Cellular therapies targeting aberrant glycosylation in solid malignancies

Abstract

Chimeric antigen receptor T cells with a lectin binding domain are targeted to carbohydrate tumor antigens, particularly T antigen.

Description

CELLULAR THERAPIES TARGETING ABERRANT GLYCOSYLATION IN SOLID MALIGNANCIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 601 ,682, filed November 21 , 2023, the disclosure of which application is herein incorporated by reference.REFERENCE TO AN ELECTRO IC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (S23-401_STAN-2155WO_Seqlist); size 19,024 bytes and Date of Creation: November 21 , 2024, is herein incorporated by reference in its entirety.BACKGROUND

[0003] Metastatic disease causes 90% of cancer deaths, and while there has been considerable progress in our mechanistic understanding of disease progression, most advanced cancers continue to have a poor prognoses. Although immunotherapy has improved overall survival in several malignancies such as melanoma and mismatch repair deficient tumors, it is currently estimated that only 20-40% of patients are responsive to any form of currently available immunotherapy. For example, current standard of care in metastatic solid malignancies such as non-small cell lung cancer (NSCLC) is typically combination chemotherapy with immunotherapy or targeted therapies, should there be an actionable targetable mutation. In metastatic melanoma, which has been a model responder to immune checkpoint blockade (ICB), approximately 50% of cancers are not responsive to first line combination immunotherapy.

[0004] Notably, patients with advanced disease have frequently undergone several rounds of chemotherapies and / or are of advanced age, and thus their endogenous immune system may not be at the functional capacity required for an effective systemic immune response. Thus, there is an unmet need for development of new treatments for the majority of patients who have relapsed, refractory carcinomas that do not harbor targetable mutations or are either unresponsive or do not have a durable response to immunotherapy.

[0005] In contrast to ICB, which requires activation of endogenous immunity, genetically modified cellular therapies have become a major treatment option for patients incapable of mounting an endogenous antitumor immune response. Such cellular therapies are now second line treatment for relapsed refractory hematologic malignancies. Potential benefits of a cell therapy approach for solid cancers are multifactorial: a long lasting response, engineered cells are programmable / modular, can interact with other cells, tissue-specific trafficking is enabled, and treatment is potentially multi-potent. However, the success of suchtherapies for the majority of carcinomas has been elusive. CAR-T cells against solid tumors have typically been engineered based upon knowledge gleaned from anti-CD19 T cells, but such advances in cell therapies for hematologic malignancies may not translate equally to solid tumors.

[0006] Challenges in engineering cellular therapies for solid malignancies broadly lie in key areas: identification of tumor antigens with limited on-target off-tumor toxicity, tissue trafficking / infiltration, T cell exhaustion, and an immunosuppressive local tumor microenvironment.

[0007] A general challenge in eliciting anti-tumor immune responses is the process of immunoediting, whereby cancer cells evade immune detection through selection of variants that do not appropriately express the antigen. By targeting antigens that are critical for tumors to function, one can render immunoediting a much less efficacious process of immune evasion. Several studies have demonstrated that solid malignancies, particularly mucinous adenocarcinomas, display aberrant glycosylation patterns and these aberrant glycopeptides are commonly used as clinical biomarkers to assess for recurrence of disease (e.g., carbohydrate antigen 19-9 (CA 19-9), alpha fetal protein (AFP), carcinoembryonic antigen (CEA)). Truncated O-linked glycans are absent in normal tissues due to further elaboration of branched glycosylation or sialylation motifs, yet are unmasked during malignant transformation. These changes in glycosylation patterns have been mechanistically implicated in vascular extravasation, cell adhesion, drug resistance, metastatic seeding, and immune inhibition, and have been correlated with overall worse prognosis.SUMMARY

[0008] Tumor associated carbohydrate antigens (TACA) exhibit a greater degree of tumor specificity and are more abundant than most protein tumor antigens. TACA are targeted for cancer therapies of the present disclosure by contacting with a glycan-specific chimeric antigen receptor (CAR), expressed on the surface of an immune cell. Truncated O-linked glycans are absent in normal tissues due to further elaboration of branched glycosylation or sialylation motifs, but are unmasked during malignant transformation. The oncofetal Thomsen- Friedenreich antigen, also known as the T antigen or CD176, is an O-linked disaccharide that has been shown to enable distant seeding by binding galectin-3 in metastatic niches. Truncated TACAs are highly specific to carcinomas.

[0009] A glycan-specific CAR construct is provided, wherein a lectin, including without limit the plant lectin protein peanut agglutinin (PNA), or a biologically active binding fragment derived therefrom, provides specificity of binding. The lectin sequence is covalently linked, e.g. conjugated or fused, to an effector domain of the CAR. In some embodiments a CAR is specific for a truncated O-linked glycan. In some embodiments a CAR is specific for T antigen(CD176), including without limitation a CAR comprising the sequence set forth in SEQ ID NO:4.

[0010] In an embodiment, an immune cell, e.g. a T cell, B cell, NK cell, macrophage etc., is engineered to express a glycan-specific CAR, which comprises a PNA sequence as a receptor, and which CAR selectively binds to CD176 with high avidity. The CAR polypeptide comprising lectin as a specific binding domain may be referred to herein as an anti-CD176 CAR. In some embodiments an anti-CD176 CAR is expressed by a human T cell. In some embodiments an anti-CD176 CAR is expressed by a human NK cell. In some embodiments an anti-CD176 CAR is expressed by a human macrophage. In some embodiments an anti- CD176 CAR is expressed by a human B cell.

[0011] In some embodiments an anti-CD176 CAR is a bi-specific CAR, where a second antigenic specificity is an antigen present on malignant cells, e.g. CD123, FLT3, TIM3, CD99, CD96, B7-H3, CD33, IL1 RAP, CLL1 (CLEC12A), etc. In other embodiments an engineered immune cell expresses an anti-CD176 CAR and a second CAR with specificity for an antigen present on malignant cells.

[0012] In some embodiments an immune cell, e.g. a macrophage, B cell, NK cell, NKT cell, T cell, etc. is genetically modified to introduce a genetic sequence encoding an anti-CD176 CAR in an ex vivo procedure, prior to transfer into a subject. In some embodiments, the genetically modified cells are expanded in vitro. An effective dose of the genetically modified cells can be administered to a patient in need thereof. T cells of interest for engineering include without limitation, naive CD8+ T cells, cytotoxic CD8+ T cells, naive CD4+ T cells, helper T cells, e.g., TH1 , TH2, TH9, TH11 , TH17, TH22, TFH; memory T cells, e.g., central memory T cells, stem cell memory T cells (TSCM), effector memory T cells, NK T cells, etc. In some embodiments a T cell for engineering is a CD8+ effector T cell. In some embodiments, engineered T cells comprise a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. In some embodiments the engineered cells are allogeneic. In some embodiments the engineered cells are autologous.

[0013] In some embodiments, a therapeutic method is provided for elimination of cancer cells expressing CD176, which cancer cells include, without limitation, solid cancer cells, e.g. lung (NSCLC and small cell carcinoma), head and neck carcinomas, gastrointestinal carcinomas, breast carcinomas, genitourinary malignancies, lymphomas, etc. Treatment of hematologic malignancies is of interest, which malignancies include, without limitation, bone marrow neoplasia, lymphomas residing in a lymph node selectively expressing the CD176, etc. Treatment of lung cancer is of interest. In some embodiments the cancer is metastatic. In some embodiments a patient is selected for treatment based on the level of T antigen expression by the cancer cells, e.g. as determined by staining of a cancer cell sample with a reagent that specifically binds to CD176.

[0014] In some embodiments a method of treatment comprises introducing into a recipient in need, e.g. an individual with cancer, an engineered cell population, wherein the cell population has been modified by introduction of a sequence encoding a glycan-specific CAR, e.g. an anti- CD176 CAR, in a dose effective to reduce the number of cancer cells present in the recipient. The cell population may be engineered ex vivo, and is usually autologous or allogeneic with respect to the recipient.

[0015] In some embodiments, an engineered immune cell is provided, e.g. an engineered T cell, B cell, macrophage, NK cell, etc., in which the cell has been modified by introduction of an anti-CD176 CAR. The engineered cell can be provided in a unit dose for therapy, and can be allogeneic, autologous, etc., with respect to an intended recipient. Introduction of the coding sequence can be performed in vivo or in vitro, using any appropriate vector, e.g., viral vectors, integrating vectors, and the like.

[0016] In some embodiments, a vector comprising a polynucleotide sequence encoding a polypeptide comprising an anti-CD176 CAR provided, where the coding sequence is operably linked to a promoter active in the desired cell. The promoter may be constitutive or inducible. Various vectors are known in the art and can be used for this purpose, e.g. viral vectors, plasmid vectors, minicircle vectors, which vectors can be integrated into the target cell genome, or can be episomally maintained. The vector may be provided in a kit.BRIEF DESCRIPTION OF THE DRAWINGS.

[0017] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0018] FIG. 1 . Schematic of cell therapy against glycosylation motifs.

[0019] FIG. 2A-2D. T-antigen increases with increased metastatic potential; T-antigen expression by flow cytometry with PNA-AlexaFluor647 (Invitrogen, L32430) by flow cytometry with 5 uL of PNA-AlexaFluor647 (Invitrogen, L32430) on cell lines 1 E6 cells of each measured on BD LSRFortessa using FACS buffer (PBS with Ca+ / Mg+) A) on mouse cell lines derived from a lung cancer mouse model using lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of KrasLSL G12D / +;p53flox / floxmice (Winslow et al. Nature 201 1 ) with 804T as non-metastatic tumor and 393M a cell line arising from liver metastasesl B) Non-small cell lung cancer (NSCLC) human derived cell lines NCI-H322 (bronchoalveolar) and A549 (lung adenocarcinoma) C) cell lines of metastatic potential derived from a spontaneous murine model of PDAC derived by hydrodynamic tail vein injection of CRISPR edited KRASG12D mutant / p537cmyc+; primary from pancreatic lesion, LN1 - firstgeneration of tumor involved lymph node lines, LN2.2-second generation, LN 3.3- third generation, pLM1 - liver metastases D) Human tissue microarray of metastatic lung adenocarcinoma in lymph nodes.

[0020] FIG. 3A-3D. Design and function of anti-T antigen CAR-T cells in vitro. A) Timeline of CAR-T generation via lentiviral transduction B) Western blot demonstrating PNA expression (n=3) C) Vector construct. D) Flow cytometry demonstrating PNA CAR expression on human CD8 and CD4 T cells respectively on day 5 after transduction, staining using PNA-lectin conjugated to AlexaFluor 647 Vector Labs ( (AS-2074-1 )) using FACS buffer (PBS with Ca+ / Mg+)

[0021] FIG. 4A-4B. Schematics of a PNA CAR and exemplary retroviral construct.

