Genetically modified cells and their use

Abstract

Genetically modified cells and uses thereof are provided. The present invention generally relates to a population of stem cells (e.g., iPSCs or HSCs) comprising nucleic acids 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.

Description

Technical Field

[0001] This application claims the benefit of priority from Australian Provisional Patent Application No. 2015904933, filed on November 27, 2015, and Australian Provisional Patent Application No. 2016901328, filed on 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 nucleic acids encoding a T cell receptor and a chimeric antigen receptor directed to a plurality of distinct antigenic determinants, such as two distinct tumor antigenic determinants. The present invention also targets a population of T cells that co-express a T cell receptor and a chimeric antigen receptor directed to a plurality of distinct antigenic determinants, such as two distinct tumor antigenic determinants. The cells of the present invention can be derived from a selected donor whose HLA type is compatible with a significant sector of the population and are useful in a variety of applications, particularly in connection with the therapeutic treatment of neoplastic conditions.

Background Art

[0003] Background of the Invention Details of the publications referred to herein by the authors are compiled alphabetically at the end of the description.

[0004] No reference in this specification to any prior publication (or information derived therefrom), or to any matter known, is to be taken as an admission or acknowledgment or any kind of suggestion that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates, nor should it be so regarded.

[0005] Malignant tumors, or cancers, grow in an uncontrolled manner, invade normal tissues, often metastasize, and grow in sites far from their place of origin. Generally, cancer originates from one or a few normal cells that have undergone a process called malignant transformation, which is not well understood. Cancer can arise from almost any tissue in the body. Cancers that originate 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, and malignant tumors of blood origin, including bone marrow, lymphocytes, and other hematopoietic cells, are called leukemias.

[0006] Cancer is one of the three leading causes of death in developed countries. As treatment of infectious diseases and prevention of cardiovascular diseases continue to improve in these countries, and average life expectancy increases, cancer is likely to become the most common fatal disease. Therefore, successfully treating cancer requires eliminating or destroying all malignant cells without causing death to the patient. 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. Once solid tumors have spread or "metastasized" throughout the body, they are usually incurable. The prognosis for metastatic solid tumors has improved only slightly in the last 50 years. The best chance of curing solid tumors is through early detection, followed by the use of localized treatments such as surgery and / or radiation therapy if the solid tumor is localized and has not spread to the lymph nodes draining the tumor or anywhere else. Nevertheless, even at this early stage, especially if the tumor has spread to the lymph nodes draining the tumor, microscopic deposits of cancer, known as micrometastases, may have already spread throughout the body, subsequently leading to the patient's death. In this sense, cancer is a systemic disease requiring systemically administered treatment.

[0008] The "Golden Bullet" approach, which attempts to attack cancer with toxin-laden antibodies, leverages the ability of antibodies to potentially target any specific molecular entity, such as carbohydrates, lipids, proteins, or combinations thereof. Once bound to cancer cells, antibodies can engage complement or FcR+NK / K cells, inducing cell lysis. Unfortunately, due to low affinity binding, low lysis efficiency, and short lifespan, antibody treatments for cancer generally achieve only moderate success. In summary, these undermine the ability of antibodies to rapidly destroy cancer cells and increase the risk of mutation and immune evasion. More recently, there have been reports of antibody-based therapies, including those based on antibodies highly targeted to cancer molecules and immune checkpoint blocker molecules. While the latter has seen some clinical success, such therapies still come with various limitations.

[0009] Therefore, the common approach to cancer treatment continues to follow the long-used protocol of surgical removal (if possible), followed by radiotherapy and / or chemotherapy as needed. The success rate of this somewhat incomplete treatment is highly variable, but generally decreases significantly as the tumor becomes more advanced and metastatic. Furthermore, these treatments are accompanied by severe side effects, including surgical scars and hemorrhages (e.g., mastectomy or limb amputation), severe nausea and vomiting from chemotherapy, and most notably, damage to normal tissues such as hair follicles, intestines, and bone marrow, induced as a result of the relatively nonspecific targeting mechanisms of toxic drugs that form part of most cancer treatments.

