WO2025004043A1

WO2025004043A1 - Combination therapy using car t cells and klotho

Info

Publication number: WO2025004043A1
Application number: PCT/IL2024/050630
Authority: WO
Inventors: Ido Wolf; Anat GLOBERSON LEVIN; Tamar Rubinek
Original assignee: Ichilov Tech Ltd.
Priority date: 2023-06-27
Filing date: 2024-06-27
Publication date: 2025-01-02

Abstract

The present invention discloses combinatory cancer therapy of immune cells engineered to express an anti-cancer chimeric antigen receptor and klotho polypeptides. The invention discloses various methods to execute the invention as well as compositions and nucleic acid construct that may be used to obtain immune cells that may be used.

Description

COMBINATION THERAPY USING CAR T CELLS AND KLOTHO

FIELD OF THE INVENTION

The present invention relates to a combinational cancer therapy using immune cells engineered to express a chimeric antigen receptor (CAR) that binds specifically a tumor- associated antigen, and a klotho polypeptide. The invention relates to different aspects of the therapy such as compositions and methods of providing the treatment.

BACKGROUND OF THE INVENTION

Therapy based on adoptive cell transfer of Chimeric Antigen Receptor (CAR)- expressing T cells (CAR T cells) has become an innovative highly effective treatment for various hematologic malignancies. CARs are genetically engineered modular single-chain structures composed of an antigen recognition unit and T cell activation moieties in tandem. For the treatment, T cells are harvested from the patient or from a suitable donor, genetically modified ex vivo to express the tumor-directed CAR and re-infused into the patient. The adoptively transferred engineered cells are now able to recognize the respective tumor antigen, and exert a tumor- specific immune response.

Despite the success of CAR T cells-based therapy in managing hematologic malignancies, its application toward solid tumors remains challenging, and efforts to design effective CAR T cells for the treatment of several solid tumors, including ovarian and breast cancers, have been unsuccessful (Kosti et al., Front Immunol 2018; 9:1104; Aharon et al, Hum Gene Ther. 2021;32(19-20): 1224-1241). A prerequisite for a success is the ability of CAR T cells to efficiently home and penetrate the desmoplastic stroma surrounding the tumor and accumulate in the tumor microenvironment. Next, cells must gain the ability to expand, persist and mediate cytotoxicity under a hostile milieu largely composed of immunosuppressive modulators. These include myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs), as well as paracrine signaling by tumor-derived IL-8, which promotes the trafficking of neutrophils and myeloid- derived suppressor cells (MDSCs) into the tumor microenvironment, further dampening anti-tumor immune responses.

The klotho gene encodes for a transmembrane protein that can be shed and act as a circulating hormone. Klotho mediates anti-aging activities, and reduced klotho levels are associated with a wide array of aging-related phenomena, including atherosclerosis, dementia, frailty in the elderly and reduced lifespan. These activities are associated with the ability of klotho to reduce inflammation, as demonstrated in the kidney, cardiovascular system, brain and other tissues. Accumulating data indicate klotho as a modulator of the immune system. Klotho knockout mice (kl/kl) show a reduced number of thymocytes, especially CD4 and CD8 double-positive cells, and of both cortical and medullary thymocytes, suggesting a positive role for klotho on the differentiation and proliferation of these cells. Klotho has also been reported to interact with RIG-I and to inhibit RIG-I-induced expression of IL-6 and IL-8 both in vitro and in vivo. It was also shown that kl/kl mice have higher IL-6 levels in the blood as well as in several tissues.

Klotho was also suggested as a tumor suppressor in various epithelial malignancies, including breast and ovarian cancers (Wolf et al., Oncogene 2008(27), 7094-7105; Ligumsky et al., Mol Cancer Res 2015, 13(10), 1398- 1407). Its expression in these tumors was reported to be epigenetically silenced, and re-introduction of klotho into cancer cells or treatment with the soluble protein inhibited their viability and aggressiveness in vitro. US 9,987,326 and US 10,555,963, to some of the inventors of the present invention, disclose the use of soluble forms of klotho (e.g., the entire extracellular domain or the KL1 domain), or proteins with at least 80% homology to the amino acid sequence of the soluble forms of klotho, in the treatment of cancer.

WO 2020/039425, to some of the inventors of the present invention, discloses compositions and methods for inhibiting tumor growth in subjects in need thereof, utilizing gene transfer vectors, such as viral vectors, comprising a nucleotide sequence encoding a klotho protein operably linked to at least one regulatory sequence directing its expression.

WO 2023/218445, to some of the inventors of the present invention, discloses polypeptides derived from klotho KL1 domain exhibiting tumor suppressor activity, and use thereof in treating cancer and inhibiting tumor development.

WO 2019111249 and WO 2019111250 to some of the inventors of the present invention, disclose immunological cells comprising dual chimeric antigen receptors and uses thereof in treating cancer.

Despite the large number of proposed strategies for cancer treatments, there is still an urgent need for the development of novel efficient therapies for cancer.

SUMMARY OF THE INVENTION

The present invention is based on unexpected results showing that a combinatory administration of CAR T cells, specifically anti-HER2 CAR T cells, and a klotho functional polypeptide provided an enhanced and even synergistic anticancer effect. Therefore, the present invention in embodiments thereof provides novel combinatory treatments for cancer comprising co-administration of engineered immune cells expressing a CAR with a klotho polypeptide. The co-administration may be realized in many different modes such as separate administration of CAR-expressing immune cells and a pharmaceutical composition comprising a klotho polypeptide or a vector encoding, or administering CAR-expressing immune cells that are further engineered to provide a klotho-based therapy. It is also contemplated that CAR and/or klotho may be expressed only under conditions induced by a tumor. In some examples, klotho may be expressed only after the T cell expressing the CAR is activated by cancer cells.

According to one aspect, the present invention provides a therapeutic combination comprising (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, for use in treating cancer, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen.

According to another aspect, the present invention provides a pharmaceutical composition comprising immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen, and (ii) a klotho polypeptide, and a pharmaceutically acceptable carrier, for use in treating cancer.

According to a further aspect, the present invention provides a therapeutic combination comprising a population of immune cells engineered to express a CAR that binds specifically to a tumor-associated antigen, and a klotho polypeptide or a vector encoding thereof, for use in improving tumor killing and/or clearance.

According to one aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising co-administering to the subject a therapeutic combination of (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor antigen associated with the cancer. According to some embodiments, the immune cells are characterized by surface expression of the CAR upon administration. According to other embodiments, the co-administering comprises a regimen selected from a sequential administering and/or a substantially simultaneous administering. According to some embodiments, the cancer expresses the tumor-associated antigen to which the CAR binds specifically. According to some embodiments, the cancer is a solid cancer.

According to another aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor antigen associated with the cancer, and (ii) a klotho polypeptide. According to some embodiments, the immune cells express the CAR constitutively. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to an inducible promoter, the promoter initiating expression of said klotho polypeptide upon activation of a T cell receptor, e.g. upon activation of the T cell receptor of the CAR. According to some embodiments, the immune cells express the klotho polypeptide upon activation of the CAR. According to some embodiments, the immune cells express the CAR and klotho polypeptide constitutively.

According to yet another aspect, the present invention provides a method of improving tumor killing and/or clearance by a population of immune cells engineered to express a CAR that binds specifically to a tumor- associated antigen, the method comprising coadministering the population of the immune cells with a klotho polypeptide or a vector encoding thereof.

According to a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding a CAR that binds specifically to a tumor- associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein each of the nucleic acid molecules is operably linked to a promoter or both nucleic acid molecules are operably linked to one promoter. According to some embodiments, both nucleic acid molecules are operably linked to one constitutive promoter. According to some such embodiments, the nucleic acid construct further comprises a cleavable nucleic acid molecule or a nucleic acid molecule encoding a self-cleaving peptide between the two nucleic acid molecules. According to alternative embodiments, the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to (i) an inducible promoter, said promoter initiates the expression of klotho polypeptide upon activation of the CAR; or (ii) a constitutive promoter.

According to another aspect, provided a vector comprising the nucleic acid construct as defined herein.

According to yet another aspect, the present invention provides a population of immune cells engineered to express (i) a chimeric antigen receptor (CAR) binding specifically to cancer cells and (ii) a klotho polypeptide. According to some embodiments, the cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR and a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide. According to some embodiments, the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to (i) an inducible promoter, said promoter initiates the expression of klotho polypeptide upon activation of the CAR; or (ii) a constitutive promoter. According to some embodiments, the cells of the population of immune cells comprise a nucleic acid construct according or the vector as described herein

According to one aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically the population of immune cells as described herein.

According to another aspect, the present invention provides a pharmaceutical composition comprising the population of immune cells as described herein, and a pharmaceutically acceptable carrier.

According to yet another aspect, the present invention provides a pharmaceutical composition comprising (i) a population of immune cells engineered to express a chimeric antigen receptor (CAR) binding specifically to a tumor-associated antigen and (ii) a klotho polypeptide.

According to some aspects, the pharmaceutical composition of the present invention is for use in treating cancer. According to some embodiments, the cancer expresses the tumor antigen associated to which the CAR binds specifically. According to some embodiments, the cancer is a solid cancer.

According to any one of the above aspects and embodiments, (i) the CAR binds specifically to a tumor-associated antigen selected from ErbB2, CD19, BCMA, CD38, CD138, EGFR, CD276, CD24, GD2, EGF, Mesothelin (MSLN), MUC-1, FAP, and MUC16; and/or (ii) the klotho polypeptide is selected from membranal klotho, secreted klotho, proteolyzed klotho, KE1 domain, KE2 domain and a KE1 active fragment (e.g., KE340); and/or (iii) the immune cells are selected from T cells, natural killer cells and tumorinfiltrating lymphocytes (TIE). According to some embodiments, the CAR binds specifically to HER2 and the klotho polypeptide is selected from a soluble human klotho and human KL1. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 40 and the klotho polypeptide comprises the amino acid sequence selected from SEQ ID NO: 3-13. According to any one of the above aspects and embodiments, the immune cells are T cells. According to some aspects, the present invention provides a method of treating cancer in a subject in need thereof comprising co-administering to the subject therapeutically effective amounts of (i) T cells and (ii) a klotho polypeptide or a vector encoding thereof, wherein the T cells are engineered to express a CAR that binds specifically to HER2 and the klotho polypeptide is selected from a soluble klotho, KL1 and an active KL1 fragment (e.g., KL340).

According to other aspects, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of T cells engineered to express a CAR that binds specifically to HER2 and a klotho polypeptide selected from a soluble klotho, KL1 and an active KL1 fragment (e.g., KL340). According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 40 and the klotho polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 3-13.

According to any one of the above aspects and embodiments, the method of the present invention provides a synergistic anticancer effect.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1A shows is a representative compression of the staining with specific aCAR antibody (aN29) and GFP expression. Both exhibit similar expression levels indicating the GFP is a good marker for measuring transduction levels. (aN29 - 61.25% ± 5.33, GFP - 71.71% ± 7.65, n=3 p-value<0.23).

Fig. IB shows HER2 expression levels on different cell lines: OVCAR3, SKOV3 (ovarian cancer), SKBR3 (breast cancer) were stained using anti-HER2 antibody and were analyzed by FACS.

Fig. 1C shows in vitro activity of human N29 CAR lymphocytes: N29 and UT cells were stimulated on HER-2+ (SKOV3, SKBR3) and HER-2- (OVCAR3) target cell lines. After 16 h, IFN-g levels in culture supernatants were measured by ELISA (SKOV3 n = 7, SKBR3 n = 3, *p-value <0.05).

Fig. ID shows the killing of target cells by the transduced lymphocytes as measured using methylene blue assay (n=5, ***p<0.005, for the differences between UT stimulated on SKOV3 and N29 stimulated on SKOV3 and between N29 stimulated on SKOV3 and N29 stimulated on OVCAR3). Killing of target cells by the transduced lymphocytes as measured using methylene blue assay (n=4, ***p<0.005 ** p<0.01, for the differences between UT stimulated on SKBR3 and N29 stimulated on SKBR3). Fig. 2 shows the effect of a soluble klotho fragment on the growth of breast and ovarian cancer cell. Cells were grown in 96-well plates, treated with klotho at indicated concentrations and 72 hours later viability was assessed using methylene blue assay. Representative results of at least 3 experiments are shown. Data are presented as the mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.005

Fig. 3A and 3B show the effect of a soluble klotho on the T cells viability. Activated and non-transduced cells (Fig. 3A) or CAR-T (Fig. 3B), were grown in 96-well plates, treated with klotho at indicated concentrations (ng/pl) and 48 hours later cells were counted using trypan blue. The experiment was repeated twice with similar results.

Fig. 4A and 4B show the effect of CAR-T and KL1 co-treatment on SKBR-3 cell growth. SKBR-3 cells were transfected with 5 (Fig. 4A) or 10 pg (Fig. 4B) of pcDNA3 or KL1- expression plasmid. Cells were grown with G418 for 2 weeks and then were re -plated in 96- well plate (10,000 cells per well) and CAR-T cells were added with decreasing CAR-T cells per cancer cells ratio. Cancer cells viability was determined after 48 hours using trypan blue. The experiment was repeated three times and a representative experiment is shown. Data are presented as the mean ± SD.

Fig. 5A and 5B show the effect of CAR-T and KL1 co-treatment on PANCI cells growth. PANCI cells were transfected with 5 pg pcDNA3 or KL1 -expression plasmid. Cells were grown with G418 for 2 weeks and then were re -plated in 96-well plate (10,000 cells per well) and CAR-T cells, from two different donors (no 56 - Fig. 5A and 64 - Fig. 5B), were added with decreasing CAR-T cells per cancer cells ratio. Cancer cells viability was determined after 48 hours using trypan blue. The experiment was repeated three times and a representative experiment is shown. Data are presented as the mean ± SD.

Fig. 6A and 6B shows the effect of CAR-T and KL1 or KL340 co-treatment on OVCAR8 ovarian cancer (Fig. 6A) or on PANCI pancreatic cancer cells (Fig. 6B). Cells were grown for a week with G418 and seeded in 96-well plates. CAR T cells expressing anti HER2 were added at indicated T:E ratio (tumor:effector, pancECAR T) and were incubated for additional 72 hrs. and viability of cancer cells was assessed using methylene blue. Each condition was seeded in five biological replicates and each of the experiments was conducted twice. * p<0.05; **, p<0.01; ***, p<0.005.

