Genetically modified cells and uses thereof

Abstract

The present invention relates generally to populations of stem cells (e.g., iPSCs or HSCs) comprising nucleic acids encoding a T cell receptor and a chimeric antigen receptor directed to a plurality of unique antigenic determinants, e.g., two unique tumor antigenic determinants. The present invention also relates to populations of T cells co-expressing a T cell receptor and a chimeric antigen receptor directed to a plurality of unique antigenic determinants, e.g., two unique tumor antigenic determinants. The cells of the present invention can be derived from a selected donor whose HLA type is compatible with a significant portion of the population, and can be used for a variety of applications, particularly in the context of therapeutic treatment of neoplastic conditions.

Description

[0001] This application is a divisional application of patent application having application number 201680069543.6, filing date 23 November 2016, priority date 27 November 2015, and the title "Genetically Modified Cells and Uses Thereof".

[0002] This application claims the benefit of priority of Australian provisional patent applications No. 2015904933 filed 27 November 2015 and No. 2016901328 filed 11 April 2016, which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present invention relates generally to populations of stem cells (e.g. iPSCs or HSCs) comprising nucleic acids encoding T cell receptors and chimeric antigen receptors directed to multiple unique antigenic determinants, such as two unique tumour antigenic determinants. The present invention also relates to populations of T cells co-expressing T cell receptors and chimeric antigen receptors directed to multiple unique antigenic determinants, such as two unique tumour antigenic determinants. The cells of the present invention can be derived from a selected donor whose HLA type is compatible with a significant portion of the population and can be used for a variety of applications, particularly in the therapeutic treatment of neoplastic conditions. BACKGROUND

[0004] The bibliographic details of the publications cited in the description by the authors are collected at the end of the description in alphabetical order.

[0005] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0006] Malignant tumours or cancers grow in an uncontrolled way, invade normal tissues, and often metastasize and grow at sites remote from the tissue of origin. Generally, a cancer is derived from one or only a few normal cells that have undergone a poorly understood process called malignant transformation. Cancers can arise from almost any tissue in the body. Those from epithelial cells, called carcinomas, are the most common type of cancer. Sarcomas are malignant tumours that arise from mesenchymal tissues such as fibroblasts, muscle cells, and fat cells. Solid malignant tumours of lymphoid tissue are called lymphomas, and bone marrow and blood borne malignant tumours of lymphocytes and other hematopoietic cells are called leukemias.

[0007] Cancer is one of the three leading causes of death in industrialized countries. With the continuous improvement in the treatment of infectious diseases and the prevention of cardiovascular diseases, and with the increase in average life expectancy, cancer can become the most common lethal disease in these countries. Therefore, successful treatment of cancer requires the removal or destruction of all malignant cells without killing the patient. The ideal method to achieve this is to induce an immune response against the tumor that can distinguish tumor cells from their normal cell counterparts. However, for over a century immunological approaches to treat cancer have been tried with unsustainable results.

[0008] Solid tumors cause the largest number of deaths from cancer. Once solid tumors have spread throughout the body or "metastasized", they are usually incurable. The prognosis for metastatic solid tumors has improved only slightly in the last 50 years. The best chance of curing a solid tumor relies on early detection, followed by local treatment such as surgery and / or radiation when the solid tumor is localized and has not yet spread to the lymph nodes or elsewhere. Nonetheless, even at this early stage, microscopic deposits of cancer, called micrometastases, can have spread throughout the body and will subsequently cause the patient's death, especially if the tumor has already spread to the draining lymph nodes. In this sense, cancer is a systemic disease that requires a systemic administration of treatment.

[0009] The "Golden Bullet" approach to attack cancer with antibodies loaded with toxins has a long history, exploiting their ability to potentially target any particular molecular entity such as a carbohydrate, a lipid or a protein or combinations thereof. Once bound to a cancer cell, antibodies can bind to complement or FcR+NK / K cells and induce cell lysis. Unfortunately, antibody therapy of cancer has generally only achieved moderate success, mainly because of low affinity binding, poor lysis efficiency and their short life span. These collectively impair the ability of antibodies to rapidly destroy cancer cells, increasing the risk of mutation and immune evasion. More recently, antibody-related therapies have been reported, including those based on antibodies against cancer molecules and immune checkpoint blockade molecules with high affinity. Despite some clinical success, especially in the case of the latter, such therapies are still associated with various limitations.

[0010] Therefore, the common approach to treat cancer continues to follow the long-standing procedure of surgical resection, if possible, followed by radiation therapy and / or chemotherapy, if necessary. The success rate of this rather primitive form of treatment is highly variable, but generally decreases significantly as the tumor becomes more advanced and metastasized. Furthermore, these treatments are associated with severe side effects, including disfigurement and scarring from surgery, e.g., mastectomy or limb amputation, severe nausea and vomiting from chemotherapy, and most importantly, damage to normal tissues such as hair follicles, intestines and bone marrow, induced by the relatively non-specific targeting mechanism of the toxic drugs that form part of most cancer treatments.

[0011] Thus, there is an urgent and continuing need to develop improved systemic treatments for cancer, particularly metastatic cancer.

[0012] Thymic production of mainstream T cells is fundamentally required for infection defense. This pool of "immunosurveilance" T cells patrols the body to remove damaged or abnormal cells, including cancer. Because thymic T cell production is characterized by random generation of the T cell receptor (TCR) repertoire, thymopoiesis must also include a very stringent selection process that eliminates or functionally silences developing thymic T cells that have the potential to attack self. Thus, this "self-tolerance" limits autoimmune disease (Fletcher et al. (2011)). However, it is this process that necessarily compromises immunosurveillance against cancer: given that non-viral induced cancers are defined as "self" diseases. This means that many T cells produced in the thymus can be eliminated before entering the blood that potentially could be reactive with tumor-associated antigens. At least they are defective in numbers, and can have low affinity TCRs. Nonetheless, T cells are clearly the main weapon against cancer - so the challenge is to improve their ability to detect cancer, expand them in numbers, or better yet, enhance their potent cytolytic capacity. While antibodies and T cells are the most logical weapons against cancer, their potential rapid and effective cancer destruction has not been realized clinically. Progress in immunotherapy has evolved by genetically engineering T cells to express novel chimeric membrane receptors composed of a cancer antigen-binding antibody fragment coupled to a T cell signaling molecule in the cytoplasm. The latter is usually one or all of the TCR zeta chain, CD28, or CD40 ligand (Corrigan-Curay et al. (2014); Fedorov et al. (2014); Perna et al. (2014); Curran et al. (2015); Curran et al. (2012); Dotti et al. (2014); Han et al. (2013)). Such chimeric antigen receptor (CAR)-expressing T cells (CAR-T) not only can harness the two most powerful anti-cancer weapons of the immune system, but also can overcome their individual deficiencies. CAR-Ts retain the potent, local cytolytic capacity and avoid the normal dependence on the TCR to detect very rare "cancer peptides" expressed in HLA breaks. The T cell repertoire specific for such nominal peptides is very rare. The antibody portion of the CAR confers cancer-seeking specificity to the T cell and overcomes the poorly known cancer-destructive efficacy of circulating antibodies. Thus, cancer binding is mediated by the antibody domain of the CAR, leading to cytoplasmic signaling that triggers the T cell lytic pathway to destroy the cancer.

[0013] While still in the early stages of clinical development, many CAR-T trials are in progress. Despite the promise, several aspects of the CAR-T technology are problematic and are impeding its clinical efficacy from being fully realized. Most apparent is the cytokine storm that occurs during T cell mediated cancer destruction and is tumor burden dependent. Fever is indicative of cancer destruction but unless carefully managed can lead to serious clinical side effects (Davila et al. (2014); Casucci et al. (2015)). Current management is through cytokine modulating therapies such as anti-IL6. In addition, there is a considerable problem of insufficient numbers of CAR-T cells being generated that not only attack the initial cancer but are also retained in sufficient supply in the event of relapse. Currently, attempts to deal with this problem are based on the overuse of proliferation inducing cytokines in vitro. Still further, for CAR-T cells to attack cancer as effectively as they do, even for CD19 + Cancer, tumor destruction is not 100% effective. While a high of 90% responsiveness has been reported for B-ALL, in other CD19 + cancers, the effectiveness of the outcome is much less. Thus, despite the encouraging observations related to CAR-T utility, there are still many significant problems to overcome before this technology can supplant the current gold standard related to cancer treatment as a reliable, effective and new one.

[0014] In the work leading to the present application, it has been determined that seemingly disparate problems existing in the current effective therapeutic application of CAR-T technology, where CAR-T cells can be derived from transfected stem cells such as adult stem cells, rather than transfected thymocytes or other transfected somatic cell types, are resolvable. For example, by transfecting stem cells, such as induced pluripotent stem cells ("iPSCs") derived from adult cells, with chimeric antigen receptors, the problem of providing a sufficient present and future supply of CAR-T cells against specific tumors is solved due to the continuous source of somatic T cells derived from these themselves renewing transfected stem cells. Moreover, these iPSCs, and thus CAR-T cells derived from them, can be selected in advance from donors expressing homozygous HLA haplotypes, particularly donors homozygous for HLA types widely expressed in the population, thereby providing a means of generating a cell bank exhibiting broad donor suitability. Still further, it has been determined that generating iPSCs from T cells exhibiting T cell receptor specificity against an antigen of interest means that the genetic rearrangement of the TCR specific for the cancer antigen will be embedded in the iPSC. All T cells induced from the iPSC will retain the anti-cancer TCR specificity. This can be followed by transfecting such iPSCs with a CAR, enabling the iPSCs to subsequently differentiate into T cells, such as CD4+ or CD8+ T cells, which stably exhibit dual specificity to the antigen the CAR is directed against and the antigen the TCR is directed against (the original T cell was directed against). Without limiting the application to any one theory or mode of action, this is believed to be due to the action of epigenetic memory. Moreover, it has also been determined that bi-specific NKT cells can similarly be generated. Thus, a continuous source of T and NKT cells can be provided, which are selectively and stably directed against multiple unique antigenic determinants, such as multiple unique tumor antigenic determinants, thereby enabling a more therapeutically effective therapeutic step. SUMMARY

[0015] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0016] As used herein, the term "derived from" shall be understood to mean that a particular integer or group of integers originates from a specified source, but is not necessarily taken directly from the specified source. Further, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0017] This specification contains amino acid sequence information made using the program PatentIn Version 3.5, which is presented herein after the list of references. Each amino acid sequence is identified in the sequence listing by a numerical designator <210> followed by a sequence identifier (e.g., <210>1, <210>2, etc.). The length of each amino acid sequence, the type of sequence (protein, etc.), and the organism of origin are indicated by the information provided in the numerical designator fields <211>, <212>, and <213>, respectively. Amino acid sequences referred to in the specification are identified by the designator SEQ ID NO:, followed by a sequence identifier (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.). Sequence identifiers referred to in the specification are related to the information provided in the numerical designator field <400> in the sequence listing, followed by a sequence identifier (e.g., <400>1, <400>2, etc.). As detailed in the specification, SEQ ID NO:1 is related to the sequence shown in <400>1 in the sequence listing.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs.

[0019] One aspect of the application relates to a genetically modified mammalian stem cell or a T cell differentiated therefrom, said cell being capable of differentiating into a T cell expressing a TCR directed to a first antigenic determinant, and comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein said receptor comprises an antigen recognition moiety directed to a second antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0020] In another aspect, there is provided a genetically modified mammalian stem cell or a T cell differentiated therefrom, said cell being capable of differentiating into a CD4 + T cell expressing a TCR directed to a first antigenic determinant, and comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein said receptor comprises an antigen recognition moiety directed to a second antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0021] In yet another aspect, there is provided a genetically modified mammalian stem cell or a T cell differentiated therefrom, said cell being capable of differentiating into a CD8 +T cells and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed against a second antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0022] In another aspect, there is provided a genetically modified mammalian stem cell or T cell differentiated therefrom, the cell being an iPSC (induced pluripotent stem cell) or HSC (hematopoietic stem cell), capable of differentiating into a T cell expressing a TCR directed against a first antigenic determinant and comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed against a second antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the genetically modified stem cell, such as an iPSC or HSC, expresses at least one homozygous HLA haplotype.

[0023] According to this aspect of the application, in one embodiment, the stem cell (e.g., iPSC) is derived from a cell in which the TCR genes have undergone rearrangement.

[0024] In another embodiment, the stem cell (e.g., iPSC) is derived from a T cell or thymocyte expressing an αβ TCR.

[0025] In yet another embodiment, the stem cell (e.g., iPSC) is derived from a T cell or thymocyte expressing a γδ TCR.

[0026] In yet another embodiment, the stem cell (e.g., iPSC) is derived from a T cell or thymocyte expressing a TCR directed against the first antigenic determinant, i.e., the same antigenic determinant as the TCR expressed on the T cell derived from the stem cell (e.g., iPSC).

[0027] In yet another embodiment, the stem cell (e.g., iPSC) is derived from a CD8 + T cell or thymocyte.

[0028] In yet another embodiment, the stem cell (e.g., iPSC) is derived from a CD4 + T cell or thymocyte.

[0029] In one embodiment, the stem cell (e.g., iPSC or HSC) is capable of differentiating into a CD4 + T cell expressing a TCR directed against a first antigenic determinant. In another embodiment, the stem cell (e.g., iPSC or HSC) is capable of differentiating into a CD8 + T cell expressing a TCR directed against a first antigenic determinant.

[0030] In yet another aspect, there is provided a genetically modified mammalian stem cell or T cell differentiated therefrom, said cell being capable of differentiating into a T cell expressing a TCR directed to a first antigenic determinant, and comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein said receptor comprises an antigen recognition moiety directed to a second antigenic determinant, operably linked to a T cell activation moiety, and wherein said antigenic determinants are selected from a tumor antigen, a microbial antigen, or a self-reactive immune cell antigen. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0031] In one embodiment, the stem cell is an iPSC. In another embodiment, the stem cell is an HSC.

[0032] In another embodiment, the stem cell is capable of differentiating into a CD4 + T cell or a CD8 + T cell.

[0033] In yet another embodiment, the TCR is an αβ TCR.

[0034] In yet another embodiment, the stem cell (e.g., iPSC) is derived from a T cell or a thymocyte, preferably a CD8 + T cell or a thymocyte. In some embodiments, the stem cell (e.g., iPSC) is derived from a CD8 + T cell or a thymocyte, the CD8 + T cell or thymocyte expresses a TCR directed to the first antigenic determinant, i.e., the same antigenic determinant as the TCR expressed on the T cell derived from the stem cell (e.g., iPSC).

[0035] In yet another aspect, there is provided a genetically modified mammalian stem cell or T cell differentiated therefrom, said cell being capable of differentiating into a T cell expressing a TCR directed to a first tumor antigenic determinant, and comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein said receptor comprises an antigen recognition moiety directed to a second tumor antigenic determinant, operably linked to a T cell activation moiety, and wherein said first antigenic determinant is selected from a peptide recognized by a TCR, such as WT-1 or EbvLMP2, and said second antigenic determinant is selected from, e.g., TAG-72, CD19, MAGE, or CD47. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0036] The genetically modified mammalian stem cells (e.g., iPSCs or HSCs) disclosed herein are capable of differentiating into T cells expressing a TCR directed to a first antigenic determinant (e.g., a first tumor antigenic determinant) and comprise a nucleic acid molecule encoding a chimeric antigen receptor comprising an antigen recognition moiety directed to a second antigenic determinant (e.g., a second tumor antigenic determinant) operably linked to a T cell activation moiety. That is, the genetically modified stem cells (e.g., iPSCs or HSCs) disclosed herein are capable of differentiating into T cells directed to multiple, i.e., at least two (i.e., two or more) antigenic determinants. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0037] Accordingly, in another aspect, there is provided a genetically modified mammalian stem cell capable of differentiating into T cells directed to more than two antigenic determinants.

[0038] According to this aspect of the application, in some embodiments, the genetically modified mammalian stem cells (e.g., iPSCs or HSCs) are capable of differentiating into T cells expressing a TCR directed to a first antigenic determinant and comprise a plurality (i.e., two or more) of nucleic acid molecules encoding a plurality of chimeric antigen receptors, wherein each chimeric antigen receptor comprises an antigen recognition moiety directed to an antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0039] In one embodiment, the plurality of antigenic determinants to which the plurality of chimeric antigen receptors are directed are each different from the first antigenic determinant to which the TCR expressed on the T cells derived from the stem cells is directed. In another embodiment, the plurality of antigenic determinants to which the plurality of chimeric antigen receptors are directed are different from each other and also different from the first antigenic determinant to which the TCR expressed on the T cells derived from the stem cells is directed.

[0040] In one embodiment, the plurality of CAR-encoding nucleic acids are comprised in one contiguous nucleic acid fragment. For example, the plurality of CAR-encoding nucleic acids are placed in one construct or vector for transfection into a cell to generate a genetically modified mammalian stem cell comprising the plurality of CAR-encoding nucleic acids. In a specific embodiment, the plurality of CAR-encoding nucleic acids can be linked to each other in one expression unit and reading frame (e.g., by making use of a self-cleaving peptide such as P2A) such that a single polypeptide comprising the plurality of CAR polypeptide sequences is initially produced and subsequently processed to generate the plurality of CARs. In another embodiment, the plurality of CAR-encoding nucleic acids are placed in separate vectors for transfection to generate a genetically modified mammalian stem cell comprising the plurality of CAR-encoding nucleic acids. Examples of CAR-encoding nucleic acid constructs are depicted in Figure 11Exemplary sequences for CARs and various domains suitable for use in CARs are provided in SEQ ID NOs: 1-2 and 7-20.

[0041] Further in accordance with an aspect of the application, there is provided a genetically modified mammalian stem cell capable of differentiating into a T cell directed against more than two antigenic determinants, in other embodiments, a genetically modified mammalian stem cell (e.g., iPSC or HSC), which optionally expresses at least one homozygous HLA haplotype, is capable of differentiating into a T cell expressing a TCR directed against a first antigenic determinant, which comprises a nucleic acid molecule encoding a chimeric antigen receptor comprising an antigen recognition moiety directed against a second antigenic determinant operably linked to a T cell activation moiety, and the T cell further comprises a nucleic acid molecule encoding an antigen binding receptor comprising an antigen recognition moiety directed against a third antigenic determinant. In accordance with these embodiments, such genetically modified stem cells are capable of differentiating into T cells directed against multiple antigenic determinants, preferably multiple antigenic determinants different from each other. Additional antigen specificities can be provided by using multiple CAR-encoding nucleic acids as described herein, and / or by utilizing multiple nucleic acids encoding antigen binding receptors.

[0042] In one embodiment, the antigen binding receptor is a non-signalling antigen binding receptor; that is, the receptor is anchored to the cell surface and binds to the third antigenic determinant, but does not transduce a signal into the cytoplasmic portion of the cell. In one embodiment, the antigen binding receptor comprises an antigen recognition moiety directed against a third antigenic determinant operably linked to a transmembrane domain, but lacks a T cell activation moiety.

[0043] In a particular embodiment, the antigen binding receptor is a non-signalling antigen binding receptor directed against CD47. For example, the antigen binding receptor is a non-signalling CD47 binding molecule, e.g., a truncated CD47 binding molecule.

[0044] Accordingly, there is provided a genetically modified mammalian stem cell (e.g., iPSC or HSC) or a T cell differentiated therefrom, which is capable of differentiating into a T cell expressing a TCR directed against a first antigenic determinant, comprising (i) a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed against a second antigenic determinant operably linked to a T cell activation moiety, and (ii) a nucleic acid molecule encoding a non-signalling CD47 binding molecule, e.g., a truncated CD47 binding molecule. In some embodiments, the genetically modified mammalian stem cell (e.g., iPSC or HSC) expresses at least one homozygous HLA haplotype.

[0045] In another aspect, there is provided a method of generating a genetically modified mammalian stem cell (e.g., iPSC or HSC) as disclosed herein.

[0046] In one embodiment, the subject method comprises obtaining a mammalian stem cell (e.g., iPSC or HSC) capable of differentiating into a T cell expressing a TCR directed to a first antigenic determinant, the stem cell (e.g., iPSC or HSC) expressing, in one embodiment, at least one homozygous HLA haplotype; and introducing into the stem cell (e.g., by transfection) one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed to an antigenic determinant operably linked to a T cell activation moiety. In another embodiment, the method further comprises introducing into the stem cell (e.g., by transfection) one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signalling antigen binding receptors), each antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant. As further disclosed herein, the plurality of receptor-encoding nucleic acids can be introduced by a single vector or separate vectors.

[0047] In another embodiment, the subject method comprises obtaining a T cell or thymocyte (preferably a CD8+ T cell or thymocyte) expressing a TCR directed to a first antigenic determinant, and, in one embodiment, further expressing at least one homozygous HLA haplotype; introducing into the T cell or thymocyte one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed to an antigenic determinant operably linked to a T cell activation moiety; and deriving a stem cell (e.g., iPSC) from the T cell or thymocyte. In another embodiment, the method further comprises introducing into the T cell or thymocyte one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signalling antigen binding receptors), each antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant, prior to the step of deriving a stem cell from the T cell or thymocyte.

[0048] In another embodiment, the method comprises obtaining HSCs (e.g., from bone marrow or blood), which in some embodiments express at least one homozygous HLA haplotype; introducing into the HSCs (i) one or more nucleic acids encoding a TCR directed to a first antigenic determinant, (ii) one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed to an antigenic determinant that is different from the first antigenic determinant, operably linked to a T cell activation moiety; and optionally (iii) one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signalling antigen binding receptors), each antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant that is different from the first antigenic determinant and different from the antigenic determinant to which the chimeric antigen receptor is directed. As disclosed herein, the multiple receptor-encoding nucleic acids can be introduced by a single vector or separate vectors. Such genetically modified HSCs can be used to generate T cells specific for multiple antigenic determinants.

[0049] In another aspect, provided is a T cell expressing a TCR directed to a first antigenic determinant and expressing one or more chimeric antigen receptors, wherein each receptor comprises an antigen recognition moiety directed to an antigenic determinant, operably linked to a T cell activation moiety. In some embodiments, the T cell further expresses an antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant. In some embodiments, the provided T cell expresses at least one homozygous HLA haplotype.

[0050] In another aspect, provided is a method of generating a T cell expressing a TCR directed to a first antigenic determinant and expressing one or more CARs, wherein each CAR comprises an antigen recognition moiety directed to an antigenic determinant, operably linked to a T cell activation moiety, and optionally further expressing one or more non-signalling antigen binding receptors, each non-signalling antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant. In some embodiments, the provided method involves generating a T cell expressing at least one homozygous HLA haplotype.

