Dual chimeric antigen receptor construct targeting CEA and epcam

Abstract

The invention relates to a nucleic acid construct, comprising: a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) polypeptide, said dual CAR polypeptide comprising a first extracellular antigen-binding domain that binds an epithelial cell adhesion molecule (EpCAM), and a second extracellular antigen-binding domain of the dual CAR that binds a carcinoembryonic antigen (CEA) protein, and a second nucleic acid sequence region encoding an immune stimulatory cytokine. The invention further relates to a dual chimeric antigen receptor (CAR) polypeptide encoded by the nucleic acid construct. The invention further relates to a genetically modified cell, comprising said nucleic acid construct and / or said dual CAR, the medical use of said cells in the treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA and / or EpCAM in a subject and a pharmaceutical composition, in the form of a therapeutic cell product, comprising said genetically modified cells and a pharmaceutically acceptable carrier. Further, the invention relates to an in vitro method for preparing said genetically modified immune cell, comprising delivering said nucleic acid construct into a cell.

Description

[0001] DUAL CHIMERIC ANTIGEN RECEPTOR CONSTRUCT TARGETING CEA AND EpCAM

[0002] DESCRIPTION

[0003] The invention is in the field of biology and medicine, in particular the field of nucleic acid constructs for genetic modification of cells, genetically modified cells and therapeutic cell products.

[0004] The invention relates to a nucleic acid construct, comprising: a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) polypeptide, said dual CAR polypeptide comprising a first extracellular antigen-binding domain that binds a carcinoembryonic antigen (CEA) protein, and a second extracellular antigen-binding domain of the CAR that binds an epithelial cell adhesion molecule (EpCAM), and a second nucleic acid sequence region encoding an immune stimulatory cytokine.

[0005] The invention further relates to a dual chimeric antigen receptor (CAR) polypeptide encoded by the nucleic acid construct. The invention further relates to a genetically modified cell, comprising said nucleic acid construct and / or said dual CAR, the medical use of said cells in the treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA and / or EpCAM in a subject and a pharmaceutical composition, in the form of a therapeutic cell product, comprising said genetically modified cells and a pharmaceutically acceptable carrier. Further, the invention relates to an in vitro method for preparing said genetically modified immune cell, comprising delivering said nucleic acid construct into a cell.

[0006] BACKGROUND OF THE INVENTION

[0007] Targeting the vulnerabilities of cancer cells is a primary goal of personalized cancer therapies, also known as "personal medicine" or "targeted therapy". Adoptive T cell immunotherapy is considered a promising targeted anti-tumor treatment. Genetically modified T cells expressing chimeric antigen receptors (CARs) eliminate tumor cells by binding to tumor antigens through antigen-antibody recognition. These chimeric antigen receptor T (CAR-T) cells directly recognize tumor cells without the process of antigen presentation and without being restricted by the major histocompatibility complex (MHC). With CAR-T cell therapeutics, a promising gene therapy concept has been introduced into haematology and oncology.

[0008] CAR-T therapy revealed an encouraging anti-tumor effect and a high rate of complete remissions in hematological CD19+ malignancies. Researchers have performed CAR-T therapy applications in solid tumors by targeting multiple tumor-associated antigens, such as human epidermal growth factor receptor 2 (HER2), carboxylic acid anhydrase IX (CAIX), carcinoembryonic antigen (CEA), disialoganglioside (GD2), and interleukin (IL)-13-receptor alpha 2 (IL-13Ra2).

[0009] Advantages of the CAR technology are based on the fact that patient autologous T cells or donor allogenic T cells are genetically engineered ex vivo in the laboratory with a recombinant surface molecule, which on the one hand has specificity for tumor cells and on the other hand mediates activation of the T cell when the tumor cell is recognized. The prototype of this surface molecule has in the extracellular part an antigen binding domain, preferably an antibody or a scFv single chain antibody or antibody fragment, for the recognition of an antigen on tumor cells, and in the intracellular part a signal chain for T cell activation, preferably the CD3 chain of the T cell receptor (TCR). A transmembrane domain anchors the molecule in the cell membrane. Since the surface molecule is composed of an antibody (fragment) on the one hand and a T cell signalling unit on the other, it is referred to as a "chimeric antigen receptor" (CAR). The CARs are therefore recombinant surface receptors which, in contrast to the natural T cell receptors (TCR), recognise their target antigens by means of an antibody and independently of the HLA (human leukocyte antigen) complex.

[0010] The genetic information for the respective CAR has so far mostly been introduced into the patient's or donor’s T cells by means of retro- or lentiviral vectors by transduction ex vivo, which then express the CAR on the surface. The CAR-T cells are reinfused into the patient and bind to specific antigens that are formed on the surface by cancer cells. A "first generation" CAR has a signalling chain for primary T cell activation, usually the CD3 signalling chain; a "second generation" CAR uses the CD3 chain together with a costimulatory unit, preferably from the CD28 family, to provide a sustained T cell activation.

[0011] Although some efficacy was observed in certain studies with these CAR generations, including complete regression of a single glioblastoma case treated with multiple infusions of IL-13Ra2 CAR-T cells, the overall outcome of CAR-T cell therapy, in particular in solid tumors, was not sufficient in a large number of studies. Another important issue of CAR-T therapy are adverse events of a cytokine release syndrome and on-target / off-tumor effects. CAR-T therapy can also cause neurologic side effects such as speech problems, tremors, delirium, and seizures. Therefore, a CAR re-design with the goal of creating a safer and more effective therapy is needed to improve safety and efficacy of the CAR-T cell therapy.

[0012] There are some aspects critical for an effective CAR-T cell strategy in the treatment of solid tumors. Among the antigens of solid tumors, that CAR-T cell therapy targets, are CEA, EGFR, EGFRvlll, GD2, HER2, IL13Ra2, PSCA, Tn-MUC1 , and PSMA. While all these antigens are either overexpressed and / or amplified in tumors compared to normal tissue, their protein expression is not clearly restricted to tumor cells, with the exception of EGFRvlll, a common oncogenic rearrangement in glioblastoma characterized by deletion of exons 2-7 of EGFR. Therefore, in contrast to CD19, a B-cell restricted antigen expressed in many B-cell malignancies, antigen targets of solid tumors give rise to significant concerns about toxicity, which may limit their usefulness in CAR-T cell therapy. Thus, tumor specificity of the antigen selected is one critical aspect in CAR design for an effective and safe CAR-T cell strategy.

[0013] Soto et. al (Frontiers in Immunology, 2023, vol.14) discloses a Dual-RevCAR system for an AND- gated combinatorial targeting of tumor cells, comprising T-cells which comprise two individual CARs binding to CEA and EpCAM, respectively. The system additionally comprises two bifunctional adaptor molecules (RevTM) each binding to one of the individual CARs and the respective antigen on the surface of the targeted cell. The T-cells are only activated in the presence of both RevTM and both antigens on the surface of a target cell. The system therefore employs a highly complex combination of components that must be simultaneously present to show a cytotoxic effect against the target cells. This results not only in a complex pharmacokinetics and treatment regime but also in a time-consuming and costly manufacturing process due to the various components of the system. In particular, both RevTM must be present to activate both CARs of the T-cells. It is disclosed in Soto et. al that, for a prolonged tumor suppression in vivo, both RevTM have to be continuously or repeatedly injected. This results in higher treatment costs and potentially reduced patient compliance and discomfort. Further, as described, one challenge in solid tumor treatment is antigen loss and / or heterogeneity of antigens within the tumor. As the system of Soto et al., is solely activated in the presence of both CEA and EpCAM, the system might not be effective against tumor cells comprising only one of both antigens on their surface due to antigen loss or heterogeneity.

[0014] Bangayan et al. (Proceedings of the National Academy of Sciences, 2023, vol. 120, no. 47) discloses an AND-NOT gate system comprising T-cells which comprise an activating CAR recognizing CEA as a tumor antigen and an inhibitory CAR (iCAR) recognizing TROP2 as a normal tissue antigen. In this manner, the CAR-T-cell can distinguish a tumor cell from a normal cell expressing the same CAR target antigen. The iCAR comprises an intracellular PD-1 signalling domain or a dual inhibitory domain comprising a PD-1 signalling domain and a LAIR-1 or SEIGLEC-9 domain.

[0015] Another difficulty in tumor treatment is tumor resistance to individual therapeutics, since the majority of tumors are heterogeneous, meaning that several antigens are present on tumour cells and the tumor cells can use various (escape) mechanisms to evade a therapy targeting one antigen. Prolonged targeting of a single drug-sensitive pathway may ultimately lead to drugresistant tumor recurrence and escape variants. Acquired or intrinsic resistance patterns have been observed after CAR-T cell therapy. CD19 CAR-T cell therapy has shown sustained clinical remissions in 70-90% of patients with B-cell malignancies, including acute lymphoblastic leukemia (ALL), but recent follow-up data from clinical trials show a common mechanism of resistance, including loss and / or downregulation of CD19 antigen in up to 70% of patients who relapse after treatment. Early clinical findings using CAR-T cells in solid tumors have observed similar resistance mechanisms of antigen leakage. There is strong evidence that a rational design combining tumor specific antigen targeting with other anti-tumor strategies will be necessary for effective disease control.

[0016] There have been approaches disclosed in the prior art for expression of two CARs in one cell (tandem cells) or dual CARs targeting two antigens, that might overcome the hurdle of tumor cell heterogeneity. Such approaches are for example disclosed in WO 2021158783 A1 , US2020115461 A1 , US 11161907 B2, WO2023158978A2 and EP4135726A1. However, such dual CARs or the expression of two different CARs in one cell are usually problematic as the CARs can interfere with each other's expression or even eliminate each other, hindering effective cancer therapy by a dual CAR or tandem CAR T cell. Further, it remains unclear from the prior art which combinations of antigens provide the most effective strategy for overcoming tumor heterogeneity and prevention of tumor escape mechanisms, in particular in the treatment of solid tumors.

[0017] An immunosuppressive tumor microenvironment is another challenge for an effective immunotherapy using a CAR. CAR vectors known in the prior art are barely able to overcome the immunosuppressive microenvironment in solid tumours. Unlike most hematological malignancies, there are no local immunosuppressive pathways that impede anti-tumor immunity and limit adoptive T cell therapies. Solid tumors can be strongly infiltrated by multiple cell types that support tumor growth, vascularization, and metastasis and may control therapeutic responses. In addition to the tumor cells themselves, the immune cells, such as regulatory T cells and myeloid suppressor cells, induce local cytokine, chemokine, and growth factor production in solid tumors, including IL-4, IL-10, VEGF and TGFp. Similarly, immune checkpoint pathways, including PD-1 and CTLA-4, can be highly active in tumors to attenuate anti-tumor immunity. There is strong evidence that the microenvironment of the tumor controls the response and resistance to immune therapies and may limit the effectiveness of CAR-T cell therapy.

[0018] There are approaches in the prior art to enhance cell proliferation and / or survival, T cell cytotoxicity and efficacy of CAR T-cell therapies in the presence of antigen-expressing target cells.

[0019] WO 2018 / 023093 A1 discloses immunomodulatory polypeptides containing a first and second peptide such as a subunit of a cytokine or chemokine connected by a joining region containing a moiety binding to a target region. Further, T-cells containing the immunomodulatory polypeptide and a genetically engineered antigen receptor, such as a CAR, are disclosed. WO 2022 / 043312 A1 discloses a recombinant nucleic acid construct comprising a first nucleic acid sequence region encoding a CAR that comprises an extracellular antigen-binding domain recognizing a CEA protein, a second nucleic acid sequence region encoding a checkpoint inhibitory molecule, and a third nucleic acid sequence region encoding an immune stimulatory cytokine.

[0020] A further challenge and major concern with CAR-T therapy is the danger of a “cytokine storm” associated with intense antitumor responses mediated by large numbers of activated T cells (Sadelain et al., Cancer Discov 3:388-98, 2013). Side effects can include high fever, hypotension and / or organ failure, potentially resulting in death. In contrast, NK cells, a distinct class of lymphocytes, offer a promising alternative for immunotherapy. NK cells, which comprise about 10-20% of peripheral blood lymphocytes, are characterized by their ability to recognize and kill tumor cells through various receptors.

[0021] Various immunotherapeutic applications of NK cells, including cytokine therapy, adoptive cell therapy (ACT), genetically engineered NK cell therapy (CAR-NK), monoclonal antibody therapy, and NK cell engagers, demonstrate their versatility in combating cancer. CAR-NK therapy, in particular, has gained attention for its improved safety profile compared to CAR-T therapy. Unlike CAR-T, NK cells exhibit inherent advantages, such as superior safety by minimizing the risk of the common adverse effect associated with CAR-T cell therapy - the cytokine release storm (CRS). This is because NK cells secrete different cytokines, such as IFN-gamma and GM-CSF, compared to the pro-inflammatory cytokines involved in CRS.

[0022] Overall, there is no CAR cell therapy in the prior art that overcomes all major hurdles of solid tumor therapy, i.e., (1) tumor antigen heterogeneity and escape, (2) inactivation by the immunosuppressive tumor microenvironment, (3) targeting accuracy, (4) low, ineffective and noncombined activation, and (5) side effects associated in particular with CAR T cell therapy such as cytokine release storm and that thus enables an effective and safe treatment of a solid tumor. In light of the disadvantages outlined above and the inherent difficulties in developing cellular cancer therapeutics that address the major challenging areas requiring attention in CAR cell therapy of solid tumors, the field is in urgent need of novel means for improving the activity and ultimately efficacy of cellular therapies, in order to overcome difficulties in solid tumor treatment.

[0023] SUMMARY OF THE INVENTION

[0024] In light of the prior art the technical problem underlying the invention was the provision of alternative or improved means for immune cell therapy of cancer, such as for the treatment of solid tumors.

[0025] A further problem underlying the invention is the provision of suitable means for immune cell therapy for the treatment of cancer, preferably solid cancer, which overcome tumor antigen heterogeneity and escape mechanisms, inactivation by the immunosuppressive tumor microenvironment and side effects associated with cell therapy, such as cytokine release storm, and have improved targeting accuracy.

[0026] Another problem of the present invention is the provision of suitable means for immune cell therapy for the treatment of solid cancer, which overcome tumor antigen heterogeneity, escape mechanisms and inactivation by the immunosuppressive tumor microenvironment and result in an efficient and long-term immune response.

[0027] A further problem underlying the invention is the provision of means for stimulation of a targeted immune response directed against a tumor tissue in a subject with less tumor escape from immunotherapy and low, inefficient, and non-combined activation of immune response.

[0028] A further problem underlying the invention is the provision of a checkpoint inhibitor and resultant stimulation of the immune response. This provides an effective local effect at the targeted tumor tissue with reduced levels of unwanted side effects.

[0029] Another object of the invention is the provision of immune cell therapies for the treatment of cancer, in particular solid tumors, that can be produced off the shelf in a time- and cost-efficient, simple and reliable manner. Another object of the invention is the provision of immune cell therapies for the treatment of cancer, in particular solid tumors, that can be administered in a simple treatment regime and do not require frequent re-administration, resulting in patient comfort and compliance.

[0030] These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided in the dependent claims.

[0031] In one aspect, the present invention relates to a nucleic acid construct, comprising: a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) polypeptide, said dual CAR polypeptide comprising a first extracellular antigen-binding domain that binds an epithelial cell adhesion molecule (EpCAM), and a second extracellular antigen-binding domain of the dual CAR that binds a carcinoembryonic antigen molecule (CEA), and a second nucleic acid sequence region encoding an immune stimulatory cytokine.

[0032] The nucleic acid construct of the present invention enables preparation of genetically modified cells that are surprisingly effective and specific in the treatment of tumors, in particular in the treatment of solid tumors. In particular, solid tumors are often antigenically diverse and cannot be effectively eliminated by immune cell therapies of the prior art, e.g., CAR-T cells designed to target one tumor antigen. One of the most challenging design aspects for solid tumor immunotherapies is thus to provide a specific therapy that targets the tumor cells but overcomes antigen loss or heterogeneity of antigens within the tumor.