Fig. 7 shows the effect of klotho on NFkB transcriptional activity. MCF7 cells were transfected with klotho, KL1 -expressing plasmid or control vector (pcDNA3) together with NFkB reporter gene (pNL3.2.NF-KB-RE vector) for 48 hrs. IL6 was added for the last 24 hrs. Luciferase activity was assessed and normalized to protein concentration. The experiment was repeated three times and a representative experiment is shown. Data are presented as the mean ± SD.

Fig. 8 shows the expression (both intracellular and secretion) of KL340 by a dual plasmid encoding KL340 and CAR.

Fig. 9A and 9B show staining of 293 lenti x cells transfected using jetprime with N29CAR- with/without KL340. Fig. 9A - unstained cells, Fig. 9B - shows staining with recombinant HER2, and an APC- human anti-HER2. The cells were analyzed using FACS canto.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to combination cancer therapy, in particular to therapeutic modalities combining the use of immune cells engineered to express a tumor- specific chimeric antigen receptor (CAR) and of klotho polypeptides or a vector encoding said peptides. The invention contemplates use of cell compositions in which a population of immune cells (e.g. T cells) is engineered to express (i) a CAR binding specifically to cancer cells and (ii) a klotho polypeptide for adoptive transfer cancer therapy. In other implementations, the invention relates to methods of treating cancer in a subject in need thereof, in which a therapeutic combination of (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, wherein the immune cells are engineered to express a CAR that binds specifically to a tumor antigen associated with the cancer, is co- administered to the subject (e.g. according to concurrent or sequential administration protocols).

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the patent specification, including definitions, will control.

General terms

The following terms are relevant to the following aspects and embodiments, and are contemplated, embedded and are part of each one of the relevant embodiments in which these terms are mentioned or used.

As used herein, the forms "a", "an" and "the" include singular as well as plural references unless the context clearly dictates otherwise. For example, the term "a protein" includes one or more copies of the recited protein.

The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

The term “treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, or ameliorating abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and/or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

The term “treating cancer” as used herein should be understood to e.g. encompass treatment resulting in a decrease in tumor size; a decrease in the rate of tumor growth; stasis of tumor size; a decrease in the number of metastasis; a decrease in the number of additional metastasis; a decrease in the invasiveness of the cancer; a decrease in the rate of progression of the tumor from one stage to the next; inhibition of tumor growth in a tissue of a mammal having a malignant cancer; control of establishment of metastases; inhibition of tumor metastases formation; regression of established tumors as well as a decrease in the angiogenesis induced by the cancer, inhibition of growth and proliferation of cancer cells and so forth. The term “treating cancer” as used herein should also be understood to encompass prophylaxis such as prevention as cancer reoccurs after previous treatment (including surgical removal) and prevention of cancer in an individual prone (genetically, due to lifestyle, chronic inflammation and so forth) to develop cancer. As used herein, “prevention of cancer” is thus to be understood to include prevention of metastases, for example after surgical procedures or after chemotherapy.

The term “cancer” comprises cancerous diseases or a tumor being treated or prevented and includes solid tumors and hematopoietic malignancies as disclosed herein. In one embodiment, the tumor is a solid tumor. In another embodiment, the cancer is a solid tumor (e.g. sarcoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, liver cancer, lung cancer, glioblastoma multiforme, glioma, melanoma, castration-resistant prostate cancer, triple negative breast cancer, squamous cell carcinoma, or colorectal cancer) .

In various embodiments the tumor is selected from the group comprising, but not limited to, mammary carcinomas, melanoma, skin neoplasms, lymphoma, leukemia, gastrointestinal tumors, including colon carcinomas, stomach carcinomas, pancreas carcinomas, colon cancer, small intestine cancer, ovarian carcinomas, cervical carcinomas, lung cancer, prostate cancer, kidney cell carcinomas and/or liver metastases. In some embodiments, the cancer is selected from breast, cervical, ovarian, pancreatic, GI cancer, melanoma, lung cancer, and glioblastoma. As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound, pharmaceutical composition, or method provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g. triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g. non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration -resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma. Additional examples include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, esophagus, liver, kidney, non-small cell lung, melanoma, mesothelioma, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells, cancer of the hepatic stellate cells, or prostate cancer. According to some embodiments, the cancer is HER2- possitive cancer (i.e. cancer expressing HER2 antigen) such as ovarian cancer, breast cancer and pancreatic cancer. According to some embodiments, the cancer express CD 19 tumor- associated antigen. According to some embodiments, the cancer is selected from a lymphoma and leukemia. According to some embodiments, the cancer express CD38 tumor- associated antigen. According to some embodiments, the cancer express CD138 tumor- associated antigen. According to some embodiments, the cancer express CD276 tumor- associated antigen. According to some embodiments, the cancer overexpresses EGFR.

As used herein, and unless otherwise specified, the term "adoptive transfer" refers to a form of passive immunotherapy where previously sensitized immunologic agents (e.g., cells or serum) are transferred to the recipients. The phrases “adoptive transfer immunotherapy”, “adoptive cell therapy” and “adoptive cell immunotherapy” are used interchangeably herein to denote a therapeutic or prophylactic regimen or modality, in which effector immunocompetent cells, such as the engineered cell compositions of the invention, are administered (adoptively transferred) to a subject in need thereof, to alleviate or ameliorate the development or symptoms of cancer or infectious diseases. The cells may be autologous or allogeneic. According to certain preferable embodiments, the cell composition is histocompatible with the subject to be treated. In some embodiments, the cells are T cells (e.g. obtained from peripheral blood or tumor infiltrating lymphocytes), that may be further manipulated or engineered as disclosed herein.

The term “therapeutically effective amount” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect, e.g., treating cancer. The full therapeutic effect does not necessarily occur by the administration of one dose and may occur only after the administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of the cognitive impairment, and the therapeutics or combination of therapeutics selected for administration, and the mode of administration. The skilled person can readily determine the effective amount for a given situation by routine experimentation.

In particular, as disclosed and demonstrated herein, the combined use of the engineered immune cells and klotho polypeptides has been demonstrated to exert a potent anti -tumor effect even when one or more of the components was used at an amount considered sub- therapeutic when used alone. Accordingly, therapeutic effective amounts as used herein further include in particular amounts that are effective when used in connection with the synergistic combinations of the invention, but provide a partial or non -significant therapeutic effect when used alone. In some embodiments, compositions and methods in accordance with the invention provide for reducing the amounts administered to a patient and/or cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more, wherein each possibility represents a separate embodiment of the invention.

As used herein, the term “synergistic” refers to a combination of therapeutic agents, which, when taken together, is more effective than the additive effects of the individual therapies. A synergistic effect of a combination of therapies (e.g., a combination of therapeutic agents) permits the use of lower dosages of one or more of the therapeutic agent(s) and/or less frequent administration of the agent(s) to a subject with a disease or disorder, e.g., a proliferative disorder. The ability to utilize a lower dosage of one or more therapeutic agents and/or to administer the therapeutic agent less frequently reduces the toxicity associated with the administration of the agent to a subject without reducing the efficacy of the therapy in the treatment of a disease or disorder. In addition, a synergistic effect can result in improved efficacy of agents in the prevention, management or treatment of a disease or disorder, e.g. a proliferative disorder. Finally, a synergistic effect of a combination of therapies may prevent or reduce adverse or unwanted side effects associated with the use of either therapeutic agent alone.

The term "co-administration" refers to administration of two or more compounds in a regimen selected from a single combined composition, separate individual compositions administered substantially at the same time, and separate individual compositions administered under separate schedules and include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “co-administration” encompasses administration of a first and second agent in an essentially simultaneous manner, such as in a single dosage form, e.g., a capsule or tablet having a fixed ratio of first and second amounts, or in multiple dosage forms for each. The agents can be administered sequentially in either order. When coadministration involves the separate administration of each agent, the agents are administered sufficiently close in time to have the desired effect (e.g., complex formation). The term “sequential manner” refers to an administration of two compounds at different times, and optionally in different modes of administration. The agents can be administered sequentially in either order. The term “substantially simultaneous manned’ refers to the administration of two compounds with only a short time interval between them. In some embodiments, the time interval is in the range of from 0.5 to 60 minutes.

The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), inhalation, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

The terms "immune cells" and "immunological cells" are used herein interchangeably and refer to cells that is involved in an immune response, e.g., promotion of an immune response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes. According to some embodiments, the term refers to cell of the immune system that are commonly used in immunotherapy such as T cells, B cells, natural killer (NK) cells and tumor infiltrating lymphocytes (TIL). The term “T cell” as used herein refers to as a thymus-derived lymphocyte that participates in a variety of cell- mediated immune reactions, as well known in art. The term “T cell” refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (a.k.a. Tx cells or CD4⁺ T cells) and subtypes, including THI, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (i.e., Tc cells, CD8⁺ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. T_reg cells or suppressor T cells) and subtypes, including CD4⁺FOXP3⁺ T_reg cells, CD4⁺FOXP3“ T_reg cells, Tri cells, Th3 cells, and T_reg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (y5 T cells), including Vy9/V62 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention. According to any one of the above embodiments, the T cell is selected are from CD4+ T-cell and a CD8+ T-cell. According to some embodiments, the T cells are a combination of CD4+ T-cell and a CD8+ T-cell.

As used herein, the term “engineered immune cell” refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of extra genetic material in the form of DNA or RNA to the total genetic material of the cell. According to embodiments herein, the engineered immune cells have been genetically modified to express a chimeric antigen receptor and/or a klotho polypeptide.

In some embodiments, the term “engineered to express” refers to cells that are modified to express a polypeptide, protein or proteins, which are not natively expressed in those cells. In one embodiment, such expression may be as a result of the integration of a sequence of interest within a genome of the cell, to facilitate such expression, or, in another embodiment, specific mutation of a sequence of interest in the genome, or in another embodiment, may be a result of extrachromosomal expression, such as via a plasmid, as will be appreciated by one skilled in the art. the term contemplates also cells expressing the polypeptide, protein or proteins. The term refers both to cell that constitutively express the polypeptide, protein or proteins or is capable of expressing it upon a stimulus, e.g., under specific conditions.

As used herein, the term “klotho protein” or "klotho polypeptide" encompasses membranal klotho, secreted klotho, proteolyzed klotho (also referred to herein as “KL1 - KL2” fragment), KL1 domain, an active KL1 fragment and KL2 domain. Each possibility represents a separate embodiment of the present invention. In some embodiments, the klotho protein is a human klotho protein (e.g. a soluble human klotho) or its active fragments as known in the art including, but not limited to KL1 and KL2. The terms "active KL1 fragment" and "KL1 active fragment" are used herein interchangeably and refer to a truncated KL1 domain which retains its anti-tumor activity, as detailed further below. A nonlimiting example of an active KL1 fragment is a truncated human KL1 identified as "KL340", in which the amino acids at positions 341-1012 of human klotho (SEQ ID NO: 3) have been deleted. KL340 includes the amino acids at positions 34-340 of human klotho and optionally a signal peptide, for example the signal peptide of human klotho, corresponding to positions 1-33 of human klotho. The term "KL340" refers to both versions, namely, with or without a signal peptide.

Human klotho mRNA and amino acid sequences are provided in accession number NM_004795.4 (mRNA sequence of human klotho is set forth herein as SEQ ID NO: 20, precursor polypeptide sequence is set forth herein as SEQ ID NO: 3). The signal peptide of human klotho is located at positions 1-33 of SEQ ID NO: 3 (underlined). It is understood, that klotho sequences provided herein may be used with or without the signal peptide, depending on e.g. whether expression in a mammalian cell is desired (for example, when the klotho polypeptide is to be expressed in the engineered immune cells). The KL1 domain of human klotho corresponds to positions 34 to 567 of SEQ ID NO: 3, and may optionally be used as a polypeptide corresponding to positions 1-567, including the signal peptide at positions 1-33 of SEQ ID NO: 3). The sequence of human KL1 domain including the signal peptide is set forth herein as SEQ ID NO: 5. The KL1 domain is encoded by the corresponding section of SEQ ID NO: 20. In particular, a nucleic acid sequence encoding the human KL1 domain is set forth as SEQ ID NO: 21 (including the sequence encoding the signal peptide, which is underlined). In various embodiments, KL1 polypeptides that may be used correspond to positions 1-567 or 34- 567 of SEQ ID NO: 3. In additional embodiments, a KL1 polypeptide that may be used is a soluble KL1 that is 549 amino acids in length, resulting from an alternative splicing, as detailed further below. The amino acid sequence of the soluble KL1 is set forth herein as SEQ ID NO: 4. The soluble KL1 may be used without the signal peptide, namely, without amino acids 1-33 of SEQ ID NO: 4.

The human KL2 domain corresponds to positions 568 to 982 of SEQ ID NO: 3. The human KL2 domain is encoded by the corresponding section of SEQ ID NO: 20. In particular, the nucleic acid sequence encoding the human KL2 domain is set forth as SEQ ID NO: 22 or 23. In some embodiments, recombinant KL2 domain polypeptides may be used, comprising e.g. the signal peptide or other heterologous sequences. Exemplary amino acid sequences of isolated KL2 domain polypeptides are set forth in SEQ ID NOs: 6 and 7.

Active KL1 fragments include truncated versions of KL1 which retain their anti-tumor activities, as detailed in WO2023218445, to some of the inventors of the present invention. In some embodiments, an active KL1 fragment is a KL1 fragment in which the amino acids C- terminal to position 340 with respect to a human klotho polypeptide sequence have been deleted. In another embodiment, the polypeptide is derived from a human klotho polypeptide having an amino acid sequence as set forth in SEQ ID NO: 3 (NM_004795.4 transcribed human klotho polypeptide), in which the amino acids at positions 341-1012 have been deleted. In other embodiments, the klotho-derived polypeptide may be derived from other naturally-occurring allelic variants of human klotho (e.g. as set forth in accession nos. BAA23382.1, KAI4063028.1, KAI2569113.1, BAA24940.1, NP_004786.2, EAX08526.1) that exert a high degree of homology thereto (typically substitutions of one or two amino acids). In the active KL1 fragments of the invention, the amino acids at positions 320-340 with respect to a human klotho polypeptide sequence are retained.

In some embodiments, the active KL1 fragment comprises a signal peptide. In various embodiments, the signal peptide is a klotho signal peptide or a heterologous signal peptide. For example, the signal peptide of human klotho as set forth at positions 1-33 of SEQ ID NO: 3. In other embodiments, the active KL1 fragment does not comprise a signal peptide. In some embodiments, the polypeptide further comprises one or more heterologous sequences, moieties or agents. For example, without limitation, the polypeptide may further comprise a protein tag (e.g. an affinity tag or an epitope tag) or a serum half-life elongating sequence (e.g. Fc). In one embodiment, the heterologous sequence is at the C end. In another embodiment, the heterologous sequence is at the N' end.

