Chimeric antigen receptor binding to ca125 for treating ovarian cancer

Abstract

The present disclosure provides targeting units for specific binding to CA125. These targeting units are suitable for implementation in chimeric antigen receptors (CARs). In contrast to what was previously predicted, it is found that CARs comprising such targeting units can efficiently recognize and kill MUC16pos tumours.

Description

[0001]Title: Chimeric antigen receptor for Ovarian cancer Field The present disclosure is related to the field of cancer therapy. In particular, it relates to novel targeting units and chimeric antigen receptors (CARs) comprising them, nucleic acids encoding the targeting units, nucleic acids encoding the CARs, immune cells expressing the CARs and their utility for treatment of cancer. Background Ovarian cancer (OC) is the eighth most common cancer in women globally, for both incidence and deaths (1), with a 5-year survival rate of 49.7% (2, 3). This is drivenby a high frequency of late-stage diagnoses - when OC is particularly aggressive -the development of chemoresistance, and evasion of host immune responses (4). Standard treatment consists of cytoreductive surgery combined with platinum-based chemotherapy. This may be followed by adjuvant maintenance treatment with anti-angiogenic agents and inhibitors of poly-ADP-ribose polymerase (PARP) (4-6).Nevertheless, a majority of patients will relapse, and treatment will shift from curative to palliative care (4).High-grade serous tubo-ovarian carcinoma (HGSC) is classified into three differentimmunological phenotypes; desert, excluded, and inflamed ovarian tumours, based on the degree of T-cell infiltration into the epithelial compartment (7). Immunotherapy in the form of immune checkpoint inhibitors performs poorly in OC (8). The general failure in T-cell priming observed in cold and excluded tumours reflects the urgent need for strategies that can deliver autologous or allogeneic effector cells into HGSC tumours. CAR T-cell therapy faces challenges similar to adoptive cell therapy in general (9), but induction of a strong anti-tumour response, plus the inclusion of additional factors in the engineered cells (e.g. 4thgeneration CARs) should help overcome these factors (10). Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, though recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed.Chimeric Antigen Receptor (CAR) T-cell therapy empowers patients’ own T-cellsto fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer-antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. The normal biological function of MUC16 is to provide a protective lubricating barrier at mucosal surfaces, but in cancer it can facilitate peritoneal metastasis by promoting proliferation and inhibiting apoptosis (13, 14). MUC16 is a single-pass integral membrane glycosylated mucin protein, with a large extracellular region comprising multiple tandem repeats. Cleavage and release of this extracellular domain results in the generation of the Cancer Antigen 125 (CA125) (15). This cleaved CA125 has three main topographic domains, can be detected in the serum of OC patients, and is used as a diagnostic marker for OCs (16, 17). For MUC16, there have been numerous efforts to target the extracellularectodomain that remains after cleavage of CA125 – sometimes referred to as MUC-CD or MUC16ecto- by various approaches; namely antibodies (25), CAR T-cells (18,26), and antibody-derived Bispecific T-cell Engagers (BiTEs, (27-30)). However, alternative technical solutions for treatment of OC is still needed. SummaryThe present disclosure provides targeting units for specific binding to CA125.These targeting units are suitable for implementation in chimeric antigen receptors (CARs). In contrast to what was previously predicted, it is found that CARscomprising such targeting units can efficiently recognize and kill MUC16postumours. Epitopes located in CA125 may be more abundant and accessible than theMUC16ectoepitopes. Furthermore, it is found that targeting of CA125 in the presence of the soluble antigen in patient serum by CAR T-cells is feasible because high levels of soluble CA125 in the immediate vicinity of the target-cells did not affect CAR T-cell activity in vitro. Without being bound by theory, re-association of CA125 with tumour cells may occur at levels sufficient to trigger a cytotoxicresponse. Cells expressing CARs comprising the claimed targeting units, e.g. K101,demonstrated superior killing efficacy against OVCAR3, compared to cellsexpressing other constructs. Accordingly, cells expressing CARs comprising theclaimed targeting units can be useful as such, but they can also complement othertherapeutic approaches targeting MUC16ecto. In particular, the cells provided hereincould be included in an earlier line of treatment than treatments based onMUC16ecto.In a first embodiment, the present disclosure provides a protein for specific bindingto an epitope located on CA125 of MUC16, comprising a light chain variabledomain (VL) and a heavy chain variable domain (VH) which together form an antigen binding unit,- wherein the VL comprises three complementarity determining regions (CDRs):CDR1, CDR2 and CDR3, which respectively comprise the amino acid sequences SEQ ID NOs: 1, 2 and 3; and- wherein the VH comprises three CDRs; CDR1, CDR2 and CDR3, whichrespectively comprise the amino acid sequences SEQ ID NOs: 4, 5 and 6. In a first aspect of the first embodiment, both the VH and VL comprises human framework sequences.In a second aspect of the first embodiment, the VL is represented by SEQ ID NO: 7or a sequence with at least 90% identity thereto, and wherein the VH is represented by SEQ ID NO: 8 or, or a sequence with at least 90% identity thereto. In a third aspect of the first embodiment, the antigen binding unit is a scFv represented by the SEQ ID NO: 11 or a sequence with at least 90% identity thereto.In a second embodiment, the present disclosure provides a chimeric antigen receptor(CAR) comprising the protein according to the first embodiment.In a first aspect of the second embodiment, the CAR comprises from N-terminal toC-terminal, a scFv, a human CD8α hinge, a human CD8α transmembrane domain, a human 4-IBB costimulatory domain and a human CD3ζ signaling domain.In a second aspect of the second embodiment, the CAR comprises an amino acidsequence represented by SEQ ID NO: 17, or a sequence with at least 90% identity thereto.In a third embodiment, the present disclosure provides a nucleic acid encoding theprotein according to the first embodiment or the CAR according to the secondembodiment.In a fourth embodiment, the present disclosure provides a cytotoxic immune cellexpressing the CAR according to the second embodiment in its cell membrane,wherein the immune cell is a T-cell or an NK-cell. In a first aspect of the fourth embodiment, the present disclosure provides a cytotoxic immune cell expressing the CAR according to the second embodiment in its cell membrane and a CAR targeting MUC16ectoin its cell membrane.In a fifth embodiment, the present disclosure provides a pharmaceuticalcomposition comprising the protein according to the first embodiment, the nucleic acid according to the third embodiment, or the cells according to the fourth embodiment.In a first aspect of the fifth embodiment, the present disclosure providespharmaceutical compositions for use in treatment of MUC16-positive cancers, suchas pancreatic cancer or ovarian cancer.In a sixth embodiment, the present disclosure provides a method of treatment ofMUC16-positive ovarian cancer or MUC16-positive pancreatic cancer comprisingthe steps, in any order:a) administering a cell expressing a CAR for specific binding to an epitope locatedon CA125 of MUC16 to a patient in need thereof, wherein the CAR comprises ascFv represented by any one of SEQ ID NO: 11 to 16, or a scFv represented by a sequence with more than 90% identity thereto (i.e. to any one of them), and b) administering a cell expressing a CAR for specific binding to an epitope locatedon MUC16ecto to the patient.In a first aspect of the sixth embodiment, step a) and b) are performed simultaneously, e.g. by infusion of a pharmaceutically acceptable cell suspension comprising both types of cells. Brief description of the FiguresFigure 1 visualizes that the binding epitopes of the antibodies X75, K93, K95, K97and K101 are located in CA125 while the binding epitope of the antibody 4H11 is located in the MUC16ecto. Figure 2a visualizes, in a simplified manner, a conventional antibody comprising two heavy chains and two light chains. Each light chain is connected to a heavy chain by a cysteine bridge, and the heavy chains are connected to each other by two cysteine bridges. The Variable domains forms two identical Fv’s. The N-terminal of the chains is represented by an N. The C-terminal of the chains is represented by a C. Each Fv comprises a VL and a VH with three CDRs (see Figure 2b). The VL and VH can be connected from C-terminal to the N-terminal by a peptide linker represented by a diagonal line, to form a scFv. The two orientations VL-linker-VH and VH-linker-VL are visualized in Figure 2c and 2d.Figure 3 visualizes a CAR, comprising from N-terminal to C-terminal, a scFv, ahuman CD8α hinge, a human CD8α transmembrane domain, a human 4-IBBcostimulatory domain and a human CD3ζ signaling domain. The mFab (anti-murinefragment antigen-binding (Fab)) is used as a universal anti-mouse antibody, todetect the CAR through its murine specific features present on the scFv.Figure 4 visualizes activation of primary T-cells from healthy donors transducedwith the different CAR constructs based on a degranulation (CD107a)-assay. Amongst the K-series CARs, K95 and K101CARs demonstrated a superior activity toward OVCAR3. As anticipated, none of the CAR T-cells reacted against HEKcells (not shown), but K101CAR T-cells demonstrated superior killing efficacyagainst OVCAR3, compared to the other constructs.Figure 5 shows a co-culture assay of CAR T-cells and target-cells in the presence ornot of 300 U / L calibration-grade CA125. We observed no difference betweenconditions for all MUC16CAR T-cell killing efficacy against the MUC16posOVCAR3 cells. As expected, no change with the