[0022] FIGS. 5A-5C. T-antigen is found in human lung tumors. T antigen, also commonly known as the Thomsen Friedenriech antigen, CD176, is an O-linked oncofetal disaccharide found on cancer cells, this is a core structure of glycosylation, that is typically has further branching or modifications, not typically seen on normal tissues. A. Schematic of O-linked glycosylation and involved glycosyltransferases starting from core glycan. B. Peanut lectin hemagglutin (PNA)-horse radish perioxidase staining of human microarray of both normal and tumor involved Lymph Node (upper panel), and normal lung parenchyma and non-small cell lung cancer (NSCLC) lower panel. C) T-antigen expression via PNA-APC staining across lung cancer cell lines by flow cytometry (white- murine, black-human). (B) and (C) from Reticker-Flynn (2014) Cancer Discovery 5(2):168-181

[0023] FIGS. 6A-6D. T-antigen is found in a wide variety of carcinomas. A) Schematic of O- linked glycosylation and involved glycosyltransferases starting from core glycan. B) T-antigen expression via PNA-APC staining across lung cancer cell lines by flow cytometry (whitemurine, black-human) C) T-antigen expression by flow cytometry with 5 uL of PNA- AlexaFluor647 (Invitrogen, L32430) using FACS buffer (PBS with Ca+ / Mg+)_on cell lines 1 E6 cells of each THP-1 (AML), Colo357 (PDAC), BXPC3(PDAC), PANC-1 (PDAC), AsPC-1 (PDAC), A549 (NSCLC) measured on BD LSRFortessa D) PNA-HRP (Sigma Aldrich, L7759) staining of human microarray of normal and tumor from commercially available human tissue array (US Biomax, GI101 ) by light microscopy on Keyance BZ-x810. (B) from Reticker-Flynn (2014) Cancer Discovery 5(2) :168-181

[0024] FIG. 7. T-antigen found in human tumors. The glycan structures on the x axis are as named in FIG. 6A. Cancer cells lines representing colon adenocarcinoma (HCT15); alveolar basal epithelial cells (A549); primary bronchioalveolar carcinoma of the lung (NCI-H322; pancreatic carcinoma of ductal cell origin (PANC1 ); and adenocarcinoma of pancreas (AsPd ) were evaluated for expression of T antigen glycans.

[0025] FIG. 8. T-antigen is expressed on lung cancer subtype tissue microarray as shown by Peanut Agglutinin staining.

[0026] FIGS. 9A-9E. T-Antigen targeting PNA-CAR T cells exhibit potent and antigen-specific anti-tumor immunity in vitro. To assess the efficacy, CAR-T cells were co-cultured with tumor cells and subjected to multiple assays. (A) To evaluate proliferation following target exposure, 1 x104CAR-T cells were labeled with CellTrace Violet (CTV) Proliferation Dye (Thermo, C34557) prior to incubation with 1x104A549 T-Antigen-expressing tumor cells in a 96 round bottom plate supplemented with DMEM + 10% FBS + glutamine. After 3 days, the fraction of proliferating CAR cells was evaluated by CTV dilution by flow cytometry on BD LSRFortessa. Anti-CD3 / CD28 beads (Thermo, 1 1 132D) were incubated with CAR cells as a positive control. T o evaluate ligand specificity, co-cultures were incubated with 10OmM of a soluble competitive ligand, N-Acetyl-galactosamine (Sigma Aldrich A2795). B. CAR-T cells exhibited significant proliferation when co-cultured with tumors, and this proliferation was markedly reduced when incubated with the soluble ligand. (C) Cytokine production was evaluated after 24 hours of coculture by cytokine bead arrays (BD Biosciences 560484). These studies demonstrated significant interferon-gamma (IFNg) production that was inhibited by co-culture with the soluble competitive ligand. (D) Anti-tumor cytotoxicity was evaluated by co-culture of CAR-T cells with A549 tumor cells at various effectortarget (CAR:tumor) ratios and evaluated by 7AAD staining. E:T ratios of 5:1 and 10:1 both exhibit significant killing of target cells. Each dot is an individual human donor. E. Schematic.

[0027] FIGS. 10A-10E. T-antigen stimulates antigen-specific CART response. Cytokine profile by cytokine bead array (BD Biosciences 560484), CAR-T cells were co-cultured with human tumor cell lines that exhibit varying degrees of T-Antigen surface expression and subjected to cytokine profiling by cytokine bead arrays. THP-1 is a monocyte leukemia that has minimal T-Antigen expression as seen by prior flow cytometry. A. T-Antigen high- expressing (A549) and low-expressing (NCI-H322) tumor cells. B. Proliferation of cells. C. CAR-T cells produce little to no cytokine alone or when cultured with THP-1 cells. Significantly elevated cytokine expression is observed with H322 and A549 cells with the greatest amounts produced in the A549 co-cultures. PMA / lonomycin as positive control (Invitrogen 00-4970-93), IL-2: interleukin-2; D. IFNy: Interferon-gamma; E. TNFrx: Tumor Necrosis Factor alpha. N=3 for 3 separate human donors

[0028] FIGS. 1 1 A-1 1 F. CART function with Granzyme B. T-Antigen targeting PNA-CAR T cells exhibit potent and antigen-specific anti-tumor immunity in vitro after 14 hour incubation with A549 as seen by flow cytometry A. Number of Granzyme B positive cells. B. % frequency of CD8+ CD3+ cells. C. MFI granzyme B with CAR and with control. D. PMA and ionomycin. E. CART. F. Control. Control is the vector but with a tDT in place of PNA. Blue shaded line is CAR-PNA, Light blue line is Control, each dot represents individual human donors. Granzyme B (Biolegend 372208), CD8 (BD Biosciences, 612755), CD3 (Biolegend 344834).

[0029] FIGS. 12A-12C. In vivo competition assay T-antigenhivs. T-antigenl0. A. NOD-scid- gamma (NSG) mice were transplanted subcutaneously (s.c.) with T-Antigen high-expressing (A549) and low-expressing (NCI-H322) tumor cells on contralateral flanks with 1 x106cells. One week after tumor implantation, mice were treated with intravenous (retrooribital) injections of CAR-T cells (at a ratio of 1 :1 CD8:CD4 T cells) at 1x106dosing. Curves indicate tumor volume above baseline (at the time of treatment) for mice injected with control CAROT cells or those specific for the T-Antigen. C. While some efficacy was observed in the low ligand expressing tumor cells, the greatest efficacy (B) was observed in the high expressing tumors (A549s). Experiment n=1 , Control is the vector but with a tDT in place of PNA.

[0030] FIG. 13. Schematic of CAR construct in a lentiviral backbone (pLVX). The CAR sequence is that of SEQ ID NO:2 (with reporter gene).DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] In order for the present disclosure to be more readily understood, certain terms and phrases are defined below as well as throughout the specification. The definitions provided herein are non-limiting and should be read in view of what one of skill in the art would know at the time of invention.Definitions

[0032] Before the present methods and compositions are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0033] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential methods andmaterials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0035] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g., polypeptides, known to those skilled in the art, and so forth.

[0036] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0001] A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, including pet and laboratory animals, e.g. mice, rats, rabbits, etc. Thus the methods are applicable to both human therapy and veterinary applications. In one embodiment the patient is a mammal, preferably a primate. In an embodiment the patient is human.

[0002] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In an embodiment, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having cancer. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, etc.

[0003] The terms “cancer,” “neoplasm,” and “tumor” are used interchangeably herein to refer to cells which exhibit autonomous, unregulated growth, such that they exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Cancers of virtually every tissue are known. The phrase “cancer burden” refers to the quantum of cancer cells or cancer volume in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the cancer volume in a subject. The term “cancer cell” as used herein refers to any cell that is a cancer cell or is derived from a cancer cell e.g. clone of a cancer cell. Many types of cancers are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, etc., and circulating cancers such as leukemias. Examples of cancer include but are not limited to ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

[0004] The types of cancer that can be treated using the subject methods of the present invention include but are not limited to adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer, non-melanoma skin cancers, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom's macroglobulinemia.

[0005] In some embodiments the cancer is a solid cancer. In some embodiments the cancer is a hematologic cancer. In some embodiments the cancer is a lung cancer.

[0006] The “pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

[0007] As used herein, the terms “cancer recurrence” and “tumor recurrence,” and grammatical variants thereof, refer to further growth of neoplastic or cancerous cells after diagnosis of cancer. Particularly, recurrence may occur when further cancerous cell growth occurs in the cancerous tissue. “Tumor spread,” similarly, occurs when the cells of a tumor disseminate into local or distant tissues and organs; therefore tumor spread encompasses tumor metastasis. “Tumor invasion” occurs when the tumor growth spread out locally tocompromise the function of involved tissues by compression, destruction, or prevention of normal organ function.

[0008] As used herein, the term “metastasis” refers to the growth of a cancerous tumor in an organ or body part, which is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part which is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from an original tumor site, and migration and / or invasion of cancer cells to other parts of the body.

[0009] The term “sample” with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells. The definition also includes sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s cancer cell, e.g., a sample comprising polynucleotides and / or polypeptides that is obtained from a patient’s cancer cell (e.g., a cell lysate or other cell extract comprising polynucleotides and / or polypeptides); and a sample comprising cancer cells from a patient. A biological sample comprising a cancer cell from a patient can also include non-cancerous cells.

[0010] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a molecular subtype of breast cancer, prostate cancer, or other type of cancer.

[0011] The term “prognosis” is used herein to refer to the prediction of the likelihood of cancer- attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning. In one example, a physician may predict the likelihood that a patient will survive, following surgical removal of a primary tumor and / or chemotherapy for a certain period of time without cancer recurrence.

[0012] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / orsymptoms of the disease. “Treatment,” as used herein, may include treatment of a tumor in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0013] Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with cancer or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0014] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic and the compounds as used herein. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0015] "Concomitant administration" with a cancer therapeutic drug means administration at such time that both the drug and CART cell will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0016] As used herein, endpoints for treatment will be given a meaning as known in the art and as used by the Food and Drug Administration.

[0017] Overall survival is defined as the time from randomization until death from any cause, and is measured in the intent-to-treat population. Survival is considered the most reliable cancer endpoint, and when studies can be conducted to adequately assess survival, it is usually the preferred endpoint. This endpoint is precise and easy to measure, documented by the date of death. Bias is not a factor in endpoint measurement. Survival improvement shouldbe analyzed as a risk-benefit analysis to assess clinical benefit. Overall survival can be evaluated in randomized controlled studies. Demonstration of a statistically significant improvement in overall survival can be considered to be clinically significant if the toxicity profile is acceptable, and has often supported new drug approval. A benefit of the methods of the invention can include increased overall survival of patients.