In some embodiments, the active KL1 fragment is selected from the group consisting of:

(a) a polypeptide derived from a human klotho polypeptide sequence as set forth in SEQ ID NO: 3, in which the amino acids at positions 341-1012 have been deleted;

(b) a polypeptide of 307-400 amino acids in length, comprising the amino acids at positions 34-340 of a human klotho polypeptide sequence; and a polypeptide having at least 90% sequence identity to (a) or (b), which exhibits tumorsuppressive activity.

Advantageously, in the KL1 active fragments of the invention, the amino acids at positions 320-340 with respect to a human klotho polypeptide sequence are retained. In some embodiments, an active KL1 fragment is a polypeptide comprising at least 280 contiguous amino acids of the human klotho polypeptide sequence as set forth in SEQ ID NO: 3, in which the amino acids at positions 341-1012 have been deleted, and at least the amino acids at positions 320-340 thereof are retained.

In some embodiments, the active KL1 fragment is KL340. KL340 corresponds to the amino acids at positions 34-340 of SEQ ID NO: 3 and may further comprise a signal peptide. According to some embodiments, KL340 comprises the amino acid sequence SEQ ID NO: 9, optionally excluding the signal peptide at positions 1-33 thereof. According to some embodiments, KL340 consists essentially of the amino acid sequence SEQ ID NO: 9, optionally excluding the signal peptide at positions 1-33 thereof. According to some embodiments, KL340 consists of the amino acid sequence SEQ ID NO: 9, optionally excluding the signal peptide at positions 1-33 thereof. According to other embodiments, KL340 comprises the amino acid sequence SEQ ID NO: 10, which comprises a C-terminus HA tag, optionally excluding the signal peptide at positions 1-33 thereof. According to some embodiments, KL340 consists essentially of the amino acid sequence SEQ ID NO: 10, optionally excluding the signal peptide at positions 1-33 thereof. According to some embodiments, KL340 consists of the amino acid sequence SEQ ID NO: 10, optionally excluding the signal peptide at positions 1-33 thereof.

In additional embodiments, the active KL1 fragment is KL365. KL365 corresponds to the amino acids at positions 34-365 of SEQ ID NO: 3 and may further comprise a signal peptide. According to some embodiments, KL365 comprises the amino acid sequence SEQ ID NO: 12, optionally excluding the signal peptide at positions 1-33 thereof (SEQ ID NO: 13). According to some embodiments, KL365 consists essentially of the amino acid sequence SEQ ID NO: 12, optionally excluding the signal peptide at positions 1-33 thereof (SEQ ID NO: 13). According to some embodiments, KL365 consists of the amino acid sequence SEQ ID NO: 12, optionally excluding the signal peptide at positions 1-33 thereof. According to some embodiments, the KL365 fragment is enclosed by a nucleic acid comprising the nucleic acid sequence selected from sEQ ID NO: 28 and 29.

The transmembrane/intracellular region of human klotho corresponds to positions 982- 1012 of SEQ ID NO: 3. It is understood, that soluble human klotho polypeptides lack this region or a portion thereof, such that they are not bound to the plasma membrane. In some embodiments, a soluble klotho polypeptide is used (e.g. expressed) wherein the expressed amino acid sequence lacks at least the transmembrane region. In other embodiments, soluble klotho polypeptides (found e.g. in blood circulation) may originate from membrane-bound klotho polypeptides that are cleaved by ADAM10/17 or other proteases. An exemplary soluble form of human klotho, which is derived from alternative RNA splicing, contains the KL1 subunit and a 15-aa C portion that is characteristic to this splice variant. The amino acid sequence of an exemplary soluble klotho variant polypeptide is set forth in SEQ ID NO: 4. The soluble form of human klotho is encoded by the corresponding section of SEQ ID NO: 20 and has a nucleic acid sequence set forth as SEQ ID NO: 21.

The KL1-KL2 fragment has an amino acid sequence corresponding to positions 34 to 981 of SEQ ID NO: 3 and is set forth in SEQ ID NO: 8. The KL1-KL2 domain is encoded by the corresponding section of SEQ ID NO: 20 and has a nucleic acid sequence set forth as 24.

The terms "chimeric antigen receptor" and "CAR" are used herein interchangeably and refer to engineered recombinant polypeptides or receptors which are grafted onto cells and comprising at least (1) an extracellular domain comprising an antigen -binding region, e.g., a single chain variable fragment of an antibody or a whole antibody, (2) a transmembrane domain to anchor the CAR into a cell, and (3) one or more cytoplasmic signaling domains (also referred to herein as “an intracellular signaling domains”). The extracellular domain comprises an antigen binding domain (ABD) and optionally a spacer or hinge region.

The term “antigen binding portion”, “antigen binding region” and” antigen binding domain” are used herein interchangeably and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi -specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb, which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term “antigen binding portion” of an antibody. In certain embodiments of the invention, scFv molecules are incorporated into a fusion protein. According to some embodiments, the antigen binding domain is a scFv. According to some embodiments, the antigen binding domain of the present invention binds specifically to a tumor-associated antigen.

The antigen binding domain of the CAR targets a specific antigen. The targeting regions may comprise full length heavy chain, Fab fragments, or single chain variable fragment (scFvs). The antigen binding domain can be derived from the same species or a different species for or in which the CAR will be used in. In one embodiment, the antigen binding domain is scFv. According to one embodiment, the CAR comprises a variable regions of light chain (VL) and variable regions of heavy chain (VH) , wherein the VL domain comprises three complementarity determining regions (CDRs) of a VL having comprises three CDRs. As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain (HC) and the light chain (LC), which are designated CDR1, CDR2 and CDR3 (or specifically HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3), for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901- 917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub -portions were designated as LI, L2 and L3 or Hl, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5) :732-45 (1996)). Still, other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs. Determination of CDR sequences from antibody heavy and light chain variable regions can be made according to any method known in the art, including but not limited to the methods known as KABAT, Chothia and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example. According to some embodiments, the CDR sequences are determined using the IMGT method. According to some embodiments, the CDRs are as determined according to the Kabat (Wu T.T and Kabat E.A., J Exp Med, 1970; 132:211-50) and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77). According to some embodiments, the CDR sequences are determined using Chothia method.

The extracellular spacer or hinge region of a CAR is located between the antigen binding domain and a transmembrane domain. Extracellular spacer domains may include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, constant domains such as CH2 region or CH3 region of antibodies, accessory proteins, artificial spacer sequences or combinations thereof.

The term "transmembrane domain" refers to the region of the CAR, which crosses or bridges the plasma membrane. The transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein, an artificial hydrophobic sequence or a combination thereof.

The term “intracellular domain” refers to the intracellular part of the CAR and may be an intracellular domain of T cell receptor or of any other receptor (e.g., TNFR superfamily member) or portion thereof, such as an intracellular activation domain (e.g., an immunoreceptor tyrosine -based activation motif (ITAM)-containing T cell activating motif), an intracellular costimulatory domain, or both.

The terms "peptide", "polypeptide" and "protein" encompass also the analogs of these peptides, polypeptide and protein. The term "analog”, “analog” and “sequence analog” are used herein interchangeably and refer to an analog of a peptide, polypeptide or protein having at least 70% sequence identity with the original peptide, wherein the analog retains the activity of the original peptide. Thus, the terms “analog” and “active analog” may be used interchangeably. The term “analog” refers to a peptide, polypeptide or protein which contains substitutions, rearrangements, deletions, additions and/or chemical modifications in the amino acid sequence of the parent peptide. According to one embodiment, the analog has about 85% to about 95%, about 90% to about 95% or about 85% to about 99% sequence identity to the original peptide. According to one embodiment, the analog has at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the original peptide. According to some embodiments, the term a peptide/polypeptide/conjugate comprising the amino acid sequence SEQ ID N0:X encompass also peptide/polypeptide/conjugate comprising an amino acid sequence having at least 85% sequence identity to SEQ ID N0:X, and having the properties such as function and structure as the peptide/polypeptide/conjugate comprising the amino acid sequence SEQ ID NO: X.

When referring to analogs of antibodies or CARs, the term analogs encompasses only those analogs in which there is no change, modification or alternation in CDRs and therefore there is no alteration in specificity of binding to the target which the CAR or antibody binds specifically.

The terms “analog” and "functional analog" refer to a polypeptide, peptide or protein which differs by one or more amino acid alterations (e.g., substitutions, additions or deletions of amino acid residues) from the original sequence, having at least 85% sequence identity to the original sequence and still maintains the properties and the functionality of the parent polypeptide, peptide or protein. According to one embodiment, the analog comprises at least one modification selected from a substitution, deletion and addition. According to some embodiments, the modification is a substitution. According to one embodiment, the substitution is a conservative substitution.

The term “conservative substitution” as used herein denotes the replacement of an amino acid residue by another, without altering the overall conformation and biological activity of the peptide, including, but not limited to, the replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like). Amino acids with similar properties are well-known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term “tumor-associated antigen ” or "tumor antigen" as used herein refers to any antigen which is found in significantly higher concentrations in or on tumor cells than on normal cells. According to some embodiments, the tumor- associated antigen is selected from AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5, BIRC7, p-catenin, -8 brc-abl, BRCA1, BORIS, CA9, CA125, carbonic anhydrase IX, caspase 1, CALR, CCR5, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CD276, CDK4, CEA, Claudin 18.2, cyclin -B l, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6- AML, PSCA, EpCAM, EphA2, Fra-1, FOLR1, GAGE, GD2, GD3, GloboH, phosphatidylinositol proteoglycan -3, GM3, gplOO, Her2, HLA/B-raf-kinases, HLA/k-ras, HLA/MAGE-A3, hTERT, IL13R a2. LMP2 k -Light, LewisY, MAGE, MART-1, Mesothelin, ML-IAP, MOv-, y, Mucl, Muc2, Muc3, Muc4, Muc5, CA-125, MUM1, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, 0X40, pl5, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA, GPC3, LMP1, CD70, RAGE protein, Ras, RGS5, Rho, R0R1, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF -p, TMPRSS2, soup-antigen, TRP-1, TRP-2, tyrosinase, urea soluble protein -3 and 5T4. According to some embodiments, the tumor-associated antigen is selected from CD19, HER2, CD38, CD138, CD276 and EGFR.

The terms "binds specifically" or "specific for" with respect to an antigen -binding domain of an antibody, of a fragment thereof or of a CAR refers to an antigen -binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. The term encompasses that the antigenbinding domain binds to its antigen with high affinity and binds other antigens with low affinity. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen -binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

According to one aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising co-administering to the subject a therapeutically effective amount of immune cells and a klotho polypeptide or a vector encoding thereof, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen.

According to one aspect, the present invention provides a combinational therapy of cancer in which a subject is treated with immune cells engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen and a klotho polypeptide or a vector encoding thereof. Different types of administration are contemplated. In some implementations of the invention, the immune cells engineered to express a CAR, and a klotho polypeptide or vector encoding thereof are administered as separated compositions. In other implementations, the immune cells engineered to express a CAR, and the klotho polypeptide or vector encoding thereof are administered as a single composition. In further implementations, the immune cells are engineered to express both the CAR and the klotho polypeptide are administered.

According to some embodiments, the present invention provides a therapeutic combination comprising (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, for use in treating cancer, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen. According to some embodiments, the immune cells of the present invention are engineered immune cells. According to some embodiments, the engineered immune cells are engineered to express a CAR. According to some embodiments, the immune cells are characterized by a surface expression of the CAR. According to some embodiments, the immune cells comprise the CAR at their surface.

According to some embodiments, the CAR used therapeutic combination of the invention binds specifically to a tumor-associated antigen selected from ErbB2, CD 19, CD38, CD138, EGFR, CD24, GD2, EGF, Mesothelin (MSLN), MUC16 and tumor- associated carbohydrate antigens. In another embodiment, the CAR binds specifically to a tumor antigen associated (TAA) with solid tumors (e.g. ErbB2, CD138, EGFR, CD24, GD2, EGF, MSLN, or MUC16). In another embodiment, the CAR binds specifically to a tumor antigen associated (TAA) with blood cancer. In another embodiment said TAA is selected from the group consisting of ErbB2, EGFR, CD24, GD2, EGF, MSLN, and MUC16. In a particular embodiment said CAR is directed to ErbB2. According to some embodiments, the TAA is HER2. According to some embodiments, the TAA is CD19. According to some embodiments, the TAA is CD38. According to some embodiments, the TAA is CD 138. According to some embodiments, the TAA is CD276. According to some embodiments, the TAA is EGFR.

The terms "ErbB2" and "HER2" may be used herein interchangeably. According to some embodiments, the CAR binds specifically to HER2. According to some embodiments, the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 40, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the VL-CDR1 comprises an amino acid selected from QDVGPN and KASQDVGPNVA, the VL-CDR2 comprises an amino acid selected from SAS and SASYLYN, the VL-CDR3 comprises the amino acid QQYNTYPFT, the VH-CDR1 comprises an amino acid selected from GFTFNTYA and TYAMN, the VH-CDR2 comprises an amino acid selected from IRSKSNNYAT and RIRSKSNNYATYYVDSVKD and the VH-CDR comprises an amino acid selected from VTSYYDYDKVLFAY and SYYDYDKVLFAY. According to some embodiments, the CAR comprises 6 CDRs comprising amino acid sequences SEQ ID NO: 43, SAS, SEQ ID NOs: 45-48. According to some embodiments, the CAR comprises 6 CDRs comprising amino acid sequences SEQ ID NOs: 49-54. According to some embodiments, the CAR comprises a VL and VH domains comprising the amino acid sequences SEQ ID NOs: 41 and 42, respectively. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 40.

According to some embodiments, the CAR binds specifically to CD19. According to some embodiments, the CAR comprises a VL and VH domains comprising the amino acid sequences SEQ ID NOs: 62 and 63, respectively. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64. According to some embodiments, the CAR comprises a VL and VH domains and comprises 6 CDRs sequences of the amino acid sequence SEQ ID NO: 60, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the CAR comprises 6 CDRs comprising amino acid sequences SEQ ID NO: 104, HTS, SEQ ID NOs: 106-109. According to some embodiments, the CAR comprises 6 CDRs comprising amino acid sequences SEQ ID NOs: 110-115.