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

[0011] The mainstream T cell thymus formation is fundamentally necessary for defense against infection. This pool of "immune surveillance" T cells circulates throughout the body, eliminating damaged or abnormal cells, including cancer cells. Since thymus-based T cell production is characterized by the random generation of a T cell receptor (TCR) repertoire, thymic proliferation must also involve a very rigorous selection process that eliminates or functionally neutralizes developing thymic T cells that have the potential to attack self. Thus, this "self-tolerance" limits autoimmune diseases (Fletcher et al., 2011). However, if non-virus-induced cancers are defined as "self" diseases, then inevitably, this process impairs immune surveillance against cancer. This means that many of the T cells generated in the thymus, which may potentially be reactive with tumor-associated antigens, can be eliminated before they enter the bloodstream. At the very least, they will be numerically insufficient and likely have low-affinity TCRs. Despite this, T cells are clearly a potentially primary weapon against cancer, and therefore the challenge is to increase their ability to detect cancer, to increase their numbers, and to maintain, improve, and enhance their potent cytolytic capacity. While antibodies and T cells are the most logical weapons against cancer, their potentially rapid and effective cancer eradication has not been clinically recognized. Advances in immunotherapy have progressed with genetically engineered T cells to express novel chimeric membrane receptors consisting of cancer antigen-binding antibody fragments cytoplasmically coupled to T cell signaling molecules. Generally, the latter are one or all of the TCRζ 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-Ts) not only leverage the immune system's two most powerful anti-cancer weapons but also overcome their individual shortcomings. CAR-Ts possess potent and localized cytolytic ability, preventing normal trust in instrinsic TCRs and detecting extremely rare "cancer peptides" expressed in HLA voids.The repertoire of T cells specific to such a small number of peptides is extremely rare. The antibody portion of CARs confers cancer-detection specificity to T cells, overcoming the well-known low cancer-destroying efficacy of blood antibodies. Thus, cancer binding is mediated by the antibody domain of CARs, leading to cytoplasmic signaling and inducing the T cell lysis pathway to destroy cancer.

[0012] Numerous CAR-T clinical trials are currently underway, though they are still in the early stages of clinical practice. As much as the potential benefits, several aspects of CAR-T technology present problems that prevent their clinical efficacy from being fully recognized. Most notably, a tumor-cell-dependent surge in cytokines occurs during T-cell-mediated cancer destruction. While fever is an indicator of cancer destruction, it can lead to severe clinical side effects if not carefully managed (Davila et al. (2014); Casucci). (et al., 2015). Current management involves cytokine modulation treatments such as anti-IL6. Furthermore, there is a significant problem related to a numerical shortage of CAR-T cells, which are not only necessary for attacking early-stage cancer but also for maintaining a sufficient supply in cases of recurrence. Currently, attempts to address this problem are based on the overuse of proliferation-inducing cytokines in vitro. Moreover, CD19 is as effective as CAR-T cells in attacking cancer. + Even against cancer, tumor eradication is not 100% effective. A response rate of up to 90% has been reported for B-ALL, but for other CD19 + The results in cancer are not very conclusive. Therefore, despite promising observations regarding the practicality of CAR-T, there are still significant problems that this technology must overcome before it can become a reliable and effective new optimal standard for cancer treatment. In work prior to the present invention, it was particularly determined that if CAR-T cells can be derived from stem cells such as transfected adult stem cells or other transfected somatic cell types, rather than transfected thymocytes, then seemingly different problems currently existing regarding the effective therapeutic application of CAR-T technology can be solved. 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 and future supply of CAR-T cells targeted to specific tumors can be solved for the continuous supply of somatic T cells derived from these autogenerating transfected stem cells. Furthermore, these iPSCs, and therefore CAR-T cells, derived from transfected stem cells can be pre-selected from donors that are homozygous for homozygous HLA haplotypes, particularly HLA types that are widely expressed in the population, thereby providing a means to generate a cell bank exhibiting broad donor compatibility. Furthermore, it was determined that the generation of iPSCs from T cells exhibiting T cell receptor specificity directed to the target antigen means that gene rearrangements for their TCRs, which are specific to the cancer antigen, are incorporated into the iPSCs. All T cells derived from these iPSCs will retain anti-cancer TCR specificity. Subsequently, such iPSCs can be transfected with CAR, and then differentiated into T cells such as CD4+ or CD8+ T cells, and these iPSCs will stably exhibit bispecificity to the TCR directed to the antigen directed by the CAR and the antigen directed to the original T cell. The present invention is not limited to any one theory or mechanism of action, but this is thought to be due to epigenetic memory. Furthermore, it was determined that bispecific NKT cells can be generated in the same way. Therefore, it is possible to provide a continuous supply of T and NKT cells selectively and stably directed to multiple distinct antigenic determinants, such as multiple distinct tumor antigen determinants, thereby enabling more therapeutically effective treatment steps. [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 otherwise required by context, throughout this specification and claims, the word “comprise,” and variations such as “comprises,” and “comprising,” will be understood to mean that they include any group of integers (singular) or steps (singular) or integers (plural) or steps (plural) that are described, but do not exclude any other group of integers (singular) or steps (singular) or integers (plural) or steps (plural).

