Use of chimeric antigen receptor modified cells to treat cancer

Abstract

Compositions, methods, and kits for treating cancer using chimeric antigen receptor (CAR) modified cells are disclosed. An isolated nucleic acid sequence encoding CAR is also disclosed. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.

Description

(19) *EP004745230A2* (11) EP 4 745 230 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 20.05.2026 Bulletin 2026 / 21 (21) Application number: 26167780.1 (22) Date of filing: 30.03.2017 (51) International Patent Classification (IPC): C12N 5 / 0783 (2010.01) (52) Cooperative Patent Classification (CPC): A61K 38 / 1774; A61K 40 / 00; A61K 40 / 11; A61K 40 / 31; A61K 40 / 4202; A61K 40 / 4214; A61K 40 / 4224; A61K 40 / 4257; C07K 14 / 4748; C07K 14 / 705; C07K 14 / 7051; C12N 5 / 0636; A61K 2039 / 505; A61K 2039 / 572; A61K 2039 / 812; (Cont.) (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 01.04.2016 US 201662317261 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 21211532.3 / 3 988 111 17773247.6 / 3 436 059 (71) Applicant: Innovative Cellular Therapeutics Holdings, Ltd. Grand Cayman, KYI - 1104 (KY) (72) Inventors: • XIAO, Lei Shanghai 201203 (CN) • CAO, Zhiyuan Shanghai 201203 (CN) • WU, Zhao Shanghai 201203 (CN) • PU, Chengfei Shanghai 201203 (CN) • SUN, He Shanghai 201203 (CN) • BI, Mao Shanghai 201203 (CN) (74) Representative: Abel & Imray LLP Westpoint Building James Street West Bath BA1 2DA (GB) Remarks: This application was filed on 25‑03‑2026 as a divisional application to the application mentioned under INID code 62. (54) USE OF CHIMERIC ANTIGEN RECEPTOR MODIFIED CELLS TO TREAT CANCER (57) Compositions, methods, and kits for treating cancer using chimeric antigen receptor (CAR) modified cells are disclosed. An isolated nucleic acid sequence encoding CAR is also disclosed. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ. EP 4 74 5 23 0 A 2 Processed by Luminess, 75001 PARIS (FR) (Cont. next page) (52) Cooperative Patent Classification (CPC): (Cont.) A61K 2039 / 82; A61K 2039 / 828; A61K 2039 / 892; A61K 2239 / 38; A61K 2239 / 49; C07K 2319 / 33 2 EP 4 745 230 A2 Description CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62317261, filed on April 1, 2016, entitled "Use of chimeric antigen receptor modified cells to treat cancer," which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0002] Thepresent disclosure relates tomodifiedcells anduses, in particularly to compositionsandmethods for treating cancer using chimeric antigen receptor (CAR) modified cells. BACKGROUND

[0003] Cancer is known as malignant tumors involving abnormal cell growth with the potential to invade or spread to other parts of the body. In humans, there aremore than one hundred kinds of cancer, for example, breast cancer occurred in epithelial tissue of breast. Since breast cancer cells lose characteristics of normal cells, the connection between breast cancer cells is loose.Once the cancer cells areexfoliated, theseexfoliated cancer cells spreadover bodies via thebloodor lymph systemsand therefore become life-threatening. Currently, breast cancer has becomeoneof the common threats to women’s physical andmental health. Immunotherapy (e.g.,CART)hasbeenproved tobeeffective for treating cancer.But There is a need to improve the immunotherapy to be more effective for certain cancer such as solid tumors. SUMMARY

[0004] Embodiments of the present disclosure relate to compositions, methods, and kits for treating cancer using chimeric antigen receptor (CAR) modified cells.

[0005] Some embodiments of the present disclosure relate to an isolated nucleic acid sequence encoding a CAR The CARmay include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domainmay bind to an antigen of a non-essential organ. For example, the antigen binding domain binds to an antigen that is expressed on the surface of a non-essential organ cell present in a microenvironment of a tumor.

[0006] Some embodiments of the present disclosure relate to an isolated CAR including an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.

[0007] Some embodiments of the present disclosure relate to a pharmaceutical composition including human Tcells. The human T cells may include a nucleic acid sequence encoding a CAR. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.

[0008] Some embodiments of the present disclosure relate to a cell including a nucleic acid sequence encoding aCAR. TheCARmay include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domain binds to an antigen of a non-essential organ. For example, the cell is selected from the group consisting of a Tcell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0009] Some embodiments of the present disclosure relate to a vector comprising a nucleic acid sequence encoding a CAR.TheCARmay include anantigenbindingdomain, a transmembranedomain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.

[0010] Some embodiments of the present disclosure relate to a method for stimulating a T cell-mediated immune response to a cell population in a non-essential organ of a subject. Themethodmay include administering to a subject an effective amount of a cell genetically modified to express a CAR. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domain is selected to recognize the cell population of the non-essential organ specifically.

[0011] Some embodiments of the present disclosure relate to a method of treating a subject with cancer. The method may include administering to the subject a cell genetically engineered to express aCAR. TheCARmay include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ. For example, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0012] In some embodiments, the antigen binding domain is an antibody, an antigen-binding fragment thereof, or a 3 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 ligand thereof For example, the antigen-binding fragment is a Fab or a scFv.

[0013] In someembodiments, the antigen is expressedonanon-essential organ cell present in amicroenvironment of a tumor.

[0014] In some embodiments, the tumor is a breast cancer. In certain embodiments, the antigen is a mammary gland antigen. For example, the mammary gland antigen is prolactin receptor (PRLR) having SEQ ID NO: 29. In some embodiments, the antigen binding domain is prolactin receptor ligand having SEQ ID NO: 20 or 44.

[0015] In someembodiments, the tumor is colorectal cancer. In certain embodiments, the antigen is a colon antigen. For example, the colon antigen is Guanylate cyclase 2C (GUCY2C) having SEQ ID NO: 33.

[0016] In someembodiments, the tumor is gastric cancer. In certainembodiments, theantigen isagastric glandantigen. For example, the gastric gland antigen is Mucin 17 (Muc17) having SEQ ID NO: 31.

[0017] In some embodiments, the tumor is a bladder cancer. In certain embodiments, the antigen is a bladder antigen. For example, the bladder antigen is CD207 having SEQ ID NO: 35.

[0018] In some embodiments, the tumor is an ovary tumor. In certain embodiments, the antigen is an ovary antigen. For example, the ovary antigen is Frizzled family receptor 10 (FZD10) having SEQ ID NO: 25.

[0019] In some embodiments, the tumor is a thyroid tumor. In certain embodiments, the antigen is a thyroid antigen. For example, the thyroid antigen is Thyroid stimulating hormone receptor (TSHR) having SEQ ID NO: 27.

[0020] In someembodiments, the costimulatory signaling regionmay include the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4‑1BB, OX40, CD30, CD40, PD‑1, ICOS, lymphocyte function-associated antigen‑1 (LFA‑1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically bindswithCD83, and any combination thereof.

[0021] In some embodiments, the antigen binding domain may include at least one of SEQ ID NOs: 2‑20 or 44.

[0022] In someembodiments, the antigen binding domainmay includeSEQ IDNO: 3 or 4, or a combination thereof, and the tumor is ovary tumor.

[0023] In someembodiments, the antigen binding domainmay includeSEQ IDNO: 6 or 7, or a combination thereof, and the tumor is thyroid tumor.

[0024] In some embodiments, the antigen binding domain may include SEQ ID NO: 9 or 10, or a combination thereof, and the tumor is breast cancer.

[0025] In some embodiments, the antigen binding domain may include SEQ ID NO: 12 or 13, or a combination thereof, and the tumor is gastric cancer.

[0026] In some embodiments, the antigen binding domain may include SEQ ID NO: 15 or 16, or a combination thereof, and the tumor is colorectal cancer.

[0027] In some embodiments, the antigen binding domain may include SEQ ID NO: 18 or 19, or a combination thereof, and the tumor is bladder cancer.

[0028] In some embodiments, the antigen binding domain may include SEQ ID NO: 20 or 44, and the tumor is breast cancer.

[0029] Someembodimentsof thepresentdisclosure relate toamethodof selectinganantigenbindingdomain foraCAR for treating a subject with tumor cells. The method may include determining an organ of cells from that the tumor cells derived, determining that the organ is a non-essential organ with respect to the subject, searching a database to identify multiplemarkers that are expressed in a cell population of the organ, selecting amarker of themultiplemarkers based ona predetermined condition, and generating cells comprising a CAR using cells from the subject. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain, and the antigen binding domain may bind to the marker.

[0030] In someembodiments, thepredetermined conditionmay include themarker is present on the cell surfaceof a cell from that tumor cells are derived at least about at least one of 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold or 1000-fold greater than a suitable control cell, and the organ is non-essential such that an injury of the organ does not produce death of the subject.

[0031] In some embodiments, the non-essential organ is a mammary gland, and the marker is PRLR. In some embodiments, the non-essential organ is a colon, and the marker is GUCY2C.

[0032] In some embodiments, the non-essential organ is a gastric gland, and the marker is Muc17.

[0033] In some embodiments, the non-essential organ is a bladder, and the marker is CD207.

[0034] In some embodiments, the non-essential organ is an ovary, and the marker is FZD10.

[0035] In some embodiments, the non-essential organ is a thyroid, and the marker is TSHR.

[0036] In some embodiments, the tumor is selected froma group consisting of breast cancer, a thyroid tumor, colorectal cancer, an ovary tumor, bladder cancer, and bladder cancer.

[0037] Someembodimentsof thepresentdisclosure relate toamodifiedcell includinganucleicacid sequenceencoding a CAR having one of SEQ ID NOs: 36‑43. For example, the cell is selected from the group consisting of a Tcell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0038] In someembodiments, theCARhasSEQ IDNO:38, andanantigenbindingdomainof theCARbinds to prolactin 4 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 receptor ligand having SEQ ID NO: 20 or 44.

[0039] In some embodiments, the CAR has SEQ ID NO: 40, and an antigen binding domain of the CAR binds to GUCY2C having SEQ ID NO: 33.

[0040] In some embodiments, the CAR has SEQ IDNO: 39, and an antigen binding domain of the CAR binds to Muc17 having SEQ ID NO: 31.

[0041] In some embodiments, the CAR has SEQ IDNO: 41, and an antigen binding domain of the CAR binds to CD207 having SEQ ID NO: 35.

[0042] In some embodiments, the CAR has SEQ IDNO: 36, and an antigen binding domain of the CAR binds to FZD10 having SEQ ID NO: 25.

[0043] In some embodiments, the CAR has SEQ ID NO: 37, and an antigen binding domain of the CAR binds to TSHR having SEQ ID NO: 27.