According to some embodiments, the CAR binds specifically to CD138. According to some embodiments, the CAR comprises a VL and VH domains comprising the amino acid sequences SEQ ID NOs: 71 and 72, respectively. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 70. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 79. According to some embodiments, the CAR comprise the amino acid sequence SEQ ID NO: 67. According to some embodiments, the CAR comprises a VL and VH domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 71 and 72, respectively, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the anti-CD138 CAR comprises 6 CDRs comprising amino acid sequences SEQ ID NO: 73, YTS, SEQ ID NOs: 75-78.

According to some embodiments, the CAR binds specifically to CD276. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 82 and 84. According to some embodiments, the CAR comprises a VL and VH domains and 6 CDRs sequences of the amino acid sequence selected from SEQ ID NO: 82 and 84, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia.

According to some embodiments, the CAR binds specifically to EGFR. According to some embodiments, the CAR comprise the amino acid sequence SEQ ID NO: 86. According to some embodiments, the CAR comprises a VL and VH domains and 6 CDRs sequences of the amino acid sequence from SEQ ID NO: 86, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia.

According to some embodiments, the CAR binds specifically to CD38. According to some embodiments, the CAR comprises a VL and VH domains comprising the amino acid sequences SEQ ID NOs: 88 and 89, respectively. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 90. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 98. According to some embodiments, the CAR comprise the amino acid sequence SEQ ID NO: 103. According to some embodiments, the CAR comprises a VL and VH domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 88 and 89, respectively, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the anti-CD138 CAR comprises 6 CDRs comprising amino acid sequences SEQ ID NO: 92, DAS, SEQ ID NOs: 94-97.

According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86.

According to any one of the above embodiments, the klotho polypeptide is selected from a membranal klotho, secreted klotho, proteolyzed klotho, a KL1 domain, a KL2 domain and an active KL1 fragment (e.g., KL340). According to any one of the above embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 3. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 4. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 5. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 6. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 7. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 8. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 9. According to some embodiments, the klotho polypeptide comprises an amino acid sequence SEQ ID NO: 12.

According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103, and 86 and the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 3. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 4. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 5. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 6. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 7. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 8. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 9. According to some embodiments, the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 12.

According to some embodiments, the CAR (anti-HER2) comprises the amino acid sequence SEQ ID NO: 40 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 3. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 40 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 5. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 40 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 8. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 40 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 9. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 40 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 11.

According to some embodiments, the CAR (anti-CD19) comprises the amino acid sequence SEQ ID NO: 60, 61, or 64and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 3. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 5. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 8. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 9. According to some embodiments, the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64 and the klotho polypeptide comprises the amino acid sequence SEQ ID NO: 11.

According to any one of the above embodiments, the vector encoding the klotho polypeptide may be a viral vector, e.g. an adeno-associated virus (AAV) vector. According to some embodiments, the vector encoding the klotho polypeptide encodes an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. According to some embodiments, the vector comprises the nucleic acid sequence selected from SEQ ID NOs: 20-29. Therefore, according to some embodiments, instead of each one of the klotho polypeptides, a vector encoding said polypeptide may be administered and contemplated herein.

According to some embodiments, the immune cells are characterized by surface expression of the CAR upon administration.

According to some embodiments, the co-administering comprises a sequential administering of the engineered immune cells and klotho polypeptide or a vector encoding the klotho polypeptide. According to other embodiments, the co-administering comprises a substantially simultaneous administering of the engineered immune cells and the klotho polypeptide.

According to any one of the above embodiments, the immune cells are selected from T cells, NK cells, and TIL. According to some embodiments, the immune cells are T cells. According to any one of the above embodiments, the T cells are selected are from CD4⁺ T- cell, CD8⁺ T-cell, and a combination thereof. In some embodiments, the cells are peripheral blood mononuclear cells (PBMC) or PBMC-derived cell populations, including, but not limited to, lymphokine-activated killer (LAK) cells and cytokine-induced killer (CIK) cells. According to some embodiments, the immune cells constitutively express the CAR on their surface. According to some embodiments, the immune cells express the CAR on their surface upon cancer-induced environment, e.g., under hypoxic conditions.

According to some embodiments, the present invention provides a therapeutic combination for use in treating cancer in a subject in need thereof the use comprises coadministering to the subject (i) a therapeutically effective amount of immune cells engineered to express a chimeric antigen receptor (CAR), and (ii) a klotho polypeptide or a vector encoding thereof, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 40, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 40, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and vector encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20- 29. According to some embodiments, the immune cells are T cells. According to some embodiments, the cancer expresses HER2 antigen. According to some embodiments, the cancer is selected from breast cancer, ovarian cancer and pancreatic cancer. In some embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 5, 8 and 9.

According to some embodiments, the present invention provides a therapeutic combination for use in treating cancer in a subject in need thereof, wherein the use comprises co-administering to the subject (i) a therapeutically effective amount of immune cells engineered to express a chimeric antigen receptor (CAR), and (ii) a klotho polypeptide or a vector encoding thereof, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 60, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and vector encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20-29. In some embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 5, 8 and 9. According to some embodiments, the immune cells are T cells. According to some embodiments, the cancer expresses CD19 antigen. According to some embodiments, the cancer is selected from leukemias and lymphomas.

According to some embodiments, the present invention provides a therapeutic combination for use in treating cancer in a subject in need thereof, wherein the use comprises co-administering to the subject (i) a therapeutically effective amount of immune cells engineered to express a chimeric antigen receptor (CAR), and (ii) a klotho polypeptide or a vector encoding thereof, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 79, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 79, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and vector encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20- 29. In some embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 5, 8 and 9. According to some embodiments, the immune cells are T cells. According to some embodiments, the cancer expresses CD138 antigen. According to some embodiments, the cancer is selected from leukemias and lymphomas.

According to some embodiments, the present invention provides a therapeutic combination for use in treating cancer in a subject in need thereof, wherein the use comprises co-administering to the subject (i) a therapeutically effective amount of immune cells engineered to express a chimeric antigen receptor (CAR), and (ii) a klotho polypeptide or a vector encoding thereof, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 82 or 84, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 82 or 84, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and vector encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20- 29. In some embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 5, 8 and 9. According to some embodiments, the immune cells are T cells. According to some embodiments, the cancer expresses CD276 antigen. According to some embodiments, the cancer is selected from lung cancer, cancer stem cells, epithelial tumor, tumors of the head and neck cells, and glioblastoma, bladder cancer, breast cancer, cervix cancer, colorectal cancer, esophageal cancer, renal cancer, hepatic cancer, ovarian cancer, pancreatic cancer, prostate cancer, biliary cancer, oral squamous cell carcinoma, intrauterine membranous cancer, squamous cell carcinoma, gastric cancer, glioma, glioblastoma, melanoma, and adrenal cancer.

According to some embodiments, the present invention provides a therapeutic combination for use in treating cancer in a subject in need thereof, wherein the use comprises co-administering to the subject (i) a therapeutically effective amount of immune cells engineered to express a chimeric antigen receptor (CAR), and (ii) a klotho polypeptide or a vector encoding thereof, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 98, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 98, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and vector encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20- 29. In some embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 5, 8 and 9. According to some embodiments, the immune cells are T cells. According to some embodiments, the cancer expresses CD38 antigen. According to some embodiments, the cancer is selected from leukemias and lymphomas [.

According to some embodiments, the present invention provides a therapeutic combination for use in treating cancer in a subject in need thereof, wherein the use comprises co-administering to the subject (i) a therapeutically effective amount of immune cells engineered to express a chimeric antigen receptor (CAR), and (ii) a klotho polypeptide or a vector encoding thereof, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 86, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 86, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and vector encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20- 29. According to some embodiments, the immune cells are T cells. In some embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 5, 8 and 9. According to some embodiments, the cancer expresses EGFR antigen. According to some embodiments, the cancer is selected from lung cancer, anal cancers glioblastoma, epithelial tumors and epithelial tumors of the head and neck cells.

According to any one of the above embodiments, the method comprises an adoptive cell transfer (ACT) therapy. In another embodiment, the immune cells are formulated as an ACT composition. In another embodiment said immune cells are administered to said subject in the form of an ACT.

According to any one of the above embodiments, the use of the therapeutic combination provides a synergistic anti-cancer effect.

According to other embodiments, the present invention provides a pharmaceutical composition comprising immune cells engineered to express both (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen and (ii) a klotho polypeptide, and a pharmaceutical acceptable carrier, for use in treating cancer. All terms, embodiments and definitions disclosed in any one of the above aspects and embodiments apply and are encompassed herein as well.

According to some embodiments, the immune cells constitutively express the CAR on their surface. According to some embodiments, the immune cells express the CAR on their surface upon cancer-induced environment, e.g., under hypoxic conditions. According to some embodiments, the immune cells constitutively express the klotho polypeptide. According to some embodiments, the immune cells express the klotho polypeptide upon activation of the CAR. According to some embodiments, the immune cells express the klotho polypeptide upon cancer-induced environment, e.g., under hypoxic conditions. Any combinations of the above embodiments are encompassed.

According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR, wherein the nucleic acid molecule is operably linked to a constitutive promoter. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR, wherein the nucleic acid molecule is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon hypoxia. According to some embodiments the CAR is as defined hereinabove in previous aspects and embodiments. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 57. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 65. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 39. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 69. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 68. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 83. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 85. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 87. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 99. According to some embodiments, the nucleic acid molecule encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 102. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon activation of a CAR. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon hypoxia. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to a constitutive promoter. According to some embodiments, the nucleic acid encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 20. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 21. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 22. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 23. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 24. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 25. According to some embodiments, the nucleic acid encoding the klotho polypeptide comprises a nucleic acid molecule sequence SEQ ID NO: 26. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 27. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 28. According to some embodiments, the nucleic acid molecule encoding the klotho polypeptide comprises a nucleic acid sequence SEQ ID NO: 29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 57 and nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 65 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 39 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 69 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 68 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20- 29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 83 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 85 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 87 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 99 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the immune cells comprise a nucleic acid molecule encoding the CAR comprising a nucleic acid sequence SEQ ID NO: 102 and a nucleic acid molecule encoding the klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20- 29. According to some embodiments, the promoter initiating expression of a polypeptide or a protein upon activation of said CAR is selected from NF AT, API, and NR4A promoters. According to some embodiments, the promoter is a NF AT promoter. In another embodiment, the promoter is a synthetic promoter comprising at least one NAFT transcription-regulating element (e.g. promoter and/or enhancer sequences). In a particular embodiment, the synthetic promoter comprises a plurality of NF AT enhancer repeats.

According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR and a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon activation the CAR. According to some embodiments, the engineered immune cells constitutively express the CAR on their surfaces and express the klotho polypeptide upon activation of T cell receptor.

According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR and a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein both nucleic acid molecules are operably linked to a constitutive promoter. According to some embodiments, the engineered immune cells constitutively express CAR on their surface and constitutively express the klotho polypeptide.

According to any one of the above embodiments, the two nucleic acid constructs may be separate constructs or located on the same vector.

According to some embodiments, the CAR and the klotho polypeptide are as described above.

According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid sequence SEQ ID NO: 66. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid sequence SEQ ID NO: 37. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid sequence SEQ ID NO: 116. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid sequence SEQ ID NO: 38. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid sequence SEQ ID NO: 117. The term "nucleic acid construct" refers to a nucleic acid molecule operably linked to a promoter. The terms “operably linked”, “operatively linked”, “operably encodes”, and “operably associated” are used herein interchangeably and refer to the functional linkage between a promoter and nucleic acid sequence, wherein the promoter initiates transcription of RNA corresponding to the DNA sequence. The terms "nucleic acid molecule" and "nucleic acid construct" encompass also their variants. The terms “homolog” “variant”, “DNA variant”, “sequence variant” and “polynucleotide variant” are used herein interchangeably and refer to a DNA polynucleotide having at least 70% sequence identity to the parent polynucleotide and encoding a the peptide(s) or protein(s) having the same functions and structure as the peptide(s) or protein(s) encoded by the parent polynucleotide. The variant may include mutations such as deletion, addition or substitution such that the mutations do not change the open reading frame and the polynucleotide encodes a peptide or a protein having substantially similar structure and function as a peptide or a protein encoded by the parent polynucleotide. According to some embodiments, the variants are conservative variants. The term “conservative variants" as used herein refers to variants in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Thus, the peptide or the protein encoded by the conservative variants has 100% sequence identity to the peptide or the protein encoded by the parent polynucleotide. According to some embodiments, the variant is a non- conservative variant encoding to a peptide or a protein being a conservative analog of the peptide of the protein encoded by the parent polynucleotide. According to some embodiments, the variant has at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the parent polynucleotide.

According to some embodiments, the CAR binds specifically to a tumor-associated antigen selected from ErbB2, CD19, CD38, CD138, EGFR, EGF, MSLN, CD24, GD2, MUC16, BCMA, CD276, MUC-1, FAP and tumor- associated carbohydrate antigen. According to some embodiments, the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 40, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 60, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the CAR comprise an amino acid sequence selected from SEQ ID NO: 40, 60, 61, or 64. According to any one of the above embodiments, the klotho polypeptide is selected from a membranal klotho, secreted klotho, proteolyzed klotho, a KL1 domain, a KL2 domain and an active KL1 fragment (e.g., KL340). According to any one of the above embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9 and 10. According to some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence selected from SEQ ID NO: 57, 39 and 65. According to some embodiments, the nucleic acid encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NO: 20-29.

According to some embodiments, the cancer is as described above. According to some embodiments, the cancer expresses HER2. According to some embodiments, the cancer is ovarian cancer, breast cancer or pancreatic cancer. According to some embodiments, the cancer is a lymphoma or leukemia.

According to any one of the above embodiments, the immune cells are selected from T cells, NK cells and TIL. According to some embodiments, the immune cells are T cells. According to any one of the above embodiments, the T cells are selected are from CD4⁺ T- cell, CD8⁺ T-cell and a combination thereof. In other embodiments, the immune cells are PBMC-derived cell populations or other immune cells as disclosed herein.

According to some embodiments, the present invention provides a pharmaceutical composition comprising immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a HER2 tumor-associated antigen and (ii) a klotho polypeptide, and a pharmaceutically acceptable carrier, for use in treating cancer, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 40, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 40, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 23 and SEQ ID NO: 57. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 21, and SEQ ID NO: 57. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 22, and SEQ ID NO: 57. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 27, and SEQ ID NO: 57. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 26 SEQ ID NO: 57. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 29, and SEQ ID NO: 57.