[0015] In this specification, the term “derived from” should be understood to indicate that a particular integer or group of integers originates from a specified species, but is not necessarily directly derived from a specified source. Furthermore, in this specification, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context otherwise explicitly indicates otherwise.

[0016] The subject specification contains amino acid sequence information prepared using the program PatentIn Version 3.5, which is presented after the references in this specification. Each amino acid sequence is represented numerically. <210> Subsequent array identifiers (for example, <210> 1. <210> 2) Identified in the sequence listing by (etc.). The length, sequence type (protein, etc.), and source organism for each amino acid sequence are indicated in the numerical column. <211> , <212> and <213> The information provided in each section indicates the amino acid sequence mentioned in the section, which is identified by the sequence number followed by the sequence identifier (e.g., SEQ ID NO: 1, SEQ ID NO: 2, etc.). The sequence identifier mentioned in the specification is identified by the number in the sequence listing that follows the sequence identifier. <400> Information provided to (for example, <400> 1. <400> (2nd class). That is, Sequence ID No. 1, detailed in the specification, is in the sequence listing. <400> It is associated with the sequence indicated as 1.

[0017] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention belongs.

[0018] One aspect of the present invention relates to genetically modified mammalian stem cells, or T cells differentiated therefrom, wherein the cells have the ability 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-recognizing portion directed to a second antigenic determinant, the antigen-recognizing portion being operably linked to a T cell activation portion. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0019] In another embodiment, genetically modified mammalian stem cells, or T cells differentiated therefrom, wherein the cells express a CD4 that is directed to a first antigenic determinant. +A cell is provided that has the ability to differentiate into a T cell and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen-recognizing portion directed to a second antigenic determinant, and the antigen-recognizing portion is operably linked to a T cell activating portion. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0020] In yet another embodiment, genetically modified mammalian stem cells, or T cells differentiated therefrom, wherein the cells express a CD8 that is directed to a first antigenic determinant. + A cell is provided that has the ability to differentiate into a T cell and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen-recognizing portion directed to a second antigenic determinant, and the antigen-recognizing portion is operably linked to a T cell activating portion. In some embodiments, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0021] In a further embodiment, there are provided genetically modified mammalian stem cells, or T cells differentiated therefrom, wherein the cells are iPSCs (induced pluripotent stem cells) or HSCs (hematopoietic stem cells), and have the ability 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, wherein the receptor comprises an antigen-recognizing portion directed to a second antigenic determinant, and the antigen-recognizing portion is operably linked to a T cell activation portion. In some embodiments, the genetically modified stem cells, such as iPSCs or HSCs, express at least one homozygous HLA haplotype.

[0022] According to one embodiment of the present invention, stem cells (e.g., iPSCs) are derived from cells in which the TCR gene has been rearranged.

[0023] In another embodiment, the stem cells (e.g., iPSCs) are derived from T cells or thymocytes expressing αβTCR.

[0024] In yet another embodiment, the stem cells (e.g., iPSCs) are derived from T cells or thymocytes expressing γδTCR.

[0025] In yet another embodiment, the stem cells (e.g., iPSCs) are derived from T cells or thymocytes that express the first antigenic determinant, i.e., a TCR that is directed to the same antigenic determinant to which the TCR expressed in T cells derived from the stem cells (e.g., iPSCs) is directed.

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

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

[0028] In one embodiment, stem cells (e.g., iPSCs or HSCs) express a CD4 that is directed to a first antigenic determinant. + It has the ability to differentiate into T cells. In another embodiment, stem cells (e.g., iPSCs or HSCs) express a CD8 that expresses a TCR directed to a first antigenic determinant. + It has the ability to differentiate into T cells.

[0029] In yet another further embodiment, there are provided genetically modified mammalian stem cells, or T cells differentiated therefrom, the cells having the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and comprising a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprising an antigen-recognizing portion directed to a second antigenic determinant, the antigen-recognizing portion being operably linked to a T cell-activating portion, and the antigenic determinant being selected from tumor antigens, microbial antigens, or autoreactive immune cell antigens. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

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

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

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

[0033] In still yet another embodiment, the stem cells (e.g., iPSCs) are T cells or thymocytes, preferably CD8 + T cells or thymocytes are derived from. In some embodiments, the stem cells (e.g., iPSCs) are the first antigen determinant, i.e., the TCR expressed in T cells derived from the stem cells (e.g., iPSCs) is directed to the same antigen determinant as the TCR expressed in CD8 + T cells or thymocytes are derived from.