MUC16negcontrol Hek was detected too and, due to its MUC16ectospecificity, 4H11CAR target recognition was not affected. Thus, K101CAR T-cells are functional even at a high concentration of CA125 and therefore not sensitive to the presence of soluble target.Figure 6 shows the efficacy of anti-MUC16CAR T-cells against OVCAR3 cell linesin mouse xenograft models. T-cells were activated, transduced, then expanded in vitro, with cell proliferation and CAR expression monitored to ensure that CAR T-cell populations were comparable. After intraperitoneal (i.p.) engraftment of theslow growing OC cell line OVCAR3, immunodeficient mice received two injections(days 3 and 11) of CAR T-cells or Mock T-cell controls. We observed thatOVCAR3 form numerous small, solid tumours, with considerable expansion of ascites in the peritoneal space. The mice receiving Mock T-cells developed a high tumour burden in approximately 30 days, whereas very little signal was detected in mice receiving the MUC16CAR T-cells until around day 95. Accordingly, weobserved prolonged survival compared with the Mock T-cell group. Ultimately, 4 / 5of the mice treated with MUC16CAR T-cells were alive at the 4-month endpoint of the experiment.Figure 7 shows the efficacy of anti-MUC16CAR T-cells against OVCAR3 cell linesin mouse xenograft models. T-cells were activated, transduced, then expanded in vitro, with cell proliferation and CAR expression monitored to ensure that CAR T-cell populations were comparable. Mice were treated twice with T-cell at day 3 and7. The HeLa tumours progressed rapidly and were more resistant to CAR T-cellcontrol than OVCAR3. Nevertheless, K101CAR T-cells results in a significant survival benefit versus Mock T-cells, whereas survival with 4H11 T-cells was not significantly different from the Mock control. Thus, K101CAR T-cells are robust and can control a fast-growing cell line, with moderate levels of MUC16 expression. Figure 8 shows evaluation of how K101CAR T-cells were performing in a complex model. We used patient-derived cells, PDX26, with confirmed expression of MUC16 which were orthotopically injected in the bursa of the animals. The mice were subsequently treated twice with either 5x106K101CAR (n=7) or Mock T-cells (n=8) i.p., at days 22 and 24 post engraftment. Tumour development was monitored by PET-CT at days 41 and 86 after PDX engraftment. Injection of K101CAR T- cells lead to a reduction in PDX tumour size and dissemination, where 6 out of 7 K101CAR T-cell-treated mice had no tumours observable by necropsy. Whilst PET- CT at day 86 indicated high signal uptake in the ovary observed in 6 / 8 mock control mice,18F-FDG uptake could only be observed in one of the treated mice (white arrow). In addition, three animals in the control group showed a large metastatic mass (black arrow) suggesting an efficient spreading of the tumour. Individual quantification supports the protective effect of K101CAR T-cells, and efficient engraftment of PDX26 in the mock control group. Finally, survival analysis demonstrates a clear protective effect of K101CAR T-cells. Taken together, thesedata show that K101CAR T-cells can control different types of MUC16pos tumour invivo, even aggressive and disseminated models.Figure 9 shows the CA125 levels in culture media, mouse serum, and peritonealsamples. Accordingly, the present disclosure has demonstrated CAR T-cells able torecognize CA125-positive cells, while being insensitive to CA125 in solution. Figure 10 shows the efficacy of anti-MUC16CAR T-cells against pancreatic cancer cell line (PANC-1) in vitro using live cell imaging (IncuCyte). (A) PANC-1 are highly MUC-16 positive when grown in 3D culture (spheroid) but weakly positive in 2D. Staining was performed using anti-MUC16 K101 antibody and fluorescent anti-mouse antibody as secondary antibody. (B) CAR T-cells were activated, transduced, then expanded in vitro, with cell proliferation and CAR expression monitored to ensure that CAR T-cell populations were comparable. The cells were then incubated at an E:T ratio of 10:1 with target cells. PANC-1 expressing GFP were used and cell killing was monitored by GFP-fluorescence detection. Figure 11 shows an in vivo experiment where two antiMUC16CAR constructs (derived from the K101 and 4H11 clones, respectively) were tested against a patient derived xenograft model of ovarian cancer. It was an orthotopic system, where thetumoural tissues were engrafted at the bursa of the animals. After 3 weeks, theanimals were treated with donor T cells redirected with either CAR constructs ormock T cells. The tumour progression was monitored using IVIS and the survival of the animals was also analysed. This aggressive model could be controlled in terms of tumour growth, where K101CAR T cells appeared more efficient than 4H11CAR T cells. Figure 12 shows the comparison between humanized K101CAR and the original K101CAR. It was found that humanized CARs (hCARs) show similar expression tomurine CAR (mCAR) after being transduced into Jurkat76-NFAT-GFP (not shown).hCARs expressed in primary human T cells efficiently eliminated cancer cells in asimilar manner to mCART cells. Here, CAR T cells were cocultured with luciferase-expressing OVCAR-3 (MUC16+) or HEK-P (MUC16-) cells in the presence of luciferin. Killing capacity was measured by the reduction inluminescence from the target cells. Shown here are the results after 10 hoursincubation, at an E:T ratio of 5:1. The killing capacity of hK101 CAR T cells is relative to that of mK101 CAR T cells. Data are means ± SD (N=3 donors), t-test.Constructs hK101-2 and -4 displayed favourable activity.The experiments included utilized a number of cell lines and patient-derivedmaterials (PDX). It was verified that these cells shed CA125, under conditions usedin the experiments in the present disclosure. Cell lines were cultured in vitro, for atleast 24 hours without changing media, and allowed to grow to at least 70% confluence. Culture media (‘supernatant’) was collected from the culture vessels, and then centrifuged (5++g for 5 minutes) to remove cellular material, and the remaining supernatant was frozen. For the in vivo study, OVCAR3 Cell lines were injected into immunodeficient (NSG) mice, or patient xenografts (PDX) wereengrafted orthotopically, also into immunodeficient mice. After at least 4 weeks ofgrowth, serum was collected from the OVCAR3-engrafted mice, and a control mouse. Serum was also collected from ODX-engrafted mice. Finally, at euthanasia, the OVCAR3-engrafted mice (and control) were given an ‘intraperitoneal wash’ with PBS. That is, the peritoneal cavity was opened, 500ul PBS was introduced via a syringe (without needle), mice were massaged, and fluids were withdrawn with the same syringe). These samples were also centrifuged to remove cellular materials before freezing. All samples were subsequently analysed using a Roche ElecSys CA125II assay. Detailed descriptionThe present disclosure provides proteins for specific binding to an epitope locatedon CA125 of MUC16. These proteins comprise an antibody light chain variabledomain (VL) and an antibody heavy chain variable domain (VH) which togetherform an antigen binding unit, also referred to as a targeting unit. Such antigen binding units, Fv’s, are well known from antibodies. Fv’s may be expressed as single chain constructs known as scFv’s. The concept of specific binding to an epitope under physiological conditions is well known. Specific binding to a target may be distinguished from non-specific binding.Specific binding to a target can be distinguished from off-target binding and non-specific binding. For example, the proteins herein bind their targets with a higher affinity than they bind other molecules (or at least most other molecules). Accordingly, the targeting units herein (e.g. Fv’s, in particular scFv’s) display a non-covalent and reversible binding to their particular epitopes under the relevant physiological conditions. They bind with a higher affinity to the target epitope than they bind non-related molecules. The binding of a targeting unit to a target epitope can be measured by conventional methods, e.g. surface plasmon resonance (SPR). The affinity between the targeting unit and its epitope can be represented by a KD- value (the equilibrium dissociation constant) for example in the range of 10-4M to 10-12M, such as 10-5M to 10-11M or 10-6M to 10-10M. By “specific binding” is meant that the protein binds specifically to its target under physiological conditions. Physiological conditions, as used herein, means the environment encountered or simulated, in a living human patient, where the disclosed proteins are intended to bind an epitope located on human target-cells. In general, the physiological conditions in most human extracellular fluids are normally ca. 37°C, pH in the range of 6.0 to 7.5. If the target-cells are cancer cells in a solid tumor, the physiological conditions are the ones found in the solid tumor, i.e. the tumor microenvironment (TME). The TME is often hypoxic and acidic. Occasionally, MUC16 has been referred to as CA125 in the prior art. However, asused herein, CA125 refers to the part of MUC16 which tends to be cleaved off fromcancer cells. Chimeric antigen receptors (CAR) are well known. As used herein, a CAR comprises, from N-terminal to C-terminal, an Fv such as a scFv, optionally a hinge, a transmembrane domain, at least one co-stimulatory domain and a signaling domain such as CD3ζ.It has been speculated that soluble CA125 may interfere with CAR reactivity, bybinding to the CAR in the absence of target-cells (18). These concerns are partially supported by the observations of interference caused by shedding of other CAR targets (e.g. 19). Soluble CA125 has been shown to reassociate with the remaining extracellular stub (31). Such reassociation is the basis of CA125-targeting in experimental immunological approaches. Notably, administration of antibodies against CA125 restricts the growth of OVCAR3 tumours in mice (32). To our knowledge, interference of CAR function by soluble CA125 has not been definitively proven. Each VL and VH herein comprises three complementarity determining regions (CDRs) flanked by framework sequences. The framework sequences may be