[0033] The nucleic acid construct of the present invention, encoding a single CAR polypeptide molecule comprising two distinct antigen binding domains binding to EpCAM and CEA, respectively, surprisingly enables the provision of genetically modified cells that are specific for tumors expressing CEA and / or EpCAM, while being surprisingly effective within the immunosuppressive microenvironment of a tumor. In contrast to the prior art, in particular to Soto et al. (Frontiers in Immunology, 2023), which discloses a complex system comprising two distinct CAR polypeptides each targeting either CEA or EpCAM, and which is solely activated when both antigens are present on the surface of a cell, the CAR polypeptide of the present invention may not only target cells comprising both CEA and EpCAM but also cells comprising either CEA or EpCAM on their surface, thereby in contrast to the prior art overcoming tumor heterogeneity and antigen escape of solid tumors and thus showing higher treatment efficiency in such tumors.

[0034] Advantageously, the CAR polypeptide encoded by the nucleic acid construct of the present invention and cells comprising such CAR polypeptide are solely activated by the presence of the antigens CEA and / or EpCAM on the surface of a target cell and thus do not require the presence or administration of any additional means for activation. In contrast the CAR-system of Soto et al. (Frontiers in Immunology, 2023) requires the additional administration of two distinct bifunctional adaptor molecules, which must simultaneously be present with the cells expressing the two distinct CARs at the site of action for activation of the cytotoxic effect of the system.

[0035] The nucleic acid construct of the present invention thus advantageously provides a simple and straight-forward, time- and cost-effective (in manufacturing) construct that can be easily transferred into the respective cells to express the CAR targeting CEA and EpCAM. There is advantageously no need for the manufacture of additional means such as multiple adaptor molecules, which require additional manufacturing steps and quality control and result in longer manufacturing time and costs. Further, by the nucleic acid construct of the present invention cells expressing the CAR encoded can be produced and administered to a subject in a simple manner resulting in high treatment efficiency without the need for administration of any additional activation means. This advantageously results in simpler pharmacokinetics and treatment regime and higher patient comfort and compliance compared to the complex systems of the prior art, such as Soto et al., requiring the additional continuous or repeated administration of two adaptor molecules.

[0036] By providing two distinct antigen targeting domains within a single CAR polypeptide molecule the CAR polypeptide can advantageously be encoded in a compact manner such that a single nucleic acid construct is provided encoding both the CAR polypeptide, the immune stimulatory cytokine and optionally further components. Thereby, the construct can be provided not only as a plasmid, commonly used for CAR expression in cells, but also other types of nucleic acids such as mRNA, which are more safely, easily, reproducible and faster produced and transfected in cells.

[0037] The inventors have discovered that the high efficiency and specificity of the inventive immune cells in comparison to the immune cell therapies of the prior art is mainly related to (1) overcoming of tumor heterogeneity and related escape mechanisms by the dual CAR specific for CEA and EpCAM and (2) modification of the local immunosuppressive tumor microenvironment by simultaneous expression of an immune stimulatory cytokine within the genetically modified cells, resulting in efficient killing and eradication particularly of heterogenous tumors. By recognizing the two distinct antigens CEA and EpCAM on the surface of cancer cells, the dual CAR mediates accurately identifying malignant cells while sparing healthy tissue. Simultaneous binding to two antigens further leads to stronger or synergistic activation of the immune response, enhancing its cytotoxic effect and leading to more efficient tumor killing. The combination of CEA and EpCAM targeting has proven to be particularly effective and advantageous, as shown in example 2.

[0038] The responsiveness of solid tumor patients to immune cell therapies such as CAR-T cell therapies of the prior art is usually low and highly variable due to intrinsic and / or acquired tumor resistance to current CAR-T regimens. In contrast, the nucleic acid construct of the present invention enables the provision of genetically modified cells making patients with solid tumors expressing CEA and / or EpCAM receptive to immune cell therapy. Thus, the invention advantageously transforms immune cell therapy for such tumors from non-curative to curative by providing a nucleic acid construct encoding for a dual anti-CEA / anti-EpCAM CAR and an immune stimulatory cytokine. Patient groups not previously treatable may now be effectively treated. Additionally, due to the increase in efficacy expected through the present approach, reduced numbers of therapeutic cells are expected to be necessary to induce the desired medical effects. Thus, the nucleic acid construct encoding the dual CAR and the immunostimulatory cytokine and the cells modified therewith, represent a novel combination of features, sufficient to distinguish the invention from the prior art. A novel clinical situation is created with direct implications for the treatment of novel patient groups and / or for administration regimes.

[0039] Further, the present invention enables the stimulation of cells involved in an anti-tumor immune response and thereby the local activation, support and / or strengthening of an anti-tumor immune response. The present invention enables an effective and therapeutically relevant dose of one or more immune stimulatory cytokines to be administered via expression from a nucleic acid construct in transplanted genetically modified cells while avoiding the significant side effects that are inherent in systemic administration of cytokines without an appropriate targeting agent. The invention therefore relates to the utilization of genetically modified cells as a targeting agent and / or vehicle for the local delivery of immune modulatory, preferably immune stimulating signals in regions of inflammation, preferably in and in proximity to tumor tissue.

[0040] The ability of tumors to escape and / or suppress the natural immune response against the tumor cells, by establishing an immunosuppressive tumor microenvironment crucially limits the effectivity of immune cell therapies against cancer, in particular against solid tumors. This phenomenon is known as tumor-mediated immunosuppression and is mediated to a large extent by the secretion of anti-inflammatory cytokines by immune cells and checkpoint proteins present in the tumor that display a regulatory phenotype (for example, regulatory T cells and monocyte- derived suppressor cells). The invention advantageously provides means to modify the tumor microenvironment, making it pro-inflammatory, promoting the activation of immune cells present in the tumor and recruitment and activation of external immune cells and thereby facilitating the broad activation of the immune system against the tumor and / or enhance the efficacy of antitumor immunotherapeutic treatments.

[0041] The expression of a stimulatory cytokine as described herein by cells modified by the nucleic acid construct according to the present invention advantageously supports an anti-tumor immune response and leads to reduction in tumor size and / or growth, and shows a distinct reduction in and / or avoidance of the side effects produced by systemic administration of such cytokines known in the art. Side effects such as nausea and vomiting, sores in the mouth or on the lips, diarrhea, drowsiness, allergic reactions, fever or chills, hives, itching, headache, coughing, shortness of breath, or swelling of the face, tongue, or throat, may be avoided by cells genetically modified by the nucleic acid construct according to the present invention.

[0042] The present invention therefore provides means for reducing the side effects of cytokine therapy, and the concomitant use of cytokines with immunotherapies, by enabling local (or locally confined) tumor-specific effects, achieved preferably by systemic administration of the cells, but exerted in a tissue specific manner via cell therapy using cells genetically modified by the nucleic acid construct according to the present invention and expressing the dual CAR that further comprise and express said cytokines under the appropriate tissue-specific conditions.

[0043] As such, the dual CAR of the present invention in combination with the expression of an immune stimulatory cytokine in a cell overall represents a surprising and beneficial approach towards the treatment of the medical conditions described herein. The employment of dual anti-CEA / anti- EpCAM CARs without the need for additional activation means such as adaptor molecules has not been previously attempted or described as a promising approach towards treating solid tumors, more preferably tumors positive for CEA and / or EpCAM. The minimal (if not non-existent) unwanted side effects, due to the selectivity of the dual CAR, also represent a beneficial and surprising aspect of the present invention. In particular, in patients, in which resistance to other solid tumor treatments have arisen, the present invention represents a very promising approach towards eradication of malignancies.

[0044] In one embodiment, the immune response-stimulating cytokine maintains or enhances the activity, survival and / or number of immune cells within and / or in proximity to tumor tissue.

[0045] In one preferred embodiment, the dual chimeric antigen receptor (CAR) preferably recognizes CEA in the membrane-bound form over the soluble form.

[0046] CEA proteins exist in a soluble and a solid form in mammalians, preferably humans. Only the solid form is found on the tumor cell membrane whereas the soluble form plays a role in endothelial cell activation and angiogenesis. A skilled person would not expect that this particular dual anti-CEA / anti-EpCAM CAR could be expressed combined with immune stimulatory cytokines in sufficient quantities to induce or enhance the desired local immune response, based on immunotherapy, and only targeting CEA positive and / or EpCAM pathogenic cells tumors, preferably solid tumor cells. The development of the dual anti-CEA / anti-EpCAM CAR described herein that distinguishes between the two CEA forms is an exceptionally and technically complex solution to the invention.

[0047] In one embodiment the first nucleic acid sequence region encoding the dual CAR comprises: a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to CEA protein, said antigen-binding domain comprising an antibody or antibody fragment, a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to EpCAM, said antigen-binding domain comprising an antibody or antibody fragment, a nucleic acid sequence linking the nucleic acid sequences encoding said extracellular antigen-binding domains, and a nucleic acid sequence encoding a hinge domain, a transmembrane domain and a signaling domain.

[0048] In any of the nucleic acid constructs encoding the first and second antigen binding domain of the dual CAR described herein, the first and second antigen binding domains of the dual CAR are connected to the transmembrane domain by a hinge region. In any of the nucleic acids encoding the transmembrane domain of the described herein, the transmembrane domain of the dual CAR comprises a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0049] In any of the nucleic acids encoding the signaling domain of the dual CAR described herein, the signaling domain comprises a costimulatory signaling domain comprising a functional signaling domain obtained from a protein selected from the group consisting of a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1 , LFA-1 (CD11a / CD18), 4-1 BB (CD137), B7-H3, CDS, ICAM-1 , ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 , ITGAM, CD11 b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, CD18, LFA-1 , ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a.

[0050] In one embodiment, at least the first nucleic acid sequence region encoding the dual CAR is constitutively expressed by a promoter or promoter / enhancer combination, preferably selected from the group consisting of a Spleen Focus Forming Virus (SFFV) promotor, EF1 alpha promoter (also termed EF1a promoter, for example the EFI alphaS promoter), PGK promoter, CMV promoter, SV40 promoters, GAG promoter and UBC promoter, more preferably a EF1a promoter.

[0051] In one embodiment the second nucleic acid sequence region encoding an immune stimulatory cytokine comprises a nucleic acid sequence encoding one or more immune stimulatory cytokines operably linked to one or more promoters, wherein at least one of said cytokines is selected from the group consisting of IL-15, IL-2, IL-7, IL-12, IL-21 , IFN gamma, and IFN beta, preferably an IL- 15 superagonist. In one embodiment the second nucleic acid sequence region encoding an immune stimulatory cytokine is operably linked to one or more constitutive promoters, preferably a human promotor, more preferably an NFAT promotor.

[0052] The use of a "tumor-specific" promoter, or promoter preferentially expressed or induced under inflammatory or "cancer-like" conditions, may show a synergistic effect in combination with the dual CAR-cells such as CAR-NK cells recruitment signal with respect to reduction of unwanted systemic effect. The dual CAR-cells of the present invention migrate towards inflammatory, in particular tumor, tissue, thereby providing effective means for avoiding systemic expression of the encoded cytokine or checkpoint inhibitor in the body of a patient. The use of a promoter for the expression of the cytokine that is preferentially expressed under conditions of inflammation or of being present in tumor tissue further enhances the reduction in systemic expression in a synergistic manner, thereby providing surprising benefits in the T cell or NK cell-based mode of administration of the cytokines described herein.

[0053] In one embodiment the immune stimulatory cytokine maintains or enhances the activity, survival and / or number of immune cells within and / or in proximity to tumor tissue.

[0054] In one embodiment the second nucleic acid sequence region encoding an immune stimulatory cytokine comprises a nucleic acid sequence encoding one or more immune stimulatory cytokines operably linked to one or more promoters, wherein at least one of said cytokines is selected from the group consisting of IL-15, IL-2, IL-7, IL-12, IL-21 , IFN gamma, and IFN beta, preferably an IL- 15 superagonist, wherein preferably the second nucleic acid sequence region encoding the immune stimulatory cytokine is operably linked to one or more constitutive promoters, more preferably a human promotor, more preferably an NFAT promotor, and wherein preferably the immune stimulatory cytokine maintains or enhances the activity, survival and / or number of immune cells within and / or in proximity to tumor tissue.

[0055] In a preferred embodiment the immune response-stimulating cytokine is IL-15. In a preferred embodiment the immune response-stimulating cytokine is an IL-15 superagonist.

[0056] In one embodiment the immune stimulatory cytokine is an IL-15 superagonist according to SEQ ID NO 18, or a sequence with at least 80% sequence identity to SEQ ID NO 18.

[0057] In embodiments, the sequence variants with 80% or more sequence identity to the immune response-stimulating cytokine sequences listed herein maintain IL-15 agonistic activity with essentially the same or similar functional properties of immune response stimulation binding with the specific sequences recited herein.

[0058] In one embodiment the nucleic acid construct additionally comprises a third nucleic acid sequence region encoding a checkpoint inhibitory molecule.

[0059] In one embodiment the checkpoint inhibitory molecule encoded by the third nucleic acid sequence region is a dominant negative polypeptide and / or an antibody inhibiting and / or blocking an immune checkpoint protein, wherein the checkpoint inhibitory molecule is preferably a dominant negative truncated PD1 polypeptide or a PD1-antibody.

[0060] In one embodiment the checkpoint inhibitor molecule is a dominant negative truncated PD1 polypeptide according to SEQ ID NO 41 , or a sequence with at least 80% sequence identity to SEQ ID NO 41. In embodiments, the sequence variants with 80% or more sequence identity to the dominant negative truncated PD1 sequences listed herein maintain PD1 binding with essentially the same or similar functional properties PD1 protein binding with the specific sequences recited herein, i.e. the PD1 binding is essentially the same or similar with respect to affinity, specificity and binding mode.

[0061] Normally, cells that are potentially cancerous are destroyed by the immune system. All cancer cells undergo changes that differentiate them from their neighbours, the most obvious change being the ability to multiply without inhibition. Cancer cells utilize mechanisms that avoid regular immune system control. Checkpoint proteins have been shown to function by communicating to the immune system that a potentially cancerous cell is not to be destroyed. There may be other molecules signalling that the cell is cancerous, but if there are enough checkpoint proteins on the cell surface, the immune system may overlook cancerous signals.

[0062] A ligand-receptor interaction that has been investigated as a target for cancer treatment is the interaction between the transmembrane programmed cell death 1 protein (PD1 ; also known as CD279) and its ligand, PD1 ligand 1 (PD-L1 ; also known as CD274). In normal physiology PD-L1 on the surface of a cell binds to PD1 on the surface of an immune cell, which inhibits the activity of the immune cell. It appears that up-regulation of PD-L1 on the cancer cell surface may allow them to evade the host immune system by inhibiting T cells that might otherwise attack the tumor cell. Antibodies that bind to either PD-1 or PD-L1 and therefore block the interaction may allow the T cells to attack the tumor.

[0063] In a preferred embodiment, the medical use of the genetically modified cells as described herein, comprising a nucleic acid construct according to the present invention, is characterized by the combined transgenic expression of a checkpoint inhibitor, preferably a PD-L1 and / or PD-1 inhibitor, with said dual CAR and immune-stimulatory cytokine.

[0064] The invention advantageously encompasses the expression of a combination of immune activating cytokine and / or checkpoint inhibitory molecules in tumors via genetically modified cell described herein, with the aim to attract immune effector and helper cells, induce immune activation, promote the maturation of memory immune cells and / or suppress the emergence and persistence of suppressive and / or regulatory immune cells in the tumor microenvironment.

[0065] The combined administration of the dual CAR-T cells expressing an immune stimulating cytokine that induces T cell proliferation and / or differentiation together with a checkpoint inhibitor leads to a synergistic effect with respect to the desired anti-cancer effect of the dual CAR-cell. The cytokine or other immune stimulator provides local enhancement of the T cell response against the cancer tissue, whilst the checkpoint inhibitor also enables the T cells to more effectively attack and destroy cancerous tissue. The effects of these two agents are combined in a synergistic manner, resulting in a technical effect greater than the sum of these two aspects when considered alone.

[0066] In one embodiment, a combination of the dual CAR targeting CEA and EpCAM, the immune stimulating cytokine(s), and / or the checkpoint inhibitory molecules is used, in order to promote the activation of different arms of the immune response, including the innate and adaptive immune response, effector, helper, and / or antigen presenting cells. On the other hand, cytokines such as IL-2, IL-7, IL-15 and IL-21 specifically activate cytotoxic lymphocytes such as T cells and NK cells that mount a specific response against tumor cells. Likewise, IL-15 will activate cytotoxic lymphocytes, but also monocytes and helper cells.