[0051] Another aspect of the application relates to a method of treating a condition characterized by the presence of an unwanted cell population in a mammal, the method comprising administering to the mammal an effective number of stem cells or T cells, as described hereinabove.

[0052] In one embodiment, the condition is a neoplastic condition, a microbial infection (such as HIV, an STD, or an antibiotic-resistant bacterium), or an autoimmune disorder.

[0053] According to this embodiment, there is provided a method of treating a neoplastic condition, the method comprising administering to the mammal an effective number of stem cells or T cells as defined above, wherein the TCR is directed to a first tumor antigenic and the CAR is directed to a second tumor antigenic determinant.

[0054] In yet another embodiment, the first tumor antigenic determinant is WT-1.

[0055] In another embodiment, the second tumor antigenic determinant is TAG-72, CD19, MAGE, or CD47.

[0056] Another aspect of the application relates to the use of stem cells or T cells as defined above in the manufacture of a medicament for treating a condition characterized by the presence of an unwanted cell population in a mammal.

[0057] The application includes the following:

[0058] Embodiment 1. A genetically modified mammalian stem cell, wherein the cell is capable of differentiating into a T cell expressing a T cell receptor (TCR) directed to a first antigenic determinant, and comprises a nucleic acid molecule encoding a chimeric antigen receptor comprising an antigen recognition moiety and a T cell activation moiety, wherein the antigen recognition moiety is directed to a second antigenic determinant and is operably linked to the T cell activation moiety.

[0059] Embodiment 2. A genetically modified stem cell, wherein the cell expresses at least one homozygous HLA haplotype.

[0060] Embodiment 3. The cell of embodiment 1 or 2, wherein the stem cell is an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell (HSC).

[0061] Embodiment 4. The cell of embodiment 3, wherein the iPSC or HSC is capable of differentiating into a CD4+ T cell or a CD8+ T cell.

[0062] Embodiment 5. The cell of embodiment 3, wherein the T cell expresses an αβ TCR or a γδ TCR.

[0063] Embodiment 6. The cell of embodiment 4, wherein the iPSC is derived from a T cell or a thymocyte.

[0064] Embodiment 7. The cell of embodiment 6, wherein the T cell or thymocyte from which the iPSC is derived is CD8+ or CD4+.

[0065] Embodiment 8. The cell of embodiment 6 or 7, wherein the T cell or thymocyte from which the iPSC is derived expresses a TCR directed to the first antigenic determinant.

[0066] Embodiment 9. The cell of embodiment 8, wherein the TCR expressed on the T cell or thymocyte from which the iPSC is derived is an αβ TCR or a γδ TCR.

[0067] Embodiment 10. The cell of embodiment 1, wherein the first and second antigenic determinants are selected from the group consisting of a tumor antigen, a microbial antigen, or a self-reactive immune cell antigen.

[0068] Embodiment 11. The cell of embodiment 10, wherein the first antigenic determinant is selected from a tumor antigen, such as WT-1.

[0069] Embodiment 12. The cell of embodiment 10 or 11, wherein the second antigenic determinant is selected from a tumor antigen, such as TAG 72, CD19, MAGE, and CD47.

[0070] Embodiment 13. The cell according to any of the preceding embodiments, wherein the antigen recognizing moiety comprises a scFv.

[0071] Embodiment 14. The cell according to any of the preceding embodiments, wherein the antigen recognizing moiety is linked to the T cell activating moiety by a hinge region and a transmembrane domain.

[0072] Embodiment 15. The cell of embodiment 14, wherein the hinge region is derived from a hinge region of IgGl, a hinge region of CD8, or a hinge region of CD28.

[0073] Embodiment 16. The cell of embodiment 14, wherein the hinge region comprises a cysteine that promotes dimerization of the chimeric antigen receptor.

[0074] Embodiment 17. The cell of embodiment 14, wherein the transmembrane domain is derived from a transmembrane domain of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4.

[0075] Embodiment 18. The cell according to any of the preceding embodiments, wherein the T cell activating moiety comprises an intracellular signaling sequence of a molecule selected from the group consisting of TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0076] Embodiment 19. The cell of embodiment 18, wherein the T cell activation moiety further comprises an intracellular signaling sequence of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-lBB (CD137), OX40, CD30, CD40, PD-1, TIM-3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0077] Embodiment 20. The cell according to any of the preceding embodiments, further comprising a further nucleic acid encoding a further chimeric antigen receptor comprising an antigen recognition moiety and a T cell activation moiety, wherein the antigen recognition moiety of the further chimeric antigen receptor is directed to a further antigenic determinant different from the first and second antigenic determinants.

[0078] Embodiment 21. The cell according to any of the preceding embodiments, further comprising a nucleic acid encoding a non-signaling antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant different from the antigenic determinant to which the first antigenic determinant and the chimeric antigen receptor are directed.

[0079] Embodiment 22. The cell of embodiment 21, wherein the antigen recognition moiety is operably linked to a transmembrane domain by a hinge region.

[0080] Embodiment 23. The cell of embodiment 21 or 22, wherein the antigen binding receptor is directed to CD47.

[0081] Embodiment 24. The cell of embodiment 23, wherein the antigen binding receptor comprises an scFv directed to CD47.

[0082] Embodiment 25. The cell of embodiment 24, wherein the hinge and transmembrane region of the antigen binding receptor is the hinge and transmembrane region of CD28.

[0083] Embodiment 26. The cell of embodiment 20, wherein the nucleic acid encoding the chimeric receptor is operably linked to the further nucleic acid encoding the further chimeric antigen receptor by a nucleotide sequence encoding a self-cleaving peptide.

[0084] Embodiment 27. The cell of embodiment 21, wherein the nucleic acid encoding the chimeric receptor is operably linked to the nucleic acid encoding the antigen binding receptor by a nucleotide sequence encoding a self-cleaving peptide.

[0085] Embodiment 28. A method of making a genetically modified mammalian stem cell, comprising:

[0086] obtaining a mammalian stem cell capable of differentiating into a T cell expressing a T cell receptor (TCR) directed to a first antigenic determinant; and

[0087] introducing into the stem cell one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed to an antigenic determinant different from the first antigenic determinant, operably linked to a T cell activation moiety; and optionally one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signalling antigen binding receptors), each antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant different from the first antigenic determinant and the antigenic determinants to which the chimeric antigen receptors are directed.

[0088] Embodiment 29. The method of embodiment 28, wherein the stem cell expresses at least one homozygous HLA haplotype.

[0089] Embodiment 30. A method of making a genetically modified mammalian stem cell, comprising:

[0090] obtaining a T cell or thymocyte expressing a T cell receptor (TCR) directed to a first antigenic determinant, wherein optionally the T cell or thymocyte is CD8+ or CD4+;

[0091] introducing into the T cell or thymocyte one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed to an antigenic determinant different from the first antigenic determinant, operably linked to a T cell activation moiety; and optionally one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signalling antigen binding receptors), each antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant different from the first antigenic determinant and the antigenic determinants to which the chimeric antigen receptors are directed; and

[0092] deriving a stem cell from the T cell or thymocyte.

[0093] Embodiment 31. The method of embodiment 30, wherein the T cell or thymocyte expresses at least one homozygous HLA haplotype.

[0094] Embodiment 32. The method according to any one of embodiments 28-31, wherein the stem cell is an iPSC.

[0095] Embodiment 33. A T cell, wherein the T cell expresses a T cell receptor (TCR) directed to a first antigenic determinant, and a chimeric antigen receptor comprising an antigen recognition moiety and a T cell activation moiety, wherein the antigen recognition moiety is directed to a second antigenic determinant and is operably linked to the T cell activation moiety.

[0096] Embodiment 34. The T cell of embodiment 33, wherein the T cell expresses at least one homozygous HLA haplotype.

[0097] Embodiment 35. The T cell of embodiment 33 or 34, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

[0098] Embodiment 36. The T cell of embodiment 33 or 34, wherein the TCR is an αβ TCR or a γδ TCR.

[0099] Embodiment 37. The T cell of embodiment 33 or 34, which is derived from a stem cell expressing the at least one homozygous HLA haplotype.

[0100] Embodiment 38. The T cell of embodiment 37, wherein the stem cell is an iPSC or an HSC.

[0101] Embodiment 39. The T cell of embodiment 38, wherein the iPSC is derived from a T cell or a thymocyte expressing a TCR against the first antigenic determinant.

[0102] Embodiment 40. The T cell of embodiment 39, wherein the TCR expressed on the T cell or thymocyte from which the iPSC is derived is an αβ TCR or a γδ TCR.

[0103] Embodiment 41. The T cell of embodiment 39, wherein the T cell or thymocyte is CD8+.

[0104] Embodiment 42. The T cell of embodiment 38, wherein the iPSC or HSC comprises the nucleic acid molecule encoding the chimeric antigen receptor.

[0105] Embodiment 43. The T cell of embodiment 33, wherein the first and second antigenic determinants are selected from the group consisting of a tumor antigen, a microbial antigen, or an autoreactive immune cell antigen.

[0106] Embodiment 44. The T cell of embodiment 43, wherein the first antigenic determinant is selected from a tumor antigen, such as WT-1 and EBV LMP2.

[0107] Embodiment 45. The T cell of embodiment 44, wherein the second antigenic determinant is selected from a tumor antigen, such as TAG 72, CD19, MAGE, and CD47.

[0108] Embodiment 46. The T cell according to any one of embodiments 33-45, wherein the antigen recognition moiety comprises a scFv.

[0109] Embodiment 47. The T cell according to any one of embodiments 33-46, wherein the antigen recognition moiety is linked to the T cell activation moiety by a hinge region and a transmembrane domain.

[0110] Embodiment 48. The T cell of embodiment 47, wherein the hinge region is derived from a hinge region of IgGl, a hinge region of CD8, or a hinge region of CD28.

[0111] Embodiment 49. The T cell of embodiment 47, wherein the hinge region comprises a cysteine that promotes dimerization of the chimeric antigen receptor.

[0112] Embodiment 50. The T cell according to embodiment 47, wherein the transmembrane domain is derived from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or the transmembrane domain of an immunoglobulin such as IgG4.

[0113] Embodiment 51. The T cell according to any one of embodiments 33-50, wherein the T cell activation moiety comprises an intracellular signaling sequence of a molecule selected from the group consisting of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0114] Embodiment 52. The T cell of embodiment 51, wherein the T cell activation moiety further comprises an intracellular signaling sequence of a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, TIM-3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0115] Embodiment 53. The T cell according to any one of embodiments 33-52, further expressing an additional chimeric antigen receptor comprising an antigen recognition moiety and a T cell activation moiety, wherein the antigen recognition moiety of the additional chimeric antigen receptor is directed to an additional antigenic determinant different from the first and second antigenic determinants.

[0116] Embodiment 54. The T cell according to any one of embodiments 33-53, further expressing a non-signaling antigen binding receptor comprising an antigen recognition moiety directed to an antigenic determinant different from the antigenic determinant to which the first antigenic determinant and the chimeric antigen receptor are directed.

[0117] Embodiment 55. The T cell of embodiment 54, wherein the antigen recognition moiety is operably linked to a transmembrane domain by a hinge region.

[0118] Embodiment 56. The T cell of embodiment 54 or 55, wherein the antigen binding receptor is directed against CD47.

[0119] Embodiment 57. The T cell of embodiment 56, wherein the antigen binding receptor comprises a scFv directed against CD47.

[0120] Embodiment 58. The T cell of embodiment 57, wherein the hinge and transmembrane region of the antigen binding receptor is the hinge and transmembrane region of CD28.

[0121] Embodiment 59. The T cell of embodiment 53, wherein the chimeric receptor and the additional chimeric antigen receptor are linked to each other by a self-cleaving peptide upon initial translation, and subsequently separated due to cleavage of the self-cleaving peptide.

[0122] Embodiment 60. The T cell of embodiment 54, wherein the chimeric receptor and the antigen binding receptor are linked to each other by a self-cleaving peptide upon initial translation, and subsequently separated due to cleavage of the self-cleaving peptide.

[0123] Embodiment 61. A T cell derived from a stem cell according to any one of embodiments 1-27.

[0124] Embodiment 62. A method of making a T cell, comprising

[0125] providing a genetically modified stem cell according to any one of embodiments 1-27, and

[0126] differentiating the genetically modified stem cell into a T cell.

[0127] Embodiment 63. A method of making a T cell, comprising

[0128] obtaining a stem cell capable of differentiating into a T cell expressing a TCR directed against a first antigenic determinant;

[0129] differentiating the stem cell into a T cell; and

[0130] introducing into the T cell one or more nucleic acids encoding one or more chimeric antigen receptors, each chimeric antigen receptor directed against an antigenic determinant different from the first antigenic determinant, and optionally one or more nucleic acids encoding one or more antigen binding receptors, each antigen binding receptor directed against an antigenic determinant different from the first antigenic determinant and the antigenic determinants against which the chimeric antigen receptors are directed.

[0131] Embodiment 64. The method of embodiment 63, wherein the stem cell expresses at least one homozygous HLA haplotype.

[0132] Embodiment 65. The method of any one of embodiments 62-64, wherein the stem cell is an iPSC or a HSC.

[0133] Embodiment 66. The method of embodiment 65, wherein the iPSC is derived from a T cell or a thymocyte.

[0134] Embodiment 67. The method of embodiment 66, wherein the T cell or thymocyte is CD8+or CD4+.

[0135] Embodiment 68. The method of embodiment 65, wherein the iPSC is derived from a T cell or thymocyte expressing a TCR directed against the same antigenic determinant as a TCR expressed on a T cell derived from the iPSC.

[0136] Embodiment 69. A method of treating a condition characterized by the presence of an unwanted cell population in a mammal, the method comprising administering to the mammal an effective number of T cells according to any one of embodiments 33-61.

[0137] Embodiment 70. The method of embodiment 69, wherein the condition is a neoplastic condition, a microbial infection (such as HIV, an STD, or an antibiotic resistant bacteria), or an autoimmune condition.

[0138] Embodiment 71. The method of embodiment 69, wherein the condition is a neoplastic condition, and the TCR is directed against a first tumor antigenic determinant and the CAR is directed against a second tumor antigenic determinant.

[0139] Embodiment 72. The method of embodiment 71, wherein the first tumor antigenic determinant is WT 1.

[0140] Embodiment 73. The method of embodiment 71 or 72, wherein the second tumor antigenic determinant is TAG 72.

[0141] Embodiment 74. The method according to any one of embodiments 71-73, wherein the cells administered to the mammal comprise a nucleic acid molecule encoding a non-signaling CD47 binding receptor. BRIEF DESCRIPTION OF DRAWINGS

[0142] Figure 1A-1O. Stimulation and expansion of cytotoxic T cells expressing TCR specific for Wilms Tumor 1 (WT-1) antigen. Cells were isolated from whole blood peripheral blood mononuclear cells (PBMCs). Cells were gated on single cells (A, F, K) with scatter plots (B, G, L) also depicted, followed by CD3 positive cells (conjugated with APC Cy7; C, H, M), then CD8 (conjugated with PE Cy7) and CD4 (conjugated with PerCp; D, I, N), and finally only on CD8 cells (E, J, O). WT-1 staining was performed using HLA-A02 tetramer specific for WT-1 37 peptide. Two different patients positive for HLA-A02 (patient 1 A-E; patient 2 F-J) are presented and compared to fluorescence minus one (FMO; this dye lacks the WT-1 tetramer dye, showing WT-1 37 specific staining (G). The proportion shown is the percentage of WT1+CD8+ cells. For both samples, the percentage of WT-1 TCR T cells increased to 1.5% and 4.5%; in unstimulated PBMCs, these cells were very low (below the level of detection using the tetramer technique herein). In other studies (e.g. Schmeid et al. (2015)), they were as few as 1 in 10 -6 -7 -3x10 -6 cells).

[0143] Figures 2A-2G . CD8+ cytotoxic T cells with TCR specific for Wilms Tumor 1 (WT-1) antigen are functional. Function is represented by production of interferon gamma (IFN-g) (Ghanekar et al., 2001). IFN-g expression was found upon WT-1 specific stimulation. Activated cells were gated on CD8+ and HLA-A02 tetramer to WT-1 37 peptide-PE conjugated fluorescent dye. These cytotoxic T cells with TCR specific for WT-1 demonstrated intracellular cytokine staining for IFN-g (conjugated with pacific blue fluorochrome) when stimulated with WT-1. Two different patients positive for HLA-A02 (WT-1 #1 and WT-1 #2) are presented (patient 1 : A-B; patient 2: C-D) and compared to fluorescence minus one (FMO; E-F; this dye lacks the WT-1 tetramer dye, showing WT-1 37 specific staining (G)). The proportion shown is the percentage of WT1+CD8+ cells. Over 80% of WT-1 TCR T cells produced IFNg. ​

[0144] Figure 2H Addition of LAG 3 inhibitor (IMP 321) increased the frequency of WT-1 specific T cells after 4 days of stimulation. In this experiment, purified but not isolated cord blood mononuclear cells were plated alone, with anti-CD28 alone, with WT-1 peptide (Miltenyi BioTech) and CD28 (1 μg / ml) or with WT-1 peptide plus IMP 321 for 24 hours and 4 days. No effect was observed at 24 hours (data not shown) but consistent with the kinetics of the effect of IMP 321 on activated dendritic cells (Brigone et al (2007)), the WT-1 specific CD8+ T cells were doubled after 4 days.

[0145] Figure 3 Generation of iPSC from cancer specific (e.g. WT-1) TCR T cells. Cancer antigen specific T cells are extremely rare in normal blood; they were revealed by in vitro stimulation with WT-1 peptide bound to autologous B cells (lymphoblastoid cell lines (LCLs) formed using EBV) acting as antigen presenting cells in the presence of cytokines. Cancer antigen specific T cells were shown as double labeled with CD8 (for cytotoxic T cells) and tetramers against HLA-WT-1 binding to the TCR of these CD8+ cells. These cells were then converted to iPSC using Yamanaka reprogramming factors. The rearranged TCR gene specific for WT-1 was inserted into the TCR locus of the iPSC.

[0146] Figure 4 Morphological progression of iPSC colonies to hematopoietic lineage and lymphoid progenitors after 1, 5, 9 and 13 days of culture on OP9 support. Note the large number of single hematopoietic-like cells by day 13.

[0147] Figure 5 Flow cytometry analysis of iPSC derived cells after 13 days of culture on OP9 cells clearly shows evidence of hematopoietic specialization in the presence of hematopoietic stem cells (HSCs) (CD34+CD43+).

[0148] Figure 6 Flow cytometry of HSCs in iPSC derived cells after 13 days of culture on OP9 cells followed by 9 days on OP9 DL-L1 cells. Cells were gated on viability, CD45 expression and then examined for HSC content of single cells by staining for CD34 and CD43. Note the decrease in HSCs from >90% of pre-OP DL-L1 culture Figure 5 to about 60% after 9 days of culture on OP9 DL-L1 cells.

[0149] Figure 7 Flow cytometry of T cell development of iPSC-derived cells after 13 days on OP9 cells followed by 9 days on OP9 DL-Ll cells. There is clear evidence of commitment to the T cell lineage with CD5 and CD7 expression and first stages of thymocyte development with immature (i.e. lack of CD3; data not shown) CD4+, CD8+ "single positive" cells and CD4+CD8+ "double positive" cells.

[0150] Figure 8 Flow cytometry of HSC and T cell differentiation in iPSC-derived cells after 13 days on OP9 cells followed by 16 days on OP9 DL-Ll cells. Immature T cells expressing CD4 and / or CD8 are still clearly present and HSCs are further reduced from about 60% to about 25%. Most importantly, mature CD8+ cells are present and express CD3, αβTCR and CD8β chain (in addition to CD8α- which is not shown).

[0151] Figure 9 . Schematic of induction of WT-1 specific TCR, CD8αβ T cells from iPSC derived from WT-1 specific TCR T cells expanded ex vivo. Treatment of CD4+CD8+ cells with (low level) anti-CD3 antibody mimics signaling that occurs within the thymus during positive selection; this increases CD8+ T cells that express both CD8α and CD8β chains.

[0152] Figure 10 . WT-1 specific TCR, CD8αβ T cells induced from iPSC derived from WT-1 specific TCR T cells expanded ex vivo retain full functionality equivalent to the original cells (e.g. cytotoxicity against WT-1 expressing targets). Effector: target ratio was 3: 1; graded concentrations of WT-1 peptide were tested.

[0153] Figure 11 . Schematic of chimeric antigen receptor and antigen binding receptor constructs. A panel of chimeric antigen receptor (CAR) constructs have been developed - for scFv against TAG 72 or CD19 (as a positive control). Constructs use human CD8 or CD28 as a hinge and transmembrane region and CD28, CD3ζ or 4-1BB as the cytoplasmic activation signaling domain. A P2A signal sequence directing proteolytic cleavage releases EGFP as a fluorescent reporter of expression in the first five constructs shown and CD8α in the last two. Figure 11 Figure 11 ​The second CAR receptor construct is shown in the lower (sixth) construct in the middle, which releases a leader (CD8)-scFv (anti-CD47)-hinge / TM (CD28)-endodomain tail (CD8), where the leader is processed to release the anti-CD47 scFv anchored on the surface by the hinge / TM, and the endodomain tail does not contain a signaling sequence. Any CD47-binding ectodomain can be used for binding to CD47 on target cells, including, for example, SIRP-alpha. The hinge region can contain a cysteine residue to direct dimerization through disulfide bond formation between adjacent hinge domains, which is characteristic of the native CD8 hinge, or can have other residues, such as serine-substituted cysteine residues, that do not form disulfide bonds and do not form covalently stable dimers. Exemplary sequences for the CAR and CD47-binding receptor, as well as various domain sequences suitable for use in constructing the CAR or antigen-binding receptor, are set forth in SEQ ID NOS: 1-20.

[0154] Figure 12 Retroviral transformation protocol. Schematic of the process for producing a CAR- containing retroviral construct. The CAR construct is cloned into the pSAMEN plasmid vector and linked to the fluorescent reporter EGFP by a P2A self-cleaving polypeptide to separate the CAR and reporter. When cell transduction is successful, P2A is expressed and cleaved, and EGFP is identified by flow cytometry and immunofluorescence microscopy.

[0155] Figure 13 Lentiviral transformation protocol. Schematic of the process for producing a CAR- containing lentiviral construct. The CAR construct is cloned into the pWP1 plasmid vector and linked to the fluorescent reporter EGFP by a P2A self-cleaving polypeptide to separate the CAR and reporter. When cell transduction is successful, P2A is expressed and cleaved, and EGFP is identified by flow cytometry and immunofluorescence microscopy.

