Genetically modified cells and uses thereof

Abstract

To provide genetically modified cells and uses thereof.SOLUTION: The present invention generally relates to a population of stem cells (e.g., iPSCs or HSCs) comprising a T cell receptor and a nucleic acid encoding a chimeric antigen receptor directed to a plurality of distinct antigenic determinants, e.g., two distinct tumor antigenic determinants. The present invention is also directed to a population of T cells co-expressing a T cell receptor and a chimeric antigen receptor directed to multiple distinct antigenic determinants, such as two distinct tumor antigenic determinants. The cells of the invention may be derived from selected donors whose HLA type matches a significant sector of the population, and are useful in a variety of applications, particularly in the context of therapeutic treatment of neoplastic conditions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority from Australian Provisional Patent Application No. 2015904933, filed November 27, 2015, and Australian Provisional Patent Application No. 2016901328, filed April 11, 2016, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention generally relates to a population of stem cells (e.g., iPSCs or HSCs) comprising a nucleic acid encoding a T cell receptor and a chimeric antigen receptor directed to multiple distinct antigenic determinants, e.g., two distinct tumor antigenic determinants. The present invention also relates to a population of T cells that co-express a T cell receptor and a chimeric antigen receptor directed to multiple distinct antigenic determinants, e.g., two distinct tumor antigenic determinants. The cells of the present invention can be derived from selected donors whose HLA type matches a significant sector of the population and are useful in a variety of applications, particularly in the context of therapeutic treatment of neoplastic conditions. [Background technology]

[0003] Background of the Invention Bibliographic details of publications mentioned by the authors in this specification are collected alphabetically at the end of the description.

[0004] Reference in this specification to any prior publication (or information derived therefrom) or any publicly known matter is not, and should not be, considered to be an acknowledgement or admission or any form of suggestion that the prior publication (or information derived therefrom) or publicly known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0005] Malignant tumors, or cancers, grow in an uncontrolled manner, invade normal tissues, and often metastasize, growing at sites distant from the tissue of origin. Generally, cancers originate from one or a small number of normal cells that undergo a poorly understood process called malignant transformation. Cancers can arise from almost any tissue in the body. Cancers originating from epithelial cells, called carcinomas, are the most common type of cancer. Sarcomas are malignant tumors of mesenchymal tissue and arise from cells such as fibroblasts, muscle cells, and adipocytes. Solid malignant tumors of lymphoid tissue are called lymphomas, while blood-borne malignant tumors of the bone marrow and lymphocytes and other hematopoietic cells are called leukemias.

[0006] Cancer is one of the three leading causes of death in developed countries. As treatments for infectious diseases and prevention of cardiovascular disease continue to improve in these countries and average expected life expectancy increases, cancer is likely to become the most common fatal disease. Therefore, successful cancer treatment requires the removal or destruction of all malignant cells without causing patient death. The ideal way to achieve this is to induce an immune response against the tumor that will distinguish tumor cells from their normal cellular counterparts. However, immunological approaches to treating cancer have been attempted for a century without sustained results.

[0007] Solid tumors are the leading cause of cancer deaths. Solid tumors are usually incurable once they have spread, or "metastasized," throughout the body. The prognosis for metastatic solid tumors has improved only slightly over the last 50 years. The best chance of curing solid tumors relies on early detection and the subsequent use of localized treatments, such as surgery and / or radiation therapy, when the solid tumor is localized and has not spread to tumor-draining lymph nodes or elsewhere. Nevertheless, even at this early stage, microscopic deposits of cancer, known as micrometastases, may have already spread throughout the body, particularly if the tumor has spread to the draining lymph nodes, subsequently resulting in the patient's death. In this sense, cancer is a systemic disease requiring systemically administered treatments.

[0008] The "Golden Bullet" approach, which seeks to attack cancer with toxin-loaded antibodies that exploit the antibody's ability to potentially target any specific molecular entity, such as carbohydrates, lipids, or proteins, or a combination thereof, has a long history. Once bound to cancer cells, antibodies can engage complement or FcR+ NK / K cells and induce cell lysis. Unfortunately, antibody treatments for cancer have generally met with only moderate success, primarily due to low affinity binding, low lytic efficiency, and short lifespan. Collectively, these compromise the antibody's ability to rapidly destroy cancer cells and increase the risk of mutation and immune evasion. Recently, there have been reports of antibody-based therapies, including those based on high-affinity antibodies directed against cancer molecules and immune checkpoint blockade molecules. While the latter, in particular, have shown some clinical success, such therapies still have various limitations.

[0009] Thus, the common method of cancer treatment continues to follow the long-established protocol of surgical removal (if possible), followed by radiation therapy and / or chemotherapy, if necessary. The success rate of this rather crude treatment varies greatly, but generally declines significantly as tumors become more advanced and metastasize. Furthermore, these treatments are associated with severe side effects, including scarring and scarring from surgery (e.g., mastectomy, or limb amputation), severe nausea and vomiting from chemotherapy, and, most notably, damage to normal tissues such as hair follicles, intestine, and bone marrow induced as a result of the relatively nonspecific targeting mechanisms of the toxic drugs that form part of most cancer treatments.

[0010] Thus, there is an urgent and ongoing need to develop improved systemic treatments for cancer, particularly metastatic cancer.

[0011] Thymic production of mainstream T cells is fundamental for defense against infection. This pool of “immune surveillance” T cells patrols the body, eliminating damaged or abnormal cells, including cancers. Because thymus-based T cell production is characterized by the random generation of T cell receptor (TCR) repertoires, thymocyte proliferation must also involve a highly stringent selection process that eliminates or functionally silences developing thymic T cells with the potential to attack the self. This “self-tolerance” therefore limits autoimmune disease (Fletcher et al., 2011). However, if non-viral-induced cancers are defined as “self” diseases, this process inevitably impairs immune surveillance against cancer. This means that many of the T cells generated in the thymus that could potentially be reactive with tumor-associated antigens may be eliminated before entering the blood. At the very least, they will be insufficient in number and likely have low-affinity TCRs. Despite this, T cells clearly represent a potentially major weapon against cancer. Therefore, the challenge is to increase their ability to detect cancer, expand their numbers, and maintain, improve, or enhance their potent cytolytic capabilities. Antibodies and T cells are the most logical weapons against cancer, but their potential for rapid and effective cancer destruction has not been clinically recognized. Advances in immunotherapy have been made by genetically engineering T cells to express novel chimeric membrane receptors consisting of cancer antigen-binding antibody fragments cytoplasmically coupled to T cell signaling molecules. The latter are typically one or all of the following: the TCR ζ chain, CD28, or CD40 ligands (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 harness the immune system's two most powerful anti-cancer weapons but also overcome their individual inadequacies: CAR-T retain potent and localized cytolytic capacity, preventing normal reliance on intrinsic TCRs to detect extremely rare "cancer peptides" expressed in HLA clefts.The repertoire of T cells specific for such a small number of peptides is extremely rare. The antibody portion of the CAR confers cancer-seeking specificity to the T cells, overcoming the notoriously low efficacy of circulating antibodies in killing cancer. Thus, cancer binding is mediated by the antibody domain of the CAR, resulting in cytoplasmic signaling that triggers the T cell lytic pathway to destroy the cancer.

[0012] Although still in the early clinical stages, numerous CAR-T clinical trials are underway. As promising as it may be, there are several aspects of CAR-T technology that pose problems and prevent its clinical efficacy from being fully realized. The most obvious is the tumor cell burden-dependent cytokine surge that occurs during T cell-mediated tumor destruction. Fever is an indicator of tumor destruction, but can lead to severe clinical side effects if not carefully managed (Davila et al., 2014; Casucci et al., 2015). (2015)). Current management relies on cytokine modulation treatments such as anti-IL6. Furthermore, there are significant problems related to the numerical insufficiency of CAR-T cells generated not only to attack the initial cancer but also to maintain an adequate supply in case of recurrence. Currently, attempts to address this problem are based on the excessive use of proliferation-inducing cytokines in vitro. Furthermore, as effective as CAR-T cells are in attacking cancer, they also need to be able to inhibit CD19 + Even for cancer, tumor destruction is not 100% effective. Responses of up to 90% have been reported for B-ALL, but other CD19 + Results in cancer have been less promising. Thus, despite encouraging observations regarding the utility of CAR-T, there are still significant challenges for this technology to overcome before it can assume the role of a reliable, effective, and novel gold standard for cancer treatment. In work preceding the present invention, it was determined, inter alia, that seemingly different problems currently existing with respect to the effective therapeutic application of CAR-T technology could be solved if CAR-T cells could be derived from stem cells, such as transfected adult stem cells, or other transfected somatic cell types, rather than from transfected thymocytes. For example, by transfecting stem cells (such as induced pluripotent stem cells ["iPSCs"] derived from adult somatic cells) with chimeric antigen receptors, the problem of obtaining a sufficient presence and future supply of CAR-T cells directed against specific tumors is solved, due to the continuous supply of somatic T cells derived from these self-renewing transfected stem cells. Furthermore, these iPSCs, and therefore CAR-T cells, derived from transfected stem cells can be pre-selected from donors homozygous for homozygous HLA haplotypes, particularly HLA types that are widely expressed in the population, thereby providing a means of generating banks of cells representing a wide range of donor compatibility. It was further determined that generating iPSCs from T cells exhibiting T cell receptor specificity directed against an antigen of interest means that a genetic rearrangement for that TCR specific for the cancer antigen will be incorporated into the iPSCs. All T cells derived from the iPSCs will retain the anti-cancer TCR specificity. Subsequently, such iPSCs can be transfected with a CAR and then differentiated into T cells, such as CD4+ or CD8+ T cells, that stably exhibit dual specificity for the TCR directed against the antigen to which the CAR is directed and the antigen to which the original T cell was directed. While not limiting the present invention to any one theory or mechanism of action, this is believed to be due to an epigenetic memory mechanism. It was also determined that bispecific NKT cells can be generated in a similar manner. Thus, a continuous supply of T and NKT cells selectively and stably directed against multiple distinct antigenic determinants, such as multiple distinct tumor antigenic determinants, can be provided, thereby enabling more therapeutically effective treatment steps to be undertaken. [Prior art documents] [Non-patent literature]

[0013] [Non-licensed document 1] 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 [Non-licensed document 2] 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 [Non-licensed document 3] 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

Non-licensed Document 4

Non-licensed Document 5

[0014] Summary of the Invention Unless the context requires otherwise, throughout this specification and the claims that follow, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean 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.

[0015] As used herein, the term "derived from" should be taken to indicate that a particular integer or group of integers originates from a specified species, but is not necessarily obtained directly from a specified source. Additionally, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0016] The subject specification contains amino acid sequence information generated using the program PatentIn Version 3.5, which is presented after the references herein. Each amino acid sequence is represented by a numerical designation. <210> Subsequent sequence identifiers (e.g., <210> 1. <210> The length, type of sequence (protein, etc.) and source organism for each amino acid sequence are identified in the sequence listing by a numeric designator. <211> , <212> and <213> The amino acid sequences referred to in are identified by the designated SEQ ID NO: followed by the sequence identifier (e.g., SEQ ID NO: 1, SEQ ID NO: 2, etc.). The sequence identifiers referred to in the specification are identified by the numeric designation box in the sequence listing followed by the sequence identifier. <400> associated with information provided to (e.g., <400> 1. <400> 2, etc.) In other words, SEQ ID NO: 1 detailed in the specification is <400> Associated with the sequence indicated as 1.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] One aspect of the present invention is directed to genetically modified mammalian stem cells, or T cells differentiated therefrom, wherein the cells have the capacity to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and comprise a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen recognition moiety directed to a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0019] In another embodiment, a genetically modified mammalian stem cell, or a T cell differentiated therefrom, is used to treat a CD4 T cell that expresses a TCR directed against a first antigenic determinant. +In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0020] In yet another embodiment, a genetically modified mammalian stem cell, or a T cell differentiated therefrom, wherein the cell expresses a CD8 TCR directed against a first antigenic determinant. + In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0021] In a further aspect, provided is a genetically modified mammalian stem cell, or a T cell differentiated therefrom, wherein the cell is an iPSC (induced pluripotent stem cell) or an HSC (hematopoietic stem cell), has the capacity to differentiate into a T cell expressing a TCR directed to a first antigenic determinant, and comprises a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen recognition moiety directed to a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In some embodiments, the genetically modified stem cell, such as an iPSC or an HSC, expresses at least one homozygous HLA haplotype.

[0022] According to aspects of the invention, in one embodiment, stem cells (eg, iPSCs) are derived from cells that have undergone TCR gene rearrangement.

[0023] In another embodiment, the stem cells (eg, iPSCs) are derived from T cells or thymocytes that express an αβTCR.

[0024] In yet another embodiment, the stem cells (eg, iPSCs) are derived from T cells or thymocytes that express a γδTCR.

[0025] 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 that directed by the TCR expressed in the T cell derived from the stem cell (e.g., iPSC).

[0026] In yet another embodiment, the stem cells (e.g., iPSCs) are CD8 + They are derived from T cells or thymocytes, which are

[0027] In yet another embodiment, the stem cells (e.g., iPSCs) are CD4 + They are derived from T cells or thymocytes, which are

[0028] In one embodiment, the stem cells (e.g., iPSCs or HSCs) are CD4+ cells expressing a TCR directed against a first antigenic determinant. + In another embodiment, the stem cells (e.g., iPSCs or HSCs) are CD8 T cells that express a TCR directed against a first antigenic determinant. + They have the ability to differentiate into T cells.

[0029] In yet another further aspect, there is provided a genetically modified mammalian stem cell, or a T cell differentiated therefrom, which cell has the capacity to differentiate 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, said receptor comprising an antigen recognition moiety directed against a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety, and said antigenic determinant being selected from a tumor antigen, a microbial antigen, or an autoreactive immune cell antigen. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0030] In one embodiment, the stem cells are iPSCs, hi another embodiment, the stem cells are HSCs.

[0031] In another embodiment, the stem cells are CD4 + T cells or CD8 + They have the ability to differentiate into T cells.

[0032] In yet another embodiment, the TCR is an αβ TCR.

[0033] In yet another embodiment, the stem cells (e.g., iPSCs) are T cells or thymocytes, preferably CD8 + In some embodiments, the stem cells (e.g., iPSCs) are derived from T cells or thymocytes. In some embodiments, the stem cells (e.g., iPSCs) are CD8+ / CD ... + Derived from T cells or thymocytes.

[0034] In yet another aspect, a genetically modified mammalian stem cell or a T cell differentiated therefrom is provided, the cell having the ability to differentiate into a T cell expressing a TCR directed against a first tumor antigenic determinant, the cell comprising a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen recognition moiety directed against a second tumor antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety, the first antigenic determinant being selected from a peptide recognized by the TCR, such as WT-1 or EbvLMP2, and the second antigenic determinant being selected from, for example, TAG-72, CD19, MAGE, or CD47. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0035] The genetically modified mammalian stem cells (e.g., iPSCs or HSCs) disclosed herein have the ability to differentiate 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 directed to a second antigenic determinant and comprising an antigen recognition portion (e.g., a second tumor antigenic determinant) operably linked to a T cell activation portion. That is, the genetically modified stem cells (e.g., iPSCs or HSCs) disclosed herein have the ability to differentiate into T cells directed to multiple, i.e., at least two (in other words, two or more), antigenic determinants. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0036] Thus, in a further aspect, there is provided a genetically modified mammalian stem cell that has the capacity to differentiate into T cells directed against more than two antigenic determinants.

[0037] According to aspects of the present invention, in some embodiments, the genetically modified mammalian stem cells (e.g., iPSCs or HSCs) have the capacity to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and comprise a plurality (i.e., two or more) nucleic acid molecules encoding a plurality of chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed to the antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0038] In one embodiment, the multiple antigenic determinants directed against the multiple chimeric antigen receptors are distinct from each other and the first antigenic determinant directed against the TCR expressed in the T cell derived from the stem cell. In another embodiment, the multiple antigenic determinants directed against the multiple chimeric antigen receptors are distinct from each other, and the first antigenic determinants directed against the TCR expressed in the T cell derived from the stem cell are also distinct.

[0039] In one embodiment, multiple CAR-encoding nucleic acids are contained in a single contiguous nucleic acid fragment. For example, multiple CAR-encoding nucleic acids are placed in a single construct or vector that is transfected into cells to generate genetically modified mammalian stem cells containing multiple CAR-encoding nucleic acids. In certain embodiments, multiple CAR-encoding nucleic acids can be linked to each other in a single expression unit and reading frame (e.g., by using a self-cleaving peptide such as P2A) so that a single polypeptide containing multiple CAR polypeptide sequences is first produced and then processed to produce multiple CARs. In another embodiment, multiple CAR-encoding nucleic acids are placed in separate vectors used for transfection to generate genetically modified mammalian stem cells containing multiple CAR-encoding nucleic acids. An example of a CAR-encoding nucleic acid construct is illustrated in Figure 11, and exemplary sequences for CARs and various domains suitable for use in CARs are provided in SEQ ID NOs: 1-2 and 7-20.

[0040] Further aspects of the present invention provide genetically modified mammalian stem cells capable of differentiating into T cells directed against more than two antigenic determinants, and in other embodiments, genetically modified mammalian stem cells (e.g., iPSCs or HSCs) (optionally expressing at least one homozygous HLA haplotype) capable of differentiating into T cells expressing a TCR directed against a first antigenic determinant, comprising a nucleic acid molecule encoding a chimeric antigen receptor directed against a second antigenic determinant and comprising an antigen recognition portion operably linked to a T cell activation portion, and further comprising a nucleic acid molecule encoding an antigen-binding receptor comprising an antigen recognition portion directed against a third antigenic determinant. According to these embodiments, such genetically modified stem cells have the ability to differentiate into T cells directed against multiple antigenic determinants, preferably multiple antigenic determinants distinct from one another. Additional antigen specificity can be provided by using multiple nucleic acids encoding CARs as described herein and / or utilizing multiple nucleic acids encoding antigen-binding receptors.

[0041] In one embodiment, the antigen-binding receptor is a non-signaling antigen-binding receptor; in other words, the receptor is anchored to the cell surface and binds to a third antigenic determinant but does not transduce a signal to the cytoplasm of the cell. In one embodiment, the antigen-binding receptor comprises an antigen recognition portion directed to a third antigenic determinant and operably linked to a transmembrane domain, but lacks a T cell activation portion.

[0042] In certain embodiments, the antigen-binding receptor is a non-signaling antigen-binding receptor directed against CD47. For example, the antigen-binding receptor is a non-signaling CD47-binding molecule, e.g., a truncated CD47-binding molecule.

[0043] Thus, provided are genetically modified mammalian stem cells (e.g., iPSCs or HSCs) or T cells differentiated therefrom, which have the ability to differentiate into T cells expressing a TCR directed against a first antigenic determinant, and which comprise: (i) a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen-recognition portion directed against a second antigenic determinant, the antigen-recognition portion operably linked to a T cell activation portion, and (ii) a nucleic acid molecule encoding a non-signaling CD47-binding molecule, e.g., a truncated CD47-binding molecule. In some embodiments, the genetically modified mammalian stem cells (e.g., iPSCs or HSCs) express at least one homozygous HLA haplotype.

[0044] In another aspect, methods of producing the genetically modified mammalian stem cells (such as iPSCs or HSCs) disclosed herein are provided.

[0045] In one embodiment, a subject method includes obtaining mammalian stem cells (such as iPSCs or HSCs) that have the potential to differentiate into T cells that express a TCR directed to a first antigenic determinant, where in one embodiment, the stem cells (e.g., iPSCs or HSCs) express at least one homozygous HLA haplotype; and introducing (e.g., by transfection) into the stem cells one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition portion directed to the antigenic determinant, the antigen recognition portion being operably linked to a T cell activation moiety. In another embodiment, the method further includes introducing (e.g., by transfection) into the stem cells one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signaling antigen binding receptors), each antigen binding receptor comprising an antigen recognition portion directed to the antigenic determinant. As further disclosed herein, multiple nucleic acids encoding the receptors can be introduced via a single vector or separate vectors.

[0046] In another embodiment, a subject method includes obtaining T cells or thymocytes (preferably CD8+ T cells or thymocytes) that express a TCR directed to a first antigenic determinant, where in one embodiment, the cells also express at least one homozygous HLA haplotype; introducing into the T cells or thymocytes one or more nucleic acid molecules encoding one or more chimeric antigen receptors, where each chimeric antigen receptor comprises an antigen recognition portion directed to the antigenic determinant, the antigen recognition portion being operably linked to a T cell activation moiety; and obtaining stem cells (e.g., iPSCs) from the T cells or thymocytes. In another embodiment, the method further includes, prior to the step of obtaining stem cells from the T cells or thymocytes, introducing into the T cells or thymocytes one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signaling antigen binding receptors), where each antigen binding receptor comprises an antigen recognition portion directed to the antigenic determinant.