[0067] A combination of the tumor specific dual CAR specific for CEA and EpCAM, the immune stimulatory cytokine, and the checkpoint inhibitory molecules therefore yields synergistic effects, as or beyond what is seen in the natural immune response, immunotolerant tumor microenvironment, and / or low rate of tumor escape. The present invention increases therapeutic efficacy, tumour targeting accuracy, and tumor regression rates.

[0068] By providing a nucleic acid construct encoding the tumor specific dual CAR, the immune stimulating cytokine(s), and the checkpoint inhibitory molecule for genetic modification of cells, such as T cells and NK cells, advantageously a locally more effective and safe anti-tumor response can be obtained by these genetically modified cells. The combination leads to unique local expression and secretion of the immune-stimulating factors that leads to a local anti-tumor response, comprising multiple arms of the immune response, without inducing systemic toxicity as is often observed when systemically applying cytokines in tumor patients.

[0069] In one embodiment the first nucleic acid sequence region encoding the dual CAR and the third nucleic acid sequence region encoding the checkpoint inhibitory molecule, are configured to encode a polycistronic mRNA comprising coding regions for the polypeptide sequences of the dual CAR and the checkpoint inhibitory molecule, and wherein an amino acid sequence comprising a polypeptide cleavage site is disposed between the dual CAR polypeptide and the checkpoint inhibitory molecule polypeptide, wherein the polypeptide cleavage site is preferably selected from the group consisting of P2A, T2A, E2A and F2A.

[0070] In one embodiment the first nucleic acid sequence region encoding the dual CAR and the third nucleic acid sequence region encoding the checkpoint inhibitory molecule, are configured to encode a polycistronic mRNA comprising coding regions for the polypeptide sequences of the dual CAR and the checkpoint inhibitory molecule, and wherein an amino acid sequence comprising the polypeptide cleavage site P2A is disposed between the dual CAR polypeptide and the checkpoint inhibitory molecule polypeptide.

[0071] In some embodiments, the polypeptide cleavage site is selected from the group consisting of P2A, T2A, E2A and F2A.

[0072] In one embodiment the checkpoint inhibitory molecule encoded by the third nucleic acid region comprises: a. a dominant negative truncated PD1 according to SEQ ID NO 41 , or a sequence with at least 80% sequence identity to SEQ ID NO 41 , b. wherein said checkpoint protein is positioned adjacently to a polypeptide cleavage site for cleaving the checkpoint inhibitory molecule from the dual CAR polypeptide, said cleavage site preferably selected from the group consisting of P2A, T2A, E2A and F2A.

[0073] Advantageously the nucleic acid construct of the present invention encodes for the dual CAR specific for CEA and EpCAM, the immune stimulatory cytokine and the checkpoint inhibitory molecule. By including nucleic acid regions encoding for polypeptide cleavage sites the multiple polypeptides (dual CAR, immune stimulatory cytokine and the checkpoint inhibitory molecule) can be delivered by a single nucleic acid construct, such as a plasmid and even more compact nucleic acids such as mRNA, advantageously ensuring delivery and expression of all polypeptides encoded by the cell to be genetically modified, while both extracellular antigenbinding domains contemporaneous efficiently target its respective antigens. It was entirely surprising for a person skilled in the art that the dual CAR, the immune stimulatory cytokine and the checkpoint inhibitory can be encoded in one nucleic acid construct and still show sufficient expression of all polypeptides encoded in a cell genetically modified by said construct.

[0074] In one embodiment, the recombinant nucleic acid expression construct comprises nucleic acid sequence regions encoding: a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) polypeptide, said dual CAR polypeptide comprising a first extracellular antigen-binding domain that binds an epithelial cell adhesion molecule (EpCAM), and a second extracellular antigen-binding domain of the dual CAR that binds a carcinoembryonic antigen (CEA) protein. a second nucleic acid sequence region encoding an immune stimulatory cytokine, and a third nucleic acid sequence region encoding a checkpoint inhibitory molecule.

[0075] In one embodiment, the recombinant nucleic acid expression construct comprises nucleic acid sequence regions encoding: a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) polypeptide, said dual CAR polypeptide comprising a first extracellular antigen-binding domain that binds an epithelial cell adhesion molecule (EpCAM), and a second extracellular antigen-binding domain of the dual CAR that binds a carcinoembryonic antigen (CEA) protein. a second nucleic acid sequence region encoding an IL-15 superagonist, and a third nucleic acid sequence region encoding a dominant negative truncated PD1 .

[0076] In one embodiment, the recombinant nucleic acid expression construct comprises nucleic acid sequence regions encoding: a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) polypeptide, said dual CAR polypeptide comprising a first extracellular antigen-binding domain that binds an epithelial cell adhesion molecule (EpCAM), and a second extracellular antigen-binding domain of the dual CAR that binds a carcinoembryonic antigen (CEA) protein. a second nucleic acid sequence region encoding an IL-15 superagonist according to SEQ ID NO 18, or a sequence with at least 80% sequence identity to SEQ ID NO 18, and a third nucleic acid sequence region encoding a dominant negative truncated PD1 according to SEQ ID NO 41 , or a sequence with at least 80% sequence identity to SEQ ID NO 41.

[0077] In one embodiment the nucleic acid construct optionally comprises: a further nucleic acid sequence region encoding a suicide gene, preferably a herpes simplex virus thymidine kinase, and / or a further nucleic acid sequence region encoding beta-2-microglobulin.

[0078] The expression of a recoded variant of beta-2-microglobulin allows seamless integration of the construct in the beta-2-microglobulin locus. This recoded beta-2-microglobulin sequence ensures low levels of cell surface MHC class I molecules sufficient to promote NK cell propagation without significant fratricide, as is the case when MHC class I is very low or missing.

[0079] The invention further provides means to optionally switch off genetically modified cell products expressing the dual CAR according to the present invention, as described here, i.e. for safe clinical use of genetically modified cell according to the present invention and cell products based thereon. This safety feature can be realized, in some embodiments, by an inducible suicide gene.

[0080] A suicide gene, as non-limiting examples, is one that codes for the thymidine kinase of an alpha herpesvirus (HHV1-3), the bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the highly toxic compound 5-fluorouracil, and inducible caspase-9 or caspase-8. Inducible caspase-9 can be activated by a specific chemical inducer of dimerization (CID). Suicide genes may also be polypeptides that are expressed on the cell surface and can make the cells sensitive to therapeutic monoclonal antibodies. The suicide gene expression may be inducible, for example by doxycyclin adapted to human cells.

[0081] In another embodiment, the genetically modified cells additionally comprise one or more immune suppression defeating proteins and / or an inducible suicide gene that provokes death of the dual CAR-cells according to the present invention allowing their selective destruction. In some cases, it may be desirable to provide a safety mechanism that allows selective deletion of the administered genetically modified cells, as the cells can spread and persist for years after administration. Therefore, the method of the invention in some embodiments may include the transformation of the cells with a recombinant suicide gene. This recombinant suicide gene is used to reduce the risk of direct toxicity and / or uncontrolled proliferation of these cells after administration to a subject. Suicide genes enable the selective deletion of transformed cells in vivo. In particular, the suicide gene has the ability to convert a non-toxic prodrug into a cytotoxic drug or to express the toxic gene expression product. In other words, "suicide gene" is preferably a nucleic acid that codes for a product, whereby the product itself or in the presence of other compounds causes cell death. In one embodiment, the suicide gene is the herpes simplex virus thymidine kinase.

[0082] In one embodiment the first nucleic acid sequence region encoding the dual CAR comprises: a. a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to CEA protein, according to SEQ ID NO 11 , or a sequence with at least 80% sequence identity to SEQ ID NO 11 ; b. a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to EpCAM, according to SEQ ID NO 9, or a sequence with at least 80% sequence identity to SEQ ID NO 9; c. a nucleic acid sequence linking the nucleic acid sequences encoding said extracellular antigen-binding domains, according to SEQ ID NO 10, or a sequence with at least 80% sequence identity to SEQ ID NO 10, and d. a nucleic acid sequence encoding a hinge domain, a transmembrane domain and a signalling domain, according to SEQ ID NO 12, or a sequence with at least 80% sequence identity to SEQ ID NO 12.

[0083] In a further aspect the invention relates to a dual chimeric antigen receptor (CAR) polypeptide encoded by the nucleic acid construct according to the present invention.

[0084] In one embodiment, the invention relates to a dual chimeric antigen receptor (CAR) polypeptide as described herein, wherein the antigen-binding domain that binds to CEA protein comprises a variable heavy chain (VH), said VH comprising: a heavy chain complementary determining region 1 (H-CDR1) according to SEQ ID NO 30 (GFTISSGYS), a heavy chain complementary determining region 2 (H-CDR2) according to SEQ ID NO 31 (IQYSGIT), and a heavy chain complementary determining region 3 (H-CDR3) according to SEQ ID NO 32 (AREDYDYHWYFDV), and a variable light chain (VL), said VL comprising: a light chain complementary determining region 1 (L-CDR1) according to SEQ ID NO 34 (SSVSY), a light chain complementary determining region 2 (L-CDR2) according to SEQ ID NO 35 (STS), and a light chain complementary determining region 3 (L-CDR3) according to SEQ ID NO 36 (HQWSSYPT).

[0085] In one embodiment, the antigen-binding domain that binds to EpCAM comprises a variable heavy chain (VH), said VH comprising: a heavy chain complementary determining region 1 (H-CDR1) according to SEQ ID NO 22 (GGTFSSYA), a heavy chain complementary determining region 2 (H-CDR2) according to SEQ ID NO 23 (IIPIFGTA), and a heavy chain complementary determining region 3 (H-CDR3) according to SEQ ID NO 24 (ARGLLWNY), and a variable light chain (VL), said VL comprising: a light chain complementary determining region 1 (L-CDR1) according to SEQ ID NO 26 (QSVSSN), a light chain complementary determining region 2 (L-CDR2) according to SEQ ID NO 27 (GAS), and a light chain complementary determining region 3 (L-CDR3) according to SEQ ID NO 28 (QQYNNWPPAYT).

[0086] In one embodiment, the invention relates to a dual chimeric antigen receptor (CAR) polypeptide as described herein, wherein the antigen-binding domain that binds to CEA protein comprises a variable heavy chain (VH) according to SEQ ID NO 29, or a sequence with at least 80% sequence identity thereto, and a variable light chain (VL) according to SEQ ID NO 33, or a sequence with at least 80% sequence identity thereto, and / or wherein the antigen-binding domain that binds to EpCAM protein comprises a variable heavy chain (VH) according to SEQ ID NO 21 , or a sequence with at least 80% sequence identity thereto, and a variable light chain (VL) according to SEQ ID NO 25, or a sequence with at least 80% sequence identity thereto.

[0087] In one embodiment, the invention relates to a dual chimeric antigen receptor (CAR) polypeptide as described herein, wherein the antigen-binding domain that binds to CEA protein comprises a variable heavy chain (VH) domain according to SEQ ID. NO 29, or a sequence with at least 80% sequence identity thereto, that comprises CDR sequences of SEQ ID NO. 30, SEQ ID No. 31 and SEQ ID NO. 32, and a variable light chain (VL) domain according to SEQ ID NO 33, or a sequence with at least 80% sequence identity thereto, that comprises CDR sequences of SEQ ID NO. 34; SEQ ID NO35, and SEQ ID NO. 36, and / or wherein the antigen-binding domain that binds to EpCAM comprises a variable heavy chain (VH) domain according to SEQ ID. NO 21 , or a sequence with at least 80% sequence identity thereto, that comprises CDR sequences of SEQ ID NO. 22, SEQ ID No. 23 and SEQ ID NO. 24, and a variable light chain (VL) domain according to SEQ ID NO 25, or a sequence with at least 80% sequence identity thereto, that comprises CDR sequences of SEQ ID NO. 26; SEQ ID NO 27, and SEQ ID NO. 28.

[0088] In embodiments, the CDR sequences may therefore be used to define the antigen binding domain of the dual CAR of the present invention, and the VH and VL domains may, in the flanking framework regions exhibit some sequence variation over the specific sequences disclosed herein. In embodiments, the CDR sequences may, on their own, be sufficient to define the inventive dual CAR, without reference to specific framework sequences.

[0089] In embodiments, the percentage sequence identity relates to the full length of variable VH and VL domains provided herein. In one embodiment, sequence variants with 80% or more sequence identity to the specific CDR sequences of SEQ ID 30 to 32 and 34 to 36 are encompassed in the invention. In embodiments, such sequence variants maintain CEA binding with essentially the same or similar functional properties as VH and VL domains with the specific CDR sequences of SEQ ID NO 30 to 32 and 34 to 36, i.e. , the CEA binding is essentially the same or similar with respect to affinity, specificity and / or epitope binding mode.

[0090] In one embodiment, sequence variants with 80% or more sequence identity to the specific CDR sequences of SEQ ID 22 to 24 and 26 to 28 are encompassed in the invention. In embodiments, such sequence variants maintain EpCAM binding with essentially the same or similar functional properties as VH and VL domains with the specific CDR sequences of SEQ ID NO 22 to 24 and 26 to 28, i.e., the EpCAM binding is essentially the same or similar with respect to affinity, specificity and / or epitope binding mode.

[0091] In some embodiments, the CDR sequences of the antigen binding domain may comprise 1 or 2 additional amino acids, in addition to those of SEQ ID NO 22 to 24, 26 to 28, 30 to 32 and 34 to 36 at N and / or C terminus end of the specific sequence of any one or more of SEQ ID NO 22 to 24, 26 to 28, 30 to 32 and 34 to 36. The additional amino acids at either one or more ends are, in embodiments, obtained from one or more of the VH and VL sequences presented herein. Thus, in embodiments, any one or more of the CDR sequences of the antigen binding domain may be 1 or 2 amino acids longer at one or both ends of the specific CDR sequences than those presented in SEQ ID NO 22 to 24, 26 to 28, 30 to 32 and 34 to 36, using additional sequences from the relevant VH and VL domains disclosed herein.

[0092] In some embodiments, the CDR sequences of the antigen binding domain may comprise 1 or 2 fewer amino acids, compared to those of SEQ ID NO 22 to 24, 26 to 28, 30 to 32 and 34 to 36, at N and / or C terminus ends of the specific sequence of any one or more of SEQ ID NO 22 to 24, 26 to 28, 30 to 32 and 34 to 36. Thus, in embodiments, any one or more of the CDR sequences may be 1 or 2 amino acids shorter, at one or both ends, of the specific CDR sequences presented in SEQ ID NO 22 to 24, 26 to 28, 30 to 32 and 34 to 36.

[0093] Any embodiment disclosed herein, reciting an “at least 80% sequence identity” or other degree of sequence identity to any given sequence, also relates to corresponding embodiments with at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the indicated sequence.

[0094] Furthermore, the order of the light and heavy chain fragments may be inverted upon the desired configuration of the antigen-binding fragment. The VH and VL fragments described herein may be arranged in multiple configurations in the dual CAR and still maintain high specificity and high affinity for the target epitope. In some embodiments, the dual CAR may be configured in the VH- VL or VL-VH configuration, with variation in the linker, hinge, transmembrane domain, costimulatory domain, and / or activation domains, and still maintain its efficacy. This feature of the invention enables greater flexibility in the design of dual CARs directed against CEA and EpCAM, thereby enabling further modification and / or optimization of the dual CAR structure on the basis of the VH and VL domains described herein, if any further development should be necessary or desired. Additionally, in some embodiments the linker sequence between heavy and light chains has been modified, for example by extending or shortening, in order to enhance the CAR function.

[0095] Additionally, the nucleic acid sequence encoding the dual CAR has been codon-optimized in order to improve expression of the dual CAR. These modifications enable sufficient surface expression on immune cells such as T cells or NK cells and still maintain proper antigen binding or recognition. High affinity and high avidity enable genetically modified cells according to the present invention such as CAR-T cells and CAR-NK cells to i) recognize, ii) be activated against, and kill tumor target cells with high, intermediate or low CEA surface expression and / or high, intermediate or low EpCAM surface expression.

[0096] The anti-CEA / anti-EpCAM dual CAR as described herein has a high affinity, high targeting accuracy, and confers high specificity and avidity to immune cells, in particular T cells and / or NK cells. These properties enable genetically modified cells according to the present invention such as dual CAR-T cells or dual CAR-NK cells to i) recognize, ii) be activated against, and iii) kill tumor target cells with high and low CEA surface expression and / or high and low EpCAM expression.