[0156] Figure 14A. Schematic of a normal second-generation CAR structure. scFv binding domain against target antigen; hinge region (stalk) that allows the CAR to integrate into the plasma membrane (hinge length can differentially affect scFv binding to target cells); cytoplasmic signaling domain that induces T cell activation upon scFv engagement. CAR structure is shown as a dimer, stabilized by disulfide bonds between adjacent cysteine residues in the hinge region.

[0157] Figure 14B. Schematic diagram of a non-signaling antigen-binding receptor, a truncated CD47 "attachment stem". The structure shows an scFv domain or a single V domain for CD47 antigen binding, which attaches to the hinge and transmembrane region, but lacks a signaling domain within the inner domain. This construct would allow CAR-T cells to have increased binding affinity for cancer cells expressing high levels of CD47. While this receptor can also bind to normal cells expressing low levels of CD47, there is no signal transduction, and therefore no harm to normal cells. The hinge region may contain cysteine ​​residues to guide dimerization via disulfide bond formation between adjacent hinge regions, or it may have cysteine ​​residues substituted with other residues such as serine, which do not form disulfide bonds and do not form covalently stable dimers.

[0158] Figure 15 Flow cytometry analysis of CAR-transduced human PBMC-derived CD3+ T cells demonstrated successful transduction using the TAG72 lentiviral CAR construct (20.8% positive, compared to <0.1% in the control) and the CD19 lentiviral CAR construct (33.9% positive).

[0159] Figure 16 Western blot analysis confirmed protein expression in T cells transfected with TAG 27 and CD19 CAR.

[0160] Figure 17 TAG-72CAR-T-mediated killing of ovarian cancer (TAG72+) target cells. Effector:target ratio (E:T) = 1:1. TAG-72CAR-T effector cells (GFP-positive cells) were isolated from CD3-activated normal blood T cells by FACS at >95% purity and subsequently stimulated in the presence of fixed αCD3 / αCD28 and IL-2 to enhance cell lysis activity for up to 72 hours before use. Changes in cell impedance (expressed as arbitrary unit cell index) were monitored over 40 hours and compared with non-transduced CD3 cells isolated from PBMCs. +ve Comparison of TAG72 CAR-T cells with those stimulated by the vector alone. TAG72 CAR-T cells showed the highest killing effect, although CD3 / CD28 activated non-CAR-T cells also showed killing effect, albeit to a much smaller degree.

[0161] Figure 18 The specificity of TAG-72 CAR-T killing was determined. TAG-72 and CD19 CAR-T were isolated separately by FACS and immediately added to 72. hi / CD19 低Target cells without in vitro stimulation (E:T = 5:1). Cell impedance was monitored over 15 hours (here expressed as arbitrary units cell index). TAG-72 CAR-T cells show strong cell line killing. CD19 CAR-T cells are identical to non-CAR T cell control.

[0162] Figures 19A-19B Flow cytometry analysis of CAR transduction of iPSC-derived WT-1 specific TCR CD8+ T cells generated from WT-1 specific T cells. Figure 19A Successful transduction of WT-1 specific TCR T cells with TAG72 lentivirus CAR construct (positive 31.3% compared to <0.1% in control). Figure 19B Successful transduction of iPSC-derived WT-1 specific TCR T cells formed from WT-1 specific TCR T cells with dual specific CAR construct of TAG 72 plus non-signalling truncated CD47 (55% transduction); 32% transduction with TAG 72 alone. These transduced T cells contain 3 anticancer specificities: WT-1 (TCR); TAG72 (CAR); truncated non-signalling CD47.

[0163] Figures 20A-20ICytotoxic function of WT-1 specific TCR T cells and dual specific TAG 72 CAR / WT-1 TCR T cells. WT-1 specific TCR T cells and dual specific TAG72 CAR / WT-1 TCR T cells were incubated with ovarian cancer cell line CAOV4 in monolayer culture for 24 hours to assess cytotoxicity. There was specific killing in the case of WT-1 TCR T cells despite a lower effector:target ratio of 2:1 (necessary due to small amounts of effector available) and this was further increased with transduction with TAG72 CAR. The technique is based on Aqua Amine which stains amines within the cell. When cells are dying or dead, the compromised cell membrane allows the dye to infiltrate the cell and stain the amines more intensely. Thus, cytotoxicity of the cells is depicted by the increased intensity of staining of the cell amines. Note: due to some amines residing on the cell surface, live cells will still give some (albeit low) positive staining. A, D, G: CAOV4 cancer cells only. B, E, H: CAOV4 cancer cells incubated with WT-1 TCR T cells. C, F, I: Dual specific TAG 72 CAR / WT-1 TCR T cells incubated with CAOV4 ovarian cancer cells. D, E, F: Gated Aqua Amine levels on CD3-ve cells (i.e. CAOVA4). G: Cancer cells alone, H: WT-1 TCR cells not CAR transfected with cancer cells, and I: TAG-72 transfected WT-1 TCR T cells and cancer cells phase contrast images. 40x magnification. WT-1 TCR T cells cause about 10% killing (above background); TAG72 CAR-T cells cause an additional 10% killing (i.e. about 20% above background). The dual anti-cancer killing mechanism is additive.

[0164] Figures 21A-21B CAR transduction of iPS. 4 days after incubation with CAR lentivirus, day 5 of growth on MEF feeder layer. CAR+ transduction (green) of TAG72, CD19 and GFP virus overlaid on bright field images at 20x magnification. The untransduced control shows no GFP signal. Images of iPSC colonies at 4x magnification demonstrate the presence of iPSC colonies on the MEF feeder layer. In each system, it is noted that some iPSC colonies appear to have started to spontaneously differentiate. Figure 21A Transduced fibroblast-derived iPSCs are depicted in the middle. Figure 21B Successful transduction of WT-1 T cell-derived iPSCs with TAG72 CAR is shown. Thus, these iPSCs are successfully printed for both WT-1 TCR and TAG 72 specificity.

[0165] Figure 22Flow cytometry analysis of chimeric antigen receptor transduced iPSCs. These iPSCs were derived from adult fibroblasts but could originate from any source, including unselected T cells, CD8+ T cells, or cancer antigen-specific (e.g., WT-1) T cells. A population of fluorescent iPSCs successfully transduced by TAG 72 or CD19 was clearly present. Coverage of transduced cells compared to untransduced controls was shown. Figure 23 middle.

[0166] Figure 23 Compare the dot plots of untransduced control cells (blue) with those of transduced iPSC cultures (green). Events within the GFP+ gate demonstrate successful transduction and are presented as the percentage frequency of non-fragmentation events.

[0167] Figure 24 Re-formation of CAR-transduced iPSC colonies after FACS sorting. CAR-transduced iPSCs can be separated by flow cytometry (GFP-positive fluorescence) and then re-plated to form stable colonies. Invention Details

[0168] This invention is partly based on the following determined predictions: T cells expressing dual TCR / CAR targeting two distinct antigenic determinants can be consistently and stably generated by, for example, transfecting CAR boxes into iPSCs derived from T cells exhibiting TCR specificity against TCRs of interest. Due to the role of exogenous memory, T cells differentiated from these iPSCs have been found to stably express both the TCR specificity of the derived iPSC somatic T cells and the CAR targeting the distinct antigenic determinant. Specificity for additional antigenic determinants can be achieved by introducing additional nucleic acids encoding molecules that bind to such additional antigenic determinants into the cells. Therefore, such multispecific cells provide more effective therapeutic outcomes than currently available results. Thus, these determinations enable the development of a sustainable source of stably transformed dual antigen-specific T cells, particularly cytotoxic CD8+αβTCR T cells, for any disease condition characterized by unwanted cell populations, such as neoplasms, viral infections, bacterial infections, or autoimmune diseases. This discovery, and the resulting cell generation, has now facilitated improvements in therapeutic treatments for conditions such as neoplasms or blood cancers (e.g., leukemia), including metastatic disease.

[0169] Accordingly, one aspect of the application relates to a genetically modified mammalian stem cell or a T cell differentiated therefrom, which is capable of differentiating into a T cell expressing a TCR directed against a first antigenic determinant and which comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed against a second antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0170] Reference to a "T cell" is understood to refer to any cell comprising a T cell receptor. In this regard, the T cell receptor can comprise any one or more of an alpha, beta, gamma or delta chain. As will be appreciated by the skilled artisan, NKT cells also express T cell receptors and thus dual specific NKT cells can also be generated according to the present application. The present application is not intended to be limited to any particular T cell subset, although in preferred embodiments, the subject T cell expresses an alpha / beta TCR dimer. Still more preferably, the T cell is a CD4 + Helper T cells, CD8 + killer T cells or NKT cells. Without wishing to limit the application to any one theory or mode of action, CD8 + T cells are also known as cytotoxic cells. As a major part of the adaptive immune system, CD8 + T cells scan the intracellular environment to target and destroy primarily infected cells. Small peptide fragments derived from intracellular contents are processed and transported to the cell surface where they are presented in the context of MHC class I molecules. However, beyond merely responding to viral infections, CD8+ T cells also provide an additional level of immunosurveillance by monitoring and removing damaged or abnormal cells, including cancer. The CD8 + T cells recognize the subject cell for destruction, typically resulting in the release of cytotoxic granules or lymphokines or activation of the apoptotic pathway through FAS / FASL interactions. On the other hand, CD4 + T cells typically recognize peptides presented by antigen presenting cells in the context of MHC class II, resulting in the release of cytokines designed to modulate B cell and / or CD8+ T cell immune responses. Thus, unlike cytotoxic T cells, T helper cells do not directly kill unwanted cells, such as cancer cells, although they can boost such responses to the extent that they are carried out by cytotoxic T cells and / or antibody-based clearance mechanisms.

[0171] Natural killer T (NKT) cells are a specialized population of T cells that express a semi-invariant T cell receptor (TCRaP) and surface antigens commonly associated with natural killer cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CDld. Most NKT cells express an invariant TCRa chain and one of a small number of TCRP chains. The TCR present on type I NKT cells recognizes the antigen a-galactosylceramide (a-GalCer). Within this group, distinguishable subsets have been identified, including CD4 + CD8 - cells, CD4 - CD8 + cells and CD4 - / CD8 - cells. Type II NKT cells (or non-invariant NKT cells) express a more diverse TCRa chain and do not recognize the a-GalCer antigen. NKT cells produce cytokines with diverse (often opposing) effects, such as promoting inflammation or inducing immune suppression, including tolerance. Thus, they can promote anti-bacterial and anti-viral immune responses, promote tumor-associated immune surveillance, and suppress or promote the development of autoimmune diseases. Like natural killer cells, NKT cells can also induce perforin, Fas and TNF-related cytotoxicity. Thus, reference to genetically modified T cells of the present application should be understood to include reference to NKT cells.

[0172] Because thymus-based T cell production is characterized by the random generation of the T cell receptor (TCR) repertoire, thymic generation must also include a very strict selection process that eliminates or functionally silences those thymic T cells in development that have the potential to attack self. Thus, this "self-tolerance" reduces the potential for autoimmune disease. However, this process necessarily compromises immune surveillance against cancer - a disease defined as "self" in the context of non-viral induced cancers. This means that many T cells produced in the thymus can have been eliminated before entering the blood, which can potentially be reactive with tumor-associated antigens. They will at least be numerically deficient and can express low affinity TCRs.

[0173] In one embodiment, a genetically modified mammalian stem cell or T cell differentiated therefrom is provided, the cell being capable of differentiating into a CD4 + T cell expressing a TCR directed to a first antigenic determinant and comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed to a second antigenic determinant operably linked to a T cell activation moiety. In one embodiment, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0174] In another embodiment, a genetically modified mammalian stem cell or T cell differentiated therefrom is provided, the cell being capable of differentiating into a CD8 + T cell and comprising a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed against a second antigenic determinant operably linked to a T cell activation moiety. In one embodiment, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0175] In some embodiments, the genetically modified cells of the application, e.g., genetically modified stem cells (e.g., iPSCs or HSCs) or T cells, are homozygous for at least one HLA haplotype. Without limiting the application to any one theory or mode of action, the major histocompatibility complex (MHC) represents a group of cell surface molecules whose primary function is to bind peptide fragments derived from antigens and present them to T cells. The MHC gene family is divided into three subgroups: class I, class II, and class III. Class I MHC molecules express a β2 subunit and thus can only be recognized by CD8 co-receptors. Class II MHC molecules do not express a β2 subunit and thus can be recognized by CD4 co-receptors. Thus, because different lymphocytes express different TCR co-receptors, MHC molecules regulate which type of lymphocyte can bind a given antigen with high affinity. The diversity of antigen presentation mediated by MHC class I and II is achieved in at least three ways:

[0176] (1) The MHC repertoire of an organism is typically polygenic (by multiple interacting genes);

[0177] (2) MHC expression is co-dominant (from both sets of genetic alleles); and

[0178] (3) MHC gene variants are highly polymorphic (a wide variety of changes among organisms within a species).

[0179] MHC molecules bind both the T cell receptor on T lymphocytes and the CD4 / CD8 co-receptors. Antigenic epitopes held in the peptide binding groove of the MHC molecule interact with the variable Ig-like domains of the TCR to trigger T cell activation. However, the MHC molecule itself can also act as an antigen and can elicit an immune response in a recipient of tissues or cells expressing foreign MHC, thereby causing graft rejection. Still further, the transplantation of immunocompetent cells can actually cause rejection of host tissues, also known as graft versus host disease. In this regard, each human cell expresses 6 MHC class I alleles (one HLA-A, -B, and -C allele from each parent) and 6 to 8 MHC class II alleles (one HLA-DP and -DQ, and one or two HLA-DR from each parent, and combinations of these). The MHC is highly variable in the human population, with at least 350 alleles of the HLA-A gene, 620 alleles of the HLA-B gene, 400 alleles of DR, and 90 alleles of DQ. Any two individuals who are not identical twins will express different MHC molecules.

[0180] All MHC molecules can mediate transplant rejection, but HLA-C and HLA-DP, which show low polymorphism, are less important. Transplant rejection can be minimized by seeking to match as many of the cell surface HLA repertoire between donor and recipient as possible. A perfect match is only possible between identical twins. However, it is highly desirable to select donors based on minimizing incompatibility with respect to one or more ranges of HLA antigens expressed on cells, and this can significantly minimize the problem of rejection. This is the particular problem addressed by the present invention, as the usual approach to managing tissue / cell rejection is to administer an immunosuppressive treatment regimen, which is not desirable in the context of a treatment regimen based on administration of genetically modified immune cells that need to function at optimal functional levels. According to the present invention, this can be achieved by utilizing cells such as iPSCs or T cells derived from iPSCs that are homozygous for one or more MHC haplotypes, the HLA alleles of interest being the major transplant antigens and preferably the HLA alleles expressed by a substantial proportion of the population, such as at least 5%, at least 10%, at least 15%, at least 17%, at least 20% or more of the population. When the homozygous HLA haplotype corresponds to a dominant MHC I or MHC II HLA type (in terms of tissue rejection), the use of such cells will result in a significant reduction in the problem of tissue rejection in a broad population receiving the cells of the present invention in the context of a treatment regimen. In terms of the present invention, the genetically modified cells can be homozygous for one HLA antigen, or they can be homozygous for more than one HLA antigen (e.g. 2, 3 or more HLA antigens). In some embodiments, the genetically modified cells are homozygous for one HLA antigen selected from the HLA antigens listed in Table 1, including for example HLA Al, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4, DQ8, or HLA A3, B7, C7, DR15, DQ6. In some embodiments, the genetically modified cells are homozygous for two or more HLA antigens selected from the HLA antigens listed in Table 1, including for example HLA Al, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4, DQ8, or HLA A3, B7, C7, DR15, DQ6.

[0181] Thus, the term "HLA type" should be understood to refer to the complement of HLA antigens present on the cells of an individual.

[0182] Obtaining suitable homozygous HLA T cells for generating iPSCs can be achieved by any suitable method, including, for example, screening a population (such as via a blood bank) to identify individuals expressing HLA homozygosity, and then screening T cells from said individuals for exhibiting TCR specificity of interest. These are typically very rare T cells that can be selectively stimulated by the specific antigen peptide that they recognize by their TCR and greatly increased in frequency (e.g. from <0.0001 to 0.2).

[0183] The skilled person will appreciate that important information is widely available in the public literature that describes the identification and utility of homozygous haplotypes in terms of minimizing donor-recipient HLA mismatches for a given population of interest, thereby enabling generation of a donor bank. See, for example, Pappas et al. (2015). In one example, Table 1 identifies the 15 highest ranked homozygous HLA haplotypes relative to the proportion of the UK population, which provides the least mismatch with the UK population. The top 8 listed homozygous HLA haplotypes are compatible with 49% of the population. Another example is outlined in Table 2, which details the top 10 ranked haplotypes compatible with a racially diverse California population. Table 2 includes the matching frequency for sub-populations, including Black or African American, Asian and Pacific Islander, White, Hispanic and American Indian and Alaska Native. Furthermore, Table 3 outlines the 50 most common haplotypes for HLA-A-B-DR, A-B, A-DR and B-DR in the Chinese Northern population. It will be appreciated that the data described in Table 3 can be used to define a set of homozygous haplotypes that will provide the least mismatch for the Chinese Northern population.

[0184] Table 1 : Utility of the 15 highest ranked homozygous HLA-A, -B, -DR types identified to provide zero HLA mismatch to the UK population.

[0185]

[0186]

[0187] Table 2: Top cis and trans matching haploline for California population. Abbreviations AFA, Black or African American; API, Asian and Pacific Islander; CAU, White (non-Hispanic); CIS, cis match benefit; f exp , expected cis match frequency; HIS, Hispanic; K i , number of matches as a percentage of subjects or total number; NAM, American Indian and Alaska Native; TRANS, trans match benefit.

[0188]

[0189] Table 3: 50 most frequent haplotypes for HLA-A-B-DR, A-B, A-DR and B-DR (top 10 -5 ) HF = haplotype frequency / 100,000

[0190]

[0191]

[0192]

[0193]

[0194] As detailed above, the present application is predicated on the determination that stem cells can be consistently and stably engineered to express dual T cell and chimeric antigen receptors against a variety of polypeptide antigens, thereby providing a sustained source of T cells that are more therapeutically effective than the cells used in currently available therapeutic cell therapy regimens. In this regard, reference to "stem cells" is understood to refer to any cell that exhibits the potential to develop in multiple lineage directions (given its particular genetic makeup), and thus form new or regenerative populations of tissues or cells of an organism. Stem cells used in accordance with the present application can be any suitable type that is capable of differentiating along two or more lineages, and include, but are not limited to, embryonic stem cells, adult stem cells, umbilical cord stem cells, hematopoietic stem cell cells (HSCs), totipotent cells, progenitor cells, precursor cells, pluripotent cells, multipotent cells, or dedifferentiated somatic cells (such as induced pluripotent stem cells). "Totipotent" means that the subject stem cell can renew itself. "Pluripotent" means that the subject stem cell can differentiate to specifically form cells of any of the three germ layers, which are the ectoderm, endoderm, and mesoderm.

[0195] In one particular embodiment, the subject stem cells are induced pluripotent stem cells (iPSCs). Without limiting the application to any one theory or mode of action, adult stem cell expansion does not necessarily rest on the occurrence of asymmetric stem cell divisions in order to achieve both stem cell renewal and differentiation along a particular somatic cell lineage. Specifically, pluripotent stem cells can be derived from T cells that are induced to transform into a multilineage potential state. The development of techniques capable of dedifferentiating adult cells is of great significance due to the otherwise difficult induction of stem cell renewal and expansion in vitro.

[0196] Thus, according to this embodiment, there is provided a genetically modified mammalian stem cell, which is an iPSC, is capable of differentiating into a T cell expressing a TCR directed to a first antigenic determinant, and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed to a second antigenic determinant operably linked to a T cell activation moiety, or a T cell differentiated therefrom. In one embodiment, the genetically modified mammalian iPSC expresses at least one homozygous HLA haplotype.

[0197] iPSCs are typically generated directly from somatic cells, but it will be appreciated that the application is not limited in this respect. That is, the subject iPSCs can be generated from cells that are not terminally differentiated; indeed, in principle, iPSCs can be induced from any nucleated cell, including, for example, mononuclear cells from blood and skin cells. For example, in the context of one embodiment of the application, the subject iPSCs can be generated from fully differentiated T cells, or they can be generated from precursor T cells, such as thymocytes. Insofar as the subject thymocytes have already rearranged their TCRs and exhibit the antigen specificity of interest in the context of the application, it can be desirable to generate iPSCs from such cells. This can be relevant, for example, in the case where the particular TCR rearrangement in question is one that is expected to be selected during thymopoiesis. It will be appreciated by the skilled person that one of the complicating factors with respect to immune responsiveness to tumor cells or self-reactive cells is the need for the immune system to direct the immune response to self cells and, thus, to self antigens in this case. Typically, such immune cells are selected during T lymphocyte differentiation in the thymus to minimize the prospect of autoimmune disease onset. However, in the context of neoplastic and autoimmune conditions, the unwanted cells are self cells, and thus, the cell surface antigens sought to be targeted will be self antigens. The application is not limited in any way, and, as discussed in more detail below, one of the advantages of using iPSCs that generate T cells expressing TCRs / CARs directed to a plurality of unique antigenic determinants is that it has been determined that the role of epigenetic memory can enhance the differentiation of iPSCs into functional T cells that express TCRs directed to the same antigens as the T cells from which the iPSCs were derived. However, insofar as the specific TCR-expressing cells from which the iPSCs are derived are selected, it can be difficult to identify suitable fully differentiated T cells, as T cells expressing functional TCRs directed to self antigens can have already been selected during thymopoiesis. Thus, it can be more feasible to screen thymocytes expressing the TCR rearrangement of interest that have not yet undergone negative selection to remove potentially self-reactive cells.

[0198] In another embodiment, the iPSCs are transfected with one or more nucleic acid molecules encoding a TCR directed to a first antigenic determinant (e.g., a tumor antigenic determinant), such as rearranged TCR genes.

[0199] In yet another embodiment, the subject stem cell is a hematopoietic stem cell (HSC). Hematopoietic stem cells (HSCs) refer to stem cells that give rise to all blood cells of the lymphoid and myeloid lineages through the process of hematopoiesis. HSCs are derived from mesoderm and can be found in adult bone marrow, peripheral blood, and umbilical cord blood. HSCs can be collected from bone marrow, peripheral blood, and umbilical cord blood by established techniques and are typically associated with CD34+expression. In some embodiments, a human HSC can be defined as CD34+CD38-CD90+CD45RA- (see Reinisch et al. (2015)). HSCs can be genetically modified, e.g., transfected, with one or more nucleic acids encoding a TCR directed to a first antigenic determinant, and then directed to differentiate into T cells. HSCs can also be introduced with nucleic acids encoding one or more CARs and optionally nucleic acids encoding one or more docking antigen binding receptors, either before or after differentiation of the HSCs into T cells.