[0047] In yet another embodiment, a subject method includes, in some embodiments, obtaining HSCs (e.g., from bone marrow or blood) that express at least one homozygous HLA haplotype; and introducing into the HSCs: (i) one or more nucleic acids encoding a TCR directed against 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 portion directed against an antigenic determinant different from the first antigenic determinant, the antigen recognition portion operably linked to a T cell activation portion; and, optionally, (iii) one or more nucleic acid molecules encoding one or more antigen-binding receptors (e.g., non-signaling antigen-binding receptors), each antigen-binding receptor comprising an antigen recognition portion directed against an antigenic determinant different from the first antigenic determinant and different from the antigenic determinant to which the chimeric antigen receptor is directed. As disclosed herein, multiple nucleic acids encoding receptors can be introduced by a single vector or separate vectors. Such genetically modified HSCs can be used to generate T cells with specificity for multiple antigenic determinants.

[0048] In a further aspect, a T cell is provided that expresses a TCR directed to a first antigenic determinant and expresses one or more chimeric antigen receptors, each receptor comprising an antigen recognition portion directed to the antigenic determinant, the antigen recognition portion operably linked to a T cell activation portion. In some embodiments, the T cell further expresses an antigen-binding receptor comprising an antigen recognition portion directed to the antigenic determinant. In some embodiments, the T cell provided therein expresses at least one homozygous HLA haplotype.

[0049] In another aspect, provided is a method for generating T cells that express a TCR directed to a first antigenic determinant and express one or more CARs, each CAR comprising an antigen recognition moiety directed to the antigenic determinant, the antigen recognition moiety operably linked to a T cell activation moiety, and optionally also expressing one or more non-signaling antigen-binding receptors, each comprising an antigen recognition moiety directed to the antigenic determinant. In some embodiments, the methods provided herein are directed to generating T cells that express at least one homozygous HLA haplotype.

[0050] Another aspect of the present invention is directed to a method of treating a condition characterized by the presence of an undesired population of cells in a mammal, comprising administering to said mammal an effective number of stem cells or T cells, as described above.

[0051] In one embodiment, the condition is a neoplastic condition, a microbial infection (such as HIV, an STD, or an antibiotic-resistant bacteria), or an autoimmune condition.

[0052] According to this embodiment, there is provided a method of treating a neoplastic condition, comprising administering to said mammal an effective number of stem cells or T cells as defined above, wherein said TCR is directed against a first tumor antigenic determinant and said CAR is directed against a second tumor antigenic determinant.

[0053] In yet another embodiment, the first tumor antigenic determinant is WT-1.

[0054] In another embodiment, the second tumor antigenic determinant is TAG-72, CD19, MAGE, or CD47.

[0055] Yet another aspect of the present invention is directed 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 undesired population of cells in a mammal. In certain embodiments, for example, the following are provided: (Item 1) 1. A genetically modified mammalian stem cell, the cell having the capacity to differentiate into a T cell expressing a T cell receptor (TCR) directed against a first antigenic determinant, and comprising a nucleic acid molecule encoding a chimeric antigen receptor comprising an antigen recognition portion and a T cell activation portion, the antigen recognition portion being directed against a second antigenic determinant and operably linked to the T cell activation portion. (Item 2) Genetically modified stem cells that express at least one homozygous HLA haplotype. (Item 3) 3. The cell of item 1 or 2, which is an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell (HSC). (Item 4) 4. The cell of item 3, wherein the iPSC or HSC has the ability to differentiate into a CD4+ T cell or a CD8+ T cell. (Item 5) The cell of item 3, wherein the T cell expresses an αβ TCR or a γδ TCR. (Item 6) 5. The cell of item 4, wherein the iPSC is derived from a T cell or a thymocyte. (Item 7) 7. The cell of item 6, wherein the T cells or thymocytes derived from the iPSCs are CD8+ or CD4+. (Item 8) 8. The cell of item 6 or 7, wherein the T cell or thymocyte derived from the iPSC expresses a TCR directed against the first antigenic determinant. (Item 9) 9. The cell of item 8, wherein the TCR expressed in the T cell or thymocyte derived from the iPSC is an αβTCR or a γδTCR. (Item 10) 2. The cell of item 1, 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. (Item 11) 11. The cell of item 10, wherein the first antigenic determinant is selected from a tumor antigen, such as WT-1. (Item 12) 12. The cell of item 10 or 11, wherein the second antigenic determinant is selected from a tumor antigen, such as TAG72, CD19, MAGE, and CD47. (Item 13) Item 10. The cell of any one of the preceding items, wherein the antigen recognition portion comprises an scFv. (Item 14) Item 1. The cell of any one of the preceding items, wherein the antigen recognition portion is linked to the T cell activation portion by a hinge region and a transmembrane domain. (Item 15) 15. The cell of item 14, wherein the hinge region is derived from an IgG1 hinge region, a CD8 hinge region, or a CD28 hinge region. (Item 16) 15. The cell of item 14, wherein the hinge region contains a cysteine ​​that promotes dimerization of the chimeric antigen receptor. (Item 17) 15. The cell of item 14, wherein the transmembrane domain is derived from the transmembrane domain of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4. (Item 18) 8. The cell of any one of the preceding items, 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. (Item 19) 19. The cell of item 18, wherein the T cell activation portion 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 CD83. (Item 20) 10. The cell of any one of the preceding items, further comprising an additional nucleic acid encoding an additional chimeric antigen receptor comprising an antigen recognition portion and a T cell activation portion, wherein the antigen recognition portion of the additional chimeric antigen receptor is directed to an additional antigenic determinant that is different from the first and second antigenic determinants. (Item 21) The cell of any one of the preceding items, 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. (Item 22) 22. The cell of claim 21, wherein the antigen recognition portion is operably linked to the transmembrane domain by a hinge region. (Item 23) 23. The cell of item 21 or 22, wherein the antigen-binding receptor is directed against CD47. (Item 24) 24. The cell of item 23, wherein the antigen-binding receptor comprises an scFv directed against CD47. (Item 25) 25. The cell of item 24, wherein the hinge and transmembrane region of the antigen-binding receptor is the hinge and transmembrane region of CD28. (Item 26) 21. The cell of item 20, wherein the nucleic acid encoding the chimeric receptor is operably linked to the additional nucleic acid encoding the additional chimeric antigen receptor via a nucleotide sequence encoding a self-cleaving peptide. (Item 27) 22. The cell of claim 21, wherein the nucleic acid encoding the chimeric receptor is operably linked to the nucleic acid encoding the antigen-binding receptor via a nucleotide sequence encoding a self-cleaving peptide. (Item 28) 1. A method for producing genetically modified mammalian stem cells, comprising: obtaining mammalian stem cells capable of differentiating into T cells expressing a TCR directed against a first antigenic determinant; introducing into the stem cells one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition portion directed to an antigenic determinant different from the first antigenic determinant, the antigen recognition portion being operably linked to a T cell activation portion; optionally one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signaling antigen binding receptors), each antigen binding receptor comprising an antigen recognition portion directed to an antigenic determinant different from the first antigenic determinant and the antigenic determinant to which the chimeric antigen receptor is directed; A method comprising: (Item 29) 29. The method of claim 28, wherein the stem cells express at least one homozygous HLA haplotype. (Item 30) 1. A method for producing genetically modified mammalian stem cells, comprising: obtaining T cells or thymocytes expressing a TCR directed against a first antigenic determinant, optionally the T cells or thymocytes being CD8+ or CD4+; 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 portion directed to an antigenic determinant different from the first antigenic determinant, the antigen recognition portion operably linked to a T cell activation portion; optionally one or more nucleic acid molecules encoding one or more antigen binding receptors (e.g., non-signaling antigen binding receptors), each antigen binding receptor comprising an antigen recognition portion directed to an antigenic determinant different from the first antigenic determinant and the antigenic determinant to which the chimeric antigen receptor is directed; deriving stem cells from T cells or thymocytes; A method comprising: (Item 31) 31. The method of claim 30, wherein the T cells or thymocytes express at least one homozygous HLA haplotype. (Item 32) 32. The method of any one of items 28 to 31, wherein the stem cells are iPSCs. (Item 33) A T cell expressing a T cell receptor (TCR) directed against a first antigenic determinant and a chimeric antigen receptor comprising an antigen recognition portion and a T cell activation portion, wherein the antigen recognition portion is directed against a second antigenic determinant and is operably linked to the T cell activation portion. (Item 34) 34. The T cell of item 33, wherein the T cell expresses at least one homozygous HLA haplotype. (Item 35) 35. The T cell of item 33 or 34, wherein the T cell is a CD4+ T cell or a CD8+ T cell. (Item 36) 35. The T cell of item 33 or 34, wherein the TCR is an αβTCR or a γδTCR. (Item 37) 35. The T cell of item 33 or 34, derived from a stem cell expressing the at least one homozygous HLA haplotype. (Item 38) 38. The T cell of item 37, wherein the stem cell is an iPSC or an HSC. (Item 39) 39. The T cell of item 38, wherein the iPSC is derived from a T cell or thymocyte expressing a TCR directed against the first antigenic determinant. (Item 40) 40. The T cell of item 39, wherein the TCR expressed in the T cell or thymocyte derived from the iPSC is an αβTCR or a γδTCR. (Item 41) 40. The T cell of item 39, wherein the T cell or thymocyte is CD8+. (Item 42) 39. The T cell of item 38, wherein the iPSCs or HSCs comprise a nucleic acid molecule encoding the chimeric antigen receptor. (Item 43) 34. The T cell of item 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. (Item 44) 44. The T cell of item 43, wherein the first antigenic determinant is selected from a tumor antigen, e.g., WT-1 and EBVLMP2. (Item 45) 45. The T cell of item 44, wherein the second antigenic determinant is selected from a tumor antigen, e.g., TAG72, CD19, MAGE, and CD47. (Item 46) 46. ​​The T cell according to any one of items 33 to 45, wherein the antigen-recognition portion comprises an scFv. (Item 47) 47. The T cell of any one of items 33 to 46, wherein the antigen recognition portion is linked to the T cell activation portion by a hinge region and a transmembrane domain. (Item 48) 48. The T cell of item 47, wherein the hinge region is derived from an IgG1 hinge region, a CD8 hinge region, or a CD28 hinge region. (Item 49) 48. The T cell of item 47, wherein the hinge region comprises a cysteine ​​that promotes dimerization of the chimeric antigen receptor. (Item 50) 48. The T cell of item 47, wherein the transmembrane domain is derived from the transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or an immunoglobulin such as IgG4. (Item 51) 51. The T cell of any one of paragraphs 33 to 50, wherein the T cell activation portion 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. (Item 52) 52. The T cell of item 51, wherein the T cell activation portion 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, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. (Item 53) 53. The T cell of any one of items 33 to 52, further expressing an additional chimeric antigen receptor comprising an antigen recognition portion and a T cell activation portion, wherein the antigen recognition portion of the additional chimeric antigen receptor is directed against an additional antigenic determinant distinct from the first and second antigenic determinants. (Item 54) 54. The T cell of any one of paragraphs 33 to 53, further expressing a non-signaling antigen-binding receptor comprising an antigen recognition portion directed to an antigenic determinant different from the antigenic determinant to which the first antigenic determinant and the chimeric antigen receptor are directed. (Item 55) 55. The T cell of item 54, wherein the antigen recognition portion is operably linked to the transmembrane domain by a hinge region. (Item 56) 56. The T cell of item 54 or 55, wherein the antigen-binding receptor is directed against CD47. (Item 57) 57. The T cell of item 56, wherein the antigen-binding receptor comprises an scFv directed against CD47. (Item 58) 58. The T cell of item 57, wherein the hinge and transmembrane region of the antigen-binding receptor is the hinge and transmembrane region of CD28. (Item 59) 54. The T cell of item 53, wherein the chimeric receptor and the additional chimeric antigen receptor, when initially translated, are linked to each other via a self-cleaving peptide and are subsequently separated as a result of cleavage of the self-cleaving peptide. (Item 60) 55. The T cell of item 54, wherein the chimeric receptor and the antigen-binding receptor, when initially translated, are linked to each other via a self-cleaving peptide and are subsequently separated as a result of cleavage of the self-cleaving peptide. (Item 61) 28. A T cell derived from the stem cell of any one of items 1 to 27. (Item 62) 1. A method of generating T cells, comprising: providing a genetically modified stem cell according to any one of items 1 to 27; and differentiating the genetically modified stem cell into a T cell. A method comprising: (Item 63) 1. A method of generating T cells, comprising: obtaining stem cells capable of differentiating into T cells that express a TCR directed against a first antigenic determinant; differentiating the stem cells into T cells; introducing into said T cells 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, optionally one or more nucleic acids encoding one or more antigen-binding receptors, each antigen-binding receptor directed against an antigenic determinant different from said first antigenic determinant and the antigenic determinant to which the chimeric antigen receptor is directed; A method comprising: (Item 64) 64. The method of claim 63, wherein the stem cells express at least one homozygous HLA haplotype. (Item 65) 65. The method of any one of items 62 to 64, wherein the stem cells are iPSCs or HSCs. How to do it. (Item 66) 66. The method of claim 65, wherein the iPSCs are derived from T cells or thymocytes. (Item 67) 67. The method of claim 66, wherein the T cells or thymocytes are CD8+ or CD4+. (Item 68) 66. The method of claim 65, wherein the iPSCs are derived from T cells or thymocytes expressing a TCR directed against the same antigenic determinant as the TCR expressed in the T cells derived from the iPSCs. (Item 69) 62. A method of treating a condition characterized by the presence of an undesired population of cells in a mammal, the method comprising administering to said mammal an effective number of T cells of any one of items 33 to 61. (Item 70) 70. The method of item 69, wherein the condition is a neoplastic condition, a microbial infection (such as HIV, an STD, or an antibiotic-resistant bacterium), or an autoimmune condition. (Item 71) 70. The method of claim 69, wherein the condition is a neoplastic condition, the TCR is directed to a first tumor epitope, and the CAR is directed to a second tumor epitope. (Item 72) 72. The method of item 71, wherein the first tumor antigen determinant is WT1. (Item 73) Item 74. The method of Item 71 or 72, wherein the second tumor antigen determinant is TAG72. 74. The method of any one of paragraphs 71 to 73, wherein the cells administered to the mammal comprise a nucleic acid molecule encoding a non-signaling CD47 binding receptor. [Brief explanation of the drawings]

[0056] [Figures 1A-1O] Stimulation and expansion of cytotoxic T cells expressing TCRs specific for the Wilms' tumor 1 (WT-1) antigen. Cells were isolated from whole blood peripheral blood mononuclear cells (PBMCs). Scatter plots are also shown (B, G, L). Cells were gated for single cells (A, F, K), then CD3-positive cells (conjugated to APCCy7; C, H, M), then CD8 (conjugated to PECy7) and CD4 (conjugated to PerCp; D, I, N), and finally CD8 cells alone (E, J, O). WT-1 staining was performed using an HLA-A02 tetramer specific for the WT-137 peptide. Shown are images from two separate HLA-A02-positive patients (Patient 1, A-E; Patient 2, F-J) and are compared with fluorescence minus one (FMO; this staining lacks WT-1 tetramer staining and shows specific staining for WT-137, K-O). The percentages shown are the percentages of CD3+ cells. The percentage of WT-1 TCR T cells increased to 1.5% and 4.5% for the two samples; in unstimulated PBMCs, these cells were very low (below the detection limit using the tetramer technology described herein). In other studies (e.g., Schmeid et al., 2015), they were found to be only 10-6 of CD8+ cells (ranging from 3 x 10-7 to 3 x 10-6 cells).

[0057] [Figures 2A-2G]CD8+ cytotoxic T cells bearing TCRs specific for the Wilms' tumor 1 (WT-1) antigen are functional. Functionality is indicated by the production of interferon gamma (IFN-γ) (Ghanekar et al., 2001). IFN-γ expression was observed after WT-1-specific stimulation. Activated cells were gated on CD8+ and HLA-A02 tetramers using a WT-137 peptide-PE conjugated fluorescent dye. These cytotoxic T cells bearing TCRs specific for WT-1 demonstrated intracellular cytokine staining for IFN-γ (conjugated to Pacific Blue fluorescent dye) upon stimulation with WT-1. Shown are images from two separate patients (WT-1 #1 and WT-1 #2) who were HLA-A02 positive (Patient 1: A-B, Patient 2: C-D) and are compared with fluorescence minus one (FMO; EF; this staining lacks WT-1 tetramer staining and shows specific staining for WT-137 (G). Percentages shown are the percentage of WT1+CD8+ cells. More than 80% of WT-1 TCR T cells produced IFNγ.

[0058] [Figure 2H] Addition of the LAG3 inhibitor (IMP321) increased the frequency of WT-1-specific T cells 4 days after stimulation. In this experiment, purified but unseparated cord blood mononuclear cells were seeded with either anti-CD28 alone, WT-1 peptide (Miltenyi BioTech) and CD28 (1 μg / ml), or WT-1 peptide plus IMP321 for 24 hours and 4 days. No effect was observed by 24 hours (data not shown), but WT-1-specific CD8+ T cells doubled after 4 days, consistent with the kinetics of IMP321's effect on dendritic cell activation (Brigone et al., 2007).

[0059] [Figure 3]Generation of cancer-specific (e.g., WT-1) TCR T cell-derived iPSCs. Cancer antigen-specific T cells are extremely rare in normal blood; they are revealed by in vitro stimulation in the presence of cytokines with WT-1 peptide bound to autologous B cells (formed into lymphoblastoid cell lines (LCLs) using EBV) acting as antigen-presenting cells. Cancer antigen-specific T cells have been shown to be double-labeled using CD8 (for cytotoxic T cells) and tetramers against HLA-WT-1, which bind to the TCR of these CD8+ cells. These cells were then converted into iPSCs using Yamanakari programming factors. A rearranged TCR gene specific for WT-1 was embedded into the TCR locus of the iPSCs.

[0060] [Figure 4] Morphological progression of iPSC colonies towards hematopoietic lineages and lymphoid progenitors after 1, 5, 9, and 13 days of culture on OP9 feeder cells. Note the numerous single hematopoietic-like cells by day 13.

[0061] [Figure 5-1] Flow cytometry analysis of iPSC-derived cells after 13 days of culture on OP9 cells clearly shows evidence of hematopoietic specialization, including the presence of hematopoietic stem cells (HSCs) (CD34+CD43+). [Figure 5-2] Flow cytometry analysis of iPSC-derived cells after 13 days of culture on OP9 cells clearly shows evidence of hematopoietic specialization, including the presence of hematopoietic stem cells (HSCs) (CD34+CD43+).

[0062] [Figure 6]Flow cytometry of iPSC-derived cells for HSCs after 13 days of culture on OP9 cells followed by 9 days of culture on OP9 DL-L1 cells. Cells were gated for viability, CD45 expression, and single cells, and then examined for HSC content by staining for CD34 and CD43. Note the reduction in HSCs from >90% in the preliminary OP9 DL-L1 culture (Figure 5) to approximately 60% after 9 days of culture on OP9 DL-L1 cells.

[0063] [Figure 7] Flow cytometry of T cell development of iPSC-derived cells after 13 days of culture on OP9 cells followed by 9 days of culture on OP9 DL-L1 cells. There is clear evidence of commitment to the T cell lineage with expression of CD5 and CD7, as well as immature (i.e., CD3-deficient; data not shown) CD4+, CD8+ "single positive" cells and CD4+CD8+ "double positive" cells.

[0064] [Figure 8-1] Flow cytometry of HSC and T cell differentiation in iPSC-derived cells after 13 days of culture on OP9 cells followed by 16 days of culture on OP9 DL-L1 cells. Immature T cells expressing CD4 and / or CD8 were still clearly present, but HSCs had been further reduced from approximately 60% to approximately 25%. Most importantly, mature CD8+ cells were present, expressing CD3, αβ TCR, and the CD8β chain (in addition to CD8α - not shown). [Figure 8-2] Flow cytometry of HSC and T cell differentiation in iPSC-derived cells after 13 days of culture on OP9 cells followed by 16 days of culture on OP9 DL-L1 cells. Immature T cells expressing CD4 and / or CD8 were still clearly present, but HSCs had been further reduced from approximately 60% to approximately 25%. Most importantly, mature CD8+ cells were present, expressing CD3, αβ TCR, and the CD8β chain (in addition to CD8α - not shown).

[0065] [Figure 9] Schematic diagram of induction of WT-1-specific TCR, CD8αβ T cells from iPSCs derived from in vitro-expanded WT-1-specific TCR T cells. Treatment of CD4+CD8+ cells with (low levels) anti-CD3 antibody mimicked the signaling that occurs in the thymus during positive selection; this expanded CD8+ T cells expressing both CD8α and CD8β chains.

[0066] [Figure 10] We demonstrate that WT-1-specific TCR, CD8αβ T cells induced from iPSCs derived from in vitro expanded WT-1-specific TCR T cells retained intact functions (e.g., cytotoxicity against WT-1-expressing targets) comparable to those of the original T cells. The effector:target ratio was 3:1; graded concentrations of WT-1 peptide were tested.