[0018] Endpoints that are based on tumor assessments include DFS, ORR, TTP, PFS, and time-to-treatment failure (TTF). The collection and analysis of data on these time-dependent endpoints are based on indirect assessments, calculations, and estimates (e.g., tumor measurements). Disease-Free Survival (DFS) is defined as the time from randomization until recurrence of tumor or death from any cause. The most frequent use of this endpoint is in the adjuvant setting after definitive surgery or radiotherapy. DFS also can be an important endpoint when a large percentage of patients achieve complete responses with chemotherapy.

[0019] Objective Response Rate. ORR is defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period. Response duration usually is measured from the time of initial response until documented tumor progression. Generally, the FDA has defined ORR as the sum of partial responses plus complete responses. When defined in this manner, ORR is a direct measure of drug antitumor activity, which can be evaluated in a single-arm study.

[0020] Time to Progression and Progression-Free Survival. TTP and PFS have served as primary endpoints for drug approval. TTP is defined as the time from randomization until objective tumor progression; TTP does not include deaths. PFS is defined as the time from randomization until objective tumor progression or death. The precise definition of tumor progression is important and should be carefully detailed in the protocol.

[0021] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0022] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeuticeffect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0023] "Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0024] "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g., Ci6alkyl esters. When there are two acidic groups present, a pharmaceutically acceptable salt or ester can be a mono-acid-mono- salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified. Compounds named in this invention can be present in unsalified or unesterified form, or in salified and / or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may be present in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.

[0025] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0026] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0027] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0037] The term "sequence identity," as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (e.g., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990).

[0038] By "protein variant" or "variant protein" or "variant polypeptide" herein is meant a protein that differs from a wild-type protein by virtue of at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified version of a WT polypeptide. Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.

[0039] By "parent polypeptide", "parent protein", "precursor polypeptide", or "precursor protein" as used herein is meant an unmodified polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a wild-type (or native) polypeptide, or a variant or engineered version of a wild-type polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.

[0040] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a- carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0041] Amino acid modifications disclosed herein may include amino acid substitutions, deletions and insertions, particularly amino acid substitutions. Variant proteins may also include conservative modifications and substitutions at other positions of the cytokine and / or receptor (e.g., positions other than those involved in the affinity engineering). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: Group I: Ala, Pro, Gly, Gin, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Vai, lie, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu. Further, amino acid substitutions with a designated amino acid may be replaced with a conservative change.

[0042] The term “isolated” refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w / w) pure, more preferably, at least 80%-90% (w / w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure. A “separated” compound refers to a compound that is removed from at least 90% of at least one component of a sample from which the compound was obtained. Any compound described herein can be provided as an isolated or separated compound.

[0028] Peanut agglutinin (PNA) is plant lectin protein derived from the fruits of Arachis hypogaea. Peanut agglutinin may also be referred to as Arachis hypogaea lectin. Lectins recognize and bind particular sugar sequences in carbohydrates; peanut agglutinin binds the carbohydrate sequence Gal-|3(1 -3)-GalNAc, which is present on T antigen. The PNA protein is 273 amino acids in length with the first 23 residues acting as a signal peptide which is subsequently cleaved. It has a Uniprot accession of P02872. It is a member of the LectinJegB PFAM family.

[0029] The mature PNA protein sequence is as follows, (SEQ ID NO:1 ) SAETVSFNFNSFSEGNPAINFQGDVTVLSNGNIQLTNLNKVNSVGRVLYAMPVRIWSSATG NVASFLTSFSFEMKDIKDYDPADGIIFFIAPEDTQIPAGSIGGGTLGVSDTKGAGHFVGVEFD TYSNSEYNDPPTDHVGIDVNSVDSVKTVPWNSVSGAVVKVTVIYDSSTKTLSVAVTNDNGD ITTIAQVVDLKAKLPERVKFGFSASGSLGGRQIHLIRSWSFTSTLITTTRRSIDNNEKKIMNMA SA.

[0043] A signal sequence may be used in combination with the mature PNA sequence, for example the native signal sequence, a mammalian signal sequence, e.g. CD8, or the like.

[0044] Aberrant glycosylation. On the cell surface there exists a variety of complex, elongated chains of sugars that decorate proteins, lipids, and even nucleic acids and are critical for day to day cell function. These sugar cores are typically masked in normal cells through further branching, and become unmasked while undergoing malignant transformation. The aberrant glycans have been shown to help to extravasate blood vessels, invade tissues, inhibit immune responses, and help spread to distant organs. In some embodiments an aberrant glycan for targeting with the methods of the disclosure is a truncated O-linked glycan. In some embodiments a truncated O-linked glycan is the Thomsen Friedenriech antigen, also called the T antigen, which is an O-linked oncofetal disaccharide, found on a range of carcinomas that do not currently have good treatments for, such as but not limited to breast, prostate, gastric, ovarian, pancreatic and lung cancers. Other cancer associated glycans include CA 19-9 - sialyl Lewis A (sLea) tumor antigen expressed in Gl tumors; CA 125- mucin glycoprotein MUC16 expressed in ovarian cancer; AFP- fucosylated form of AFP for hepatocellular carcinoma; CA 15-3 (MUC1 ) - O-glycosylated and moderately N-glycosylated protein expressed in breast cancer.

[0045] Galectins are a family of soluble glycan-binding proteins that preferentially recognize N-acetyl-lactosamine (Gal|31 -4GlcNAc) residues present in both complex N-glycans and core 1 / 2 O-glycans on different cell surface receptors. There are fifteen in mammals (1 1 in humans). Although they lack a typical secretory signal, galectins are secreted through an unconventional ER / Golgi-independent pathway. Extracellularly, galectins can establish multivalent interactions with glycosylated receptors (often termed ‘lattices’) and control receptor segregation, endocytosis and signaling, leading to modulation of immune cell activation, differentiation, trafficking and survival.

[0046] In an embodiment, a glycan of interest for targeting is T-antigen, which is found on carcinomas including lung adenocarcinoma, non-small cell lung cancer, squamous cell lung cancer, mixed adeno / bronchial cancer and bronchoalveolar cancer; and carcinomas of the stomach, colon and pancreas among others.

[0047] Chimeric antigen receptor (CAR). A CAR is comprised of the general structure where a binding domain, e.g. PNA or a fragment derived therefrom, is linked to T cell receptor with effector functions. The term refers to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell. An exemplary CAR is diagrammed in Figure 3A, and with a sequence as shown in SEQ ID NO:4. A CAR will generally comprise PNA as a binding domain, as described herein, linker, transmembrane domain and cytoplasmicsignaling domain. In some instances, a CAR will include one or more co-stimulatory domains and / or one or more co-inhibitory domains.

[0048] A spacer (linker) region links the antigen binding domain to the transmembrane domain. It should be flexible enough to allow the binding domain to orient in different directions to facilitate antigen recognition. Suitable linker sequences include hinge sequences, such as the CD8a hinge, hinge regions from an immunoglobulin, e.g. the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. In some embodiments the linker comprises the amino acid sequence (G4S)nwhere n is 1 , 2, 3, 4, 5, etc., and in some embodiments n is 3.

[0049] The CAR transmembrane domain (TM) is frequently derived from type I membrane proteins, such as CD3 , CD4, CD8, CD28, etc.

[0050] A cytoplasmic signaling domain, such as those derived from the T cell receptor -chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory or co-inhibitory molecules may be included in the cytoplasmic signaling portion of the CAR.

[0051] The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which are incorporated herein by reference in their entirety. Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0052] The term “co-inhibitory domain” refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigenspecific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell coinhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12. In some embodiments, co-inhibitory domains homodimerize. A co-inhibitory domain can be an intracellular portion of a transmembrane protei. Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1.

[0053] A first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor forIgE FctRly, or the CD3^ chain. The signaling domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T-cell activation. The ITAM- based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.

[0054] Second-generation CARs include a co-stimulatory signal in addition to the CD3^ signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain is usually membrane proximal relative to the CD3^ domain. Third-generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3^, 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR T-cells are further gene modified to express or block molecules and / or receptors to enhance immune activity.

[0055] CAR variants include split CARs wherein the extracellular portion, the CAR and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e. , CAR variants) are described, e.g., in PCT Application Nos. US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1 ):91 -106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0056] CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens. iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains

[0057] The dual recognition of different epitopes by two CARs diversely designed to either deliver killing through ^-chain or costimulatory signals, e.g. through CD28 allows a more selective activation of the reprogrammed T cells by restricting tandem CAR’s activity to cancercell expressing simultaneously two antigens rather than one. The potency of delivered signals in engineered T cells will remain below threshold of activation and thus ineffective in absence of the engagement of costimulatory receptor. The combinatorial antigen recognition enhances selective tumor eradication and protects normal tissues expressing only one antigen from unwanted reactions.

[0058] Inhibitory CARs (iCARs) are designed to regulate CAR-T cells activity through inhibitory receptors signaling modules activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.

[0059] In some embodiments a CAR construct is a logic gated construct, see for example et al. (2022) Journal for ImmunoTherapy of Cancer 10:e004185, herein specifically incorporated by reference. Logic gating allows either multi-antigen targeting, or combinatorial treatment with biological agents (such as checkpoint inhibitors or cytokines). Biological AND-gating occurs when two input signals or events must occur before the outcome. In OR-gating, there may be multiple possible input signals and any of them can trigger the desired outcome. NOT- gating occurs when the signal input results in an inhibitory signal being generated.

[0060] In an ON switche system, the administration of small molecules enables the completion of a circuit to allow T signalling, for example when the antigen-binding domain and the signaling domain, containing the CD3^ domain and ITAMs are two separate molecules that only associate in the presence of a small molecule, such as rapamycin or a derivative thereof. In the presence of the target antigen and small molecule, a functional CAR unit is formed.

[0061] AND-gating requires two distinct antigens to be detected to trigger the outcome or downstream event; one to induce the logic gated system, the other to kill the target cell, for example the ‘AND-gated’ Synthetic Notch receptor (SynNotch). On ligation of the binding domain with its target antigen, a transcription factor is cleaved from the receptor, which drives the transcription of the desired cellular program. The SynNotch system has been applied to multiple cancer models

[0062] OR-gating is a multi-antigen approach requiring the recognition of either one or more of the targeted CAR T cell antigens. This strategy expands the CAR T cell antigen repertoire and therefore increases the likelihood of tumor eradication for heterogeneous tumors. There are a variety of different approaches to the OR-gated CAR T cell concept, including pooling a combination of single antigen targeting CAR T cells or engineering a single CAR receptor totarget multiple antigens termed tan- or biCARs. CAR T cells can also be engineered to express CARs targeting two different antigens (dual CAR T), as well as three or more antigens such as triCARs or quad-CARs.