[0200] Accordingly, reference to a "T cell receptor" (TCR) is understood to refer to a heterodimer found on the surface of T cells or NKT cells that recognizes a peptide presented by an MHC. Specifically, CD8+T cells recognize peptides presented in the context of MHC class I, while CD4+T cells recognize peptides presented in the context of MHC class II. Without limiting the application to any one theory or mode of action, in the majority of human T cells, the TCR comprises an alpha and beta chain, while a small population of cells express a TCR comprising a gamma delta heterodimer. The TCR is a disulfide-linked, membrane anchored heterodimeric protein. The gamma, delta, alpha, and beta chains are composed of two extracellular domains: a variable (V) region and a constant (C) region, which both form part of the immunoglobulin superfamily and fold to form an anti-parallel beta-sheet. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex.

[0201] The variable domains of both the TCR a and β chains each express three hypervariable regions or complementarity determining regions (CDRs), while the variable region of the β-chain has an additional hypervariable region (HV4) that does not normally contact antigen and is therefore not considered a CDR. The process of generating TCR diversity is based primarily on gene recombination of DNA coding segments in precursor T cells - somatic V(D)J recombination using the RAG1 and RAG2 recombinases or gene conversion using cytidine deaminases. Each recombined TCR possesses a unique antigen specificity, which is determined by the structure of the antigen binding site formed by the a and β chains in the case of aβ T cells or the γ and δ chains in the case of γδ T cells. TCR a chains are generated by VJ recombination, while β chains are generated by VDJ recombination. Likewise, TCR γ chains are generated by VJ recombination, while TCR δ chains occur by VDJ recombination. The interaction of these specific regions (V and J for a or γ chains; V, D, and J for β and δ chains) corresponds to the CDR3 region important for peptide / MHC recognition. The even greater diversity of T cell receptor specificity for processed antigenic peptides is the unique combination of segments at this region, as well as palindromic and random nucleotide additions.

[0202] Thus, reference to a TCR "against" an antigenic determinant is to be understood as a TCR that has undergone rearrangement and exhibits specificity for an antigenic determinant, preferably a self (in particular a self-cancer) antigenic determinant.

[0203] In one embodiment, the iPSC is derived from a cell expressing a rearranged TCR, preferably a rearranged aβ TCR. Examples of cells suitable for generating the iPSC of the application include, but are not limited to, CD4 + T cells, CD8 + T cells, NKT cells, thymocytes, or other forms of precursor T cells. In another embodiment, the cell expresses a rearranged γδ TCR.

[0204] Thus, there is provided a genetically modified mammalian iPSC or HSC, or a T cell differentiated therefrom, which is capable of differentiating into a T cell expressing a TCR against a first antigenic determinant, is derived from a cell in which the TCR gene has undergone rearrangement, or has been transduced with said rearranged gene, and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety against a second antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian iPSC or HSC expresses at least one homozygous HLA haplotype.

[0205] In one embodiment, the iPSC is derived from a T cell or a thymocyte.

[0206] In another embodiment, the iPSCs are derived from a T cell or thymocyte expressing an αβ TCR.

[0207] In yet another embodiment, the iPSCs are derived from a T cell or thymocyte expressing a γδ TCR.

[0208] The subject stem cells can have been freshly isolated from the individual who is the subject of the treatment, or they can have been obtained from a non-fresh source, e.g., from a culture (e.g., in which the number of cells is expanded and / or the cells are cultured to subject them to differentiation signals) or a frozen stock of cells that has been isolated from the individual or another source at some earlier point in time. It will also be appreciated that the subject cells can have been subjected to some other form of treatment or manipulation prior to being subjected to differentiation, such as but not limited to purification, alteration of cell cycle status, or formation of a cell line such as an embryonic stem cell line. Thus, the subject cells can be primary cells or secondary cells. Primary cells are cells that have been isolated from an individual. Secondary cells are cells that have been subjected to some form of in vitro manipulation after their isolation prior to the application of the methods of the application, such as the preparation of an embryonic stem cell line.

[0209] Insofar as the stem cells of the application are iPSCs, methods for generating iPSCs are well known to those of skill in the art. In this regard, as detailed above, iPSCs are cells that have been derived from a more mature cell type, such as a somatic cell, that has been converted / dedifferentiated back to a pluripotent state.

[0210] Without limiting the application to any one theory or mode of action, iPSCs can be derived by introducing a specific set of pluripotency-associated genes or "reprogramming factors" into somatic cell types. The most commonly used set of reprogramming factors, also known as Yamanaka factors, is the genes Oct4 (Pou5fl), Sox2, cMyc, and Klf4. Yamanaka showed in 2006 that transfection of these four specific genes encoding transcription factors to convert adult cells into pluripotent cells. While this combination is the most routine combination used to generate iPSCs, each factor can be functionally replaced by a related transcription factor, miRNA, small molecule, or even an unrelated gene such as a lineage specifier. For example, iPSC induction after transfection with retroviral systems using Oct 3 / 4, Sox2, Klf4, and c-Myc has been achieved, as has transfection with lentiviral systems using Oct4, Sox2, Nanog, and Lin28. The former set of transcription factors is known as Yamanaka factors, while the latter is commonly known as Thomson factors. As will be appreciated by those skilled in the art, extensive modifications have been made to the basic reprogramming factor expression vectors, and new modes of delivery have been designed to improve efficiency and minimize or remove vector sequences that can otherwise integrate into the reprogrammed iPSC genome. These methods are well known to those skilled in the art and include, but are not limited to:

[0211] (i) single cassette reprogramming vectors with Cre-Lox mediated transgene excision;

[0212] (ii) reprogramming by non-integrating viruses such as adenovirus or Sendai virus. Alternatively, expression of the reprogramming factors as proteins provides a means of generating iPSCs that have not undergone integration of the introduced vector DNA into the germline.

[0213] Non-viral reprogramming methods have also been developed. These include, but are not limited to:

[0214] (i) mRNA transfection - the ability to express reprogramming factors in mRNA provides a means of generating iPSCs that do not undergo chromosomal integration of viral vectors. Warren transcribed mRNA to express reprogramming factors effectively (Warren et al. (2010)). Efficiency can be improved by adding Lin28 to the Yamanaka reprogramming factor protocol, culturing at 5% 02, and including valproic acid in the cell culture medium. Reprogramming factor mRNA is commercially available.

[0215] (ii) miRNA infection / transfection - Several miRNA clusters are strongly expressed in embryonic stem cells. When synthetic mimics of mature miR-302b and / or miR-372 were added to MRC5 and BJ-1 fibroblasts along with four lentiviral Yamanaka factors, the reprogramming efficiency was increased 10- to 15-fold compared to the four lentiviral factors alone (Subramanyam et al. (2011)). Certain miRNAs were also found to reprogram cells at high efficiency in the absence of Yamanaka factors.

[0216] (iii) PiggyBac - PiggyBac is a mobile genetic element (transposon) that can integrate into chromosomal TTAA sites in the presence of transposase and is subsequently excised from the genome upon re-expression of the transposase. Yamanaka factors can reprogram cells 14-25 days post-transfection when cloned into piggyBac vectors and co-transfected into MEFs (Kaji et al. (2009); Woltjen et al. (2009)). The piggyBac vectors can be excised from the iPSCs upon re-expression of the transposase.

[0217] (iv) minicircle vectors - Minicircle vectors are minimal vectors containing only a eukaryotic promoter and the cDNA to be expressed. Minicircle vectors expressing Lin28, GFP, Nanog, Sox2, and Oct4 in human adipose stromal cells were able to reprogram cells (Narsinh et al. (2011)).

[0218] (v) episomal plasmids - Transient expression of reprogramming factors as episomal plasmids allows for the generation of iPSCs. For example, an oriP / EBNA vector can be constructed with Yamanaka factors in one cassette plus Lin28 and another oriP / EBNA vector containing SV40 large T antigen (Chuo et al. (2011)). These vectors have been shown to express in CD34+ cord blood, peripheral blood, and bone mononuclear cells in medium supplemented with sodium butyrate, generating iPSC colonies within 14 days. The transfected plasmids are eventually lost.

[0219] In another aspect, one skilled in the art will also be familiar with additional methods known to enhance the efficiency of cell programming. For example, iPSC efficiency can vary between cells even when the same methods are used. Various small molecules have been shown to enhance reprogramming efficiency (Table 4).

[0220] Table 4

[0221] Compounds that increase iPSC reprogramming efficiency

[0222] Treatment Processes affected Valproic acid Histone deacetylase inhibition Sodium butyrate Histone deacetylase inhibition PD0325901 MEK inhibition A-83-01 TGF -inhibition SB43152 TGF -inhibition Vitamin C Enhancing epigenetic modifiers, promoting antioxidant effects on survival Thiazovivin ROCK inhibitor, promoting cell survival PS48 P13K / Akit activation, promoting glycolysis 5% oxygen Promoting glycolysis

[0223] Several known mechanisms enable these molecules to promote reprogramming, including inhibition of histone deacetylation (Mali et al. (2010); Huangfu et al. (2008)), blockade of TGFβ and MEK signaling pathways (Lin et al. (2009); Ichida et al. (2009)), enhancement of epigenetic modifier function (Esteban et al. (2010)), inhibition of the ROCK pathway (Noggle et al. (2011)), and induction of glycolysis (Zhu et al. (2010)). Among these small molecules, the histone deacetylase inhibitors valproic acid and sodium butyrate are most commonly used in reprogramming protocols. It should also be noted that culturing cells in 5% oxygen during reprogramming can also improve iPSC derivation efficiency (Yoshida et al. (2009)). For cells that are particularly difficult to reprogram, the addition of small molecules and culturing under hypoxic conditions can yield improvements. Another option is to use embryonic stem cell conditioned medium (ESCM) to induce expression of endogenous reprogramming factors (Balasubramanian et al. (2009)). Efficiency can be further improved by the addition of valproic acid. Such strategies can also be used to enhance the ability of exogenously introduced reprogramming factors to improve reprogramming efficiency.

[0224] In case the stem cells of the application are HSCs, methods for generating or preparing HSCs are well known to the person skilled in the art. HSCs can be obtained by direct extraction from bone marrow after release of HSCs from the bone marrow, e.g. after treatment with specific molecules such as GM-CSF. HSCs can then be purified by their plasma membrane expression of CD34, e.g. by using anti-CD34 coated magnetic beads or by cell sorting after labelling with fluorescent anti-CD34 by flow cytometry. These so purified HSCs can be induced to T cell differentiation using the OP9 / OP9DL-L1 system outlined in Example 3 and Figures 3-10 Example 4. The HSCs of the application can also be obtained from peripheral blood, e.g. by apheresis.

[0225] Reference to a subject stem cell, particularly an iPSC or HSC, being "capable" of differentiating into a T cell expressing a TCR directed against an antigenic determinant, is understood to refer to a cell that is either already or has the capacity to transcribe and translate the subject TCR gene, and then assemble the TCR heterodimer as a functional receptor on the cell surface. As will be appreciated by the skilled person, in most cases a stem cell such as an iPSC will not express a TCR in its undifferentiated form. It is generally expected that TCR expression will occur once directed differentiation along a T cell lineage has been induced. In one embodiment, the cell is one that can be induced to differentiate into a T cell expressing a functional TCR, with or without a CAR genetic modification. It will be appreciated that the ability of a cell to express a TCR of a particular specificity can be achieved by any suitable means. For example, the cell can have been transfected with genes encoding both TCR chains (e.g. alpha and beta chains) which when expressed will associate to form a TCR heterodimer. Alternatively, in the context of the preferred embodiments of the application, the stem cell of the application is one that has been generated from a T cell, thymocyte or other cell in which the TCR genes have been rearranged. It has been determined that iPSCs generated from such cells will express the same TCR antigen specificity as the somatic T cell from which the iPSC was derived upon directed differentiation of the iPSC into a CD4 + or CD8 + T cell under appropriate cell culture conditions. It is further important, and as discussed in more detail below, that it has been determined that T cells differentiated therefrom are capable of stably expressing both a functional TCR and one or more CARs (and optionally one or more antigen binding receptors) upon transfection of the iPSC or HSC with or without one or more nucleic acids encoding one or more CARs or the alpha and beta chains of a CAR encoding an antigen / MHC class I specific TCR, and thus involve two or more distinct antigenic determinants. Thus, based on the fact that this will occur if the iPSC or HSC is provided with the appropriate differentiation signals, such stem cells are considered to be "capable" of differentiating into a T cell and expressing the requisite TCR. In this regard, since rearrangement of TCR genes is a completely independent genomic event, the selection of the T cell subpopulation from which the iPSC is generated need not necessarily be the same as the subpopulation of T cells sought to be generated ultimately through directed differentiation of the iPSC. For example, a CD4 + T cell exhibiting an appropriate TCR specificity can be selected to generate an iPSC. However, once the iPSC has been generated, the skilled person can seek to differentiate the iPSC into a CD8 + T cell. In this case, by virtue of epigenetic memory, the newly generated CD8 + T cell will exhibit the functionality of a CD8 + T cell, but the TCR specificity will be that of the CD4 + T cell from which the iPSC was derived. The reverse is also true.

[0226] Reference to "conversion" of somatic cells, such as T cells, to a multi-lineage potential phenotype, such as iPSCs, is understood to refer to the induction of genetic, morphological and / or functional changes that are required to change the somatic cell phenotype to a multi-lineage (pluripotent) phenotype of the type defined herein.

[0227] Insofar as it can be chosen, the iPSCs that are capable of producing a TCR by transfection of the cells with DNA encoding the TCR, it is understood that this transfection can occur at any point in time, for example, prior to the generation of the iPSCs of the application, after the generation of the iPSCs, or it can occur simultaneously with the CAR transfection.

[0228] As detailed above, somatic cells, in particular T cells or thymocytes, can be induced to convert to a stem cell, i.e. a functional state of multi-lineage differentiation potential. Thus, reference to a cell that exhibits "multi-lineage differentiation potential" or "multi-lineage potential" is understood to refer to a cell that exhibits potential to develop along more than one somatic cell differentiation pathway. For example, a cell can be able to give rise to a limited range of somatic cell types, such cells are often referred to as pluripotent or multipotent. These cells exhibit potential to commit to a more limited range of lineages than totipotent cells, the latter being cells that can develop in any of the intrinsic possible differentiation directions, including all somatic lineages and gametes.

[0229] Based on the following, cells classically referred to as "progenitor" or "precursor" cells fall within the definition of "multi-lineage differentiation potential" in that they can give rise to cells of more than one somatic lineage under appropriate stimulation conditions. Insofar as reference is made herein to "stem cells" in terms of cells produced by the methods of the application, this is understood to refer to cells as defined herein that exhibit multi-lineage differentiation potential.

[0230] Insofar as the application is concerned, it is understood that an important feature of the subject stem cells is that the multi-lineage differentiation potential exhibited by the cells includes the ability to differentiate into T cells and to express a TCR that exhibits specificity for an antigen of interest. Whether or not TCR specificity is induced prior to or after the generation of the stem cells, e.g. by transfection of the stem cells with DNA encoding a TCR of interest, is immaterial. It is understood that the stem cells claimed herein encompass all stem cells that exhibit the requisite differentiation potential, regardless of when or how the ability is introduced. Still further, it is also understood that the subject stem cells need not be totipotent. As long as they exhibit the ability to differentiate along more than one somatic lineage and as long as one of these lineages is the T cell lineage, the cells fall within the scope of the application.

[0231] As detailed above, the stem cells provided by the present application are genetically modified. By "genetically modified" is meant that the subject cell is derived from some form of molecular manipulation relative to what would be observed in a corresponding unmodified cell background. In the context of the present application, the subject stem cells comprise a nucleic acid molecule encoding a chimeric antigen receptor, and optionally also comprise a nucleic acid molecule encoding an antigen binding receptor. As disclosed herein, the nucleic acid encoding the receptor (whether a chimeric antigen receptor or an antigen binding receptor) can be introduced into a stem cell such as an iPSC or HSC, or into a cell from which the stem cell is derived (e.g., a T cell); and in both cases, the resulting stem cell comprising the nucleic acid encoding the receptor is considered herein to be a genetically modified stem cell. T cells differentiated from genetically modified stem cells and T cells engineered to contain a nucleic acid encoding a genetically engineered CAR or antigen binding receptor are also considered herein to be genetically modified T cells.

[0232] Reference to a "nucleic acid molecule" is understood to refer to both deoxyribonucleic acid and ribonucleic acid. The subject nucleic acid molecule can be in any suitable form of nucleic acid molecule, including, for example, genomic, cDNA, or ribonucleic acid molecules. In this regard, the term "expression" refers to the transcription and translation of DNA or the translation of RNA, resulting in the synthesis of a peptide, polypeptide, or protein. For example, a DNA construct corresponds to a construct that can be sought to be transfected into a cell for subsequent expression, whereas an example of an RNA construct is an RNA molecule transcribed from a DNA construct that only needs to be translated to produce a protein of interest. Reference to an "expression product" refers to the product produced from the transcription and translation of a nucleic acid molecule.

[0233] Reference to a "chimeric antigen receptor" (also known as "artificial T cell receptors," "chimeric T cell receptors," and "chimeric immunoreceptors") is understood to refer to an engineered receptor that grafts an antigen binding moiety onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell; transfection of their coding sequence is facilitated by a retroviral vector. More specifically, and without limiting the application in any way, the most common form of these molecules is a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta chain transmembrane and endodomain. Such molecules result in the transmission of a CD3-zeta chain signal in response to recognition of their target by the scFv. When T cells express this chimeric molecule, they recognize and kill target cells expressing the antigen against which the scFv is directed. For example, to target malignant B cells, the specificity of T cells is redirected using a chimeric immunoreceptor specific for the B lineage molecule CD19.

[0234] The variable portions of the immunoglobulin heavy and light chains are often fused by a flexible linker to form a scFv. This scFv is often preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression, which is ultimately cleaved. The flexible spacer allows the scFv to orient in different directions to enable antigen binding. The transmembrane domain is typically a hydrophobic alpha helix, which is often derived from the initial molecule of a signaling endodomain that projects into the cell and transmits the desired signal. Thus, reference to an "antigen recognition moiety" is understood to refer to the extracellular portion of a receptor that recognizes and binds to an antigenic determinant of interest (i.e., the target-specific binding element). The antigen recognition domain is typically a scFv. However, there are many alternatives. For example, antigen recognition moieties from native T cell receptor (TCR) alpha and beta single chains have also been used, as have simple extracellular domains (e.g., CD4 extracellular domain recognizes HIV infected cells) and other recognition components such as linked cytokines, which lead to recognition of cells bearing cytokine receptors. In fact, any moiety that binds a given target with sufficient high affinity can be used as an antigen recognition domain. Such molecules are well known to those of skill in the art, and selecting a suitable molecule for use is well within the skill of one of skill in the art. In terms of designing a chimeric antigen receptor, particularly the extracellular domain, the skilled artisan can include other moieties that are useful in terms of enabling efficient expression or functional performance. For example, as detailed earlier, the nucleic acid molecule expressing the CAR can be designed to express a signal peptide at the N-terminus of the antigen recognition moiety. Without limiting the application to any one theory or mode of action, the signal peptide directs the nascent protein into the endoplasmic reticulum. This is necessary if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence can be used. Often, the signal peptide native to the amino terminus is used (e.g., in scFvs having the orientation light chain-linker-heavy chain, the native signal of the light chain is used). In another example, the extracellular domain can also include a spacer region, which can be used to link the antigen recognition domain to the transmembrane domain. It should be sufficiently flexible to allow the antigen recognition domain to orient in different directions to facilitate antigen recognition and binding. The simplest form of a spacer region is the hinge region from IgGl. Alternatives include the CH2 CH3 region of immunoglobulin and portions of CD3. For most scFv-based constructs, the IgGl hinge is sufficient. Thus, the term "spacer region" refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular domain or the cytoplasmic domain in a polypeptide chain. The spacer region can comprise up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. In yet another example, the hinge region can be modified to alter its length and thereby achieve additional functional benefits. For example, in traditional CARs that include the CD8 or CD28 hinge, a single cysteine (Cys) can be left in the hinge to stabilize dimerization on the surface of the T cell. Thus, two scFvs are typically displayed (bivalent).In another example, a Cys can be substituted for (Ser) such that a stabilizing disulfide bond cannot form, thus preventing dimerization and thus premature activation. The Cys can also be removed altogether. Another design is to display only the VH domain on one CAR and the VL domain on the other, so the Cys pairs align the VH / VL pair to form a functional monovalent Fv that targets the antigen of interest.

[0235] The antigen recognition portion of the subject chimeric antigen receptors can be operably linked to a T cell activation portion. A "T cell activation portion" refers to the subregion of a receptor that, after antigen recognition and binding, is responsible for transmitting a signal into the T cell to effect its activation and induction of effector mechanisms. The T cell activation portion of a CAR is typically located within the intracellular domain (or "endodomain") of the CAR; thus, the intracellular domain of a CAR molecule will typically also comprise or be an "intracellular signaling domain." A commonly used endodomain component is the CD3-zeta intracellular domain, which contains 3 ITAMs. This transmits an activation signal into the T cell after antigen binding. CD3-zeta can not provide a fully competent activation signal, and additional costimulatory signaling is desirable. For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit a proliferation / survival signal, or all three can be used together. It will be appreciated that this intracellular signaling domain of the CAR is responsible for activating at least one normal effector function of the immune cell, preferably a T cell that has expressed the CAR. The term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While it is generally possible to use the entire intracellular signaling domain, in many cases it is not necessary to use the entire domain. In terms of using a truncated portion of the intracellular signaling domain, such a truncated portion can be used in place of the complete chain so long as it transduces the effector function signal. Thus, the term "intracellular signaling domain" is meant to include any truncated portion of the intracellular domain that is sufficient to transduce the effector function signal.

[0236] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0237] It is known that the signal generated by the TCR alone is insufficient to fully activate a T cell, and that a secondary or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct types of cytoplasmic signaling sequences: a cytoplasmic signal sequence that initiates antigen-dependent primary activation of the TCR complex (primary cytoplasmic signaling sequence) and a cytoplasmic signal sequence that acts in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequence). Primary cytoplasmic signaling sequences modulate primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of particularly useful primary cytoplasmic signaling sequences containing ITAMs include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3-zeta.