According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide and comprising a vector comprising the nucleic acid sequence SEQ ID NO: 66. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide and comprising a vector comprising the nucleic acid sequence SEQ ID NO: 37. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide and comprising a vector comprising the nucleic acid sequence SEQ ID NO: 38. According to some embodiments, the cancer is selected from breast cancer, ovarian cancer and pancreatic cancer.

According to some embodiments, the present invention provides a pharmaceutical composition comprising immune cells engineered to express (i) a CD 19 chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen and (ii) a klotho polypeptide, and a pharmaceutical acceptable carrier, for use in treating cancer, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 60, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13. According to some embodiments, the cancer expresses CD19 antigen. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 23 and SEQ ID NO: 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 21 and SEQ ID NO: 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 22 and SEQ ID NO: 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 27 and SEQ ID NO: 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 26 and SEQ ID NO: 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 29 and SEQ ID NO: 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide and comprising a vector comprising the nucleic acid sequence SEQ ID NO: 116. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide and comprising a vector comprising the nucleic acid sequence SEQ ID NO: 117. According to some embodiments, the immune cells are T cells.

According to some embodiments, the present invention provides a pharmaceutical composition comprising immune cells engineered to express (i) a CD 138 chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen and (ii) a klotho polypeptide, and a pharmaceutical acceptable carrier, for use in treating cancer, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 70, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia and wherein, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13. According to some embodiments, the cancer expresses CD 138 antigen. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 23 and SEQ ID NO: 69 or 68. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 21 and SEQ ID NO: 69 or 68. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 22 and SEQ ID NO: 69 or 68. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 27 and SEQ ID NO: 69 or 68. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 26 and SEQ ID NO: 69 or 68. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 29 and SEQ ID NO: 69 or 68. According to some embodiments, the immune cells are T cells.

According to some embodiments, the present invention provides a pharmaceutical composition comprising immune cells engineered to express (i) a CD38 chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen and (ii) a klotho polypeptide, and a pharmaceutical acceptable carrier, for use in treating cancer, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 90, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia and wherein, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13. According to some embodiments, the cancer expresses CD 138 antigen. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 23, and SEQ ID NO: 99 or 102. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 21 , and SEQ ID NO: 99 or 102. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 22, and SEQ ID NO: 99 or 102. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 27, and SEQ ID NO: 99 or 102. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 26, and SEQ ID NO: 99 or 102. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 29, and SEQ ID NO: 99 or 102. According to some embodiments, the immune cells are T cells.

According to some embodiments, the present invention provides a pharmaceutical composition comprising immune cells engineered to express (i) chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen selected from CD276 and EGFR, and (ii) a klotho polypeptide, and a pharmaceutical acceptable carrier, for use in treating cancer. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 23 and a nucleic acid sequence selected from SEQ ID NO: 83, 85, and 87. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 21 and a nucleic acid sequence selected from SEQ ID NO: 83, 85, and 87. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 22 and a nucleic acid sequence selected from SEQ ID NO: 83, 85, and 87. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 27 and a nucleic acid sequence selected from SEQ ID NO: 83, 85, and 87. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 26 and a nucleic acid sequence selected from SEQ ID NO: 83, 85, and 87. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid comprising a nucleic acid sequence SEQ ID NO: 29 and a nucleic acid sequence selected from SEQ ID NO: 83, 85, and 87. According to some embodiments, the immune cells are T cells.

According to some embodiments, the nucleic acid construct comprising a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide further comprises a sequence encoding a selfcleaving peptide e.g. 2A, such as P2A, E2A, F2A, and T2A. According to some embodiments, the self-cleaving peptide is T2A. According to some embodiments, the selfcleaving peptide comprises amino acid sequence SEQ ID NO: 100. According to some embodiments, the self -cleaving peptide is encoded by the nucleic acid sequence SEQ ID NO: 101. Alternatively, the nucleic acid construct may comprise an internal ribosome entry site (IRES) sequence between the two nucleic acid molecules.

According to any one of the above embodiments, the use comprises an adoptive cell transfer therapy.

According to some embodiments, the use provides a synergistic anti-cancer effect.

According to some embodiments, the present invention provides therapeutic combination comprising a population of immune cells engineered to express a CAR that binds specifically to a tumor-associated antigen, and a klotho polypeptide or a vector encoding thereof, for use in improving tumor killing and/or clearance. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to some embodiments, the CAR, the klotho polypeptide, and the vector encoding the klotho polypeptide is as described in any one of the above embodiments and aspects.

According to another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding a CAR that binds specifically to a tumor- associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein each of the nucleic acid molecules is operably linked to a promoter or both nucleic acid molecules are operably linked to one promoter. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to some embodiments, the nucleic acid construct of the present invention comprises a nucleic acid molecule encoding a CAR that binds specifically to a tumor- associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein each of the nucleic acid molecules is operably linked to a promoter. According to some embodiments, the promoters may be the same promoter or different promoters. According to some embodiments, the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon activation of the CAR. According to some embodiments, the nucleic acid molecule encoding the CAR and the nucleic acid molecule encoding the klotho polypeptide are each operably linked to a constitutive promoter. According to some embodiments, the promoter initiating expression of a polypeptide or a protein upon activation of the CAR is selected from NF AT, API, and NR4A. According to some embodiments, the promoter is NFAT.

According to some embodiments, the nucleic acid construct comprises a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide, both nucleic acid molecules are operably linked to one promoter. According to some embodiments, the promoter is a constitutive promoter. According to some embodiments, the promoter in a tumor-inducible promoter. According to such embodiments, the nucleic acid construct typically further comprises a cleavable nucleic acid molecule or a nucleic acid molecule encoding a self-cleaving peptide between the two nucleic acid molecules. According to some embodiments, the self-cleaving peptide is e.g. 2A, such as P2A, E2A, F2A, and T2A. According to some embodiments, the self-cleaving peptide is T2A. According to some embodiments, the self -cleaving peptide comprises amino acid sequence SEQ ID NO: 100. According to some embodiments, the selfcleaving peptide is encoded by the nucleic acid sequence SEQ ID NO: 101. Alternatively, the nucleic acid construct may comprise an internal ribosome entry site (IRES) sequence between the two nucleic acid molecules. Each possibility represents a separate embodiment of the invention. According to some embodiments, the CAR and the klotho polypeptide are as defined hereinabove in any one of the above aspects and embodiments. According to some embodiments, the CAR binds specifically to a tumor-associated antigen selected from ErbB2, CD19, CD38, CD138, EGFR, EGF, MSLN, CD24, GD2, MUC16 and tumor- associated carbohydrate antigen. According to some embodiments, the CAR comprise a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 40, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the CAR comprise an amino acid sequence SEQ ID NO: 40. According to any one of the above embodiments, the klotho polypeptide is selected from a membranal klotho, secreted klotho, proteolyzed klotho, a KL1 domain, a KL2 domain, and KL340 polypeptide. According to any one of the above embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9 and 10. According to some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 57. According to some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 29. According to some embodiments, the nucleic acid encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein the nucleic acid construct comprises the nucleic acid sequence SEQ ID NO: 66. According to some embodiments, the nucleic acid construct comprises a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein the nucleic acid construct comprises the nucleic acid sequence SEQ ID NO: 37. According to some embodiments, the nucleic acid construct comprises a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein the nucleic acid construct comprises the nucleic acid sequence SEQ ID NO: 116. According to some embodiments, the nucleic acid construct comprises a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein the nucleic acid construct comprises the nucleic acid sequence SEQ ID NO: 38. According to some embodiments, the nucleic acid construct comprises a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein the nucleic acid construct comprises the nucleic acid sequence SEQ ID NO: 117.

According to some embodiments, the nucleic acid construct comprises a nucleic acid molecule encoding a CAR comprising a nucleic acid sequence selected from SEQ ID NO: 57, 65, 39, 69, 68, 83, 85, 87, 102 and 99 and a nucleic acid molecule encoding a klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises nucleic acid sequence SEQ ID NO: 57 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, t the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 65 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, t the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 39 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 69 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 68 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 83 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 85 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 87 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 99 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the nucleic acid construct comprises a nucleic acid sequence SEQ ID NO: 102 and a nucleic acid sequence selected from SEQ ID NO: 20-29.

According to some embodiments, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding a CAR and a nucleic acid molecule encoding a klotho polypeptide, both nucleic acid molecules are operably linked to one promoter, wherein the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 60, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 60, 61, or 64, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and/or comprising a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a comprises a nucleic acid sequence SEQ ID NO: 57 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a comprises a nucleic acid sequence SEQ ID NO: 65 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a comprises a nucleic acid sequence SEQ ID NO: 39 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 66. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 37. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 38. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 116. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 117.

According to another aspect, the present invention provides a vector comprising the nucleic acid construct of the present invention as described in any one of the above aspects and embodiments. According to some embodiments, the nucleic acid construct is according to any one of the above embodiments and aspects. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 57 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 65 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 39 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 69 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 68 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 83 or 85 and a nucleic acid sequence selected from SEQ ID NOs: 20- 29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 89 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 87 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to another aspect, the present invention provides a vector comprising the nucleic acid sequence selected from SEQ ID NO: 66, 37, 38, 116 and 117.

The terms “vector” and “expression vector” are used herein interchangeably and refer to any viral or non-viral vector such as plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), phage, binary vector in double or single stranded linear or circular form, or nucleic acid, sequence which is able to transform host cells and optionally capable of replicating in a host cell. The vector may be integrated into the cellular genome or may exist extra-chromosomally (e.g., autonomous replicating plasmid with an origin of replication). The vector may contain an optional marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. According to some embodiments, the vector is a viral vector. The term "viral vector" as used herein refers to a nucleic acid vector that includes at least one element of a virus genome and the nucleic acid fragment to be transferred and may be packaged into a viral particle. The terms "virus", "virions", "viral particles" and "viral vector particle" are used interchangeably to refer to viral particles that are formed when the nucleic acid vector is transduced into an appropriate cell or cell line according to suitable conditions allowing the generation of viral particles. In the context of the present invention, the term "viral vector" has to be understood broadly as including nucleic acid vector (e.g. DNA viral vector) as well as viral particles generated thereof. The term "infectious" refers to the ability of a viral vector to infect and enter into a host cell or subject. According to some embodiments, the vector is a virus, e.g. a modified or engineered virus. The modification of a vector may include mutations, such as deletion or insertion mutation, gene deletion or gene inclusion. In particular, a mutation may be done in one or more regions of the viral genome. Such mutations may be introduced in a region related to internal structural proteins, replication, or reverse transcription function. Other examples of vector modification are deletion of certain genes constituting the native infectious vector such as genes related to the virus' pathogenicity and/or to its ability to replicate. Any virus can be attenuated by the methods disclosed herein. The virus can be a dsDNA virus (e.g. Adenoviruses, Herpesviruses, Poxviruses), a single stranded “plus” sense DNA virus (e.g., Parvoviruses) a double stranded RNA virus (e.g., Reoviruses), a single stranded +sense RNA virus (e.g. Picornaviruses, Togaviruses), a single stranded “minus” sense RNA virus (e.g. Orthomyxoviruses, Rhabdoviruses), a single stranded +sense RNA virus with a DNA intermediate (e.g. Retroviruses), or a double stranded reverse transcribing virus (e.g. Hepadnaviruses). In certain non-limiting embodiments of the present invention, the virus is poliovirus (PV), rhinovirus, influenza virus including avian flu (e.g. H5N1 subtype of influenza A virus), severe acute respiratory syndrome (SARS) coronavirus, Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), infectious bronchitis virus, ebolavirus, Marburg virus, dengue fever virus (Flavivirus serotypes), West Nile disease virus, Epstein-Barr virus (EBV), yellow fever virus, Ebola (ebolavirus), chickenpox (varicella-zoster virus), measles (a paramyxovirus), mumps (a paramyxovirus), rabies (Lyssavirus), human papillomavirus, Kaposi's sarcoma-associated herpesvirus, Herpes Simplex Virus (HSV Type 1), or genital herpes (HSV Type 2). According to some embodiments, the vector is a virus selected from lentivirus, adenovirus, modified adenovirus and retrovirus. In one particular embodiment, the vector is lentivirus.

According to some embodiments, the vector construct comprises a nucleic acid sequence selected from SEQ ID NO: 57 and 65 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. In one embodiment, the vector is lentivirus.

According to another aspect, the present invention provides a cell comprising the nucleic acid construct or vector of the present invention. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. According to some embodiments, the cell is selected from a bacterial, yeast and mammalian cell.

According to yet another aspect, the present invention provides a population of immune cells engineered to express (i) a chimeric antigen receptor (CAR) binding specifically to cancer cells and (ii) a klotho polypeptide. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to some embodiments, the cells population of immune cells comprises a nucleic acid construct comprising a nucleic acid molecule encoding the CAR and a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide. According to some embodiments, the nucleic acid constructs are as defined in any one of the above embodiments. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon activation of a T cell receptor (TCR). According to some embodiments, the promoter initiating expression of a polypeptide or a protein upon activation of TCR is selected from NF AT, API, and NR4A promoters. According to some embodiments, the promoter is NF AT. In another embodiment, said inducible promoter initiates expression of the klotho polypeptide upon activation of the immune cell via specific binding of the CAR to its antigen target. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon hypoxia. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to a constitutive promoter. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR, wherein the nucleic acid molecule is operably linked to a constitutive promoter. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR, wherein the nucleic acid molecule is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon hypoxia. According to some embodiments, the immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the CAR and a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to an inducible promoter, said promoter initiates expression of the klotho polypeptide upon activation of a T cell receptor. According to some embodiments, the engineered immune cells constitutively express the CAR on their surfaces and express the klotho polypeptide upon activation of T cell receptor.

According to some embodiments, the cells of the population of engineered immune cells comprise the nucleic acid construct and/or the vector comprising the nucleic acid construct of the present invention as defined in any one of the above embodiments and aspects. According to some embodiments, the CAR binds specifically to a tumor-associated antigen selected from ErbB2, CD19, CD38, CD138, EGFR, EGF, MSLN, CD24, GD2, BCMA, CD276, MUC-1, FAP MUC16 and tumor-associated carbohydrate antigen. According to some embodiments, the CAR comprise a VH and VE domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 40, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the CAR comprises an amino acid sequence SEQ ID NO: 40. According to some embodiments, the CAR comprises a VH and VL domains each comprising 3 CDRs sequences of the amino acid sequence SEQ ID NO: 60, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia. According to some embodiments, the CAR comprises an amino acid sequence SEQ ID NO: 60, 61, or 64. According to any one of the above embodiments, the klotho polypeptide is selected from a membranal klotho, secreted klotho, proteolyzed klotho, a KL1 domain, a KL2 domain, and KL340 polypeptide. According to any one of the above embodiments, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9 and 10. According to some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 57. According to some embodiments, the nucleic acid encoding the CAR comprises a nucleic acid sequence SEQ ID NO: 29. According to some embodiments, the nucleic acid encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20-29.