[0067] [Figure 11]Schematic diagram of chimeric antigen receptor and antigen-binding receptor constructs. A panel of chimeric antigen receptor (CAR) constructs bearing scFvs against either TAG72 or CD19 (as a positive control) was developed. The constructs used either human CD8 or CD28 as the hinge and transmembrane domains, and CD28, CD3 zeta chain, or 4-1BB cytoplasmic activation signaling domains. P2A is a signal sequence directing proteolytic cleavage; the top five constructs shown in Figure 11 release EGFP as a fluorescent reporter of expression, while the bottom (sixth) construct shown in Figure 11 releases a second CAR receptor construct, shown as leader(CD8)-scFv(anti-CD47)-hinge / TM(CD28)-endodomain tail(CD8), where the leader is processed to release the anti-CD47 scFv anchored to 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 may contain a cysteine ​​residue to direct dimerization by disulfide bond formation between adjacent hinge domains, as is characteristic of the native CD8 hinge, or may have a cysteine ​​residue substituted with another residue, such as serine, which does not form disulfide bonds and does not form covalently stabilized dimers. Exemplary sequences of CARs and CD47-binding receptors, as well as various domain sequences suitable for use in constructing CARs or antigen-binding receptors, are set forth in SEQ ID NOs: 1-20.

[0068] [Figure 12] Retroviral transformation schematic. Schematic of the steps undertaken to generate 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 the reporter. Upon successful cell transduction, P2A is expressed and cleaved, and EGFP is identified by flow cytometry and immunofluorescence microscopy.

[0069] [Figure 13] Schematic of lentiviral transformation. Schematic of the process undertaken to generate a CAR-containing lentiviral construct. The CAR construct is cloned into the pWP1 plasmid vector and linked to the fluorescent reporter EGFP by the P2A self-cleaving polypeptide, which separates the CAR and the reporter. Upon successful transduction of cells, P2A is expressed and cleaved, and EGFP is identified by flow cytometry and immunofluorescence microscopy.

[0070] [Figures 14A-14B] Figure 14A. Schematic of a typical second-generation CAR structure. It includes an scFv binding domain to the target antigen; a hinge region (stalk) that allows the CAR to integrate into the plasma membrane (hinge length can affect scFv binding to target cells); and a cytoplasmic signaling domain that induces T cell activation through scFv engagement. The CAR structure is shown as a dimer stabilized by disulfide bonds between adjacent cysteine ​​residues in the hinge region. Figure 14B. Schematic of a non-signaling antigen-binding receptor, the truncated CD47 "attached stalk." The structure shows the scFv domain or single V-domain for CD47 antigen binding attached to the hinge and transmembrane regions, but with no signaling domain present in the endodomain. This construct can increase the binding affinity of CAR-T cells to cancer cells expressing high levels of CD47. This receptor also binds to normal cells expressing low levels of CD47, without signaling and therefore without damage to the normal cells. The hinge region can contain cysteine ​​residues that direct dimerization by disulfide bond formation between adjacent hinge domains, or can have cysteine ​​residue substitutions with other residues, such as serine, that do not form disulfide bonds and do not form covalently stabilized dimers.

[0071] [Figure 15]Flow cytometry analysis of CAR-transduced human PBMC-derived CD3+ T cells demonstrating successful transduction with the TAG72 lentiviral CAR construct (20.8% positive, compared to <0.1% in controls) and the CD19 lentiviral CAR construct (33.9% positive).

[0072] [Figure 16] Western blot analysis confirming protein expression in TAG27 and CD19 CAR-transfected T cells.

[0073] [Figure 17] TAG72 CAR-T-mediated killing of ovarian cancer (TAG72+) target cells. The effector:target ratio (E:T) was 1:1. TAG72 CAR-T effector cells (GFP-positive cells) developed from CD3-activated normal blood T cells were isolated via FACS at >95% purity and then stimulated for 72 hours in the presence of immobilized αCD3 / αCD28 and IL-2 prior to use to enhance cytolytic activity. Changes in cell impedance (expressed herein as cell index in arbitrary units) were monitored over 40 hours and compared with stimulated untransduced CD3+ve cells isolated from PBMCs and stimulated vector-only CAR-T cells. TAG72 CAR-T cells demonstrated the highest killing, although CD3 / CD28-activated non-CAR-T cells also exhibited killing, albeit to a much lesser extent.

[0074] [Figure 18] Determination of the specificity of TAG72 CAR-T killing. TAG72 and CD19 CAR-T were each isolated via FACS and immediately added to TAG72hi / CD19low target cells (E:T=5:1) without in vitro stimulation. Changes in cell impedance (expressed herein as cell index in arbitrary units) were monitored over 15 hours. TAG72 CAR-T cells demonstrated robust killing of the cell line. CD19 CAR-T cells were comparable to non-CAR T cell controls.

[0075] [Figure 19A] 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. WT-1-specific TCR T cells were successfully transduced with the TAG72 lentiviral CAR construct (31.3% positive, compared with <0.1% in the control). Figure 19B. iPSC-derived WT-1-specific TCR T cells formed from WT-1-specific TCR T cells were successfully transduced with a bispecific CAR construct against TAG72 and unsignaled truncated CD47 (55% transduction); 32% transduction with TAG72 alone. These transduced T cells contained three anti-cancer specificities: WT-1 (TCR); TAG72 (CAR); and unsignaled truncated CD47. [Figure 19B] 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. WT-1-specific TCR T cells were successfully transduced with the TAG72 lentiviral CAR construct (31.3% positive, compared with <0.1% in the control). Figure 19B. iPSC-derived WT-1-specific TCR T cells formed from WT-1-specific TCR T cells were successfully transduced with a bispecific CAR construct against TAG72 and unsignaled truncated CD47 (55% transduction); 32% transduction with TAG72 alone. These transduced T cells contained three anti-cancer specificities: WT-1 (TCR); TAG72 (CAR); and unsignaled truncated CD47.

[0076] [Figures 20A-20I]Cytotoxic function of WT-1-specific TCR T cells and bispecific TAG72 CAR / WT-1 TCR T cells. WT-1-specific TCR T cells and bispecific TAG72 CAR / WT-1 TCR T cells were incubated in monolayer culture with the ovarian cancer cell line CAOV4 for 24 hours to assess cytotoxicity. Despite a low effector:target ratio of 2:1 (consequentially due to the paucity of effectors obtained), there was specific killing of WT-1 TCR T cells, which was further increased by transduction with the TAG72 CAR. The technique is based on AquaAmine, which stains intracellular amines. When cells are dead or dying, the compromised cell membrane allows the dye to penetrate the cell, resulting in more intense staining of amines. Thus, cellular cytotoxicity is indicated by increased staining intensity of cellular amines. Note: Because some amines are present on the cell surface, live cells also produce 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 TAG72 CAR / WT-1 TCR T cells incubated with CAOV4 ovarian cancer cells. D, E, F: AquaAmine levels in gated CD3-ve cells (i.e., CAOV4). Phase contrast images: G: cancer cells only, H: non-CAR-transfected WT-1 TCR cells with cancer cells, and I: TAG72-transfected WT-1 TCR T cells with cancer cells. 40x magnification. WT-1 TCR T cells cause approximately 10% killing (above background); TAG72 CAR-T cells cause an additional 10% killing (i.e., approximately 20% above background). The dual anti-cancer killing mechanisms are additive.

[0077] [Figure 21A]Figures 21A-21B. CAR transduction of iPS cells. Day 5 of growth on an MEF feeder layer. After 4 days of incubation with CAR lentivirus. CAR+ transduction of TAG72, CD19, and GFP viruses (green) was overlaid on a brightfield image at 20x magnification. The untransduced control did not show any GFP signal. Images of iPSC colonies at 4x magnification demonstrate the presence of iPSC colonies on the MEF feeder layer. It is noteworthy that in each line, some iPSC colonies appeared to have begun to differentiate spontaneously. Transduced fibroblast-derived iPSCs are shown in Figure 21A. Figure 21B demonstrates successful transduction of WT-1 T cell-derived iPSCs with the TAG72 CAR. These iPSCs were therefore successfully imprinted with both WT-1 TCR and TAG72 specificity. [Figure 21B] Figures 21A-21B. CAR transduction of iPS cells. Day 5 of growth on an MEF feeder layer. After 4 days of incubation with CAR lentivirus. CAR+ transduction of TAG72, CD19, and GFP viruses (green) was overlaid on a brightfield image at 20x magnification. The untransduced control did not show any GFP signal. Images of iPSC colonies at 4x magnification demonstrate the presence of iPSC colonies on the MEF feeder layer. It is noteworthy that in each line, some iPSC colonies appeared to have begun to differentiate spontaneously. Transduced fibroblast-derived iPSCs are shown in Figure 21A. Figure 21B demonstrates successful transduction of WT-1 T cell-derived iPSCs with the TAG72 CAR. These iPSCs were therefore successfully imprinted with both WT-1 TCR and TAG72 specificity.

[0078] [Figure 22-1]Flow cytometry analysis of chimeric antigen receptor transduction of iPSCs. These iPSCs were derived from adult fibroblasts but could be of any origin, including unselected T cells, CD8+ T cells, or cancer antigen-specific (e.g., WT-1) T cells. There is clearly a population of fluorescent iPSCs successfully transduced with TAG72 or CD19. An overlay of transduced cells compared to untransduced controls is shown in Figure 23. [Figure 22-2] Flow cytometry analysis of chimeric antigen receptor transduction of iPSCs. These iPSCs were derived from adult fibroblasts but could be of any origin, including unselected T cells, CD8+ T cells, or cancer antigen-specific (e.g., WT-1) T cells. There is clearly a population of fluorescent iPSCs successfully transduced with TAG72 or CD19. An overlay of transduced cells compared to untransduced controls is shown in Figure 23.

[0079] [Figure 23-1] Dot plot overlay comparing non-transduced control cells (blue) with transduced iPSC cultures (green). Events in the GFP+ gate demonstrate successful transduction and are expressed as percent frequency of non-debris events. [Figure 23-2] Dot plot overlay comparing non-transduced control cells (blue) with transduced iPSC cultures (green). Events in the GFP+ gate demonstrate successful transduction and are expressed as percent frequency of non-debris events.

[0080] [Figure 24] Re-formation of CAR-transduced iPSC colonies after FACS sorting. CAR-transduced iPSCs can be isolated by flow cytometry (GFP-positive fluorescence) and replated to form stable colonies. DETAILED DESCRIPTION OF THE INVENTION

[0081] Detailed Description of the Invention The present invention is based, in part, on the determination that TCR / CAR dual-expressing T cells directed against two distinct antigenic determinants can be consistently and stably generated, for example, by transfecting a CAR cassette into iPSCs derived from T cells exhibiting TCR specificity directed against a desired antigenic determinant. It was found that, through the action of epigenetic memory, T cells differentiated from these iPSCs stably express both the TCR specificity of the somatic T cell from which the iPSCs were derived and a CAR directed against a distinct antigenic determinant. Specificity for additional antigenic determinants can be achieved by introducing into the cells additional nucleic acids encoding molecules that bind to such additional antigenic determinants. Such multispecific cells thereby provide more effective therapeutic outcomes than currently available cells. These determinations therefore now enable the development of a continuous supply of stably transformed dual antigen-specific T cells, particularly cytotoxic CD8+ αβTCR T cells, for use in connection with any disease state characterized by a population of unwanted cells, such as neoplastic conditions, viral infections, bacterial infections, or autoimmune conditions. This discovery and the generation of cells based thereon has now facilitated the improvement of therapeutic treatment regimes aimed at treating such conditions, particularly neoplastic conditions such as solid tumors or hematological cancers (e.g., leukemia), including metastatic disease.

[0082] Thus, one aspect of the present invention is directed to genetically modified mammalian stem cells, or T cells differentiated therefrom, which have the capacity to differentiate into T cells expressing a TCR directed against a first antigenic determinant, and which comprise a nucleic acid molecule encoding a chimeric antigen receptor, said receptor comprising an antigen recognition moiety directed against a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0083] Reference to "T cells" should be understood as a reference to any cell that contains a T cell receptor. In this regard, a T cell receptor can include any one or more of the α, β, γ, or δ chains. As will be appreciated by those skilled in the art, NKT cells also express T cell receptors, and therefore bispecific NKT cells can also be generated by the present invention. The present invention is not intended to be limited to any particular subclass of T cells, but in preferred embodiments, the subject T cells express an α / β TCR dimer. Even more preferably, the T cells are CD4 + Helper T cells, CD8 + Killer T cells or NKT cells. Without limiting the present invention to any one theory or mechanism of action, CD8 + T cells, also known as cytotoxic cells, are a major part of the adaptive immune system. + T cells scan the intracellular environment to primarily target and destroy infected cells. Small peptide fragments derived from intracellular contents are processed and delivered to the cell surface where they are presented in the context of MHC class I molecules. However, T cells are not limited to responding to viral infections; CD8 + T cells also provide an additional level of immune surveillance by monitoring and eliminating damaged or abnormal cells, including cancer cells. + T cell recognition usually leads to the destruction of the target cell by activating the apoptotic pathway through the release of cytotoxic granules or lymphokines or through FAS / FASL interactions. + T cells generally recognize peptides presented by antigen-presenting cells in the context of MHC class II and are expressed by B cells and / or CD8 + They result in the release of cytokines designed to regulate T cell immune responses. Thus, unlike cytotoxic T cells, helper T cells do not directly kill unwanted cells, such as cancer cells, but can augment such responses insofar as they are killed by cytotoxic T cells and / or antibody-mediated clearance mechanisms.

[0084] Natural killer T (NKT) cells are a specialized population of T cells that express the semi-invariant T cell receptor (TCRαβ) and a surface antigen commonly associated with natural killer cells. The TCR on NKT cells is distinct in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. Most NKT cells express the invariant TCR alpha chain and one of a few TCR beta chains. The TCR present on type 1 NKT cells recognizes the antigen alpha-galactosylceramide (alpha-GalCer). Within this group, CD4 + CD8 - cells, CD4 - CD8 + cells and CD4 - / CD8 - Distinct subpopulations, including type 2 NKT cells, have been identified. Type 2 NKT cells (or non-invariant NKT cells) express a broader range of TCRα chains and do not recognize the alpha-GalCer antigen. NKT cells produce cytokines that have multiple, often opposing, effects, such as either promoting inflammation or inducing immunosuppression, including tolerance. As a result, they can contribute to antibacterial and antiviral immune responses, promote tumor-associated immune surveillance, and inhibit or promote the development of autoimmune diseases. Like natural killer cells, NKT cells can also induce perforin, Fas, and TNF-related cytotoxicity. Therefore, reference to genetically modified T cells of the present invention should be understood to include reference to NKT cells.

[0085] Because thymus-based T cell production is characterized by the random generation of T cell receptor (TCR) repertoires, thymocyte proliferation must also involve a highly stringent selection process that eliminates or functionally silences developing thymic T cells with the potential to attack the self. This "self-tolerance" therefore reduces the potential for autoimmune disease. However, if non-viral-induced cancers are defined as "self" diseases, this process necessarily impairs immune surveillance against cancer. This means that many of the T cells generated in the thymus that could potentially be reactive with tumor-associated antigens may be eliminated before entering the blood. At the very least, they will be insufficient in number and likely express low-affinity TCRs.

[0086] In one embodiment, a genetically modified mammalian stem cell, or a T cell differentiated therefrom, is provided, wherein the cell expresses a CD4 TCR directed against a first antigenic determinant. + Cells are provided that have the ability to differentiate into T cells and that comprise a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen recognition moiety directed to a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In one embodiment, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0087] In another embodiment, a genetically modified mammalian stem cell, or a T cell differentiated therefrom, is provided, wherein the cell expresses a CD8 TCR directed against a first antigenic determinant. + Cells are provided that have the ability to differentiate into T cells and that comprise a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen recognition moiety directed to a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In one embodiment, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0088] In some embodiments, the genetically modified cells of the present invention, e.g., genetically modified stem cells (such as iPSCs or HSCs) or T cells, are homozygous for at least one HLA haplotype. Without limiting the present invention to any one theory or mechanism of action, the major histocompatibility complex (MHC) represents a set of cell surface molecules whose primary function is to bind antigen-derived peptide fragments 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 the β2 subunit and can therefore only be recognized by the CD8 coreceptor. Class II MHC molecules do not express the β2 subunit at all and can therefore be recognized by the CD4 coreceptor. Thus, because different lymphocytes express different TCR coreceptors, 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: (1) An organism's MHC repertoire is usually polygenic (due to multiple, interacting genes); (2) MHC expression is codominant (due to both sets of inherited alleles); (3) MHC gene variants are highly diverse (vary widely between organisms within a species).

[0089] MHC molecules bind to both the T cell receptor and the CD4 / CD8 coreceptors on T lymphocytes. Antigen epitopes held in the peptide-binding groove of MHC molecules interact with the variable Ig-like domain of the TCR to trigger T cell activation. However, MHC molecules can also act as antigens themselves, triggering an immune response in recipients of tissues or cells expressing foreign MHC, thus causing transplant rejection. Furthermore, transplantation of immunocompetent cells can actually result in rejection of host tissues, also known as graft-versus-host disease. In this regard, each human cell expresses six MHC class I alleles (one HLA-A, -B, and -C allele from each parent) and six to eight MHC class II alleles (one HLA-DP and -DQ from each parent, and one or two HLA-DR, as well as combinations of these). MHC variation in the human population is high, with at least 350 alleles for the HLA-A gene, 620 alleles for HLA-B, 400 alleles for DR, and 90 alleles for DQ. Any two individuals who are not identical twins express different MHC molecules.

[0090] All MHC molecules can mediate transplant rejection, but HLA-C and HLA-DP, which exhibit low polymorphism, are less important. Transplant rejection can be minimized by attempting to match as many cell surface HLA repertoires as possible between the donor and recipient. A perfect match is only possible between identical twins. However, donor selection based on minimizing incompatibility in one or more of the ranges of HLA antigens expressed on cells is highly desirable and can significantly minimize rejection problems. The usual method of managing tissue / cell rejection is the administration of immunosuppressive treatment regimens, which is undesirable in the context of treatment regimens based on the administration of genetically modified immune cells that need to function at optimal levels of functionality, and this represents a particular problem solved by the present invention. According to the present invention, this can be achieved by utilizing cells, such as iPSCs or cells, such as T cells from which iPSCs are derived, that are homozygous for one or more MHC haplotypes, where the HLA allele of interest is a major transplantation antigen and is preferably expressed by a significant 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. If the homozygous HLA haplotype corresponds to a predominant MHC I or MHC II HLA type (in terms of tissue rejection), the use of such cells will significantly reduce problems with tissue rejection in the wider population receiving the cells of the present invention in the context of a treatment regimen. In the context of the present invention, genetically modified cells may be homozygous for one cellular HLA antigen, or they may be homozygous for more than one HLA antigen, for example, two, three, or more HLA antigens. In some embodiments, the genetically modified cells are homozygous for one HLA antigen selected from the antigens listed in Table 1, including, for example, HLA A1, 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 antigens listed in Table 1, including, for example, HLA A1, B8, C7, DR17, DQ2, or HLA A2, B44, C5, DR4, DQ8, or HLA A3, B7, C7, DR15, DQ6.

[0091] The term "HLA-type" should therefore be understood to refer to the complement of HLA antigens present on the cells of an individual.

[0092] Obtaining suitable homozygous HLA T cells for use in generating iPSCs can be accomplished by any suitable method, for example, a method that involves screening a population (such as by blood banking) to identify individuals expressing HLA homozygotes, and then screening T cells from those individuals that exhibit the desired TCR specificity. These normally very rare T cells can be selectively stimulated with the specific antigenic peptide recognized by their TCR and greatly increased in frequency (e.g., <0.0001 to 0.2).

[0093] Those skilled in the art will recognize that significant information regarding the identification and utility of homozygous haplotypes in terms of minimizing donor-recipient HLA mismatches across a given population of interest, thereby enabling the generation of donor banks, is widely available in the published literature. See, e.g., Pappas et al. (2015). In one example, Table 1 identifies the 15 homozygous HLA haplotypes that rank highest relative to the proportion of the UK population that provides minimal mismatch. The first eight of the listed homozygous HLA haplotypes are compatible with 49% of the population. A further example is outlined in Table 2, which details the first ten ranked haplotypes that are compatible with racially diverse California populations. Table 2 includes match frequencies for subpopulations, including Black or African American, Asian and Pacific Islander, Caucasian, Hispanic, and Native American and Alaska Native. Furthermore, Table 3 outlines the 50 most frequent haplotypes for HLA-AB-DR, AB, A-DR, and B-DR in the North Chinese population. It will be appreciated that one skilled in the art will understand that the data illustrated in Table 3 can be used to define a set of homozygous haplotypes that provide minimal mismatches for the North Chinese population.