human, humanized or murine sequences. The six CDRs comprise or consist of the following sequences: VL CDR1 (SEQ ID NO: 1): QSLLYTSIQKNY VL CDR2 (SEQ ID NO: 2): WAS VL CDR3 (SEQ ID NO: 3): QQYYTYPWT VH CDR1 (SEQ ID NO: 4): GYTFTDYY VH CDR2 (SEQ ID NO: 5): INPNNGDN VH CDR3 (SEQ ID NO: 6): AKDGDYAMDY Framework sequences are structurally conserved regions that normally tend to form a β-sheet structure positioning the CDRs for specific binding to the target epitope under physiological conditions. The first framework sequence is N-terminal to the CDR1, the second framework sequence is located between CDR1 and CDR2, while the third framework sequence is located between CDR2 and CDR3. Accordingly, both a VL and VH can be roughly visualized as follows, with the CDRs boxed and the N-terminal indicated as N-: N-FRAMEWORK1CDR1FRAMEWORK2CDR2FRAMEWORK3CDR3FRAMEWORK4 In one embodiment, the antigen binding unit comprises or consists of VL-linker-VH(from N- to C-terminus). In another embodiment, the antigen binding unit comprisesor consists of VH-linker-VL (from N- to C- terminus). Such antigen binding unitsare often referred to as single chain Fv’s (scFv’s).The linker needs a certain length in order to allow the VH and VL to form afunctional antigen binding unit. In one embodiment, the linker comprises 10 to 30 amino acid residues. In one embodiment, the linker comprises 15 to 25 amino acidresidues, in particular glycine and / or serine residues. In a particular embodiment,the linker is a G4S linker, i.e. a peptide linker comprising repeating units with thesequence GGGGS. For example, suitable linkers may comprise 3, 4 or 5 adjoiningrepeating G4S units. In antigen binding units, the framework sequences may tolerate variation without destroying the specificity and affinity to the target antigen. For example, substitutions of amino acid residues may be tolerated better than deletions or additions of amino acid residues. Replacing murine framework sequences with human framework sequences, preferably of similar length, is known as humanization. The term "conservative amino acid substitution", as used herein, refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acids with similar side chains tend to have similar properties, and thus a conservative substitution of an amino acid important for the structure or function of a polypeptide may be expected to affect polypeptide structure / function less than a non-conservative amino acid substitution at the same position. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. asparagine, glutamine, serine, threonine, tyrosine), non-polar side chains (e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative amino acid substitution may be considered to be a substitution in which a particular amino acid residue is substituted for a different amino acid residue in the same family. In particular, the products comprising a conservative amino acid substitution relative to a reference sequence are covered by the terminology. In one embodiment, each VL and VH herein comprises three CDRs flanked by human framework sequences. Human framework sequences are structurally conserved regions that normally tend to form a β-sheet structure delicately positioning the CDRs for specific binding to the target antigen under physiological conditions. Many human framework sequences are available from known human antibodies and from the international ImMunoGeneTics information system (IMGT) online database (see Giudicelli et al, Nucleic Acids Research, 2006, Vol.34, Database issue D781-D784), but the term also covers human framework sequences comprising amino acid substitutions. Each of the human framework sequences may optionally comprise 0 to 5 amino acid substitutions relative to the natural sequence. An amino acid substitution is a sequence wherein an amino acid residue in a specific position is substituted for a different amino acid residue at the corresponding position, apparent when the sequences are aligned. Each of the human framework sequences may optionally comprise 1 amino acid substitution. Each of the human framework sequences may optionally comprise 2 or up to 2 amino acid substitutions. Each of the human framework sequences may optionally comprise 3 or up to 3 amino acid substitutions. Each of the human framework sequences may optionally comprise 4 or up to 4 amino acid substitutions. Each of the human framework sequences may optionally comprise 5 or up to 5 amino acid substitutions. The substitutions may be conservative substitutions. Even if such framework sequences are not necessarily previously known from human antibodies, they may provide lower immunogenic risk compared to a murine framework sequence. In one embodiment, 0 to 5 amino acid residues in the human framework sequences are substituted with the corresponding amino acid residue(s) from the murine parent sequences. Collectively, scFv’s comprising CDRs from a murine antibody and human framework sequences which each may optionally comprise 0 to 5 substitutions, are referred to as humanized scFv’s. In one embodiment, the human framework sequences are mature human framework sequences available from known human antibodies. Without being bound by theory, such framework sequences may convey very low risk of triggering unwanted immunogenic responses against the antigen binding unit, and at the same time increase the likelihood of obtaining stable binding units which are expressed well in cellular systems.Generally speaking, in humanized VH and VL sequences, the CDRs are retained, asin the parental VH and VL sequences. However as is known in the art, different programs, or schemes, are available to determine CDRs, and these may not in all cases give exactly co-incident results. Thus, different CDR identification schemes may yield different CDRs. For example, they may be shorter or longer, or positioned slightly differently in the VH or VL sequences (e.g. in a second schemethe CDR may be partially displaced up- or downstream relative to a first scheme).However, in relation to the present disclosure, the CDRs shall be determined usingthe well-known IMGT system.Humanization may involve grafting CDRs from murine VL and / or VH into selectedhuman VL and / or VH, i.e. replacing the human CDRs. Alternatively, human framework sequences may replace murine framework sequences in the murine VL and / or VH, and thus retain the murine CDRs. Accordingly, provided herein are humanized targeting units suitable for specific binding to an epitope located on CA125 of MUC16, comprising a VL selected from the group consisting of SEQ ID NO: 18, 20, 22, 24 and 26 or sequences with more than 80% sequence identity thereto, and a VH selected from the group consisting of SEQ ID NO: 19, 21, 23, 25 and 27 or sequences with more than 80% sequence identity thereto, provided -the VL comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 1, 2 and 3, respectively, and -the VH comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 4, 5 and 6, respectively. Accordingly, provided herein are humanized targeting units suitable for specific binding to an epitope located on CA125 of MUC16, comprising a VL selected from the group consisting of SEQ ID NO: 18, 20, 22, 24 and 26 or sequences with more than 90% sequence identity thereto, and a VH selected from the group consisting of SEQ ID NO: 19, 21, 23, 25 and 27 or sequences with more than 90% sequence identity thereto, provided-the VL comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 1, 2 and 3,respectively, and-the VH comprises CDR1, CDR2 and CDR3 represented by SEQ ID NO: 4, 5 and 6,respectively. Provided herein are also humanized targeting units suitable for specific binding toan epitope located on CA125 of MUC16 comprisinga VL selected from the group consisting of SEQ ID NO: 18, 20, 22, 24 and 26 and a VH selected from the group consisting of SEQ ID NO: 19, 21, 23, 25 and 27.For efficient expression of the claimed CARs in immune cells, a conventionalleader peptide may be introduced N-terminally for facilitating location in the cellmembrane. The leader peptide is believed to be trimmed off and will likely not bepresent in the functional CARs in the cell membrane.Soluble proteins provided herein (i.e. proteins lacking transmembrane domains, e.g. scFv’s) may be linked or conjugated to a therapeutic or diagnostic agent, or to a carrier which comprises or contains a therapeutic or diagnostic agent. A therapeutic agent is an agent used in therapy. By therapy is meant the treatment or prevention of a disease. The therapeutic agent may be an agent useful in the treatment of a neoplastic condition, particularly cancer. Also provided herein are recombinant nucleic acid molecules encoding the proteins. The nucleic acid molecules provided herein may be an isolated nucleic acid molecule and may include DNA or RNA or chemical derivatives of DNA or RNA.The term "nucleic acid molecule" specifically includes single- and double-strandedforms of DNA and RNA. The nucleic acid (e.g. DNA or RNA) may be circular or linear. A “recombinant” nucleic acid molecule is a nucleic acid molecule synthesized using recombinant techniques, e.g. molecular cloning.The recombinant nucleic acid molecules provided herein may encode the proteinprovided herein. In particular, such recombinant nucleic acids may have promoters allowing expression of the proteins in cellular systems.The recombinant nucleic acid molecules or constructs provided herein may beprovided within a vector. The term "vector" as used herein refers to a vehicle into which the nucleic acid molecule or construct provided herein may be introduced (e.g. be covalently inserted) from which the specific binding molecule encoded by the nucleic acid molecule may be expressed and / or the nucleic acid molecule / construct cloned. The vector may accordingly be a cloning vector or an expression vector.The recombinant nucleic acid molecules provided herein may be provided within arecombinant construct comprising the recombinant nucleic acid molecule linked to a heterologous nucleic acid sequence. By “heterologous” as used herein is meant a nucleic acid sequence which is not natively linked to the nucleic acid molecule described herein, i.e. which is not linked to the nucleic acid molecule described