According to some embodiments, the cells of the population of cells comprise a construct comprising a nucleic acid sequence SEQ ID NO: 57 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 65 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 39 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 69 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 68 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 83 or 85 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 89 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the construct comprises a nucleic acid sequence SEQ ID NO: 87 and a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the present invention provides a population of immune cells comprising a construct comprising a nucleic acid sequence SEQ ID NO: 66. According to some embodiments, the present invention provides a population of immune cells comprising a construct comprising a nucleic acid sequence SEQ ID NO: 37. According to some embodiments, the present invention provides a population of immune cells comprising a construct comprising a nucleic acid sequence SEQ ID NO: 38. According to some embodiments, the present invention provides a population of immune cells comprising a construct comprising a nucleic acid sequence SEQ ID NO: 116. According to some embodiments, the present invention provides a population of immune cells comprising a construct comprising a nucleic acid sequence SEQ ID NO: 117.

According to any one of the above embodiments, the immune cells are selected from T cells, NK cells and TIL. According to some embodiments, the immune cells are T cells. According to any one of the above embodiments, the T cells are selected are from CD4⁺ T- cell, CD8⁺ T-cell and a combination thereof.

According to some embodiments, the present invention provides a population of immune cells engineered to express a (i) CAR and (ii) a klotho polypeptide, wherein the CAR comprises a VH and VL domains each comprising 6 CDRs sequences of the amino acid sequence SEQ ID NO: 40 or 60, wherein the CDRs sequences are determined using the method of Kabat, IMGT or Chothia or wherein the CAR comprises the amino acid sequence SEQ ID NO: 40 or 60, the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3-13 and vector encoding the klotho polypeptide comprises a nucleic acid sequence selected from SEQ ID NOs: 20-29. According to some embodiments, the immune cells are T cells.

According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid molecule comprising a nucleic acid sequence SEQ ID NO: 23 and SEQ ID NO: 57 or 65. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid molecule comprising a nucleic acid sequence SEQ ID NO: 21 and SEQ ID NO: 57 or 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid molecule comprising a nucleic acid sequence SEQ ID NO: 22 and SEQ ID NO: 57 or 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid molecule comprising a nucleic acid sequence SEQ ID NO: 27 and SEQ ID NO: 57 or 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid molecule comprising a nucleic acid sequence SEQ ID NO: 26 and SEQ ID NO: 57 or 65 or 39. According to some embodiments, the immune cells engineered to express both a CAR and a klotho polypeptide comprise a nucleic acid molecule comprising a nucleic acid sequence SEQ ID NO: 29 and SEQ ID NO: 57 or 65 or 39.

According to yet another aspect, the present invention provides a pharmaceutical composition comprising the population of immune cells as defined in any one of the above aspects and embodiments, and a pharmaceutically acceptable carrier. According to some embodiments, the population of immune cells are engineered to express a chimeric antigen receptor (CAR) binding specifically to a tumor-associated antigen and a klotho polypeptide. According to some embodiments, the population of immune cells comprises the construct of the present invention. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence selected from SEQ ID NO: 57, 65, 69, 83, 85, 87 and 99 and a nucleic acid molecule encoding a klotho polypeptide comprising a nucleic acid sequence selected from SEQ ID NO: 20-29.

According to some embodiments, the CAR binds specifically to a tumor-associated antigen selected from ErbB2, CD19, CD38, CD138, EGFR, CD24, GD2, EGF, MSLN, BCMA, CD276, MUC-1, FAP, MUC16 and tumor-associated carbohydrate antigen.

According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 57 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 65 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 39 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 69 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 68 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 83 or 85 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 89 and a nucleic acid sequence selected from SEQ ID NO: 20-29. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 87 and a nucleic acid sequence selected from SEQ ID NO: 20-29.

According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 66. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 37. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 38. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 116. According to some embodiments, the pharmaceutical composition comprises a population of cells comprising a nucleic acid sequence SEQ ID NO: 117.

According to some embodiments, the pharmaceutical composition comprises a population of cells expressing a CAR and a klotho polypeptide as described above.

According to some embodiments, the pharmaceutical composition comprises a population of cells expressing a CAR comprising an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 98, 79, 82, 84, 64, 67, 103 and 86 and the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. According to some embodiments, the pharmaceutical composition comprises a population of cells expressing a CAR comprising an amino acid SEQ ID NO: 40 and a klotho polypeptide comprising an amino acid sequence SEQ ID NO: 9 or 11. According to some embodiments, the pharmaceutical composition comprises a population of cells expressing a CAR comprising an amino acid SEQ ID NO: 40 and a klotho polypeptide comprising an amino acid sequence SEQ ID NO: 5. According to some embodiments, the pharmaceutical composition comprises a population of cells expressing a CAR comprising an amino acid SEQ ID NO: 40 and a klotho polypeptide comprising an amino acid sequence SEQ ID NO: 8.

According to any one of the above embodiments, the immune cells are selected from T cells, NK cells and TIL. According to some embodiments, the immune cells are T cells. According to any one of the above embodiments, the T cells are selected are from CD4⁺ T- cells, CD8⁺ T-cells and combinations thereof

The term “pharmaceutical composition” as used herein refers to a composition comprising at least one active agent, such as immune cells and/or klotho polypeptide as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

Formulation of the pharmaceutical composition may be adjusted according to applications. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration to mammals. For example, the formulation may be any one selected from among plasters, granules, lotions, liniments, lemonades, aromatic waters, powders, syrups, ophthalmic ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, fluidextracts, emulsions, suspensions, decoctions, infusions, ophthalmic solutions, tablets, suppositories, injections, spirits, capsules, creams, troches, tinctures, pastes, pills, and soft or hard gelatin capsules.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds providing supplemental, additional, or enhanced therapeutic functions, solid carriers or excipients such as, for example, lactose, starch or talcum or liquid carriers such as, for example, water, fatty oils or liquid paraffins.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application typically include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol (or other synthetic solvents), antibacterial agents (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, phosphates), and agents that adjust tonicity (e.g., sodium chloride, dextrose). The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, for example. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose glass or plastic vials.

Pharmaceutical compositions adapted for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injectable solutions or suspensions, which can contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Such compositions can also comprise water, alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Such compositions preferably comprise a therapeutically effective amount of a compound of the invention and/or other therapeutic agent(s), together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

According to yet another aspect, the present invention provides a pharmaceutical composition comprising (i) a population of immune cells engineered to express a chimeric antigen receptor (CAR) binding specifically to a tumor-associated antigen and (ii) a klotho polypeptide or vector encoding the klotho polypeptide. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

According to any one of the embodiments and aspects, the pharmaceutical composition of the present invention is for use in treating cancer. It would be clear that the type of cancer is related to the CAR expressed by the CAR T cell. The use of cells expressing specific CARs is defined in the above embodiments. According to some embodiments, the pharmaceutical composition is for use in adoptive cell therapy. According to some embodiments, the cancer is selected from lung cancer, anal cancers, glioblastoma, epithelial tumors, epithelial tumors of the head and neck cells, lymphoma and leukemia, breast cancer, ovarian cancer, pancreatic cancer, cancer stem cells, bladder cancer, cervix cancer, colorectal cancer, esophageal cancer, renal cancer, hepatic cancer, prostate cancer, biliary cancer, oral squamous cell carcinoma, intrauterine membranous cancer, squamous cell carcinoma, gastric cancer, glioma, melanoma, and adrenal cancer. According to some embodiments, the pharmaceutical composition comprising a population of immune cells engineered to express and anti-HER2 CAR, either further expressing a klotho polypeptide or comprising the klotho polypeptide, is for use in treating breast cancer, ovarian cancer, or pancreatic cancer. According to some embodiments, the pharmaceutical composition comprising a population of immune cells engineered to express and anti-HER2 CAR, either further expressing a klotho polypeptide or comprising the klotho polypeptide, is for use in treating lymphoma and/or leukemia.

According to another aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising co-administering to the subject a therapeutic combination of (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen.

According to another aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen, and (ii) a klotho polypeptide. According to some embodiments, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of immune cells expressing (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen, and (ii) a klotho polypeptide

According to another aspect, the present invention provides a method of improving tumor killing and/or clearance by a population of immune cells engineered to express a CAR that binds specifically to a tumor- associated antigen, the method comprising coadministering the population of the immune cells with a klotho polypeptide or a vector encoding thereof.

According to another aspect, the present invention provides use of (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, in preparation of a medicament for treating cancer, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen.

According to another aspect, the present invention provides use of immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen, and (ii) a klotho polypeptide, in preparation of a medicament for treating cancer.

According to another aspect, the present invention provides use of a population of immune cells engineered to express a CAR that binds specifically to a tumor-associated antigen, and a klotho polypeptide or a vector encoding thereof, for preparing a medicament for improving tumor killing and/or clearance

The pharmaceutical composition of the present invention may be administered in any known method. According to some embodiments, the pharmaceutical composition is administered parenterally. According to some embodiments, the pharmaceutical composition is administered IV or IM.

In another aspect there is provided a process for preparing a cell composition adapted for ACT, the process comprising: a) providing a sample of immune cells (e.g. blood-derived immune cells), b) activating the cells (e.g. with T-cell activating agents including, but not limited to, TCR/CD3 activators, co stimulating agents and/or cytokines) c) engineering the cells to express a CAR and a klotho polypeptide as disclosed herein, and d) expanding the cells (e.g. in the presence of T-cell activating cytokines such as IL- 2) so as to obtain an effective amount of cells expressing the CAR and klotho polypeptide. In one embodiment, the blood-derived immune cells are peripheral blood mononuclear cells (PBMC). In another embodiment, step b) is performed in the presence of anti CD3 and anti CD28 antibodies for 24-72 hours and optionally IL2. In another embodiment step c) is performed using transfection and/or transduction methods known in the art, e.g. transfected by electroporation, using Nucleofector technology, liposome-mediated transfer, or transduced using a viral vector as disclosed herein. In another embodiment step d) may be performed by expanding the cells in the presence of IL-2, e.g. for at least 3, 5, or 8 days and up to e.g. 10, 12 or 15 days.

In various embodiments, the engineering the cells to express the CAR may be performed prior to, concomitantly with, or subsequent to engineering said cells to express the klotho polypeptide. In another embodiment, step c) may be at least partly overlapping with step d). For example, without limitation, step c) may include engineering the cells to express the CAR (e.g. by transduction), while a second, subsequent step of engineering the cells to express the klotho polypeptide (e.g. by transfection) may be performed during the expansion step. Each possibility represents a separate embodiment of the invention.

In another aspect there is provided a process for preparing a cell composition adapted for ACT, the process comprising: a) providing a PBMC sample, b) activating the cells for 24-72 hours in the presence of (i) anti CD3 and anti CD28 antibodies and optionally IL2, or (ii) antiCD3 antibodies or (iii) anti CD3 antibodies and IL2, c) engineering the cells to express a CAR and a klotho polypeptide as disclosed herein, and d) expanding the cells in the presence of IL-2 (e.g. 100-350U/ML) so as to obtain an effective amount of cells expressing the CAR and klotho polypeptide.

In another exemplary embodiment, there is provided a process for preparing a cell composition adapted for ACT, the process comprising: a) providing a PBMC sample, b) activating the cells in the presence of anti CD3 and anti CD28 antibodies for 24- 72 hours and optionally IL2, c) engineering the cells to express a CAR as disclosed herein, d) expanding the cells in the presence of IL-2 (e.g. 100-350U/ML for 24-72 hours), e) engineering said cells to express a klotho polypeptide as disclosed herein, and f) further expanding said cells in the presence of IL-2 (e.g. 100-350U/ML) so as to obtain an effective amount of cells expressing the CAR and klotho polypeptide.

In another embodiment, the expanded cells are formulated in the form of an ACT composition comprising about 10⁷-10⁹ or 1O⁸-1O¹⁰ cells and a pharmaceutically acceptable carrier.

In another aspect, there is provided a method of treating cancer in a subject in need thereof, comprising: a) obtaining a sample of immune cells (e.g. PBMC) form the subject, of from an allogeneic donor (histocompatible or non-histocompatible), b) activating the cells (e.g. with T-cell activating agents as disclosed herein), engineering the cells to express a CAR and a klotho polypeptide as disclosed herein, c) expanding the cells (e.g. in the presence of IL-2 as disclosed herein) so as to obtain an effective amount of cells expressing the CAR and klotho polypeptide, d) harvesting the expanded cells, and e) adoptively transferring the cells to the subject so as to treat cancer in said subject.

According to yet another aspect, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically amount of the immune cells of the present invention. According to some embodiments, the method comprises administering the population of cells according to any one of the above embodiments and aspect.

Table 1. Partial list of sequences listed in the application

Clause 1. A method of treating cancer in a subject in need thereof comprising co-administering to the subject a therapeutic combination of (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor antigen associated with the cancer.

Clause 2. The method of any clauses herein, particularly clause 1, wherein the immune cells are characterized by surface expression of the CAR upon administration. Clause 3. The method of any clauses herein, particularly clause 1 or 2, wherein the co-administering comprises a regimen selected from a sequential administering or a substantially simultaneous administering.

Clause 4. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor antigen associated with the cancer, and (ii) a klotho polypeptide.

Clause 5. The method of any clauses herein, particularly clause 4, wherein the immune cells express the CAR constitutively.

Clause 6. The method of any clauses herein, particularly clause 4 or 5, wherein said immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to an inducible promoter, the promoter initiating expression of said klotho polypeptide upon activation of a T cell receptor.

Clause 7. The method of any clauses herein, particularly clause 6, wherein the immune cells express the klotho polypeptide upon activation of the CAR.

Clause 8. The method of any clauses herein, particularly clause 8, wherein the immune cells express the CAR and klotho polypeptide constitutively.

Clause 9. A method of improving tumor killing and/or clearance by a population of immune cells engineered to express a CAR that binds specifically to a tumor- associated antigen, the method comprising co-administering the population of the immune cells with a klotho polypeptide or a vector encoding thereof.