[0238] In a preferred embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain itself or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the application. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, TIM3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, etc. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the application can be linked to one another in random or defined order. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. Glycine-serine doublets provide particularly suitable linkers. In one embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28.

[0239] As detailed above, the antigen recognition moiety is operably linked to the T cell activation moiety. "Operably linked" means that the antigen recognition moiety is linked to, associated with, or otherwise connected to the T cell activation moiety such that upon binding of the antigen recognition moiety to an antigenic determinant, a signal is induced through the T cell activation moiety to activate the subject T cell and enable its effector functions to be activated. This is achieved, for example, by designing the transmembrane domain.

[0240] In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of these domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain can be derived from natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region can be derived from (i.e., comprise at least) the transmembrane region of the T cell receptor CD28, CD3 epsilon, CD45, CD4, CD5, CD8 alpha, beta or zeta chain, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or from an immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably, a triphenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form the junction between the transmembrane domain and the cytoplasmic signaling domain of the CAR. Glycine-serine doublets provide particularly suitable linkers. Typically, the transmembrane domain is a hydrophobic alpha helix that spans the membrane. Transmembrane domains from the most membrane proximal component of the endodomain are typically used.

[0241] Reference to an "antigen binding receptor" is understood to refer to an engineered receptor that is anchored to the surface of a cell and binds an antigen. Similar to the chimeric antigen receptors disclosed herein, the antigen binding receptors disclosed herein also comprise an antigen recognition moiety directed to an antigenic determinant. As described herein, the antigen recognition moiety in the antigen binding receptor can take the same form and be designed in the same manner as the antigen recognition moiety of the chimeric antigen receptor. Also similar to the chimeric antigen receptors disclosed herein, the antigen recognition moiety in the antigen binding receptor is operably linked (e.g., by a spacer sequence such as a hinge region) to a transmembrane domain, such that the antigen binding receptor is anchored to the surface of a cell. As described above, the spacer sequence and transmembrane domain in the antigen binding receptor can also be designed in the same manner as the spacer sequence and transmembrane domain of the chimeric antigen receptor. However, unlike the chimeric antigen receptors, the antigen binding receptors as defined herein are typically non-signaling and can include an intracellular sequence that lacks a T cell activation domain. Such non-signaling antigen binding receptors can bind an antigen but do not trigger any signal transduction in a T cell and are therefore also referred to as "acceptors" or "anchoring receptors." Certain embodiments of antigen binding receptors such as non-signaling CD47 binding receptors are further described below.

[0242] Figure 11Examples of nucleic acid constructs encoding CARs and / or antigen binding receptors are depicted in the Figures, and exemplary sequences for CARs and antigen binding receptors, as well as various domains suitable for use in CARs and / or antigen binding receptors, are provided in SEQ ID NOs: 1-20.

[0243] Those skilled in the art will appreciate that the mechanism by which these genetic modifications are introduced into the cells can take any suitable form known and appreciated by those skilled in the art. For example, genetic material is conveniently introduced into cells by the use of expression constructs, typically.

[0244] In one embodiment, a cell capable of differentiating into a TCR-expressing T cell (i.e., a stem cell such as an iPSC or HSC) or a TCR-expressing cell from which a stem cell such as an iPSC can be derived is transfected with an expression construct encoding a CAR. The expression construct can comprise one or more DNA regions comprising a promoter operably linked to a nucleotide sequence encoding a CAR and, optionally, a second DNA region encoding a selection marker and, optionally, a third DNA region encoding a suicide protein. In this regard, it will be appreciated that as a matter of routine procedure, constructs can be designed with any one or more additional components (e.g., suicide genes) that those skilled in the art deem useful. In the context of cells of the application that are proposed for use in the treatment of patients in vivo, the ability to control the killing of the genetically modified cells of the application and, thus, their elimination from the in vivo environment is highly desirable. Without limiting the application to any one theory or mode of action, the adoptive transfer of cells of the application, particularly insofar as they can be directed against a "self" antigen such as a tumor antigen expressed on an autoreactive cell or an antigen that can cross-react with a self antigen, is not without risk. In such a case, an outcome similar to graft versus host disease can occur, in which these cells attack healthy (non-diseased) cells. In the overall therapeutic regimen, these side effects can still be more desirable than non-specific systemic killing of healthy tissue characteristic of treatments such as chemotherapy or autoimmune disorders, uncontrolled killing of healthy tissue. Nonetheless, killing of cancer cells is of paramount importance, but the ability to control the elimination of the cells of the application is highly desirable and can be routinely achieved by very well known and widely used techniques of constructing inducible suicide genes into the genetic construct that is introduced into the stem / T cells of the application.

[0245] The subject promoters can be constitutive or inducible. Where the subject constructs express more than one protein of interest, they can be under the control of separate promoters, or they can be under the control of a single promoter, as in the context of the use of a bicistronic vector that utilizes an IRES sequence to facilitate translation of more than one protein product from a single RNA transcript in non-fused form. In addition, the subject constructs can be designed to facilitate inducible gene expression systems using Cre recombinase-mediated splicing.

[0246] Reference to a nucleic acid "expression construct" is understood to refer to a nucleic acid molecule that can be delivered to a cell and designed to undergo transcription. From its transcription, an RNA molecule is then produced. Typically, expression constructs are also referred to by a number of alternative terms that are used interchangeably and broadly, including "expression cassettes" and "vectors."

[0247] To introduce nucleic acids encoding multiple receptors, whether the receptors are CARs, antigen binding receptors, or combinations thereof, multiple receptor-encoding nucleic acids can be placed in one construct that is transfected into a cell. In one embodiment, the multiple receptor-encoding nucleic acids can be contained in a multicistronic vector that utilizes an IRES sequence to facilitate translation of multiple receptor proteins. In another embodiment, the multiple receptor-encoding nucleic acids can be linked to one another within one expression unit and reading frame, for example, by utilizing a self-cleaving peptide (e.g., P2A) such that a single polypeptide containing multiple receptor sequences is initially produced and then processed to produce multiple receptors. In another embodiment, the multiple receptor-encoding nucleic acids are placed in separate constructs for transfection.

[0248] The expression constructs of the present application can be produced by any suitable method, including recombinant or synthetic techniques. To this end, the subject constructs can be built from first principles, as would occur in the context of utilizing a completely synthetic approach, or it can be built by appropriately modifying an existing vector. In the context of the latter approach, the range of vectors that can be used as a starting point is broad, including but not limited to:

[0249] (i) Plasmids: Plasmids are small, independently replicating, cytoplasmic DNA segments, typically found in prokaryotic cells, that are capable of autonomous replication. Plasmids are often used in the context of molecular cloning because they are capable of being transferred from one organism to another. Without limiting the present application to any one theory or mode of action, plasmids can remain episomal or they can become incorporated into the host's genome. Examples of plasmids that can be utilized include the bacterial-derived pBR322 and pUC.

[0250] (ii) Bacteriophage: Bacteriophages are viruses that infect and replicate in bacteria. They generally consist of a nucleic acid core encapsulated within a protein coat, known as a capsid. Depending on the type of bacteriophage, the nucleic acid can be DNA (single- or double-stranded) or RNA (single-stranded), and they can be linear or circular. Bacteriophages can be filamentous, polyhedral, or polyhedral and tailed, with a tail of one or more tubular fibers attached. Bacteriophages can generally accommodate larger foreign DNA fragments than, for example, plasmids. Examples of bacteriophages include, but are not limited to, E. coli lambda phage, P1 phage, and T-even phage (e.g., T4).

[0251] (iii) Baculovirus: They are any of a group of DNA viruses that replicate only in invertebrates and are generally classified in the family Baculoviridae. Their genome consists of double-stranded circular DNA.

[0252] (iv) Mammalian viruses: Examples of viruses that infect mammals include lentiviruses, Sendai viruses, retroviruses, and vaccinia viruses.

[0253] (v) Artificial chromosomes: Artificial chromosomes, such as yeast artificial chromosomes or bacterial artificial chromosomes.

[0254] (vi) Hybrid vectors such as cosmids, phagemids, and plasmids: Cosmids are generally derived from plasmids but also contain the cos sites of lambda phage, while phagemids represent chimeric phage plasmid vectors. Plasmids generally also represent plasmid-phage chimeras, but are defined by the fact that they contain functional origins of replication of both. Thus, plasmids can be propagated as plasmids or phages in appropriate host strains.

[0255] (vii) Commercial vectors, which are either completely synthetically generated or are modified versions of naturally occurring vectors such as viral vectors.

[0256] One skilled in the art will appreciate that the selection of a vector suitable for modification depends on many factors, including the end use for which the genetically modified cell is placed, in choosing to do this rather than synthetically generate the construct. For example, the use of certain types of vectors, such as viral vectors, can be less desirable in the case where the cell is to be administered to a human in vivo. Furthermore, it is necessary to consider the amount of DNA sought to be introduced into the construct. It is generally understood that certain vectors are more easily transfected into certain cell types. For example, the range of cell types that can serve as hosts for a given plasmid can vary from one plasmid type to another. In yet another example, the larger the DNA insert required for insertion, the more limited the selection of vectors for producing the expression constructs of the present application. For this purpose, the size of the inserted DNA can vary depending on factors such as the size of the DNA sequence encoding the protein of interest, the number of proteins sought to be expressed, the number of selection markers used, and the incorporation of features such as linearized polylinker regions.

[0257] The expression construct for use in the present application can be in any form, including circular or linear. In this context, a "circular" nucleotide sequence should be understood to refer to a circular portion of a nucleotide molecule. For example, the nucleotide sequence can be entirely circular, such as a plasmid, or it can be partially circular, such as a circular portion of a nucleotide molecule produced during rolling circle replication (this can be relevant, for example, where the construct is initially replicated by this type of process rather than by a cell-based cloning system prior to its introduction into a population of cells). In this context, a "circular" nucleotide sequence corresponds to the circular portion of the molecule. A "linear" nucleotide sequence should be understood to refer to any nucleotide sequence that is in essentially linear form. A linear sequence can be a linear nucleotide molecule, or it can be a linear portion of a nucleotide molecule that also contains non-linear portions, such as circular portions. Examples of linear nucleotide sequences include, but are not limited to, plasmid-derived constructs that have been linearized in order to facilitate their integration into the chromosome of a host cell or constructs that have been produced synthetically in linear form. For this purpose, it should also be understood that the configuration of the construct of the present application can or can not remain constant. For example, a circular plasmid-derived construct can be transfected into a cell where it remains a stable circular episome that is replicated and transcribed in such form. However, in another example, the subject construct can be one that is transfected into a cell in circular form but undergoes intracellular linearization prior to chromosomal integration. This is not necessarily a desirable situation, as such linearization can occur in a random fashion and potentially cleave the construct in a critical region, rendering it ineffective.

[0258] The nucleic acid molecules for use in the methods of the present application can be derived from any human or non-human source. Non-human sources contemplated by the present application include primates, domesticated animals (e.g., sheep, swine, cattle, goats, horses, donkeys), laboratory test animals (e.g., mice, hamsters, rabbits, rats, guinea pigs), household companion animals (e.g., dogs, cats), avian species (e.g., chickens, geese, ducks and other domestic fowl, game birds, cranes, ostriches), captive wild or domesticated animals (e.g., cattle, kangaroos, wild ducks), reptiles, fish, insects, prokaryotes, or synthetic nucleic acids.

[0259] It should be understood that the constructs of the application encoding receptors can comprise nucleic acid material from more than one source. For example, while a construct can be derived from a particular microorganism, in modifying the construct to incorporate features defined herein, nucleic acid material from other microbial sources can be incorporated. These sources can include, for example, viral or bacterial DNA (e.g., IRES DNA), mammalian DNA (e.g., DNA encoding a CAR), or synthetic DNA (e.g., to incorporate specific restriction endonuclease sites). Additionally, the cell type in which the subject construct is expressed can again vary, as it does not correspond to the same organism as all or part of the nucleic acid material of the construct. For example, a construct consisting essentially of DNA derived from bacteria and viruses can still be expressed in a mammalian stem cell contemplated herein.

[0260] Without in any way limiting the application, the application preferably uses DNA constructs comprising CAR sequences, wherein the sequences comprise nucleic acid sequences of an antigen binding moiety operably linked to nucleic acid sequences of an intracellular domain. For example, intracellular domains that can be used in a subject CAR include, but are not limited to, an intracellular domain of CD3-zeta. In another embodiment, the intracellular domain of a CAR comprises an intracellular domain of CD28 operably linked to an intracellular domain of CD3-zeta; and in another embodiment, the intracellular domain of a CAR comprises intracellular domains of CD3-zeta, CD28, and OX40 operably linked to each other.

[0261] Vectors derived from retroviruses such as lentivirus are one example of vectors suitable for achieving long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Other suitable viruses include Sendai virus and vaccinia virus. The vector should be suitable for replication and integration into eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and a promoter that can be used to regulate expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and described in, for example, Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0262] A number of viral-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject’s stem cells. Numerous retroviral systems are known in the art.

[0263] Additional promoter elements, such as enhancers, modulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the initiation site, although recently it has been shown that many promoters contain functional elements downstream of the initiation site as well. The spacing between promoter elements is often flexible, such that a promoter functions when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function synergistically or independently to activate transcription.

[0264] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous Sarcoma Virus promoter, and human gene promoters such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, the construct should not be limited to the use of constitutive promoters. The use of inducible promoters is also contemplated. The use of inducible promoters provides a molecular switch that can turn on such expression of a CAR polynucleotide sequence operably linked thereto when expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0265] To assess expression of the CAR polypeptide or portion thereof, the expression vector to be introduced into the cell can also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked on both sides by appropriate regulatory sequences to effect expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like. Epitope tags can also be included in the extracellular domain of the CAR molecule, such as the short polypeptide c-myc or FLAG often used, preferably located within the hinge region, to identify CAR expression by epitope-specific targeting agents such as, for example, antibodies used in combination with flow cytometry.

[0266] Reporter genes are used to identify potentially transfected cells and to assess functionality of the regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and encodes a polypeptide whose expression is expressed by some readily detectable property, such as enzymatic activity. Following introduction of the DNA into the recipient cells, expression of the reporter gene is assayed at a suitable time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al, 2000 FEBS Letters 479:79-82). Suitable expression systems are well known, and can be prepared using known techniques or obtained commercially. Generally, a construct having the smallest 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to assess the ability of an agent to modulate promoter-driven transcription. Those skilled in the art will appreciate that a reporter such as eGFP (enhanced green fluorescent protein) can be incorporated as a C-terminal polypeptide extension of the CAR, separated by a self-cleaving peptide such as P2A, which will release the reporter intracellularly.

[0267] Methods of introducing and expressing genes in cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells by physical, chemical or biological means.

[0268] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0269] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, among others. See, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0270] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0271] In cases where the use of a non-viral delivery system is sought, an exemplary delivery vehicle is a liposome. It is contemplated that lipid formulations be used to introduce nucleic acids into host cells. In another aspect, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, linked to a liposome through a linker molecule that binds both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or have a "collapsed" structure. They can also simply be interspersed in solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include the fat droplets naturally occurring in cytoplasm and the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0272] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidyl choline ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoyl phosphatidyl glycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that includes various single- and multilamellar lipid carriers formed from closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of an aqueous solution. The lipid components self- rearrange into a bilayer structure with the water and dissolved solutes trapped between the layers (Ghosh et al. (1991)). However, compositions having structures other than normal vesicular structures in solution are also included. For example, the lipids can assume micellar structures or exist simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0273] Regardless of the method used to introduce foreign nucleic acids into a host cell, to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays can be performed. Such assays include, for example, Southern and Northern blotting, RT-PCR and PCR, or detection of the presence or absence of a particular peptide by, for example, immunological means (ELISA and Western blotting).

[0274] In some embodiments the TCR and CAR as well as the antigen binding receptor of the present cell are each directed against an antigenic determinant. Reference to an "antigenic determinant" is to be understood as reference to any proteinaceous or non-proteinaceous molecule expressed by a cell sought to be targeted by the T cell expressing the receptor of the present application. It is to be understood that these are molecules which can be "self" molecules in that they are normally expressed in the patient (e.g. as expected on certain tumor cells or autoreactive cells) or they can be non-self molecules as expected in the case of a cell infected with a microorganism (e.g. viral proteins). It is also to be understood that the subject antigen is not limited to an antigen (whether or not self) which is naturally capable of eliciting a T or B cell immune response. Rather, in the context of the present application, reference to an "antigen" or "antigenic determinant" refers to any proteinaceous or non-proteinaceous molecule sought to be targeted. As detailed above, the target molecule can be a target molecule which is naturally tolerated by the immune system, such as a tumor antigen or an autoreactive immune cell antigen. However, it can be desirable (even taking into account potential collateral damage) to still target this antigen, for example to minimize potential even more severe side effects which can be observed in highly non-specific and systemic treatments such as chemotherapy or immunosuppression, or to reduce the duration of treatment via a highly targeted treatment and / or to maximize the prospect of killing all unwanted cells. Preferably, the molecule is expressed on the cell surface.

[0275] The skilled person will understand that in the context of TCR binding, the subject antigenic determinant will take the form of a peptide derived from an antigen, expressed in the context of MHC I or MHC II. In the context of a CAR, since the design of this receptor is based on the use of immunoglobulin variable region binding domains, the receptor will recognize an epitope present on the antigen in its native form. The subject epitope can be linear or conformational. It is to be understood that the subject antigenic determinant can be any molecule expressed by the cell sought to be targeted. That is, the targeting molecule can be expressed only by the target cell, or it can also be expressed by non-target cells. Preferably, the subject antigenic determinant is a non-self antigenic determinant or an antigenic determinant which is expressed only by the cell sought to be targeted, or at significantly higher levels than normal cells. However, as discussed above, depending on the disease condition to be treated, it can not always be possible to identify and target a non-self antigenic determinant.

[0276] References herein to TCR / CAR receptors directed to a "first" and a "second" antigenic determinant are to be understood as referring to the fact that the subject receptors are directed to two different antigenic regions. However, in this regard, it is to be understood that the receptors can be directed to epitopes on two completely different cell surface molecules, or the receptors can be directed to two different regions / epitopes of the same cell surface molecule. In embodiments referring to TCRs together with multiple CARs or to TCRs with one or more CARs and one or more antigen binding receptors, it is to be understood that each of the receptors is directed to an antigenic determinant and that the antigenic determinants are preferably different from each other, i.e. correspond to different epitope regions of the same or different molecules.

[0277] Thus, in one embodiment, there is provided a genetically modified mammalian stem cell or a T cell differentiated therefrom, said cell expressing at least one homozygous HLA haplotype, being capable of differentiating into a T cell expressing a TCR directed to a first antigenic determinant and comprising at least one, i.e. one or more, nucleic acid molecule encoding a chimeric antigen receptor, wherein said receptor comprises an antigen recognition moiety directed to a second antigenic determinant operably linked to a T cell activation moiety and optionally further comprising a nucleic acid encoding an antigen binding receptor directed to a third antigenic determinant, and wherein said antigenic determinants are selected from tumor antigens, microbial antigens or autoreactive immune cell antigens.

[0278] In one embodiment, the stem cell is an iPSC. In another embodiment, the stem cell is an HSC.

[0279] In yet another embodiment, the stem cell is capable of differentiating into a CD4 + T cell or a CD8 + T cell.

[0280] In yet another embodiment, the TCR is an αβ TCR.

[0281] In yet another embodiment, the stem cell, such as an iPSC derived from a T cell or a thymocyte, preferably a CD8 + T cell or a thymocyte.

[0282] As will be appreciated by those skilled in the art, the identification of tumor- specific antigens is an important area of research, but progress in this regard has been limited. Since tumor cells are generally self-cells (as opposed to, for example, tumors arising from transplanted tissue), the situation is that the antigens they express are not only self-antigens, but can also be expressed by non-neoplastic cells of the tissue from which the tumor is derived. Since this can lead to an anti-neoplastic therapeutic regimen that, while targeting such antigens, is not ideal due to the inevitable side effects in terms of destruction of non-neoplastic tissue, this is clearly less than ideal. Nonetheless, some progress has been made in identifying target tumor antigens that are expressed, if not only by tumor cells, at lower levels, or in other cases less frequently on non-neoplastic cells.

[0283] The choice of antigen binding moiety of the present application will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, MAGE, LMP-2, CD19, CD20, WT1, MART-1 glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, tumor-associated glycoprotein 72 (TAG 72), alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, Prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, Prostein, PSMA, Her2 / neu, survivin and telomerase, prostate carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin. CD47 ("don't eat me" receptor) is also a tumor target, as it is typically highly expressed in cancer cells compared to normal cells, and prevents these cancer cells from being attacked by cells of the immune system, including and in particular scavenger macrophages.

[0284] In one embodiment, the tumor antigen comprises one or more epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, WT-1, tyrosinase, and GP100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation- associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Another group of target antigens are the oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute a truly tumor-specific immunoglobulin antigen unique to the individual tumor. B-cell differentiation antigens such as CD 19, CD20, and CD37 are other candidates for target antigens in B-cell lymphomas.

[0285] Non-limiting examples of antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens derived from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigens EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large antigens based on proteins include CD47, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl 85 erbB2, pl80 erbB-3, cMet, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0286] The cells of the application are designed to target multiple, i.e. two or more, antigenic determinants. As detailed herein, in some embodiments, the multiple antigenic determinants can be or include multiple epitopes of one molecule, or in other embodiments, epitopes of multiple completely unique molecules. The selection of which multiple antigenic determinants should be targeted and further whether they should be targeted by a TCR or a CAR is within the skill of the artisan. In one embodiment, the cells of the application are designed to clear tumor cells, and the TCR / CAR is directed to a tumor antigen, in particular TAG 72, MAGE and WT1. In another embodiment, the cells are designed to clear autoreactive immune cells, and the TCR / CAR is directed to an idiotypic T cell or B cell receptor.

[0287] Thus, in one embodiment, there is provided a genetically modified mammalian stem cell or T cell differentiated therefrom, said cell being capable of differentiating into a T cell expressing a TCR directed to a first tumor antigenic determinant, and comprising one or more nucleic acid molecules encoding one or more chimeric antigen receptors, wherein each chimeric antigen receptor comprises an antigen recognition moiety directed to a tumor antigenic determinant operably linked to a T cell activation moiety, and wherein the antigenic determinant is selected from the group consisting of TAG 72, CD47, CD19, WT-1, MAGE and EBV LMP2.

[0288] Preferably, the genetically modified cell is directed to TAG72 and WT-1. Still more preferably, the CAR is directed to TAG72 and CD47, and the TCR is directed to WT-1.