[0063] Inhibitory NOT- logic gates prevents T cell activation on target recognition. NOT-gating can be applied to tumor antigens that lack high tumor specific and display a low level of expression on healthy cells. Inhibitory CARs (iCARs) are designed with inhibitory signaling domains such as those found in PD-1 and CTLA-4. iCARs can be used in combination with CARs specific for TAAs to reduce the likelihood of healthy tissue damage. Biological function (cytotoxic T cell responses) will only occur if the TAA-CAR AND-NOT- the healthy antigen CAR are ligated. In this way, the biological gating system is a combination of AND-NOT systems.

[0064] In some embodiments a CAR is designed for expression by a macrophage, see for example Hadiloo et al. (2023) Biomarker Research volume 1 1 , Article number: 103, herein specifically incorporated by reference. The CAR structure for macrophages and T cells is generally the same but differs in the intracellular domain. For example, CD147, FcRy, and Megfl 0 may be used in CAR-Ms, while CD3^ is a common intracellular domain utilized in both. The manufacturing process of CAR-M cells is like that of CAR-T and CAR-NK cells. First, the cells arere collected from a promising source, and with unique gene-modified methods, the CAR-gene is inserted into the cell, and the achieved cells were transformed to the culture environment for cell expansion.

[0065] CAR-NK cells have a similar structure to a CAR designed for T cells, see for example Wang, et al. (2024) Cell Death Discov. 10, 40, herein specifically incorporated by reference. CAR-NK achieves gene transduction through various methods, including retroviruses, lentiviruses, electroporation, liposomes, and DNA transposons. Electroporation and liposome transfection can also effectively introduce exogenous genes into NK cells, and the transferred genes express quickly, the level of apoptosis is low, and the inter-individual variability is small. DNA transposons are mobile DNA elements that can be efficiently transposed between vectors and chromosomes through a “cut and paste” mechanism, resulting in CAR-iPSC-NK cells stably expressing CAR molecules. NK cells have cytotoxic activity and function most similarly to CD8 + T cells. The low risk of rejection of NK cells allows NK cells in CAR-NK to be generated from a variety of sources. NK cells originate from the spleen, liver, secondary lymphoid organs, thymus, intestine, tonsil, and uterus. Unlike B cells and T cells, NK cells can non-specifically kill tumor cells and virus-infected cells without prior sensitization.

[0066] Effector anti-CD176 CAR cells include autologous or allogeneic immune cells having cytolytic activity against a target cell expressing CD176, including malignant cancer cells. The effector cells may have cytolytic activity that does not require recognition through the T cellantigen receptor. In some embodiments, a T cell, NK cell, macrophage, etc. is engineered to express an anti-CD176 CAR. The term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and / or T cell antigen receptor.

[0067] In some embodiments, the engineered cells comprise a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy- Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01 174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”.

[0068] In other embodiments, the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2018) Cells. 7(10) E155. In some embodiments an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor and a cellular product suitable for all patients independent of HLA type provides an alternative. A universal ‘off the shelf CAR T cell product provides advantages in uniformity of harvest and manufacture.

[0069] Allogeneic T cells can be genetically modified to reduce graft v host disease. For example, the TCRap receptor can be knocked out by different gene editing techniques. TCRap is a heterodimer and both alpha and beta chains need to be present for it to be expressed. A single gene codes for the alpha chain (TRAC), whereas there are 2 genes coding for the beta chain, therefore TRAC loci KO has been deleted for this purpose. A number of different approaches have been used to accomplish this deletion, e.g. CRISPR / Cas9; meganuclease; engineered l-Crel homing endonuclease, etc. See, for example, Eyquem et al. (2017) Nature 543:1 13-117, in which the TRAC coding sequence is replaced by the CAR coding sequence; and Georgiadis et al. (2018) Mol. Ther. 26:1215-1227, which linked CAR expression with TRAC disruption by clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 without directly incorporating the CAR into the TRAC loci. An alternative strategy to prevent GVHD modifies CAR-T cells to express an inhibitor of TCRap signaling, for example using a truncated form of CD3 as a TCR inhibitory molecule.

[0070] Allogeneic T cells may be administered in combination with intensification of lymphodepletion to allow CAR-T cells to expand and clear malignant cells prior to host immune recovery, e.g. by administration of Alemtuzumab (monoclonal anti-CD52), purine analogs, etc. The allogeneic T cells may be modified for resistance to Alemtuzumab, and currently in clinicaltrials. Gene editing has also been used to prevent expression of HLA class I molecules on CAR-T cells, e.g. by deletion of [32-microglobulin, see NCT03166878.

[0071] In addition to modifying T cells, induced pluripotent stem (iPS) CAR-T cells can provide a source of allogeneic CAR-T cells. For example, transducing donor T cells with reprogramming factors can restore pluripotency, and are then re-differentiated to T effector cells.

[0072] Immune cells for engineering as described above are collected from a subject or a donor, and may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[0073] Techniques for affinity separation may include magnetic separation, using antibody- coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the selected cells. The affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above. In addition to antibody reagents, peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.

[0074] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal calf serum (FCS).

[0075] The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0076] The engineered cells may be infused to the subject in any physiologically acceptable medium by any convenient route of administration, normally intravascularly, although they may also be introduced by other routes, where the cells may find an appropriate site for growth. Usually, at least 1 x10scells / kg will be administered, at least 1 x107cells / kg, at least 1 x108cells / kg, at least 1 x109cells / kg, at least 1 x101° cells / kg, or more, usually being limited by the number of T cells that are obtained during collection.

[0077] Expression construct: The glycan-specific anti-CD176 CAR construct coding sequence may be introduced on an expression vector into a cell to be engineered. The viral delivery of CRISPR components has been extensively demonstrated using lentiviral and retroviral vectors. Alternatively, a CAR coding sequence may be introduced into the site of the endogenous T cell receptor, e.g. TRAC gene, e.g., using CRISPR technology (see, for example Eyquem et al. (2017) Nature 543:113-1 17; Ren et al. (2017) Protein & Ceil 1 -10; Ren et al. (2017) Oncotarget 8(10):17002-17011 ). CRISPR / Cas9 system or variants thereof can be directly applied to human cells, e.g. by transfection with a plasmid that encodes Cas9 and sgRNA, transfection with TRAC targeting by preloading an sgRNA onto Cas protein (RNP) and transfection of the complex, etc. Gene editing with CRISPR encoded by non-integrating virus, such as adenovirus and adenovirus-associated virus (AAV), also finds use. Recent discoveries of smaller Cas proteins have enabled and enhanced the combination of this technology with vectors that have gained increasing success for their safety profile and efficiency, such as AAV vectors.

[0078] The nucleic acid encoding an anti-CD176 CAR construct is inserted into a vector for expression and / or integration. Many such vectors are available. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like.

[0079] Expression vectors may contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.

[0080] Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that signals the secretion of thepolypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[0081] Expression vectors will contain a promoter that is recognized by the host organism and is operably linked to the anti-CD176 CAR construct coding sequence. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known.

[0082] Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus LTR (such as murine stem cell virus), hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, or from heat-shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The EF1 a promoter for lentiviral integration find use, or the LTR itself for gamma-retrovirus (MSGV).

[0083] Transcription by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp in length, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, oc-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.

[0084] Expression vectors for use in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequencesare commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the abovelisted components employs standard techniques.

[0085] Suitable host cells for cloning or expressing an anti-CD176 CAR construct are the prokaryotic, yeast, or other eukaryotic cells described above. Examples of useful mammalian host cell lines are mouse L cells (L-M[TK-], ATCC#CRL-2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0086] Host cells, including T cells, stem cells, etc. can be transfected with the abovedescribed expression vectors for anti-CD176 CAR construct expression. Cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Mammalian host cells may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.Polypeptide and Polynucleotide Compositions

[0087] Polypeptide constructs and compositions are provided, which comprise PNA covalently linked, e.g. as a single polypeptide fused in frame, to an effector polypeptide of a CAR. In some embodiments an anti-CD176 CAR is expressed by a human immune cell. In some embodiments an anti-CD176 CAR is a bi-specific CAR, where a second antigenic specificity may be an antigen present on hematologic malignant cells, e.g. CD123, FLT3, TIM3, CD99, CD96, B7-H3, etc. In other embodiments an engineered cell expresses an anti-CD176 CAR and a second CAR with specificity for an antigen present on hematologic malignant cells.

[0088] Also provided are isolated nucleic acids encoding the anti-CD176 CAR and constructs thereof, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the polypeptide constructs. Nucleic acids of interest encode a polypeptide that is at least about 80% identical to the provided polypeptide sequences, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or identical. Polynucleotide sequences may encode any or all of the provided CDR sequences, or may encode a complete variable region, an scFv, a complete polypeptide construct such as a CAR, and antibody, and the like. As is known in the art, a variable region sequence may be fused to any appropriate constant region sequence.

[0089] In some embodiments, a vector comprising a coding sequence that encodes an anti- CD176 CAR or anti-CD176 CAR construct is provided, where the coding sequence is operably linked to a promoter active in the desired cell; or is provided in a vector suitable for genomic insertion, e.g., by CRISPR. Various vectors are known in the art and can be used for this purpose, e.g., viral vectors, plasmid vectors, minicircle vectors, which vectors can be integrated into the target cell genome, or can be episomally maintained.

[0090] Compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Proteins can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0091] The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0092] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such asoctadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0093] In another embodiment of the invention, an article of manufacture containing in isolated polypeptide or polynucleotide is provided. The article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a polypeptide or polynucleotide composition, which may be a therapeutic composition, e.g. for treatment of cancer, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). A label on or associated with the container may indicate that the composition is used for treating the condition of choice. Further container(s) may be provided with the article of manufacture which may hold, for example, a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.Cell Compositions

[0094] In some embodiments, an engineered cell is provided, in which the cell has been modified by introduction of an anti-CD176 CAR. In some embodiments the cell is a T cell, including without limitation naive CD8+ T cells, cytotoxic CD8+ T cells; etc. In other embodiments, the engineered cell is a stem cell, e.g. a hematopoietic stem cell, or an iPSC. In some embodiments, the cell is genetically modified in an ex vivo procedure, prior to transfer into a subject. The engineered cell can be provided in a unit dose for therapy, and can be allogeneic, autologous, etc. with respect to an intended recipient. In other embodiments the engineered cell is a macrophage, NK cell, B cell, etc.