[0044] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in theDetailedDescription. ThisSummary is not intended to identify key featuresor essential featuresof theclaimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] TheDetailedDescription isdescribedwith reference to theaccompanyingfigures.Theuseof thesame reference numbers in different figures indicates similar or identical items. FIG. 1 illustrates multiple tumors and antigens of non-essential organs as well as corresponding genes thereof. FIG. 2 shows the construction of antigen overexpressed K562 cell lines and includes images showing established target tumor cell lines. FIG. 3 includes schematic diagrams illustrating the construction of CARs. FIG. 4 shows assay results demonstrating that anti-CD207 CAR Tcell lines were established. FIG. 5 shows assay results demonstrating that anti-Muc17 CAR T cell lines were established. FIG. 6 shows assay results demonstrating that anti-TSHR CAR Tcell lines were established. FIG. 7 shows assay results demonstrating that anti-PRLR CAR T cell lines were established. FIG. 8 shows assay results demonstrating that anti-FZD10 CAR Tcell lines were established. FIG. 9 shows assay results demonstrating that anti-GUCY2C CAR T cell lines were established. FIG. 10 shows co-cultivation assays demonstrating that anti-FZD10 CAR Tcells recolonize specific tumor cells and release IFN-γ accordingly. FIG. 11 shows co-cultivation assays demonstrating that anti-GUCY2CCARTcells recolonize specific tumor cells and release IFN-γ accordingly. FIG. 12 shows co-cultivation assays demonstrating that anti-Muc17 CAR Tcells recolonize specific tumor cells and release IFN-γ accordingly. FIG. 13 shows co-cultivation assays demonstrating that anti-CD207 CAR Tcells recolonize specific tumor cells and release IFN-γ accordingly. FIG. 14 shows co-cultivation assays demonstrating that anti-TSHR CAR Tcells recolonize specific tumor cells and release IFN-γ accordingly. FIG. 15 shows co-cultivation assays demonstrating that anti-PRLR CAR T cells and PRLR ligand CAR T cells recolonize specific tumor cells and release IFN-γ accordingly. FIG. 16 shows killing assay results based on co-cultivation of anti-THSR CAR T cells and TSHR-K562 cells. FIG. 17 shows killing assay results based on co-cultivation of anti-PRLR CAR T cells and PRLR-K562 cells. FIG.18 showskillingassay results basedonco-cultivationofPRLR ligandCARTcells andPRLR-K562cells. Ligand1 indicates humanwild-type prolactin receptor ligand (SEQ IDNO: 20), and ligand2 indicatesmodified humanprolactin receptor ligand (SEQ ID NO: 44). FIG. 19 is an image illustrating fluorescent signals observed from3t3 cells expressing TSHRand fluorescent proteins (RFP). FIG. 20 shows killing assay results based on co-cultivation of anti-TSHR CAR T cells and TSHR‑3T3 cells. FIG. 21 is an image illustrating fluorescent signals observed from3t3 cells expressing PRLRand fluorescent proteins (RFP). FIG. 22 shows killing assay results based on co-cultivation of anti-PRLR CAR T cells and PRLR‑3T3 cells. FIG. 23 shows killing assays results based on co-cultivation of PRLR ligand CAR T cells and PRLR‑3T3 cells. DETAILED DESCRIPTION

[0046] The present disclosure relates to compositions and methods for treating cancer among other diseases. The 5 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 embodiments of the present disclosure include constructing a CAR including an antigen binding domain that binds to an antigencorresponding toa target gene.The target gene is specifically expressed inacertain tissue (e.g., agroupof cells or anorgan) or expressed in the tissuemore than expressed in other tissues. In someembodiments, theCAR is expressed in modifiedcells (e.g., T-cells orNKcells),whichareadministrated toasubject havinga tumorderived fromcells of the tissue. Because theantigen isexpressed in the tumor, themodifiedcellsmay identify and thencause these tumor cells tobekilled. Themodified cells are able to replicate and expand in vivo, and therefore are present long-term in the body of the subject, leading to sustained tumor control. In these instances, non-tumor cells that express the antigen may also be killed or damagedby the transferredCART / NKcells. However, since the tissue is selected fromnon-essential tissueswith respect to the subject, the killing of normal cells of the tissue does not cause a life-threatening event (e.g., complications) to the subject. Examples of the non-essential tissues include organs such as prostate, breast, or melanocyte.

[0047] In someembodiments, antigensselected forCARsaremainlyexpressed in cancer cells and their primaryorgans (e.g., non-essential organs). In certain embodiments, a non-essential organ may be removed, before treating of CAR T cells, from a subject who has cancer in an advanced stage derived from the non-essential organ. For example, treatment for stage IV thyroid cancer is usually a combination of treatment techniques including surgery and radioactive iodine treatment. In these instances, the entire thyroid is removed, and procedure is called a total thyroidectomy. Since selected antigensaremainly expressed in thyroidnormal cells and thyroid cancer cells, the risk ofCARTcells attackingnormal cells is reduced.

[0048] Generally, treatment of breast cancer includesa topical therapyandsystemic therapy.Topical treatment includes mastectomyand radiotherapy,whichbringpatients great sufferingandareonly available for treating breast cancer in early stages.Systemic treatment includes chemotherapy, endocrine therapy, and targetedmolecular therapies.Chemotherapy causes patients great pain, and its efficacy is poor. Endocrine therapy mainly applies to postmenopausal women. Molecular targeted therapy is one of themost active areas of research in recent years. However, its efficiency is relatively low.Forexample, recurrence ratesof typicalHER2monoclonal antibodyHerceptinmonotherapy in the treatmentof breast cancer are 15% to 30%.

[0049] Ovarian cancer is one of the common malignant tumors of female genital mutilation. Among ovarian cancer, epithelial cancer and malignant germ cell tumors are common. Most epithelial ovarian tumors will spread to the uterus, bilateral annex, omentum and pelvic organs, causing a serious threat to women’s lives. Different pathological types of ovarian cancer have different treatment options. Therapies combined with surgery and chemotherapy may be used for treating ovarian cancer. For ovarian cancer in early stages, surgeries may be available and include comprehensive surgery and preserving fertility surgery. Because the complexity of ovarian embryonic development, tissue anatomy, and endocrine functions, symptoms for early stages are not typical; therefore, diagnosis of benign and malignant as well as tissue types is quite difficult. In the caseof pelvic tumormetastasis, especiallymalignant germcell tumors of patients in late stages,most of thesepatients cannot remove the lesion. In these instances, radiation andchemotherapy canonly beused as adjunctive therapy, and their effect is limited.

[0050] Endometrial cancer occurs in a group of endometrial tumors, also called endometrial cancer. Tumors originated in endometrial glands are known as endometrial adenocarcinoma. These cancer cells mainly spread directly or via lymphatic metastasis, while for those in late stages, the cancer cells may spread to the lung, liver, and bone via blood metastasis. Treatment plans may be decided based on clinical stages, levels of tumor differentiation, and general conditions of patients. Generally, the treatment includes surgical treatment as well as radiotherapy, hormone, and chemotherapy. Conservative surgery may cause a relapse, while radical resection may cause symptoms of premature menopause. Radiation and chemotherapy can only be used as adjunctive therapies, and their effect is limited. Progestin therapy is effective using progesterone drugs to control the development of cancer, but the dosage is large, and long-term progestin therapies may impair liver functions. For patients with advanced tumor metastases, no effective treatment is available.

[0051] Cervical cancer is the most common form of female genital malignancies, including carcinoma in situ and invasive carcinoma. Cervical cancer is limited to the mucosa within the cortex, known as carcinoma in situ when no infiltration. When cancer stromal invasion is under mucous membranes, it is called invasive cancer. Cervical cancer can cause infertility in women. Since invasion and metastasis of advanced cancer, the symptomsmay appear corresponding parts of the body as well. Generally, treatment plans are decided based on clinical stages, levels of tumor differentiation, and general conditions of patients. Generally, the treatment includes surgical treatment, as well as radiotherapy and chemotherapy as adjuvant therapy. Surgical treatment includes Radical hysterectomy and pelvic lymph node surgery Elimination, and there are recurrence risks. Radiation and chemotherapy can only be used as adjunctive therapies, and their effect is limited. For patients with advanced tumor metastases, currently, no effective treatment is available.

[0052] Thyroid cancer is the most common thyroid malignancy. There are four common thyroid cancer types: papillary (e.g., mixed papillary-follicular carcinoma), follicular, medullary (e.g., an entity with amyloidosis thyroid tumors) and undifferentiated carcinoma. Generally, treatment of papillary and follicular carcinoma is relatively effective, while medullary carcinoma isoften transferredalong lymphatic andblood roadsandundifferentiated carcinoma is lesscommon. Advanced thyroid cancer may produce hoarseness, breathing, difficulty swallowing, and Horner syndrome and sympa- 6 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 thetic nerve compression caused by cervical plexus violations occur ear, pillows, shoulder pain, etc. and regional lymph nodes and distant metastasis, etc. There is a need for improving the level of prognosis and treatment of thyroid cancer. Currentmodes of treatment for thyroid cancer include surgery, postoperative radionuclide therapy, and endocrine therapy after surgery, wherein the surgical treatment is the first choice. In choices of surgery methods for treating differentiated thyroid cancer, many aspects of postoperative radioiodine treatment and TSH (thyroid stimulating hormone, TSH) suppression therapy, etc. have controversy. Also, there are no unified and standardized treatment guidelines, resulting in incomplete or excessive treatment. For advanced differentiated thyroid cancer, there is no effective treatment.

[0053] Colorectal cancer is a type of common gastrointestinal cancer, a serious threat to life and health. There is a need for improving the level of prognosis and treatment of colorectal cancer. Generally, clinical treatment programs for colorectal cancer include local excision, chemotherapy, and biologic therapy. The use of local excision of metastatic lesions: the standard of biological disease (example: synchronization and anisotropy) is important but difficult to be evaluated. For example, 75% of patients will relapse after liver metastases resection of the disease. Most of the relapsing occurs in livers, and its efficacy is limited. Commonly used chemotherapy regimens include FOLFOX, FOLFIRI, CapeOX, and FOLFOXIRI (2B), and other, However, combined with FOLFOX regimen 7% ‑8% of patients can extend 3-year PFS, while overall survival rates are not significant. Recent experimental data suggests that adding cetuximab to FOLFOX application for possible transfer patientswith resectable lesions is harmful. Based on the current research data, combined with biological agents (e.g., bevacizumab, cetuximab, and panitumumab) treatment with these drugs have been recommended.

[0054] However, monoclonal antibody drugs generally have problems such as efficacy and persistence. Patients need long-term, repeated drug uses. At the same time, due to the combined treatment methods are generally stronger than monotherapy; therefore, whether other types of drugs will affect treatment and prognosis is still a question.