Clause 10. A nucleic acid construct comprising a nucleic acid molecule encoding a CAR that binds specifically to a tumor-associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein each of the nucleic acid molecules is operably linked to a promoter or both nucleic acid molecules are operably linked to one promoter.

Clause 11. The nucleic acid construct of any clauses herein, particularly clause 10, wherein both nucleic acid molecules are operably linked to one constitutive promoter.

Clause 12. The nucleic acid construct of any clauses herein, particularly clause 11, further comprising a cleavable nucleic acid molecule or a nucleic acid molecule encoding a self-cleaving peptide between the two nucleic acid molecules, preferably wherein the construct comprises the nucleic acid sequence selected from SEQ ID NO: 66, 37, 38, 116, and 117. Clause 13. The nucleic acid construct of any clauses herein, particularly clause 10, wherein the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to (i) an inducible promoter, said promoter initiates the expression of klotho polypeptide upon activation of the CAR; or (ii) a constitutive promoter.

Clause 14. A vector comprising the nucleic acid construct according to any one of clauses 10 to 13.

Clause 15. A population of immune cells engineered to express (i) a chimeric antigen receptor (CAR) binding specifically to cancer cells and (ii) a klotho polypeptide.

Clause 16. The population of immune cells of any clauses herein, particularly clause 15, comprising a nucleic acid construct comprising a nucleic acid molecule encoding the CAR and a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide.

Clause 17. The population of immune cells of any clauses herein, particularly clause 16, wherein the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to (i) an inducible promoter, said promoter initiates the expression of klotho polypeptide upon activation of the CAR; or (ii) a constitutive promoter.

Clause 18. The population of immune cells of any clauses herein, particularly clause 15, comprising a nucleic acid construct according to any one of claims 10 to 13 or the vector according to claim 14.

Clause 19. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically the population of immune cells according to any one of clauses 15 to 18.

Clause 20. A pharmaceutical composition comprising the population of immune cells according to any one of clauses 15 to 18, and a pharmaceutically acceptable carrier.

Clause 21. A pharmaceutical composition comprising (i) a population of immune cells engineered to express a chimeric antigen receptor (CAR) binding specifically to a tumor-associated antigen and (ii) a klotho polypeptide.

Clause 22. The pharmaceutical composition according to clause 20 or 21, for use in treating cancer.

Clause 23. The method, the nucleic acid construct, the population of immune cells, or the pharmaceutical composition of any above clauses, wherein (i) the CAR binds specifically to a tumor-associated antigen selected from ErbB2, CD19, CD38, CD138, EGFR, CD24, GD2, EGF, BCMA, CD276, MUC-1, FAP, Mesothelin (MSLN), and MUC16; and/or (ii) the klotho polypeptide is selected from membranal klotho, secreted klotho, proteolyzed klotho, KL1 domain, KL2 domain and an active KL1 fragment; and/or (iii) the immune cells are selected from T cells, natural killer cells and tumor infiltrating lymphocytes (TIL).

Clause 24. The method, the nucleic acid construct, the population of immune cells, or the pharmaceutical composition of any clauses herein, particularly clause 23, wherein the CAR binds specifically to HER2 or CD 19 and the klotho polypeptide is selected from soluble human klotho, human KL1 and an active KL1 fragment.

Clause 25. The method, the nucleic acid construct, the population of immune cells, or the pharmaceutical composition of any clauses herein, particularly clauses 23 or 24, wherein the active KL1 fragment is KL340.

Clause 26. The method, the nucleic acid construct, the population of immune cells, or the pharmaceutical composition of any clauses herein, particularly clauses 24 or 25, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60 and 28 and the klotho polypeptide comprises the amino acid sequence selected from SEQ ID NO: 3-13.

Clause 27. The method, the nucleic acid construct, the population of immune cells, or the pharmaceutical composition of any clauses herein, particularly clauses 23 to 26, wherein the immune cells are T cells.

Clause 28. A method of treating cancer in a subject in need thereof comprising co-administering to the subject therapeutically effective amounts of (i) T cells and (ii) a klotho polypeptide or a vector encoding thereof, wherein the T cells are engineered to express a CAR that binds specifically to HER2 or CD 19 and the klotho polypeptide is selected from a soluble klotho, KL340 and KL1.

Clause 29. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of T cells engineered to express (i) a CAR that binds specifically to HER2 or CD19 and (i) a klotho polypeptide selected from a soluble klotho, KL340 and KL1.

Clause 30. The method, the nucleic acid construct, the population of immune cells, or the pharmaceutical composition of any clauses herein, particularly clauses 28 and 29, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60 and 61 and the klotho polypeptide comprise amino acid sequence selected from SEQ ID NOs: 3-13.

Clause 31. The method of any clauses herein, wherein the method provides a synergistic anticancer effect. Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

Materials and methods

Tumor cells: The HER2 -positive breast cancer cell line SKBR-3, HER2 -positive ovarian cancer cells OVCAR-8 or HER2-expressing pancreatic cancer PANCI cells or 0VCAR3 HER2 negative control cell line were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin and 1% L-glutamine solution.

CAR T cells: Peripheral human blood mononuclear cells (PBMC) were isolated from the blood of healthy human donors by density gradient centrifugation on Ficoll-Paque (Axisshield, Oslo, Norway). PBMC were activated in non-tissue culture-treated 6-well plates, precoated with both purified anti-human CD3 and purified anti-human CD28 for 48 hours at 37°C. Activated lymphocytes were harvested and subjected to two consecutive retroviral transductions in RetroNectin pre-coated, non-tissue culture-treated 6-well plates supplemented with human IL-2 (100 lU/mL). After transduction, cells were cultured in the presence of 350 lU/mL IL-2 for 24-72 hours. Transduction efficiency was monitored by flow cytometry. Activated but non-infected cells were included as T cell controls.

Colony assay: Tumor cells were plated in triplicates in 12-well plates and transfected with 2 pg KL1 expression vector (encoding the KL1 active domain of klotho) or empty vector pcDNA3 as control, using JetPEI (Polyplus Transfection). Two days later, cells were replated in 6-well plates and G418 (750 pg/ml) was added to the culture media. Media containing G418 was replaced every 3-4 days and after 10-14 days cells were fixed and stained using crystal violet (Sigma- Aldrich).

Viability assay:

Soluble klotho - Cells were seeded in 96-well plate (3000 cells per well) and a day later were treated with a soluble klotho polypeptide (KL1-KL2 fragment, R&D) for 48 or 72 hours. Methylene blue assay was conducted to determine viability.

Combined treatment - Tumor cells were seeded in 6cm dishes and transfected with pcDNA3 or KL1 expression vector (5pg or lOpg per well). Two days later G418 was added (750 pg/ml) and media was replaced twice a week with fresh G418-containing media. After 2 weeks cells were trypsinized and 10,000 cells of either the pcDNA3 or KL1 transfected groups were seeded in 96-well plate, and CAR-T cells were added at decreasing ratio 1:1, 1:0.5, 1:0.25, 1:0.13, 1:0.06 cancer cells to CAR-T. Viability was determined after 72 hours using methylene blue assay.

Drug interaction analysis: The Bliss Independence method was employed to determine drug interaction. The model predicts that if individual drugs have the inhibitory effects fi and/2 then the expected combined effect of the two drugs is:

E(f₁₂) = 1 - (1 -fi) (1 -f₂) =fi +f₂ -fif₂

Excess over Bliss eob) is calculated by eob =f - E(fi₂), where //? is the observed combined effect. A positive, negative, or null value, is used to determine a synergistic, antagonistic or no interaction, respectively.

Luciferase assay: Tumor cells were seeded in 24-well plates and transfected with either pcDNA3 or klotho/KLl expressing vectors, along with pGL4.32[luc2P/NF-KB-RE/Hygro] vector (Promega). After 24 hours IL-6 was added, and a day later luciferase assay was conducted using the Luciferase Assay System kit (Promega) according to the manufacturer’s instructions. Luciferase units were normalized to total protein concentration. Data are presented as mean values ± SD’s for at least three independent experiments done in triplicates.

Sequences: The sequences corresponding to or encoding klotho, its fragments, the CAR and additional elements of the constructs, are presented in Table 1 above.

The klotho-expressing vector encodes a polypeptide having the amino acid sequence SEQ ID NO: 3.

The KL1 -expressing vector encodes a polypeptide having the amino acid sequence SEQ ID NO: 5.

KL340- expressing vector encodes a polypeptide having the amino acid sequence SEQ ID NO: 9, 10 or 11.

The soluble KL1-KL2 fragment used in Examples 2 and 3 corresponds to positions 34-981 of SEQ ID NO: 8. Example 1. Anti-HER2 CAR T cells characterization and their cytotoxic effect on breast and ovarian cancers.

The cytotoxic activity of CAR-T cells in the presence of target tumor cells was evaluated and analyzed as follows. CAR-T cells, directed against HER-2 tumor antigen, were produced using the N29 CAR and which co-expresses a GFP reporter gene (SEQ ID NO: 58). T cells were isolated from healthy human donors and transduced to express the anti-ErbB2 (N29) CAR. The average transduction rate, demonstrated with anti-idiotype antibody staining was 61.25% (Fig. 1A). Similarly, the average expression of the GFP reporter gene was 70.77% (p-value=0.23, Fig. 1A). GFP was expressed by the same cells that were stained with antiidiotype antibody. Therefore, the transduction rate in the following experiments was evaluated by measuring GFP expression. At day 7 of activation as detailed above, 97-98% of the transduced cells were CD3 positive, with 44.38% expressing CD4, and 45.76% expressing CD8.

CAR-T cells were then stimulated with SKOV3 (ovarian) and SKBR3 (breast) ErbB2- expressing target cells. The OVCAR3 (ovarian cancer cell line) line is ErbB2- negative, and therefore served as a negative control. SKOV3 (ovarian cancer) and SKBR3 (breast cancer) showed high ErbB2 levels (Fig. IB). These cell lines were then used as target cells for anti- ErbB2 CAR-T cells or non-transduced cells (UT, as control), and were co-cultured for 16 hours (hrs) at a 2:1 CAR-T, effector cells (E): target cells (T) ratio.

As can be seen in Fig. 1C, CAR-T cell stimulation with SKOV3 or SKBR3 cells, both expressing high ErbB2 levels, induced high IFNy secretion (with the average of 30,000pg/ml; p-value<0.05, 47,000pg/ml; p-value<0.05 respectively), indicating an enhanced tumor- specific response. The killing activity of ErbB2 CAR-T cells was assessed by methylene -blue assay. Culture of ErbB2 CAR-T cells with ErbB2 positive target cells at an 8: 1 ratio resulted in the killing of up to 80% of SKOV3 cells (Fig. ID) and 65% of SKBR3 cells. However, anti-ErbB2 CAR-T cells had no effect on ErbB2 negative cells (OVCAR3). Thus, the results demonstrate that the ErbB 2- specific CAR-T cells induce an effective tumor- specific immune response against the ErbB 2 -expressing tumor cell lines. Under the experimental conditions tested, an E:T ratio of at least 1:1 was sufficient for inducing a statistically significant response in all tested cell lines. Accordingly, E:T ratios of 0.5: 1 or less were determined as sub-lethal doses when used in the combination experiments. Example 2. Klotho decreases viability of breast and ovarian cancer cells.

It was previously shown that overexpression of klotho reduces viability and colony formation of estrogen receptor (ER)-positive MCF-7 and of the triple negative MDA-MB231 breast cancer (BC) cells (Wolf et al., 2008, Ligumsky et al., 2015). In order to reveal the effect of klotho on HER2-positive BC cells, colony assay was conducted by overexpressing klotho in HER2-positive BC cell line SK-BR-3 cells. The inhibitory effect of klotho and KL1 on colony formation of ovarian cancer (OC) cell lines SKOV33, ES2 and OVCA432 was previously shown by Lojkin et al., (PMID: 25827069).

The viability of SK-BR-3, MCF-7 and the ovarian cancer cells SKOV33 in the presence of different concentrations of soluble klotho (KL1-KL2 fragment) was studied using methylene blue assay. The results (Fig. 2) showed that soluble klotho moderately decreases the viability of these cells, with maximal effect at lOng/ml.

Example 3. Effect of klotho on CAR T cells.

As klotho inhibits growth and viability of highly-proliferating cells, it was examined whether it may affect the viability of CAR-T cells. To this end, control T cells (non-transfected PBMC activated and expanded as described above) or anti-HER2 CAR T cells (100,000 and 200,000 cells/well, 24 well plate) were treated with gradually elevating levels of a soluble klotho (KL1-KL2 fragment, R&D) for 24 and 48 hrs, and then counted with Trypan blue. The results, presented in Fig. 3A-3B, show that klotho did not decrease the viability of the cells. Rather, treatment with the soluble klotho polypeptide unexpectedly increased dose- dependently (at 200,000 cells/well) the number of viable cells upon incubation with both control (Fig. 3A) and CAR T cells (Fig. 3B).

Example 4. Co-treatment with klotho and CAR-T cells is effective against cancer cells and provides a synergistic effect.

High HER2 expressing cells:

The anti-cancer effect of a combinatory treatment of klotho and CAR-T cells on tumor cells expressing high HER2 levels was studied.

SK-BR-3 breast cancer cells express high HER2 protein levels. These cells were transfected with two concentrations of KL1 expression vector (5 and lOpg, referred to as "low dose KL1" and "high dose KL1, respectively) or control empty vector (pcDNA3) and a G418 selection was performed for two weeks to enrich for klotho-expressing cells. Next, about IxlO⁵ of the selected cells were seeded and a day later CAR-T cells, at 1:1, 1:0.5, 1:0.25, 1:0.13, 0:0.06 cancer cells : CAR-T ratios, were added for 48 hours and the viability was determined using methylene blue. The results are presented in Figs. 4A-4B for high and low KL1 -expressing cells, respectively.

As can be seen, the combined treatment was more effective than each of the single treatments (Fig. 4A-4B). Remarkably, the combined treatment resulted in significantly reduced viability of the tumor cells even at sub-lethal doses of CAR T cells treatment (lower than 0.5: 1 E:T, as determined in Example 1).

Bliss score was assessed using the formula (Ea + Eb - Ea x Eb), Ea = fractional inhibition obtained by KL1, Eb = fractional inhibition obtained by CAR-T. Excess over Bliss (eoZ?) was calculated by the observed combined effect compared with the Bliss score. Using the formula, a synergistic effect upon co-treatment with KL1 and CAR-T was observed for all tested concentrations.