herein in nature. The term “linked” as used herein with respect to the construct may simply mean that the nucleic acid molecule is directly joined to a heterologous nucleic acid sequence. In one embodiment, in the recombinant construct the nucleic acid molecule provided herein is operatively linked to a heterologous expression control sequence. The term “expression control sequence” refers to nucleotide sequences located upstream of, within, or downstream of a coding sequence, and which influence transcription, RNA processing or stability, or translation of the associated coding sequence (i.e. which influence any aspect of expression of the encoded specific binding molecule). Expression control sequences include promoters, operators, enhancers, and other such cis-elements. In one particular embodiment herein, the recombinant nucleic acid molecule comprises a cDNA molecule. By “cDNA” as used herein is meant cDNA in its true and original sense (i.e. DNA synthesized by reverse transcription of mRNA), DNAamplified from original cDNA, and also DNA that is equivalent to cDNA. DNA thatis equivalent to cDNA is DNA that encodes a protein as provided herein and that lacks introns, such that it resembles a protein-coding sequence obtained by reverse transcription of mRNA. In one particular embodiment herein, the nucleic acid molecule encoding a protein as provided herein is codon-optimized, in particular the binding protein may be encoded by a codon-optimized cDNA sequence. It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a cell”, is understood to represent one or more cells, and the term of course includes cell populations. As any skilled person will understand, a cellexpressing a CAR will normally express numerous such CARs. As such, the terms“a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein. As used herein, when referring to “sequence identity” of proteins, an amino acid sequence having at least x% identity to a second amino acid sequence means that x% represents the number of amino acid residues in the first sequence which are identical to their matched amino acid residues of the second sequence when both sequences are optimally aligned via a global alignment, relative to the total length of the second amino acid sequence. Both sequences are optimally aligned when x is maximum by using the comparison matrix BLOSUM62 with gap costs: existence 11, extension 1. The pharmaceutical compositions herein can be a composition suitable foradministration of therapeutic proteins, nucleic acids or cells to a patient.Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a neutral pH. Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a physiological pH. Suitable cells for expressing the CARs herein include T-cells and NK-cells, butother immune cells can also be used. It is generally preferred to use immuneproviding at least one effector function (e.g. cytotoxic cell killing activity, secretionof cytokines, induction of ADCC and / or CDC). Such cells thus include T-lymphocytes, in particular cytotoxic T-cells and helper T-cells. Other immune cells include NK cells, NKT cells, neutrophils, and macrophages. The cells may be primary cells or cell lines. The pharmaceutical compositions herein can be a composition suitable for administration of therapeutic cells to a patient. The most common administration route for therapeutic cells is intravenous administration. Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a neutral pH. Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a physiological pH. The sterile pharmaceuticalcompositions may be a cell suspension for infusion comprising from 1 x 106 cells to1 x 1010cells, such as 1 x 107cells to 1 x 109cells.Such compositions can be for example be supplied in an infusion bag containingapproximately 30 to 100 mL of a frozen suspension of the cells in 5% DMSO and 2.5% human serum albumin. The most common administration route for T-cells is intravenous administration. For example, a patient’s peripheral blood mononuclear cells may be obtained via a standard leukapheresis procedure. The mononuclear cells may be enriched for T- cells, before transducing them with a lentiviral vector or mRNA encoding the proteins disclosed herein, in particular CARs. Said cells may then be activated with anti-CD3 / CD28 antibody coated beads. The transduced T-cells may be expanded in cell culture, washed, and formulated into a sterile suspension, which can be cryopreserved. If so, the product is thawed prior to administration.Antibodies from three hybridomas from our hospital’s collection (33) were adaptedto a CAR format. Of these, K101CAR induced the strongest response againstMUC16-positive (MUC16pos) tumour cells in vitro and was selected for furthertesting. Soluble CA125 did not interfere with K101CAR T-cell cytotoxicity, and the functional activity of this CAR was superior to existing anti-MUC16ectoCAR in vitro, and showed significant impairment of tumour growth in vivo. K101CAR represents a promising candidate for clinical CAR-based therapy.OC is a common gynaecological cancer with high mortality rates. This is due in partto typically late diagnosis, limited treatment options, and high rates of recurrence (4). Conventional therapies are limited in efficacy and range, and new treatment modalities are needed. CAR therapy has the potential to direct immune cells to targets in immunologically-cold tumour microenvironments. In this disclosure, opposite to what was previously predicted (18), we demonstrate that a CAR thattargets CA125 – the cleaved domain of MUC16 - can efficiently recognize and killMUC16postumours. This also confirms the re-association of CA125 with tumour cells at levels sufficient to trigger a cytotoxic response (31). Another argument precluding the targeting of CA125 was the presence of the soluble antigen in the patient serum which could inhibit a CAR by competing with the cellular CA125.While the replication of “true” in situ conditions can be challenging, we also showthat high levels of soluble CA125 in the immediate vicinity of the target-cells didnot affect CAR T-cell activity in vitro. OVCAR3 cells, which are often used as a validation model, do present all the expected features; they are slow growing and MUC16high, and, as expected, were easily killed by the anti-MUC16CARs. However, the cervical tumour-derived HeLa cells were markedly harder to control, especially in 2D culture. We suspect that this was due to [1] heterogeneity of MUC16 expression for which we confirmed the reports of previous authors (54), and / or [2] the influence of culture conditions on CA125 expression and distribution (55). Although K101CAR T-cells could kill HeLa cells grown in 3D and in vivo, these observations challenge the validity of the in vitro systems widely used by the CAR community, while supporting previous propositions that 3D systems are more representative or predictive of the in vivo context (56, 57). We found that the advantage of our CAR carried over into testing on CA125-positive patient-derived samples; K101CARs typically showed stronger reactivity than did the 4H11CARs in vitro, although the nature of the samples prevented us from directly comparing cytotoxicity against them. Nevertheless,testing of the K101CAR in vivo in an orthotopic PDX model confirmed killingunder these conditions. To our knowledge, this is the first demonstration of CAR- mediated killing of patient-derived tubo-ovarian carcinomas in vivo. Amongst CAR-based immunotherapeutic to treat OC, the 4H11CAR has been main the focus of CAR research, progressing to clinical trials in multiple formats (9, 58).This focus upon the targeting of the MUC16ecto (18) is evidently based upon theassumptions CA125 is not associated (or reassociated) with the tumour cells at levels sufficient for targeting, and / or that the shed (soluble) CA125 interferes withCAR T-cell functionality. These questions provided a context for the current study,and we have answered both. First, CA125 is detectable on tumour cells lines and primary tumour samples, at levels sufficient for targeting by CAR T-cells both invitro and in vivo. Secondly, we found no evidence that soluble CA125, even at highconcentrations, had any effect on CAR T-cell cytotoxicity nor specificity in vitro. In comparison to the ectodomain-targeting 4H11-based CAR, the efficient killing of MUC16postarget-cells by the K101CAR T-cells indicates that the extracellular repeat domain is indeed an attractive target. Accordingly, cytotoxic immune cells expressing a CAR for specific binding to anepitope located on CA125 of MUC16, as provided herein, in their cell membrane,and a CAR targeting MUC16ecto in their cell membrane will represent a robust cell-therapeutic approach for ovarian cancer. Because cells expressing the CARscomprising the targeting unit herein were not significantly hampered by thepresence of soluble CA125, such cells can provide cytotoxic effect to the cancercells expressing MUC16 even if CA125 is cleaved off. The present disclosure thusprovides alternative solutions for treatment of OC based on targeting of MUC16 even in case of partial loss of CA125 antigen. Furthermore, the present disclosure provides a method of treatment of ovarian cancer comprising the steps,a) administering a therapeutic protein, or a nucleic acid encoding it, for specificbinding to an epitope located on CA125 of MUC16 to a patient in need thereofand subsequently b) administering a therapeutic protein, or a nucleic acid encoding it, for specific binding to an epitope located on MUC16ectoto the patient. Furthermore, the present disclosure provides a method of treatment of ovarian cancer comprising the steps, a) administering a cell expressing a CAR for specific binding to an epitope locatedon CA125 of MUC16 to a patient in need thereof, wherein the CAR comprises ascFv as provided herein, and subsequently b) administering a therapeutic protein, or a nucleic acid encoding it, for specific binding to an epitope located on MUC16ectoto the patient. Furthermore, the present disclosure provides a method of treatment of ovarian cancer comprising the steps, a) administering a cell expressing a CAR for specific binding to an epitope locatedon CA125 of MUC16 to a patient in need thereof, wherein