[0055] Stomach cancer (gastric cancer) is amalignant gastric epithelial origin, one of themost commongastrointestinal malignancies. Pain andweight loss are themost common clinical symptoms of advanced gastric cancer. Proliferation and metastasis pathway of gastric cancer cells include lymphatic metastasis, direct invasion, hematogenousmetastasis, and peritoneal metastasis. There is a need for improving the level of prognosis and treatment of gastric cancer. Generally, gastric cancer treatment modes include surgery, chemotherapy, radiotherapy, immunotherapy, etc. But most cases of advanced gastric cancer have unresectable primary or metastatic lesions. Tumor metastasis and recurrence of gastric cancer are the leading causes of poor prognosis

[0056] Esophageal cancer (esophageal cancer, EC) is originated in the esophageal mucosal epithelial malignancies and is a commonclinicalmalignant tumor. Tumormetastasis and recurrenceof esophageal cancer are the leading causes of poor prognosis. Due to incidence andmortality, esophageal cancer is among the top ten inmalignant tumors. There is a need for improving the level of prognosis and treatment of esophageal cancer. The current mode of treatment of esophageal cancer is a surgical combined therapy. There are problems in the staging of esophageal cancer, surgical treatment mode selection, surgical approach selection, lymphadenectomy way, and postoperative adjuvant therapy, etc.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understoodby thoseof ordinary skill in the art towhich thedisclosurebelongs.Althoughanymethodsandmaterials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferredmethods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

[0058] Thearticles "a" and "an" are usedherein to refer to oneor tomore thanone (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0059] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0060] The term "activation," as used herein, refers to the state of a Tcell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers to, among other things, T cells that are undergoing cell division.

[0061] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full- length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. The antibodies of the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies andhumanizedantibodies (Harlowet al., 1999, In:UsingAntibodies:A LaboratoryManual, ColdSpringHarbor LaboratoryPress,NY;Harlowet al., 1989, In:Antibodies:ALaboratoryManual, ColdSpringHarbor,NewYork;Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879‑5883; Bird et al., 1988, Science 242:423‑426).

[0062] "Antibody fragments" compriseaportionof a full-lengthantibody, generally theantigen-bindingor variable region of the antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0063] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and - binding site. This fragment consists of a dimer of one heavy‑ and one light-chain variable region domain in tight, non-covalent association. 7 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, evenasingle variable domain (or half of aFv includingonly three complementarity determining regions (CDRs) specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two major antibody light chain isotypes.

[0064] By the term, "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0065] The term "antigen" as used herein is defined as a molecule that provokes an immune response, which may involve either antibody production, or the activation of specific immunologically competent cells, or both. Antigens may include anymacromolecule, including virtually all proteins or peptides, ormolecules derived from recombinant or genomic DNA. For example, DNA including a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response, therefore, encodes an "antigen" as that term is used herein. Furthermore, an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. Further, an antigen can be generated, synthesized or derived from a biological sample including a tissue sample, a tumor sample, a cell or a biological fluid.

[0066] The term "anti-tumor effect" as used herein, refers to a biological effect associated with a decrease in tumor volume, adecrease in thenumber of tumor cells, a decrease in thenumber ofmetastases, an increase in life expectancyof a subject having tumor cells, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the disclosure in the prevention of the occurrence of tumor in the first place.

[0067] The term "autoantigen" refers to anantigenmistakenly recognizedby the immunesystemasbeing foreign. Auto- antigens include cellular proteins, phosphoproteins, cellular surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0068] The term "autologous" is used to describe amaterial derived from the same individual to which it is later to be re- introduced into the individual.

[0069] "Allogeneic" is used to describe agraft derived fromadifferent animal of the samespecies. "Xenogeneic" is used to describe a graft derived from an animal of a different species.

[0070] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstreamand lymphatic system to other parts of the body. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer et al.

[0071] Throughout this specification, unless the context requires otherwise, the words "comprise," "includes" and "including" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0072] By "consistingof" ismeant including, and limited to,whatever follows thephrase "consistingof." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory and that no other elements may be present.

[0073] By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements areoptional andmayormaynot bepresent depending uponwhether or not theyaffect the activity or action of the listed elements.

[0074] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0075] By "corresponds to" or "corresponding to" is meant (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence of a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.

[0076] "Co-stimulatory ligand," includes amolecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, et 8 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 al.) that specifically binds a cognate co-stimulatorymolecule on a Tcell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including proliferation, activation, differentiation, et al. A co-stimulatory ligand can include CD7, B7‑1 (CD80), B7‑2 (CD86), PD-L1, PD-L2, 4‑1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM),CD30L,CD40,CD70,CD83,HLA-G,MICA,MICB,HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligandalsoencompasses, inter alia, anantibody that specifically binds to aco-stimulatorymolecule present on a T cell, such as CD27, CD28, 4‑1BB, OX40, CD30, CD40, PD‑1, ICOS, lymphocyte function associated antigen‑1 (LFA‑1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0077] A "co-stimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as proliferation. Co-stimulatory molecules include an MHC class I molecule, BTLA, and a Toll-like receptor.

[0078] A "co-stimulatory signal" refers to a signal, which in combinationwith aprimary signal, suchasTCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0079] As used herein, the terms "disease" and "condition" may be used interchangeably or may be different in that the particularmalady or conditionmay not have a known causative agent (so that etiology has not yet beenworked out), and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians. As used herein, a "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate. In contrast, a "disorder" in a subject is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0080] As used herein, the term "effective" means adequate to accomplish a desired, expected, or intended result. For example, an "effective amount" may be an amount of a compound sufficient to produce a therapeutic or prophylactic benefit.

[0081] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA andmRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequenceofwhich is identical to themRNAsequenceand is usually provided in sequence listings, and thenon- coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0082] With regard to polynucleotides, the term "exogenous" refers to a polynucleotide sequence that does not naturally occur in awild-type cell or organismbut is typically introduced into the cell bymolecular biological techniques.Examples of exogenous polynucleotides include vectors, plasmids, and / or man-made nucleic acid constructs encoding the desired protein. With regard to polynucleotides, the term "endogenous" or "native" refers to naturally-occurring polynucleotide sequences that may be found in a given wild-type cell or organism. Also, a particular polynucleotide sequence that is isolated from a first organism and transferred to the second organism by molecular biological techniques is typically considered an "exogenous" polynucleotidewith respect to the second organism. In specific embodiments, polynucleotide sequences can be "introduced" by molecular biological techniques into a microorganism that already contains such a polynucleotide sequence, for instance, to create one or more additional copies of an otherwise naturally-occurring polynucleotide sequence, and thereby facilitate overexpression of the encoded polypeptide.

[0083] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0084] "Expression vector" refers to a vector including a recombinant polynucleotide including expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) andviruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinant polynucleotide.

[0085] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared×100. For example, if 6 of 10 of the positions in two sequencesarematchedor homologous then the two sequencesare 60%homologous. Byway of example, theDNAsequencesATTGCCandTATGGCshare50%homology.Generally, a comparison ismadewhen two 9 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 sequences are aligned to give maximum homology.

[0086] The term "immunoglobulin" or "Ig," refers to a class of proteins, which function as antibodies. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is themost commoncirculatingantibody. IgM is themain immunoglobulin produced in theprimary immune response inmost subjects. It is themost efficient immunoglobulin in agglutination, complement fixation, andother antibody responses,and is important indefenseagainstbacteriaandviruses. IgD is the immunoglobulin that hasnoknown antibody function but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensi- tivity by causing the release of mediators from mast cells and basophils upon exposure to the allergen.

[0087] By "isolated" is meant a material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide," as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and / or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell.

[0088] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0089] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes aprotein or anRNAmayalso include introns to theextent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0090] A "lentivirus" as used herein refers to a genus of the retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer themeans to achieve significant levels of gene transfer in vivo.

[0091] By the term "modulating," as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0092] The nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressedasapreprotein that participates in the secretionof thepolypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positionedsoas to facilitate translation.Generally, "operably linked"means that theDNAsequencesbeing linked are contiguous, and, in the caseof a secretory leader, contiguous and in reading phase.However, enhancers donot have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0093] The term "overexpressed" tumor antigen or "overexpression" of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organof the patient relative to the level of expression in anormal cell from that tissueor organ.Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0094] "Parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intracisternal injection, or infusion techniques.

[0095] The terms "patient," "subject," "individual," et al. areused interchangeablyherein, and refer toanyanimal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is ahuman. In someembodiments, the term "subject" is intended to include livingorganisms in which an immune response canbeelicited (e.g.,mammals). Examples of subjects include humans, dogs, cats,mice, rats, and transgenic species thereof.

[0096] The recitation "polynucleotide" or "nucleic acid" as used herein designatesmRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA and RNA.

[0097] The terms "polynucleotide variant" and "variant" and the like refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize to a reference sequence 10 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides that are distin- guished froma referencepolynucleotideby theaddition, deletionor substitutionof at least onenucleotide.Accordingly, the terms "polynucleotide variant" and "variant" include polynucleotides inwhich oneormore nucleotides havebeenadded or deleted, or replacedwith different nucleotides. In this regard, it iswell understood in theart that certain alterations inclusive of mutations, additions, deletions, and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide or has increased activity in relation to the reference polynucleotide (i.e., optimized). Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages in between, e.g., 90%, 95%, or 98%) sequence identity with a reference polynucleotide sequence described herein. The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants and orthologs that encode these enzymes.

[0098] "Polypeptide," "polypeptide fragment," "peptide" and "protein" are used interchangeably herein to refer to a polymerofaminoacid residuesand tovariantsandsyntheticanaloguesof thesame.Thus, these termsapply toaminoacid polymers inwhich one ormore amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In certain aspects, polypeptides may include enzymatic polypeptides, or "enzymes," which typically catalyze (i.e., increase the rate of) various chemical reactions.

[0099] The recitation polypeptide "variant" refers to polypeptides that are distinguished from a reference polypeptide sequenceby theaddition, deletionor substitutionofat least oneaminoacid residue. In certainembodiments, apolypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non- conservative. In certain embodiments, the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.

[0100] The term "promoter" as used herein is defined as aDNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0101] The term "bind," "binds," or "interacts with" means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. By the term "specifically binds," as used herein with respect to an antibody, ismeant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen fromone speciesmay also bind to that antigen fromone ormore species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigenmay also bind to different allelic forms of the antigen. However, such cross-reactivity doesnot itself alter the classification of anantibodyas specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A," the presence of a molecule containing epitopeA (or free, unlabeledA), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

[0102] A "soluble receptor" is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors may include additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have naturally occurred, soluble counterparts that are produced by proteolysis. Soluble receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.

[0103] By "statistically significant," it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p- value,which is the frequencyor probabilitywithwhich theobservedeventwouldoccur if thenull hypothesiswere true. If the obtainedp-value is smaller than thesignificance level, then thenull hypothesis is rejected. In simplecases, thesignificance level is defined at a p-value of 0.05 or less. A "decreased" or "reduced" or "lesser" amount is typically a "statistically significant" or a physiologically significant amount, andmay include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or level described herein. 11 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55

[0104] By the term "stimulation," is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand therebymediating a signal transduction event, such as signal transduction via theTCR / CD3 complex. Stimulation canmediate altered expression of certainmolecules, such as downregulation of TGF- β, and / or reorganization of cytoskeletal structures et al.