Example 5. Effect of a Co-treatment of klotho and anti-HER2 CAR-T cells on cells expressing moderate HER2 levels

Moderate HER2 expressing cells:

Next, the effect of the combinatory treatment on pancreatic cancer PANCI cells which express moderate HER2 levels was studied. Cells were treated as above. Briefly, PANCI cells were transfected with 5 pg pcDNA3 or KL1 -expression plasmid. Cells were grown with G418 for 2 weeks and then were replated in 96 -well plate (10,000 cells per well) and anti-HER2 (N29) CAR-T cells, from two different donors (Nos 56 and 64), were added with decreasing CAR-T cells per cancer cells ratio. Cancer cells viability was determined after 48 hours using trypan blue. The results are presented in Figs. 5A-5B.

The results show that the combined treatment was more effective than either single treatment (Figs. 5A-5B), and exerted a significant anti-tumor response even at sub-lethal doses.

As observed with SKBR3 cells, a synergistic effect upon co-treatment with KL1 and CAR- T was observed also with PANCI cells.

Accordingly, the results demonstrate synergistic effect of the combined treatment against tumors expressing various levels of the specific tumor antigen. A remarkable anti-tumor effect was observed even when using low doses of the treatment, providing for enhanced safety and reduced potential for developing adverse effects.

The experiment was repeated using a shorter fragment of Klotho - KL340. To this aim, ovarian cancer cells OVCAR8 and pancreatic cancer cells Panc-1 were used, that express moderate HER2 protein levels. These cells were transfected with either KL1, KL340 expression vectors or pcDNA3 empty vector control and grown for a week with G418 to enrich for klotho-expressing cells. IxlO⁴ cells were seeded in 96 well plates and two hours later CAR-T cells expressing anti-HER2 N29 CAR were added at different ratios: 1: 1, 1:0.5, 1:0.25, 1:0.13, 0:0.06 cancer cells : CAR-T ratio for OVCAR8 cells or 1:2, 1:1, 1:0.5, 1:0.25, 1:0.13 for Panc-1 cells. Viability was determined after 72 hours using methylene blue assay. The results show that KL1 and KL340 efficacy was similar, and that the combined treatment with CAR T was more effective than either single treatment, in both OVCAR8 and Panc-1 cells (Figs. 6A and 6B).

Example 6. Klotho decreases inflammatory pathways in breast cancer cells.

MCF7 cells were transfected with a plasmid expressing klotho or its active fragment KL1, or control vector (pcDNA3) together with NFKB reporter gene (pNL3.2.NF-KB-RE vector) for 48 hrs. IL6 was added for the last 24 hrs as indicated and luciferase activity was measured and normalized to protein concentration. The results show that klotho and KL1 effectively reduced both IL6-induced and non-induced NFKB transcriptional activity (Fig. 7).

Example 7. Co-treatment of klotho and anti-CD19 CAR-T cells

The effect of the combinatory treatment on cancer cells which express CD 19 tumor- associated antigen is studied. Cells are treated as described above. Briefly, the cells are transfected with 5 pg pcDNA3 or with a vector encoding klotho, KL1, KL1-KL2 or KL340- expression plasmid. Cells are grown with G418 for 2 weeks and then replated in 96-well plate (10,000 cells per well) and anti-CD19 CAR-T cells, from two different donors, are added with decreasing CAR-T cells per cancer cells ratios. In alternative examples, cells are incubated with KL1, KL1-KL1 or KL340 peptide in the presence of anti-CD19 CAR-T cells. The klotho polypeptide comprise the amino acid sequence selected from SEQ ID NOs: 5, 8 and 9 and the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 60 or 61 or 64.

Example 8. Co-treatment of klotho and anti-CD138 CAR-T cells

The effect of the combinatory treatment on cancer cells which express CD 138 tumor- associated antigen is studied. Cells are treated as described above. Briefly, the cells are transfected with 5 pg pcDNA3 or with a vector encoding klotho, KL1, KL1-KL2 or KL340- expression plasmid. Cells are grown with G418 for 2 weeks and then replated in 96-well plate (10,000 cells per well) and anti-CD138 CAR-T cells, are added with decreasing CAR- T cells per cancer cells ratios. The klotho polypeptide comprise the amino acid sequence selected from SEQ ID NOs: 5, 8 and 9 and the anti-CD138 CAR comprises the amino acid sequence SEQ ID NO: 79.

Example 9. Co-treatment of klotho and anti-CD38 CAR-T cells

The effect of the combinatory treatment on cancer cells which express CD38 tumor- associated antigen is studied. Cells are treated as described above. Briefly, the cells are transfected with 5 pg pcDNA3 or with a vector encoding klotho, KL1, KL1-KL2 or KL340- expression plasmid. Cells are grown with G418 for 2 weeks and then replated in 96-well plate (10,000 cells per well) and anti-CD38 CAR-T cells, are added with decreasing CAR- T cells per cancer cells ratios. The klotho polypeptide comprise the amino acid sequence selected from SEQ ID NOs: 5, 8 and 9 and the anti-CD38 CAR comprises the amino acid sequence SEQ ID NO: 98 or 103.

Example 10. Co-treatment of klotho and anti-CD276 CAR-T cells

The effect of the combinatory treatment on cancer cells which express CD276 tumor- associated antigen is studied. Cells are treated as described above. Briefly, the cells are transfected with 5 pg pcDNA3 or with a vector encoding klotho, KL1, KL1-KL2 or KL340- expression plasmid. Cells are grown with G418 for 2 weeks and then replated in 96-well plate (10,000 cells per well) and anti-CD276 CAR-T cells are added with decreasing CAR- T cells per cancer cells ratios. The klotho polypeptide comprise the amino acid sequence selected from SEQ ID NOs: 5, 8 and 9 and the anti-CD276 CAR comprises the amino acid sequence SEQ ID NO: 82 or 84.

Example 11. Co-treatment of klotho and anti-EGFR CAR-T cells

The effect of the combinatory treatment on cancer cells which express EGFR tumor- associated antigen is studied. Cells are treated as described above. Briefly, the cells are transfected with 5 pg pcDNA3 or with a vector encoding klotho, KL1, KL1-KL2 or KL340- expression plasmid. Cells are grown with G418 for 2 weeks and then replated in 96-well plate (10,000 cells per well) and anti-EGFR CAR-T cells are added with decreasing CAR-T cells per cancer cells ratios. The klotho polypeptide comprise the amino acid sequence selected from SEQ ID NOs: 5, 8 and 9 and the anti-EGFR CAR comprises the amino acid sequence SEQ ID NO: 86. Example 12. Expression of KL340 and CAR using a single construct

HEK293 LentiX cells were transfected (or not) with each of the plasmids plasmid encoding to (a) N29 CAR (anti-HER2 SEQ ID NO: 119), (b) N29-flag-KL340-HA (SEQ ID NO: 118, HA stands for hemagglutinin tag), (c) KL340-HA (SEQ ID NO: 120), and (d) CD19-flag- KL340-HA (SEQ ID NO: 121). After 48 hours from the transfection media, was replaced with serum-free media. Six hours later media was collected and proteins were precipitated, whereas cells protein was extracted with RIPA buffer. Protein from cells and media were resolved on SDS-PAGE, and probed with anti-HA to detect KL340. The expression of KL340 is clearly shown Fig. 8 presenting a longer (upper raw) and shorter (2nd raw) exposure time.

Further, HEK293 LentiX cells were transfected (or not) with each of the plasmids as described above. After 48 hrs from the transfection, cells transfected with N29-flag-KL340- HA, N29 CAR, CD19-flag-KL340-HA and untransfected cells were stained with recombinant HER2, and an APC- human a-HER2 (the latter is shows in Fig. 9A and 9B, in this staining, the CD19CAR also served as negative control). We detected high level of anti- HER2 CAR (see Fig. 9A and 9B).

Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims

Claims

1. A therapeutic combination comprising (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, for use in treating cancer, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor- associated antigen.

2. The therapeutic combination for use according to claim 1, wherein the immune cells are characterized by surface expression of the CAR upon administration.

3. The therapeutic combination according to claim 1 or 2, wherein the use comprises coadministering the engineered immune cells and a klotho polypeptide or a vector encoding thereof in a regimen selected from a sequential administering or a substantially simultaneous administering.

4. A pharmaceutical composition comprising immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen, and (ii) a klotho polypeptide, and a pharmaceutical acceptable carrier, for use in treating cancer.

5. The pharmaceutical composition for use according to claim 4, wherein the immune cells express the CAR constitutively.

6. The pharmaceutical composition for use according to claim 4 or 5, wherein said immune cells comprise a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide, wherein the nucleic acid molecule is operably linked to an inducible promoter, and wherein the promoter initiates the expression of said klotho polypeptide upon activation of a T cell receptor.

7. The pharmaceutical composition for use according to claim 6, wherein the immune cells express the klotho polypeptide upon activation of the CAR.

8. The pharmaceutical composition for use according to claim 4, wherein the immune cells express the CAR and klotho polypeptide constitutively.

9. A therapeutic combination comprising a population of immune cells engineered to express a CAR that binds specifically to a tumor-associated antigen, and a klotho polypeptide or a vector encoding thereof, for use in improving tumor killing and/or clearance.

10. A nucleic acid construct comprising a nucleic acid molecule encoding a CAR that binds specifically to a tumor- associated antigen and a nucleic acid molecule encoding a klotho polypeptide, wherein each one of the nucleic acid molecules is operably linked to a promoter or both nucleic acid molecules are operably linked to one promoter.

11. The nucleic acid construct according to claim 10, wherein both nucleic acid molecules are operably linked to one constitutive promoter.

12. The nucleic acid construct according to claim 11, further comprising a cleavable nucleic acid molecule or a nucleic acid molecule encoding a self-cleaving peptide between the two nucleic acid molecules, preferably wherein the construct comprises the nucleic acid sequence selected from SEQ ID NO: 66, 37, 38, 116 and 117.

13. The nucleic acid construct according to claim 10, wherein the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to (i) an inducible promoter, said promoter initiates the expression of klotho polypeptide upon activation of the CAR; or (ii) a constitutive promoter.

14. A vector comprising the nucleic acid construct according to any one of claims 10 to 13.

15. A population of immune cells engineered to express (i) a chimeric antigen receptor (CAR) binding specifically to a tumor-associated antigen and (ii) a klotho polypeptide.

16. The population of immune cells according to claim 15, comprising a nucleic acid construct comprising a nucleic acid molecule encoding the CAR and a nucleic acid construct comprising a nucleic acid molecule encoding the klotho polypeptide.

17. The population of immune cells according to claim 16, wherein the nucleic acid molecule encoding the CAR is operably linked to a constitutive promoter and the nucleic acid molecule encoding the klotho polypeptide is operably linked to (i) an inducible promoter, said promoter initiates the expression of klotho polypeptide upon activation of the CAR; or (ii) a constitutive promoter.

18. The population of immune cells according to claim 15, comprising a nucleic acid construct according to any one of claims 10 to 13 or the vector according to claim 14.

19. A pharmaceutical composition comprising the population of immune cells according to any one of claims 15 to 18, and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition according to claim 20, for use in treating cancer.

21. A pharmaceutical composition comprising (i) a population of immune cells engineered to express a chimeric antigen receptor (CAR) binding specifically to a tumor- associated antigen, (ii) a klotho polypeptide or a vector encoding thereof, and (iii) a pharmaceutically acceptable carrier.

22. The pharmaceutical composition according to claim 21, for use in treating cancer.

23. A method of treating cancer in a subject in need thereof comprising co -administering to the subject a therapeutic combination of (i) engineered immune cells and (ii) a klotho polypeptide or a vector encoding thereof, wherein the immune cells are engineered to express a chimeric antigen receptor (CAR) that binds specifically to a tumor- associated antigen.

24. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of immune cells engineered to express (i) a chimeric antigen receptor (CAR) that binds specifically to a tumor-associated antigen, and (ii) a klotho polypeptide.

25. A method of improving tumor killing and/or clearance by a population of immune cells engineered to express a CAR that binds specifically to a tumor-associated antigen, the method comprising co-administering the population of the immune cells with a klotho polypeptide or a vector encoding thereof.

26. A method of treating cancer in a subject in need thereof comprising administering to the subject a population of immune cells according to any one of claims 15 to 18.

27. The therapeutic combination for use according to any one of claims 1 to 3 and 9, the pharmaceutical composition for use according to any one of claims 4-8, 20 and 22, the nucleic acid construct according to any one of claims 10 to 13, the nucleic acid construct according to claim 14, the population of immune cells according to any one of claims 15 to 18, the pharmaceutical composition according to claim 19 or 21, or the method according to any one of claims 23 to 26, wherein (i) the CAR binds specifically to a tumor-associated antigen selected from ErbB2, CD19, CD38, CD138, EGFR, CD276, CD24, GD2, EGF, BCMA, MUC-1, FAP, Mesothelin (MSEN), and MUC16; and/or (ii) the klotho polypeptide is selected from a membranal klotho, secreted klotho, proteolyzed klotho, KE1 domain, KE2 domain, KE1-KE2 domain, and an active KE1 fragment; and/or (iii) the immune cells are selected from T cells, natural killer cells and tumor infiltrating lymphocytes (TIE).

28. The therapeutical combination for use, the pharmaceutical composition, the pharmaceutical composition for use, the population of immune cells, the nucleic acid construct, the vector or the method according to claim 27, wherein the CAR binds specifically to HER2 or CD 19 and the klotho polypeptide is selected from soluble human klotho, human KL1 and an active KL1 fragment.

29. The therapeutical combination for use, the pharmaceutical composition, the pharmaceutical composition for use, the population of immune cells, the nucleic acid construct, the vector or the method according to claim 27 or claim 28, wherein the active KL1 fragment is KL340.

30. The therapeutical combination for use, the pharmaceutical composition, the pharmaceutical composition for use, the population of immune cells, the nucleic acid construct, the vector or the method according to any one of claims 27 or 30, wherein the CAR comprises an amino acid sequence selected from SEQ ID NO: 40, 60, 61, 64, 79, 67, 82, 84, 86, 98, and 103 and the klotho polypeptide comprises an amino acid sequence selected from SEQ ID NO: 3-13.

31. The therapeutical combination for use, method, the population of immune cells, the pharmaceutical composition, or the pharmaceutical composition for use according to any one of claims 1 to 9 and 15 to 30, wherein the immune cells are T cells.

32. The therapeutical combination for use, method, or the pharmaceutical composition for use according to any one of claims 1 to 9 and 20 to 30, wherein the use or the treating provides a synergistic effect.