[0097] In one embodiment the nucleic acid construct as described herein can be administered into human cells. The invention therefore relates to a nucleic acid construct according to the present invention, comprising a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) and corresponding cells expressing said construct, preferably dual CAR-T cells or dual CAR-NK cells with a high cytotoxic activity against defined tumors, while sparing non- pathogenic cells within the tissue surrounded by the tumor, such as pancreatic, lung, colon or liver cells. In preferred embodiments all T cells, B cells, NK cells are likewise spared, as the CAR- T cell product of the present invention shows no or negligible activity against these cells.

[0098] The invention also encompasses the expression of a combination of immune activating cytokine and / or checkpoint inhibitory molecules in tumors via genetically modified cells such as dual CAR- T cells or dual CAR-NK cells described herein, with the aim to attract immune effector and helper cells, induce immune activation, promote the maturation of memory immune cells and / or suppress the emergence and persistence of suppressive and / or regulatory immune cells.

[0099] In a further aspect the invention relates to a cell, preferably a genetically modified cell, comprising a nucleic acid construct according to the present invention and / or the dual CAR polypeptide according to the present invention.

[0100] The present invention therefore relates to a cell, preferably a genetically modified cell, expressing a recombinant nucleic acid construct comprising a first nucleic acid sequence region encoding dual a chimeric antigen receptor (CAR) that recognizes a carcinoembryonic antigen (CEA) protein and an epithelial cell adhesion molecule (EpCAM).

[0101] In one embodiment, the cell is an immune cell, preferably selected from the group consisting of induced pluripotent stem cells (iPSC), preferably iPSC line ND50039, immortalized immune cells including NK-92 and YT cells, primary immune cells including a natural killer (NK) cells, cytokine- induced killer cells (CIK), T lymphocytes wherein the T lymphocytes are preferably CD4 or CD8 T cells, more preferably cytotoxic T lymphocytes or T helper cells or tumor infiltrating lymphocytes (TIL).

[0102] In one embodiment, the cell is a T cell. In one embodiment the genetically modified immune cell comprising a nucleic acid molecule or vector as described herein, and / or expressing a dual CAR as described herein, is characterized in that it is a CD4+ or a CD8+ T cell. In one embodiment a therapeutic composition according to the present invention preferably comprises a mixture of CD4+ and CD8+ T cells which have been genetically modified and comprise a nucleic acid molecule or vector as described herein, and / or expressing a dual CAR as described herein. These T cells, and preferably the composition comprising both CD4+ and CD8+ transformed cells, show particularly effective cytolytic activity against various solid and liquid tumors, such as colorectal cancer, preferably against those cells and / or the associated medical conditions described herein.

[0103] In one embodiment the therapeutic composition comprises CD4+ and CD8+ T cells, preferably in a ration of 1 : 10 to 10:1 , more preferably in a ratio of 5: 1 to 1 :5, 2: 1 to 1 :2 or 1 : 1 . Administration of dual CAR-T cells expressing the dual CAR described herein at the ratios mentioned, preferably at a 1 :1 CD4+ / CD8+ ratio, lead to beneficial characteristics during treatment of the diseases mentioned herein, for example these ratios lead to improved therapeutic response and reduced toxicity.

[0104] In one embodiment, the cell is a natural killer (NK) cell.

[0105] In one embodiment the cell is a natural killer (NK) cell, preferably derived from induced pluripotent stem cells (iPSC), more preferably derived from the iPS cell line ND50039.

[0106] In embodiment, the progenitor iPS cell line ND50039 is cultured under define culture conditions to become immune effector cells.

[0107] In one embodiment the cell is an immune cell derived from iPSC that has been genetically modified with the nucleic acid construct before differentiation into a NK cell.

[0108] NK cells advantageously operate independently of HLA-matching, allowing them to function as allogeneic effectors without being sourced from a particular patient or a donor with a specific HLA match. NK cell-mediated cytotoxicity is regulated by a repertoire of activating and inhibitory receptors (Guillerey et al., 2016; Morvan and Lanier, 2016). Activating receptors include but are not limited to natural cytotoxic receptors (NCRs), NKG2D, CD16 (FcyRllla), FasL, TRAIL, and costimulatory receptors such as LFA-1 , CD244 (2B4), and CD137 (41 BB). These activating cell surface receptors have the capacity to trigger cytolytic programs, as well as cytokine and chemokine secretion via intra-cytoplasmic ITAMs such as in 2B4, 41 BB, and / or via other transmembrane signaling adaptors (Li Y et al., Cell Stem Cell, 2018).

[0109] It is encompassed within the invention that administration of genetically modified cells expressing a dual CAR according to the present invention will trigger lysing of tumor-transformed cells in a major histocompatibility class I or II independent manner. NK cells are capable of directly lysing tumor-transformed cells and can also act as bridge between the innate and adaptive immune responses to enhance recognition and destruction of tumors by adaptive immune cells. Distinct from the mechanism by which T-cells lyse tumor cells, which requires recognition of tumor antigens presented in the context of major histocompatibility class I or II by a specific T-cell receptor, NK cells are able to kill tumor cells without prior sensitization to tumor antigens. NK cells act as a first line of defense against newly transformed cells. NK cells kill tumor targets through receptor-mediated cytotoxicity. This process is dependent on the presence of tumor specific antibodies bound to tumor surface antigens. This feature of the dual CAR-NK cells according to the present invention allows to reduce risks for a graft versus host disease in an allogeneic cell context. The dual CAR-NK cells according to the present invention can thus advantageously be prepared and used as allogeneic and "off the shelf' product in a time- and cost-efficient and simple manner.

[0110] In one embodiment, the cells are allogeneic cells with respect to a subject into which said cells are delivered.

[0111] In one embodiment, the cells are allogeneic T-cells, NK-cells or T cells with respect to a subject into which said cells are delivered.

[0112] In one embodiment, the cells are autologous cells with respect to a patient into which said cells are delivered.

[0113] In one embodiment, the cells are autologous NK cells with respect to a patient into which said cells are delivered.

[0114] T ransplantation of autologous or allogeneic cells comprising the nucleic acid construct and / or the dual CAR polypeptide described herein is feasible. For example, when reintroduced back to patients after autologous cell transplantation, the NK cells modified with the dual CAR of the invention as described herein may recognize and kill tumor cells. As would be understood by the skilled person, other cells may also be used as immune effector cells with the dual CARs as described herein. In particular, immune effector cells also include NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into dual CAR-T effector cells in vivo or in vitro. Progenitors can be iPS cells that become immune effector cells under defined culture conditions.

[0115] In another embodiment, the genetically modified cells additionally comprise one or more immune suppression defeating proteins and / or an inducible suicide gene that provokes death of the dual CAR-T cells or dual CAR-NK cells according to the present invention allowing their selective destruction. In some cases it may be desirable to provide a safety mechanism that allows selective deletion of the administered genetically modified cells, as the cells can spread and persist for years after administration. Therefore, the method of the invention in some embodiments may include the transformation of the cells with a recombinant suicide gene. This recombinant suicide gene is used to reduce the risk of direct toxicity and / or uncontrolled proliferation of these cells after administration to a subject. Suicide genes enable the selective deletion of transformed cells in vivo. In particular, the suicide gene has the ability to convert a non-toxic prodrug into a cytotoxic drug or to express the toxic gene expression product. In other words, "suicide gene" is preferably a nucleic acid that codes for a product, whereby the product itself or in the presence of other compounds causes cell death. In one embodiment, the suicide gene is the herpes simplex virus thymidine kinase.

[0116] An additional and surprising aspect of the invention is an improved stability of the dual CAR as disclosed herein. The dual CAR polypeptide can readily be stored for extended periods under appropriate conditions without any loss of binding affinity.

[0117] In a further aspect the invention relates to the genetically modified cell according to the present invention for use in the treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA and / or EpCAM in a subject. The invention relates to a new dual chimeric antigen receptor (CAR), wherein the receptor recognizes (preferably specifically recognizes) the antigens CEA and EpCAM on cancer cells. In a preferred embodiment, the genetically modified cell expressing the dual anti-CEA / anti-EpCAM- CAR is an immune cell that recognizes the CEA antigen and the EpCAM antigen on cancer cells and lyse cancer cells carrying the antigen CEA and / or EpCAM. The invention therefore relates to a cell product, such as an advanced therapy medicinal product, designed and manufactured for medical use, comprising a next generation dual CEA / EpCAM-CAR transduced immune cell that can be used in the treatment of cancer.

[0118] The invention provides a means for efficient and specific therapy of malignant diseases. Preferably the invention provides means for efficient and specific therapy of malignancies associated with the presence of pathogenic cells expressing CEA and / or EpCAM. Preferably the invention provides means for efficient and specific therapy of solid malignancies associated with the presence of pathogenic cells expressing CEA and / or EpCAM.

[0119] In one embodiment the pathogenic cells expressing CEA and / or EpCAM are cancer cells, preferably cancer cells of solid malignancies, more preferably cancer cells of breast cancer, pancreatic cancer, colon cancer, rectal cancer, lung cancer, liver cancer, stomach cancer and / or ovarian cancer.

[0120] For example, CEA is highly expressed in a variety of cancerous malignancies, in particular solid cancers, including colon cancer, ovarian cancer, thyroid cancer, pancreatic cancer and breast cancer. For example, EpCAM is highly expressed in a variety of cancerous malignancies, in particular solid cancer, including colon cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer and prostate cancer. CEA-expressing and / or EpCAM-expressing cells are known to a skilled person, and can be identified by further screening of cancers or other pathogenic cells.

[0121] The genetically modified cell according to the present invention as described herein is in preferred embodiments applicable to the treatment of solid tumor patients who are not eligible for other therapies. More specifically, embodiments of the invention relate to the treatment of the following patient collectives: i) patients with multidrug resistances, ii) patients not eligible for allogeneic stem cell transplantation, iii) patients with co-morbidities that preclude further chemotherapies, iv) patients suffering from solid and / or liquid cancers, v) aged patients who do not tolerate chemotherapies, vi) the dual CAR is applicable for salvage therapies even after progressive disease and multiple lines of other standard of care therapies have failed, vii) it is applicable even at low antigen density on target tumor cells, where antibodies can fail, and / or viii) it is applicable as a monotherapy which is not the case for antibodies ix) it is applicable in combination with one or more anti-cancer treatments, anti-cancer medicaments or transplantation. The dual anti-CEA / anti-EpCAM CAR described herein confers high avidity to T cells or NK cells, necessary for anti-tumor efficacy. The present invention has an unprecedented low off-target reactivity on other tissues.

[0122] Treatment with the genetically modified cells according to the invention can in some embodiments be combined with one or more anti-cancer treatments or medicaments, preferably selected from the group of antibody therapy, vaccines, oncolytic viral therapy, chemotherapy, radiation therapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, immune suppression or transplantation.

[0123] The invention relates further to methods of treatment of the medical conditions described herein, comprising typically the administration of a therapeutically effective amount of the dual CAR, or genetically modified cells expressing said dual CAR, to a patient in need of said treatment.

[0124] The present invention provides the technical solution for an efficient tumor treatment using the dual CAR construct described herein that selectively recognizes the tumor specific antigens CEA and EpCAM, actively stimulates immune cells, especially T cells and NK cells, and encourages an immune stimulating tumor microenvironment by an efficient immune stimulatory cytokine and an checkpoint inhibitor.

[0125] In a further aspect the invention relates to a pharmaceutical composition, in the form of a therapeutic cell product, comprising genetically modified cells according to the present invention and a pharmaceutically acceptable carrier.

[0126] The composition described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally.

[0127] In some embodiments, the pharmaceutically acceptable carrier is, for example, prepared in the form of a therapeutic cell product.

[0128] In one embodiment, the therapeutic cell product of the pharmaceutical composition is for use in the treatment and / or prevention of a medical disorder described herein.

[0129] In one embodiment, the pharmaceutical composition can be administered to patients before, after and / or in combination with one or more anti-cancer treatments or medicaments including antibody therapy, vaccines, oncolytic viral therapy, chemotherapy, radiation therapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, B-cell ablative therapy, T-cell ablative therapy immune suppression, peripheral blood stem cell transplantation or bone marrow stem cell transplantation.

[0130] In a further aspect the invention relates to an in vitro method for preparing a genetically modified immune cell according to the present invention, comprising delivering a nucleic acid construct according to the present invention into a cell.

[0131] Said method can be employed with one or more gene transfer techniques including a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, an alphavirus vector, a chemical transfection, an electroporation, and a mRNA transfection, preferably the adeno-associated viral vector. In a preferred embodiment, the immune cells intended for administering in treatment of the diseases mentioned herein are genetically modified with a nucleic acid construct as described herein, encoding and expressing the dual CAR as described herein, using a viral vector, in particular, a retroviral vector. Retroviral vectors have demonstrated high transduction efficiency in particular in expanded NK cells, facilitating the development of dual CAR-NK cells. Additionally, the CRISPR / Cas9 technique provides a targeted and precise approach for gene integration, offering the potential to enhance NK cell anti-tumor functions by modifying specific genes involved in NK cell regulation. Further method for transferring the nucleic acid construct according to the present invention into a cell in order to genetically modify said cell include without limitation non-viral methods such as DNA-based transposons and polymeric and lipid vectors and direct transfer of DNA or RNA by electroporation. Electroporation has proven to be particularly advantageous for the production of the dual CAR immune cells, particularly dual CAR NK cells, and genetically modified iPS cells, compared to the prior art, such as avoiding the risk of insertional mutagenesis associated with retroviral and lentiviral approaches, as the genetic material is not randomly integrated into the genome of the host cell, and unwanted interference with other genes and the associated oncogenic events. The risk of viral contamination or transmission of infectious agents to the patient is avoided, which increases safety for clinical applications, simplifies regulatory requirements and processes and thus reduces costs. In addition, electroporation of the nucleic acid construct disclosed herein offers high efficiency, simplicity and speed.

[0132] All features described in the present specification may be employed to define any other embodiment or aspect of the invention, for example, features used to describe the nucleic acid construct may for example be used to describe the dual CAR polypeptide, the genetically modified cell, the genetically modified cell for use in the treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA and / or EpCAM, the pharmaceutical composition, or the method for preparing said cell, and vice versa. Similarly, features used to describe the methods of the invention may be used to describe the cells, nucleic acid constructs or compositions, and vice versa.

[0133] DETAILED DESCRIPTION

[0134] An important function of the immune system is to recognize and eliminate tumors. Tumor antigens are either specifically expressed on tumor cells and not found on non-pathogenic cells or abnormally expressed, e.g. at least twice above the level found in non-pathogenic cells. Antigens, specifically found on tumor cells, may appear foreign to the immune system and their presence may cause the immune cells to attack the transformed tumor cells. Some antigens are derived from oncogenic viruses such as the human papilloma virus, which causes cervical cancer. One example of an abnormally expressed protein is an enzyme called tyrosinase which, when expressed in high amounts, converts certain skin cells (e.g. melanocytes) into tumors called melanomas. Another possible source of tumor antigens are proteins that are normally important for the regulation of cell growth and survival and that mutate into molecules called oncogenes, which often cause cancer. However, a native immune response often fails to eliminate tumors and a therapeutic invention is urgently needed. The main response of the immune system to tumors is to destroy the abnormal cells with the help of killer T cells, sometimes with helper T cells. Tumor antigens are presented on MHC class I molecules in a similar way to viral antigens. This enables killer T cells to recognize the tumor cell as abnormal. NK cells also kill tumor cells in a similar way, especially if the tumor cells have fewer MHC class I molecules on their surface than normal; this is a common phenomenon in tumors. Some tumor cells also release products that inhibit the immune response, for example by secreting the cytokine TGF-p, which suppresses the activity of macrophages and lymphocytes. Cytokine-induced killer cells (CIK) are a group of immune effector cells that exhibit a hybrid T- and NK-like phenotype. They are produced by ex vivo incubation of mononuclear cells from human peripheral blood (PBMC) or cord blood with interferon-gamma (IFN-y), anti-CD3 antibody, recombinant human interleukin (IL-) 1 and recombinant human interleukin (I L)-2.