[0105] A "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

[0106] A "stimulatory ligand" refers to a ligand that when present on an antigen presenting cell (e.g., anAPC, a dendritic cell, aB-cell, et al.) can specifically bindwith a cognate binding partner (referred to herein as a "stimulatorymolecule") on a T cell, thereby mediating a primary response by the T cell, including activation, initiation of an immune response, proliferation, et al. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0107] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to a cell that has been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0108] The term "therapeutic" as used hereinmeans a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0109] The term "therapeutically effective amount" refers to the amount of the subject compound that will elicit the biological or medical response of tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or another clinician. The term "therapeutically effective amount" includes that amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to someextent, one ormore of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0110] To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0111] The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferredor introduced into thehost cell. A "transfected" or "transformed" or "transduced" cell is onewhich has been transfected, transformed or transduced with exogenous nucleic acid The cell includes the primary subject cell and its progeny.

[0112] Thephrase "under transcriptional control" or "operatively linked" as usedhereinmeans that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0113] A "vector" is a composition of matter which includes an isolated nucleic acid andwhich can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, et al. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors et al. For example, lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV‑1, HIV‑2, and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by attenuating theHIV virulence genes, for example, the genes env, vif, vpr, vpu, and nef are deleted making the vector biologically safe.

[0114] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges suchas from1 to 3, from1 to 4, from1 to 5, from2 to 4, from2 to 6, from3 to 6 etc., aswell as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0115] The present disclosure relates to isolated nucleic acid sequences, vectors including the isolated nucleic acid sequences, cells including the isolated nucleic acid sequences and methods of treating cancer using these cells. Compositions and Therapeutic Application

[0116] Some embodiments of the present disclosure relate to an isolated nucleic acid sequence encoding a CAR. The 12 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 CARmay include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domainmay bind to an antigen of a non-essential organ. For example, the antigen binding domain binds to an antigen that is expressed on the surface of a non-essential organ cell present in a microenvironment of a tumor.

[0117] As used herein, a non-essential organ refers to an organ of a subject, and the organ is non-essential such that an injury of the organ does not produce death of the subject. In some embodiments, an injury of the organ does not visibly affect the subject’s health. For example, the prostate may be a non-essential organ for amalemammal, while breast may be a non-essential organ for a female mammal.

[0118] In certain embodiments, a non-essential organ may be removed, before treating of CAR Tcells, from a subject who has cancer in an advanced stage derived from the non-essential organ. In these instances, the impact of treating of CAR T cells targeting antigens of the non-essential organ on the subject is substantially reduced. Examples of non- essential organs include a mammary gland, a colon, a gastric gland, an ovary, and a thyroid.

[0119] Some embodiments of the present disclosure relate to an isolated CAR including an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.

[0120] Some embodiments of the present disclosure relate to a pharmaceutical composition including human Tcells. The human T cells may include a nucleic acid sequence encoding a CAR. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.

[0121] Some embodiments of the present disclosure relate to a cell including a nucleic acid sequence encoding aCAR. TheCARmay include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domain binds to an antigen of a non-essential organ. For example, the cell is selected from the group consisting of a Tcell, a natural killer (NK) cell, NK‑92 cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0122] Some embodiments of the present disclosure relate to a vector comprising a nucleic acid sequence encoding a CAR.TheCARmay include anantigenbindingdomain, a transmembranedomain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ.

[0123] Some embodiments of the present disclosure relate to a method for stimulating a T cell-mediated immune response to a cell population in a non-essential organ of a subject. Themethodmay include administering to a subject an effective amount of a cell genetically modified to express a CAR. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and aCD3 zeta signaling domain. The antigen binding domain is selected to recognize the cell population of the non-essential organ specifically.

[0124] In someembodiments, theTcellsmaybemodified to haveadisruption in an endogenousgene associatedwith a biosynthesis or transportation pathway of one or more proteins. Examples of the one or more proteins include Programmed cell death protein 1 (PD‑1) gene and Major Histocompatibility Complex I (MHC I). For example, Major Histocompatibility Complex of T cells may be disrupted by modification of beta‑2-microglobulin (B2M) gene, antigen presentation 1 (TAP1) gene, and TAP-associated glycoprotein (TAPBP) gene in the Tcells. In certain embodiments, the disruption may be introduced into a Tcell before or after the Tcell is transferred with a nucleic acid sequence encoding a CAR.

[0125] Some embodiments of the present disclosure relate to a method of treating a subject with cancer. The method may include administering to the subject a cell genetically engineered to express aCAR. TheCARmay include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain. The antigen binding domain may bind to an antigen of a non-essential organ. For example, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, NK‑92 cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0126] CARs are molecules generally including an extracellular and intracellular domain. The extracellular domain includes a target-specific binding element. The intracellular domain (e.g., cytoplasmic domain) includes a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR including the intracellular domain of a costimulatorymolecule. Costimulatory molecules are cell surfacemolecules other than antigens receptors or their ligands that are required for an efficient response of lymphocytes to antigen.

[0127] Between the extracellular domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term "spacer domain" generally means any oligo‑ or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain of the polypeptide chain. A spacer domain may include up to 300 amino acids, preferably 10 to 100 amino acids, andmost preferably 25 to 50 amino acids.

[0128] In some embodiments, the target-specific binding element of the CAR in the present disclosuremay recognize a tumor antigen. Tumor antigens areproteins that are producedby tumor cells that elicit an immune response, particularly T- cell mediated immune responses. Tumor antigens arewell known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, 13 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 thyroglobulin,RAGE‑1,MN-CA IX, human telomerase reverse transcriptase,RU1,RU2 (AS), intestinal carboxylesterase, mut hsp70‑2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO‑1, LAGE‑1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate carcinoma tumor antigen‑1 (PCTA‑1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)‑I, IGF-II, IGF-I receptor and mesothelin.

[0129] In some embodiments, the tumor antigen includes HER2, CD19, CD20, CD22, Kappa or light chain, CD30, CD33, CD123, CD38, ROR1, ErbB3 / 4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL‑13 receptor α 2, IL‑11 receptor α, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO‑1, PSC1, folate receptor-α, CD44v7 / 8, 8H9, NCAM, VEGF receptors, 5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, TEM8, or viral-associated antigens expressed by the tumor.

[0130] In some embodiments, the binding element of theCARmay include any antigen bindingmoiety that when bound to its cognate antigen, affects a tumor cell such that the tumor cell fails to grow, or is promoted to die or diminish.

[0131] In some embodiments, the antigen binding element of the CAR of the disclosure targets an antigen of a non- essential organ. In some instances, the antigen binding element of the CAR of the disclosure includes anti-antigen including the amino acid sequence set forth at least at one of SEQ ID NOs: 2‑20 or 44.

[0132] In some embodiments, internal ribosome entry sites (IRES) elements are used to create multigene, or poly- cistronic, ormessages. For example, an IRESelementmay link a nucleic acid sequence encodingCARand anucleic acid sequence encoding one of the various antigens (See FIG. 1 and Table 2). In other embodiments, other tools such as 2A may be used to create multigene, or polycistronic, or messages.

[0133] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0134] The embodiments of the present disclosure further relate to vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0135] The expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to one or more promoters and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0136] In some embodiments, the antigen binding domain is an antibody, an antigen-binding fragment thereof, or a ligand thereof. For example, the antigen-binding fragment is a Fab or a scFv.

[0137] In someembodiments, the antigen is expressedonanon-essential organ cell present in amicroenvironment of a tumor.

[0138] In some embodiments, the tumor is a breast cancer. In certain embodiments, the antigen is a mammary gland antigen. For example, the mammary gland antigen is prolactin receptor (PRLR) having SEQ ID NO: 29.

[0139] As used herein, "a mammary gland antigen" refers to an antigen expressed on or by a mammary gland cell. Examples of mammary gland cells include catheter epithelial cells / foam cells.

[0140] The prolactin receptor (PRLR) is involved in the growth and differentiation of various cells. Prolactin receptors have been identified in a number of cells and tissues, including the mammary gland, organs of the reproductive system, central nervous system, pituitary, adrenal cortex, skin, bone, lung, heart, liver, pancreas, GI tract, kidney and lymphoid tissues. Human growth hormone (hGH), human prolactin (hPRL) and human placental lactogen (hPL) all specifically bind the prolactin receptor with high affinity.

[0141] As used herein, "PRLR" refers to human prolactin receptor. The term should be construed to include not only human prolactin receptor but variants, homologs, fragments and portions thereof to the extent that such variants, homologs, fragments and portions thereof retain the ability of prolactin receptor to bind to antibodies or ligands of human prolactin receptor as disclosed herein.

[0142] In some embodiments, the nucleotide sequence encoding at least a portion of the human prolactin receptor is shown in SEQ ID NO: 28 and at least a portion of the human prolactin receptor are shown in SEQ ID NO: 29.

[0143] In someembodiments, thepresent disclosure isparticularlywell-suited todeliver agents tocells thatoverexpress the prolactin receptor differentially. As used herein, a prolactin receptor is "overexpressed" when it is present on the surfaceofacell (e.g., amammaryglandcell) inanamount that is statistically significantly greater thanasuitable control cell (e.g., a brain cell). In someembodiments, the prolactin receptor is present on the cell surfaceat least about 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold or 1000-fold greater than a suitable control cell. For example, over 80% of breast tumors overexpress the prolactin receptor by as much as 10‑ to 1000-fold over normal breast tissue. Accordingly, some 14 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 embodiments of the present disclosure are particularly suitable for delivery of an agent (e.g., CAR T / NK cells) to breast cancer cells of a subject. In these instances, non-tumor cells that express the prolactin receptor may also be killed by the transferredCART / NK cells. However, if themammary gland is non-essential tissueswith respect to the subject, the killing of normal cells of the mammary gland does not cause a life-threatening event (e.g., complications) to the subject.

[0144] In some embodiments, the antigen binding domain is prolactin receptor ligand having SEQ ID NO: 20 or 44.

[0145] A prolactin receptor ligand, as used herein, refers to an entity that binds the prolactin receptor, regardless of whether downstream biological effects of prolactin receptor binding are observed. As will be appreciated, no particular level of binding specificity is required, and acceptable levels of specificity will depend on the application. Suitable prolactin receptor ligands include human placental lactogen and variants thereof (including truncated or modified forms), prolactin and variants thereof (including truncated or modified forms) and human growth hormone (hGH) (including truncated or modified forms). Selection of a suitable prolactin receptor ligand will depend on, e.g., the agent to be delivered, the coupling strategy to be used, and the degree of receptor activation desired, if any.

[0146] In some embodiments, at least a portion of prolactin receptor ligand is shown in SEQ ID NO: 20 or 44.

[0147] In someembodiments, the tumor is colorectal cancer. In certain embodiments, the antigen is a colon antigen. For example, the colon antigen is Guanylate cyclase 2C (GUCY2C) having SEQ ID NO: 33.

[0148] As used herein, "a colon antigen" refers to an antigen expressed on or by a colon cell. Examples of colon cells include goblet cells and enterocytes.