[0094] [Table 1]

[0095] [Table 2]

[0096] [Table 3-1] [Table 3-2] [Table 3-3]

[0097] As detailed above, the present invention is based on the determination that stem cells can be consistently and stably engineered to express dual T cells and chimeric antigen receptors directed against multiple distinct antigens, thereby providing a continuous supply of more therapeutically effective T cells than cells used in currently available therapeutic cell treatment regimens. In this regard, reference to a "stem cell" should be understood as a reference to any cell that develops along multiple lineages, imparting a specific genetic makeup and thus exhibiting the potential to form a new organism or regenerate the tissue or cell population of an organism. Stem cells utilized by the present invention can be of any suitable type that has the ability to differentiate into two or more lineages, including, but not limited to, embryonic stem cells, adult stem cells, umbilical cord stem cells, hematopoietic stem cells (HSCs), totipotent cells, progenitor cells, progenitor cells, pluripotent cells, multipotent cells, or dedifferentiated somatic cells (such as induced pluripotent stem cells). "Totipotency" means that the subject stem cells are capable of self-renewal. By "pluripotent" is meant that the subject stem cells are capable of differentiating to form cells of any one of the three germ layers: ectoderm, endoderm, and mesoderm, among others.

[0098] In a specific embodiment, the target stem cells are induced pluripotent stem cells (iPSCs). While not limiting the present invention to any one theory or mechanism, the expansion of adult stem cells does not necessarily involve both stem cell regeneration and differentiation into specific somatic cell lineages based on the occurrence of asymmetric stem cell division. In particular, pluripotent stem cells can be derived from T cells induced to transform into a multilineage potential stage. The development of techniques that enable dedifferentiation of adult cells is of significant importance, particularly due to the difficulty of inducing stem cell regeneration and expansion in vitro.

[0099] According to this embodiment, there is thus provided a genetically modified mammalian stem cell, or a T cell differentiated therefrom, wherein the stem cell is an iPSC, has the capacity to differentiate into a T cell expressing a TCR directed against a first antigenic determinant, and comprises a nucleic acid molecule encoding a chimeric antigen receptor, said receptor comprising an antigen recognition moiety directed against a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In one embodiment, the genetically modified mammalian iPSC expresses at least one homozygous HLA haplotype.

[0100] While iPSCs are typically generated directly from somatic cells, it should be understood that the present invention is not limited in this respect. That is, subject iPSCs can be generated from cells that are not terminally differentiated; indeed, iPSCs can, in principle, be derived from any nucleated cell, including, for example, blood-derived mononuclear cells and skin cells. For example, in the context of one embodiment of the present invention, subject iPSCs can be generated from fully differentiated T cells or from precursor T cells such as thymocytes. As long as the subject thymocyte rearranges its TCR and exhibits the antigen specificity of interest in the context of the present invention, it is possible to attempt to generate iPSCs from this cell. This may be the case, for example, when the particular TCR rearrangement in question can be expected to be selected during thymocyte proliferation. Those skilled in the art will recognize that one complicating factor associated with immune responsiveness to tumor cells or autoreactive cells is the need for the immune system to direct the immune response to self-cells and, therefore, self-antigens in this situation. Such immune cells are typically selected during T lymphocyte differentiation in the thymus to minimize the likelihood of developing autoimmune disease. In the context of neoplasia and autoimmune conditions, however, the unwanted cells are autologous cells, and therefore the cell surface antigens that can be targeted will be autoantigens. While not limiting the present invention in any way, as discussed in more detail below, one advantage of using iPSCs to generate TCR / CAR-expressing T cells directed against multiple distinct antigenic determinants is that it has been determined that epigenetic memory effects can enhance the differentiation of iPSCs into functional T cells expressing TCRs directed against the same antigen as the T cells from which the iPSCs were derived. However, with regard to the selection of the specific TCR-expressing cells from which the iPSCs were derived, it can be difficult to identify adequately differentiated T cells, since T cells expressing functional TCRs directed against autoantigens can be selected during thymocyte expansion. Thus, it may be more feasible to screen thymocytes that express the desired TCR rearrangement and have not yet undergone negative selection to remove potentially autoreactive cells.

[0101] In another embodiment, the iPSCs are transfected with one or more nucleic acid molecules encoding a TCR (such as a rearranged TCR gene) directed against a first antigenic determinant (e.g., a tumor antigenic determinant).

[0102] In yet another embodiment, the target stem cells are hematopoietic stem cells (HSCs). 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 blood production. HSCs are derived from the mesoderm and can be found in adult bone marrow, peripheral blood, and umbilical cord blood. HSCs can be harvested from bone marrow, peripheral blood, and umbilical cord blood using established techniques and are generally associated with CD34+ expression. In some embodiments, human HSCs can be defined as CD34+CD38-CD90+CD45RA- (see Reinisch et al. (2015)). HSCs can be genetically modified, for example, transfected with one or more nucleic acids encoding TCRs directed against a first antigenic determinant, and then directed to differentiate into T cells. Nucleic acids encoding one or more CARs, optionally one or more docking antigen-binding receptors, can be introduced into HSCs before or after their differentiation into T cells.

[0103] Thus, reference to a "T cell receptor" (TCR) should be understood as a reference to the heterodimer found on the surface of T cells or NKT cells that recognize peptides presented by MHC. In particular, CD4+ T cells recognize peptides presented in the context of MHC class II, whereas CD8+ T cells recognize peptides presented in the context of MHC class I. Without limiting the present invention to any one theory or mechanism of action, in the majority of human T cells, the TCR comprises an α and β chain, while a minority population of cells expresses a TCR comprising a γδ heterodimer. TCRs are disulfide-linked, membrane-anchored heterodimeric proteins. The γ, δ, α, and β chains are composed of two extracellular domains: a variable (V) region and a constant (C) region, both of which form part of the immunoglobulin superfamily and fold to form an antiparallel β-sheet. The constant regions flank the cell membrane and are followed by a transmembrane region and a short cytoplasmic tail, while the variable regions bind to the peptide / MHC complex.

[0104] The variable domains of the TCR α and β chains each express three hypervariable or complementarity-determining regions (CDRs), while the variable region of the β chain possesses an additional area of ​​hypervariability (HV4) that does not normally contact antigen and is therefore not considered a CDR. The process of generating TCR diversity is primarily based on the genetic recombination of DNA-encoded segments in precursor T cells, either by somatic V(D)J recombination using RAG1 and RAG2 recombinases or by gene conversion using cytidine deaminase. Each recombined TCR possesses distinct antigen specificities determined by the structure of the antigen-binding site formed by the α and β chains in the case of αβ T cells, or the γ and δ chains in the case of γδ T cells. TCR α chains are generated by VJ recombination, while β chains are generated by VDJ recombination. Similarly, the generation of the TCR γ chain involves VJ recombination, while the generation of the TCR δ chain occurs by VDJ recombination. The intersection of these specific regions (V and J for α or γ chains; V, D and J for β and δ chains) corresponds to the CDR3 region that is important for peptide / MHC recognition. It is the distinct combinations of segments in this region, along with palindromes and random nucleotide additions, that account for the even greater diversity of T cell receptor specificity for processed antigenic peptides.

[0105] Thus, reference to a TCR "directed" against an antigenic determinant should be understood as a reference to a TCR that has undergone rearrangement and exhibits specificity for an antigenic determinant, preferably a self (particularly a self-cancer) antigenic determinant.

[0106] In one embodiment, the iPSCs are derived from cells expressing a rearranged TCR, preferably a rearranged αβ TCR. Examples of cells suitable for use in generating the iPSCs of the present invention include CD4 + T cells, CD8 + In another embodiment, the cells are rearranged γδ T cells, including, but not limited to, T cells, NKT cells, thymocytes, or other forms of precursor T cells. Expresses TCR.

[0107] Thus, provided are genetically modified mammalian iPSCs or HSCs, or T cells differentiated therefrom, wherein the iPSCs or HSCs have the capacity to differentiate into T cells expressing a TCR directed against a first antigenic determinant, and the TCR gene is derived from a cell that has undergone rearrangement or has been converted with the rearranged gene and comprises a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen recognition moiety directed against a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In some embodiments, the genetically modified mammalian iPSCs or HSCs express at least one homozygous HLA haplotype.

[0108] In one embodiment, said iPSCs are derived from T cells or thymocytes.

[0109] In another embodiment, the iPSCs are derived from T cells or thymocytes that express an αβTCR.

[0110] In yet another embodiment, the iPSCs are derived from T cells or thymocytes that express a γδ TCR.

[0111] The subject stem cells may be freshly isolated from the individual to be treated, or may be provided from a non-fresh source, such as a culture of cells isolated at some earlier time from the individual or another source (e.g., cultured to expand the cell number and / or make the cells receptive to differentiation signals) or a frozen stock. It should also be understood that the subject cells may have undergone some other form of treatment or manipulation before undergoing differentiation, such as, but not limited to, purification, alteration of the cell cycle state, or formation of a cell line, such as an embryonic stem cell line. Thus, the subject cells can be primary or secondary cells. Primary cells are those isolated from an individual. Secondary cells are those that have undergone some form of in vitro manipulation after their isolation, such as preparation of an embryonic stem cell line, before applying the methods of the present invention.

[0112] To the extent that the stem cells of the present invention are iPSCs, methods for generating iPSCs are well known to those skilled in the art. In this regard, and as detailed above, iPSCs are cells that are derived from more mature cell types, such as somatic cells, and converted / dedifferentiated to a pluripotent stage.

[0113] Without limiting the present invention to any one theory or mechanism 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 (Pou5f1), Sox2, cMyc, and Klf4. In 2006, Yamanaka demonstrated that transfection of these four specific genes encoding transcription factors converts human adult cells into pluripotent cells. While this combination is the most commonly used to produce iPSCs, each of the factors can be functionally replaced by unrelated genes, such as related transcription factors, miRNAs, small molecules, or lineage-specific factors. For example, iPSC induction has been achieved following transfection of Oct3 / 4, Sox2, Klf4, and cMyc using a retroviral system, as well as by transfection of Oct4, Sox2, Nanog, and Lin28 using a lentiviral system. The former set of transcription factors are known as Yamanaka factors, and the latter are 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, new mechanisms of delivery have been designed to increase efficiency, and vector sequences that might otherwise be integrated into the reprogrammed iPSC genome have been minimized or removed. These methods are well known to those skilled in the art: (i) Single-cassette reprogramming vector containing Cre-Lox-mediated transgene excision; (ii) Reprogramming with non-integrating viruses, such as, but not limited to, adenovirus or Sendai virus. Alternatively, expression of reprogramming factors as proteins provides a means of generating iPSCs that do not undergo integration of vector DNA introduced into the germline.

[0114] Non-viral reprogramming methods have also been developed. These include, but are not limited to: (i) mRNA transfection - The ability to express reprogramming factors as mRNA provides a method for generating iPSCs without chromosomal integration of viral vectors. Warren et al. have transcribed mRNA to efficiently express reprogramming factors (Warren et al., 2010). Efficiency can be increased by adding Lin28 to the Yamanaka reprogramming factor protocol, culturing at 5% O2, and including valproic acid in the cell culture medium. Reprogramming factor mRNA is commercially available. (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 plus four lentiviral Yamanaka factors are added to MRC5 and BJ-1 fibroblasts, there is a 10- to 15-fold increase in reprogramming efficiency compared to the four lentiviral factors alone (Subramanyam et al., 2011). In particular, It was also found that certain miRNAs can reprogram cells with high efficiency without Yamanaka factors. (iii) PiggyBac - PiggyBac is a mobile genetic element (transposon) that can be integrated into chromosomal TTAA sites in the presence of a transposase and subsequently excised from the genome by re-expression of the transposase. When cloned into the piggyBac vector and co-transfected into MEFs, Yamanaka factors can reprogram cells 14 to 25 days after transfection (Kaji et al., 2009; Woltjen et al., 2009). The piggyBac vector is a transgenic vector. It can be excised from iPSCs by re-expression of posease. (iv) Minicircular vectors—Minicircular vectors are minimal vectors containing only a eukaryotic promoter and the cDNA to be expressed. Lin28, GFP, Nanog, Sox2, and Oct4 minicircular vectors expressed in human adipose stromal cells can reprogram cells (Narsinh et al., 2011). (v) Episomal Plasmids—Transient expression of reprogramming factors as episomal plasmids allows for the generation of iPSCs. For example, oriP / EBNA vectors can be constructed with Yamanaka factors and Lin28 in one cassette, and another oriP / EBNA vector contains the SV40 large T antigen (Chuo et al., 2011). These vectors have been shown to be expressed in CD34+ umbilical cord blood, peripheral blood, and bone mononuclear cells in medium supplemented with sodium butyrate, resulting in iPSC colonies on day 14. The transfected plasmids are eventually lost. Includes:

[0115] In another aspect, those skilled in the art are also familiar with known auxiliary methods that can enhance the reprogramming efficiency of cells.For example, even if the same method is used, there may be variations in iPSC efficiency between cells.Various small molecules have been shown to enhance reprogramming efficiency (Table 4).

[0116] [Table 4]

[0117] Through several known mechanisms, these molecules act by inhibiting histone deacetylation (Mali et al., 2010; Huangfu et al., 2008), TGFβ and MEK signaling pathways. Blocking (Lin et al., 2009; Ichida et al., 2009), enhancing the function of epigenetic modifiers (Esteban et al. (2010)), inhibition of the ROCK pathway (Noggle et al. (2011)) and This may facilitate reprogramming, including the induction of glycolysis (Zhu et al., 2010). These small molecules, histone deacetylase inhibitors valproic acid and sodium butyrate, are the most commonly used in reprogramming protocols. It is also noteworthy that culturing cells in 5% oxygen during the reprogramming process can increase the efficiency of iPSC derivation (Yoshida et al., 2009). For particularly difficult cells, the addition of small molecules and culturing under hypoxic conditions may yield improvements. Another option is to use embryonic stem cell conditioned medium (ESCM) to induce the expression of endogenous reprogramming factors (Balasubramanian et al., 2009). Efficiency varies depending on the cell type. Further improvement can be achieved by the addition of luproic acid. Such a strategy can also be used to enhance the potency of exogenously introduced reprogramming factors, thereby increasing reprogramming efficiency.

[0118] To the extent that the stem cells of the present invention are HSCs, methods for generating or preparing HSCs are well known to those skilled in the art. HSCs can be obtained by direct extraction from bone marrow or from blood after HSCs are released from bone marrow following treatment with specific molecules, such as GM-CSF. HSCs can then be purified for plasma membrane expression of CD34, for example, by flow cytometric cell sorting after labeling with anti-CD34-coated magnetic beads or fluorescent anti-CD34. These purified HSCs can be induced to differentiate into T cells using the OP9 / OP9 DL-L1 system outlined in Example 3 and Figures 3 to 10.

[0119] Reference to a subject stem cell, particularly an iPSC or HSC, "capable" of differentiating into a T cell expressing a TCR directed against an antigenic determinant should be understood as a reference to any cell that transcribes and translates the subject TCR gene and then assembles a TCR heterodimer as a functional receptor on the cell surface, or is capable of doing so. As will be appreciated by those skilled in the art, in most situations, stem cells, such as iPSCs, will not express a TCR in their undifferentiated form. Once directed differentiation along a T cell lineage is induced, TCR expression is generally expected to occur. In one embodiment, cells can be induced to differentiate into T cells expressing a functional TCR, with or without CAR gene modification. It should be understood that the ability of a cell to express a TCR of a particular specificity can be achieved by any suitable means. For example, cells may be transfected with genes encoding two TCR chains (e.g., α and β chains) that, when expressed, associate to form a TCR heterodimer. Alternatively, and in the context of a preferred embodiment of the present invention, the stem cells of the present invention are generated from T cells, thymocytes, or other cells that have rearranged TCR genes. iPSCs generated from such cells can be expressed under appropriate cell culture conditions as CD4 + or CD8 +It has been determined that when directed to differentiate into T cells, the iPSCs will express the same TCR antigen specificity as the somatic T cells from which they were derived. Even more importantly, and as discussed in more detail below, it has been determined that T cells differentiated therefrom, with or without transfection of the iPSCs or HSCs with one or more nucleic acids encoding one or more CARs, or the α and β chains of an antigen / MHC class I-specific TCR, have the ability to stably express both a functional TCR and one or more CARs (and optionally one or more antigen-binding receptors), and are therefore directed against two or more distinct antigenic determinants. Thus, such stem cells are considered "competent" to differentiate into T cells and express the required TCR, provided that this occurs when the iPSCs or HSCs are provided with the appropriate differentiation signals. In this regard, because TCR gene rearrangements are entirely independent genomic events, the selection of a T cell subpopulation for generating iPSCs need not necessarily be identical to the T cell subpopulation ultimately to be produced by the directed iPSCs. For example, CD4 T cells exhibiting the appropriate TCR specificity may be used. + T cells can be selected to generate iPSCs. However, once the iPSCs are generated, one skilled in the art can select CD8 + One can try to direct the differentiation of iPSCs into T cells, where epigenetic memory can direct the differentiation of newly generated CD8 + T cells are CD8 + The T cell functionality will be determined by the TCR specificity of the CD4 iPSCs derived from the iPSCs. + That of T cells is also true.

[0120] Reference to inducing the "conversion" of a somatic cell, such as a T cell, to a multilineage potential phenotype, such as an iPSC, should be understood as a reference to inducing the genetic, morphological and / or functional changes necessary to change the somatic phenotype to a multilineage (pluripotent) phenotype of the type defined herein.

[0121] It will be appreciated that, to the extent that one chooses to provide iPSCs with the capacity to produce a TCR by transfection of the cells with DNA encoding a TCR, this transfection can occur at any time, such as before generation of the iPSCs of the invention, after generation of the iPSCs, or can occur simultaneously with CAR transfection.

[0122] As detailed above, somatic cells, particularly T cells or thymocytes, can be induced to transform into stem cells, i.e., functional stages of multilineage differentiation potential. Thus, reference to cells exhibiting "multilineage differentiation potential" or "multilineage potential" should be understood as a reference to cells exhibiting the potential to develop along more than one somatic differentiation pathway. For example, cells can have the ability to generate a limited range of somatic cell types; such cells are typically referred to as being pluripotent or multipotent. These cells exhibit a more limited range of lineage-committed potential than totipotent cells, which are cells that can develop into essentially every possible differentiation direction, including all somatic lineages and gametes.

[0123] Cells classically referred to as "progenitor" or "precursor" cells are included within the definition of "multilineage differentiation potential," provided that, under appropriate stimulatory conditions, they can give rise to cells of more than one somatic lineage. To the extent that reference to "stem cells" is made herein with respect to cells produced by the methods of the present invention, this should be understood as a reference to cells that exhibit multilineage differentiation potential as defined herein.

[0124] It should be understood that, with respect to the present invention, an important feature of the subject stem cells is that the multilineage differentiation potential exhibited by the cells includes the ability to differentiate into T cells and express a TCR that exhibits specificity for a desired antigen. It is irrelevant whether the TCR specificity is induced before or after the stem cells are generated (e.g., by transfecting the stem cells with DNA encoding the TCR of interest). It should be understood that the stem cells claimed herein encompass all stem cells that exhibit the requisite differentiation potential, regardless of when or how that potential was introduced. It should also be understood that the subject stem cells need not be totipotent. However, if they exhibit the ability to differentiate along more than one somatic cell lineage, and one of these lineages is the T cell lineage, then such cells are within the scope of the present invention.

[0125] As detailed above, the stem cells provided by the present invention are genetically modified. "Genetically modified" means that the subject cells are obtained as a result of some form of molecular manipulation compared to the cells observed in the context of corresponding unmodified cells. In the context of the present invention, the subject stem cells comprise a nucleic acid molecule encoding a chimeric antigen receptor, and optionally further comprise a nucleic acid molecule encoding an antigen-binding receptor. As disclosed herein, a nucleic acid encoding a receptor, whether a chimeric antigen receptor or an antigen-binding receptor, can be introduced into stem cells such as iPSCs or HSCs, or cells from which stem cells are derived (e.g., T cells); in both cases, the resulting stem cells containing a nucleic acid encoding the receptor are considered herein to be genetically modified stem cells. 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.

[0126] Reference to a "nucleic acid molecule" should be understood as a reference to deoxyribonucleic acid and its ribonucleic acid. The subject nucleic acid molecule can be in any suitable form of nucleic acid molecule, for example, genome, cDNA, or ribonucleic acid molecule. Thus, the term "expression" refers to the transcription and translation of DNA or the translation of RNA, which results in the synthesis of a peptide, polypeptide, or protein. A DNA construct corresponds to, for example, a construct that can be transfected into a cell for subsequent expression, while an example of an RNA construct is an RNA molecule that is transcribed from a DNA construct, and an RNA construct only requires translation to produce a protein of interest. Reference to an "expression product" refers to a product produced from the transcription and translation of a nucleic acid molecule.

[0127] References to "chimeric antigen receptors" (also known as "artificial T cell receptors," "chimeric T cell receptors," and "chimeric immune receptors") should be understood as references to engineered receptors that graft antigen-binding moieties onto immune effector cells. Generally, these receptors are used to graft the specificity of monoclonal antibodies onto T cells; transfection of their coding sequences is facilitated by retroviral vectors. More specifically, and without limiting the present invention in any way, the most common of these molecules are fusions of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a transmembrane CD3 zeta chain and endodomain. Such molecules transmit CD3 zeta chain signals 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 was directed. For example, to target malignant B cells, T cell specificity has been redirected using a chimeric immune receptor specific for the B-lineage molecule CD19.