[0135] The present invention therefore provides means to enable and / or enhance an anti-tumor immune response by simultaneous expression of a dual chimeric antigen receptor (CAR) directed against the tumor antigens CEA and EpCAM and an immune-stimulatory cytokine, e.g. IL-15, in a genetically modified cell, e.g. T lymphocyte, cytokine-induced killer cell (CIK), NK cell, as described herein. Further, additionally an immune checkpoint inhibitory molecule such as PD1 might be expressed in said genetically modified cell.

[0136] Immunotherapy is to be understood in the context of the present invention to encompass any therapeutic agent that uses the immune system to treat cancer. Immunotherapy exploits the fact that cancer cells have subtly different molecules on their surface that can be detected by the immune system. These molecules, known as cancer antigens, are most commonly proteins, but also include molecules such as carbohydrates, lipids, and lipoproteins. Immunotherapy provokes or enhances the immune system in attacking the tumor cells by using these antigens as targets. Further, the present invention exceptionally combines the dual CAR described herein with an immune stimulatory cytokine to induce activation and proliferation of T and / or natural killer (NK) cells and with a checkpoint inhibitory molecule to enhance the endogenous anti-tumor activity of the immune system.

[0137] Immunotherapy encompasses, without limitation, cellular and antibody therapy. Cellular therapies typically involve the administration of immune cells isolated from the blood or from a tumor of the patient. Immune cells directed towards the tumor to be treated are activated, cultured and returned to the patient where the immune cells attack the cancer. Cell types that can be used in this way are, without limitation, natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, and dendritic cells. Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens. Dendritic cells present antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen.

[0138] Antibodies are proteins produced by the immune system that bind to a target antigen on the cell surface. Those that bind to cancer antigens may be used to treat cancer. Cell surface receptors are common targets for antibody therapies and include for example CD19, CD44, CD20, CD274, and CD279. Once bound to a cancer antigen, antibodies can induce antibody-dependent cell- mediated cytotoxicity, activate the complement system, or prevent a receptor from interacting with its ligand, all of which can lead to cell death. Multiple antibodies are approved to treat cancer, including Alemtuzumab, Ipilimumab, Nivolumab, Ofatumumab, and Rituximab.

[0139] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of attack by the immune system that requires antibodies to bind to target cell surfaces. Antibodies are formed of a binding region (Fab) and the Fc region that can be detected by immune cells via their Fc surface receptors. Fc receptors are found on many immune system cells, including natural killer cells. When natural killer cells encounter antibody-coated cells, the latter's Fc regions interact with their Fc receptors, leading to the release of perforin and granzyme B. These two chemicals programmed cell death (apoptosis) in the tumor cell. Effective antibodies include Rituximab, Ofatumumab, and Alemtuzumab.

[0140] The complement system includes blood proteins that can cause cell death after an antibody binds to the cell surface. Generally, the system deals with foreign pathogens, but can be activated with therapeutic antibodies in cancer. The system can be triggered if the antibody is chimeric, humanized or human; as long as it contains the IgG 1 Fc region. Complement can lead to cell death by activation of the membrane attack complex, known as complement-dependent cytotoxicity; enhancement of antibody-dependent cell-mediated cytotoxicity; and CR3-dependent cellular cytotoxicity. Complement-dependent cytotoxicity occurs when antibodies bind to the cancer cell surface, the C1 complex binds to these antibodies and subsequently protein pores are formed in the cancer cell membrane.

[0141] The dual CAR of the present invention is capable of enabling and / or enhancing the immunotherapies described herein through their unique properties derived through the affinity to CAE and EpCAM and the combination with immune-stimulating transgene cytokine. Further, the checkpoint inhibitory molecule enhances anti-tumor activity. Overall anti-tumor activity is increased by overcoming tumor heterogeneoity and the immunosuppressive microenvironment of tumors, in particular solid tumors.

[0142] The tumor immune microenvironment (TIME) is the environment within tumor, including the tumor cells, surrounding blood vessels, immune cells, fibroblasts, signaling molecules and the extracellular matrix (ECM) of the tumor. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of cancerous cells. Immune cells infiltrate into the tumor microenvironment, interact with each other and tumor cells, and then harbor an immunosuppressive phenotype that is responsible for the immune escape of tumor cells and the following tumor progression. These immunosuppressive cells include MDSCs, M2-macrophages, Tregs, N2-TANs, mast cells, B regs, dendritic cells. They secrete cytokines such as IL-2, IL-10, and TGF-p, growth factors like VEGF, express checkpoint ligands like PD-L1 or express checkpoints on the cell surface like PD-1 , TIM-3 on Tregs, that negatively regulate the anti-tumor immune response, remodel the extracellular matrix, and promote angiogenesis. As a result, these immunosuppressive cells and their interaction generate an immunosuppressive microenvironment (TIME) and promote the proliferation, evasion, and migration of tumor cells.

[0143] The immune stimulatory cytokines described herein may relate to any mammalian cytokine corresponding to the cytokines named herein or any molecule acting agonistic on the receptor of the cytokines named herein, e.g., an IL-15 superagonist. Preferably, the cytokines relate to human cytokines, or mouse cytokines. Cancer immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Initially, immunotherapy treatments involved administration of immunostimulatory cytokines such as “Interleukin”, as described herein. Interleukin 15 (IL-15) is a cytokine with structural similarity to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 induces cell proliferation of natural killer cells; cells of the innate immune system whose principal role is to kill virally infected or cancerous cells. IL-15 has been shown to enhance the anti-tumor immunity of CD8+ T cells in pre-clinical models (Klebanoff CA, et al. Proc. Natl. Acad. Sci. U.S.A. 101 (7): 1969-74).

[0144] Checkpoint inhibitors also termed “immune checkpoint inhibitor molecule”, also known as immune checkpoint modulators, are designed to lessen the effectiveness of checkpoint proteins. They may have a variety of mechanisms of action, but if effective, they enable the immune system to recognize other molecules on the surface of the cancer cells. Preferably the checkpoint inhibitor is selected from the group consisting of: PD1 , PD-L1 , CTLA4, TIM3, CEACAM (e.g., CEACAM-1 , CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.

[0145] Chimeric Antigen Receptors:

[0146] According to the present invention, a chimeric antigen receptor polypeptide (CAR), comprises an extracellular antigen-binding domain, comprising an antibody or antibody fragment that binds a target antigen, a transmembrane domain, and an intracellular domain. A ’’dual CAR” according to the present invention comprises at least one extracellular antigen-binding domain comprising an antibody or antibody fragment that binds a first target antigen, i.e., a CEA protein, and at least a second extracellular antigen-binding domain comprising an antibody or antibody fragment that binds a second target antigen, i.e. an EpCAM protein, a transmembrane domain, and an intracellular domain. CARs such as dual CARs are typically described as comprising one or more extracellular ectodomains (antigen-binding domains) derived from an antibody and an endodomain comprising signaling modules derived from T cell signaling proteins.

[0147] In a preferred embodiment, the one or more ectodomains preferably comprise variable regions from the heavy (VH) and light chains (VL) of an immunoglobulin configured as a single-chain variable fragment (scFv). The scFv is preferably attached to a hinge region that provides flexibility and transduces signals through an anchoring transmembrane moiety to an intracellular signaling domain. The transmembrane domains originate preferably from either CD8a or CD28. In the first generation of CARs the signaling domain consists of the CD3 zeta chain of the TCR complex. The term “generation” refers to the structure of the intracellular signaling domains. Second generation CARs are equipped with a single costimulatory domain originated from CD28 or 4- 1 BB. Third generation CARs already include two costimulatory domains, e.g. CD28, 4-1 BB, ICOS or 0X40, CD3 zeta. The present invention preferably relates to a second or third generation CAR.

[0148] In various embodiments, genetically engineered receptors that redirect cytotoxicity of immune effector cells toward cancerous cells of solid tumors are provided. These genetically engineered receptors referred to herein as chimeric antigen receptors (CARs) or a dual CAR when comprising a first antigen binding domain against CEA and a second antigen binding domain against EpCAM. CARs, e.g., dual CARs are molecules that combine antibody-based specificity 1 for one or more desired antigens with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits an antigen specific cellular immune activity. As used herein, the term, "chimeric," describes being composed of parts of different proteins or DNAs from different origins.

[0149] Dual CARs contemplated herein, comprise two extracellular domains (also referred to as a binding domain or antigen-binding domain) that bind to CEA and EpCAM respectively, a transmembrane domain, and an intracellular domain, or intracellular signaling domain (also termed signaling domain). Engagement of at least one of the two antigen binding domains of the dual CAR on the surface of a target cell results in clustering of the dual CAR and delivers an activation stimulus to the dual CAR-containing cell. The main characteristics of CARs such as dual CARs are their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility complex (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific co-receptors.

[0150] In various embodiments, a CAR such as a dual CAR comprises one or more extracellular binding domains that comprise a humanized antigen-specific binding domain; a transmembrane domain; one or more intracellular signaling domains. In particular embodiments, a dual CAR comprises two extracellular binding domains that each comprise a humanized antigen binding fragment thereof; one or more spacer domains; a transmembrane domain; one or more intracellular signaling domains.

[0151] The terms "extracellular antigen-binding domain" or "extracellular binding domain" are used interchangeably and provide a CAR such as a dual with the ability to specifically bind to the target antigens of interest, i.e., CEA and EpCAM. The binding domains may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. Preferred are scFv domains.

[0152] “Specific binding” is to be understood as via one skilled in the art, whereby the skilled person is clearly aware of various experimental procedures that can be used to test binding and binding specificity. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. Some cross-reaction or background binding may be inevitable in many protein-protein interactions; this is not to detract from the “specificity” of the binding between CAR such as the dual CAR according to the present invention and epitope. “Specific binding” describes binding of an antibody or antigen binding fragment thereof (or a CAR such as a dual CAR comprising the same) to a target antigen at greater binding affinity than background binding. The term “directed against” is also applicable when considering the term “specificity” in understanding the interaction between antibody and epitope.

[0153] An "antigen (Ag)" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal. In particular embodiments, the target antigen is an epitope of a desired polypeptide. In particular embodiments, the target antigens or the dual CAR according to the present invention are an epitope of CEA and an epitope of EpCAM. An "epitope" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0154] Tumor associated antigens targeted via the dual CAR of the present invention include in particular CEA and EpCAM. The number of CEA or EpCAM antigens expressed on the surfaces of tumor cells can be quantified by using an anti-CEA or anti-EpCAM antibody coupled to a fluorescent-dye in conjunction with Quantibrite beads (from BD). The preferred method applied to quantify CEA or EpCAM antigens expressed on the surfaces of tumor cells is "fluorescence activated cell sorting / cell analysis" (FACS). Fluorescence intensity of beads correlates exactly with the numbers of fluorescent antibodies bound to cells, and this is a measure for the number of CEA molecules on cells.

[0155] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain and in either orientation {e.g., VL-VH or VH-VL). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. In preferred embodiments, a dual CAR contemplated herein comprises antigen-specific binding domain that is an scFv and may be a murine, human or humanized scFv. Single chain antibodies may be cloned from the V region genes of a hybridoma specific for a desired target. scFv can be also obtained from phage display libraries, thus bypassing the tradtional hybridoma technology. In particular embodiments, the antigen-specific binding domain that is a humanized scFv that binds a human target antigen polypeptide.

[0156] In particular embodiments, the dual CAR comprises an antigen-specific binding domain that is a humanized scFv that binds a human CEA polypeptide. An illustrative example of a variable heavy chain that is suitable for constructing a dual anti-CEA / anti-EpCAM CAR contemplated herein include, but are not limited to the amino acid sequence set forth in SEQ ID NO: 29. An illustrative example of a variable light chain that is suitable for constructing anti-CEA CARs contemplated herein include, but is not limited to the amino acid sequence set forth in SEQ ID NO: 33.

[0157] In particular embodiments, the dual CAR comprises an antigen-specific binding domain that is a humanized scFv that binds a human EpCAM polypeptide. An illustrative example of a variable heavy chain that is suitable for constructing a dual anti-CEA / anti-EpCAM CARs contemplated herein include, but are not limited to the amino acid sequence set forth in SEQ ID NO: 21 . An illustrative example of a variable light chain that is suitable for constructing anti-EpCAM / anti-CEA / CARs contemplated herein include, but is not limited to the amino acid sequence set forth in SEQ ID NO. 25

[0158] Antibodies and antibody fragments:

[0159] The dual CAR comprises two extracellular antigen-binding domains, comprising an antibody or antibody fragment that binds a target polypeptide. Antibodies or antibody fragments of the invention therefore include, but are not limited to polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain fragments (scFv), single variable fragments (ssFv), single domain antibodies (such as VHH fragments from nanobodies), Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies and epitope-binding fragments or combinations thereof of any of the above, provided that they retain similar binding properties of the dual CAR described herein, preferably comprising the corresponding CDRs, or VH and VL regions as described herein. Also mini-antibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptabodies can be used in a method of the invention. The immunoglobulin molecules of the invention can be of any class (i.e. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules. Thus, the term antibody, as used herein, also includes antibodies and antibody fragments comprised by the dual CAR of the invention, either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.

[0160] As used herein, an "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Where the term “antibody” is used, the term “antibody fragment” may also be considered to be referred to. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (L) (about 25 kD) and one "heavy" (H) chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains respectively. Optionally, the antibody or the immunological portion of the antibody, can be chemically conjugated to, or expressed as, a fusion protein with other proteins.

[0161] The dual CARs of the invention are intended to bind against mammalian, in particular human, protein targets. The use of protein names may correspond to either mouse or human versions of a protein.

[0162] Affinities of binding domain polypeptides and dual CAR proteins according to the present disclosure can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or displacement assays using labeled ligands, or using a surface-plasmon resonance device such as the Biacore.

[0163] Humanized antibodies comprising one or more CDRs of antibodies of the invention or one or more CDRs derived from said antibodies can be made using any methods known in the art. For example, four general steps may be used to humanize a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains (2) designing the humanized antibody, i.e. , deciding which antibody framework region to use during the humanizing process (3) the actual humanizing methodologies / techniques and (4) the transfection and expression of the humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331 ,415; 5,530,101 ; 5,693,761 ; 5,693,762; 5,585,089; 6,180,370; 5,225,539; 6,548,640.

[0164] The term humanized antibody means that at least a portion of the framework regions, and optionally a portion of CDR regions or other regions involved in binding, of an immunoglobulin is derived from or adjusted to human immunoglobulin sequences. The humanized, chimeric or partially humanized versions of the mouse monoclonal antibodies can, for example, be made by means of recombinant DNA technology, departing from the mouse and / or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques (Queen et al., 1989; WO 90 / 07861). Alternatively, the monoclonal antibodies used in the method of the invention may be human monoclonal antibodies. Human antibodies can be obtained, for example, using phagedisplay methods (WO 91 / 17271 ; WO 92 / 01047).

[0165] As used herein, humanized antibodies refer also to forms of non-human (e.g. murine, camel, llama, shark) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.

[0166] As used herein, human or humanized antibody or antibody fragment means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies known in the art or disclosed herein. Human antibodies or fragments thereof can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. The humanized antibodies of the present invention surprisingly share the useful functional properties of the mouse antibodies to a large extent. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using amyloid fibrillar and / or non-fi brillar polypeptides or fragments thereof as an affinity reagent. Monoclonal antibodies can be obtained from serum according to the technique described in WO 99 / 60846.

[0167] Variable Regions and CDRs

[0168] A variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies.

[0169] There are a number of techniques available for determining CDRs, such as an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991 , National Institutes of Health, Bethesda Md.)); and an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al. (1997) J. Molec. Biol. 273:927-948). Alternative approaches include the IMGT international ImMunoGeneTics information system, (Marie-Paule Lefranc). The Kabat definition is based on sequence variability and is the most commonly used method. The Chothia definition is based on the location of the structural loop regions, wherein the AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software (refer www.bioinf.org.uk : Dr. Andrew C.R. Martin's Group). As used herein, a CDR may refer to CDRs defined by one or more approach, or by a combination of these approaches.