[0149] Guanylyl cyclase 2C (GUCY2C) is principally expressed in intestinal epithelial cells. GUCY2C is the receptor for diarrheagenic bacterial enterotoxins (STs) and the gut paracrine hormones, guanylin, and uroguanylin. These ligands regulate water and electrolyte transport in the intestinal and renal epithelia and are ultimately responsible for acute secretory diarrhea.

[0150] As used herein, "GUCY2C" refers to human Guanylyl cyclase 2C. The term should be construed to include not only humanGuanylyl cyclase 2C, but variants, homologs, fragments and portions thereof to the extent that such variants, homologs, fragmentsandportions thereof retain theabilityofGuanylyl cyclase2C tobind toantibodiesor ligandsofhuman Guanylyl cyclase 2C as disclosed herein.

[0151] In some embodiments, the nucleotide sequence encoding at least a portion of GUCY2C is shown in SEQ IDNO: 32 and the amino acid sequence of at least a portion of GUCY2C are shown in SEQ ID NO: 33.

[0152] In someembodiments, thepresent disclosure isparticularlywell-suited todeliver agents tocells thatoverexpress theGUCY2Cdifferentially. Asusedherein, aGUCY2C is "overexpressed"when it is present on the surfaceof a cell (e.g., a colon cell) in an amount that is statistically significantly greater than a suitable control cell (e.g., a brain cell and a pancreas cell). In some embodiments, theGUCY2C is present on the cell surface at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 100- fold or 1000-fold greater than a suitable control cell. Accordingly, some embodiments of the present disclosure are particularly suitable for delivery of anagent (e.g., CART / NKcells) to colorectal cancer cells. In these instances, non-tumor cells that express theGUCY2Cmayalsobekilledby the transferredCART / NKcells.However, if the colon is non-essential tissues with respect to the subject, the killing of normal cells of the colon does not cause a life-threatening event (e.g., complications) to the subject.

[0153] In someembodiments, the tumor is gastric cancer. In certainembodiments, theantigen isagastric glandantigen. For example, the gastric gland antigen is Mucin 17 (Muc17) having SEQ ID NO: 31.

[0154] Asusedherein, "a gastric glandantigen" refers to anantigen expressedonor by agastric gland cell. Examples of gastric gland cells include gastric parietal cells, mucous cells, and surface epithelial cells.

[0155] Muc17 is a type 1 membrane protein comprising 4,493 amino acids. Muc17 belongs to the membrane-form mucin family, andmost part of its extracellular domain comprises a tandem repeat of a serine‑, threonine‑, and proline-rich 59-mer sequence and is glycosylated.

[0156] As used herein, "Muc17" refers to human Mucin 17. The term should be construed to include not only human Mucin17but variants, homologs, fragmentsandportions thereof to theextent that suchvariants, homologs, fragmentsand portions thereof retain the ability of Mucin 17 to bind to antibodies or ligands of human Mucin 17 as disclosed herein.

[0157] In someembodiments, the nucleotide sequence encoding at least a portion ofMuc17 is shown in SEQ IDNO: 30 and the amino acid sequence of at least a portion of Muc17 are shown in SEQ ID NO: 31.

[0158] In someembodiments, thepresent disclosure isparticularlywell-suited todeliver agents tocells thatoverexpress the Muc17 differentially. As used herein, a Muc17 is "overexpressed" when it is present on the surface of a cell (e.g., a gastric gland cell) in an amount that is statistically significantly greater than a suitable control cell (e.g., a brain cell and a pancreas cell). In some embodiments, the Muc17 is present on the cell surface at least about 1.5-fold, 2-fold, 5-fold, 10- fold, 100-fold or 1000-fold greater than a suitable control cell. Accordingly, some embodiments of the present disclosure are particularly suitable for delivery of an agent to gastric cancer cells. In these instances, non-tumor cells that express the Muc17 may also be killed by the transferred CAR T / NK cells. However, if the gastric gland is non-essential tissues with respect to the subject, the killing of normal cells of the colon does not cause a life-threatening event (e.g., complications) to the subject.

[0159] In some embodiments, the tumor is a bladder cancer. In certain embodiments, the antigen is a bladder antigen. 15 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 For example, the bladder antigen is CD207 having SEQ ID NO: 35.

[0160] As used herein, "a bladder antigen" refers to an antigen expressed on or by a bladder cell. Examples of bladder cells include transitional cells and mucosal epithelial cells.

[0161] CD207 (langerin or Cluster of Differentiation 207) is a protein which in humans is encoded by the CD207 gene. CD207 is a type II transmembrane,C-type lectin receptor onLangerhans cells. CD207 is localized in theBirbeck granules, organelles present in the cytoplasm of Langerhans cells and including superimposed and zippered membranes.

[0162] As used herein, "CD207" refers to human CD207. The term should be construed to include not only human CD207 but variants, homologs, fragments and portions thereof to the extent that such variants, homologs, fragments and portions thereof retain the ability of human CD207 to bind to antibodies or ligands of human CD207 as disclosed herein.

[0163] In someembodiments, thenucleotidesequenceencodingat least aportionof thehumanCD207 isshown inSEQ ID NO: 34 and the amino acid sequence of at least a portion of the human CD207 are shown in SEQ ID NO: 35.

[0164] In someembodiments, thepresent disclosure isparticularlywell-suited todeliver agents tocells thatoverexpress the CD207 differentially. As used herein, a CD207 is "overexpressed" when it is present on the surface of a cell (e.g., a bladder cell) in anamount that is statistically significantly greater thana suitable control cell (e.g., an endocrine tissue cell). In some embodiments, the CD207 is present on the cell surface at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold or 1000-fold greater than a suitable control cell. Accordingly, some embodiments of the present disclosure are particularly suitable for delivery of an agent to bladder cancer cells. In these instances, non-tumor cells that express the CD207 may alsobekilledby the transferredCART / NKcells.However, if thebladder is non-essential tissueswith respect to thesubject, the killing of normal cells of the colon does not cause a life-threatening event (e.g., complications) to the subject.

[0165] In some embodiments, the tumor is an ovary tumor. In certain embodiments, the antigen is an ovary antigen. For example, the ovary antigen is Frizzled family receptor 10 (FZD10) having SEQ ID NO: 25.

[0166] As used herein, "an ovary antigen" refers to an antigen expressed on or by an ovary cell. Examples of ovary cells include follicular cells, granulosa cells, and germinal epithelium.

[0167] A human Fz gene family member, Frizzled‑10 (FZD10), has been cloned and characterized. Analysis of the FZD10 nucleotide sequence showed that the human FZD10 gene encodes a seven-transmembrane-receptor of 581 amino acids, including an amino-terminal cysteine-rich domain and a carboxy-terminal Ser / Thr-Xxx-Val motif. FZD10- encoding mRNA (4.0 kb) was detected in placenta, fetal kidney, fetal lung, and brain. In adult brain, FZD10 mRNA was abundant in the cerebellum. The FZD10 gene was mapped to human chromosome 12q24.33. FZD10 shares 65.7% amino-acid identity with Frizzled‑9 (FZD9). FZD10 and FZD9 constitute a subfamily of the Frizzled genes. FZD10 is the receptor for the Wnt ligand proteins WNT7a and WNT7b. There is 93% identity between mouse and human FZD10.

[0168] As used herein, "FZD10" refers to human FZD10. The term should be construed to include not only human FZD10 but variants, homologs, fragments and portions thereof to the extent that such variants, homologs, fragments and portions thereof retain the ability of human FZD10 to bind to antibodies or ligands of human FZD10 as disclosed herein.

[0169] In someembodiments, thenucleotide sequenceencodingat least aportionof thehumanFZD10 is shown inSEQ ID NO: 24 and the amino acid sequence of at least a portion of the human FZD10 are shown in SEQ ID NO: 25.

[0170] In someembodiments, thepresent disclosure isparticularlywell-suited todeliver agents tocells thatoverexpress the FZD10 differentially. As used herein, an FZD10 is "overexpressed" when it is present on the surface of a cell (e.g., an ovary cell) in anamount that is statistically significantly greater thanasuitable control cell (e.g., an endocrine tissue cell). In some embodiments, the FZD10 is present on the cell surface at least about 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold or 1000-fold greater than a suitable control cell. Accordingly, some embodiments of the present disclosure are particularly suitable for delivery of anagent to ovary cancer cells. In these instances, non-tumor cells that express theFZD10mayalso be killed by the transferred CAR T / NK cells. However, if the ovary is non-essential tissues with respect to the subject, the killing of normal cells of the colon does not cause a life-threatening event (e.g., complications) to the subject.

[0171] In some embodiments, the tumor is a thyroid tumor. In certain embodiments, the antigen is a thyroid antigen. For example, the thyroid antigen is Thyroid stimulating hormone receptor (TSHR) having SEQ ID NO: 27.

[0172] Asusedherein, "a thyroid antigen" refers toanantigenexpressedonor bya thyroid cell. Examplesof thyroid cells include follicular cells and parafollicular cells.

[0173] A human TSHR is a receptor for thyroid-stimulating hormone (TSH) which is present on the thyroid membrane. WhenTSHsecreted from thepituitaryglandbinds toTSHRon the thyroid follicle cellmembrane, the thyroidglandsecretes T3 and T4 having metabolic functions. TSHR is a seven-transmembrane receptor having a molecular weight of about 95,000 to 100,000. It was reported that the human thyrotropin receptor (TSHR) includes three domains: a leucine-rich domain (LRD; amino acids 36‑281), a cleavage domain (CD; amino acids 282‑409), and transmembrane domain (TMD; aminoacids 410‑699).Human thyrotropin (hTSH)α chainswere found tomake contactwithmanyaminoacidson theLRD surface and CD surface.

[0174] As used herein, "TSHR" refers to human thyroid stimulating hormone receptor. The term should be construed to include not only human thyroid stimulating hormone receptor, but variants, homologs, fragments and portions thereof to the extent that such variants, homologs, fragments and portions thereof retain the ability of human thyroid stimulating hormone receptor to bind to antibodies or ligands of human thyroid stimulating hormone receptor as disclosed herein. 16 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55

[0175] In some embodiments, the nucleotide sequence encoding at least a portion of the human thyroid stimulating hormone receptor is shown in SEQ ID NO: 26 and the amino acid sequence of at least a portion of the human thyroid stimulating hormone receptor are shown in SEQ ID NO: 27.

[0176] In someembodiments, thepresent disclosure isparticularlywell-suited todeliver agents tocells thatoverexpress theTSHRdifferentially. Asusedherein, aTSHR is "overexpressed"when it is present on thesurfaceofa cell (e.g., a thyroid cell) in an amount that is statistically significantly greater than a suitable control cell (e.g., a brain cell or a pancreas cell). In someembodiments, theTSHR ispresenton thecell surfaceat least about 1.5-fold, 2-fold, 5-fold, 10-fold, 100-foldor1000- fold greater than a suitable control cell. Accordingly, some embodiments of the present disclosure are particularly suitable for deliveryof anagent to thyroid cancer cells. In these instances, non-tumor cells that express theTSHRmayalsobekilled by the transferred CART / NK cells. However, if the thyroid is non-essential tissues with respect to the subject, the killing of normal cells of the colon does not cause a life-threatening event (e.g., complications) to the subject.