[0095] Methods may include a step of obtaining desired cells, e.g., T cells, hematopoietic stem cells, macrophages, NK cells, etc., which may be isolated from a biological sample, or may be derived in vitro from a source of progenitor cells, e.g. iPSC. The cells are transducedor transfected with a vector comprising a sequence encoding the receptor, which step may be performed in any suitable culture medium. As discussed above, the vector may integrate a CAR coding sequence into the genomic site of a TCR chain, e.g. the TCRA site, or may provide for expression from an exogenous promoter.

[0096] For example, cells may be collected from a cancer patient, modified ex vivo to express an anti-CD176 CAR, and reintroduced into the subject. The cells collected from the subject may be collected from any convenient and appropriate source, including e.g., peripheral blood (e.g., the subject’s peripheral blood), a biopsy (e.g., a tumor biopsy from the subject), and the like. In some instances, the cells collected may be tumor infiltrating lymphocytes (TILs), e.g., TILs collected from a tumor of a subject.

[0097] Where the use of autologous cells is not desirable, e.g. where a patient has insufficient T cells for modification, where there is insufficient time to expand autologous cells, etc., allogeneic cells may be used, e.g. T cells or stem cells from a healthy donor. As discussed herein, such allogeneic cells can be genetically modified to reduce GVHD, to reduce host versus graft responses, etc.

[0098] In some instances, modification of cells to generate CAR cells will be limited to introduction of an anti-CD176 CAR. In other instances, the immune cell is modified to express a second CAR, e.g. a tandem CAR, an iCAR, and the like. The second CAR can provide for improved specificity to tumor cells, by reducing activity of the CAR-T cell to normal cells expressing CD176.

[0099] Engineered cells can be provided in pharmaceutical compositions suitable for therapeutic use, e.g. for human treatment. Therapeutic formulations comprising such cells can be frozen, or prepared for administration with physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions. The cells will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0100] The cells can be administered by any suitable means, usually parenteral. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration.Methods of Treatment

[0101] The invention further provides methods for reducing growth of cancer cells, e.g., hematologic malignancies. The methods provide for decreasing the number of cancer cellsexpressing CD176. In general, the methods comprise contacting a cancer cell with an anti- CD176 CAR cells, usually contacting in vivo under conditions that cause cell death of the CD176 expressing cancer cells, e.g. by cell mediated cytotoxicity, in a dose sufficient to reduce cancer cell growth and treat the cancer.

[0102] "Reducing growth of cancer cells" includes, but is not limited to, reducing proliferation of cancer cells, and reducing the numbers of viable cancer cells in a patient tissue, e.g. blood, bone marrow, lymph nodes, etc. Whether a substance, or a specific amount of the substance, is effective in treating cancer can be assessed using any of a variety of known diagnostic assays for cancer, including, but not limited to biopsy, contrast radiographic studies, CAT scan, and detection of a tumor marker associated with cancer in the blood or biopsy of the individual.

[0103] In some embodiments, the methods may include administering to a subject in need thereof an effective amount of immune cells expressing an anti-glycan CAR, e.g. an anti- CDD176 CAR. In some embodiments the immune cells are CD8+ T cells. In one embodiment, a subject having a malignancy is administered an effective amount of autologous or allogeneic engineered immune cells expressing an anti-CD176 CAR to treat the cancer. The CAR cell population may be engineered and expanded ex vivo. In addition to CD176, the CAR or a second CAR present in the cell, may recognize a second antigen present on the surface of the cancer cells such that, upon binding of both the second antigen and CD176, the immune cell expressing the CAR is activated. Individual immune cells of the population may express the CAR and a second CAR, e.g. an iCAR or TAN-CAR. An immune response may be manifest as an increase in the cytolytic response of cells towards the target cells present in the recipient, e.g. towards elimination of tumor cells; and the like.

[0104] Where the contacting is performed in vivo, an effective dose of engineered cells is infused to the recipient. Dosage and frequency may vary depending on the agent; mode of administration; nature of the cytokine; and the like. It will be understood by one of skill in the art that such guidelines will be adjusted for the individual circumstances. The dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. intramuscularly (i.m.), intraperitoneally (i.p.), intravenously (i.v.), and the like. Generally at least about 104engineered cells / kg, at least about 105engineered cells / kg; at least about 106engineered cells / kg, at least about 107engineered cells / kg, or more are administered to the recipient.

[0105] Administration of a CAR cell may be combined with additional therapeutic agents. The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours,1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0106] Chemotherapeutic agents that can be administered in combination with an anti-CD176 CAR engineered cell include, without limitation, abitrexate, adriamycin, adrucil, amsacrine, asparaginase, anthracyclines, azacitidine, azathioprine, bicnu, blenoxane, busulfan, bleomycin, camptosar, camptothecins, carboplatin, carmustine, cerubidine, chlorambucil, cisplatin, cladribine, cosmegen, cytarabine, cytosar, cyclophosphamide, cytoxan, dactinomycin, docetaxel, doxorubicin, daunorubicin, ellence, elspar, epirubicin, etoposide, fludarabine, fluorouracil, fludara, gemcitabine, gemzar, hycamtin, hydroxyurea, hydrea, idamycin, idarubicin, ifosfamide, ifex, irinotecan, lanvis, leukeran, leustatin, matulane, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, mithramycin, mutamycin, myleran, mylosar, navelbine, nipent, novantrone, oncovin, oxaliplatin, paclitaxel, paraplatin, pentostatin, platinol, plicamycin, procarbazine, purinethol, ralitrexed, taxotere, taxol, teniposide, thioguanine, tomudex, topotecan, valrubicin, velban, vepesid, vinblastine, vindesine, vincristine, vinorelbine, VP-16, and vumon.

[0107] Targeted therapeutics that can be administered in combination with an anti-CD176 CAR engineered cell may include, without limitation, tyrosine-kinase inhibitors, such as Imatinib mesylate (Gleevec, also known as STI-571 ), Gefitinib (Iressa, also known as ZD1839), Erlotinib (marketed as Tarceva), Sorafenib (Nexavar), Sunitinib (Sutent), Dasatinib (Sprycel), Lapatinib (Tykerb), Nilotinib (Tasigna), and Bortezomib (Velcade), Jakafi (ruxolitinib); Janus kinase inhibitors, such as tofacitinib; ALK inhibitors, such as crizotinib; Bcl-2 inhibitors, such as obatoclax, venclexta, and gossypol; FLT3 inhibitors, such as midostaurin (Rydapt), IDH inhibitors, such as AG-221 , PARP inhibitors, such as Iniparib and Olaparib; PI3K inhibitors, such as perifosine; VEGF Receptor 2 inhibitors, such as Apatinib; AN-152 (AEZS-108) doxorubicin linked to [D-Lys(6)]-LHRH; Braf inhibitors, such as vemurafenib, dabrafenib, and LGX818; MEK inhibitors, such as trametinib; CDK inhibitors, such as PD- 0332991 and LEE011 ; Hsp90 inhibitors, such as salinomycin; and / or small molecule drug conjugates, such as Vintafolide; serine / threonine kinase inhibitors, such as Temsirolimus (Torisel), Everolimus (Afinitor), Vemurafenib (Zelboraf), Trametinib (Mekinist), and Dabrafenib (Tafinlar).

[0108] An anti-CD176 CAR engineered cell may be administered in combination with an immunomodulator, such as a cytokine, a lymphokine, a monokine, a stem cell growth factor, a lymphotoxin (LT), a hematopoietic factor, a colony stimulating factor (CSF), an interferon(IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, a transforming growth factor (TGF), such as TGF-oc or TGF- , insulin-like growth factor (IGF), erythropoietin, thrombopoietin, a tumor necrosis factor (TNF) such as TNF-a or TNF-fS, a mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), an interferon such as interferon-a, interferon-p, or interferon-y, S1 factor, an interleukin (IL) such as IL-1 , IL-1 cc, IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL- 18 IL-21 or IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, and LT.

[0109] T umor specific monoclonal antibodies that can be administered in combination with an anti-CD176 CAR engineered cell may include, without limitation, Rituximab (marketed as MabThera or Rituxan), Alemtuzumab, Panitumumab, Ipilimumab (Yervoy), etc.Kits

[0110] Also provided are kits for use in the methods. The subject kits may include an expression vector encoding the anti-CD176 CAR construct. In some embodiments, the components are provided in a dosage form (e.g., a therapeutically effective dosage form), in liquid or solid form in any convenient packaging (e.g., stick pack, dose pack, etc.). Reagents for the selection or in vitro derivation of cells may also be provided, e.g. growth factors, differentiation agents, tissue culture reagents; and the like.

[0111] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

[0112] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.EXPERIMENTALEXAMPLE 1

[0113] Our innovation lies in incorporating a novel lectin-based targeting domain specific for a carbohydrate antigen within a cell therapy receptor construct, and developing the first cell therapy that targets the T-antigen, in a peptide agnostic manner (Figure 1 ). The therapy provides new insights into the ability to target glycosylation motifs on carcinomas for treatment of these tumors. Our CAR approach exploits the high valency of the cancer-associated motif by leveraging the high avidity interactions between lectins and glycans. Given cytotoxic activation of CAR-T cells increases with high avidity binding, our cell based therapy will generate a robust anti-tumor response that would be difficult to achieve using a monoclonal antibody or targeted drug conjugate approach alone.

[0114] Unlike most engineered cell therapies, our CAR-T directly targets a functional mediator of disease progression, and in doing so, renders evolutionary escape through loss of the antigen, as such adaptations would result in reduced metastatic capacity of the tumor. Finally, targeting metastases in lymph nodes and distant metastases may disrupt metastatic tolerance yielding a more effective endogenous immune response to immunotherapy.

[0115] Tumor-associated carbohydrate motifs are expressed on carcinomas and metastases. The expression of T-antigen is increased on both murine and human lung carcinoma cell lines with increasing metastatic potential (Figure 2 A,B). The T-antigen is highly expressed on several lung adenocarcinoma cell lines from patients of varying ethnic backgrounds suggesting a conserved mechanism of tumorigenesis and disease progression. We have also shown that T-antigen has high expression in several human pancreatic cell lines, in comparison to negative control THP-1 (AML) and positive control A549 (lung adenocarcinoma) (Figure 2C). The T-antigen is highly specific for lung adenocarcinomas and associated lymph node metastases in comparison to normal lung parenchyma and lymph node tissues (Figure 2D). The high degree of tumor specificity supports means that targeting this antigen can preferentially treat metastatic lesions in distant organs and lymph nodes with minimal toxicity to normal tissues.