[0170] In some embodiments, the invention provides an antibody or fragment thereof incorporated into a dual CAR, wherein said antibody or fragment thereof comprises at least one CDR, at least two, at least three, or more CDRs that are substantially identical to at least one CDR, at least two, at least three, or more CDRs of the antibody of the invention. Other embodiments include antibodies which have at least two, three, four, five, or six CDR(s) that are substantially identical to at least two, three, four, five or six CDRs of the antibodies of the invention or derived from the antibodies of the invention. In some embodiments, the at least one, two, three, four, five, or six CDR(s) are at least about 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two or three CDRs of the antibody of the invention. It is understood that, for purposes of this invention, binding specificity and / or overall activity is generally retained, although the extent of activity may vary compared to said antibody (may be greater or lesser).

[0171] Additional components of the CAR

[0172] In certain embodiments, the dual CARs contemplated herein may comprise linker residues between the various domains, added for appropriate spacing and conformation of the molecule, for example a linker comprising an amino acid sequence that connects the VH and VL domains and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. Dual CARs contemplated herein, may comprise one, two, three, four, or five or more linkers. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids.

[0173] Illustrative examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art, such as the Whitlow linker. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CARs described herein.

[0174] In particular embodiments, the binding domain of the dual CAR is followed by one or more "spacers” or “spacer polypeptides," which refers to the region that moves the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG 1 or lgG4. In one embodiment the Fc-binding domain of such a spacer / hinge region is mutated in a manner that prevents binding of the CAR- To Fc-receptors expressed on macrophages and other innate immune cells.

[0175] Each of the binding domains of the dual CAR may in some embodiments be followed by one or more "hinge domains," which play a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. A dual CAR may comprise one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the dual CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1 , CD152, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In another embodiment, the hinge domain comprises a PD1 , CD152, or CD8 alpha hinge region.

[0176] The "transmembrane domain" is the portion of the dual CAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR-To the plasma membrane of the immune effector cell. The TM domain may be derived either from a natural, synthetic, semi- synthetic, or recombinant source. The TM domain may be derived from the alpha, beta or zeta chain of the T-cell receptor, CD3E, CD3 , CD4, CD5, CD8 alpha, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In one embodiment, the CARs contemplated herein comprise a TM domain derived from CD8 alpha or CD28

[0177] In particular embodiments, CARs contemplated herein comprise an intracellular signaling domain, also termed “signaling domain”. A "signaling domain," refers to the part of a CAR such as a dual CAR that participates in transducing the message of effective binding to a human target polypeptide into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the dual CAR-bound target cell, or other cellular responses elicited with one or more antigens binding to the extracellular dual CAR domain. The term "effector function" refers to a specialized function of an immune effector cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the term "signaling domain" refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. Dual CARs contemplated herein comprise one or more co-stimulatory signaling domains to enhance the efficacy, expansion and / or memory formation of cells expressing dual CAR receptors. As used herein, the term, "costimulatory signaling domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen.

[0178] The co-stimulatory molecule is further a cell surface molecule other than an antigen receptor or its ligand that contributes to an efficient immune response. Co-stimulatory molecules include MHC class I molecules, BTLA and Toll ligand receptors, 0X40, CD27, CD28, CDS, ICAM-1 , LFA- 1 (CD11 a / CD18), ICOS (CD278), and 4-1 BB (CD137). Including, but not limited to, further examples of such co-stimulatory molecules are CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta , IL2Rbeta, IL2Rgamma, IL7Ralpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49f, ITGAD, CD11 d, ITGAE, CD103, ITGAL, CD11 a, LFA-1 , ITGAM ITGB1 , CD29, ITGB2, CD18, LFA-1 , ITGB7, NKG2D, NKG2C, TNFR2, DNAM1 (CD 26), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEAC (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 ( (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG I Cbp, CD19a and ligands that specifically bind to CD83.

[0179] In one embodiment, the dual CAR comprises an intracellular domain, which comprises a co- stimulatory domain and a signalling (activation) domain. The dual CAR construct may therefore include an intracellular signaling domain (CD3 zeta) of the native T cell receptor complex and one or more co-stimulatory domains that provide a second signal to stimulate full immune cells activation, such as T cell activation. Co-stimulatory domains are thought to increase CAR-T cell cytokine production and facilitate immune cell replication and immune cell persistence, in particular T cell replication and T cell persistence. Co-stimulatory domains have also been shown to potentially prevent CAR-T cell exhaustion, increase T cell antitumor activity, and enhance survival of CAR-T cells in patients. As a non-limiting example, dual CAR constructs with the 4- 1 BB co-stimulatory domain have been associated with gradual, sustained expansion and effector function, increased persistence, and enriched central memory cells (TCM) in the T cell subset composition in preclinical studies. 4-1 BB is a member of the tumor necrosis factor (TNF) superfamily, and it is an inducible glycoprotein receptor in vivo that is primarily expressed on antigen-activated CD4 and CD8 T cells. As a non-limiting example, CD28 is member of the immunoglobulin (Ig) superfamily. It is constitutively expressed on resting and activated CD4 and CD8 T cells and plays a critical role in T cell activation by stimulating the PI3K-AKT signal transduction pathway. In one embodiment, the intracellular domain comprises both 4-1 BB and CD28 co-stimulatory domains. Other co-stimulatory domains comprise ICOS and 0X40 that can be combined with the CD3 zeta signalling (activation) domain.

[0180] "Peptide" "polypeptide”, “polypeptide fragment" and "protein" are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence or a fragment of a full length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0181] In various embodiments, the dual CAR polypeptides contemplated herein comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post- translationally directs transfer of the protein. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides contemplated herein specifically encompass the CARs of the present disclosure, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acid of a dual CAR as disclosed herein.

[0182] An "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and / or purification of a peptide or polypeptide molecule from a cellular environment, and from association with other components of the cell, i.e., it is not significantly associated with in vivo substances. Similarly, an "isolated cell" refers to a cell that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.

[0183] Nucleic acids

[0184] As used herein, the terms "polynucleotide", “nucleic acid construct” or "nucleic acid molecule" refers to a polymeric form of nucleotides (nt) of any length, wherein the nucleotides can be either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. The terms include without limitation ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or any mimetic or structurally modified nucleic acid thereof, including, without limitation DNA, messenger RNA (mRNA), RNA, genomic RNA (gRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), single guide RNA (sgRNA), short hairpin RNA (shRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), antisense oligonucleotides (ASO), recombinant polynucleotides, branched polynucleotides, plasmids, nucleic acid probes and primers. Polynucleotides include single and double stranded polynucleotides. Preferably, polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the variant maintains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, and compositions, and cells comprising the same. In one embodiment the nucleic acid construct is a plasmid or an mRNA molecule.

[0185] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of well- established techniques known and available in the art. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, can be inserted into appropriate vector. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus {e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus {e.g., SV40). Examples of expression vectors are pCIneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5- GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequences of the chimeric proteins disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells. The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector - origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters may be used.

[0186] An “exogenous nucleic acid”, “exogenous genetic element”, or “transgenic” nucleic acid or construct relates to any nucleic acid introduced into the cell, which is not a component of the cells “original” or “natural” genome or pool of nucleic acids found “naturally” in an unmodified cell. Exogenous nucleic acids may be integrated or non-integrated in the genetic material of the target cell or relate to stably transduced nucleic acids. Delivery of an exogenous nucleic acid may lead to genetic modification of the initial cell through permanent integration of the exogenous nucleic acid molecule in the initial cell. However, delivery of the exogenous nucleic acid may also be transient, meaning that the delivered genetic material for provision of the one or more TF disappears from the cell after a certain time. Nucleic acid molecule delivery and potentially genetic modification of a biological cell, i.e. a NK cell, can be performed and determined by a skilled person using commonly available techniques. For example, for detecting genetic modification sequencing of the genome or parts thereof of a cell is possible, thereby identifying if exogenous nucleic acids are present. Alternatively, other molecular biological techniques may be applied, such as the polymerase chain reaction (PCR), to identify / amplify exogenous genetic material. Exogenous nucleic acids may be detected by vector sequences, or parts of vector sequences, e.g. those remaining at the site of genetic modification. In cases where vector sequences (for example vector sequences flanking a therapeutic transgene) can be removed from the genome or do not remain after modification, for example by CRISPR technology, the addition of a transgene may still be detected by sequencing efforts by detecting sequences comprising an exogenous sequence at a “non-natural” position in the genome.

[0187] Embodiments of the invention relate to genetically modified cells comprising one or more exogenous nucleic acid molecules encoding a dual CAR according to the present invention. In embodiments of the invention, the exogenous nucleic acid represents a nucleic acid sequence not found naturally in a cell, based on for example comparisons with an unmodified human genome sequence. In embodiments of the invention, the nucleic acid construct is a nucleic acid sequence coding dual CAR, for which the coding sequence is not found naturally in a cell, based on for example comparisons with an unmodified human genome sequence. In some embodiments, the sequence is present at a “non-natural location” of the genome. In some embodiments, the targeting construct comprises or consists of a non-naturally occurring sequence, i.e. a synthetic sequence designed and created using recombinant or other molecular biological techniques.

[0188] "poly-A site", “poly-A signal” or "poly-A sequence", as used herein, refers to a nucleic acid sequence that signals both, termination and polyadenylation, of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can enhance mRNA stability by adding a poly-A tail at the 3' end of the coding sequence, thus contributing to increased translation efficiency. Efficient polyadenylation of the recombinant transcript is preferable because transcripts without a poly-A tail are unstable and degrade rapidly. A “stop codon” is also a sequence of three consecutive nucleotides (termed triplet) within the messenger RNA that signals the termination of a protein translation process at the ribosome.

[0189] Methods for amplification of the nucleic acid constructs of the present invention are known to a person skilled in the art and include polymerase chain reaction (PCR)-based methods such as real-time PCR, quantitative real-time PCR (qPCR), quantitative real time reverse transcription PCR (qRT-PCR) and digital PCR using suitable primers hybridizing with the nucleic acid constructs.

[0190] The term “primer” refers to an oligonucleotide whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH value. A primer thus can serve as a starting point for DNA-replicating enzymes such as DNA polymerase. The primer or oligonucleotide is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligonucleotide, more preferably an oligodeoxyribonucleotide. The primer or oligonucleotide must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides

[0191] Hybridization is the process of establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex or DNA double strand in the case of DNA. The terms “binding” or “annealing” may be used in place of hybridization in the present invention. The hybrids may be dissociated by thermal denaturation, also referred to as melting. Here, the solution of hybrids is heated to break the hydrogen bonds between nucleic bases, after which the two strands separate. In the absence of external negative factors, the processes of hybridization and melting may be repeated in succession indefinitely, which lays the ground for PCR.

[0192] As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm (melting temperature) of the formed hybrid, and the G:C ratio within the nucleic acids. As used herein the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. In an illustrative example, hybridization under “highly stringent condition” can mean hybridization at 65 °C in 5X SSPE and 50% formamide and washing at 65 °C in 0.5X SSPE. In another illustrative example, “highly stringent condition” can mean hybridization at 55°C in a hybridization buffer consisting of 50% formamide (vol / vol); 10% dextran sulfate; 1 x Denhard”s solution; 20 mM sodium phosphate, pH 6.5; 5 x SSC; and 200 pg of salmon sperm DNA per ml of hybridization buffer for 18 to 24 hours, and washing four times (5 min each time) with 2 x SSC; 1 % SDS at room temperature and then washing for 15 min at 50- 55°C with 0.1 x SSC. In another illustrative example Conditions for high stringency hybridization are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporated herein by reference. In some illustrative aspects, hybridization occurs along the full- length of the nucleic acid. Detection of highly stringent hybridization in the context of the present invention indicates strong structural similarity or structural homology (e.g., nucleotide structure, base composition, arrangement or order) to, e.g., the nucleic acids provided herein.

[0193] Vectors and gene editing

[0194] In particular embodiments, a cell (e.g., an immune effector cell, such as a NK cell) is transduced with an adeno-associated viral vector, a retroviral vector, e.g., a lentiviral vector, encoding a dual CAR. For example, an immune effector cell is transduced with a vector encoding a dual CAR that comprises a humanized anti-CEA antibody or antigen binding fragment that binds a CEA polypeptide and a humanized anti-EpCAM antibody or antigen binding fragment that binds EpCAM, with a transmembrane and intracellular signalling domain, such that these transduced cells can elicit a CAR-mediated cytotoxic response.

[0195] In some embodiments, a particular advantage of the invention is the use of AAVs for the genetic transfer of the recombinant nucleic acid construct of the present invention due to its high safety and transduction efficiency in vivo. Variants of AAV, such as AAV and capsid variants, can provide or transfer polynucleotides and / or proteins that offer desired or therapeutic benefits and thereby treat various diseases. For example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , Rh10, Rh74 or AAV-2i8 and variants thereof and AAV capsid variants (e.g. 4-1) are a useful vector for providing a therapeutic gene to treat cells, tissues and organs. Recombinant viruses and AAV vectors of the invention containing the vector genome (virus or AAV) (encapside and encapsidate) contain additional factors which function in cis or trans. The AAV vector is selected from a group including AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , Rh10, Rh74 or AAV-2i8 AAV capsid sequences, or AAV1 , AAV2, Capsid variants of AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , Rh10, Rh74 or AAV-2i8 are included. Retroviruses are a common tool for gene delivery. In particular embodiments, a retrovirus is used to deliver a polynucleotide encoding a dual chimeric antigen receptor (CAR) to a cell. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules, which encode the structural proteins and enzymes needed to produce new viral particles.

[0196] Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and lentiviruses. As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 , and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones (i.e., HIV cis-acting sequence elements) are preferred. In particular embodiments, a lentivirus is used to deliver a polynucleotide ecoding a dual CAR to a cell.

[0197] The term "vector" is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cellular DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses. In further embodiments of the invention, CRISPR / Cas and TALEN-mediated insertion of the dual anti-CEA / anti-EpCAM CAR encoding nucleic acid may be employed. Appropriate vectors for CRISPR / Cas and TALEN- mediated insertion are known to a skilled person.

[0198] As will be evident to one of skill in the art, the term "viral vector" is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s).

[0199] The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.

[0200] In a preferred embodiment the invention therefore relates to a method for transfecting cells with an expression vector encoding a dual CAR using electroporation. Electroporation is a physical method, which creates pores in the cell membrane by applying an electric shock to the cell. These pores allow the increased diffusion of materials into the cell. This increased permeability allows for easier transfection. Sonoporation is similar to electroporation except it uses ultrasound to stimulate the cell membrane. The ultrasound also creates turbulence in the fluid surrounding the cell, which increases the rate of diffusion across the membrane.

[0201] In a preferred embodiment the invention therefore relates to a method for transfecting cells with an expression vector encoding a dual CAR using chemical transfection. Chemical transfection refers to the calcium phosphate transfection which is well known by skilled persons. Calcium phosphate transfection uses calcium phosphate bonded to DNA (A Watson and D Latchman, “Gene Delivery into Neuronal Cells by Calcium Phosphate-Mediated Transfection”, Methods, Volume 10, Issue 3, December 1996, Pages 289-291). It has been suggested to use calcium phosphate particles as agents for transfection of therapeutic polynucleotides in gene therapy. See U.S. Pat. No. 5,460,831 . DNA or RNA is attached to the particulate core and delivered to a target cell, resulting in expression of therapeutic proteins.

[0202] In some embodiments, the genetically transformed cells are further transfected with a transposase that facilitates integration of a dual CAR coding sequence into the genome of the transfected cells. In some embodiments the transposase is provided as DNA expression vector. However, in preferred embodiments, the transposase is provided as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells. For example, in some embodiments, the transposase is provided as an mRNA (e.g., an mRNA comprising a cap and poly- A tail). Any transposase system may be used in accordance with the embodiments of the present invention. However, in some embodiments, the transposase is salmonid-type Tel -like transposase (SB). For example, the transposase can be the so called "Sleeping beauty" transposase, see e.g., U.S. Patent 6,489,458, incorporated herein by reference. In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, without limitation, SB 10, SB 1 1 or SB 100X transposase (see, e.g., Mates et al, 2009, Nat Genet. 41 (6):753-61 , or US9228180, herein incorporated by reference). For example, a method can involve electroporation of cells with an mRNA encoding an SB 10, SB 1 1 or SB 100X transposase.

[0203] The term “promoter” is used for a DNA sequence that initiates and regulates transcription from the DNA downstream of by binding of enzymes reading and transcribing the DNA. The resulting transcribed RNA may encode a protein. Promoters are preferably located near the transcription start sites of genes, upstream of the coding DNA, typically before the beginning of the openreading frame (towards the 5' region of the sense strand).