[0177] In someembodiments, the costimulatory signaling regionmay include the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 4‑1BB, OX40, CD30, CD40, PD‑1, ICOS, lymphocyte function associated antigen‑1 (LFA‑1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

[0178] In some embodiments, the antigen binding domain may include at least one of SEQ ID NOs: 2‑20 or 44.

[0179] In someembodiments, the antigen binding domainmay includeSEQ IDNO: 3 or 4, or a combination thereof, and the tumor is ovary tumor.

[0180] In someembodiments, the antigen binding domainmay includeSEQ IDNO: 6 or 7, or a combination thereof, and the tumor is thyroid tumor.

[0181] In some embodiments, the antigen binding domain may include SEQ ID NO: 9 or 10, or a combination thereof, and the tumor is breast cancer.

[0182] In some embodiments, the antigen binding domain may include SEQ ID NO: 12 or 13, or a combination thereof, and the tumor is gastric cancer.

[0183] In some embodiments, the antigen binding domain may include SEQ ID NO: 15 or 16, or a combination thereof, and the tumor is colorectal cancer.

[0184] In some embodiments, the antigen binding domain may include SEQ ID NO: 18 or 19, or a combination thereof, and the tumor is bladder cancer.

[0185] In some embodiments, the antigen binding domain may include SEQ ID NO: 20 or 44, and the tumor is breast cancer.

[0186] Someembodimentsof thepresentdisclosure relate toamethodof selectinganantigenbindingdomain foraCAR for treating a subject with tumor cells. The method may include determining an organ of cells from that the tumor cells derived, determining that the organ is a non-essential organ with respect to the subject, searching a database to identify multiplemarkers that are expressed in a cell population of the organ, selecting amarker of themultiplemarkers based ona predetermined condition, and generating cells comprising a CAR using cells from the subject. The CAR may include an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain, and the antigen binding domainmay bind to themarker. Examples of an organ includemammary gland, gastric gland, etc. In certain embodiments, the organ does not include a blood tissue.

[0187] In someembodiments, thepredetermined conditionmay include themarker is present on the cell surfaceof a cell from that tumor cells are derived at least about at least one of 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold or 1000-fold greater than a suitable control cell, and the organ is non-essential such that an injury of the organ does not produce death of the subject.

[0188] In some embodiments, the non-essential organ is a mammary gland, and the marker is PRLR.

[0189] In some embodiments, the non-essential organ is a colon, and the marker is GUCY2C.

[0190] In some embodiments, the non-essential organ is a gastric gland, and the marker is Muc17.

[0191] In some embodiments, the non-essential organ is a bladder, and the marker is CD207.

[0192] In some embodiments, the non-essential organ is an ovary, and the marker is FZD10.

[0193] In some embodiments, the non-essential organ is a thyroid, and the marker is TSHR.

[0194] In some embodiments, the tumor is selected froma group consisting of breast cancer, a thyroid tumor, colorectal cancer, an ovary tumor, bladder cancer, and is bladder cancer.

[0195] Someembodimentsof thepresentdisclosure relate toamodifiedcell includinganucleicacid sequenceencoding a CAR having one of SEQ ID NOs: 36‑43. For example, the cell is selected from the group consisting of a Tcell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

[0196] In someembodiments, theCARhasSEQ IDNO:38, andanantigenbindingdomainof theCARbinds to prolactin receptor ligand having SEQ ID NO: 20 or 44.

[0197] In some embodiments, the CAR has SEQ ID NO: 40, and an antigen binding domain of the CAR binds to GUCY2C having SEQ ID NO: 33.

[0198] In some embodiments, the CAR has SEQ IDNO: 39, and an antigen binding domain of the CAR binds to Muc17 17 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 having SEQ ID NO: 31.

[0199] In some embodiments, the CAR has SEQ IDNO: 41, and an antigen binding domain of the CAR binds to CD207 having SEQ ID NO: 35.

[0200] In some embodiments, the CAR has SEQ IDNO: 36, and an antigen binding domain of the CAR binds to FZD10 having SEQ ID NO: 25.

[0201] In some embodiments, the CAR has SEQ ID NO: 37, and an antigen binding domain of the CAR binds to TSHR having SEQ ID NO: 27.

[0202] Additional information related to expression synthetic nucleic acids encoding CARs and gene transfer into mammalian cells is provided in U.S. Pat. No. US8,906,682, incorporated by reference in its entirety.

[0203] The embodiments further relate to methods for treating a patient for an illness including administering to the patient an effective amount of the engineered cells of the present disclosure. Various illnesses can be treated according to the present methods including cancer, such as ovarian carcinoma, breast carcinoma, colon carcinoma, glioblastoma multiforme, prostate carcinoma and leukemia. In some embodiments, the method includes administering to a human patient apharmaceutical composition includinganeffectiveantitumoramountof apopulationofhumanTcells,wherein the humanTcells of the population include humanT-cells that comprise the nucleic acid sequence as described in the present disclosure.

[0204] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may include non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may include solid tumors. Types of cancers to be treated with the CARs of the disclosure include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoidmalignancies, benign and malignant tumors, and malignancies, e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0205] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0206] Solid tumors are abnormalmasses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamouscell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocyto- mas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,medulloblastoma,Schwannomacraniopharyngioma, ependymoma,pinealoma,hemangioblastoma, acous- tic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases).

[0207] Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the engineered cells of the present disclosure are used in the treatment of cancer. In certain embodiments, the cells of the present disclosure are used in the treatment of patients at risk of developing cancer. Thus, the present disclosure provides methods for the treatment or prevention of cancer comprising administering to a subject in need thereof, a therapeutically effective amount of the engineered T cells of the present disclosure.

[0208] The engineered T cells of the present disclosure may be administered either alone or as a pharmaceutical composition in combinationwith diluents and / or with other components such as IL‑2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present disclosuremay include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are preferably formulated for intravenous administration.

[0209] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease tobe treated (orprevented). Thequantity and frequencyofadministrationwill bedeterminedbysuch factorsas the 18 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[0210] When "an immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or "therapeutic amount" is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the Tcells described herein may be administered at a dosage of 104 to 109cells / kg body weight, preferably 105 to 106 cells / kgbodyweight, includingall integer valueswithin those ranges. Tcell compositionsmayalsobe administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0211] In certain embodiments, it may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present disclosure, and reinfuse the patient with these activated and expanded Tcells. This process can be carried out multiple times every few weeks. In certain embodiments, Tcells can be activated fromblood draws of from10 cc to 400 cc. In certain embodiments, Tcells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multiple blood draw / multiple reinfusion protocols, may select out certain populations of T cells.

[0212] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscu- larly, by intravenous (i. v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present disclosure are preferably administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.

[0213] In certain embodiments of the present disclosure, cells activated and expanded using the methods described herein, or othermethods known in the art where Tcells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatmentwith agents suchasantiviral therapy, cidofovir and interleukin‑2,Cytarabine (also knownasARA-C) or natalizumab treatment forMSpatients or efalizumab treatment for psoriasis patients or other treatments for PMLpatients. In further embodiments, the Tcells of the present disclosure may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate,mycophenolate, andFK506, antibodies, or other immunoablationagents suchasCAMPATH,anti-CD3antibodiesor other antibody therapies, cytotoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor-induced signaling (rapamycin). (Liu et al., Cell 66:807‑815, 1991; Henderson et al., Immun 73:316‑321, 1991; Bierer et al., Curr. Opin. Immun 5:763‑773, 1993; Isoniemi (supra)). In a further embodiment, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present disclosure are administered following B-cell ablative therapy such asagents that reactwithCD20, e.g.,Rituxan. For example, in oneembodiment, subjectsmayundergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present disclosure. In an additional embodiment, expanded cells are administered before or following surgery.

[0214] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for CAMPATH, for example, will generally be in the range 1 to about 100mg for an adult patient, usually administered daily for a period of 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Pat. No. 6,120,766, incorporated by reference in its entirety).

[0215] Additional information on the methods of cancer treatment using engineer T cells is provided in U.S. Pat. No. US8,906,682, incorporated by reference in its entirety.

[0216] Some embodiments of the present disclosure relate to a nucleic acid sequence encoding a CAR. For example, theCAR includes an antigen binding domain, a transmembrane domain, and an intracellular domain. The antigen binding domain binds to an organ lineage antigen, and the organ lineage antigen is expressed in a tumor and normal tissue from which the tumor is derived.

[0217] The terms "tumor associated antigens" as used herein refer to antigens selectively expressed or overexpressed 19 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 by malignant cells as compared with normal adult tissue. The tumor associated antigens include various groups such as tumor specific antigens, oncogetal antigens, oncogeneproducts, organ lineageantigens, viral antigens, etc. For example, oncogene and suppressor gene products, such as nonmutated HER‑2 / neu and p53, are analogous to oncofetal antigens in that they can be overexpressed in tumors and may be expressed in some fetal tissues.

[0218] The term "tumor specific antigens" as used herein refers to antigens that are uniquely expressed in tumors, such as point-mutated ras oncogenes, p53 mutations, anti-idiotype antibodies (Abs), and products of ribonucleic acid (RNA) splice variants and gene translocations.

[0219] The term "organ lineage antigen" as used herein is defined an antigen expressed in a tumor of a given type and the normal organ from which the tumor is derived. Examples of organ lineage antigen include prostate-specific antigen (PSA) and the melanocyte antigens, such as MART‑1 / Melan A, tyrosinase, gp100, and TRP‑1 / gp75. Organ lineage antigens may be targets for immunotherapy if the normal organ in which they are expressed is not essential, such as the prostate, breast, ormelanocyte.Asusedherein, anorgan refers toan integratedgroupof cellswithacommonstructure, an intercellular material, and / or a function.

[0220] Some embodiments of the present disclosure relate to a vector including the nucleic acid sequence. In some embodiments, the vector is an expression vector.

[0221] Some embodiments of the present disclosure relate to a pharmaceutical composition comprising an effective antitumor amount of a population of humanTorNK cells. The humanTorNK cells of the population include humanTorNK cells that include the nucleic acid sequence as described above.

[0222] Some embodiments of the present disclosure relate to a method of treating the tumor in a human patient, the method comprising administering to the human patient the pharmaceutical composition as described above.

[0223] In some embodiments, the tumor is a breast tumor, and the organ lineage antigen comprises PRLR.

[0224] In some embodiments, the tumor is a colorectal tumor, and the organ lineage antigen comprises at least one of CLCA1, NFUC12, GUCY2C, or GPR35.

[0225] In some embodiments, the tumor is a gastric tumor, and the organ lineage antigen comprisesCR1L and / or MUC 17.

[0226] In someembodiments, the tumor isanesophageal tumor, and theorgan lineageantigencomprisesat least oneof TMPRSS11B, MUC21, or TMPRSS11E.