the CAR comprises ascFv as provided herein, and subsequently b) administering a cell expressing a CAR for specific binding to an epitope locatedon MUC16ecto to the patient.MATERIALS AND METHODS Analysis of RNA-Seq data RNA-Seq data from The Cancer Genome Atlas (TCGA, released July 27, 2022) project for OC for 378 tumour samples was used. The analysis was performed in R (https: / / www.r-project.org / (accessed in 2022)) using Bioconductor (https: / / www.bioconductor.org) packages. The corresponding OC clinical data aswell as raw counts of gene expression for tumour-associated samples weredownloaded through the TCGA biolinks package. The Harmonized database (https: / / portal.gdc.cancer.gov / ), which is mapped to the reference genome GRCh38 (hg38), was used. Clinical staging in OC, using the FIGO (International Federation of Gynaecology and Obstetrics) system, categorised patients into eight distinct groups (I, II, IIIA, IIIB, IIIC, IVA, IVB and unknown stage). In this staging, three factors of tumour size, lymph node, and spread of metastasis, were used to classifysamples. To identify differential expression of genes on the TCGA cohort, the Rpackage DESeq2 was used for normalisation of raw counts. Pre-filtering wasapplied to keep only genes that have at least 10 reads in total. Differential expression and design of the analysis were also performed with the DEseq2 package (34). The clinical stage of tumour samples was used in the design of the analysis. For FDR correction, adjusted p-values less than 0.05 were selected. Differential expression of genes was considered to be statistically significant if their |log2 fold change| ≥ 2 and FDR < 0.05. Since the MUC16 gene is known to be highly expressed in OC tissues, we focussed upon a selection of genes (MUC16, MUC1, MSLN, FOLR1) in order to show the significant differential expression of MUC16 in the TCGA database (35). To visualise the results of differential expression analyses of selected genes (MUC16, MUC1, MSLN, FOLR1), pheatmap was used. Cell culture and cell linesStandard in vitro culture was at 37oC, with 5% CO2, in a humidified environment.Cell lines, OVCAR3 (HTB-161), and HeLa (CCL-2.2), were obtained from ATCC and cultured in RPMI-1640 (Biowest, Nuaillé, France) with 10% FCS (Thermo Fisher Scientific, USA), and 10 µg / mL gentamycin (Thermo Fisher Scientific), except HEK-293 (CRL-1573) cells, also from ATCC, were cultured in DMEM (Sigma, USA) with 10% HyClone FBS (GE Healthcare Life Sciences, USA) and 10 µg / mL gentamycin. For regular passaging, adherent-cells were washed twice with PBS, then detached with Trypsin / EDTA (Sigma-Aldrich, USA, T3924-100ML). Prior to co-culture experiments, target-cells were detached with EDTA alone (5 mM in PBS, without Trypsin). Spheroids composed of tumour cells were generated using standard methods. Briefly, flat bottom 96-well plates were coated with 1% agarose dissolved in PBS. After 2 hours at room temperature, 2000 tumour cells in 200 µL of culture medium were added per well. PBMCs were isolated from whole blood of healthy donors, under an approved institutional protocol, and were cultured in X-vivo 15 (Lonza, Switzerland), supplemented with 5% human serum (TCS Biosciences, Buckingham MK182LR, UK), and IL-2 (Clinigen, UK) at 100 U / mL. For activation, 24-well culture plates were pre-coated with 500 µL / well of PBS containing 0.5µg anti-CD3 (functional grade OKT3, eBioscience, USA, #16-0037-85) and 0.5µg anti-CD28 (functional grade CD28.6, eBioscience, USA, #16-0288-85) antibodies, for 2 hours at room temperature. This was removed, and PBMCs were added at 106cells per mL, per well. After 2-3 days, cells were counted, and available for transduction. Production of retroviral vectors, transduction, and protein production Production of retroviral particles, and transduction of T-cells, were performed as described previously (36). Briefly, human embryonic kidney (HEK) cells were transiently transfected with accessory plasmids and a retroviral vector bearing the CAR sequence. After two days, the supernatant was harvested and applied to J76- NFAT-GFP cells, or activated PBMCs, in one or two rounds of transduction, respectively. Flow Cytometry Testing of antibodies raised against CA125 was performed as follows. Target-cell lines were detached with EDTA (see above), and then incubated with hybridoma supernatant diluted 1 / 20, for 15 minutes at RT. As a positive control, we used an anti-CA125 antibody (clone X75, ThermoFisher, USA, #MA1-90039) at a dilution of 1 / 200. Cells were then washed twice with flow buffer, and incubated with goat anti-mouse IgG secondary antibody (minimal cross-reactivity, BioLegend, USA, #405308), for 15 minutes at RT. Cells labelled with secondary antibody-alone were used as negative controls. Cells were washed once more, and resuspended in flow buffer, for flow cytometry analysis. Expression of the anti-MUC16CARs was detected by flow cytometry. T-cells were labelled with a biotinylated anti-mFab antibody (1 / 200 dilution, Jackson ImmunoResearch, USA #115-066-072) followed by incubation with a Streptavidin- APC secondary antibody (1 / 200-1 / 400. BioLegend, USA, #405207). Cells were acquired on a FACSCanto 10 instrument (BD Biosciences, USA) and analysed using FlowJo software version 10 (BD Biosciences, USA). Sequencing of hybridomas and generation of CAR constructs CA125 contains three topographically distinct antigenic determinants, with most monoclonal antibodies grouped as OC125-like (group A), or M11-like (group B), while OV197 alone constitutes a third category, group C (37). The hybridomas producing the anti-CA125 antibodies were generated at The Department of Medical Biochemistry, Oslo University Hospital, Radium Hospital, Oslo, Norway. We selected hybridomas K93 and K95 (both group A), and K97 and K101 (both group B) (38). Sequencing of hybridoma samples was performed as in (39). Briefly, mRNA was extracted from frozen, pelleted hybridoma cells. This was used for 5’- RACE sequencing, and the resulting heavy and light chain sequences were codon- optimised and adapted as a single chain variable fragment (scFv) using the orientation of light chain-linker-heavy chain. The CAR constructs were finalised by fusing the scFv to a CD8 hinge, CD8 transmembrane domain, a 4-1BB co- stimulatory domain, and a CD3ζ intracellular domain, as described in (40). The anti-CD19 scFv derived from FMC63 was incorporated in the same arrangement.The sequence of the anti- MUC16ecto clone 4H11 heavy and light chains wasobtained from patent WO2011119979A2.To generate luciferase-expressing cell lines for bioluminescent in vitro and in vivoassays, we adapted a construct incorporating the firefly luciferase-GFP (41) intothe retroviral vector pMP71 (36). Target-cell lines were transduced with this vector,and GFP-positive cells were sorted by FACS. The NFAT-GFP construct used in the reporter assays (pSIRV-NFAT-eGFP) was a gift from Peter Steinberger (Addgene plasmid # 118031)(42). Co-culture assays For the reporter assays, Jurkat-76 cells were transduced with the NFAT-GFP reporter construct (42), to generate a J76-NFAT-GFP clone that expressed GFP in response to T-cell activation signals (43). This clone was subsequently transduced with the CAR vectors, then co-cultured with GFP-negative target-cells at an Effector: Target (E: T) ratio of 1:2, overnight in RPMI with 10% FCS. CAR- mediated reactivity was assessed by flow cytometry of GFP expression. CAR T-cell functionality was assessed by using CD107a as a degranulation marker. CAR transduced T-cells were co-cultured with target-cells (E: T = 1:2) for 5 hours, in the presence of anti-CD107a (BD Bioscience, USA, catalogue number 555802) at the recommended concentration. Degranulation was assessed by flow cytometry. Here, effector and target-cells were distinguished by GFP expression (GFP+ target- cells), or by pre-labelling with Cell Trace Violet, as per the manufacturer’s methods (Thermo Fisher Scientific, USA). Bioluminescence-based cytotoxicity (BLI) assays were performed as previously described (44). Briefly, luciferase-expressing tumour cells were co-cultured with CAR T-cells in a 96-well plate in the presence of D-Luciferin (75 µg / mL, PerkinElmer, Norway) at indicated effector: target (E: T) ratios. Cells were placed in anincubator at 37 °C, 5% CO2 and bioluminescence was measured with a luminometer (VICTOR Multilabel Plate Reader, Perkin Elmer) at several time points up to 24 hours. For killing assays using the IncuCyte (IncuCyte S3, Sartorius Lab Instruments)target-cell lines, at 2000 cells / well, were seeded as spheroids for 5 days. T-cellswere added (10,000 cells / well), along with Annexin V red, at the recommended concentration (Sartorius, USA, catalogue number 4641). Annexin V signal was recorded hourly, and the Total Red Object Integrated Intensity (RCU x µm² / Image) was calculated. For examination of cytokine secretion, CAR T-cells were co-cultured with target- cells (E: T = 1:2) overnight in serum-free RPMI or X-vivo 15. After centrifugation (500xg for 5 minutes), the supernatant was removed and immediately frozen at - 80oC until use. Samples were prepared as per the manufacturer’s methods (Bio-Plex Pro Human Cytokine Group I panel, 17-plex kit, Bio-Rad Laboratories, USA). Patient samples Samples were obtained from patients undergoing primary debulking surgery at the Department of Gynaecological Oncology, Oslo University Hospital (n=4), or from frozen patient peritoneal effusions (n=2) from the Department of Pathology, Oslo University Hospital. The samples from debulking surgery were dissociated by physical and enzymatic means. Briefly, tumour samples were washed with RPMI with 10% FCS and reduced to 1-2mm3-sized pieces by cutting with scalpels. This was centrifuged for 5 minutes at 100xg, and the supernatant was removed. Samples were resuspended in 10% RPMI, supplemented with collagenase II (Sigma Aldrich, C6885-500MG, at 4.7 mg / mL final) and DNase (Sigma Aldrich, DN25-100MG, at 0.57 mg / mL final), and incubated at 37oC with constant rotation for 1 hour, or longer if necessary. Samples were then passed successively through 100 µm, 70 µm, and 40 µm cell strainers. Samples were centrifuged at 500xg (5 minutes), then resuspended