[0204] Sequence Variants

[0205] Sequence variants of the claimed nucleic acids, proteins, antibodies, antibody fragments and / or CARs such as dual CARs, for example those defined by % sequence identity, that maintain similar binding properties of the invention are also included in the scope of the invention. Such variants, which show alternative sequences, but maintain essentially the same binding properties, such as target specificity, as the specific sequences provided are known as functional analogs, or as functionally analogous. Sequence identity relates to the percentage of identical nucleotides or amino acids when carrying out a sequence alignment.

[0206] The recitation "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. , the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0207] Any suitable methods of alignment of sequences for determination the percent sequence identity may be employed and are known to a person skilled in the art. The determination of percent identity between any two or more sequences can be accomplished using a publicly available mathematical algorithm. Computer software implementations of these mathematical algorithms include but are not limited to: ClustalW algorithm (VNTI software, InforMax Inc.), ALIGN (Version 2.0), GAP (Genetics Computer Group, software; now available via Accelrys on http: / / www.accelrys.com), BESTFIT, BLAST®, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), Madison, Wis., USA) and Multiple sequence alignments MUSCLE (EMBL’s European Bioinformatics Institute (EMBL-EBI), Cambridgeshire, UK). Alignments using these programs can be performed using the default parameters. Software for performing BLAST® analyses is publicly available through the National Center for Biotechnology Information. A sequence database can be searched using the nucleic acid sequence of interest. Algorithms for database searching are typically based on the BLAST software (Altschul et al., 1990).

[0208] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology or sequence identity to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions and other changes in sequence that fall under the described sequence identity are also encompassed in the invention.

[0209] Protein sequence modifications, which may occur through substitutions, are also included within the scope of the invention. Substitutions as defined herein are modifications made to the amino acid sequence of the protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein which contains a different amino acid sequence than the primary protein. Substitutions may be carried out that preferably do not significantly alter the function of the protein. Like additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions may be made without significantly altering the protein's function. This is particularly true when the modification relates to a "conservative" amino acid substitution, which is the substitution of one amino acid for another of similar properties. Such "conserved" amino acids can be natural or synthetic amino acids which because of size, charge, polarity and conformation can be substituted without significantly affecting the structure and function of the protein. Frequently, many amino acids may be substituted by conservative amino acids without deleteriously affecting the protein's function. In general, the non-polar amino acids Gly, Ala, Vai, lie and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gin, Asn and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu, represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can substitute for each other even though they belong to different groups.

[0210] Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but framework alterations are also contemplated. If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in the table immediately below, or as further described below in reference to amino acid classes, may be introduced and the products screened.

[0211] Potential Amino Acid Substitutions:

[0212] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0213] Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids are omega amino acids of various chain lengths and cyclohexyl alanine which are neutral non-polar analogs; citrulline and methionine sulfoxide which are neutral non-polar analogs, phenylglycine which is an aromatic neutral analog; cysteic acid which is a negatively charged analog and ornithine which is a positively charged amino acid analog. Like the naturally occurring amino acids, this list is not exhaustive, but merely exemplary of the substitutions that are well known in the art.

[0214] Genetically modified cells and Immune cells

[0215] The present invention contemplates, in particular embodiments, cells genetically modified to express the dual CARs contemplated herein, for use in the treatment of medical conditions. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell. The terms, "genetically modified cells”," genetically modified immune cells”, "modified cells," and, "redirected cells," are used interchangeably. As used herein, the term "gene therapy" refers to the introduction-permanently or transiently- of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects, or modifies expression of a gene, or for the purpose of expressing a therapeutic polypeptide, e.g., a dual CAR. In particular embodiments, the dual CARs contemplated herein are introduced and expressed in immune effector cells so as to redirect their specificity to a target antigen of interest, e.g., a CEA polypeptide and a EpCAM polypeptide.

[0216] An "immune cell" or "immune effector cell" are any cells of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). An Immune effector cells can be also differentiated from iPSCs (induced pluripotent stem cells) or derived from peripheral human blood and / or human cord blood.

[0217] “Natural Killer (NK) cells,” also known as large granular lymphocytes (LGL), are a vital component of the innate immune system, comprising 5-20% of circulating lymphocytes in humans. Functionally analogous to cytotoxic T cells in adaptive immunity, NK cells respond rapidly to virus-infected cells and tumors, acting approximately three days after infection. Unlike most immune cells, NK cells do not require antibodies or major histocompatibility complex (MHC) for target recognition, allowing for a swift immune response. Identified by the presence of CD56 and absence of CD3, NK cells differentiate from CD127+ common innate lymphoid progenitors and mature in various tissues before entering circulation. CD56bright NK cells, predominant in bone marrow and lymphoid tissues, exhibit immunoregulatory roles, while CD56dim NK cells in peripheral blood excel in cell killing. There are various sources from which NK cells can be derived, e.g., peripheral blood mononuclear cells, cord blood, immortalized cell lines, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). All sources can provide clinically meaningful cell doses, are amenable to CAR receptor engineering, and have transitioned into in-human studies. NK cells express distinct receptors, including inhibitory receptors recognizing self-MHC class I and activating receptors (Raftery MJ, Ann Rev of Cancer Biol, 2023). Activating molecules may include the natural cytotoxicity receptors, NKG2D, some CD94 / NKG2 complexes, and CD16 (Fcylll). Activating molecules recognize partner ligands on cells except for CD16, a low-affinity Fc receptor responsible for antibodydependent cellular cytotoxicity (ADCC). Inhibitory molecules may include the killer cell immunoglobulin-like receptor (KIR) family of molecules, some CD94 / NKG2 complexes, and leukocyte Ig-like receptors (LIRs). These receptors govern NK cell activity, adjusting to environmental cues. NK cells contribute to both innate and adaptive immunity, demonstrating antigen-specific immunological memory. The NK-cell may be autologous with respect to an individual to whom it is to be administered. The NK-cell may be allogeneic with respect to an individual to whom it is to be administered.

[0218] For example, when reintroduced back to patients after autologous cell transplantation, the NK cells modified with the dual CAR of the invention as described herein may recognize and kill tumor cells expressing CEA and / or EpCAM on the cell surface. iPS cell lines can also be ordered from public vendors and cell repositories, such as the NINDS Human Cell and Data Repository (https: / / stemcells.nindsgenetics.org / ). For example, publically available iPCS cells can be selected from a group of iPS cell line NDS00159; NDS00249; NDS00250, NDS00251 , NDS00252, NDS00253, NDS00254, NDS00255, NDS00256, NDS00257, NDS00258, NDS00259, NDS00260, NDS00261 , NDS00262, NDS00263, ND50039. In one embodiment, the iPS cell line NDS00159 is used.

[0219] “Immortalized”, as used herein, refers to immortalized cells as a population of cells that would not normally reproduce for an unlimited number of cell cycles. Due to a mutation, immortal cells escape from a normal cellular senescence and instead keep undergoing the cell proliferation. The mutation can occur spontaneously or induced by UV light, genetic manipulation, or entry by a virus, toxin or bacteria into the cell. The cells can therefore be cultivated in vitro over a longer period of time for experimental, therapeutic or medical purposes. Said immortalized immune cells include K13, ap T cells, yb T cells, NK cells, NKT cells, NK-92 and YT cells, stem cells, or stem cell-derived cells including cells of the aforementioned immune system, preferably NKT cells, NK- 92 cells, YT cells, and NK cells. An immortalized T cell line may retains its lytic function.

[0220] As would be understood by the skilled person, other cells may also be used as cells with the dual CARs as described herein. In particular, such cells include T cells, NK cells, neutrophils, and macrophages. These cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Progenitors can be iPSCs that become immune effector cells under defined culture conditions.

[0221] A "T cell" also termed "T lymphocyte" is an immune cell belonging to the group of lymphocytes. A T cell can be a thymocyte, immature T lymphocyte, mature T lymphocyte, resting T lymphocyte, cytokine-induced killer cell (CIK cell) or activated T lymphocyte. T cells originate from the bone marrow and migrate via the blood stream to the thymus, where they generate T cell receptors (TCR) and undergo a positive and negative selection in which the cells that show high affinity to endogenous proteins are degraded. T cells may be a T helper (Th; CD4+ T cell) cell, for example a T helper (Th) cell, such as a TH1 , TH2, TH3, TH17, TH9 or TFH cell. The T cell can be a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4 CD8 T cell, or a regulatory T cell (reg) or any other subset of T cells such as a cytokine-induced killer (CIK) cell which is typically a CD3- and CD56-positive, non-major histocompatibility complex (MHC)- restricted, natural killer (NK)- like T lymphocyte. The T cell can be a naive, effector, memory, effector storage, central storage, memory stem T cell. The T cell can be an umbilical cord blood cell. The T cell can be a peripheral lymphocyte. A T-cell can be derived and expanded from peripheral mononuclear blood cells (PBMNCs). The T-cell may be autologous with respect to an individual to whom it is to be administered. The T-cell may be allogeneic with respect to an individual to whom it is to be administered.

[0222] The cells of the invention can be autologous / autogeneic ("self’) or non-autologous ("non- self," e.g., allogeneic, syngeneic or xenogeneic). “Autologous”, as used herein, refers to cells from the same subject, and represent a preferred embodiment of the invention. "Allogeneic," as used herein, refers to cells of the same species that differ genetically to the cell in comparison. "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. "Xenogeneic," as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells of the invention are autologous or allogeneic.

[0223] The present invention provides methods for making the genetically modified cells that express the dual CAR contemplated herein. In one embodiment, the method comprises transfecting or transducing cells isolated from an individual such that the cells express one or more dual CARs as described herein. In certain embodiments, the cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly readministered into the individual. In further embodiments, the cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a dual CAR. In this regard, the cells may be cultured before and / or after being genetically modified (i.e. , transduced or transfected to express a dual CAR contemplated herein).

[0224] In particular embodiments, prior to in vitro manipulation or genetic modification of the cells described herein, the source of cells is obtained from a subject. In one embodiments, the dual CAR-modified cells comprise NK cells. NK cells can be obtained from several sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, NK cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLL™ separation, magnetic bead labeling, antibody-conjugated bead-based methods such as MACSTM separation (Miltenyi). In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. The cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most, if not all, other divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be accomplished by methods known to those in the art, such as using a semiautomated flow through a centrifuge, for example, the Cobe 2991 cell processor, the Baxter Cyto Mate, or the like. After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed in the cell directly resuspended culture media.

[0225] In certain embodiments, NK cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Positive or negative selection techniques can further isolate a specific subpopulation of NK cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. PBMC may be directly genetically modified to express CARs using methods contemplated herein. In certain embodiments, after isolation of PBMC, NK cells are further isolated.

[0226] In some embodiments, the immune cells of the present invention, for example, the NK cells described herein, can be obtained from inducible pluripotent stem cells (iPSCs) using methods known to a skilled person.

[0227] Direct in vitro differentiation of engineered NK cells from pluripotent stem cells, such as inducible pluripotent stem cells, provides an unlimited source of cells that can be genetically modified to express the dual CAR of the present invention. In some embodiments, a so-called master iPSC line can be maintained, representing a renewable source for consistently and repeatedly manufacturing homogeneous cell products. In some embodiments, the transformation of a master iPS cell line with the dual CAR encoding nucleic acid is contemplated prior to expansion and differentiation to the desired immune cell, preferably NK cell. NK cells can, for example, be generated from iPSCs, such that iPSCs could be modified with the dual CAR encoding nucleic acids and subsequently expanded and differentiated to NK cells for administration to the patient. Differentiation to the appropriate immune cell, such as an NK cell, could also be conducted from the iPSCs before transformation with dual CAR encoding nucleic acids and expansion prior to administration. All possible combinations of iPSC expansion, genetic modification, and expansion to provide suitable numbers of cells for administration are contemplated in the invention.

[0228] In one embodiment, the transformation of the master iPS cell line ND50039 with the dual CAR encoding nucleic acid construct is contemplated, before expansion and differentiation to the desired immune cell, preferably T cells or NK cells. T lymphocytes can for example be generated from iPSCs or prior genetically modified iPSCs, such that iPSCs could be modified with the dual CAR encoding nucleic acids and subsequently expanded and differentiated to T cells for administration to the patient.

[0229] The immune effector cells, such as NK cells, can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In a particular embodiment, the immune effector cells, such as NK cells, are genetically modified with the chimeric antigen receptors contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a dual CAR) and then are activated and expanded in vitro. In various embodiments, NK cells can be activated and expanded before or after genetic modification to express a dual CAR using methods known to a skilled person.

[0230] In a further embodiment, a mixture of, e.g., one, two, three, four, five, or more, different expression vectors can be used in genetically modifying a donor population of immune effector cells wherein each vector encodes a different chimeric antigen receptor protein as contemplated herein. The resulting modified immune effector cells form a mixed population of modified cells, with a proportion of the modified cells expressing more than one different dual CAR protein.

[0231] In one embodiment, the invention provides a method of storing genetically modified murine (rat), human, or humanized dual CAR protein expressing immune effector cells that target a CEA protein and an EpCAM protein, comprising cryopreserving the immune effector cells such that the cells remain viable upon thawing. A fraction of the immune effector cells expressing the dual CAR proteins can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with T cell-related conditions. The cryopreserved transformed immune effector cells can be thawed, grown, and expanded for more such cells when needed.

[0232] Compositions and Formulations

[0233] The compositions contemplated herein may comprise one or more polypeptides, polynucleotides, vectors comprising the same genetically modified cells, etc., as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or an animal, either alone or in combination with one or more other therapy modalities. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0234] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0235] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatine; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.

[0236] AAV vectors, lentiviral vectors and / or other compositions, agents, drugs, biologies (proteins) can be incorporated into pharmaceutical compositions, eg, pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions are particularly useful for administration and delivery to a subject in vivo or ex vivo In particular embodiments, compositions of the present invention comprise an amount of dual CAR- expressing cells contemplated herein. As used herein, the term "amount" refers to "an amount effective" or "an effective amount" of a genetically modified therapeutic cell, e.g., T cell, NK cell, CIK cell, to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0237] A "prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount. The term prophylactic does not necessarily refer to a complete prohibition or prevention of a particular medical disorder. The tern prophylactic also refers to the reduction of risk of a certain medical disorder occurring or worsening in its symptoms.

[0238] A "therapeutically effective amount" of genetically modified immune cells may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject {e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0239] Generally, a pharmaceutical composition comprising the NK cells described herein may be administered at a dosage of 102to 1010cells / kg body weight, preferably 105to 107cells / kg body weight, including all integer values within those ranges. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For uses provided herein, the cells are generally in a volume of a liter or less, 500 mLs or less, or even 250 mLs or 100 mLs or less. Hence, the density of the desired cells is typically greater than 106cells / ml and generally greater than 107cells / ml, generally 108cells / ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012cells. In some aspects of the present invention, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells may be administered. Dual CAR-expressing cell compositions may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.

[0240] Generally, compositions comprising the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, compositions comprising the dual CAR-modified T cells contemplated herein are used in the treatment of cancer, more preferably solid malignancies, more preferably rectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, stomach cancer, and ovarian cancer, more preferably metastatic tumor cells positive for CEA and / or EpCAM. The dual CAR-modified T cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with carriers, diluents, excipients, and / or with other components such as interleukins or other immune response stimulating cytokines e.g. IL-15 and / or checkpoint inhibitory molecules, e.g. PD1 polypeptide or a PD1- antibody, or cell populations. In particular embodiments, pharmaceutical compositions contemplated herein comprise an amount of genetically modified cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0241] Pharmaceutical compositions of the present invention comprising a dual CAR-expressing cell population, such as T cells or NK cells or CIK cells, 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., aluminium hydroxide); and preservatives. Compositions of the present invention are preferably formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0242] The liquid pharmaceutical compositions, whether they are solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerine, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0243] In a particular embodiment, compositions contemplated herein comprise an effective amount of dual CAR-expressing immune effector cells, alone or in combination with one or more therapeutic agents. Thus, the dual CAR-expressing immune effector cell compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents.