[0128] The variable portions of immunoglobulin heavy and light chains are commonly fused via a flexible linker to form scFvs. These scFvs typically contain a signal peptide that directs the nascent protein to the endoplasmic reticulum and subsequently to surface expression; the signal peptide is eventually cleaved. The flexible spacer allows the scFvs to orient in different directions, enabling antigen binding. The transmembrane domain generally protrudes intracellularly and is a typical hydrophobic alpha-helix, typically derived from the original molecule's signaling endodomain, which transmits the desired signal. Therefore, reference to an "antigen recognition portion" should be understood as a reference to the extracellular portion of the receptor that recognizes and binds to the antigenic determinant of interest, i.e., the target-specific binding element. The antigen recognition domain is typically an scFv. However, many other options exist. For example, antigen recognition portions from natural T cell receptor (TCR) alpha and beta single chains have also been used, as they have simple ectodomains (e.g., the CD4 ectodomain for recognizing HIV-infected cells) and linked recognition components, such as cytokines, which provide recognition of cells bearing cytokine receptors. In fact, any moiety that binds a given target with sufficiently high affinity can be used as the antigen recognition domain. Such molecules are well known to those skilled in the art, and selecting an appropriate molecule for use would be well within the skill of the art. When designing a chimeric antigen receptor, particularly an extracellular domain, those skilled in the art can include additional moieties useful for efficient expression or functionalization. For example, as detailed above, a nucleic acid molecule expressing a CAR can be designed to express a single peptide at the N-terminus of the antigen recognition moiety. While not limiting the present invention to any one theory or mechanism of action, the single peptide directs the nascent protein to the endoplasmic reticulum. This is necessary if the receptor is glycosylated and anchored to the cell membrane. Any eukaryotic single peptide sequence can be used. Generally, a single peptide originally attached to the amino terminus is used (e.g., in an scFv with a light chain-linker-heavy chain configuration, the natural signal of the light chain is used).In another example, the extracellular domain can also contain a spacer region that can be used to link the antigen-recognition domain to the transmembrane domain. The spacer region should be sufficiently flexible to orient the antigen-recognition domain in different directions to facilitate antigen recognition and binding. The simplest form of spacer region is the hinge region of IgG1. Alternatives include the CH2CH3 region of immunoglobulins and portions of CD3. For the majority of scFv-based constructs, the IgG1 hinge is sufficient. Thus, the term "spacer" refers to any oligo- or polypeptide that functions to link the transmembrane domain to either the extracellular or cytoplasmic domain in the polypeptide chain. The spacer domain can contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. In yet another example, the hinge region can be modified to vary its length, thereby achieving additional functional benefits. For example, in conventional CARs containing a CD8 or CD28 hinge, one cysteine ​​(Cys) can be left in the hinge to stabilize dimerization on the T cell surface. Thus, two scFvs are typically displayed (bivalent). In another example, Cys can be substituted (with Ser) so that a stabilizing disulfide bond cannot be formed, thereby preventing dimerization and therefore premature activation. Cys can also be removed entirely. Another design is to display only the VH domain on one CAR and the VL domain on the other, so that Cys pairing aligns the VH / VL to form a functional monovalent Fv that targets the antigen of interest.

[0129] The antigen recognition portion of a subject chimeric antigen receptor is operably linked to a T cell activation portion. The "T cell activation portion" refers to a subregion of a receptor that, after antigen recognition and binding, is responsible for transmitting a signal to a T cell, enabling its activation and induction of effector mechanisms. The T cell activation portion of a CAR is generally located in the intracellular domain (or "endodomain") of the CAR; thus, the intracellular domain of a CAR molecule also generally contains or is an "intracellular signaling domain." A commonly used endodomain component is the intracellular domain of CD3 zeta, which contains three ITAMs. After antigen binding, this domain transmits an activation signal to T cells. CD3 zeta cannot provide a sufficiently potent activation signal, and additional costimulatory signaling is desirable. For example, chimeric CD28 and OX40 can be used together with CD3 zeta to transmit proliferation / survival signals, or all three can be used together. It should be understood that this intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of immune cells, preferably T cells in which the CAR is expressed. The term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs the cell to carry out a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. When a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire chain, so long as it transduces the effector function signal. Thus, the term "intracellular signaling domain" is intended to include any truncated portion of the intracellular domain sufficient to transduce an effector function signal.

[0130] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and coreceptors that act together to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional qualities.

[0131] It is known that signals generated by the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate 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 ITAM-containing primary cytoplasmic signaling sequences that are particularly useful 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.

[0132] In a preferred embodiment, the cytoplasmic domain of the CAR can be designed to include a CD3 zeta signaling domain by itself or can be combined with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for the effective response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-lBB (CD137), OX40, CD30, CD40, PD-1, TIM3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker. In one embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3 zeta and the signaling domain of CD28.

[0133] 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, bound, or otherwise associated with the T cell activation moiety such that binding of the antigen recognition moiety to an antigenic determinant results in a signal being induced by the T cell activation moiety to activate the target T cell and activate its effector function. This can be achieved, for example, by engineering a transmembrane domain.

[0134] In one embodiment, a transmembrane domain that naturally associates 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 prevent such domain from binding to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex. The transmembrane domain can be derived from a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-binding or transmembrane protein. For example, the transmembrane region can be derived from (i.e., at least the transmembrane region of) an immunoglobulin such as 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 IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it will primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. Generally, the transmembrane domain is a hydrophobic alpha helix that spans the membrane. Generally, the transmembrane domain from the component closest to the membrane of the endodomain is used.

[0135] Reference to an "antigen-binding receptor" should be understood as a reference to an engineered receptor that is anchored to a cell surface and binds to an antigen. Similar to the chimeric antigen receptors disclosed herein, the antigen-binding receptors disclosed herein also include an antigen recognition portion directed to an antigenic determinant. The antigen recognition portion in the antigen-binding receptor can take the same form and be designed in the same manner as the antigen recognition portion of the chimeric antigen receptors described herein. Also similar to the chimeric antigen receptors disclosed herein, the antigenic recognition portion in the antigen-binding receptor is operably linked to a transmembrane domain (e.g., by a spacer sequence such as a hinge region) so that the antigen-binding receptor is anchored to the cell surface. The spacer sequence and the 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 chimeric antigen receptors, the antigen-binding receptors defined herein are generally non-signaling and can include an intracellular sequence lacking a T cell activation domain. Such non-signaling antigen-binding receptors can bind to antigen but do not induce any signal transduction in T cells, and are therefore also referred to as "docking receptors" or "anchoring receptors." Certain embodiments of antigen binding receptors, such as non-signaling CD47 binding receptors, are further described herein below.

[0136] Examples of nucleic acid constructs encoding CARs and / or antigen-binding receptors are illustrated in FIG. 11, and exemplary sequences of CARs and antigen-binding receptors, as well as various domains and / or antigen-binding receptors suitable for use in CARs, are shown in SEQ ID NOs: 1 to 20.

[0137] Those skilled in the art will recognize that the mechanism by which these genetic modifications are introduced into cells can take any suitable form that would be well known and understood by those of skill in the art. For example, genetic material is commonly conveniently introduced into cells through the use of expression constructs.

[0138] In one embodiment, cells capable of differentiating into TCR-expressing T cells (i.e., stem cells such as iPSCs or HSCs) or cells expressing a TCR capable of deriving stem cells such as iPSCs are transfected with an expression construct encoding a CAR. The expression construct can include one or more DNA regions comprising a promoter operably linked to a nucleotide sequence encoding a CAR, optionally a second DNA region encoding a selectable marker, and optionally a third DNA region encoding a suicide protein. In this regard, it should be understood that, as a matter of routine, constructs can be designed with any additional component or components that one skilled in the art would deem useful, such as a suicide gene. In the context of cells of the present invention proposed for in vivo use to treat patients, the ability to control the death of genetically modified cells of the present invention and thus effect their elimination from the in vivo environment is highly desirable. While not limiting the present invention to any one theory or mechanism of action, adoptive transfer of cells of the present invention is not without risk, particularly insofar as they can be directed against "self" antigens such as tumor antigens or antigens expressed in autoreactive cells or antigens that may cross-react with autoantigens. In this situation, if these cells attack healthy (disease-free) cells, an outcome similar to graft-versus-host disease may occur. In the overall therapeutic scheme, these side effects may be even more desirable than the nonspecific systemic killing of healthy tissue characteristic of treatments such as chemotherapy or the uncontrolled killing of healthy tissue in autoimmune disorders. However, while killing cancer cells is of utmost importance, the ability to control the elimination of the cells of the present invention is highly desirable and can be routinely achieved by the well-known and widely used technique of incorporating an inducible suicide gene into the genetic construct introduced into the stem / T cells of the present invention.

[0139] The target promoter can be constitutive or inducible. If the target construct expresses 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 occurs in the context of a bicistronic vector that uses an IRES sequence to promote the translation of more than one protein product in unfused form from a single RNA transcript. The target construct can additionally be designed to facilitate the use of a Cre recombinase-mediated splicing-inducible gene expression system.

[0140] Reference to a nucleic acid "expression construct" should be understood as a reference to a nucleic acid molecule that is designed to be transducible to a cell and undergo transcription. An RNA molecule is then transcribed from it. Expression constructs in general are also referred to by a number of alternative terms that are widely used interchangeably, including "expression cassette" and "vector."

[0141] For the purpose of introducing nucleic acids encoding multiple receptors, whether the receptors are CARs, antigen-binding receptors, or a combination thereof, the nucleic acids encoding multiple receptors can be placed in one construct that is transfected into cells. In one embodiment, the nucleic acids encoding multiple receptors can be included in a polycistronic vector that uses an IRES sequence to promote the translation of multiple receptor proteins. In another embodiment, the nucleic acids encoding multiple receptors can be linked to each other in one expression unit and reading frame, for example, by using a self-cleaving peptide (e.g., P2A), so that a single polypeptide containing multiple receptor sequences is first produced and then processed to produce multiple receptors. In another embodiment, the nucleic acids encoding multiple receptors are placed in separate constructs used in transfection.

[0142] The expression constructs of the present invention may be produced by any suitable method, including recombinant or synthetic techniques. To this end, the constructs may be constructed from first principles, in which case a completely synthetic approach is utilized, or by appropriately modifying existing vectors. When the latter approach is adopted, the range of vectors available as a starting point is wide, and includes but is not limited to: (i) Plasmid: Plasmids are independently replicating pieces of cytoplasmic DNA, generally found in prokaryotic cells, and capable of autonomous replication. Plasmids are commonly used in the context of molecular cloning due to their ability to transfer from one organism to another. Without limiting the present invention to any theory or mode of action, plasmids may remain episomal or they may be integrated into the genome of the host. Examples of plasmids that can be used include bacterial-derived pBR322 and pUC. (ii) Bacteriophages: Bacteriophages are viruses that infect and replicate in bacteria. Generally, they consist of a core of nucleic acid enclosed within a protein coat (called a capsid). Depending on the type of phage, the nucleic acid may be either DNA (single- or double-stranded) or RNA (single-stranded), and they may be either linear or circular. Phages may be filamentous, polyhedral, or tailed polyhedral, with one or more tubular tail fibers attached to a tubular tail. Generally, phages can accommodate longer pieces of foreign DNA than, for example, plasmids. Examples of phages include, but are not limited to, E. coli lambda phage, P1 bacteriophage, and T-even phages (e.g., T4). (iii) Baculoviruses: A diverse group of DNA viruses that grow only in invertebrates, generally classified as Baculoviridae. Their genome consists of double-stranded circular DNA. (iv) Mammalian viruses: Examples of such viruses that infect mammals include lentiviruses, Sendai viruses, retroviruses, and vaccinia viruses; (v) Artificial chromosomes: Artificial chromosomes such as yeast artificial chromosomes or bacterial artificial chromosomes; (vi) Hybrid vectors such as cosmids, phagemids and phasmids: Cosmids are generally derived from plasmids but also contain cos sites for lambda phage, while phagemids represent chimeric phage-plasmid vectors. Phasmids generally also represent plasmid-phage chimeras, but are defined by the fact that they contain both functional origins of replication. Phasmids can therefore be propagated in appropriate host strains as either plasmids or phages. (vii) commercially available vectors that are either entirely synthetically produced or are modified versions of naturally occurring vectors such as viral vectors; Includes:

[0143] Those skilled in the art will understand that the selection of an appropriate vector for modification, to the extent that this is preferred over synthetically producing the construct, depends on numerous factors, including the ultimate use of the genetically modified cell in which it will be placed. For example, if the cells will be administered to humans in vivo, it may be less desirable to utilize certain types of vectors, such as viral vectors. Furthermore, the amount of DNA to be introduced into the construct must also be considered. It is generally understood that certain vectors are more easily transfected than certain cell types. For example, the range of cell types that can function as hosts for a given plasmid may vary from one type of plasmid to another. In yet another example, the larger DNA inserts that need to be inserted further limit the choice of vectors in which the expression constructs of the present invention are generated. To this end, the size of the inserted DNA may vary depending on factors such as the size of the DNA sequence encoding the protein of interest, the number of proteins to be expressed, the number of selectable markers used, and the incorporation of features such as linearized polylinker regions.

[0144] The expression constructs used in the present invention may be in any form, including circular or linear. In this context, a "circular" nucleotide sequence should be understood to refer to the circular nucleotide sequence portion of any nucleotide molecule. For example, the nucleotide sequence may be fully circular, such as a plasmid, or partially circular, such as a circular portion of a nucleotide molecule generated during rolling circle replication (which may be relevant, for example, if the construct is initially replicated by this type of method rather than via a cell-based cloning system prior to its introduction into a cell population). In this context, a "circular" nucleotide sequence corresponds to the circular portion of the molecule. A "linear" nucleotide sequence, which is essentially in linear form, should be understood to refer to any nucleotide sequence. A linear sequence may be a linear nucleotide molecule, or it may be a linear portion of a nucleotide molecule that also includes a non-linear portion, such as a circular portion. Examples of linear nucleotide sequences include, but are not limited to, a plasmid-derived construct that has been linearized to facilitate integration into the host cell chromosome, or a construct that is synthetically generated in linear form. To this end, it should also be understood that the configuration of the constructs of the present invention may or may not remain constant. For example, a circular plasmid-derived construct may be transfected into a cell as a stable circular episome that undergoes replication and transcription in this form. In another example, however, the construct of interest may be transfected into a cell in a circular form, but undergo linearization within the cell prior to integration into the chromosome. This is not necessarily an ideal situation, as such linearization may occur in a random manner, truncating the construct in critical regions and thereby rendering it ineffective.

[0145] The nucleic acid molecules utilized in the methods of the present invention can be derived from any human or non-human source. Non-human sources contemplated by the present invention include primates, livestock animals (e.g., sheep, pigs, cows, goats, horses, donkeys), laboratory animals (e.g., mice, hamsters, rabbits, rats, guinea pigs), domestic companion animals (e.g., dogs, cats), birds (e.g., chickens, geese, ducks, and other poultry, game birds, emus, ostriches), wild or domesticated captive animals (e.g., bulls, kangaroos, dingoes), reptiles, fish, insects, prokaryotes, or synthetic nucleic acids.

[0146] It should be understood that a construct encoding a receptor of the present invention may contain nucleic acid material from more than one source. For example, while a construct may be derived from a particular microorganism, nucleic acid material from other microbial sources may be introduced to modify the construct to introduce the characteristics defined herein. These sources 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 introduce a specific restriction endonuclease site). Furthermore, the cell type proposed to express the subject construct may further differ so that the nucleic acid material of the construct, in whole or in part, does not correspond to the same organism. For example, a construct consisting essentially of bacterial and viral DNA may nevertheless be expressed in mammalian stem cells as contemplated herein.

[0147] Without limiting the present invention in any way, the present invention preferably uses a DNA construct comprising a CAR sequence, which comprises the nucleic acid sequence of an antigen-binding portion operably linked to the nucleic acid sequence of an intracellular domain. For example, the intracellular domain that can be used in the subject CAR includes, but is not limited to, the intracellular domain of CD3 zeta. In another embodiment, the intracellular domain of the CAR comprises the intracellular domain of CD3 zeta operably linked to the intracellular domain of CD28; in a further embodiment, the intracellular domain of the CAR comprises the intracellular domains of CD3 zeta, CD28 and OX40 operably linked to each other.

[0148] Retrovirus-derived vectors, such as lentivirus, are an example of a vector suitable for achieving long-term gene transfer, as they allow for 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 in eukaryotes. Typical cloning vectors contain transcription and translation termination factors, initiation sequences, and promoters useful for controlling the expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and is described, for example, in 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.Suitable vectors generally contain a replication origin, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers that are functional in at least one organism (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0149] A number of virus-based systems have been developed for gene transfer into mammalian cells.For example, retrovirus provides a convenient platform for gene delivery systems.Selected genes can be inserted into vectors using techniques known in the art and packaged into retroviral particles.The recombinant virus can then be isolated and delivered to target stem cells.A number of retroviral systems are known in the art.

[0150] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the start site, although recent studies have shown that many promoters contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription.

[0151] 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 to it. Another example of a suitable promoter is the elongation growth factor Ia (EF-Ia). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, constructs should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated. The use of an inducible promoter provides a molecular switch capable of turning on expression of an operably linked CAR polynucleotide sequence when such expression is desired and turning off expression when such expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progenitor promoters, and the like. Examples include the steroid promoter and the tetracycline promoter.

[0152] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells can contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that have been transfected or infected with a viral vector. In other embodiments, 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 by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include antibiotic resistance genes, such as neo. Epitope tags can also be included in the extracellular domain of CAR molecules, such as the commonly used short polypeptide c-myc or FLAG, and are preferably placed in the hinge region to identify CAR expression using epitope-specific targeting agents, such as antibodies, used in combination with flow cytometry.

[0153] Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is not present or expressed in the recipient organism or tissue and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (see, e.g., Ui-Tei et al., 2000). FEBS Letters vol. 479:79-82). Suitable expression systems are well known and can be prepared by methods known in the art. These promoters can be prepared using techniques or commercially available. Generally, the construct with the smallest 5' flanking region that shows the highest expression level of the reporter gene is identified as the promoter. Such promoter region can be linked to a reporter gene and used to evaluate drugs for their ability to regulate promoter-driven transcription. It is understood by those skilled in the art that reporters such as eGFP (enhanced green fluorescent protein) can be separated by a self-cleaving peptide such as P2A and incorporated into CAR as a C-terminal polypeptide extension, releasing reporters such as eGFP into cells.

[0154] Methods for introducing and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells by physical, chemical, or biological means.

[0155] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing nucleotides is calcium phosphate transfection.

[0156] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.Viral vectors, and especially 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 lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0157] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular 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).

[0158] When a non-viral delivery system is attempted, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells. In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids and mixed with the lipid, combined with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, such as a micelle, or in a "collapsed" structure. They may simply be dispersed in the solution, or they may form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0159] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("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; dimyristyl phosphatidylglycerol ("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 approximately -20°C. Chloroform is used as the only solvent, as it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess amount of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991). However, they have a structure in solution that differs from the normal vesicle structure. Compositions are also encompassed. For example, lipids can be assumed to be micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Also contemplated are lipofectamine nucleic acid complexes.

[0160] Regardless of the method used to introduce exogenous nucleic acid into host cells, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells, including, for example, Southern and Northern blotting, RT-PCR and PCR, or by detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot).

[0161] In some embodiments, the TCR and CAR of the cell and the antigen-binding receptor are each directed against an antigenic determinant. Reference to an "antigenic determinant" should be understood as a reference to any proteinaceous or non-proteinaceous molecule expressed by the cell that is to be targeted by the receptor-expressing T cells of the present invention. It is understood that these molecules may be "self" molecules normally expressed in the patient's body (such as those expressed in some tumor cells or autoreactive cells) or may be non-self molecules (e.g., viral proteins) such as would be expected if the cell were infected with a microorganism. It should also be understood that the target antigen is not limited to antigens (whether self or not) that can naturally induce a T or B cell immune response. Rather, in the context of the present invention, reference to an "antigen" or "antigenic determinant" refers to any proteinaceous or non-proteinaceous molecule that is to be targeted. As detailed above, the target molecule may be one to which the immune system is naturally tolerant, such as a tumor antigen or an autoreactive immune cell antigen. However, targeting this antigen may nevertheless be desirable (also in terms of collateral damage) to minimize the potential for more severe side effects that may be observed with highly non-specific and systemic treatments such as chemotherapy or immunosuppression, or to reduce the duration of treatment through highly targeted treatment, and / or to maximize the potential killing of all unwanted cells. Preferably, the molecule is expressed on the cell surface.