[0227] In some embodiments, the tumor is a bladder carcinoma, and the organ lineage antigen comprises CD207.

[0228] In some embodiments, the tumor is a pancreatic tumor, and the organ lineage antigen comprises SLC30A8 and / or CFC1.

[0229] In some embodiments, the tumor is a cervical tumor, and the organ lineage antigen comprises SLC12A3 and / or SSTR1.

[0230] In some embodiments, the tumor is an ovary tumor, and the organ lineage antigen comprises GPR27 and / or FZD10.

[0231] In some embodiments, the tumor is a thyroid tumor, and the organ lineage antigen comprises TSHR.

[0232] Table 1 below lists multiple tumor and organ lineage antigens as well as corresponding genes. Cancer types Gene Gene Abbreviation Corresponding Protein / antigen Breast Cancer Prolactin receptor PRLR PRLR colorectal Cancer Chloride channel accessory 1 CLCA1 CLCA1 colorectal Cancer Mucin 12 MUC12 MUC12 colorectal Guanylate cyclase GUCY2C GUCY2C Cancer 2C colorectal Cancer G protein-coupled receptor 35 GPR35 GPR35 Gastric Cancer Complement component (3b / 4b) re- ceptor 1-like CRlL CR1L Gastric Cancer Mucin 17, cell surface associated MUC 17 MUC 17 esophageal Cancer Transmembrane protease, serine 11B TMPRSS11B TMPRSSIIB esophageal Cancer Mucin 21 MUC21 MUC21 esophageal Cancer Transmembrane protease, serine 11E TMPRSS11E TMPRSS11E bladder Cancer CD207 CD207 CD207 20 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Cancer types Gene Gene Abbreviation Corresponding Protein / antigen pancreatic Cancer Solute carrier family 30 (zinc trans- porter), member 8 SLC30A8 SLC30A8 pancreatic Cancer Cripto, FRL‑ 1, cryptic family 1 CFC1 CFC1 Cervical Cancer Solute carrier family 12 (sodium / chloride transporters) member 3 SLC12A3 SLC12A3 Cervical tumor Somatostatin receptor 1 SSTR1 SSTR1 Ovary tumor G protein-coupled receptor 27 GPR27 GPR27 Ovary tumor Frizzled family receptor 10 FZD10 FZD10 Thyroid Tumor Thyroid stimulating hormone recep- tor TSHR TSHR Examples

[0233] The present disclosure is further described with reference to the following examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Construction of antigen-expressed K562 cell lines

[0234] K562 cells were transduced with lentivirus including nucleic acid sequences encoding various antigens (FIG. 1) to establish target tumor cell lines. The lentivirus included the IRES-mCherry (red) construct, which encodes red fluorescence for confirmation of antigen expression. Red fluorescent signals were observed from these cell lines, indicating that target solid tumor cell lines were successfully established (FIG. 2). Techniques of construction of cell linesmay be found at "Chimeric Receptors Containing CD137 Signal Transduction Domains Mediate Enhanced Survival of T Cells and Increased Antileukemic Efficacy In Vivo Molecular Therapy vol. 17 no. 8, 1453‑1464 Aug. 2009," which is incorporated herein by reference. K562 cells were obtained from American Type Culture Collection (ATCC). Construction of CAR T cells

[0235] Primary T cells were transduced with lentivirus including various CARs to establish different CAR T cell lines targeting different antigens listed in FIG. 1. These cells were obtained fromhealthy human donors. As illustrated in FIG. 3, the lentivirus included nucleic acid sequence encoding CAR molecules, respectively, and further included the IRES- mCherry (green) construct, which encodes green fluorescence for confirmation of CAR expression. Taking anti- CD207 / anti-Muc17 CAR T cell as examples in FIG.3A, and expression of CARs was measured to confirm that CAR T-cell lines express specific anti-antigen molecules (See above boxes 302) in FIGS. 4‑9. Techniques related to cell cultures, construction of lentiviral vectors, and flow cytometry may be found in "Treatment of Advanced Leukemia in Mice with mRNA-Engineered T Cells, HUMAN GENE THERAPY 22:1575‑1586 (December 2011)", which is incorporated herein by reference.

[0236] Table 2 below lists various sequence identifiers and their sequences for establishing various anti-antigenCART cells. SEQ ID NO: Identifier Target tumors 2 scFv FZD10 Ovary tumor 5 scFv TSHR Thyroid Tumor 8 scFv PRLR Breast cancer 11 scFv Muc 17 Gastric Cancer 14 scFv GUCY2C colorectal Cancer 17 scFv CD207 bladder Cancer 21 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Target tumors 36 CAR FZD10 Ovary tumor 37 CAR TSHR Thyroid Tumor 38 CAR PRLR Breast cancer 39 CAR Muc 17 Gastric Cancer 40 CAR GUCY2C colorectal Cancer 41 CAR CD207 bladder Cancer 42 CAR Prolactin Breast cancer 44 CAR modified Prolactin Breast cancer IFN-γ release in co-cultivation assays

[0237] Each type of CAR Tcells and the corresponding type of antigen-expressed K562 cells were co-cultured (See, Table 2), and CAT T cells’ responses induced by the antigen-express K562 cells were measured. A ratio of E: T 1:1 / 3:1 / 10:1 / 30:1 (i.e., CAR Tcells: target tumor cells) of CAR Tcells and target tumor cells were co-culture for 24 hours. The supernatant was collected then, and release of IFN-γ was measured. Various levels of IFN-γ release were observed whenCARTcells and their correspondingantigen-expressK562cellswere co-cultured. IFN-γ release is not obviouswhen the CARTcells andwild-type K562were co-cultured. This observation shows that the CART cells specifically identify the corresponding antigen-express K562 cells and attack these cells by releasing IFN-γ (see FIGS. 10‑15). As illustrated in box404ofFIG. 4B,Tcells includingprolactin-CARalso recognize target tumor cells expressingPRLRand release IFN-γ in response to co-culturing the prolactin-CARTcells and the antigen-expressK562 cells. As compared to box 402 in FIG. 15 (scFv anti-PRLR CAR T cells), prolactin-CAR T cells achieve a similar effect. Techniques related to cell cultures, construction of cytotoxic T-lymphocyte assay may be found in "Control of large, established tumor xenografts with genetically retargeted humanTcells containingCD28 andCD137 domains. 3360‑3365PNASMarch 3, 2009, vol. 106 no. 9", which is incorporated herein by reference. CAT T cell killing assay

[0238] CAR T cell killing assays were conducted to measure the effectiveness of CAR T cells. Primary T cells were obtained from blood samples of healthy human donors. These T cells were transduced with a nucleic acid sequence encoding various CARs (See Table 2 and FIG. 1), respectively, and CAR expression on T-cells was measured using flow cytometry techniques.

[0239] K562 cells were transducedwith nucleic acid sequences encoding corresponding human antigens (See FIG. 1), respectively, and antigen expression was measured using flow cytometry techniques. Further antigen-expression K562 cells were transduced with nucleic acid sequence encoding fluorescent proteins (RFP) for killing assay analysis. Various CARTcells were incubated with corresponding K562 cells for 24 hours in various E:T ratios (30:1, 10:1, 3:1, 1:1), and red fluorescencesignals fromco-culturedcellswereobserved.For example,CARTcells expressinganti-THSRCAR (SEQ ID NO: 5) were co-cultured with K562 expressing human THSR (SEQ ID NO: 26) for at least five days. As compared with normal Tcells, CARTcells significantly reduced numbers of antigen-expressionK562 cells. Examples of anti-PRLRCAR, anti-THSR CAR, and Prolactin CAR Tcells were provided in FIGS 16‑18. In these examples, red fluorescence signals of co-cultured cells were observed at day five after co-culturing the CAR Tcells and the corresponding antigen-expression K562 cells.

[0240] CARTcell killinganalysiswas further performedusing3T3murinefibroblasts (ATCC). 3T3cellswere transduced with various human antigens (See FIG. 1), and antigen expression on 3T3 cells was measured using flow cytometry techniques. Further, 3T3 cells were transducedwith nucleic acid sequence encoding fluorescent proteins (RFP) for killing assay analysis. 3T3 cells expressing human target antigens / RFP and corresponding CAR Tcells were co-cultured at an E:T 30:1 or 10:1, respectively, and then fluorescent signals were observed from the co-cultured cells for at least five days. As comparedwith normal Tcells, CARTcells significantly reduced numbers of antigen-expression 3T3 cells. Examples of anti-PRLR CAR, anti-PRLR CAR, and Prolactin CAR T cells were provided in FIGS 19‑23. In these examples, red fluorescencesignalsof co-culturedcellswereobservedatdayfiveafter co-culturing theCARTcellsand thecorresponding antigen-expression 3T3 cells.

[0241] Table 3 below lists various sequence identifiers and their sequences. 22 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 SEQ ID NO: Identifier Sequence 1 SP MALPVTALLLPLALLLHAARP 2 scFv FZD10 3 2L-FZD10 4 2H-FZD10 5 scFv TSHR 6 5L-TSHR 7 5H-TSHR 8 scFv PRLR 9 8L-PRLR 10 8H-PRLR 11 scFv Muc 17 12 11L-Muc 17 13 11H-Muc 17 23 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 14 scFv GUCY2C 15 14L-GUCY2C 16 14H-GUCY2C 17 scFv CD207 18 17L-CD207 19 17H-CD207 20 Prolactin (li- gand) 21 Hinge & trans- membra ne domain 22 Co-stimulatory region KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 23 CD3-zeta 24 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 24 A-FZD10 25 A-FZD10 25 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 26 B-TSHR 26 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 27 B-TSHR 28 C-PRLR 27 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 29 C-PRLR 30 D-Muc17 28 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 31 D-Muc17 32 E-GCC 29 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 30 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 33 E-GCC 34 F-CD207 31 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 35 F-CD207 36 CAR FZD 10 37 CAR TSHR 38 CAR PRLR 39 CAR Muc 17 32 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) SEQ ID NO: Identifier Sequence 40 CAR GUCY2C 41 CAR CD207 42 CAR Prolactin 43 CAR modified Prolactin 44 Prolactin (mu- tation)