[0289] In one embodiment, the stem cell is an iPSC. In another embodiment, the stem cell is an HSC.

[0290] In yet another embodiment, the stem cell is capable of differentiating into a CD4 + T cell or a CD8 + T cell.

[0291] In yet another embodiment, the TCR is an αβ TCR.

[0292] In yet another embodiment, the stem cell, such as an iPSC, is derived from a T cell or a thymocyte, preferably a CD8 + T cell or a thymocyte.

[0293] In connection with the cells of the application being directed to the treatment of neoplasia in one embodiment, a large number of CARs have been developed to target known tumor antigens. A non-limiting overview of some of these CARs and the structure of the receptors is provided in Table 5 below:

[0294] Table 5

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] In some embodiments, the CAR comprises an antigen recognition domain consisting of a scFv against CD 19 or TAG-72, and a hinge (stalk) region and transmembrane region both derived from CD28 or CD8, and an intracytoplasmic domain also derived from CD28 or CD8 and comprising a T cell activation moiety. The CAR can comprise a reporter protein (e.g. EGFP) as a C-terminal polypeptide extension linked together by a P2A self-cleaving polypeptide to release EGFP post-translationally. See, e.g. Figure 11 and 14.

[0301] In related aspects, it has been further determined that if the cells of the application are engineered to express a non-signal transducing antigen binding receptor, such as a CD47 binding molecule that is incapable of effecting signal transduction, they can be made particularly effective. Expression of a CD47 binding molecule on the cell surface anchors the cells of the application to the neoplastic cells against which they are directed, thereby facilitating improved interaction of the TCR and CAR with their respective ligands. In particular, with respect to the treatment of solid tumors, the increased stability and binding affinity of the interaction of the subject cells results in improved functional outcome with respect to neoplastic cell killing relative to cells that do not express the subject CD47 binding molecule.

[0302] Thus, in related aspects of the application, there is provided a genetically modified mammalian stem cell or T cell differentiated therefrom, the cell being capable of differentiating into a T cell expressing a TCR against a first antigenic determinant, and comprising (i) a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety against a second antigenic determinant operably linked to a T cell activation moiety, and (ii) a nucleic acid molecule encoding a non-signal transducing antigen binding receptor, such as a non-signal transducing CD47 binding receptor. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0303] Without limiting the application to any one theory or mode of action, CD47 (also known as integrin-associated protein) is a transmembrane protein encoded by the CD47 gene in humans. CD47 belongs to the immunoglobulin superfamily. CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion and migration. In addition, it plays a key role in immune and angiogenic responses. CD47 is ubiquitously expressed in human cells and has been found to be overexpressed in many different tumour cells.

[0304] CD47 is a 50 kDa membrane receptor that contains an extracellular N-terminal IgV domain, 5 transmembrane domains and a short C-terminal intracellular tail. There are four alternatively spliced isoforms of CD47, which differ only in the length of their cytoplasmic tail. Isoform 2 is the most widely expressed isoform found in all circulating and immune cells. The second most abundant isoform is isoform 4, which is primarily expressed in the brain and peripheral nervous system. Only keratinocytes express significant amounts of isoform 1. These isoforms are highly conserved between mouse and human, suggesting an important role for the cytoplasmic domain in CD47 function.

[0305] CD47 is a receptor for thrombospondin-1 (TSP-1), a secreted glycoprotein that plays a role in vascular development and angiogenesis. Binding of TSP-1 to CD47 influences several fundamental cellular functions, including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in regulating angiogenesis and inflammation. CD47 also interacts with signal-regulatory protein alpha (SIRPα), an inhibitory transmembrane receptor found on myeloid cells. The CD47 / SIRPα interaction results in bidirectional signalling, leading to different intercellular responses, including inhibition of phagocytosis (promoting cancer cell escape), stimulation of cell-cell fusion and T cell activation. Further, CD47 interacts with several membrane integrins, most commonly integrin avb3. These interactions result in a CD47 / integrin complex that influences a range of cellular functions, including adhesion, spreading and migration.

[0306] However, despite the ubiquitous expression of CD47, it has been determined that an increase in the level of expression of CD47 on a neoplastic cell is sufficient to promote improved responsiveness to a molecule targeting CD47 and clearance of the neoplastic cell by the molecule targeting CD47 prior to any substantial adverse impact on non-neoplastic cells.

[0307] Reference to a "binding receptor" for CD47 is to be understood as reference to any receptor that interacts with CD47. This can take the form of a CD47 binding receptor, such as a surface displayed antibody fragment, and is preferably devoid of signalling function.

[0308] According to this embodiment, there is provided a genetically modified mammalian stem cell or T cell differentiated therefrom, which is capable of differentiating into a T cell expressing a TCR directed to a first antigenic determinant, and which comprises (i) a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety directed to a second antigenic determinant operably linked to a T cell activation moiety, and (ii) a nucleic acid molecule encoding a non-signal transducing antigen binding receptor, wherein the receptor comprises an antigen recognition moiety directed to CD47. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0309] As detailed above, the subject CD47 binding receptor is a non-signal transducing receptor. By "non-signal transducing" it is meant that upon binding of the subject receptor to CD47 on a target cell, there is no signal transduction that would change the functional implementation of the cell of the application. Rather, the purpose of the CD47 binding receptor is to provide improved anchoring of the subject cell to the target cell, thereby improving the effectiveness of binding of CARs and TCRs directed to target antigenic moieties, such as tumor antigenic moieties.

[0310] For example, in one design of a non-signal transducing antigen binding receptor, the extracellular domain of the receptor comprises an antigen recognition moiety having binding specificity for CD47, a hinge (stalk) domain, a transmembrane domain, and an intracellular domain, which is completely devoid of cytoplasmic signaling function. Such a non-signal transducing CD47 binding receptor can simply serve to attach, rather than to signal, and thus it can drive T cell docking to cancer cells through CD47 binding, and if bound to normal CD47 expressing cells, there is no unwanted activation and killing.

[0311] In some embodiments, the antigen recognition portion of a non-signaling CD47 binding receptor includes antibody-like domains such as scFv, Fv, Fab, etc., and any CD47-targeting V domain, including single human and mammalian V domains and their equivalents (VhH or vNAR) domains, or may include “protein-based alternative targeting scaffolds” known in the art, including but not limited to Darpins, Anticalins, Knottin, ImmE7s, Affibodies, Fn3 fibronectin domain, etc. The antigen recognition portion may also include one or more V-like domains of SIRPα (the natural ligand of CD47). In one embodiment, the antigen recognition portion may include one natural V-like domain of SIRPα. In another embodiment, the antigen recognition portion may include all three natural V-like domains of SIRPα. In other embodiments, a molecule suitable for providing an antigen recognition portion in a non-signaling CD47 binding receptor is the Hu5F9-G4 scFv molecule (described in U.S. Patent Application Serial No. 14 / 656,431). Hu5F9 has been designed with three different forms of VH (1, 2, 3) and three different forms of VL (11, 12, 13), as in U.S. Patent Application Serial No. 14 / 656,431. Figure 12 As shown in A, 12B, its publication is US 20150183874 A1. Liu et al. (PLOS One (2015) Sep21; 10(9):e0137345) described Hu5F9-G4, in which the selected V-domain is the heavy VH-2 with four unique residue variations in the frame (distinguishing VH-2 from VH-1, 3) and the light chain VL-12 with two unique residue variations in the frame (distinguishing VL-12 from VL-11, 13).

[0312] In some implementations, the hinge region of the non-signaling CD47-binding receptor can be a natural SIRPα hinge sequence, or a CD8 or CD28 hinge as typically used in CARs, or alternative hinges known in the art, such as a CD4 domain or a mucin peptide hinge. The hinge region can be designed to contain one or more cysteine ​​(Cys) residues to allow receptor dimerization. CD28 is a natural dimer structure linked by a single Cys residue in the stem region. Therefore, when using the stem region of CD28 as the hinge of the non-signaling CD47-binding receptor, the introduction of an additional Cys residue may not be necessary, but can provide additional stability to the dimer.

[0313] A skilled artisan will appreciate that introduction of nucleic acids encoding a CAR and a non-signalling antigen binding receptor, such as a non-signalling CD47 binding receptor, into a cell, e.g. a T cell or iPSC, can be accomplished using two separate transfection vectors or a single bicistronic vector or a single gene encoding an internal cleavage signal to separate the CAR from the antigen binding receptor. In one embodiment, the internal cleavage signal is P2A, a peptide sequence directing self-cleavage to separate the CAR from the antigen binding receptor. In a specific embodiment, the non-signalling CD47 binding receptor is expressed as a C-terminal extension of the CAR and is separated by a P2A self-cleavage peptide to separate the CAR and CD47 binding receptor post-translationally.

[0314] Means for modifying stem cells of the application so that they also express a non-signalling CD47 binding molecule have been described in considerable detail above in relation to expression of a chimeric antigen receptor directed against a tumour antigen moiety. The skilled artisan will appreciate that the transfection and other methods described herein to achieve receptor expression are equally applicable to the case of the subject CD47 binding molecules.

[0315] In one embodiment, the stem cell is an iPSC. In another embodiment, the stem cell is an HSC.

[0316] In another embodiment, the stem cell is capable of differentiating into a CD4 + T cell or CD8 + T cell.

[0317] In yet another embodiment, the TCR is an αβ TCR.

[0318] In yet another embodiment, the stem cell, such as an iPSC, is derived from a T cell or thymocyte, preferably a CD8 + T cell or thymocyte, and in some embodiments, has an endogenous TCR for a tumour antigen of a CD8 + T cell or thymocyte.

[0319] In yet another embodiment, the stem cell is directed against TAG 72 and WT 1. Still more preferably, the CAR is directed against TAG 72 and the TCR is directed against WT 1.

[0320] In a further aspect, methods of generating genetically modified mammalian stem cells are provided. Various means for generating genetically modified mammalian stem cells, in particular iPSCs, have been described above.

[0321] In another aspect, provided is a T cell expressing a TCR against a first antigenic determinant and a chimeric antigen receptor, wherein the receptor comprises an antigen recognition moiety against a second antigenic determinant operably linked to a T cell activation moiety. In some embodiments, the T cell expresses at least one homozygous HLA haplotype.

[0322] In one embodiment, the T cell expresses a plurality of chimeric antigen receptors, wherein each chimeric antigen receptor comprises an antigen recognition moiety against an antigenic determinant operably linked to a T cell activation moiety.

[0323] In one embodiment, the plurality of antigenic determinants against which the plurality of chimeric antigen receptors are directed are each different from the first antigenic determinant against which the TCR expressed on the subject T cell is directed. In another embodiment, the plurality of antigenic determinants against which the plurality of chimeric antigen receptors are directed are different from each other and also different from the first antigenic determinant against which the TCR expressed on the subject T cell is directed.

[0324] In one embodiment, the plurality of CARs are encoded by a contiguous nucleic acid fragment. For example, the plurality of CARs are encoded by a plurality of nucleic acids placed in one vector, which is transfected into a cell to ultimately produce the subject T cell. In a specific embodiment, the plurality of CAR-encoding nucleic acids can be linked to each other within one expression unit and reading frame (e.g., by use of a self-cleaving peptide such as P2A), such that a single polypeptide comprising the plurality of CAR polypeptide sequences is initially produced and then processed to provide the plurality of CARs. In another embodiment, the plurality of CAR-encoding nucleic acids are placed in separate vectors, which are used for transfection to produce the subject T cell.

[0325] In another embodiment, the T cell expressing one or more CARs also expresses at least one (i.e., one or more) antigen binding receptor comprising an antigen recognition moiety against a third antigenic determinant.

[0326] In one embodiment, the antigen binding receptor is a non-signaling antigen binding receptor; that is, the receptor anchors to the cell surface of the subject T cell and binds to the third antigenic determinant, but does not transduce a signal to the cytoplasmic portion of the T cell that would affect the function of the T cell (hence also referred to as a non-T cell signaling antigen binding receptor). In one embodiment, the antigen binding receptor comprises an antigen recognition moiety against the third antigenic determinant operably linked to a transmembrane domain, but lacks a T cell activation moiety.

[0327] In a specific embodiment, the antigen binding receptor is a non-signaling antigen binding receptor against CD47. For example, the antigen binding receptor is a non-signaling CD47 binding molecule.

[0328] In some embodiments, the T cells provided herein are CD4+. In other embodiments, the T cells are CD8+.

[0329] In some embodiments, the T cells provided herein express an ab TCR. In other embodiments, the T cells provided herein express a gd TCR.

[0330] In some embodiments, the plurality of antigenic determinants against which the subject T cells are directed, i.e., the first antigenic determinants against which the TCRs are directed, the antigenic determinants against which the chimeric antigen receptors are directed, and the antigenic determinants against which the antigen binding receptors are directed, if such antigen binding receptors are present, can be selected from tumor antigens, microbial antigens, or self-reactive immune cell antigens. In certain embodiments, the antigenic determinants are selected from tumor antigens. In specific embodiments, the antigenic determinants against which the TCRs are directed are selected from peptides recognized by the TCRs such as WT-1 or EbvLMP2. In other specific embodiments, the antigenic determinants against which the chimeric antigen receptors and the antigen binding receptors are directed can be selected from, for example, TAG-72, CD19, MAGE, or CD47.

[0331] In some embodiments, the subject T cells derived from iPSCs or HSCs express a TCR directed against a first antigenic determinant and express a chimeric antigen receptor comprising an antigen recognition moiety directed against a second antigenic determinant, the antigen recognition moiety operably linked to a T cell activation moiety.

[0332] In one embodiment, the iPSCs or HSCs from which the subject T cells are derived are genetically modified iPSCs or HSCs that are capable of differentiating into T cells expressing a TCR directed against the first antigenic determinant and comprise one or more nucleic acids encoding one or more chimeric antigen receptors, and optionally one or more nucleic acids encoding an antigen binding receptor. In another embodiment, the iPSCs or HSCs from which the subject T cells are derived are capable of differentiating into T cells expressing a TCR directed against the first antigenic determinant; and one or more nucleic acids encoding one or more chimeric antigen receptors, and optionally one or more nucleic acids encoding an antigen binding receptor, are introduced after the iPSCs or HSCs have differentiated into T cells. In some embodiments, the iPSCs or HSCs from which the subject T cells are derived express at least one HLA haplotype, and the T cells derived from such iPSCs or HSCs also express the at least one HLA haplotype.

[0333] In one embodiment, the iPSC from which the subject T cells are derived is itself derived from a T cell or a thymocyte. In one embodiment, the iPSC is derived from a CD8+ T cell or a thymocyte. In one embodiment, the iPSC is derived from a T cell or a thymocyte that expresses a TCR directed to a first antigenic determinant, i.e., the same antigenic determinant to which the TCR of the subject T cell derived from the iPSC is directed.

[0334] The value of the cells of the application presages the guidance of the subject stem cells to differentiate into CD4 + or CD8 + T cells. In this regard, reference to the "guidance" of stem cells to differentiate into T cells is understood to refer to the application of a cell culture system that induces the commitment of the stem cells to the T cell lineage and differentiation along that lineage into mature T cells. Means for effecting the directed differentiation of stem cells along the T cell lineage are well known to those of skill in the art. For example, and as exemplified herein, it is known to direct the Notch-dependent signaling into the culture system to effect the directed differentiation of stem cells along the T cell lineage. Moreover, particularly effective differentiation is achieved if this signaling is provided to the stem cells in the context of co-culture on OP-9 feeder cell layers. Examples of Notch ligands suitable for use include, but are not limited to, delta-like 1 and delta-4. In this regard, OP-9 cells have been engineered to express delta-like 1 (OP9-DL1), thereby providing a very convenient means for the generation of T cells from stem cells. In another example, and as exemplified herein, the subject stem cells are first cultured in feeder-free conditions to generate mesoderm, and then co-cultured on OP9-DL1 cell lines. Particularly preferred methods for effecting the directed differentiation into CD8 + T cells are exemplified herein.

[0335] In another aspect, methods are provided for generating T cells that express a TCR directed to a first antigenic determinant and express one or more CARs and, optionally, one or more antigen binding receptors. In some embodiments, the T cells further express at least one homozygous HLA haplotype.

[0336] In one embodiment, the method comprises obtaining a genetically modified stem cell (e.g., a genetically modified iPSC or HSC) that is capable of differentiating into a T cell that expresses a TCR directed to a first antigenic determinant, and comprises one or more nucleic acids encoding one or more chimeric antigen receptors each directed to an antigenic determinant (preferably different from the first antigenic determinant), and, optionally, further comprises one or more nucleic acids encoding one or more antigen binding receptors each directed to an antigenic determinant (preferably different from the first antigenic determinant); and differentiating such genetically modified stem cell into a T cell. In some embodiments, the genetically modified stem cell further expresses at least one homozygous HLA haplotype.

[0337] In another embodiment, the method comprises obtaining a stem cell (e.g., iPSC or HSC) capable of differentiating into a T cell expressing a TCR directed to a first antigenic determinant; differentiating the stem cell into a T cell; introducing into the T cell one or more nucleic acids encoding one or more chimeric antigen receptors, each chimeric antigen receptor directed to an antigenic determinant (preferably different from the first antigenic determinant), and optionally also one or more nucleic acids encoding one or more antigen binding receptors, each antigen binding receptor directed to an antigenic determinant (preferably different from the first antigenic determinant). In some embodiments, the genetically modified stem cell (e.g., iPSC or HSC) also expresses at least one homozygous HLA haplotype.

[0338] Regardless of whether the nucleic acid encoding the CAR is introduced into the stem cell prior to differentiation into a T cell or into the T cell after differentiation from the stem cell, the stem cell (e.g., iPSC) itself can be derived from a T cell or a thymocyte. Such T cells and thymocytes can have a TCR specific for a nominal antigen (e.g., a tumor antigen). In one embodiment, the stem cell is an iPSC. In one embodiment, the iPSC is derived from a CD8+ T cell or a thymocyte. In another embodiment, the iPSC is derived from a T cell or a thymocyte expressing a TCR directed to the same antigenic determinant to which the TCR expressed by the T cell derived from the iPSC is directed.

[0339] Reference to a "mammal" shall be understood to include a reference to a mammal such as, but not limited to, a human, a primate, a livestock animal (e.g., a sheep, a cow, a horse, a donkey, a pig), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rabbit, a rat, a guinea pig, a hamster), a captive wild animal (e.g., a fox, a deer). Preferably, the mammal is a human or a primate. Most preferably, the mammal is a human.

[0340] The discovery underlying the present invention facilitates the development of means for treating disease conditions characterized by the presence of unwanted cell populations, such as neoplastic cell populations, virally infected cells, autoreactive immune cells, or microorganisms, such as antibiotic resistant bacteria. More specifically, the cells of the present invention provide means for eradicating these cells in a more targeted manner than current highly non-specific approaches, such as chemotherapy to treat neoplastic conditions, anti-inflammatory therapy to treat symptoms of autoimmune disease, or immunosuppression to manage autoimmunity. In this regard, reference to a "disease condition characterized by the presence of unwanted cell populations" should be understood to refer to any condition whose symptoms or etiology are attributable to the presence or functional exertion of a cell population that can be targeted by virtue of an expressed cell surface antigen, and the elimination of some or all of the cells would be beneficial to the patient. Treatment of the subject conditions is achieved by administering T cells differentiated from the stem cells of the present invention, the T cells being dual TCR / CAR TCR / CAR directed to two or more antigenic determinants expressed by the cells sought to be eradicated.

[0341] It will be appreciated that the "cells" sought to be eradicated by the T cells of the present invention can be any cell, whether self or non-self. For example, in the case of T cells of the present invention designed to treat a disease condition such as neoplasia, viral infection, or autoimmune disease, the target cell population sought to be eradicated is a self cell. However, in the case of a condition sought to be treated that is an infection caused by a microorganism, such as an antibiotic resistant bacterium or a parasite, the "cells" to be eradicated are foreign cells. In this regard, the cells can be in suspension (such as leukemia cells present in circulation), or they can be part of a mass (e.g., a tumor or a tissue). In the case of a condition treated that is a microbial infection, the cells can correspond to single-celled microorganisms (such as many bacteria), or they can be part of a multi-cellular organism. The T cells of the present invention can be used to target any type of cell present in any form.

[0342] Accordingly, another aspect of the present invention relates to a method of treating a condition characterized by the presence of unwanted cell populations in a mammal, the method comprising administering to the mammal an effective number of stem cells or T cells differentiated therefrom as defined above.

[0343] In one embodiment, the condition is a neoplastic condition, a microbial infection (such as HIV, an STD, or an antibiotic resistant bacterium), or an autoimmune condition.

[0344] In another embodiment, the stem cells are iPSCs or HSCs.

[0345] In yet another embodiment, the stem cells are capable of differentiating into CD4 + T cells or CD8 +T cells.

[0346] In yet another embodiment, the TCR is an αβ TCR.

[0347] In yet another embodiment, the stem cell, such as an iPSC, is derived from a T cell or a thymocyte.

[0348] In yet another embodiment, the cell further comprises a nucleic acid molecule encoding a non-signalling antigen binding receptor, wherein the receptor comprises an antigen recognition moiety directed against CD47.

[0349] According to these embodiments, in a particular aspect, there is provided a method of treating a neoplastic condition, the method comprising administering to the mammal an effective number of stem cells or T cells differentiated therefrom as defined above, wherein the TCR is directed against a first tumor antigenic determinant and the CAR is directed against one or more additional tumor antigenic determinants.

[0350] In one embodiment, the first tumor antigenic determinant is WT1.

[0351] In another embodiment, the second tumor antigenic determinant is TAG72.

[0352] In another embodiment, the cell further comprises a nucleic acid molecule encoding a non-signalling antigen binding receptor, wherein the receptor comprises an antigen recognition moiety directed against CD47.

[0353] In another embodiment, the genetically modified stem cell further expresses at least one homozygous HLA haplotype.

[0354] Reference to a "neoplastic condition" is to be understood as reference to a condition characterized by the encapsulated or unencapsulated growth or aggregation of neoplastic cells. Reference to a "neoplastic cell" is to be understood as reference to a cell exhibiting abnormal growth. The term "growth" is to be understood in its broadest sense and includes reference to enlargement in size as well as proliferation of neoplastic cells.

[0355] In this context the phrase "abnormal growth" is intended to refer to cell growth that exhibits one or more of the following relative to normal cell growth: an increase in individual cell size and nuclear / cytoplasmic ratio, an increase in the rate of cell division, an increase in the number of cell divisions, a decrease in the length of the cell division time period, an increase in the frequency of cell division phase, or uncontrolled proliferation and evasion of apoptosis. Without in any way limiting the application, the common medical meaning of the term "neoplasia" refers to "new cell growth" due to loss of responsiveness to normal growth controls, e.g., neoplastic cell growth. Neoplasia includes "tumors" which can be benign, premalignant, or malignant. The term "neoplasm" is understood to refer to a lesion, tumor or other encapsulated or unencapsulated mass or other form of growth or cell aggregation comprising neoplastic cells.