in 5 mL ACK lysis buffer (Lonza, #10-548E) for 30 seconds, before 5 mL of 10% RPMI was added. Samples were centrifuged at 400xg (10 minutes), then resuspended and frozen, or used fresh. To test the reactivity of CAR T-cells against OC samples, the samples were labelled with commercial anti-CA125 antibody (X75 at 1 / 200 dilution. Thermo Fisher, #MA1-90039) followed by a secondary antibody (APC goat anti-mouse IgG,BioLegend #405308, at 1 / 200 dilution), and labelling intensity was normalisedagainst the same samples labelled with the secondary antibody only. For clarity, samples were grouped as CA125-low / negative, medium, or high. CAR T-cells and patient samples were co-cultured overnight at an E: T ratio of 1:2, in the presence of anti-CD107a (BD Bioscience, USA, catalogue number 555802). CD107a labelling was compared to CAR T-cells cultured under the same conditions, but without patient-cells present. An established patient-dervied xenograft (PDX) model derived from primary patient tumours, categorised as a stage IV high-grade serous tubo-ovarian carcinoma (HGSC), was used in this study. This PDX was generated from a treatment-naïve patient tumour, sampled from primary debulking surgery (Gynaecologic Cancer Biobank, Women’s Clinic, Haukeland University Hospital, Bergen, Norway). This tumour sample was cut into small tissue pieces of 1-2 mm3in size. Tissue pieces were enzymatically dissociated with collagenase II (300 U / mL, cat#17101015, Gibco, USA) and DNase (0.1 mg / mL, # 07900, STEMCELL Technologies, UK) supplemented with calcium chloride (3 mM) for two hours with constant agitation (250 rpm) at 37^C. Digested tumour tissue was washed in PBS, strained through a 40 ^m cell strainer and centrifuged. Cell viability was determined with trypan blue staining. Samples were cryopreserved or immediately injected orthotopically into the bursa of the ovary (45). After successful engraftment into NSG mice, the passaged PDX material (passage 2) was cryopreserved before further use in in vivopreclinical MUC16 CAR T-cell treatment studies. PET-CT imaging and analysis For quantitation of the PDX model, PET-CT scans were acquired using theintegrated nanoScan PC PET / CT (Mediso Ltd, Hungary) featuring spatial resolutionof 800 μm and 300 μm of the PET and CT detector systems, respectively. The field of view (FOV) of the stacked images was 9.6 axial × 10 cm transaxial allowing whole-body 3D imaging of the mice. Animals were scanned using a dual mouse bed with integrated heating (37 °C). Each PET scan was conducted over 20 minutes, 45 minutes after intravenous administration of18F-Fluorodeoxyglucose (18F-FDG; 5-12 MBq / mouse). Prior to PET acquisition, a whole-body CT scan (helical projections with tube energy of 50 kVp, exposure time 300 ms, 7.2 projections, max FOV, binning 1-4) was performed providing anatomical information. PET images were reconstructed using the Nucline software and parameters were static normal, whilst CT images were reconstructed using a RamLak filter. The PET and CT images were co-registered automatically. Images were reconstructed with a voxel size of 0.25×0.25×0.25 mm3for CT, and 0.4×0.4×0.4 mm3for PET. Data analyses were performed using InterView Fusion version 3.03.078.0000 (Mediso Ldt). Standard uptake value (SUV) was calculated using the equation: SUV = CPET(T) / (ID / BW), where CPET(T) was the measured activity in tissue, ID the injected dose measured in kBq (corrected using https: / / www.cyclotron.nl / decay- calculator / ), and BW the mouse's body weight in kg. For each scan, a signal above 2.5 SUV was regarded as a true tumour signal and a spherical volume of interest (VOI) was drawn semi-automatically for estimation and calculation of SUVmeanand SUVmax. Soluble CA125 To represent cleaved CA125 in our experiments, we used human calibration-grade CA125 protein (#abx060961, Abbexa, UK). Where this was used in co-culture assays (BLI cytotoxicity and CD107a functional assays), it was added in solution, at the indicated concentrations. Dot Blots Proteins of interest were applied to a nitrocellulose membrane and allowed to dry. The membrane was then blocked with 5% Bovine Serum Albumin (BSA) in Tris- Buffered Saline (TBS), before incubation with the primary antibody (various). Next the membrane was washed three times in TBS-Tween, then incubated with Horse Radish Peroxidase-conjugated anti-mouse IgG secondary antibody (#62-6520, ThermoFisher). After another three washes with TBS-Tween, the membrane was incubated for 1 minute with Enhanced ChemiLuminescent substrate, then the luminescent signal was read on an Amersham Imager 600 (G.E. Healthcare, USA). Semi-quantitative analysis was performed using GelQuant software (BiochemLabSolutions.com). In vivo Assays Female NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ, OUS and UiB) and NXG (NOD- Prkdcscid-IL2rgTm1 / Rj, OUS) mice were maintained in pathogen-free conditions under the respective institutional animal care protocols. All animal experiments were conducted in compliance with the procedures from the Norwegian State Commission for Laboratory Animals and approved by the Norwegian Food Safety Authority. For the cell-line study, luciferase-expressing OVCAR3 or HeLa cells weretrypsinised and washed twice in PBS. Six- to 10-week-old NXG mice were injectedintraperitoneally (i.p.) with 1x106luciferase-expressing OVCAR3 or HeLa cells in 200 µl PBS. After 3 days, 200 µL D-luciferin (20 mg / mL, #122799-5, Perkin Elmer, Waltham, USA) was injected i.p., and engraftment was confirmed by bioluminescence using the IVIS Spectrum In Vivo Imaging System (Perkin Elmer). Mice were allocated to treatment groups based on comparable tumour loads. The same day 5x106T-cells (Mock, K101CAR, or 4H11CAR) were injected i.p. This was followed by a second injection of the same cells, 8 days later for the OVCAR3 experiment, and 4 days later for the HeLa experiment. For the PDX study, 6-8-week-old NSG mice were injected with 1x105PDX P2 cells orthotopically into the bursa of the right ovary (45). After 22 days, engrafted mice received 5x106Mock (n=8) or K101CAR T-cells (n=7) i.p., in 200 µL serum-free RPMI. A second injection was delivered 2 days later. At 41 days (19 days after firstT-cell injection), mice were examined by PET-CT. This was repeated at 86 days (64days after T-cell injection). The humane endpoint was reached on day 276.Statistical analysis Statistical analyses as indicated below (Paired t-tests, One-way and Two-way ANOVAs, Simple linear regression, Mann-Whitney, and Log-rank tests), were calculated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). MUC16 is expressed in a large proportion of OC samples Although already recognized as a valid OC target, MUC16 distribution was analysed from a TCGA cohort of 375 ovarian tumour samples (46). We comparedMUC16 expression to that of other OC-validated targets (MSLN, FOLR1 andMUC1) (47-49) and included the clinical stage information. We conducted DESeq2analysis on the TCGA tumor samples, confirming that MUC16 was expressed in the majority of OC samples. MUC16 was compared to the three other targets but none of these genes were in the top 100 for RNA expression, they however all show positive fold changes throughout the different clinical staging groups. These data confirm that MUC16 is a marker for OC and therefore a potential drug target for treating advanced patients. Anti-CA125 scFv leads to functional CAR constructs Supernatants from four anti-CA125 hybridomas (K-series, (33)) were tested against CA125-positive and CA125-negative cell lines. The commercial anti-CA125 antibody, X75, was included for reference. Labelling with each of the supernatants of the K series closely matched the commercial X75 antibody signal. We next identified the heavy and light variable chains sequences from each of the K- hybridomas, except K93, and designed scFv. In order to compare K-series CAR toan anti-MUC16 validated product, we also cloned the 4H11CAR (18) which isspecific to the MUC16ecto. First, a J76-NFAT-GFP reporter cell clone (43, 50) wastransduced with each of the CAR constructs, and their expression was confirmed by staining for mFab . These reporter cells were then co-cultured with different MUC16pos(HeLa, OVCAR3) or -negative (HEK) cell lines and GFP signal was detected. Only the MUC16poscells induced GFP expression, suggesting that theCAR constructs were functional and specific. Then, primary T-cells from healthydonors were transduced with the different CAR constructs, and degranulation(CD107a) was used as a marker of T-cell activation. Amongst the K-series CARs,K95 and K101CARs demonstrated a superior activity toward OVCAR3 and HeLa cells and no reactivity against the MUC16-negative (MUC16neg) HEK cells. However, K95CAR T-cells reacted against MUC16negBL41 lymphoma cell line and was therefore eliminated from the study. In addition, K93CAR showed weakreactivity in the different assays and further development was abandoned. On theother hand, 4H11CAR weakly activated J76 cells in the reporter assay, anddemonstrated a non-significant primary T-cell CD107a response against MUC16poscells, although the CAR was well detected on effector cells, suggesting that MUC16ectomight not be well displayed in these experimental designs. Thus in vitro studies of MUC16 CAR targeting is not trivial and even validated constructs mightnot properly respond. We then studied the cytotoxic function of K101- and4H11CAR T-cells by Bioluminescence (BLI)-based cytotoxicity assays at a range of E: T ratios. We included an irrelevant CAR as a control, anti-CD19 FMC63. As anticipated, none of the CAR T-cells reacted against HEK cells, but K101CAR T- cells demonstrated superior killing efficacy against OVCAR3, compared to the other constructs. Of note, 4H11CAR T-cells were also able to efficiently kill OVCAR3 cells despite the low CD107a detection. Intriguingly, although CD107a was detected on K101CAR T-cells incubated with HeLa cells, no killing was observed with any of the construct. We speculated that the 2D cell growth may influence MUC16 distribution which might prevent proper killing. We thus testedCAR T-cell cytotoxicity in a 3D-based live-cell imaging assay. We observed anincrease in killing of HeLa spheroids with both