[0162] It is understood by those skilled in the art that in the context of TCR binding, the target antigenic determinant takes the form of an antigen-derived peptide expressed in either the context of MHC I or MHC II. In the context of CAR, since the design of this receptor is based on the use of an immunoglobulin variable region binding domain, the receptor recognizes an epitope present in the natural form of the antigen. The target epitope can be either linear or conformational. It should be understood that the target antigenic determinant can be any molecule expressed by the cells to be targeted. That is, the targeted molecule may be exclusively expressed by target cells, or may also be expressed by non-target cells. Preferably, the target antigenic determinant is a non-self antigenic determinant, or an antigenic determinant that is exclusively expressed by the cells to be targeted or at a significantly higher level than that expressed by normal cells. However, as already discussed herein, depending on the disease state to be treated, it is not always possible to identify and target a non-self antigenic determinant.

[0163] Reference herein to a TCR / CAR receptor directed against a "first" antigenic determinant and a "second" antigenic determinant should be understood as a reference to the fact that the subject receptor is directed against two different epitopic regions. In this regard, it should be understood that the receptors may be directed against epitopes on two completely different cell surface molecules, or that the receptors may be directed against two different regions / epitopes of the same cell surface molecule. In embodiments referring to a TCR in conjunction with multiple CARs, or a TCR in conjunction with one or more CARs and one or more antigen-binding receptors, it should be understood that each receptor is directed against an antigenic determinant, and that the antigenic determinants are preferably different from each other, i.e., antigenic determinants corresponding to different epitopic regions of the same or different molecules.

[0164] Thus, in one embodiment, a genetically modified mammalian stem cell or a T cell differentiated therefrom is provided, the cell expressing at least one homozygous HLA haplotype and having the capacity to differentiate into a T cell expressing a TCR directed against a first antigenic determinant, and comprising at least one (i.e., one or more) nucleic acid molecule encoding a chimeric antigen receptor, said receptor comprising an antigen recognition moiety directed against a second antigenic determinant, the antigen recognition moiety operably linked to a T cell activation moiety, and optionally further comprising a nucleic acid encoding an antigen-binding receptor directed against a third antigenic determinant, wherein said antigenic determinant is selected from a tumor antigen, a microbial antigen, or an autoreactive immune cell antigen.

[0165] In one embodiment, the stem cells are iPSCs, hi another embodiment, the stem cells are HSCs.

[0166] In yet another embodiment, the stem cells are CD4 + T cells or CD8 + They have the ability to differentiate into T cells.

[0167] In yet another embodiment, the TCR is an αβ TCR.

[0168] In yet another embodiment, said stem cells, such as iPSCs, are T cells or thymocytes, preferably CD8 + It is of T cell or thymocyte origin.

[0169] As will be appreciated by those skilled in the art, the identification of tumor-restricted antigens is an important area of ​​research, but progress in this area has been limited. Because tumor cells are usually autologous cells (as opposed to, for example, tumors arising from transplanted tissue), it is true that the antigens they express are not only autoantigens, but are also likely to be expressed by non-neoplastic cells in the tissue from which the tumor originates. This is clearly a less-than-ideal situation due to the unavoidable side effects (in terms of destruction of non-neoplastic tissue) that may arise when anti-neoplastic treatment regimens target such antigens. Nevertheless, some progress has been made in identifying target tumor antigens that are expressed at low levels or poorly expressed in non-neoplastic cells, if not exclusively by tumor cells.

[0170] The choice of antigen-binding moiety of the present invention will depend on the specific 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), β-human chorionic gonadotropin, tumor-associated glycoprotein 72 (TAG72), alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut These include hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin, and telomerase, prostate cancer tumor antigen 1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin. CD47 (a "denial of phagocytosis" receptor) is also a tumor target because it is often highly expressed on cancer cells compared to normal cells, preventing them from being attacked by cells of the immune system, specifically scavenger macrophages.

[0171] In one embodiment, the tumor antigen comprises one or more epitopes associated with a malignant tumor. Malignant tumors express numerous 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 and 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-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are distinct for each individual tumor. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas.

[0172] Non-limiting examples of antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gplOO (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; distinct tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and the like; and viral antigens such as the Epstein-Barr virus antigen EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include CD47, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, cMet, nm-23Hl, PSA, TAG72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, and B These include TAA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0173] The cells of the present invention are designed to be directed against multiple antigenic determinants, i.e., two or more. As detailed herein, multiple antigenic determinants may be or include, in some embodiments, multiple epitopes of a single molecule, or in other embodiments, epitopes of multiple completely different molecules. The selection of which of the multiple antigenic determinants to target, and whether they should be targeted by a TCR or CAR, is well within the skill of those in the art. In one embodiment, the cells of the present invention are designed to eliminate tumor cells, and the TCR / CAR is directed against tumor antigens, specifically TAG72, MAGE, and WT1. In another embodiment, the cells are designed to eliminate autoreactive immune cells, and the TCR / CAR is directed against an idiotypic T cell or B cell receptor.

[0174] Thus, in one embodiment, a genetically modified mammalian stem cell or a T cell differentiated therefrom is provided, wherein the cell has the capacity to differentiate into a T cell expressing a TCR directed against a first tumor antigenic determinant, and comprises one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed against a tumor antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety, wherein said antigenic determinant is selected from TAG72, CD47, CD19, WT-1, MAGE, and EBVLMP2.

[0175] Preferably, the genetically modified cells are directed against TAG72 and WT-1. Even more preferably, the CAR is directed against TAG72 and CD47, and the TCR is directed against WT-1.

[0176] In one embodiment, the stem cells are iPSCs, hi another embodiment, the stem cells are HSCs.

[0177] In yet another embodiment, the stem cells are CD4 + T cells or CD8 + They have the ability to differentiate into T cells.

[0178] In yet another embodiment, the TCR is an αβ TCR.

[0179] In yet another embodiment, the stem cells (such as iPSCs) are T cells or thymocytes, preferably CD8 + It is of T cell or thymocyte origin.

[0180] In one embodiment, to the extent that the cells of the present invention are directed to treating neoplasms, a wide range of CARs have been developed to target known tumor antigens. A non-limiting summary illustrating some of these CARs, along with the receptor structures, is provided below in Table 5: [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0181] In some embodiments, the CAR comprises an antigen recognition domain consisting of an scFv directed against CD19 or TAG72, a hinge (stalk) region and a transmembrane region, both of which are derived from CD28 or CD8, and a cytoplasmic endodomain containing a T cell activation moiety, also derived from CD28 or CD8. The CAR can comprise a reporter protein (such as EGFP) as a C-terminal polypeptide extension, connected to each other by a P2A self-cleaving polypeptide to release EGFP after translation. See, for example, Figures 11 and 14.

[0182] In a related embodiment, it has been further determined that the cells of the present invention are particularly effective when engineered to express a non-signaling antigen-binding receptor, such as a CD47 binding molecule that cannot affect signal transduction.Expression of a CD47 binding molecule on the cell surface anchors the cells of the present invention to the neoplastic cells to which they are directed, thereby promoting improved interaction between their respective ligands and TCR and CAR.In particular, for the treatment of solid tumors, the increased stability and binding affinity of the target cell interaction allows for improved functional outcomes in terms of killing neoplastic cells compared to cells that do not express the target CD47 binding molecule.

[0183] Thus, in a related aspect of the invention, genetically modified mammalian stem cells, or T cells differentiated therefrom, are provided, the cells having the capacity to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and comprising: (i) a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen recognition portion directed to a second antigenic determinant, the antigen recognition portion operably linked to a T cell activation portion, and (ii) a nucleic acid molecule encoding a non-signaling antigen binding receptor, such as a non-signaling CD47 binding receptor. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0184] Without limiting the present invention to any 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 various intracellular processes, including apoptosis, proliferation, adhesion, and migration. Furthermore, it plays an important role in immune and angiogenic responses. CD47 is ubiquitously expressed in human cells and has been found to be overexpressed in many different tumor cells.

[0185] CD47 is a 50 kDa membrane receptor containing an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. There are four alternatively spliced ​​isoforms of CD47 that differ only in the length of their cytoplasmic tails. Form 2 is the most widely expressed form found in all circulating and immune cells. The second most abundant isoform is form 4, which is expressed primarily in the brain and peripheral nervous system. Only keratinocytes express significant amounts of form 1. These isoforms are highly conserved between mice and humans, suggesting an important role for the cytoplasmic domain in CD47 function.

[0186] CD47 is the receptor for thrombospondin-1 (TSP-1), a secreted glycoprotein that plays a role in blood vessel development and angiogenesis. The 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 the regulation of angiogenesis and inflammation. CD47 also interacts with signal-regulatory protein alpha (SIRPα), an inhibitory transmembrane receptor present on myeloid cells. CD47 / SIRPα interactions result in bidirectional signal transduction, resulting in various intercellular responses, including inhibition of phagocytosis (facilitating cancer cell escape), stimulation of cell-cell fusion, and T cell activation. Furthermore, CD47 interacts with several membrane integrins, most commonly integrin avb3. These interactions result in CD47 / integrin complexes that influence a wide range of cellular functions, including adhesion, spreading, and migration.

[0187] However, although CD47 is ubiquitously expressed, it has been determined that increased levels of CD47 expression on neoplastic cells is sufficient to promote improved responsiveness of said neoplastic cells to molecules targeting CD47 and to eliminate them prior to any substantial adverse effects on non-neoplastic cells.

[0188] Reference to a "binding receptor" directed to CD47 should be understood as a 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 preferably lacks signaling function.

[0189] The present embodiments provide genetically modified mammalian stem cells, or T cells differentiated therefrom, which have the capacity to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and which comprise: (i) a nucleic acid molecule encoding a chimeric antigen receptor, said receptor comprising an antigen recognition portion directed to a second antigenic determinant, the antigen recognition portion operably linked to a T cell activation portion, and (ii) a nucleic acid molecule encoding a non-signaling antigen-binding receptor, said receptor comprising an antigen recognition portion directed to CD47. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0190] As detailed above, the target CD47 binding receptor is a non-signal transduction receptor.By " non-signal transduction ", it is meant that there is no signal transduction that causes changes in the function of the cell of the present invention after the target receptor is bound to the CD47 on target cell.Rather, the purpose of CD47 binding receptor is to provide improved anchoring of target cell to target cell, thereby improving the binding efficiency of TCR and CAR that are directed to target antigen moiety, such as tumor antigen moiety.

[0191] For example, in one design of a non-signaling antigen-binding receptor, the extracellular domain of the receptor comprises an antigen recognition portion with binding specificity for CD47, a hinge (stalk) domain, a transmembrane domain, and an intracellular domain that completely lacks cytoplasmic signaling function. Such a non-signaling CD47-binding receptor can be used solely for attachment rather than signaling, thereby driving the docking of T cells to cancer cells via CD47 binding without the undesired activation and killing that occurs when engaging normal CD47-expressing cells.

[0192] In some embodiments, the antigen recognition portion of a non-signaling CD47-binding receptor may comprise any CD47-targeting V-domain, including antibody-like domains such as scFv, Fv, Fab, and single human and mammalian V-domains and their equivalents (VhH or vNAR) domains, or may comprise "alternative protein-based targeting scaffolds" known in the art, including, but not limited to, darpins, anticalins, knottins, ImmE7s, affibodies, Fn3 fibronectin domains, and the like. The antigen recognition portion may also comprise one or more of the V-like domains of SIRPα (the natural ligand for CD47). In one embodiment, the antigen recognition portion may comprise the native V-like domain of SIRPα. In another embodiment, the antigen recognition portion may comprise all three native V-like domains of SIRPα. In other embodiments, a molecule suitable for use to provide an antigen recognition portion in a non-signaling CD47-binding receptor is the Hu5F9-G4 scFv molecule (described in U.S. Patent Application No. 14 / 656,431). Hu5F9 was designed with three different versions of VH (1, 2, 3) and three different versions of VL (11, 12, 13), as shown in Figures 12A and 12B of U.S. Patent Application No. 14 / 656,431, published as U.S. Patent No. 20150183874 A1. Liu et al. (PLOS One (2015) September 21; Vol. 10(9): e0137345) described Hu5F9-G4, in which the selected V-domains were heavy chain VH-2, which contains four distinct residue changes in the framework (differentiating VH-2 from VH-1 and VH-3), and light chain VL-12, which contains two distinct residue changes in the framework (differentiating VL-12 from VL-11 and VL-13).

[0193] In some embodiments, the hinge region of the non-signaling CD47-binding receptor may be a natural SIRPα hinge sequence, or an alternative hinge known in the art, such as a CD8 or CD28 hinge typically used in CARs, or a CD4 domain or mucin peptide hinge. The hinge region may be designed to contain one or more cysteine ​​(Cys) residues to enable receptor dimerization. CD28 is a natural dimeric structure linked via a single Cys in the stalk region. Therefore, when the stalk region of CD28 is used as the hinge of a non-signaling CD47-binding receptor, the introduction of an additional Cys may not be necessary, but can provide additional stabilization to the dimer.

[0194] It will be understood by those skilled in the art that introduction of nucleic acids encoding a CAR and a non-signaling antigen-binding receptor (such as a non-signaling CD47-binding receptor) into cells (e.g., T cells or iPSCs) can be achieved 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 that directs self-cleavage to separate the CAR from the antigen-binding receptor. In a specific embodiment, the non-signaling CD47-binding receptor is expressed as a C-terminal extension of the CAR and is separated by the P2A self-cleaving peptide to separate the CAR and the CD47-binding receptor after translation.

[0195] The means for modifying stem cells of the present invention to also express non-signaling CD47-binding molecules have been described in great detail herein above in terms of affecting expression of chimeric antigen receptors directed against tumor antigen moieties. The transfection and other methods for achieving receptor expression described herein will be understood by those skilled in the art as being equally applicable in the context of the subject CD47-binding molecules.

[0196] In one embodiment, the stem cells are iPSCs, hi another embodiment, the stem cells are HSCs.

[0197] In another embodiment, the stem cells are CD4 + T cells or CD8 + They have the ability to differentiate into T cells.

[0198] In yet another embodiment, the TCR is an αβ TCR.

[0199] In yet another embodiment, said stem cells, such as iPSCs, are T cells or thymocytes, preferably CD8 + CD8 derived from T cells or thymocytes, with endogenous TCRs that, in some embodiments, are directed against tumor antigens + It is of T cell or thymocyte origin.

[0200] In yet another embodiment, the stem cells are directed against TAG72 and WT 1. Even more preferably, the CAR is directed against TAG72 and the TCR is directed against WT 1.

[0201] In a further aspect, a method for producing genetically modified mammalian stem cells is provided. Various means for producing genetically modified mammalian stem cells, particularly iPSCs, are described hereinabove.

[0202] In a further aspect, a T cell and a chimeric antigen receptor are provided that express a TCR directed to a first antigenic determinant, said receptor comprising an antigen recognition moiety directed to a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety. In some embodiments, the T cell expresses at least one homozygous HLA haplotype.

[0203] In one embodiment, the T cell expresses multiple chimeric antigen receptors, each chimeric antigen receptor comprising an antigen recognition moiety directed to an antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety.

[0204] In one embodiment, the antigenic determinants against which the multiple chimeric antigen receptors are directed are each different from the first antigenic determinant against which the TCR expressed on the subject's T cells is directed. In another embodiment, the antigenic determinants against which the multiple chimeric antigen receptors are directed are each different and also different from the first antigenic determinant against which the TCR expressed on the subject's T cells is directed.

[0205] In one embodiment, multiple CARs are encoded by one adjacent nucleic acid fragment. For example, multiple CARs are encoded by multiple nucleic acids placed in one vector, which are transfected into cells to ultimately generate target T cells. In a specific embodiment, multiple CAR encoding nucleic acids can be linked together in one expression unit and reading frame (for example, by using a self-cleaving peptide such as P2A), so that a single polypeptide comprising multiple CAR polypeptide sequences is first produced, which is then processed to provide multiple CARs. In another embodiment, multiple CAR encoding nucleic acids are placed in separate vectors, which are used in transfection to generate target T cells.

[0206] In another embodiment, the T cells expressing one or more CARs further express at least one (i.e., one or more) antigen-binding receptors comprising an antigen recognition moiety directed against a third antigenic determinant.

[0207] In one embodiment, the antigen-binding receptor is a non-signaling antigen-binding receptor; i.e., the receptor is anchored on the cell surface of the subject T cell and binds to a third antigenic determinant, but does not transduce a signal to a cytoplasmic portion of the T cell that affects T cell function (hence, also referred to as a non-T cell signaling antigen-binding receptor). In one embodiment, the antigen-binding receptor comprises an antigen recognition portion directed to a third antigenic determinant and operably linked to a transmembrane domain, but lacks a T cell activation portion.

[0208] In a specific embodiment, the antigen binding receptor is a non-signaling antigen binding receptor directed against CD47, e.g., the antigen binding receptor is a non-signaling CD47 binding molecule.

[0209] In some embodiments, the T cells provided herein are CD4+. In other embodiments, the T cells are CD8+.

[0210] In some embodiments, the T cells provided herein express an αβ TCR. In other embodiments, the T cells provided herein express a γδ TCR.

[0211] In some embodiments, the target T cells are directed against multiple antigenic determinants, i.e., the first antigenic determinant against which the TCR is directed, the antigenic determinant(s) against which the chimeric antigen receptor(s) are directed, and the antigen-binding receptor(s), if such antigen-binding receptor(s) are present, may be selected from a tumor antigen, a microbial antigen, or an autoreactive immune cell antigen. In certain embodiments, the antigenic determinant is selected from a tumor antigen. In specific embodiments, the antigenic determinant against which the TCR is directed is selected from a TCR-recognized peptide such as WT-1 or EbvLMP2. In other specific embodiments, the antigenic determinant against which the chimeric antigen receptor and the antigen-binding receptor are directed may be selected from, for example, TAG72, CD19, MAGE, or CD47.

[0212] In some embodiments, the subject T cells expressing a TCR directed to a first antigenic determinant and expressing a chimeric antigen receptor comprising an antigen recognition moiety directed to a second antigenic determinant, operably linked to a T cell activation moiety, are derived from iPSCs or HSCs.

[0213] In one embodiment, the iPSCs or HSCs from which the subject's T cells are derived are genetically modified iPSCs or HSCs that express a TCR directed against the first antigenic determinant, contain one or more nucleic acids encoding one or more chimeric antigen receptors, and are capable of differentiating into T cells that optionally contain one or more nucleic acids encoding antigen-binding receptor(s). In another embodiment, the iPSCs or HSCs from which the subject's T cells are derived are capable of differentiating into T cells that express a TCR directed against the first antigenic determinant; the one or more nucleic acids encoding one or more chimeric antigen receptors and, optionally, one or more nucleic acids encoding antigen-binding receptor(s) are introduced into the iPSCs or HSCs after they have differentiated into T cells. In some embodiments, the iPSCs or HSCs from which the subject's T cells are derived express at least one HLA haplotype, and such iPSC- or HSC-derived T cells also express the at least one HLA haplotype.

[0214] In one embodiment, the iPSCs from which the subject T cells are derived are themselves derived from T cells or thymocytes. In one embodiment, the iPSCs are derived from CD8+ T cells or thymocytes. In one embodiment, the iPSCs are derived from T cells or thymocytes that express a TCR directed against a first antigenic determinant, i.e., the TCR of the subject T cells derived from the iPSCs is directed against the same antigenic determinant.

[0215] The value of the cells of this invention is + or CD8 +This method is based on directing the differentiation of a subject stem cell into a T cell. In this context, "directing" stem cell differentiation into a T cell should be understood to mean applying a cell culture system that induces the stem cell's commitment to the T cell lineage and its differentiation into mature T cells along that lineage. Means for effecting directed differentiation of stem cells along a T cell lineage are well known to those skilled in the art. For example, as exemplified herein, the introduction of Notch-dependent signaling into a culture system is known to result in directed differentiation of stem cells along a T cell lineage. Furthermore, particularly efficient differentiation is achieved when this signaling is provided to stem cells in the context of their co-culture on an OP-9 feeder cell layer. 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 highly convenient means of generating T cells from stem cells. In another example, and as exemplified herein, a subject stem cell is first cultured in feeder-free conditions to generate mesoderm, followed by co-culture with the OP9-DL1 cell line. CD8 + A particularly preferred method for achieving directed differentiation into T cells is exemplified herein.

[0216] In another aspect, methods are provided for generating T cells that express a TCR directed to a first antigenic determinant, one or more CARs, and optionally one or more antigen-binding receptors. In some embodiments, the T cells also express at least one homozygous HLA haplotype.

[0217] In one embodiment, a method comprises obtaining genetically modified stem cells (such as genetically modified iPSCs or HSCs) capable of differentiating into T cells that express a TCR directed against a first antigenic determinant, comprising one or more nucleic acid(s) encoding one or more chimeric antigen receptors, each directed against an antigenic determinant (preferably different from the first antigenic determinant), optionally one or more nucleic acids encoding one or more antigen-binding receptor(s), each directed against an antigenic determinant (preferably different from the first antigenic determinant); and differentiating such genetically modified stem cells into T cells. In some embodiments, the genetically modified stem cells also express at least one homozygous HLA haplotype.