[0356] In the context of the present application, the term "neoplasm" is understood to include reference to all types of cancerous growths or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathology type or state of invasiveness.

[0357] The term "carcinoma" is art-recognized and refers to a malignant growth of epithelial or endocrine tissue, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testes, breast, prostate, endocrine system and melanoma. The term also includes carcinosarcomas, e.g., malignant tumors composed of carcinoma and sarcoma tissues. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which tumor cells form recognizable glandular structures.

[0358] The neoplastic cells comprising the neoplasm can be of any cell type derived from any tissue, such as epithelial or nonepithelial cells. Reference to the terms "malignant neoplasm" and "cancer" and "carcinoma" herein are understood to be interchangeable.

[0359] The term "neoplasm" shall be understood to refer to a lesion, tumor or other encapsulated or unencapsulated mass or other form of growth or cell aggregation comprising neoplastic cells. The neoplastic cells that make up a neoplasm can be of any cell type derived from any tissue, such as epithelial or non-epithelial cells. Examples of neoplasms and neoplastic cells encompassed by the present application include, but are not limited to, central nervous system tumors, retinoblastomas, neuroblastomas, pediatric tumors, head and neck cancers (e.g., squamous cell carcinoma), breast and prostate cancers, lung cancers (both small cell lung cancer and non-small cell lung cancer), kidney cancers (e.g., renal cell adenocarcinoma), esophagogastric cancers, hepatocellular carcinomas, pancreaticobiliary neoplasias (e.g., adenocarcinoma and islet cell tumors), colorectal cancers, cervical and anal cancers, uterine and other reproductive tract cancers, urethral cancers (e.g., ureter and bladder cancers), germ cell tumors (e.g., testicular germ cell tumors or ovarian germ cell tumors), ovarian cancers (e.g., ovarian epithelial cancers), cancers of unknown primary, human immunodeficiency-related malignancies (e.g., Kaposi's sarcoma), lymphomas, leukemias, carcinomas, sarcomas, endocrine tumors (e.g., thyroid tumors), mesotheliomas and other pleural or peritoneal tumors, neuroendocrine tumors, and carcinoid tumors.

[0360] In one particular embodiment, the neoplastic condition is a leukemia or lymphoma.

[0361] In another embodiment, the neoplastic condition is metastatic.

[0362] The subject being treated or prevented can be any human or animal in need of therapeutic or prophylactic treatment. In this regard, references herein to "treatment" and "prevention" are to be considered in their broadest context. The term "treatment" does not necessarily imply that a mammal is treated to complete recovery. Similarly, "prevention" does not necessarily mean that a subject does not ultimately contract a disease condition. Thus, treatment and prevention include ameliorating symptoms of a particular condition or preventing a particular condition or otherwise reducing the risk of developing a particular condition. The term "prevention" can be considered to reduce the severity of onset of a particular condition. "Treatment" can also reduce the severity of an existing condition.

[0363] It will therefore be appreciated that the present application encompasses reducing or otherwise ameliorating a condition in a mammal. This will be understood to mean reducing or ameliorating any one or more symptoms of the disease. While achieving a cure of the disease is always most desirable, there is still significant clinical value in slowing the progression of the disease. For example, in the case of a viral infection such as HIV or an STD, even if a complete cure cannot be achieved, a reduction in viral load and the extent of transmission can provide a means of controlling the infection such that, for example, severe HIV immunodeficiency (which is ultimately fatal) is not experienced and a relatively normal lifespan can be achieved, without the severe side effects characteristic of the current cocktail of antiviral drugs that the patient would need to take. In the specific case of a neoplastic condition, the T cells of the present application, when administered to a patient, downregulate the growth of the neoplasm. Reference to "growth" of a cell or neoplasm will be understood to mean proliferation, differentiation and / or maintenance of viability of the subject cell, and "downregulating the growth of a cell or neoplasm" means either the process of senescence of the cell or reduction, prevention or inhibition of proliferation, differentiation and / or maintenance of viability of the subject cell. In a preferred embodiment, the subject growth is proliferation and the subject downregulation is CD8 + T cell mediated killing. In this regard, killing can be evidenced by a reduction in tumor mass size or inhibition of further growth of the tumor or by slowing of tumor growth. In this regard, and without limiting the present application to any one theory or mode of action, killing of neoplastic cells can occur through any suitable mechanism, such as direct lysis or apoptosis induction or can occur through some other mechanism facilitated by CD4 + or CD8 + T cells, or T cells lacking these CD4 and CD8 markers. It will therefore be appreciated that the present application encompasses reducing or otherwise ameliorating a neoplastic condition in a mammal. This will be understood to mean preventing, reducing or ameliorating any one or more symptoms of the neoplastic condition. Symptoms can include, but are not limited to, pain at the site of tumor growth or impaired metabolic or physiological bodily function as a result of the neoplastic condition. It will be appreciated that the methods of the present application can reduce the severity of any one or more symptoms or eliminate the presence of any one or more symptoms. The methods of the present application also extend to preventing the onset of any one or more symptoms.

[0364] Accordingly, the methods of the present application are useful both in terms of treatment and palliation. In this regard, reference to "treatment" is understood to encompass both therapy and palliation. As will be appreciated by those skilled in the art, while cure of the neoplastic condition is always the most desirable outcome, there is still significant benefit in being able to slow or stop the progression of the tumor, even if it is not completely cured. Without in any way limiting the present application, there are some neoplastic conditions which, if they are sufficiently down-regulated in terms of cell division, will not be fatal to the patient, and the patient can still have a reasonable quality of life. Still further, it will be appreciated that the present methods provide a useful alternative to existing treatment regimens. For example, in some cases the therapeutic outcome of the present methods can be equivalent to chemotherapy or radiation, but the benefit to the patient is a treatment regimen which induces fewer side effects or a shortened period of side effects, and thus can be much better tolerated by the patient. As detailed above, it will also be appreciated that the term "treatment" does not necessarily imply that the subject is treated to complete recovery. Thus, as detailed above, treatment includes reducing the severity of an existing condition or ameliorating the symptoms of a particular disorder or palliation. In this regard, where the treatment of the present application is applied in the context of treating a primary tumor, it can effectively function as a prophylactic agent to prevent the onset of metastatic cancer. For example, for some types of solid tumors, surgical removal of the tumor can still be the most desirable. However, there is always the possibility that the entire tumor is not successfully removed, or there can be some risk of neoplastic cells escaping. In such a case, by applying the methods of the present application to lyse any such neoplastic cells, the methods effectively function as a prophylactic agent to prevent metastatic spread.

[0365] According to this aspect of the present application, the subject cells are preferably autologous cells which are isolated ex vivo and genetically modified, and transplanted back into the individual from which they were originally harvested. However, it will be appreciated that the present application also extends to the use of cells derived from any other suitable source, wherein the subject cells exhibit a similar histocompatibility profile to the individual who is the subject of the treatment, such that the transplanted cells can perform their function of removing unwanted cells before being rejected by the host. Thus, effectively, such cells are autologous in that they do not lead to histocompatibility problems which are normally associated with the transplantation of cells which exhibit a foreign MHC profile. Such cells are understood to fall within the definition of histocompatibility. For example, in some cases it can be desirable, necessary or of practical significance to derive the subject cells from a genetically identical twin or from an embryo which has been produced using a ligand derived from or cloned from the individual who is the subject of the treatment (in which case the cells can correspond to stem cells which have been subjected to directed differentiation into a suitable somatic cell type). The use of such cells can also be engineered to exhibit the desired major histocompatibility profile. The use of such cells overcomes the difficulties inherent in the context of tissue and organ transplants.

[0366] However, in cases where isolation or generation of autologous or histocompatible cells is not possible or feasible, it can be necessary to utilize allogeneic cells. "Allogeneic" cells are cells isolated from the same species as the subject being treated, but exhibit a different MHC profile. While use of these cells in a therapeutic context can result in graft versus host problems or host graft rejection, this problem is minimized by using cells exhibiting a MHC profile similar to that of the subject being treated, such as cells isolated / generated from a relative, such as a sibling, parent or child, or in other cases cell populations generated according to the methods exemplified herein.

[0367] It will be appreciated that in preferred embodiments, the cells used are autologous. However, due to the circumstances of a given case, it can not always be possible to generate an autologous stem cell population. This can be due to issues such as the urgency of commencing treatment or the availability of facilities to effect transformation and directed differentiation. In such cases, and as detailed above, it can be desirable or necessary to use syngeneic or allogeneic cells, such as cells that have been previously transfected and are available as frozen stocks in a cell bank. Such cells, although syngeneic, can be selected for transformation based on the expression of MHC haplotypes that exhibit lower immunogenicity than some haplotypes known to be highly immunogenic or in other cases generated according to the methods exemplified herein.

[0368] Reference to an "effective number" refers to the number of cells necessary to at least partially achieve the desired effect or delay its onset, inhibit the onset or completely stop the onset or progression of the particular condition being treated. Of course, such amount will depend on the particular condition being treated, the severity of the condition, and individual patient parameters including age, physical condition, size, body mass, physiological condition, concurrent treatment, medical history, and parameters relating to the disease in question. Determining the appropriate amount of cells of the application to be administered, and the optimal mode of administration, is within the skill of those in the art once presented with the teachings herein, and this latter question is discussed further below. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally to use the maximum safe number of cells, i.e., the highest safe number according to sound medical judgment. However, one of ordinary skill in the art will appreciate that a lower number of cells can be administered for medical reasons, psychological reasons, or for any other reason.

[0369] As discussed above, it will also be appreciated that, although the methods of the application are predicated on the introduction of genetically modified cells into an individual having a condition as defined herein, it can not be necessary that every cell of the population introduced into the individual has acquired or will maintain the subject modification and differentiation. For example, where a population of transfected and expanded cells is administered in total (i.e. without enrichment for successfully modified or differentiated cells), there can be a proportion of the population of cells that have not acquired or retained the genetic modification and / or the desired T cell cell differentiation. Thus, the application is achieved if a relevant proportion of the cells thus introduced constitute an "effective number" as defined above. However, in particularly preferred embodiments, the population of cells subjected to differentiation will be subjected to identification of successfully modified and differentiated cells, their selective isolation.

[0370] In the context of this aspect of the application, the subject cells need to be introduced into the subject individual. To this end, the cells can be introduced by any suitable method. For example, the cell suspension can be introduced by direct injection or inside a blood clot, whereby the cells are immobilized on the clot, thus facilitating engraftment. The cells can also be introduced by surgical implantation. For example, this can be necessary where the cells are present in the form of a tissue graft. The site of engraftment can be any suitable site, for example subcutaneous. Without wishing to limit the application to any one theory or mode of action, where the cells are administered as an encapsulated cell suspension, the cells will coalesce into a mass. It will also be appreciated that the cells can continue to divide after engraftment. In this regard, as discussed above, the introduction of a suicide gene provides a convenient means of controlling ongoing division.

[0371] The cells administered to the patient can be administered in a single dose or in multiple doses, by any suitable route. Preferably, and where possible, a single administration is used. Depending on the type of treatment required, administration by injection can be directed to various regions of a tissue or organ.

[0372] According to the methods of the application, other proteinaceous or non-proteinaceous molecules can be co-administered with the transfected cells. By "co-administration" is meant simultaneous administration in the same or different formulations, via the same or different routes, or via sequential administration via the same or different routes. By "sequential" administration is meant a time difference of seconds, minutes, hours or days between engraftment of the cells and administration of the proteinaceous or non-proteinaceous molecule. For example, depending on the nature of the condition being treated, it can be necessary to maintain the patient on medication to alleviate the symptoms of the condition until the engrafted cells become fully integrated and fully functional (e.g. in the case of an HIV patient, administration of anti-viral drugs). Alternatively, in treating a condition, it can be necessary to initiate long-term use of a drug to prevent recurrence of the condition. For example, where the injury to the subject is caused by an autoimmune disease, once the autoreactive cells have been destroyed, it can be necessary to continue use of a low level of immunosuppressive drug.

[0373] It will also be appreciated that the methods of the application can be performed alone to treat the condition in question, or it can be performed in conjunction with one or more additional techniques designed to facilitate or enhance the subject treatment. As detailed above, these additional techniques can take the form of co-administration of other proteinaceous or non-proteinaceous molecules or surgery.

[0374] Yet another aspect of the application relates to the use of a stem cell or a T cell differentiated therefrom as defined above in the manufacture of a medicament for the treatment of a condition characterized by the presence of an unwanted cell population in a mammal.

[0375] In another embodiment, the stem cell is an iPSC or an HSC.

[0376] In yet another embodiment, the stem cell is capable of differentiating into a CD4 + T cell or a CD8 + T cell.

[0377] In yet another embodiment, the TCR is an αβ TCR.

[0378] In yet another embodiment, the stem cell, such as an iPSC, is derived from a T cell or a thymocyte, preferably a CD8 + T cell or a thymocyte.

[0379] In yet another embodiment, the cell further comprises a nucleic acid molecule encoding a non-signalling antigen binding receptor, wherein the receptor comprises an antigen recognition moiety directed against CD47.

[0380] Reference herein to a "cell" is understood to refer to an isolated cell or an isolated or substantially purified population of cells. In the context of a population of cells, "substantially pure" means that the relevant cell type comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of all cells in the population of cells. For example, a population of cells is substantially pure with respect to a relevant T cell if such T cells comprise at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of the cells in the population.

[0381] The application is further described by reference to the following non-limiting examples. Examples

[0382] The present application is further illustrated by the following examples, which demonstrate the formation of certain embodiments of the application, including dual anticancer specific T cells derived from iPSC cells or HSCs. These examples should in no way be construed as limiting.

[0383] Example 1 : Enrichment of cancer peptide antigen specific T cells from blood

[0384] WT-1 specific TCR T cell stimulation and expansion

[0385] WT-1 specific T cells are very rare in normal human blood, but can be expanded and enriched for detection. In this context, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density gradient centrifugation. Freshly isolated PBMCs were resuspended in tissue culture medium supplemented with human AB serum, L-glutamine, and CD28 monoclonal antibody, which acts as a co-stimulator of T cells in the presence of WT-1; anti-CD28 alone does not activate T cells. Then, PBMCs were stimulated overnight with Wilman's tumor 1 (WT-1) peptide at a concentration of 0.6 ml / ml for each of the four WT-1 peptides: WT-1 37 (VLDFAPPGA, SEQ ID NO:22), WT-1 126 (RMFPNAPYL, SEQ ID NO:23), WT-1 187 (SLGEQQYSV, SEQID NO:24), and WT-1 235 (CMTWNQMNL, SEQ ID NO:25) represents a major HLA class I binding motif. The data presented in this application use WT-1 peptide 1-37 as a representative of this WT-1 peptide family. WT-1 specific T cells can be identified using HLA-WT-1 specific tetramers or by early induction of the molecule CD137 on the surface of stimulated but not resting T cells. CD137 is a member of the tumor necrosis factor (TNF) receptor family. It is also known as 4-1BB. CD137-positive cells (i.e., WT-1-stimulated T cells) were magnetically separated using a magnetic cell separator 24-36 hours later. CD137-positive (WT-1 specific TCR) cells were cultured in a T cell expansion medium consisting of X-Vivo-15 basal medium supplemented with human AB serum, recombinant interleukin-7, interleukin-15, and interleukin-21. The corresponding CD137-negative cells were further subjected to CD3 magnetic separation. CD3-negative cells (mainly B cells) were treated with mitomycin C and used as antigen-presenting feeder cells loaded with WT-1 peptide to induce a CD137-positive population. The remaining CD3-positive cells (non-WT-1 specific) were cultured in culture to serve as control T cell types for downstream functional assays. Culture medium containing recombinant cytokines was supplemented every other day.

[0386] For flow cytometry analysis, cells were resuspended in FAC buffer: every 10 6μl FcR blocking reagent was added to the cells for 5 minutes at room temperature. 10 μl of HLA-A02 WT-1 tetramer was added and the cells were incubated for 20 minutes at 4°C in the dark. 50 μl of "T cell activation" mix was added and the cells were incubated for 20 minutes at 4°C in the dark. 100 μl of FACs buffer plus 2 μl Aqua Amine was added and the cells were incubated for 5 minutes, followed by centrifugation at 150 x g for 5 minutes. The supernatant was aspirated or decanted and the pellet was resuspended in 100 μl of BD Cytofix / Cytoperm solution per sample and the cells were incubated for 20 minutes at 4°C. The cells were washed in BD / Perm wash buffer. IFN-γ antibody was diluted 1 / 100 in BD / Perm wash buffer and incubated with the cells for 30 minutes at 4°C in the dark. The cells were washed in BD / Perm wash buffer and resuspended in FACs buffer before flow cytometry analysis. FACS data acquisition was done on a Miltenyi Quant cytometer.

[0387] T cells with TCRs specific for WT-1 peptides are usually very low in frequency (e.g. Schmeid et al. (2015) showed they are as few as 1 in 10 -6 CD8+ cells (ranging from 3 x 10 -7- 3 x 10 -6 cells). After the stimulation protocol described above, WT-1 TCR specific T cells increased about 100-fold to about 3.0% (WT-1 patient #1 1.5%; WT-1 patient #2 4.0%; Figure 1 ).

[0388] Functional analysis of WT-1 TCR T cells

[0389] In addition, the in vitro expanded T cells were stimulated with autologous antigen presenting cells (EBV transformed B cells) and primed with a pool of WT-1 peptides: WT-1 37 (VLDFAPPGA), WT-1 126 (RMFPNAPYL), WT-1 187 (SLGEQQYSV), and WT-1 235 (CMTWNQMNL). Interferon gamma (IFNy) production by the T cells was detected by flow cytometry using a fluorescent bead assay. Cells were double labeled for WT-1 peptide specificity by binding to WT-1 peptide-HLA tetramers (see Figure 2).

[0390] T cells stimulated with WT-1 clearly express (80-90%) interferon gamma (IFNy) (Figure 2), which is a well-recognized measure of T cell function (e.g. Ghanekar et al. (2001)). To potentially increase the level of CD8 T cell activation (targeting WT-1 T cells), the LAG3 inhibitor IMP321 was used. LAG3 is generally a "checkpoint block" that inhibits the stimulatory function of dendritic cells (DC) and the response of CD8 T cells to DCs as antigen presenting cells. When added to the WT-1 specific T cell activation assay, there was no effect of IMP 321 at 24 hours, but after 4 days, there was a rare doubling of CD8+ WT-1 specific TCR T cells Figure 2H

[0391] Example 2: Generation of iPSCs from human blood T cells

[0392] To derive iPSCs from human blood T cells, there are a number of methods that have varying degrees of faithful preservation of the initial T cell properties. iPSCs have been generated from a broad collection of peripheral blood T lymphocyte pools (T-iPSCs) from normal healthy people. For example, T cells were pre-activated with the mitogen PHA or anti-CD3 and anti-CD28 antibodies. Using dual retroviral vector cassettes each containing two of the Yamanaka reprogramming factors (Oct4, Sox 2, KLF, cMyc), multiple T-iPSC clones were generated that were verified at the cellular and molecular level, including flow cytometry and qRT-PCR for a panel of markers including Nanog, Oct3 / 4, SSEA 3,4, TRA-1-60 and TRA-1-81. Their pluripotency was confirmed by teratoma formation after injection into NOD-SCID-IL common gamma chain- / - (NSG mice). Confirmation of T cell origin was confirmed by showing that TCR genes were rearranged.

[0393] Figure 3 Generation of iPSCs from WT-1 specific blood T cells is summarized in Example 1.

[0394] Example 3: Induction of human T cells from iPSCs

[0395] This example shows that true T cells are generated from iPSCs. These T cells are shown to express key features of typical T cells, as T cells that are normally generated by the thymus. They are shown to express the mainstream T cell alpha beta TCR and CD8 with both beta and alpha chains.

[0396] ​T cells have been induced from either adult whole liquid T cells or pre-selected CD8+ T cells, or antigen-specific T cells (e.g., those specific for WT-1) (T-iPSCs), or adult fibroblast-derived iPSCs. There are two basic stages of commitment to the hematopoietic (hematopoietic stem cell or "HSC") and partially lymphoid lineage, by culture on OP9 cells; these cultured cells are transferred to an OP cell line genetically modified to express the Notch signaling molecule Delta-like ligand 1 (OP9-DL-L1) for subsequent T cell differentiation induction.

[0397] Stage 1: Preparation of OP9 support cells and iPSC colonies

[0398] Day -8: Mitomycin-treated mouse embryonic fibroblast feeder layer plated at 0.3 x 105cells / mL in 3 mL MEF media (DMEM + 15% FCS + 1% pen / strep L-glutamine) onto a 10 cm TC plate coated with 0.1% gelatin and incubated overnight. 6 (14,250 cells / cm 2 ) plated onto 10 cm TC plates coated with 0.1% gelatin and incubated overnight. 6 OP9 cells were pre-prepared by plating at 0.25 x 105cells / mL in 11 mL OP9 media (MEMM + 20% FCS + 1% pen / strep) onto a 10 cm TC plate coated with 0.1% gelatin.

[0399] Day -7: iPS cells were thawed and plated onto MEF cells and cultured for 7 days at 37°C 5% CO2.

[0400] Stage 2: Conversion of iPSCs to hematopoietic cells

[0401] Day 0: Initiation of hematopoietic commitment. iPS colonies were dissociated and plated onto OP9 for HSC differentiation. Colony suspensions were added drop-wise for even distribution onto the OP9 plate. Fresh differentiation media was added on days 1, 5, and 9.

[0402] Day 13 harvest-induced HSC precursors for T cell differentiation

[0403] Cells cultured on OP9 cell lines were gently removed by collagenase (working solution 100 pg / mL collagenase / HBSS; 37°C for 1:15 hours) and further colony disruption into single cells by trypsin / EDTA 0.05% at 37°C for 30 minutes. Cells were gently washed and examined by phase microscopy ( Figure 4 ) and flow cytometry ( Figure 5 ). Hematopoietic identity of cells was confirmed by flow cytometry ( Figure 5 ).

[0404] Stage 3: Induction of iPSC-derived HSCs into T cells

[0405] Day 13: Induction of T cell differentiation: Day 13 OP9 conditioned (hematopoietic induced) cells are transferred to OP9 DL-L1 cells.