anti-MUC16CARs, whereas no killing was observed against HEK spheroids. Thus, HeLa cells might change their MUC16 distribution following culture conditions. In order to complete the in vitro evaluation of K101CAR T-cells, we examined their cytokine responses when co- cultured with target-cells for 24 hours by Bioplex assay. We found that K101CAR T-cells stimulated a strong release of IL-2, IL-17, and IFN-γ, whereas 4H11CAR T- cells lead to TNF-α release. Together, these data support the specificity and effectiveness of K101CAR T-cells against MUC16postargets. In order to validate the robustness of K101CAR T-cells, we tested them against a series of primary patient samples, including peritoneal effusions and samples derived from debulking surgery of solid tumours. MUC16 expression was measured by anti-CA125 (X75)labelling and correlated to MUC16CAR T-cell reactivity assessed by degranulationand anti-CD107a staining. From a total of 6 patient-derived samples, we observed that K101CAR T-cells reactivity followed MUC16 presence, whereas 4H11CAR T- cells did not react with these samples. From these data K101CAR appears more potent than 4H11CAR to redirect T-cells against MUC16postargets.Soluble CA125 does not alter K101CAR T-cell activityAn important concern when targeting a cleaved antigen, such as CA125 which is released in the serum, is the risk of reducing the CAR activity. This can happen by competition mechanisms as we previously reported with an anti-IgΚCAR blocked by serum IgGs (51). We first confirmed K101 antibody binding to calibration-grade CA125 protein. Dot blot experiments were designed with top CA125 concentration fixed to 300 kU / L which would mimic a diagnostic situation for OC and other gynaecological cancers where the lower cut-off is set to 20 kU / L (52, 53). For the staining, the commercial X75 antibody was used as a positive control and the background level was fixed using an irrelevant hybridoma supernatant, producing an anti-CEA antibody, CEA10. We then tested the indicated amounts or dilutions ofantibodies and observed K101 binding to CA125. Thus, K101 also reacts withCA125 in a cell-free context, suggesting a risk of potential inhibition of K101CAR by serum CA125. We next performed a co-culture assay of CAR T-cells and target- cells in the presence or not of 300 U / L calibration-grade CA125. We observed nodifference between conditions for all MUC16CAR T-cell killing efficacy against theMUC16posOVCAR3 cells. As expected, no change with the MUC16negcontrol Hek was detected too and, due to its MUC16ectospecificity, 4H11CAR target recognition was not affected. Thus, K101CAR T-cells are functional even at a high concentration of CA125 and therefore not sensitive to the presence of soluble target. K101CAR T-cells are efficient in vivo We tested the efficacy of anti-MUC16CAR T-cells against OVCAR3 and HeLa cell lines in mouse xenograft models. T-cells were activated, transduced, then expanded in vitro, with cell proliferation and CAR expression monitored to ensure that CART-cell populations were comparable. After intraperitoneal (i.p.) engraftment of theslow growing OC cell line OVCAR3, immunodeficient mice received two injections(days 3 and 11) of CAR T-cells or Mock T-cell controls. We observed that OVCAR3 form numerous small, solid tumours, with considerable expansion of ascites in the peritoneal space. The mice receiving Mock T-cells developed a high tumour burden in approximately 30 days, whereas very little signal was detected in mice receiving the MUC16CAR T-cells until around day 95. Accordingly, weobserved prolonged survival compared with the Mock T-cell group. Ultimately, 4 / 5of the mice treated with MUC16CAR T-cells were alive at the 4-month endpoint of the experiment. In order to assess the robustness of the MUC16CAR constructs, we tested the cervical cancer cell line HeLa because it expresses lower levels of MUC16 at the surface and grow at a faster rate in vitro. As for OVCAR3 assay,mice were treated twice with T-cell but the injections were performed closer in time(at day 3 and 7). As predicted, the HeLa tumours progressed rapidly and were moreresistant to CAR T-cell control than OVCAR3. Nevertheless, K101CAR T-cellsresults in a significant survival benefit versus Mock T-cells, whereas survival with 4H11 T-cells was not significantly different from the Mock control. Thus, K101CAR T-cells are robust and can control a fast-growing cell line, with moderate levels of MUC16 expression. We finally wanted to evaluate how K101CAR T-cells were performing in a complex model. We thus used patient-derived cells, PDX26, with confirmed expression of MUC16 which were orthotopically injected in the bursa of the animals. The mice were subsequently treated twice with either 5x106K101CAR (n=7) or Mock T-cells (n=8) i.p., at days 22 and 24 post engraftment. Tumour development was monitored by PET-CT at days 41 and 86 after PDX engraftment. Injection of K101CAR T-cells lead to a reduction in PDX tumour size and dissemination, where 6 out of 7 K101CAR T-cell-treated mice had no tumours observable by necropsy. Whilst PET-CT at day 86 indicated high signal uptake in the ovary observed in 6 / 8 mock control mice,18F-FDG uptake could only be observed in one of the treated mice (white arrow). In addition, three animals in the control group showed a large metastatic mass (black arrow) suggesting an efficient spreading of the tumour. Individual quantification supports the protective effect of K101CAR T-cells, and efficient engraftment of PDX26 in the mock control group. Finally, survival analysis demonstrates a clear protective effect of K101CAR T- cells. Taken together, these data show that K101CAR T-cells can control differenttypes of MUC16pos tumour in vivo, even aggressive and disseminated models.Sequences VL CDR1 (SEQ ID NO: 1): QSLLYTSIQKNY VL CDR2 (SEQ ID NO: 2): WAS VL CDR3 (SEQ ID NO: 3): QQYYTYPWT VH CDR1 (SEQ ID NO: 4): GYTFTDYY VH CDR2 (SEQ ID NO: 5): INPNNGDN VH CDR3 (SEQ ID NO: 6): AKDGDYAMDY Murine VL (SEQ ID NO: 7) DIVMSQSPSSLAVSVGEKVTLSCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPWTFGGGTKLEIKMurine VH (SEQ ID NO: 8)EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGEINPNNGD NFYNQKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCAKDGDYAMDYWGQGTSVT VSS Leader peptide (SEQ ID NO: 9) METDTLLLWVLLLWVPGSTG Linker (SEQ ID NO: 10 GGGGSGGGGSGGGGSGGGGSMurine K101 scFv (SEQ ID NO: 11)DIVMSQSPSSLAVSVGEKVTLSCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPWTFGGGTKLEIKG GGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVK QSHGKSLEWIGEINPNNGDNFYNQKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYY CAKDGDYAMDYWGQGTSVTVSSHumanized scFv K101-1 (SEQ ID NO: 12)DIVMTQSPDSLAVSLGERATINCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYTYPWTFGGGTKVEIKG GGGSGGGGSGGGGSGGGGSEVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVR QAPGKSLEWIGEINPNNGDNFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYY CAKDGDYAMDYWGQGTTVTVSSHumanized scFv K101-2 (SEQ ID NO: 13)EIVMTQSPATLSLSPGERATLSCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGIPDRFSGSGSGTDFTLTISRVEPEDFAVYYCQQYYTYPWTFGGGTKVEIKG GGGSGGGGSGGGGSGGGGSEVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVR QAPGKSLEWIGEINPNNGDNFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYY CAKDGDYAMDYWGQGTTVTVSSHumanized scFv K101-3 (SEQ ID NO: 14)DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQYYTYPWTFGGGTKVEIKG GGGSGGGGSGGGGSGGGGSEVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVR QAPGKSLEWIGEINPNNGDNFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYY CAKDGDYAMDYWGQGTTVTVSSHumanized scFv K101-4 (SEQ ID NO: 15)DIVMTQSPDSLAVSLGERATINCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYTYPWTFGGGTKVEIKG GGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVR QAPGKSLEWIGEINPNNGDNFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYY CAKDGDYAMDYWGQGTTVTVSSHumanized scFv K101-5 (SEQ ID NO: 16)DIVMTQSPDSLAVSLGERATINCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYTYPWTFGGGTKVEIKG GGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVR QAPGKSLEWIGEINPNNGDNFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYY CAKDGDYAMDYWGQGTTVTVSSK101CAR including leader peptide in bold (SEQ ID NO: 17)METDTLLLWVLLLWVPGSTGDIVMSQSPSSLAVSVGEKVTLSCKSSQSLLYTSIQKN YLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC QQYYTYPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASV KMSCKASGYTFTDYYMKWVKQSHGKSLEWIGEINPNNGDNFYNQKFKGKATLTVDKS SSTAYMQLNSLTSEDSAVYYCAKDGDYAMDYWGQGTSVTVSSSDPFVPVFLPAKPTT TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PRK101-1 humanized VL (SEQ ID NO: 18)DIVMTQSPDSLAVSLGERATINCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYTYPWTFGGGTKVEIKK101-1 humanized VH (SEQ ID NO: 19) EVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGKSLEWIGEINPNNGD NFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYYCAKDGDYAMDYWGQGTTVT VSS K101-2 humanized VL (SEQ ID NO: 20) EIVMTQSPATLSLSPGERATLSCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGIPDRFSGSGSGTDFTLTISRVEPEDFAVYYCQQYYTYPWTFGGGTKVEIK K101-2 humanized VH (SEQ ID NO: 21) EVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGKSLEWIGEINPNNGD NFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYYCAKDGDYAMDYWGQGTTVT VSS K101-3 humanized VL (SEQ ID NO: 22) DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCQQYYTYPWTFGGGTKVEIK K101-3 humanized VH (SEQ ID NO: 23) EVQLQQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGKSLEWIGEINPNNGD NFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYYCAKDGDYAMDYWGQGTTVT VSS K101-4 humanized VL (SEQ ID NO: 24) DIVMTQSPDSLAVSLGERATINCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYTYPWTFGGGTKVEIK K101-4 humanized VH (SEQ ID NO: 25) EVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGKSLEWIGEINPNNGD NFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYYCAKDGDYAMDYWGQGTTVT VSS K101-5 humanized VL (SEQ ID NO: 26) DIVMTQSPDSLAVSLGERATINCKSSQSLLYTSIQKNYLAWYQQKPGQSPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYTYPWTFGGGTKVEIK K101-5 humanized VH (SEQ ID NO: 27) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMKWVRQAPGKSLEWIGEINPNNGD NFYNQKFQGRATLTVDKSSSTAYMELSSLRSEDTAVYYCAKDGDYAMDYWGQGTTVT VSS References1. 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Claims

