino acid sequence IYPGDSDT (SEQ ID No. 21) and a third complementarity determining region (HCDR3) comprising the amino acid sequence ARRKGPHYGSGAIFDY (SEQ ID No. 22) and- wherein said light chain variable region of an antibody (VL) comprises a first complementarity determining region (LCDR1) comprising an amino acid sequence NIGSKS (SEQ ID No. 36), a second complementarity determining region (LCDR2) comprising the amino acid sequence DDS (SEQ ID No. 37) and a third complementarity determining region( LCDR3) having the amino acid sequence QVWDSSSDPNYV (SEQ ID No. 38)2. A protein according to claim 1 wherein said heavy chain variable region of an antibody (VH) comprises a first framework region (HFR1) comprising an amino acid sequence of SEQ ID No. 23, a second framework region (HFR2) comprising an amino acid sequence of SEQ ID No. 24, a third framework region (HFR3) comprising an amino acid sequence of SEQ ID No. 25 and a fourth framework region (HFR4) comprising an amino acid sequence of SEQ ID No. 26 and wherein said light chain variable region of an antibody (VL) comprises a first framework region (LFR1) comprising an amino acid sequence of SEQ ID No. 39, a second framework region (LFR2) comprising an amino acid sequence of SEQ ID No. 40, a third framework region (LFR3) comprising an amino acid sequence of SEQ ID No. 41 and a fourth framework region (LFR4) comprising an amino acid sequence of SEQ ID No.

423. A protein according to any of the claims 2-3, wherein said antigen binding domain comprises a heavy chain variable region of an antibody (VH) comprising the amino acid sequence SEQ ID No: 19 and a light chain variable region of an antibody (VL) comprising the amino acid sequence SEQ ID No:

354. A protein according to any of the claims 1-3, wherein said antigen binding domain comprises the amino acid sequence SEQ ID No:

805. A protein according to any of the claims 1-4, wherein said protein is a chimeric antigen receptor (CAR) comprising said antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain6. A protein according to claim 5, wherein said spacer domain comprises the hinge domain of CD8 alpha.

7. A protein according to any of the claims 5-6, wherein said CAR comprises the transmembrane domain of CD8 alpha and wherein said intracellular signaling domain comprises the costimulatory domain of CD137 and the stimulatory domain of CD3zeta.

8. A protein according to any of the claims 5-7, wherein said CAR comprises the amino acid sequence SEQ ID No:44.

9. A protein according to any of the claims 1-4, wherein said protein comprises said antigen binding domain and a detection moiety10. A protein according to any of the claims 9, wherein the detection moiety is selected from the group of: fluorescent dyes, fluorescent proteins or biotin11. A method of detecting FolRl expressing cells in a biological sample, comprising the steps: a. contacting a sample comprising FolRl expressing cells with a protein according to claim 9 or 10 b. Detecting said protein bound to said FolRl expressing cells

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