[0116] Generation of CAR-T cells binding T-antigen. Using lentiviral constructs, a second- generation 4-1 BBz CAR-T cell was generated against the T antigen (Figure 3A) that expresses our targeting receptor on both transduced human CD8+ and CD4+ T cells (Figure 3B). We subsequently demonstrated CAR-T cell proliferation and interferon gamma (I FNy) production in response to co-culture with tumor cells, and this response was blunted when the competitive receptor N-Acetyl-galactosamine (GalNac) was added to the co-culture, thus demonstrating specificity for the T-Antigen (Figure 3C,D).

[0117] Additionally, T-antigen specificity is shown in a cytotoxicity assay where tumor cytotoxicity correlates with increasing CAR-T cells to tumor cell ratios in comparison to ourcontrol CAR-T (Figure 3E). Thus, we have designed and generated a cell therapy with a novel lectin-based targeting receptor that specifically targets the T-antigen in human lung adenocarcinoma in a peptide agnostic manner.

[0118] It is believed that the lectin-based targeting receptor against the T-antigen activates T cells via high avidity multivalent binding, thus enabling cytotoxic killing in the majority of metastatic carcinomas where the T-antigen is highly expressed. As this truncated glycan motif is not seen on the surface of normal tissues or at low density, with high avidity T cell activation due to high TACA expression, we avoid any potential on-target, off-tumor cytotoxic effects.

[0119] Valency dependence for activation of T-antigen CAR-T cells. T o determine the degree to which the CAR-T is reliant upon varying epitope expression, we test for T antigen CAR-T cell activation in response to increasing concentrations of plate-immobilized T-antigen via Cell- Trace Violet cell proliferation assays, intracellular cytokine (interleukin-2 (IL-2), interferon gamma (IFNy), tumor necrosis factor a (TNFa), CD107a) staining by flow cytometry, and serum cytokine measurement with cytometric bead array (IL-12p70, TNF, IL-2, IFNy, IL-6). In comparison, we also test for TACA specificity of our CAR-T cells against other immobilized truncated O-glycans such as sTn and Tn antigen.

[0120] Differences in T-antigen CAR-T activation in cell lines with varying T antigen surface expression. To determine the anti-tumor response of CAR-T cells in low versus high TACA expression in murine models, activation of murine CD8+ and CD4+ CAR-T cells is tested in vitro against previously developed murine cell lines of non-metastatic T-antigenlowand metastatic T-antigenhi9hlung adenocarcinoma (804T4, 393M4 respectively) derived from the autochthonous KrasLSL G12D / +;p53,lox / floxgenetically engineered mouse model (GEMM). We measure cytotoxicity via IncuCyte® live cell imaging, which dynamically quantifies CAR-T tumor cytotoxicity over 72-96 hour time intervals. We similarly evaluate for activation of CAR- T cells in vitro when co-cultured with human cell lines of T antigen10™ lung adenosquamous versus T-antigenh'9hlung adenocarcinoma (NCI-H322, A549). We quantify preferential cell killing via a competitive cytotoxicity assay using time-lapsed confocal microscopy over 12 hours with Cell-trace Violet labeled NCI-H322 vs. CellTracker Red CMTPX labeled A549. Additionally, we assess for proliferation, intracellular cytokines, and effector cytokine release.

[0121] T-antigen CAR-T cell anti-tumor response against primary tumor vs. metastases in vivo. In order to recapitulate effects of the immune system on tumors, CAR-T cell responses against primary vs. metastatic disease in immune competent murine models of metastases in both lung adenocarcinoma (804T4, 393M4) and transplantable syngeneic melanoma LN metastasis model (B16-F0, LN9) are assessed. On Day 0, 1 -1 .5 x 106non-metastatic vs. metastatic cells are orthotopically implanted on contralateral flanks and tumor growth is monitored. On day 2, 2-4 x 106autologous mWasabi-expressing anti-T-antigen CAR-T cellscombined CD8+ and CD4+ cells at 1 :1 ratio are adoptively transfered, where tumor growth kinetics and overall survival are endpoints. We assess tumor size reduction, serum metabolic electrolytes, and cytokines via Luminex pro-inflammatory murine panel at 24 and 48 hour time points via retro-orbital blood draw to monitor for electrolyte abnormalities and cytokine release syndrome. We determine whether on-target, off-tumor toxicities exist by histologic analyses of organs and staining for the CAR-T cells. Proliferation and persistence of these CAR-Ts are analyzed by flow cytometry of serum, spleen, bone marrow, and tumor at day 60 post infusion after adoptive transfer of 5x106CAR-T cells. We evaluate the ability of the CAR-T cells to elicit functional memory responses by performing tumor rechallenge experiments wherein we transplant metastatic cell lines into mice that have previously cleared their tumors 70 days later. Tumors are harvested in 10 days to determine total numbers of CD3+ mWasabi labeled CAR-T cells.

[0122] Evaluate anti-tumor response of T-antigen CAR-T in clearance of metastases in vivo. To assess efficacy against metastatic tumors, an experimental liver metastasis model for lung adenocarcinoma and lung metastasis model for melanoma, respectively, is employed. For lung adenocarcinoma model, we perform intrasplenic injections of 5x106393M4 cells, allowing for seeding of the liver, followed by treatment with our T-antigen CAR-T cells or control CAR- T cells on day 10. For the melanoma model, we perform intravenous injections of 5 x105LN9 cells, allowing for seeding of the lungs, followed by treatment with our T-antigen CAR-T cells or control CAR-T cells. Four weeks following CAR-T infusion, we euthanize the mice and evaluate liver and lung metastatic burden respectively.

[0123] Evaluation of T-antigen CAR-T cell efficacy in patient derived xenograft models. Autologous T-antigen CAR-T cells are generated to test against individual patient derived tumors of non- small cell lung adenocarcinoma. Through our Human Tissue Bank, we have access to deidentified patient derived fresh tumors with matched blood for CAR-T synthesis. Using our Stanford Transgenic, Knockout, and Tumor Model core, we subcutaneously implant 1 -3 x106patient derived tumor cells in NSG mice on day 0 and adoptively transfer 2-4 x 106autologous anti-T antigen CAR T cells generated from matched blood sample on day 14 after ex vivo CAR-T expansion. Tumor volume with Tantigen specific CAR-T vs. control is monitored longitudinally via 2-perpindicular caliper measurements and metastatic burden is evaluated in distant tissues by histology. Markers of exhaustion are determined prior to infusion of CAR-Ts and in tumors of responders and nonresponders.

[0124] Screening of T-antigen binding on human tumors and normal tissue microarrays. In order to select tumor type and help determine patient eligibility criteria for a clinical trial, several tumor types are screened for T-antigen expression, including, lung (NSCLC and small cell carcinoma), head and neck, gastrointestinal, breast, genitourinary malignancies, using both commercially available sources (US Biomax) and internally developed tissue microarrays.Additionally, we screen tissue microarrays of normal human tissue to determine whether there is receptor binding in normal tissues (US Biomax FDA999 series).Methods:

[0125] Generation of CAR-T cells. Human T cells are isolated from de-identified leukocyte reduced chambers on the day of blood donation by magnetic labeled negative selection (StemCell EasySep). They are stimulated for 24 hours with CD3 / CD8 Dynabeads (ThermoFisher Scientific) in T-cell media (TCM) supplemented with 30 lU / mL IL-2 (Peprotech). HEK293GP2(retrogamma vector) or HEK293FT(lentiviral vector) cells are cultured in collagen coated plates and transfected with Lipofectamine 3000 (Thermo Fisher Scientific), VSV-g plasmid, and our CAR-T constructs with ViralBoost (AllstemBio). Viral supernatant will be collected at 48 hours and concentrated via spinnoculation prior to transduction with RetroNectin (Takara Bio) in TCM supplemented with 10 ng / mL IL7 and IL-15 (R&D Biosciences). mWasabi labeled cells are sorted by FACs Aria II for in vitro assays or to further expand for in vivo assays. Insertion site analysis will be conducted with Takara Lenti-X™ Integration Site Analysis Kit.

[0126] Flow cytometry. For cell surface staining, monoclonal antibodies conjugated to BUV496, BUV 737, BUV805, PERCP, BV605, PE-Cy7, and Live / Dead Zombie Aqua. Antibody panel for the intracellular evaluation: CD3 (UCHT1 ), CD45 (HI30), CD8 (SK1 ), CD4 (A161 A1 ), CD107a (LAMP-1 ), TNFa (Mab1 1 ), INFg (XMG1.2), CD69 (FN50) from BioLegend and BD Biosciences. Memory panel will have antibodies: CD3 (UCHT1 ), CD4 (A161 A1 ), CD8 (SK1 ), CD45RA (HI100), CD45RO (UCHL1 ), CCR7 (G04387), CD62L (DREG-56), CD95 (DX2). Antibody exhaustion panel: CD3 (UCHT1 ), CD4 (A161 A1 ), CD8 (SK1 ), LAG3 (11 C3C65), PD-1 (EH12.2H7), TIM-3 (F38-2E2), and CD39 (FN50). T-antigen receptor staining is conjugated to AlexaFluor647.

[0127] CART constructs have mWasabi labeling, while negative CAR-T control have control dTomato instead of a targeting receptor. For in vitro assays, positive control using PMA- lonomycin is utilized, where cells will be activated for 4 hours. Flow cytometry is performed on BD Fortessa and datasets are analyzed using FlowJo software (Tree Star, Inc.).

[0128] Immunohistochemistry. Paraffin-embedded tissues microarrays undergo antigenretrieval in citrate buffer and blocked with Carbo-Free Blocking Solution (Vector Laboratories). Endogenous peroxidase activity was blocked using the Dual Endogenous Enzyme Block (Dako). Subsequently, tissues are stained followed by overnight receptor-HRP staining. Tissues are developed using DAB and counterstained with hematoxylin. Sections are examined under a Zeiss Laser Scanning Confocal Microscope. CAR-T infiltration is conducted by staining for human CD4, CD8, and CD3 on excised mouse and human tumor specimens.Images are collected using a Zeiss 700 confocal laser scanning microscope, and analyzed using ZEN software (Carl Zeiss Microscopy).