[0218] In another embodiment, the method includes obtaining stem cells (such as iPSCs or HSCs) that have the capacity to differentiate into T cells that express a TCR directed against a first antigenic determinant; differentiating the stem cells into T cells; and introducing into the T cells one or more nucleic acid(s) encoding one or more chimeric antigen receptors, each directed against an antigenic determinant (preferably different from the first antigenic determinant), and optionally one or more nucleic acids encoding one or more antigen-binding receptor(s), each directed against an antigenic determinant (preferably different from the first antigenic determinant). In some embodiments, the genetically modified stem cells (such as iPSCs or HSCs) also express at least one homozygous HLA haplotype.

[0219] Regardless of whether the CAR-encoding nucleic acid is introduced into the stem cell before differentiation into a T cell or after differentiation from the stem cell, the stem cell (such as an iPSC) may itself be derived from a T cell or a thymocyte. Such T cells and thymocytes may have a TCR specific for a nominal antigen, for example, 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 against the same antigenic determinant to which the TCR expressed on the iPSC-derived T cell is directed.

[0220] Reference to "mammal" should be understood to include, but is not limited to, reference to mammals such as humans, primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), companion animals (e.g., dogs, coats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). Preferably, the mammal is a human or a primate. Most preferably, the mammal is a human.

[0221] The development of the present invention now facilitates the development of means for treating disease states characterized by the presence of unwanted cell populations, such as neoplastic populations of cells, virally infected cells, autoreactive immune cells, or infections with microorganisms such as antibiotic-resistant bacteria. More specifically, the cells of the present invention provide a means for eliminating these cells in a more targeted manner than current, highly nonspecific methods, such as chemotherapy for treating neoplastic conditions, anti-inflammatory therapy for treating symptoms of autoimmune disease, or immunosuppression for managing autoimmunity. In this regard, reference to a disease state "characterized by the presence of unwanted cell populations" should be understood to refer to any condition whose symptom or cause is the presence or function of a cell population that can be targeted by the nature of the cell surface antigens expressed, and whose removal of some or all of the cells is beneficial to the patient. Treatment of the target condition is achieved by administering T cells differentiated from the stem cells of the present invention, whose dual TCR / CAR is directed against two or more antigenic determinants expressed by the cells sought to be eliminated.

[0222] It should be understood that the "cells" attempted to be eliminated by the T cells of the present invention can be any cells, whether autologous or non-autologous. For example, to the extent that the T cells of the present invention are designed to treat a disease state such as a neoplasm, a viral infection, or an autoimmune disease, the target population of cells attempted to be eliminated are autologous cells. However, to the extent that the condition attempted to be treated is an infection by a microorganism, such as, for example, an antibiotic-resistant bacterium or a parasite, the "cells" to be eliminated are foreign cells. In this regard, the cells may be in suspension (such as leukemia cells present in the circulation) or part of a mass (such as a tumor or tissue). To the extent that the condition to be treated is a microbial infection, the cells may represent a unicellular microorganism (such as many bacteria) or may be part of a multicellular organism. The T cells of the present invention are useful for targeting any type of cell present in any type of structure.

[0223] Accordingly, another aspect of the present invention is directed to a method of treating a condition characterized by the presence of an undesired population of cells, as defined above, in a mammal, said method comprising administering to said mammal an effective amount of stem cells or T cells differentiated therefrom.

[0224] 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.

[0225] In another embodiment, the stem cells are iPSCs or HSCs.

[0226] In yet another embodiment, the stem cells are CD4 + T cells or CD8 + They have the ability to differentiate into T cells.

[0227] In yet another embodiment, the TCR is an αβ TCR.

[0228] In yet another embodiment, said stem cells, such as iPSCs, are derived from T cells or thymocytes.

[0229] In yet another embodiment, the cell further comprises a nucleic acid molecule encoding a non-signaling antigen-binding receptor, said receptor comprising an antigen recognition moiety directed against CD47.

[0230] In one more specific aspect of these embodiments, there is provided a method of treating a neoplastic condition, said method comprising administering to said mammal an effective number of stem cells or T cells differentiated therefrom, as defined above, wherein said TCR is directed against a first tumor epitope and said CAR is directed against one or more additional tumor epitope(s).

[0231] In one embodiment, the first tumor antigenic determinant is WT1.

[0232] In another embodiment, the second tumor antigenic determinant is TAG72.

[0233] In another embodiment, the cell further comprises a nucleic acid molecule encoding a non-signaling antigen-binding receptor, said receptor comprising an antigen recognition moiety directed against CD47.

[0234] In another embodiment, the genetically modified stem cells also express at least one homozygous HLA haplotype.

[0235] Reference to a "neoplastic condition" should be understood as referring to a condition characterized by the presence or development of encapsulated or non-encapsulated growths or aggregations of neoplastic cells. Reference to "neoplastic cells" should be understood as referring to cells exhibiting abnormal growth. The term "growth" should be understood broadly and includes reference to an increase in size and proliferation of neoplastic cells.

[0236] In this context, the phrase "abnormal growth" is intended to refer to cell growth that exhibits one or more of the following, compared to normal cell growth: an increase in the size and nuclear / cytoplasmic ratio of individual cells; an increased rate of cell division; an increased number of cell divisions; a decrease in the length of cell division periods; an increase in the frequency of cell division periods or occurrences of uncontrolled proliferation; and avoidance of apoptosis. Without limiting the present invention in any way, the general medical meaning of the term "neoplasm" refers to "new cell growth," e.g., neoplastic cell growth, that occurs as a lack of responsiveness to normal growth controls. Neoplasms include "tumors," which may be benign, premalignant, or malignant. The term "neoplasm" should be understood as referring to a lesion, tumor, or other encapsulated or non-encapsulated mass or other form of growth or cellular aggregate containing neoplastic cells.

[0237] In the context of the present invention, the term "neoplasm" should be understood to include reference to all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type or invasive status.

[0238] The term "cancer" is recognized by those skilled in the art to refer to malignant tumors of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. The term also includes carcinosarcomas, including malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to cancers derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0239] The neoplastic cells comprising the neoplasm may be of any cell type derived from any tissue, such as epithelial or non-epithelial cells. References herein to the terms "malignant neoplasm" and "cancer" and "carcinoma" should be understood to be interchangeable.

[0240] The term "neoplasm" should be understood as referring to a lesion, tumor or other encapsulated or non-encapsulated mass or other form of growth or cellular aggregate comprising neoplastic cells. The neoplastic cells comprising a neoplasm may 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 invention include, but are not limited to, central nervous system tumors, retinoblastoma, neuroblastoma, pediatric tumors, head and neck cancer (e.g., squamous cell carcinoma), breast and prostate cancer, lung cancer (both small cell and non-small cell lung cancer), kidney cancer (e.g., renal cell adenocarcinoma), esophagogastric cancer, hepatocellular carcinoma, pancreatic and biliary neoplasms (e.g., adenocarcinoma and pancreatic islet cell tumors), colorectal cancer, cervical and anal cancer, uterine and other reproductive system cancers, urinary tract cancer (e.g., ureter and bladder), germ cell tumors (e.g., testicular germ cell tumors or ovarian germ cell tumors), ovarian cancer (e.g., ovarian epithelial carcinoma), cancer of unknown primary origin, human immunodeficiency-associated malignancies (e.g., Kaposi's sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumors (e.g., thyroid), mesothelioma and other pleural or peritoneal tumors, neuroendocrine tumors, and carcinoid tumors.

[0241] In a specific embodiment, said neoplastic condition is leukemia or lymphoma.

[0242] In another embodiment, the neoplastic condition is metastatic.

[0243] The subject undergoing treatment or prevention may be any human or animal in need of therapeutic or prophylactic treatment. In this regard, "treatment" and "prevention" as referred to herein may be considered in their broadest contexts. The term "treatment" does not necessarily mean that a mammal is treated until complete recovery. Similarly, "prevention" does not necessarily mean that a subject will not eventually contract a disease state. Thus, treatment and prevention include ameliorating the symptoms of a particular condition, or preventing or otherwise reducing the risk of developing a particular condition. The term "prevention" may be considered as reducing the severity of the onset of a particular condition. "Treatment" may also reduce the severity of an existing condition.

[0244] Thus, the present invention should be understood to encompass reducing or otherwise ameliorating a condition in a mammal. This should be understood as referring to the reduction or amelioration of any one or more symptoms of a disease. While achieving a cure of a disease is always most desirable, slowing the progression of a disease also has significant clinical value. In the context of viral infections, such as HIV or STDs, even if a complete cure cannot be achieved, reducing the viral load and extent of spread can provide a means of managing the infection so that, for example, the severe immunodeficiency of HIV, which is ultimately fatal, is not experienced and a relatively normal lifespan can be achieved without the severe side effects characteristic of the antiviral drug cocktails that patients must take. In the specific context of neoplastic conditions, the T cells of the present invention, when administered to a patient, downregulate the growth of the neoplasm. Reference to the "growth" of a cell or neoplasm should be understood as referring to the proliferation, differentiation, and / or maintenance of viability of the subject cell, while "downregulating the growth" of a cell or neoplasm refers to reducing, preventing, or inhibiting the aging process of the cell or the maintenance of proliferation, differentiation, and / or viability of the subject cell. In a preferred embodiment, the growth in the subject is proliferation and the downregulation in the subject is CD8 +In this regard, killing can be evidenced either by a reduction in the size of the tumor mass, or by inhibition of further growth of the tumor, or by a delay in tumor growth. Without limiting the invention to any theory or mode of action in this regard, neoplastic cells can be killed by direct lysis or induction of apoptosis or by CD4 T cell-mediated killing. + or CD8 + The tumor cells may be killed by any suitable mechanism, such as T cells or some other mechanism that can be promoted by T cells lacking these CD4 and CD8 markers. Therefore, the present invention should be understood to encompass reducing or otherwise ameliorating a neoplastic condition in a mammal. This should be understood as referring to the prevention, reduction, or amelioration of any one or more symptoms of a neoplastic condition. Symptoms include, but are not limited to, pain at the tumor growth site or metabolic or physiological bodily dysfunction caused by a neoplastic condition. It should be understood that the method of the present invention can reduce the severity of any one or more symptoms, or eliminate the presence of any one or more symptoms. The method of the present invention also extends to preventing the onset of any one or more symptoms.

[0245] Therefore, the method of the present invention is useful from both a therapeutic and palliative perspective. For this purpose, reference to "treatment" should be understood to encompass both therapeutic and palliative care. As will be understood by those skilled in the art, while curing a neoplastic condition is always the most desirable outcome, being able to slow or stop the progression of the neoplasm, even if not completely cured, is of significant benefit. Without limiting the present invention in any way, there are some neoplastic conditions that, provided they are sufficiently downregulated in terms of cell division, are not fatal to the patient and allow the patient to still have a reasonable quality of life. Furthermore, it should be understood that the present method provides a useful alternative to existing treatment regimens. For example, in some situations, the therapeutic results of the present method may be equivalent to chemotherapy or radiation, but a treatment regimen that induces fewer side effects or a shorter duration of side effects and is therefore better tolerated by the patient is a benefit to the patient. As detailed above, it should be understood that the term "treatment" does not necessarily mean that the subject is treated until complete recovery. Thus, as detailed above, treatment includes reducing the severity of an existing condition or inducing recovery or alleviation of the symptoms of a specific condition. In this regard, when the treatment of the present invention is applied when treating a primary tumor, it can effectively function as a preventative measure to prevent the onset of metastatic cancer. For example, for certain types of solid tumors, surgical removal of the tumor may still be the most desirable option. However, there is always a risk that the entire tumor may not be successfully removed or that some neoplastic cells may escape. In this case, by applying the method of the present invention to lyse any such neoplastic cells, the method can be effectively applied as a preventative measure to prevent metastatic spread.

[0246] According to this aspect of the invention, the target cells are preferably autologous, isolated, genetically modified ex vivo, and transplanted back into the individual from whom they were originally obtained. However, it should be understood that the present invention extends to the use of cells from any other suitable source, provided that the target cells exhibit a histocompatibility profile similar to that of the individual being treated, thereby allowing the transferred cells to perform their function of removing unwanted cells before they are subject to immune rejection by the host. Such cells are therefore effectively autologous and do not pose the histocompatibility problems typically associated with the transplantation of cells exhibiting a foreign MHC profile. Such cells should be understood as falling within the definition of histocompatible. For example, under certain circumstances, it may be desirable, necessary, or indeed important for the target cells to be isolated from a genetically identical twin or from an embryo generated using gametes derived from the target individual, or cloned from the target individual (in which case the cells may correspond to stem cells that have been directed to differentiate into the appropriate somatic cell type). The cells may also be engineered to exhibit a desired major histocompatibility profile. The use of such cells overcomes difficulties inherently encountered in tissue and organ transplantation settings.

[0247] However, when it is not possible or feasible to isolate or generate autologous or histocompatible cells, it may be necessary to utilize allogeneic cells. "Allogeneic" cells are those isolated from the same species as the subject being treated, but exhibit a different MHC profile. While the use of such cells in a therapeutic setting may result in graft-versus-host problems or graft rejection by the host, this problem may nevertheless be minimized by the use of cells exhibiting an MHC profile similar to that of the subject being treated, such as a cell population isolated / generated from a relative, such as a sibling, parent, or child, or otherwise generated by the methods exemplified herein.

[0248] It is understood that in a preferred embodiment, the cells used are autologous. However, depending on the circumstances of a given situation, it may not always be possible to generate an autologous stem cell population. This may be due to issues such as the urgency of starting treatment or the effectiveness of promoting transformation and differentiation direction. In this case, and as detailed above, it may be desirable or necessary to use syngeneic or allogeneic cells, such as cells that have already been transfected and are available as cryopreserved products in cell banks. Although allogeneic, such cells may be selected for transformation based on the expression of MHC haplotypes that are known to be highly immunogenic or otherwise less immunogenic than some haplotypes generated by the methods exemplified herein.

[0249] Reference to an "effective number" refers to the number of cells necessary to at least partially achieve a desired effect or to delay the onset, inhibit progression, or completely halt the onset or progression of the specific condition being treated. Such amounts will, of course, depend on the specific condition being treated, the severity of the condition, and individual patient parameters, including age, general condition, size, weight, physiological status, concomitant therapy, medical history, and parameters related to the disorder in question. Those skilled in the art can determine the number of cells of the present invention that constitute an effective dose and the optimal mode of administration thereof without undue experimentation; the latter issue is discussed further herein below. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred to use the maximum number of cells, i.e., the highest safe number according to sound medical judgment. However, it will be understood by those skilled in the art that a lower number of cells may be administered for medical, psychological, or any other reason.

[0250] As discussed above, the method of the present invention is based on the introduction of genetically modified cells into an individual suffering from a condition defined herein. However, it should be understood that not all cells in the population introduced into the individual need to acquire and maintain the targeted modification and differentiation. For example, if a transfected and expanded cell population is administered in its entirety (i.e., not enriched for well-modified and differentiated cells), there may be a population of cells that do not acquire or maintain the genetic modification and / or desired T cell differentiation. Thus, the present invention is achieved when a relevant portion of the cells introduced thereby constitutes an "effective number" as defined above. However, in a particularly preferred embodiment, the population of differentiated cells is subjected to the identification and selective isolation of well-modified and differentiated cells.

[0251] In the context of this aspect of the present invention, the target cells require introduction into the target individual. For this purpose, the cells may be introduced by any suitable method. For example, a cell suspension may be introduced by direct injection or within a blood clot, whereby the cells are immobilized within the clot and facilitate engraftment. The cells may also be introduced by surgical implantation. This may be necessary, for example, when the cells are present in the form of a tissue graft. The implantation site may be any suitable site, for example, subcutaneous. Without limiting the present invention to any theory or mode of action, when the cells are administered as an encapsulated cell suspension, the cells coalesce into a mass. It should be understood that the cells can continue to divide after implantation. In this regard, as previously described herein, the introduction of a suicide gene provides a convenient means for managing continued division.

[0252] The cells administered to the patient may be administered in single or multiple doses by any suitable route. Preferably, and where possible, a single dose is utilized. Administration via injection may be directed to various regions of the tissue or organ depending on the type of treatment required.

[0253] According to the method of the present invention, other proteinaceous or non-proteinaceous molecules may be co-administered with the introduction of transformed cells. By "co-administration" is meant simultaneous administration via the same or different routes in the same or different formulations, or sequential administration via the same or different routes. By "sequential" administration is meant a time lag of a few seconds, minutes, hours, or days between the transplantation of these cells and the administration of the proteinaceous or non-proteinaceous molecules. For example, depending on the nature of the condition being treated, it may be necessary to maintain the patient on drug therapy to alleviate the symptoms of the condition (e.g., administration of antiviral drugs in the case of HIV patients) until the transplanted cells are integrated and fully functional. Alternatively, once the condition is treated, it may be necessary to begin long-term drug therapy to prevent the recurrence of the condition. For example, if the damage to the subject is caused by an autoimmune condition, the continued use of low-level immunosuppressants may be necessary from the time the autoreactive cells are destroyed.

[0254] It should also be understood that the methods of the present invention may be performed alone to treat the condition in question, or may be performed in conjunction with one or more additional techniques designed to facilitate or enhance treatment of the subject. These additional techniques may be in the form of co-administration with other proteinaceous or non-proteinaceous molecules or surgery, as detailed above.

[0255] Yet another aspect of the present invention is directed to the use of stem cells or T cells differentiated therefrom, as defined above, in the manufacture of a medicament for the treatment of a condition characterized by the presence of an undesired population of cells in a mammal.

[0256] In another embodiment, the stem cells are iPSCs or HSCs.

[0257] In yet another embodiment, the stem cells are CD4 + T cells or CD8 + They have the ability to differentiate into T cells.

[0258] In yet another embodiment, the TCR is an αβ TCR.

[0259] In yet another embodiment, said stem cells, such as iPSCs, are T cells or thymocytes, preferably CD8 + It is of T cell or thymocyte origin.

[0260] In yet another embodiment, the cell further comprises a nucleic acid molecule encoding a non-signaling antigen-binding receptor, said receptor comprising an antigen recognition moiety directed against CD47.

[0261] It should be understood that references herein to "cells" refer to isolated cells or to a population of isolated or substantially purified cells. By "substantially pure," when referring to a cell population, it is meant that the relevant cell type accounts for at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of all cells in the cell population. For example, a cell population is substantially pure with respect to relevant T cells if such T cells account for at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of all cells in the cell population.

[0262] The invention will now be further described with reference to the following non-limiting examples. [Example]

[0263] The present specification is further presented by the following examples, which demonstrate the development of certain embodiments of the invention, including dual anti-cancer specific T cells derived from iPSC cells or HSCs, and should not be construed as limiting in any way.

[0264] Example 1 Enrichment of blood-borne cancer peptide antigen-specific T cells WT-1-specific TCR T cell stimulation and expansion WT-1-specific T cells are very rare in normal human blood, but they 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 and supplemented with L-glutamine and CD28 monoclonal antibody, which act as costimulators for T cells in the presence of WT-1; anti-CD28 alone does not activate T cells. PBMCs were then incubated with Wilms' tumor 1 (WT-1) peptides, four WT-1 peptides representing the major HLA class I binding motif: WT-1 37 (VLDFAPPGA, SEQ ID NO: 22), WT-1 126 (RMFPNAPYL, SEQ ID NO: 23), WT-1 187 (SLGEQQYSV, SEQ ID NO: 24) and WT-1 235 (CMTWNQMNL, SEQ ID NO: 25) overnight. Data presented in the Examples of this application use WT-1 peptide 1-37 as a representative of this family of WT-1 peptides. WT-1-specific T cells can be identified using HLA WT-1-specific tetramers or by the early induction of the surface molecule CD137 on 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. After 24–36 hours, CD137-positive cells (which represent T cells stimulated with WT-1) were magnetically separated using a magnetic cell separator. CD137-positive (WT-1-specific TCR) cells were cultured in T cell expansion medium consisting of X-Vivo-15 base medium supplemented with human AB serum, recombinant interleukin-7, interleukin-15, and interleukin-21. Corresponding CD137-negative cells were further subjected to CD3 magnetic separation. CD3-negative cells (primarily B cells) were subjected to mitomycin C treatment and used as WT-1 peptide-loaded antigen-presenting feeder cells to derive the CD137-positive population, while the remaining CD3-positive cells (non-WT-1 specific) were grown in culture to serve as a control T cell type for downstream functional assays. Medium containing recombinant cytokines was replenished every other day.

[0265] For flow cytometry analysis, cells were resuspended in FACs buffer: 10 cells 6 30 μl per sample. 10 μl of 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, protected from light. 50 μl of "T cell activation" cocktail was added, and the cells were incubated for 20 minutes at 4°C, protected from light. 100 μl of FACs buffer was added along with 2 μl of AquaAmine, and the cells were incubated for 5 minutes, then centrifuged 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 with BD / Perm wash. IFN-γ antibody was diluted 1 / 100 in BD / Perm wash solution, and the cells were incubated for 30 minutes in the dark at 4°C. Cells were washed with BD / Perm wash and resuspended in FACS buffer prior to flow cytometry analysis. FACS data acquisition was performed on a Miltenyi Quant cytometer.