[0406] In a preferred embodiment, to enhance the efficiency of contact with OP9 DL-L1 cells, the cells are sorted for CD34 + CD43 + (HSC) purified OP9 conditioned cells are then plated onto OP9 DLL-1 cells for the first stage of T cell differentiation. A key component of the methods disclosed herein is the collection of cells that initially grow under OP9 DL-L1 cells.

[0407] Cells collected from OP9 cultures are resuspended in T cell differentiation media (OP9 media, SCF 5 ng / mL, Flt3 5 ng / mL, IL-7 5 ng / mL, and Vitamin C 100 mM) and the suspension is added dropwise to OP9 DLL-1 cells and incubated at 37°C. Cells are harvested after 2, 9, 16, 23, and 30 days of culture on OP9 DLL-1 and flow cytometry analysis is performed Figure 6 and Figure 7 ).

[0408] When these cultures are examined for T cell development, there is clear evidence of expression of early markers CD7 and CD9 and later markers of T cell development with CD4 and CD8 expression Figure 7 ). Even at this early stage, there are about 10% of cells expressing both CD4+ and CD8+; these CD4+CD8+ cells are characteristic of T cells normally developing in the thymic cortex (Heng et al. (2010)).

[0409] Flow cytometry shows gradual development of T cells from initial expression of CD5, CD7+, and then CD8+. Most importantly, the induced T cells express the phenotype of "best, thymus-produced" CD8 T cells. In addition to the CD8a chain, they also express the CD8b chain (other reported T cell induction systems do not induce the best signaling CD8 chain; e.g., Themeli et al. (2013)). As an indication of function, they also express ab TCR with CD3. Furthermore, these cells exist as early as day 16 of culture on OP9 DL-L1 cells, compared to day 30 in other reported systems.

[0410] Stage 4 Formation of mature T cells

[0411] Seven days later (13 days total on OP9 cells, then 16 days on OP DL-L1 cells), these developing T cells make a critical transition to express T cell receptor complex with CD8+ T cells that are clearly positive on CD3 and a TCR; in addition, these cells express important CD8- these are CAR-T desirable cells. There is a corresponding further reduction of CD34+ CD43+ HSCs Figure 8

[0412] Thus, this induction system successfully generates mature CD8 T cells from iPSCs after 13 days of culture on OP9 cells, followed by 16 days of culture on OP9 DL-L1 cells.

[0413] Using the methods described above, T cells expressing a TCR specific for WT-1 were generated from iPSCs themselves derived from WT-1 TCR CD8+ T cells Figure 9 These iPSC-derived WT-1 T cells have cytotoxic function equivalent to the original T cells from which the iPSCs were derived Figure 10

[0414] Example 4: Development of CAR constructs

[0415] The components of chimeric antigen receptor (CAR)-T cells are the antigen recognition component of the CAR mediated by a scFv extracellular domain, represented by a single chain Fv (scFv) anchored by a CD8 or CD28 hinge, and includes a transmembrane (TM) region and signal transduction by the CAR through a cytoplasmic domain - represented by CD28, 4-1BB, and CD3 zeta (CD3 zeta) chains. There are two suitable viral delivery systems - retrovirus and lentivirus. Exemplary CAR and CD47 binding receptor constructs are shown in Figure 11

[0416] Example 5: Chimeric antigen receptor vector cloning strategy

[0417] Figures 12-13 An exemplary chimeric antigen receptor vector cloning strategy is shown in FIG. 14. FIG. 14 shows the strategy for our second generation CAR and non-signaling anti-CD47 constructs. Exemplary sequences for the chimeric antigen receptor, non-signaling antigen binding receptor, and various domains thereof are provided in SEQ ID NOs: 1-20.

[0418] Example 6: Chimeric antigen receptor transduction of T cells

[0419] Lentivirus production

[0420] 293T cells were plated onto 175 cm2flasks coated with poly-L-lysine (Sigma) at 50% confluency the day before transfection. The following day, the cells were transfected with 10 ug of pMD2.G, 10 ug of psPAX2, and 20 ug of the lentiviral vector using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer’s instructions. The medium was changed 24 hours after transfection and the supernatant was collected 48 and 72 hours after transfection. The supernatant was filtered through a 0.45 pm filter and stored at -80°C until use. 2 ​​​Flasks. Two hours prior to transfection, the medium was replaced with DMEM supplemented with 10% FCS. The lentiviral transfer vector DNA was combined with the packaging and envelope plasmid DNA and mixed with Lipofectamine 2000. The solution was vortexed briefly and incubated at room temperature for 30 minutes. After this time, the solution was mixed again and then added dropwise to the cells. The flasks were returned to the incubator. After 6 hours, fresh growth medium was added. The viral supernatant was collected after 48 hours and clarified by centrifugation at 1500 rpm for 5 minutes at 4°C, then passed through a 0.45 μιη diameter PVDF Millex-HV filter (Millipore). Concentration of lentivirus was performed using ultracentrifugation with a Sorval Discovery 100 SE centrifuge using an AH-629 rotor. Thirty mL of filtered viral supernatant was added to a 36 mL heteroconic polymer conical tube (Beckman). Centrifugation was performed at 20000 g for 90 minutes. The supernatant was completely removed and the viral pellet was resuspended in 300 μΐ^of PBS and stored at -80°C until use.

[0421] Generation of CAR-T cells

[0422] Figure 11 And SEQ ID NOs: 1-6 show a set of chimeric antigen receptors (CARs) and CD47 binding receptor constructs that have been developed - with scFv specific for TAG 72 or CD19 (as a positive control). These constructs use human CD8 or CD28 as a hinge region and CD28, CD3 zeta chain or 4-1BB cytoplasmic activation signaling domains. The CARs and CD47 binding receptor constructs were cloned into lentiviral vectors as described in the preceding paragraph.

[0423] Optimal lentiviral transduction of T cells involves their activation at the TCR and costimulatory receptors. Therefore, on day 0, fresh PBMCs were collected by apheresis from a healthy donor, and activated T cells were enriched using anti-CD3 and anti-CD28 antibodies bound to paramagnetic beads (Dynabeads ClinExVivo CD3 / CD28, Invitrogen, Camarillo, CA, USA) at a 3: 1 ratio (beads:cells). Cells and beads were co-incubated at room temperature for 1 hour, and CD3+ cell enrichment was performed using a magnet (Invitrogen). Cells in the CD3+ fraction were plated at 1 x 105cells / mL in T cell expansion medium with 100 IU / ml IL-2. The cells were incubated at 37°C in a 5% C02humidified incubator. The medium was replaced every 3-4 days with fresh T cell expansion medium with 100 IU / ml IL-2. After 10-14 days, the cells were harvested and used for lentiviral transduction. 6Cells were resuspended at a concentration of 0.5-1 x 10 6 cells / mL in fresh T cell proliferation media. Cultures were maintained to day 14 and fed every other day with fresh expansion media to maintain cell concentration at 1 x 10 6 cells / mL.

[0424] Blood-derived human T cells were first subjected to CAR transduction, the success of which was measured by flow cytometry, depicting eGFP+ cells Figure 15 This was also confirmed by Western blot analysis Figure 16 .

[0425] CAR-T cells were evaluated for function.

[0426] TAG-72 CAR-T cells (created from normal PBMCs) were examined for their ability to kill target cells expressing TAG72 in vitro. The real-time cell monitoring system (xCELLigence) was employed to determine CAR-T cell killing efficiency in vitro. 10,000 - 2 x 10 6 target cells / 100 μΐ, (e.g. TAG72+ ovarian cancer cell line CaOV4) were deposited into RTCA plates. In some cases, target cells can need to be tethered by pre-coating the plates with anti-hCD40 or with human fibronectin. Target cells were maintained at 37°C, 5% C02for 3-12 hours to allow cell attachment. After target cell attachment, CAR-T effector cells were added at variable effector: target ratios (ranging from 1:1 to 10:1). In some experiments, CAR-T effector cells were isolated based on their GFP expression by FACS prior to use. Co-cultures were maintained for at least 12 hours under optimal growth conditions. Cell impedance was monitored throughout; a decrease in impedance indicates cell shedding and eventual cell death.

[0427] Figure 17Results from this experiment are shown for monitoring over 40 hours. The ovarian cancer cell line CaOV4 grew consistently over this time period (blue line). In contrast, cultures supplemented with TAG-72 specific CAR T cells showed significantly less initial growth period than the initial growth period of target cells alone, followed by gradual elimination of target cells over time (purple line). To overcome non-specific killing due to CD3 / CD28 activation, TAG 72 CAR-T cells were isolated by flow cytometry and compared to CD19 CAR-T cells and non-CAR-T cells (without prior CD3 / CD28 activation) Figure 18 Figure 18 Data shown in FIG. 6 indicate strong antigen specificity of TAG-72 CAR-T cells within the first 24 hours of culture with TAG-72 expressing cancer cells, as negative controls of vector transfected T cells and untransfected T cells showed no cancer cell killing within this time frame.

[0428] The above studies were performed on polyclonal T cells derived from peripheral blood. To demonstrate CAR-transduction of monospecific T cells expressing TCRs specific for nominal cancer peptide antigens, WT-1 TCR specific T cells were transduced by TAG 72 CAR lentivirus, which were derived from iPSCs formed from WT-1 specific TCRs. Figure 19A Success of CAR transduction of these WT-1 specific TCR CD8+ T cells, which themselves were derived from iPSCs generated from WT-1 specific T cells, is shown. The CAR contains specificity for TAG 72. Most importantly, Figure 19B Success of transduction of WT-1 specific TCR CD8+ T cells, which themselves were derived from iPSCs generated from WT-1 specific T cells, with CAR constructs against both TAG 72 and CD47 is shown. This indicates that T cells with three cancer specificities can be generated: WT-1 (TCR), TAG 72 (CAR), and CD47 (truncated CD47 binding receptor).

[0429] These results demonstrate the formation of dual specificity CAR transduced cancer specific TCRs (WT-1) derived from iPSCs, which themselves were derived from WT-1 specific TCR T cells from normal adult blood.

[0430] ​Figure 20 shows that the two components of the bispecific T cell (containing a WT-1 TCR and a TAG72 CAR) can contribute to killing of cancer cells. When corrected for spontaneous cell death, the WT-1 cells caused about 10% cell killing, then the addition of the TAG72 CAR by transduction caused an additional 10% killing, even at a low effector-target ratio (here, it was 2 effectors to 1 target cell).

[0431] Example 7: Chimeric Antigen Receptor Transduction of iPSCs

[0432] Generation of multispecific CAR-T cells can be achieved by a variety of methods, including CAR transduction of existing blood T cells Figure 15 ) or by transduction of iPSCs, which are then induced to be T cells (expressing a cancer-specific TCR and CAR) (e.g., SEQ ID NOs: 1-6). Progress has been made using a variety of iPSC lines for CAR-T transduction. These iPSCs can be derived from non-T cells, or cancer antigen-specific T cells that will retain TCR gene rearrangements (e.g., WT-1). These iPSCs are derived from adult fibroblasts or T cells with endogenous TCRs specific for a particular cancer antigen (WT-1 peptide).

[0433] As shown in Figure 14, iPSCs were stably transduced with a single cistron, where the CAR ectodomain contains a scFv specific for TAG72 (or CD19 as a control). The hinge (stalk) region and transmembrane region are derived from CD28 or CD8, and the intracytoplasmic domain containing the T cell signaling domain is derived from CD28 and TCR zeta chain. The CAR has a C-terminal extension encoding EGFP linked by a P2A self-cleaving polypeptide that separates the CAR and the reporter. Upon viral integration, the P2A is cleaved, and the success of transduction is quantified by measuring the fluorescence of the released EGFP reporter. GFP fluorescence is indicative of successful transduction. It can be used to show in situ transduction (Figure 21) or to identify and isolate CAR-transduced iPSCs Figure 22 by flow cytometry (23).

[0434] These studies clearly show the ability to transduce iPSCs with lentiviral CAR constructs. Figure 21A It is shown that iPSCs derived from human fibroblasts were successfully transduced with a CAR encoding TAG 72 or CD19 Figure 21A . Figure 21B It is shown that iPSCs derived from WT-1 TCR-specific T cells were successfully transduced with TAG72. Thus, any T cells from this line will express a dual anti-cancer specificity (WT-1 by TCR; TAG 72 by CAR).

[0435] Transduced iPSCs can also be isolated by fluorescence-based cell sorting. Positive cells can be collected and replated to successfully form (CAR-transduced) iPSC colonies Figure 24

[0436] Those skilled in the art will appreciate that the application herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the application includes all such variations and modifications. The application also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0437] Bibliography

[0438] Balasubramanian S, Babai N, Chaudhuri A, Qiu F, Bhattacharya S, Dave BJ,

[0439] Parameswaran S, Carson SD, Thoreson WB, Sharp JG, et al. (2009) Non cell- autonomous reprogramming of adult ocular progenitors: generation of pluripotent stem cells without exogenous transcription factors. Stem Cells. ;27:3053-3062 [PubMed: 19859985]

[0440] Brignone, C, C. Grygar, M. Marcu, K. Schakel, and F. Triebel. 2007. A soluble form of lymphocyte activation gene-3 (IMP321) induces activation of a large range of human effector cytotoxic cells. J Immunol 179:4202.

[0441] ​Casucci M, Hawkins RE, Dotti G, Bondanza A. (2015). Overcoming the toxicity hurdles of genetically targeted T cells. Cancer Immunol Immunother.; 64(1): 123-30

[0442] Chuo BK, Mali P, Huang X, Ye Z, Dowey SN, Resar LM, Zou C, Zhang YA,

[0443] Tong J, Cheng L. (2011) Efficient human iPS cell derivation by a

[0444] non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res.; 21 :518-529 [PubMed: 21243013]

[0445] Corrigan-Curay J, Kiem HP, Baltimore D, O'Reilly M, et al, Kohn DB. (2014). T-cell immunology: looking forward. Mol Ther.; 22(9): 1564-74

[0446] Curran KJ, Pegram HJ, Brentjens RJ. (2012). Chimeric antigen receptors for T cell immunotherapy: current understanding and future directions. J Gene Med.; 14(6):405-15

[0447] Curran KJ, Seinstra BA, Nikhamin Y, Yeh R et al Brentjens RJ (2015)

[0448] Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression. Mol Ther.; Jan 13. doi: 10.1038 / mt.2015.4. [Epub ahead of print]

[0449] Davila ML, Riviere I, Wang X, Bartido S, et al Brentjens R. (2014). Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med.; 6(224): 224

[0450] Dotti G, Gottschalk S, Savoldo B, Brenner MK. (2014). Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev.; 257(1): 107-26

[0451] Esteban MA, Wang T, Qin B, Yang J, Qin D, Cai J, Li W, Weng Z, Chen J, Ni S, et al. (2010) Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell.; 6: 71-79 [PubMed: 20036631]

[0452] Fedorov VD, Sadelain M, Kloss CC. (2014). Novel approaches to enhance the specificity and safety of engineered T cells. Cancer J; 20(2): 160-53.

[0453] Fletcher AL, Calder A, Hince MN, Boyd RL, Chidgey AP. (2011). The contribution of thymic stromal abnormalities to autoimmune disease. Crit Rev Immunol; 7(12):954-63

[0454] Ghanekar SA, Nomura LE, Suni MA, Picker LJ, Maecker HT, Maino VC. (2001). Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65. Clin Diagn Lab Immunol. 8(3):628-31

[0455] Gargett, T, Brown MP. (2014). He inducible caspase 9 suicide gene systems as a'safety switch" to limit on-target, off-tumour toxicities of chimeric antigen receptor T cells. Front. Pharmacol 28:5:235

[0456] Ghosh et al., 1991 Glycobiology 5:505-10

[0457] Han EQ, Li XL, Wang CR, Li TF, Han SY. (2013). Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges. J Hematol Oncol.; 8; 6:47

[0458] Heng, T.S.P, Chidgey, A.P., and Boyd, R.L., (2010) Getting back at nature: understanding thymic development and overcoming its atrophy. Curr Opin Pharmacol, 10:425-433

[0459] Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, Melton DA. (2008) Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol.; 26: 1269-1275 [PubMed: 18849973]

[0460] Ichida JK, "Blanchard J, Lam K, Son EY, Chung JE, Egli D, Loh KM, Carter AC, DiGiorgio FP, Kiszka K, et al. (2009) A small-molecule inhibitor of TGF-β signalling replaces Sox2 in reprogramming by inducing nanog. Cell Stem Cell.; 5: 491-503 [PubMed: 19818703]

[0461] Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. (2009)

[0462] Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature. 458: 771-775

[0463] Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Aburjarour R, Lin X, Hahm HS, Hao E, Hayek, A, Ding S. (2009) A chemical platform for improved induction of human iPSCs. Nat Methods; 6: 805-808 [PubMed: 19838168]

[0464] Liu J et al. (PLOS One (2015) Sep 21; 10(9): e0137345)

[0465] Mali P, Chuo BK, Yen J, Ye Z, Zou J, Dowey S, Brodsky RA, Ohm JE, Yu W, Baylin SB, et al. (2010) Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes. Stem Cells; 28:713-720. [PubMed: 20201064] Narsihn KH, Jia F, Robbins RC, Kay MA, Longaker MT, Wu JC. (2011)

[0466] Generation of adult human induced pluripotent stem cells sing nonviral minicircle DNA vectors. Nature Protoc.; 6: 78-88. [PubMed: 21212777] Noggle S, Fung H-L, Gore A, Martinez H, Satriani KS, Prosser R, Oum K, Paull D, Druckenmiller S, Freeby M, et al. (2011) Human oocytes reprogram somatic cells to a pluripotent state. Nature.; 478: 70-75 [PubMed: 21979046] Pappas DJ, Gourraud, R-A, Le Gall C, Laurent J, Trounson A, DeWitt N and Talib S. (2015) Proceedings: Human Leukocyte Antigen Haplo-Homozygous Induced Pluripotent Stem Cell Haplobank Modeled After the California Population: Evaluating Matching in a Multiethnic and Admixed Population. Stem Cells Translational Medicine 4:413-418

[0467] Perna SK, Savoldo B, Dotti G (2014). Genetic modification of cytotoxic T lymphocytes to express cytokine receptors. Methods Mol Biol.; 1139: 189-200 "Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York)

[0468] Schmied,S,Gostsick E,PriceDA,AbkenH,AssenmacherM,RichterA.(2015).Analysis of the functional WT1 specific T-cell repertoire in healthydonorsreveals a discrepancy between CD4+and CD*+memoryformation.Immunology145:558-569

[0469] Subramanyam D,Lamouille S,Judson RL,Liu Jy,Bucay N,Derynck R,BlellocR.(2011)Multiple targets of miR-302 and mi$-372 promote reprogrammingofhuman fibroblasts to induced pluripotent stem cells.Nature Biotechnol.;29:443-448.[PubMed:21490602]

[0470] Themeli M,Kloss CC,Ciriello G et al M,Sadelain M(2013)NatBiotechnol31(10):928-33

[0471] Ui-Tei et al,2000 FEBS Letters 479:79-82

[0472] Warren L,Manos PD,Ahfeldt,Loh YH,Li H,Lau F,Ebina W,Mandal PK,SmithZD,Meissner A,et al.(2010)Highly efficient reprogramming to pluripotency anddirected differentiation of human cells with synthetic modified mRNA.CellStem Cell.;7:618-630.[PubMed:20888316]

[0473] Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R, Cowling R, Wang W, Liu P, Gertsenstein M, et al. (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature; 458:766-770. [PubMed: 19252478]

[0474] Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S. (2009) Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell; 5:237-241 [PubMed: 19716359]

[0475] Zhu S, Li w, Zhou H, Wei W, Ambasudhan R, Lin T, Kim J, Zhang K, King S. (2010) Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell; 7:651-655 [PubMed: 21112560]

[0476] U.S. Patent No. 5,350,674

[0477] U.S. Patent No. 5,585,362

[0478] WO 2001 / 96584

[0479] WO 2001 / 29058

[0480] U.S. Patent No. 6,326,193

[0481] U.S. Patent Application Serial No. 14 / 656,431, published as US 20150183874 Al.

Claims

1. A genetically modified mammalian stem cell, which cell is capable of differentiating into a T cell expressing a T cell receptor (TCR) directed against a first antigenic determinant, and which comprises a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen recognition moiety directed against a second antigenic determinant operably linked to a T cell activation moiety by a hinge region and a transmembrane domain; wherein: (1) the antigen recognition moiety comprises the amino acid sequence of SEQ ID NO: 8; (2) the hinge region is a CD8 hinge or a CD28 hinge; (3) the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain; and (4) the T cell activation moiety comprises (a) a 4-1BB signaling domain or a CD28 signaling domain, and (b) a TCR zeta signaling domain.

2. The cell according to claim 1, wherein the CD8 hinge comprises the amino acid sequence of SEQ ID NO: 12, the CD28 hinge comprises the amino acid sequence of SEQ ID NO: 14 or 15, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 13, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or 17, the 4-1BB signaling domain comprises the amino acid sequence of SEQ ID NO: 19, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO: 18, and / or the TCR zeta signaling domain comprises the amino acid sequence of SEQ ID NO:

20.

3. The cell according to claim 1 or 2, wherein the cell is selected from an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell (HSC).

4. The cell according to claim 1 or 2, further comprising a nucleic acid encoding a non-signaling antigen binding receptor comprising an antigen recognition moiety directed against an antigenic determinant different from the first antigenic determinant and the second antigenic determinant.

5. The cell according to claim 3, further comprising a nucleic acid encoding a non-signaling antigen binding receptor comprising an antigen recognition moiety directed against an antigenic determinant different from the first antigenic determinant and the second antigenic determinant.

6. The cell according to claim 4, wherein the non-signaling antigen binding receptor comprises an antigen recognition moiety directed against CD47.

7. The cell according to claim 5, wherein the non-signaling antigen binding receptor comprises an antigen recognition moiety directed against CD47.

8. A method of generating a T cell, comprising: (i) obtaining a stem cell capable of differentiating into a T cell expressing a TCR directed against a first antigenic determinant; (ii) differentiating the stem cell into a T cell; and (iii) introducing into the T cell one or more nucleic acids encoding one or more chimeric antigen receptors directed against an antigenic determinant different from the first antigenic determinant, wherein at least one of the nucleic acids is a nucleic acid encoding a chimeric antigen receptor as defined in claim 1 or 2. ​ ​ ​ ​ ​ ​ ​ 9. The method according to claim 8, further comprising introducing into the T cell one or more nucleic acids encoding one or more antigen binding receptors, each antigen binding receptor directed against an antigenic determinant different from the first antigenic determinant and the antigenic determinant against which the chimeric antigen receptor is directed.

10. Use of the cell according to any one of claims 1-7 in the manufacture of a medicament for treating a condition in a mammal, wherein the condition is ovarian cancer.

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