[0129] Statistics. Animal sample sizes are determined based on power analyses in order to reach statistical significance when comparing k means using One-Way ANOVA pairwise comparisons with 2-sided equality using software provided by HyLown Consulting. The power is set to 0.9 and the Type I error rate is set to 0.05, errors approximated from previously reported studies. Estimates of variances is approximated from our data and reports in the literature. Cell populations are compared by flow cytometry using a probability binning test (FlowJo, Tree Star Inc.). Whenever non-Gaussian distributions exist, non-parametric tests are used including the Wilcoxon rank-sum test for continuous variables and Fisher’s Exact test for categorical analyses. All statistics will be evaluated using either Prism (GraphPad Software) or the statistics package. Multiple comparisons are evaluated by ANOVA with a Tukey or Bonferroni post-hoc test. TumGrowth is utilized for statistical analysis of survival curves.Example 2

[0130] T-antigen is an O-linked oncofetal disaccharide found on cancer cells. It is a core structure of glycosylation, that is not typically seen on normal tissues. Shown in FIG. 5A is a schematic of O-linked glycosylation and glycosyltransferases, starting from a core glycan. Peanut lectin agglutin (PNA)-horse radish perioxidase staining of a human microarray of both normal and tumor involved lymph node, and normal lung parenchyma and non-small cell lung cancer (NSCLC) show expression of T-antigen. T-antigen expression via PNA-APC staining across lung cancer cell lines by flow cytometry (white- murine, black-human) is shown in FIG. 5C. While T-antigen is not or lowly expressed on healthy tissues it has elevated expression on a wide variety of carcinomas, including colorectal carcinoma cells, pancreatic adenocarcinoma cells and non-small cell lung carcinoma cells (FIG. 6). Shown in FIG. 7 are a panel of human tumors evaluated for transcriptional expression of glycosyltransferases. Expression in lung cancer is further confirmed with PNA staining, shown in FIG. 8.

[0131] T-Antigen targeting PNA-CAR T cells exhibit potent and antigen-specific anti-tumor immunity in vitro. To assess the efficacy, CAR-T cells were co-cultured with tumor cells and subjected to multiple assays. To evaluate proliferation following target exposure, 1x104CAR- T cells were labeled with CellTrace Violet (CTV) Proliferation Dye (Thermo, C34557) prior to incubation with 1 x104A549 T-Antigen-expressing tumor cells in a 96 round bottom plate supplemented with DMEM + 10% FBS + pen / strep+ glutamine. After 3 days, the fraction of proliferating CAR cells was evaluated by CTV dilution by flow cytometry on BD LSRFortessa. Anti-CD3 / CD28 beads (Thermo, 1 1 132D) were incubated with CAR cells as a positive control. T o evaluate ligand specificity, co-cultures were incubated with 10OmM of a soluble competitive ligand, N-Acetyl-galactosamine (Sigma Aldrich A2795). CAR-T cells exhibited significantproliferation when co-cultured with tumors, and this proliferation was markedly reduced when incubated with the soluble competing ligand (FIG. 9A-B). Cytokine production was evaluated after 24 hours of co-culture by cytokine bead arrays (BD 560484). These studies demonstrated significant interferon-gamma (IFNy) production that was inhibited by co-culture with the soluble competitive ligand (FIG. 9C). Anti-tumor cytotoxicity was evaluated by co-culture of CAR-T cells with A549 tumor cells at various effectortarget (CAR:tumor) ratios and evaluated by 7AAD staining. E:T ratios of 5:1 and 10:1 both exhibit significant killing of target cells. Each dot is an individual human donor, shown in FIG. 9D.

[0132] T-antigen stimulates antigen-specific CART response. Cytokine profiles were determined by cytokine bead array (BD Biosciences 560484). CAR-T cells expressing the anti- CD176 CAR were co-cultured with human tumor cell lines that exhibit varying degrees of T- Antigen. The CART cells were then subjected to cytokine profiling by cytokine bead arrays. Shown in FIGS. 10A-10B shows the proliferation of the CART cells in the presence of T- Antigen high-expressing (A549) and low-expressing (NCI-H322) tumor cells. These CAR-T cells produce little to no cytokine alone or when cultured with THP-1 AML cells (with low T- antigen expression). Significantly elevated cytokine expression was observed with NCI-H322 and A549 cells with the greatest amounts produced in the A549 co-cultures, shown in FIGS. 10C-E.

[0133] CART function with Granzyme B. T-Antigen targeting PNA-CAR T cells exhibit potent and antigen-specific anti-tumor immunity in vitro after 14 hour incubation with A549 as seen by flow cytometry (FIG. 1 1 A-11 E), shown by an increase in Granzyme B positive cells in the presence of T-antigen expressing tumor cells. FIG. 11 A. Fraction of Granzyme B positive cells. FIG. 1 1 B. % frequency of CD8+ CD3+ cells. FIG. 1 1 C. MFI granzyme B with CAR and with control. FIG. 1 1 D-F. % of CD8 T cells that are Granzyme B+. FIG. 11 D. PMA and ionomycin stimulated CD8 T cells. FIG. 1 1 E. CART cells cultured with A549. FIG. 11 F. Control T cells cultured with A549. Control is the vector but with a tDT in place of PNA.