[0242] The current disclosure also includes: §1. A pharmaceutical composition comprising human T cells, wherein the human T cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR comprises an antigen binding domain, a transmem- 33 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 branedomain, a costimulatory signaling region, andaCD3zeta signalingdomain, theantigenbindingdomainbinds to an antigen that is expressed on the surface of a non-essential organ cell present in a microenvironment of a tumor. §2. The pharmaceutical composition of clause 1, wherein the antigen binding domain comprises SEQ IDNOs: 2, 5, 8, 11, 14, 17, 44, or 20, or the CAR comprises SEQ ID NOs: 36‑43. §3. The pharmaceutical composition of clause 2, wherein the antigen binding domain comprises SEQ ID NO: 2, and the tumor is an ovary tumor. §4. The pharmaceutical composition of clause 2, wherein the antigen binding domain comprises SEQ ID NO: 5, and the tumor is a thyroid tumor. §5. The pharmaceutical composition of clause 2, wherein the CAR comprises SEQ ID NO: 8, and the tumor is breast cancer. §6. The pharmaceutical composition of clause 2, wherein the antigen binding domain comprises SEQ IDNO: 11, and the tumor is gastric cancer. §7. The pharmaceutical composition of clause 2, wherein the antigen binding domain comprises SEQ IDNO: 14, and the tumor is colorectal cancer. §8. The pharmaceutical composition of clause 2, wherein the antigen binding domain comprises SEQ IDNO: 17, and the tumor is bladder cancer. §9. Thepharmaceutical composition of clause2,wherein theantigenbindingdomain comprisesSEQ IDNO:42or 43, and the tumor is breast cancer. §10. The isolated pharmaceutical composition of clause 1,wherein the tumor is breast cancer, the antigen is prolactin receptor (PRLR) having SEQ ID NO: 29. §11. The pharmaceutical composition of clause 1, wherein the tumor is colorectal cancer, the antigen is Guanylate cyclase 2C (GUCY2C) having SEQ ID NO: 33. §12.Thepharmaceutical compositionof clause1,wherein the tumor isgastric cancer, theantigen isMucin17 (Muc17) having SEQ ID NO: 31. §13. The pharmaceutical composition of clause 1, wherein the tumor is bladder cancer, the antigen is CD207 having SEQ ID NO: 35. §14. The pharmaceutical composition of clause 1, wherein the tumor is an ovary tumor, the antigen is Frizzled family receptor 10 (FZD10) having SEQ ID NO: 25. §15. The pharmaceutical composition of clause 1, wherein the tumor is a thyroid tumor, the antigen is Thyroid stimulating hormone receptor (TSHR) having SEQ ID NO: 27. §16. The pharmaceutical composition of clause 1, wherein the tumor is selected from a group consisting of breast cancer, a thyroid tumor, colorectal cancer, an ovary tumor, bladder cancer, and is bladder cancer. §17. A method for stimulating a T cell-mediated immune response to a cell population expressing the antigen, the method comprising contacting the cell populationwith an effective amount of the humanTcells of any of clauses 1‑16. §18. A modified T cell comprising a nucleic acid sequence encoding a CAR having one of SEQ ID NOs: 36‑43. §19. ThemodifiedTcell of clause 18,wherein theCARhasSEQ IDNO: 38, andanantigen bindingdomain of theCAR binds to prolactin receptor ligand having SEQ ID NO: 20 or 44. §20. ThemodifiedTcell of clause 18,wherein theCARhasSEQ IDNO: 40, andanantigen bindingdomain of theCAR binds to GUCY2C having SEQ ID NO: 33. 34 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 §21. ThemodifiedTcell of clause 18,wherein theCARhasSEQ IDNO: 39, andanantigen bindingdomain of theCAR binds to Muc17 having SEQ ID NO: 31. §22. ThemodifiedTcell of clause 18,wherein theCARhasSEQ IDNO: 41, andanantigen bindingdomain of theCAR binds to CD207 having SEQ ID NO: 35. §23. ThemodifiedTcell of clause 18,wherein theCARhasSEQ IDNO: 36, andanantigen bindingdomain of theCAR binds to FZD10 having SEQ ID NO: 25. §24. ThemodifiedTcell of clause 18,wherein theCARhasSEQ IDNO: 37, andanantigen bindingdomain of theCAR binds to TSHR having SEQ ID NO: 27. §25. A method for stimulating a T cell-mediated immune response to a cell population expressing the antigen, the method comprising contacting the cell population with an effective amount of the human T cells comprising the modified T cell of any of clauses 9‑24. Claims 1. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, a CD3 zeta signaling domain, and a co-stimulatory signaling region, wherein the antigen binding domain comprises anti- GUCY2C single chain variable fragment (scFv) comprising a heavy chain variable region (VH) comprising SEQ ID NO: 16 and a light chain variable region (VL) comprising SEQ ID NO: 15. 2. The CAR of claim 1, wherein the anti-GUCY2C scFv comprises SEQ ID NO: 14. 3. The CAR of claim 1 or 2, wherein the co-stimulatory signaling region comprises an intracellular domain of a co- stimulatory molecule selected from CD27, CD28, 4‑1BB, OX40, CD30, CD40, ICOS, CD2, CD7, LIGHT, and combinations thereof. 4. The CAR of claim 3, wherein the co-stimulatory signaling region comprises an intracellular domain of 4‑1BB. 5. The CAR of claim 4, wherein the co-stimulatory signaling region comprises SEQ ID NO: 22. 6. The CAR of any one of claims 1‑5, comprising a hinge domain, wherein the hinge and transmembrane domains comprise SEQ ID NO: 21 or a portion thereof. 7. A CAR comprising anti-GUCY2C scFv comprising SEQ ID NO: 14, hinge and transmembrane domains comprising SEQ ID NO: 21 or a portion thereof, a CD3 zeta signaling domain, and a co-stimulatory signaling region comprising SEQ ID NO: 22. 8. A nucleic acid molecule encoding the CAR of any one of claims 1‑7. 9. A vector comprising the nucleic acid molecule of claim 8, optionally wherein the vector is a viral vector, further optionally wherein the vector is a lentiviral vector. 10. ATcell comprising the CAR of any one of claims 1‑7, the nucleic acid molecule of claim 8, or the vector of claim 9. 11. Apharmaceutical composition comprisinghumanTcells comprising theCARof anyoneof claims1‑7, thenucleic acid molecule of claim 8, or the vector of claim 9. 12. A T cell of claim 10 or the pharmaceutical composition of claim 11 for use as a medicament. 13. A T cell of claim 10 or the pharmaceutical composition of claim 11 for use in treating colorectal cancer in a human patient. 14. A T cell of claim 10 or the pharmaceutical composition of claim 11 for use in stimulating a T cell-mediated immune response in a human subject, optionally wherein the human subject has colorectal cancer. 35 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 15. TheTcell or pharmaceutical composition for use of claim13or 14, wherein theTcell or pharmaceutical composition is administered intravenously to the patient or subject, optionally wherein the administration is by infusion. 36 EP 4 745 230 A2 5 10 15 20 25 30 35 40 45 50 55 37 EP 4 745 230 A2 38 EP 4 745 230 A2 39 EP 4 745 230 A2 40 EP 4 745 230 A2 41 EP 4 745 230 A2 42 EP 4 745 230 A2 43 EP 4 745 230 A2 44 EP 4 745 230 A2 45 EP 4 745 230 A2 46 EP 4 745 230 A2 47 EP 4 745 230 A2 48 EP 4 745 230 A2 49 EP 4 745 230 A2 50 EP 4 745 230 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description • US 62317261 A

[0001] • US 8906682 B

[0202]

[0215] • FR 901228

[0213] • US 6120766 A

[0214] Non-patent literature cited in the description • HARLOW et al. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1999

[0061] • HARLOW et al. Antibodies: A Laboratory Manual. Cold Spring Harbor, 1989

[0061] • HOUSTON et al. Proc. Natl. Acad. Sci. USA, 1988, vol. 85, 5879-5883

[0061] • BIRD et al. Science, 1988, vol. 242, 423-426

[0061] • ROSENBERG et al. New Eng. J. of Med., 1988, vol. 319, 1676

[0210] • LIU et al. Cell, 1991, vol. 66, 807-815

[0213] • HENDERSON et al. Immun, 1991, vol. 73, 316-321

[0213] • BIERER et al. Curr. Opin. Immun, 1993, vol. 5, 763- 773

[0213] • ChimericReceptorsContainingCD137Signal Trans- duction Domains Mediate Enhanced Survival of T Cells and Increased Antileukemic Efficacy. Vivo Molecular Therapy, August 2009, vol. 17 (8), 1453- 1464

[0234] • Treatment of Advanced Leukemia in Mice with mRNA-Engineered T Cells. HUMAN GENE THER- APY, December 2011, vol. 22, 1575-1586

[0235] • Control of large, established tumor xenografts with genetically retargetedhumanTcells containingCD28 andCD137 domains.PNAS, 03March 2009, vol. 106 (9), 3360-3365

[0237] 摘要 本发明公开了利用嵌合抗原受体（CAR）修饰细胞治疗癌症的组合物、方法和试剂 盒。本发明也公开了一种编码 CAR 的分离核酸序列。所述 CAR 可包括抗原结合 域、跨膜结构域、共刺激信号传导区及 CD3ζ 信号传导结构域。所述抗原结合域可 与非必需器官的抗原结合。

Claims

1. A chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, a CD3 zeta signaling domain, and a co-stimulatory signaling region, wherein the antigen binding domain comprises anti-GUCY2C single chain variable fragment (scFv) comprising a heavy chain variable region (VH) comprising SEQ ID NO: 16 and a light chain variable region (VL) comprising SEQ ID NO: 15.

2. The CAR of claim 1, wherein the anti-GUCY2C scFv comprises SEQ ID NO: 14.

3. The CAR of claim 1 or 2, wherein the co-stimulatory signaling region comprises an intracellular domain of a co-stimulatory molecule selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, CD2, CD7, LIGHT, and combinations thereof.

4. The CAR of claim 3, wherein the co-stimulatory signaling region comprises an intracellular domain of 4-1BB.

5. The CAR of claim 4, wherein the co-stimulatory signaling region comprises SEQ ID NO: 22.

6. The CAR of any one of claims 1-5, comprising a hinge domain, wherein the hinge and transmembrane domains comprise SEQ ID NO: 21 or a portion thereof.

7. A CAR comprising anti-GUCY2C scFv comprising SEQ ID NO: 14, hinge and transmembrane domains comprising SEQ ID NO: 21 or a portion thereof, a CD3 zeta signaling domain, and a co-stimulatory signaling region comprising SEQ ID NO: 22.

8. A nucleic acid molecule encoding the CAR of any one of claims 1-7.

9. A vector comprising the nucleic acid molecule of claim 8, optionally wherein the vector is a viral vector, further optionally wherein the vector is a lentiviral vector.

10. A T cell comprising the CAR of any one of claims 1-7, the nucleic acid molecule of claim 8, or the vector of claim 9.

11. A pharmaceutical composition comprising human T cells comprising the CAR of any one of claims 1-7, the nucleic acid molecule of claim 8, or the vector of claim 9.

12. A T cell of claim 10 or the pharmaceutical composition of claim 11 for use as a medicament.

13. A T cell of claim 10 or the pharmaceutical composition of claim 11 for use in treating colorectal cancer in a human patient.

14. A T cell of claim 10 or the pharmaceutical composition of claim 11 for use in stimulating a T cell-mediated immune response in a human subject, optionally wherein the human subject has colorectal cancer.

15. The T cell or pharmaceutical composition for use of claim 13 or 14, wherein the T cell or pharmaceutical composition is administered intravenously to the patient or subject, optionally wherein the administration is by infusion.

Images