CLAIMS1. A protein for specific binding to an epitope located on CA125 of MUC16,comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) which together form an antigen binding unit,- wherein the VL comprises three complementarity determining regions (CDRs):CDR1, CDR2 and CDR3, which respectively comprise the amino acid sequences SEQ ID NOs: 1, 2 and 3; and- wherein the VH comprises three CDRs; CDR1, CDR2 and CDR3, whichrespectively comprise the amino acid sequences SEQ ID NOs: 4, 5 and 6.

2. The protein according to claim 1, wherein both the VH and VL comprises humanor humanized framework sequences.

3. The protein according to claim 1 or 2, wherein the VL is represented by SEQ IDNO 7 or a sequence with at least 90% identity thereto, and wherein the VH is represented by SEQ ID NO: 8 or, or a sequence with at least 90% identity thereto.

4. The protein according to any of claim 1 to 3, wherein the antigen binding unit is ascFv represented by the SEQ ID NO: 11 or a sequence with at least 90% identitythereto.

5. A chimeric antigen receptor (CAR) comprising the protein as defined in any oneof claims 1 to 4.

6. A chimeric antigen receptor (CAR) comprising a scFv represented by SEQ ID NO: 13 or SEQ ID NO: 15.

7. The CAR according to claim 5 or 6, comprising from N-terminal to C-terminal, ascFv, a human CD8α hinge, a human CD8α transmembrane domain, a human 4-IBBcostimulatory domain and a human CD3ζ signaling domain.

8. The CAR according to claim 5 or 7, comprising an amino acid sequencerepresented by SEQ ID NO: 17, or a sequence with at least 90% identity thereto.

9. A nucleic acid encoding the protein according to any one of claims 1 to 4 or theCAR according to any one of claims 5 to 8.

10. A cytotoxic immune cell expressing the CAR according to any one of claims 5to 8 in its cell membrane, wherein the immune cell is a T-cell or an NK-cell.

11. A pharmaceutical composition comprising the protein according to any one ofclaims 1 to 4, the nucleic acid according to claim 9, or the cells according to claim10.

12. A pharmaceutical composition comprising the protein according to any one ofclaims 1 to 4, the nucleic acid according to claim 9, or the cells according to claim10 for use in treatment of MUC16-positive cancers.

13. A cytotoxic immune cell expressing the CAR according to any one of claims 5to 8 in its cell membrane, for use in treatment of MUC16-positive cancers.

14. A cytotoxic immune cell expressing the CAR according to any one of claims 5to 8 in its cell membrane, for use in treatment of MUC16-positive pancreatic canceror ovarian cancer.

15. A method of treatment of ovarian or pancreatic cancer comprising the steps, a) administering a cell expressing a CAR for specific binding to an epitope locatedon CA125 of MUC16 to a patient in need thereof, wherein the CAR comprises ascFv represented by any one of SEQ ID NO: 11 to 16, or a scFv represented by a sequence with more than 90% identity thereto, and b) administering a cell expressing a CAR for specific binding to an epitope locatedon MUC16ecto to the patient.

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