[0134] Activity of anti-CD176 CART cells in vivo. The in vivo activity of the engineered T cells was shown in an in vivo competition assay between T-antigenhiand T-antigenl0human tumor cells in a xenograft model. NOD-scid-gamma (NSG) mice were transplanted subcutaneously (s.c.) with T-Antigen high-expressing (A549) and low-expressing (NCI-H322) tumor cells on contralateral flanks with 1 x106cells. One week after tumor implantation, mice were treated with intravenous (retrooribital) injections of anti-CD176 CAR-T cells at a ratio of 1 :1 CD8:CD4 T cells, at 1 x106dosing. Curves indicate tumor volume above baseline (at the time of treatment) for mice injected with control CAR0T cells or those specific for the T-Antigen. C. Some efficacy was observed in the low ligand expressing tumor cells, but the greatest efficacy (FIG. 12B) was observed in the high expressing tumors (A549s). These data show a nearly 2log decrease in tumor size for T-antigenhiexpressing cancer cells grown in vivo in the presence of anti-CD176 CART cells.SequencesSEQ ID NO:2 CAR construct with Wasabi reporter sequenceALPVTALLLPLALLLHAARPSAETVSFNFNSFSEGNPAINFQGDVTVLSNGNIQLTNLNKVNS VGRVLYAMPVRIWSSATGNVASFLTSFSFEMKDIKDYDPADGIIFFIAPEDTQIPAGSIGGGT LGVSDTKGAGHFVGVEFDTYSNSEYNDPPTDHVGIDVNSVDSVKTVPWNSVSGAVVKVTVI YDSSTKTLSVAVTNDNGDITTIAQVVDLKAKLPERVKFGFSASGSLGGRQIHLIRSWSFTSTL ITTTRRSIDNNEKKIMNMASATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP RGSGAT / VFSLL QAGDt / EEA / PGPSSVSKGEETTMGVIKPDMKIKLKMEGNVNGHAFVIEGE GEGKPYDGTNTINLEVKEGAPLPFSYDILTTAFSYGNRAFTKYPDDIPNYFKQSFPEGYSWE RTMTFEDKGIVKVKSDISMEEDSFIYEIHLKGENFPPNGPVMQKETTGWDASTERMYVRDG VLKGDVKMKLLLEGGGHHRVDFKTIYRAKKAVKLPDYHFVDHRIEILNHDKDYNKVTVYEIAVARNSTDGMDELYK*CD8a Signal Sequence residues 1 -20; Peanut Agglutinin residues 21 -271 ; CD8 hinge residues 272-340; 4-1 BB residues 341 -382; CD3zeta residues 383-494; P2A residues 495- 516; mWasabi residues 517 753.SEQ ID NO:3Nucleotide Sequence (including Kozak): gccgccaccatggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtcagccgaaaca gtttccttcaacttcaactctttcagcgaaggaaaccccgcaataaatttccaaggtgacgtcactgttctttcaaatggcaatataca actcacgaacctcaacaaggtaaatagcgtcggccgtgttctttatgccatgccggtgcgcatttggagcagtgccaccggcaat gtcgccagcttcctcacctccttctctttcgagatgaaggatatcaaagattatgatcctgccgacggtatcatctttttcattgcaccg gaagatacgcagattcctgccggcagtattggtggtggaaccttaggcgtctctgacactaaaggggcgggtcactttgttggagt ggagtttgatacctattccaacagtgagtacaacgatccacccactgatcacgttggaattgatgtaaatagcgtggattcggtga agaccgtgccatggaatagtgtgagtggagcagtggtgaaagtgactgtgatatatgactcttcaacaaagacattgagtgttgct gtgaccaacgacaatggcgatattaccaccattgctcaagttgttgatttgaaggcgaagcttccggagagggtcaagttcggtttt tctgcctccggctcccttggcggtcgtcagatacatctcatccgttcatggtctttcacttcaaccttgataacaacaaccagaagaa gcatcgacaataacgaaaagaaaataatgaatatggcaagtgcaaccacgacgccagcgcctcgccctcctactcctgcccct actatcgccagtcaacccctcagtttgagacccgaggcctgtcgacccgccgctgggggggccgtgcacacaaggggactcg acttcgcttgtgatatatatatatgggcacccctcgctggaacttgtggcgtccttcttctctcacttgtgatcacattgtactgtaagcggggacggaagaagttgctgtacattttcaagcagccatttatgagacctgtccagacaacccaggaggaagatggatgctcttgcc ggttcccagaggaggaggagggtggctgcgagttgcgcgtaaaattctccagatccgctgacgctcctgcatacaaacagggc caaaaccaactctataacgagctgaatcttgggaggcgggaggaatacgacgtgttggacaaaaggcgcggccgagatcctg agatgggtggtaagccacgacgaaaaaacccacaagaagggctctataacgaacttcagaaagacaaaatggccgaggcc tacagtgaaattggaatgaaaggcgagcgcagaaggggaaaaggccacgatggactgtaccaaggcctttccacagccact aaggatacctacgatgcacttcacatgcaggcactccctccccggggaagtggagccactaacttttccctcttgaaacaggctg gagatgtcgaagagaatccagggcctagtagcgtctctaagggtgaggaaactactatgggcgtaatcaagcccgacatgaa aattaaattgaaaatggagggcaacgttaacggtcacgcatttgtgatcgagggggagggagaaggcaagccctacgatgga acaaatacaattaacctcgaggtgaaggaaggcgcacctcttccctttagttacgacattctgactactgcatttagctacggtaac agggcctttactaaatatcccgacgatatacctaattattttaagcaatcattccctgaggggtattcttgggagaggactatgacatt tgaggacaagggtatagtaaaggtaaagtccgatatcagcatggaggaagacagttttatatatgagatacaccttaaagggga gaattttccaccaaatggtccagtgatgcaaaaagagactactggttgggatgccagtaccgagcggatgtatgtgcgcgatggt gtgcttaaaggagacgtaaagatgaagctgctcctcgaaggcggaggacatcacagagtagactttaaaacaatatatagagc caaaaaggctgtgaaattgccagattaccatttcgtagatcaccgcattgaaattctcaaccacgataaggattataacaaagtca ctgtctatgaaatagccgtggcccgcaactccactgacggtatggacgagctctacaagtaaSEQ ID N0:4, CAR constructMALPVTALLLPLALLLHAARPSAETVSFNFNSFSEGNPAINFQGDVTVLSNGNIQLTNLNKVN SVGRVLYAMPVRIWSSATGNVASFLTSFSFEMKDIKDYDPADGIIFFIAPEDTQIPAGSIGGG TLGVSDTKGAGHFVGVEFDTYSNSEYNDPPTDHVGIDVNSVDSVKTVPWNSVSGAVVKVT VIYDSSTKTLSVAVTNDNGDITTIAQVVDLKAKLPERVKFGFSASGSLGGRQIHLIRSWSFTS TLITTTRRSIDNNEKKIMNMASATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PRCD8a Signal Sequence residues 1-20; Peanut Agglutinin residues 21 -271 ; CD8 hinge residues 272-340; 4-1 BB residues 341 -382; CD3zeta residues 383-494; P2A residues 495- 516.SEQ ID NO:5. DNA sequence of DNA sequence for plasmid of FIG. 13 tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagca gaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggt agaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagt gttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatc gagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcaga tcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatcc ctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagagga gctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaat tttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaa aaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcg cagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcag aagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagcttt agacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacctggag gaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccacc aaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcag gaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatt tgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctg tggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatg ctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaa gcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggca agtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaata gtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacc cgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgac ggtatcgcctttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacata caaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttatcgatg agtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtagaacccagag atcgctgcgttcccgccccctcacccgcccgctctcgtcatcactgaggtggagaagagcatgcgtgaggctccggtgcccgtca gtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtg gcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagta gtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctcttta cgggttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgg gtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttt tttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggccc gtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctg gccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttg cgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcg ggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtcc aggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactga gtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctca agcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaggatctatttccggtgaattcctcgagactagtgccgccac catggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgtcagccgaaacagtttccttcaacttcaactctttcagcgaaggaaaccccgcaataaatttccaaggtgacgtcactgttctttcaaatggcaatatacaactcacga acctcaacaaggtaaatagcgtcggccgtgttctttatgccatgccggtgcgcatttggagcagtgccaccggcaatgtcgccag cttcctcacctccttctctttcgagatgaaggatatcaaagattatgatcctgccgacggtatcatctttttcattgcaccggaagatac gcagattcctgccggcagtattggtggtggaaccttaggcgtctctgacactaaaggggcgggtcactttgttggagtggagtttga tacctattccaacagtgagtacaacgatccacccactgatcacgttggaattgatgtaaatagcgtggattcggtgaagaccgtgc catggaatagtgtgagtggagcagtggtgaaagtgactgtgatatatgactcttcaacaaagacattgagtgttgctgtgaccaac gacaatggcgatattaccaccattgctcaagttgttgatttgaaggcgaagcttccggagagggtcaagttcggtttttctgcctccg gctcccttggcggtcgtcagatacatctcatccgttcatggtctttcacttcaaccttgataacaacaaccagaagaagcatcgaca ataacgaaaagaaaataatgaatatggcaagtgcaaccacgacgccagcgcctcgccctcctactcctgcccctactatcgcc agtcaacccctcagtttgagacccgaggcctgtcgacccgccgctgggggggccgtgcacacaaggggactcgacttcgcttgt gatatatatatatgggcacccctcgctggaacttgtggcgtccttcttctctcacttgtgatcacattgtactgtaagcggggacggaa gaagttgctgtacattttcaagcagccatttatgagacctgtccagacaacccaggaggaagatggatgctcttgccggttcccag aggaggaggagggtggctgcgagttgcgcgtaaaattctccagatccgctgacgctcctgcatacaaacagggccaaaacca actctataacgagctgaatcttgggaggcgggaggaatacgacgtgttggacaaaaggcgcggccgagatcctgagatgggt ggtaagccacgacgaaaaaacccacaagaagggctctataacgaacttcagaaagacaaaatggccgaggcctacagtga aattggaatgaaaggcgagcgcagaaggggaaaaggccacgatggactgtaccaaggcctttccacagccactaaggatac ctacgatgcacttcacatgcaggcactccctccccggggaagtggagccactaacttttccctcttgaaacaggctggagatgtcg aagagaatccagggcctagtagcgtctctaagggtgaggaaactactatgggcgtaatcaagcccgacatgaaaattaaattg aaaatggagggcaacgttaacggtcacgcatttgtgatcgagggggagggagaaggcaagccctacgatggaacaaataca attaacctcgaggtgaaggaaggcgcacctcttccctttagttacgacattctgactactgcatttagctacggtaacagggccttta ctaaatatcccgacgatatacctaattattttaagcaatcattccctgaggggtattcttgggagaggactatgacatttgaggacaa gggtatagtaaaggtaaagtccgatatcagcatggaggaagacagttttatatatgagatacaccttaaaggggagaattttcca ccaaatggtccagtgatgcaaaaagagactactggttgggatgccagtaccgagcggatgtatgtgcgcgatggtgtgcttaaa ggagacgtaaagatgaagctgctcctcgaaggcggaggacatcacagagtagactttaaaacaatatatagagccaaaaag gctgtgaaattgccagattaccatttcgtagatcaccgcattgaaattctcaaccacgataaggattataacaaagtcactgtctatg aaatagccgtggcccgcaactccactgacggtatggacgagctctacaagtaaacgcgtctggaacaatcaacctctggattac aaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattg cttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgt ggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccct ccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtg gtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccct tcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacg agtcggatctccctttgggccgcctccccgcctggaattaattctgcagtcgagacctagaaaaacatggagcaatcacaagtag caatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggt acctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaagaggggactggaagggctaattcac tcccaacgaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagcagaactacacaccaggg ccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtgttagagtggaggtttg acagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatcgagcttgctacaaggg actttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatcctgcatataagcag ctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtc agtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgaga ggccttgacattgctagcgtttaccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatcc gctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaat tgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagagg cggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcact caaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag tcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgacc ctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtg taggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgag tccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtg ctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacctt cggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgc gcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagta aacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccc cgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggc tccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtct attaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtc acgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagc ggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctctt actgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagtt gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggg gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactt tcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttga atactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaata aacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcgggagatcaacttgtttattgcagctt ataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactca tcaatgtatcttatcatgtctggatcaactggataactcaagctaaccaaaatcatcccaaacttcccaccccataccctattaccac tgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaa acttagtagt

Claims

What is Claimed is:1 . A polypeptide comprising (i) a chimeric antigen receptor (CAR) effector domain, and (ii) a carbohydrate recognition domain (CRD) of peanut agglutinin (PNA) as a binding domain.

2. The polypeptide of claim 1 , wherein the (ii) carbohydrate recognition domain (CRD) is present in a mature PNA sequence.

3. The polypeptide of claim 1 or claim 2, wherein the (i) CAR effector domain comprises a transmembrane domain joined by a linker to a cytoplasmic signaling domain.

4. The polypeptide of claim 3, wherein the cytoplasmic signaling domain comprises an endodomain of human TCR .

5. The polypeptide of any of the preceding claims, further comprising an endodomain of an immune costimulatory protein.

6. The polypeptide of claim 5, wherein the immune costimulatory protein is one or more of 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

7. The polypeptide of claim 6, wherein the immune costimulatory protein is CD28.

8. The polypeptide of claim 6, wherein the immune costimulatory protein is 4-1 BB.

9. The polypeptide of any of the preceding claims, further comprising an endodomain of an immune co-inhibitory protein.

10. The polypeptide of claim 9, wherein the immune co-inhibitory protein is one or more of CTLA-4 and PD-1.1 1 . The polypeptide of any of the preceding claims, wherein the CAR is bi-specific for a second antigen other than CD176.

12. The polypeptide of any of the preceding claims, in combination with a second CAR specific for a second antigen, which forms a tandem CAR.

13. The polypeptide of claim 11 or 12, wherein the second antigen is an antigen present on a cancer cell.

14. The polypeptide CAR of any of claims 1 -13, wherein the CAR is a split CAR wherein the CAR and the cytoplasmic signaling domain of the CAR are present on two separate molecules.

15. A nucleic acid encoding the polypeptide according to any of the preceding claims.

16. A nucleic acid vector comprising the nucleic acid of claim 15.

17. The nucleic acid vector of claim 16, wherein the vector is an integrating vector.

18. The nucleic acid vector of claim 17, wherein the vector is suitable for genomic insertion by CRISPR.

19. The nucleic acid vector of claim 16, wherein the vector is episomally maintained.

20. A mammalian cell genetically engineered ex vivo to comprise the vector of any of claims 16-19, wherein the vector encodes a polypeptide comprising a CAR specific for human CD176 or a sialylated form thereof, and which cell expresses the CAR polypeptide.21 . The cell of claim 20, wherein the cell is a human cell.

22. The cell of claim 21 wherein the cell is an immune cell.

23. The cell of claim 22, wherein the cell is a T cell.

24. The cell of claim 23, wherein the T cell is a CD8+ T cell.

25. The cell of claim 20, wherein the cell is isolated from an individual with a malignancy.

26. The cell of claim 20, wherein the cell is isolated from a healthy donor.

27. The cell of claim 20, wherein the cell comprises a genetic modification to reduce graft versus host disease.

28. The cell of claim 20, wherein the cell comprises a genetic modification to reduce graft rejection.

29. The cell of any of claims 20-28, wherein the cell is present in a population of cells expanded in ex vivo culture.

30. A pharmaceutical formulation comprising a population of cells according to any of claims 20-29.31 . The pharmaceutical formulation of claim 30 in a unit dose formula.

32. A method of treating an individual for cancer, the method comprising: administering to an individual in need thereof an effective dose of population of cells according to any of claims 20-29 or a pharmaceutical formulation of claim 30 or 31 .

33. The method of claim 32, wherein the population of cells is autologous to the individual.

34. The method of claim 32, wherein the population of cells is allogeneic to the individual.

35. The method of any of claims 32-34, wherein the cancer is a carcinoma, melanoma or lymphoma.

36. The method of any of claims 32-34, wherein the cancer is an adenocarcinoma.

37. The method of claim 36, wherein the adenocarcinoma is lung cancer.

38. The method of any of claims 32-34 wherein the cancer has high expression of T antigen.

39. The method of claim 38 wherein the cancer is selected from known breast, ovarian, gastric, colorectal, pancreatic, esophageal, and prostate cancer.

Images