[0266] T cells with TCR specific for WT-1 peptides are usually very rare (e.g., Schmeid et al., 2015), and they account for only 10% of CD8+ cells. -6 in It has been shown that (cells 3 × 10 -7 From 3 x 10 -6 Following the stimulation protocol described above, WT-1 TCR-specific T cells increased approximately 100-fold to approximately 3.0% (WT-1 patient #1, 1.5%; WT-1 patient #2, 4.0%; Figure 1).

[0267] Functional analysis of WT-1 TCR T cells In vitro expanded T cells were further stimulated with autologous antigen-presenting cells (EBV-transformed B cells) to express a broad range of WT-1 peptides:WT-1 37 (VLDFAPPGA), WT-1 126(RMFPNAPYL), WT-1 187 (SLGEQQYSV) and WT-1 235 T cells were pre-stimulated with WT-1 peptide-specific HLA-HLA (CMTWNQMNL). T cells were examined for interferon-gamma (IFNγ) production by flow cytometry using a fluorescence-based assay. Cells were double-labeled for WT-1 peptide specificity via binding to WT-1 peptide-HLA tetramers (see Figure 2).

[0268] WT-1 stimulated T cells clearly expressed interferon gamma (IFNγ) (80-90%) (Figure 2), a well-accepted measure of T cell function (e.g., Ghanekar et al., 2001). This potentially increased the level of CD8 T cell activation (WT-1). To target T cells, we used the LAG3 inhibitor IMP321. LAG3 is a "checkpoint blockade" inhibitor that inhibits the stimulatory function of dendritic cells (DCs) and the CD8+ T cell response to DCs as antigen-presenting cells. When added to a WT-1-specific T cell activation assay, IMP321 had no effect at 24 hours, but by 4 days, the number of rare CD8+ WT-1-specific TCR T cells doubled (Figure 2H).

[0269] Example 2 Generation of human blood T cell-derived iPSCs There are numerous approaches for deriving iPSCs from human blood T cells that precisely maintain the properties of the original T cells at various levels. iPSCs were generated from a broad repertoire of peripheral blood T lymphocyte pools (T-iPSCs) derived from healthy humans. T cells were preactivated, for example, with the mitogen PHA or anti-CD3 and anti-CD28 antibodies. Using a dual retroviral vector cassette containing two Yamanakari programming factors (Oct4, Sox2, KLF, cMyc), multiple T-iPSC clones were generated and validated at the cellular and molecular levels, including flow cytometry and qRT-PCR for a wide range of markers, including Nanog, Oct3 / 4, SSEA3,4, 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 the T cell source was confirmed by demonstrating rearranged TCR genes.

[0270] The generation of WT-1-specific blood T cell-derived iPSCs is summarized in FIG.

[0271] Example 3 Induction of iPSC-derived human T cells This example demonstrates the generation of iPSC-derived bona fide T cells. These T cells were shown to express key characteristics of typical T cells as normally produced by the thymus. They were shown to express the mainstream T cell αβ TCR and CD8 with both β and α chains.

[0272] T cells were derived from adult whole blood T cells or preselected CD8+ T cells, or antigen-specific T cells (e.g., specific for WT-1) (T-iPSCs), or iPSCs derived from adult fibroblasts. There are two basic stages of differentiation: hematopoietic (hematopoietic stem cells or "HSCs") and, in part, lymphoid lineages; culture in OP9 cells; and subsequent transfer of these cultured cells to an OP cell line genetically modified to express the Notch signaling molecule Delta-like ligand 1 (OP9-DL-L1) for induction of T cell differentiation.

[0273] Phase 1. Preparation of OP9 feeder cells and iPSC colonies Day -8: Mitomycin-treated mouse embryonic fibroblast feeder layers were plated onto 0.1% gelatin-coated TC plates at 0.3 × 10 cells in 3 mL of MEF medium (DMEM + 15% FCS + 1% pen / strep L-glutamine). 6 (14,250 cells / cm 2 ) and incubated overnight. OP9 cells were plated on a 0.1% gelatin-coated 10 cm TC plate at 0.25 × 10 cells in 11 mL of OP9 medium (αMEM + 20% FCS + 1% pen / strep). 6 The seeds were pre-prepared by sowing individually.

[0274] Day -7: iPS cells were thawed and plated onto MEF cells and incubated at 37°C, 5% CO2 for 7 days.

[0275] Phase 2 iPSC conversion to hematopoietic cells Day 0: Initiation of hematopoietic differentiation. iPS colonies were dissociated and plated onto OP9 for HSC differentiation. The colony suspension was added dropwise for even distribution onto OP9 plates. Fresh differentiation medium was added on days 1, 5, and 9.

[0276] Day 13: Harvesting of induced HSC precursors for T cell differentiation

[0277] Cells cultured on the OP9 cell line were gently removed with collagenase (working solution 100 μg / mL collagenase / HBSS; 37°C, 1:15 h), and colonies were further broken down into single cells with trypsin / EDTA 0.05% at 37°C for 30 min. The cells were gently washed and examined by phase contrast microscopy (Figure 4) and flow cytometry (Figure 5). The hematopoietic nature of the cells was confirmed by flow cytometry (Figure 5).

[0278] Phase 3 - Induction of iPSC-derived HSCs into T cells Day 13: Induction of T cell differentiation: Transfer of OP9-conditioned (hematopoietic-induced) cells to OP9DL-L1 cells on day 13

[0279] In a preferred embodiment, OP9-conditioned cells are transfected with CD34 to enhance the efficiency of contact with OP9DL-L1 cells. + CD43 + The cells were purified for T cell differentiation (HSC) and then plated onto OP9 DLL-1 cells for the first step of T cell differentiation. A critical component of the process disclosed herein was the harvesting of cells initially grown under OP9 DL-L1 cells.

[0280] Cells recovered from OP9 cultures were resuspended in T cell differentiation culture medium (OP9 medium, SCF 5 ng / mL, Flt3 5 ng / mL, IL-7 5 ng / mL, and vitamin C 100 μM), and the suspension was added dropwise to OP9 DLL-1 cells and incubated at 37°C. Cells were harvested after 2, 9, 16, 23, and 30 days of culture in OP9 DL-L1 and subjected to flow cytometry analysis (Figures 6 and 7).

[0281] When these cultures were examined for T cell development, there was clear evidence of expression of the early markers of T cell development, CD7 and CD9, along with CD4 and CD8 expression (Figure 7). Even at this early stage, there were already approximately 10% of cells expressing both CD4 and CD8; these CD4CD8 cells are characteristic of T cells that develop normally in the thymic cortex (Heng et al., 2010).

[0282] Flow cytometry demonstrated progressive T cell development from initial CD5+, CD7+, and then CD8+ expression. Most importantly, the induced T cells expressed the "optimally thymogenic" CD8 T cell phenotype. They expressed the CD8β chain in addition to the CD8α chain (other reported T cell induction systems do not induce the optimally signaling CD8β chain; e.g., Themeli et al., (2013)). As a functional indicator, they also expressed CD3 with the αβTCR. Furthermore, these cells were present as early as day 16 of culture on OP9 DL-L1 cells compared to day 30 in other reported systems.

[0283] Phase 4 Mature T cell development

[0284] After an additional 7 days (a total of 13 days on OP9 cells followed by 16 days on OP DL-L1 cells), these developing T cells underwent a definitive transition to express the T cell receptor complex, including CD8+ T cells clearly positive for CD3 and αβ TCR; additionally, these cells expressed significant CD8β—the desired cells for CAR-T. There was a corresponding further reduction in CD34+ CD43+ HSCs (Figure 8).

[0285] Thus, this induction system successfully produced mature CD8 T cells from iPSCs after 13 days of culture on OP9 cells followed by 16 days of culture on OP9DL-L1 cells.

[0286] Using the process described above, T cells expressing WT-1-specific TCRs were generated from iPSCs that were themselves derived from WT-1 TCR CD8+ T cells (Fig. 9). These iPSC-derived WT-1 T cells had cytotoxic function comparable to that of the original T cells from which the iPSCs were derived (Fig. 10).

[0287] Example 4 Development of CAR constructs The components of chimeric antigen receptor (CAR)-T cells are the antigen recognition component of the CAR mediated by the single-chain Fv (scFv) ectodomain anchored by a CD8 or CD28 hinge and containing a transmembrane (TM) region, and the signal transduction component of the CAR via the cytoplasmic endodomain represented by the CD28, 4-1BB, and CD3 zeta (CD3ζ) chains. There are also two suitable viral delivery systems: retrovirus and lentivirus. An exemplary CAR and CD47-binding receptor construct is shown in Figure 11.

[0288] Example 5 Chimeric antigen receptor vector cloning strategy Exemplary chimeric antigen receptor vector cloning strategies are illustrated in Figures 12-13. Figure 14 shows our strategies for second-generation CAR and non-signaling anti-CD47 constructs. Exemplary sequences of chimeric antigen receptors, non-signaling antigen-binding receptors, and their various domains are provided in SEQ ID NOs: 1-20.

[0289] Example 6 Chimeric antigen receptor transduction of T cells Lentivirus production 293T cells were cultured on a 175cm thick poly-L-lysine (Sigma)-coated plate. 2 The cells were plated in a flask. Two hours before 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. Following this, the solution was mixed again and then added dropwise to the cells. The flask was returned to the incubator. After 6 hours, fresh growth medium was added. After 48 hours, the viral supernatant was collected and clarified by centrifugation at 1500 rpm for 5 minutes at 4°C, then passed through a 0.45 μm pore PVDF Millex-HV filter (Millipore). Lentivirus concentration using ultracentrifugation was performed using a Sorval Discovery 100 SE centrifuge with an AH-629 rotor. 30 mL of the filtered viral supernatant was added to a 36 mL polyallomer conical tube (Beckman). Centrifugation was carried out for 90 minutes at 20,000 g, the supernatant was completely removed, and the virus pellet was resuspended in 300 μL PBS and stored at −80°C until use.

[0290] Generation of CAR-T cells Figure 11 and SEQ ID NOS: 1-6 show a panel of chimeric antigen receptor (CAR) and CD47-binding receptor constructs developed, along with scFvs specific for either TAG72 or CD19 (as a positive control). These constructs use either human CD8 or CD28 as the hinge region and the CD28, CD3 ζ chain, or 4-1BB cytoplasmic activation signaling domain. The CAR and CD47-binding receptor constructs are cloned into lentiviral vectors as described in the previous section.

[0291] Optimal lentiviral transduction of T cells involves their activation with the TCR and costimulatory receptors. Therefore, on day 0, fresh PBMCs were collected by apheresis from healthy donors and enriched for activated T cells using anti-CD3 and anti-CD28 antibodies coupled to paramagnetic beads (Dynabeads ClinExVivo CD3 / CD28, Invitrogen, Camarillo, CA, USA) at a ratio of 3:1 (beads:cells). Cells and beads were co-incubated for 1 h at room temperature, and CD3+ cell enrichment was performed using a magnet (Invitrogen). Cells in the CD3+ fraction were added to starting medium at a concentration of 1 × 10 cells. 6 The cells were resuspended in T cell expansion medium containing 100 IU / ml IL-2 at a concentration of 100 cells / ml. On day 1, RetroNectin was used to coat cell culture dishes at a concentration of 2 mg / cm² in a 10 mg / mL solution in PBS overnight at 4°C. On day 2, the RetroNectin solution was aspirated, and an equal volume of blocking solution consisting of 0.5% human serum albumin in PBS was added to each bag and incubated at room temperature for 30 minutes. The blocking solution was aspirated, and each bag was washed with PBS. The lentiviral supernatant was rapidly thawed and added to each dish containing T cell expansion medium containing 300 IU / ml IL-2. The cultures were returned to the incubator and left undisturbed for at least 24 hours. On day 4, transduction was stopped; cells were resuspended in fresh T cell expansion medium at a concentration of 0.5–1 × 10 cells. 6 The culture was maintained for up to 14 days, with the cell concentration being 1 x 10 cells / mL. 6 Fresh expansion medium was given every other day to maintain the cell density at 100 cells / mL.

[0292] First, blood-derived human T cells were subjected to CAR transduction, and the success was measured by flow cytometry, which revealed eGFP+ cells (Figure 15), which was also confirmed by Western blot analysis (Figure 16).

[0293] Evaluation of CAR-T cell function TAG72 CAR-T cells (generated from normal PBMCs) were examined for their ability to kill TAG72-expressing target cancer cells in vitro. A real-time cell monitoring system (xCELLigence) was used to determine the killing efficiency of CAR-T cells in vitro. Target cells (e.g., TAG72+ ovarian cancer cell line CaOV4) were 10,000–2 × 10 6 100 μL of cells / ml were placed into RTCA plates. In some cases, tethering of target cells with anti-hCD40 or pre-coating of the plate with human fibronectin may be necessary. Target cells were maintained at 37°C and 5% CO2 for 3-12 hours to allow cell attachment. Following target cell attachment, CAR-T effector cells were added at various effector:target ratios (ranging from 1:1 to 10:1). In some experiments, CAR-T effector cells were isolated via FACS prior to use based on GFP expression on CAR-T cells. Co-cultures were maintained for at least 12 hours under optimal growth conditions. Cell impedance was monitored throughout; a decrease in impedance is indicative of cell detachment and ultimately cell death.

[0294] Figure 17 shows the results from this experiment, monitored over a 40-hour period. The ovarian cancer cell line, CaOV4, grew consistently during this period (blue line). In contrast, cultures supplemented with TAG72-specific CAR T cells showed an initial growth phase that was significantly less than that of target cells alone, followed by a gradual elimination of target cells over time (purple line). To exclude nonspecific killing due to CD3 / CD28 activation, TAG72 CAR-T cells were isolated by flow cytometry and compared to CD19 CAR-T cells and non-CAR-T cells without preceding CD3 / CD28 activation (Figure 21). The data shown in Figure 21 demonstrate the strong antigen specificity of TAG72 CAR-T cells during the first 24 hours of culture with TAG72-expressing cancer cells, as T cells transfected with vector alone and negative controls of non-transfected T cells showed no cancer cell killing during this period.

[0295] The above studies were performed with peripheral blood-derived polyclonal T cells. To demonstrate CAR transduction of monospecific T cells expressing TCRs specific for nominal cancer peptide antigens, iPSC-derived WT-1 TCR-specific T cells generated from WT-1-specific TCRs were transduced with TAG72 CAR lentivirus. Figure 22A shows successful CAR transduction of these WT-1-specific TCR CD8+ T cells derived from iPSCs generated from WT-1-specific T cells. The CAR had specificity for TAG72. Most importantly, Figure 22B shows successful transduction of iPSC-derived WT-1-specific TCR CD8+ T cells generated from WT-1-specific T cells with both TAG72 and CD47 CAR constructs. This demonstrates that T cells can be generated with three specificities for cancer: WT-1 (TCR), TAG72 (CAR), and CD47 (truncated, CD47-binding receptor).

[0296] The results demonstrated the development of an iPSC-derived bispecific CAR transduced cancer-specific TCR(WT-1), which was itself derived from WT-1-specific TCR T cells from healthy adult blood.

[0297] Figure 20 shows that both components of bispecific T cells (containing WT-1 TCR and TAG72 CAR) can contribute to cancer cell killing. When corrected for natural cell death, even at a low effector-target ratio (here, 2 effectors to 1 target cell), WT-1 cells resulted in approximately 10% cell killing, and then the addition of TAG72 CAR by transduction resulted in an additional 10% killing.

[0298] Example 7 Chimeric antigen receptor transduction of iPSCs The production of multispecific CAR-T cells can be achieved by several approaches, including CAR transduction of existing blood T cells (Figure 15) or transduction of iPSCs (expressing a cancer-specific TCR and CAR) (e.g., SEQ ID NOS: 1-6) that are then induced into T cells. Multiple iPSC lines have been used to advance CAR-T transduction. These iPSCs can be derived from non-T cells or from cancer antigen-specific T cells carrying TCR gene rearrangements (e.g., WT-1). These iPSCs were either derived from adult fibroblasts or from T cells with an endogenous TCR specific for a specific cancer antigen (WT-1 peptide).

[0299] iPSCs were stably transduced with a single cistron in which the CAR ectodomain contained a scFv specific for TAG72 (or CD19 as a control), as shown in Figure 14. The hinge (stalk) and transmembrane regions were derived from CD28 or CD8, and the cytoplasmic endodomain containing the T cell signaling domain was derived from CD28 and the TCR ζ chain. The CAR has a C-terminal extension encoding EGFP linked by a P2A self-cleaving polypeptide to separate the CAR and the reporter. After viral integration, P2A was cleaved, and successful transduction was quantified by measuring the fluorescence of the released EGFP reporter. GFP fluorescence indicates successful transduction. It can be used to demonstrate in situ transduction (Figure 21) or to identify and isolate CAR-transduced iPSCs via flow cytometry (Figures 22 and 23).

[0300] These studies clearly demonstrated the ability to transduce iPSCs using lentiviral CAR constructs. Figure 21A shows the successful transduction of human fibroblast-derived iPSCs with CARs encoding TAG72 or CD19 (Figure 21A). Figure 21B shows the successful transduction of WT-1 TCR-specific T cell-derived iPSCs with TAG72. Thus, any T cells derived from this cell line express dual anti-cancer specificity (WT-1 via TCR; TAG72 via CAR).

[0301] It is also possible to isolate transduced iPSCs by fluorescence-based cell sorting. Positive cells can be collected and plated to successfully form (CAR-transduced) iPSC colonies (Figure 24).

[0302] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications. The invention also includes all steps, properties, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations of any two or more of said steps or properties. Bibliography Balasubramanian S, Babai N, Chaudhuri A, Qiu F, Bhattacharya S, Dave BJ, 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 oxogenous transcription factors. Stem Cells.; 27:3053-3062 [PubMed: 19859985] Brignone, C., C. Grygar, M. Marcu, K. Schakel, and F. Triebel. 2007. 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Claims

1. A genetically modified mammalian stem cell, the cell having the ability to differentiate into a T cell expressing a T cell receptor (TCR) directed to a first antigenic determinant, the cell comprising nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen recognition moiety directed to a second antigenic determinant, the antigen recognition moiety being operably linked to a T cell activation moiety by a hinge region and a transmembrane domain; (1) The antigen recognition portion 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; (4) A genetically modified mammalian stem cell, wherein the T cell activation portion comprises (a) a 4-1BB signaling domain or a CD28 signaling domain, and (b) a TCR zeta signaling domain.

2. The stem cell of 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. A stem cell described in claim 1 or 2, wherein the cell expresses at least one homozygous HLA haplotype.

4. A stem cell described in any one of claims 1 to 3, wherein the cell is selected from an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), or a lymphocyte progenitor cell.

5. A stem cell described in any one of claims 1 to 4, wherein the hinge region contains a cysteine ​​that promotes dimerization of the CAR.

6. A stem cell described in any one of claims 1 to 5, further comprising a nucleic acid encoding a non-signaling antigen-binding receptor comprising an antigen recognition portion directed against a third antigenic determinant.

7. The stem cell described in claim 6, wherein the second antigenic determinant and the third antigenic determinant are different from each other.

8. A stem cell described in claim 6 or 7, wherein the non-signaling antigen-binding receptor comprises an antigen recognition portion directed against CD47.

9. A stem cell described in any one of claims 6 to 8, wherein the third antigen recognition portion is operably linked to the transmembrane domain by a hinge region.

10. The stem cell described in claim 9, wherein the non-signaling antigen-binding receptor has either a removed or substituted cysteine ​​residue in the hinge region, preventing dimer formation.

11. A T cell derived from the stem cell according to any one of claims 1 to 10.

12. A method for producing genetically modified mammalian stem cells, comprising:

3. The method of claim 1, wherein the mammalian stem cells have the ability to differentiate into T cells expressing a TCR directed against a first antigenic determinant.

13. A method for producing genetically modified mammalian stem cells, said method comprising: (i) introducing into T cells or thymocytes one or more nucleic acids 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, the antigen recognition moiety being operably linked to a T cell activation moiety, at least one of the nucleic acids being a nucleic acid encoding the chimeric antigen receptor of claim 1 or 2, wherein the T cells or thymocytes express a TCR directed to the first antigenic determinant; (ii) deriving stem cells from said T cells or thymocytes.

14. The method of claim 13, wherein step (i) further comprises introducing into said T cells or thymocytes one or more nucleic acids encoding at least one non-signaling antigen-binding receptor comprising an antigen recognition portion directed against an additional antigenic determinant.

15. A method for producing T cells, comprising the steps of creating genetically modified stem cells according to the method of claim 12 or 13, and differentiating the genetically modified stem cells into T cells.

16. A composition for use in treating a condition in a mammal, the composition comprising a cell according to any one of claims 1 to 11.

17. The composition of claim 16, wherein the condition is a neoplastic condition.

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