[0244] A combined immunotherapy encompasses simultaneous treatment, co-treatment or joint treatment, and includes the administration of genetically modified immune cells expressing a nucleic acid construct encoding a dual CAR combined with immunotherapies, such as checkpoint inhibitors and / or immune stimulatory cytokines, whereby treatment may occur within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. A combination medicament, comprising one or more of said genetically modified immune cells with another immunotherapeutic, may also be used in order to co-administer the various components in a single administration or dosage. ic Methods

[0245] The genetically modified cells contemplated herein provide improved methods of adoptive immunotherapy for use in the treatment of medical disorders associated with the presence of pathogenic cells expressing CEA and / or EpCAM that include preferably solid malignancies.

[0246] As use herein, "medical disorders associated with the presence of pathogenic cells expressing CEA and / or EpCAM refer to medical conditions, such as a cancer or autoimmune disease, in which the cells involved in pathophysiology of the disease demonstrate expression of CEA and / or EpCAM, and preferably presentation of CEA and / or EpCAM on the cell surface. The expression of CEA and / or EpCAM can be determined by various methods known to a skilled person, for example by isolating cells from a patient and assessing these by PCR using primers directed CEA or EpCAM transcripts, immune-staining with anti-CEA or anti-EpCAM antibodies, or by analysis by flow cytometry. Such pathogenic cells may typically be tumor stem cells and / or solid tumor cells and / or metastatic cancer cells.

[0247] "Cancer", as used herein, is a disease characterized by the uncontrolled growth of abnormal cells. Cancer refers to any type of cancerous growth or carcinogenic process, metastatic tissue or malignant transformed cells, tissues or organs, regardless of histopathological type or invasive stage. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Cancer cells spreading to other parts to the body are termed “metastatic cells” or “metastatic tumor cells”. The terms "tumor" and "cancer", used herein, are utilized interchangeably, e.g. both terms include solid and liquid, e.g. general or circulating tumors, premalignant and malignant cancers and tumors.

[0248] Examples of solid tumors are liver, lung, breast, lymphatic system, digestive organs (e.g. colon), urogenital organs (e.g. kidney, urothelial cells), prostate and throat, malignant organ systems including tumors such as sarcomas, adenocarcinomas and cancer. Examples for breast cancer that can be treated with the are ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), invasive ductal carcinoma (IDC), invasive ductal carcinoma including tubular, medullary, mucinous, papillary, and cribriform carcinomas, invasive lobular carcinoma (ILC), inflammatory breast cancer, male breast cancer, Paget’s Disease of the nipple, phyllodes tumors of the breast, recurrent and / or metastatic breast cancer. Adenocarcinoma includes most malignant tumors such as colon cancer, rectal cancer, renal cell cancer, liver cancer, non-small cell lung cancer, small intestine cancer and esophageal cancer. In certain forms the cancer is a melanoma, e.g. an advanced stage melanoma. Metastatic lesions of the cancer can also be treated or prevented with the methods and compositions of the invention. Examples of other types of cancer that can be treated are bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, faropius duct cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myelocytic leukaemia, acute lymphoblastic leukaemia, chronic or acute leukaemia including chronic lymphatic leukaemia, solid tumor in childhood, lymphatic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasm of the central nervous system (CNS) primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, asbestos-induced T cell lymphoma Including a combination of environmental cancer and cancer including things T reatment of metastatic cancer, e.g. metastatic cancer that expresses PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144) can be performed with the inhibitory molecules described in this invention.

[0249] Sarcomas as defined in the context of the present invention include, but are not limited to a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

[0250] Melanomas according to the present invention include, but are not limited to include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.

[0251] In the context of the present invention the term solid tumor further refers without limitation to acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticurn, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0252] In particular embodiments, compositions comprising dual CAR-modified T cells or comprising dual CAR-modified NK cells contemplated herein are used in the treatment of cancer, including but not limited to solid malignancies, such as, for example, breast cancer, pancreatic cancer colon cancer, rectal cancer, lung cancer, breast cancer, liver cancer, stomach cancer and / or ovarian cancer.

[0253] As used herein, the terms "individual", "subject" and “patient” are often used interchangeably and refer to any animal that exhibits a symptom of a solid tumor that can be treated with the cellbased therapeutics, and methods disclosed elsewhere herein. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. Typical subjects include human patients that have a solid cancer, have been diagnosed with a cancer, preferably solid cancer, or are at risk or having a cancer, preferably a solid cancer.

[0254] As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated.

[0255] T reatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0256] As used herein, "prevent," and similar words such as "prevented," "preventing" or “prophylactic” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition.

[0257] In one embodiment, a method of treating a cancer related condition in a subject in need thereof comprises administering an effective amount, e.g., therapeutically effective amount of a composition comprising genetically modified cells contemplated herein. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0258] The compositions contemplated herein may be administered conveniently, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases "parenteral administration" and "administered parenterally," as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection. SEQUENCES

[0259] Table 1 : Preferred nucleic acid sequences of the present invention

[0260] Table 2: Preferred amino acid sequences of the invention FIGURES

[0261] The invention is further described by the following figures. These are not intended to limit the scope of the invention but represent preferred embodiments of aspects of the invention provided for greater illustration.

[0262] Brief description of the figures

[0263] Fig.1 : NFkB activity (GFP) of Jurkat CAR cells stimulated with target cells (MDA-MB-468, HCC1937, SKOV-3).

[0264] Fig.2: Cytotoxicity of dual EpCAM CEA CAR-transduced NK cells towards LS174t cells.

[0265] Fig. 3: Cytotoxicity of mRNA transfected NK92 cells expressing dual EpCAM CEA CAR towards MDA-MB-468 cells.

[0266] Detailed description of the figures

[0267] Fig.1 : NFkB activity (GFP) of Jurkat CAR cells stimulated with target cells (MDA-MB-468, HCC1937, SKOV-3). Jurkat cells expressing GFP under the control of the NFkB promoter were seeded with a lentivirus-encoded control CAR construct, a CEA-CAR construct, an EpCAM-CAR construct or a dual EpCAM CEA-CAR construct. Cells were delivered to target cells and 1 day later positive cells were assessed by flow cytometry.

[0268] Fig.2: Cytotoxicity of dual EpCAM CEA CAR-transduced NK cells towards LS174t and HCC1937 cells. Human peripheral blood NK cells were transduced with a lentivirus-encoding Control-CAR construct, a CEA-CAR construct, an EpCAM-CAR construct or a dual EpCAM CEA-CAR construct. The cells were delivered to target cells LS174t and cytotoxicity was determined 18 hours later.

[0269] Fig. 3: Cytotoxicity of mRNA transfected NK92 cells expressing dual EpCAM CEA CAR towards MDA-MB-468 cells. (A) Schematic presentation of a NL92 cell expressing the dual CAR. (B) Flow cytometry measurement of the expression of CEA and EpCAM on the surface of the MDA-MB- 468 target cells. (C) NK92 cells were transfected with an mRNA encoding for a dual EpCAM CEA-CAR according to the present invention or a control CAR construct. The transfected cells (NK92 DUAL EpCAM CEA CAR mRNA) or control cells (NK92 CTRL CAR mRNA) were delivered to target cells MDA-MB-468 in a 2D-model at different effector cells to target cell ration (E:T ratio) and cytotoxicity was determined 18 hours later.

[0270] EXAMPLES

[0271] The invention is demonstrated through the examples disclosed herein. The examples provided represent particular embodiments and are not intended to limit the scope of the invention. The examples are to be considered as providing a non-limiting illustration and technical support for carrying out the invention.

[0272] Example 1 : NF-kB promoter activity of anti-CEA / anti-EpCAM CAR expressing Jurkat cells Lentiviral vectors encoding the anti-CEA / anti-EpCAM CAR, a checkpoint inhibitor, the dominant negative truncated PD-1 protein, an immunostimulatory cytokine IL-15, are introduced into a immune cell line Jurkat expressing GFP under control of the NF-kB promoter. The cells are transduced by using lentiviral gene transfer.

[0273] The Jurkat cell line modified in this way was stimulated with the cancer cell lines MDA-MB-468, HCC1937 or SKOV-3., expressing CEA and EpCAM on the cellular surface.

[0274] GFP expression under the control of the NF-kB promoter was determined in the modified Jurkat cells transduced with a lentiviral vector encoding (1) a dual anti-CEA / anti-EpCAM CAR construct combined with checkpoint inhibitor dntPDI opt and IL-15 transgene 15R15 (Dual EpCAM CEA CAR) or (2) an anti-EpCAM CAR (EpCAM CAR), and anti-CEA CAR (CEA CAR) or a (4) a control CAR. The transduced Jurkat cells were transferred to MDA-MB-468, HCC1937 or SKOV- 3target cells.

[0275] After 1 day, GFP positive cells were determined by flow cytometry. The Jurkat cells expressing the dual EpCAM CEA CAR show substantially higher GFP expression compared to cell expressing the CEA CAR, the EpCAM CAR or the control CAR, thus demonstrating a synergistic effect of the dual CAR over the CEA CAR or the EpCAM CAR alone described in this invention (Figure 1).

[0276] Example 2: Cytotoxicity of dual EpCAM CEA CAR-transduced NK cells towards LS174t cells.

[0277] Human peripheral blood NK cells were transduced with a lentivirus-encoding Control-CAR construct, an EpCAM-CAR construct, a CEA-CAR construct or a dual EpCAM CEA-CAR construct. The cells were delivered to target cells LS174t or HCC1937 expressing CEA and EpCAM on the cell surface and cytotoxicity was determined 18 hours later.

[0278] The NK cells expressing the dual EpCAM CEA CAR show substantially higher cytotoxicity towards LS174t compared to cells expressing the CEA CAR, the EpCAM CAR or the control CAR, thus demonstrating a synergistic effect of the dual CAR over the EpCAM CAR described in this invention (Figure 2).

[0279] Example 3: Cytotoxicity of mRNA transfected NK92 cells expressing dual EpCAM CEA CAR towards MDA-MB-468 cells.

[0280] NK92 cells were transfected with with an mRNA encoding for a Control-CAR construct or a dual EpCAM CEA-CAR construct by electroporation. The transfected cells were delivered to target cells MDA-MB-468 expressing high amounts of EpCAM and lower amounts of CEA (Fig. 3 B and C). Different effector cells to target cell ration (E:T ratio) were evaluated (Figure 3C). Cytotoxicity was determined 18 hours later.

[0281] The NK cells expressing the dual EpCAM CEA CAR show substantially higher cytotoxicity towards MDA-MB-468 cells compared to cells expressing the control CAR, thus surprisingly demonstrating that the dual EpCAM CEA CAR can be provided and efficiently transfected as an mRNA construct even in NK cells. This is particularly surprising as NK cells are usually sensitive to mRNA transfection and electroporation and show low transfection efficiencies. By providing the nucleic acid construct of the present invention as an mRNA construct advantageously even difficult to transfect cells can by efficiency transfected to express the dual EpCAM CEA-CAR in a simple, safe and cost-efficient manner.

Claims

CLAIMS1 . A nucleic acid construct, comprising: a first nucleic acid sequence region encoding a dual chimeric antigen receptor (CAR) polypeptide, said dual CAR polypeptide comprising a first extracellular antigen-binding domain that binds an epithelial cell adhesion molecule (EpCAM) and a second extracellular antigen-binding domain of the dual CAR that binds a carcinoembryonic antigen (CEA) protein, and a second nucleic acid sequence region encoding an immune stimulatory cytokine.

2. The nucleic acid construct according to claim 1 , wherein the first nucleic acid sequence region encoding the dual CAR comprises: a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to CEA protein, said antigen-binding domain comprising an antibody or antibody fragment, a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to EpCAM, said antigen-binding domain comprising an antibody or antibody fragment, a nucleic acid sequence linking the nucleic acid sequences encoding said extracellular antigen-binding domains, and a nucleic acid sequence encoding a hinge domain, a transmembrane domain and a signalling domain.

3. The nucleic acid construct according to any one of the preceding claims, wherein the second nucleic acid sequence region encoding an immune stimulatory cytokine comprises a nucleic acid sequence encoding one or more immune stimulatory cytokines operably linked to one or more promoters, wherein at least one of said cytokines is selected from the group consisting of IL-15, IL-2, IL-7, IL-12, IL-21 , IFN gamma, and IFN beta, preferably an IL-15 superagonist, wherein preferably the second nucleic acid sequence region encoding the immune stimulatory cytokine is operably linked to one or more constitutive promoters, more preferably a human promotor, more preferably an NFAT promotor, and wherein preferably the immune stimulatory cytokine maintains or enhances the activity, survival and / or number of immune cells within and / or in proximity to tumor tissue.

4. The nucleic acid construct according to any one of the preceding claims, additionally comprising a third nucleic acid sequence region encoding a checkpoint inhibitory molecule.

5. The nucleic acid construct according to the preceding claim, wherein the checkpoint inhibitory molecule encoded by the third nucleic acid sequence region is a dominant negative polypeptide and / or an antibody inhibiting and / or blocking an immune checkpoint protein, wherein the checkpoint inhibitory molecule is preferably a dominant negative truncated PD1 polypeptide or a PD1 -antibody.

6. The nucleic acid construct according to according to any of the preceding claims, wherein the first nucleic acid sequence region encoding the dual CAR and the third nucleic acid sequence region encoding the checkpoint inhibitory molecule, are configured to encode a polycistronic mRNA comprising coding regions for the polypeptide sequences of the dual CAR and the checkpoint inhibitory molecule, and wherein an amino acid sequence comprising a polypeptide cleavage site is disposed between the dual CAR polypeptide and the checkpoint inhibitory molecule polypeptide, wherein the polypeptide cleavage site is preferably selected from the group consisting of P2A, T2A, E2A and F2A.

7. The nucleic acid construct according to any one of the preceding claims, optionally comprising: a further nucleic acid sequence region encoding a suicide gene, preferably a herpes simplex virus thymidine kinase, and / or a further nucleic acid sequence region encoding beta-2-microglobulin.

8. The nucleic acid construct according to any one of the preceding claims, wherein the first nucleic acid sequence region encoding the dual CAR comprises: a. a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to CEA protein, said antigen-binding domain comprising VH and VL sequences according to SEQ ID NO 29 and 33, respectively, or sequences with at least 80% sequence identity to SEQ ID NO 29 and 33; and b. a nucleic acid sequence encoding an extracellular antigen-binding domain that binds to EpCAM, said antigen-binding domain comprising VH and VL sequences according to SEQ ID NO 21 and 25, respectively, or sequences with at least 80% sequence identity to SEQ ID NO 21 and 25; c. a nucleic acid sequence linking the nucleic acid sequences encoding said extracellular antigen-binding domains, preferably according to SEQ ID NO 10, or a sequence with at least 80% sequence identity to SEQ ID NO 10, and d. a nucleic acid sequence encoding a hinge domain, a transmembrane domain and a signalling domain, preferably according to SEQ ID NO 12, or a sequence with at least 80% sequence identity to SEQ ID NO 12.

9. The nucleic acid construct according to any one of the preceding claims, wherein the immune stimulatory cytokine is a IL-15 superagonist according to SEQ ID NO 18, or a sequence with at least 80% sequence identity to SEQ ID NO 18.

10. A dual chimeric antigen receptor (CAR) polypeptide encoded by the nucleic acid construct according to any one of claims 1 to 9.11 . A cell, comprising a nucleic acid construct according to any one of claims 1 to 9 and / or the dual CAR polypeptide according to claim 10.

12. The cell according to claim 11 , whereina. the cell is a natural killer (NK) cell, preferably derived from induced pluripotent stem cells (iPSC), more preferably derived from the iPS cell line ND50039, and / or b. wherein the cell is an immune cell derived from iPSC that has been genetically modified with the nucleic acid construct before differentiation into a NK cell.

13. The cell according to any one of claims 11 to 12 for use in the treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA and / or EpCAM in a subject.

14. The cell for use according to the preceding claim, wherein the pathogenic cells expressing CEA and / or EpCAM are cancer cells, preferably cancer cells of solid malignancies, more preferably cancer cells of breast cancer, pancreatic cancer colon cancer, rectal cancer, lung cancer, liver cancer, stomach cancer and / or ovarian cancer.

15. A pharmaceutical composition, in the form of a therapeutic cell product, comprising cells according to any of claims 11 to 12 and a pharmaceutically acceptable carrier.

16. An in vitro method for preparing a genetically modified immune cell according to any of claims 11 to 12, comprising delivering a nucleic acid construct according to any one of claims 1 to 9 into a cell.

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