[0034] In yet another aspect, a genetically modified mammalian stem cell, or a T cell differentiated therefrom, wherein the cell has the ability to differentiate into a T cell expressing a TCR directed to a first tumor antigen determinant, and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen recognition portion directed to a second tumor antigen determinant, and the antigen recognition portion is operably linked to a T cell activation portion, wherein the first antigen determinant is selected from peptides recognized by TCRs such as WT-1 or EbvLMP2, and the second antigen determinant is selected from, for example, TAG-72, CD19, MAGE, or CD47, and a cell is provided. 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 include a nucleic acid molecule encoding a chimeric antigen receptor that includes an antigen-recognition moiety (e.g., a second tumor antigenic determinant) directed to a second antigenic determinant and operably linked to a T cell activation moiety. 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] Therefore, in a further embodiment, genetically modified mammalian stem cells are provided that have the ability to differentiate into T cells directed to more than two antigenic determinants.

[0037] In some embodiments of the present invention, genetically modified mammalian stem cells (e.g., iPSCs or HSCs) have the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and comprise a plurality of (i.e., two or more) nucleic acid molecules encoding a plurality of chimeric antigen receptors, each chimeric antigen receptor comprising an antigen-recognizing portion directed to an antigenic determinant, the antigen-recognizing portion being operably linked to a T cell activation portion. 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 to the multiple chimeric antigen receptors are each distinct from the first antigenic determinant directed to the TCR expressed in the T cells derived from the stem cells. In another embodiment, the multiple antigenic determinants directed to the multiple chimeric antigen receptors are each distinct, and the first antigenic determinant directed to the TCR expressed in the T cells derived from the stem cells is also distinct.

[0039] In one embodiment, multiple nucleic acids encoding CARs are contained within a single adjacent nucleic acid fragment. For example, multiple nucleic acids encoding CARs are placed in a single construct or vector to be transfected into cells to generate genetically modified mammalian stem cells containing multiple nucleic acids encoding CARs. In certain embodiments, multiple nucleic acids encoding CARs can be linked together within a single expression unit and reading frame (e.g., by utilizing self-cleaving peptides 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 nucleic acids encoding CARs are placed in separate vectors used for transfection to generate genetically modified mammalian stem cells containing multiple nucleic acids encoding CARs. Examples of nucleic acid constructs encoding CARs are illustrated in Figure 11, and typical sequences for CARs and various domains suitable for use in CARs are provided in Sequence IDs 1 to 2 and 7 to 20.

[0040] Furthermore, according to aspects of the present invention, genetically modified mammalian stem cells are provided having the ability to differentiate into T cells directed to more than two antigenic determinants. In other embodiments, the genetically modified mammalian stem cells (e.g., iPSCs or HSCs) (optionally expressing at least one homozygous HLA haplotype) have the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and further comprise a nucleic acid molecule encoding a chimeric antigen receptor having an antigen recognition moiety operably linked to an antigen-recognizing moiety directed to a second antigenic determinant, and further comprise a nucleic acid molecule encoding an antigen-binding receptor having an antigen-recognizing moiety directed to a third antigenic determinant. According to these embodiments, such genetically modified stem cells have the ability to differentiate into T cells directed to 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 immobilized on the cell surface and binds to a third antigenic determinant but does not translate the signal to the cytoplasm of the cell. In one embodiment, the antigen-binding receptor is directed to a third antigenic determinant and operably linked to a transmembrane domain, but includes an antigen-recognition moiety that lacks a T cell activation moiety.

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

[0043] Accordingly, there are provided genetically modified mammalian stem cells (e.g., iPSCs or HSCs) or T cells differentiated therefrom, the cells having the ability 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, wherein the receptor includes an antigen-recognizing portion directed to a second antigenic determinant, the antigen-recognizing portion being operably linked to a T cell activation portion, and (ii) a non-signaling CD47-binding molecule, for example, a nucleic acid molecule encoding a shortened 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, a method for producing genetically modified mammalian stem cells (such as iPSCs or HSCs) as disclosed herein is provided.

[0045] In one embodiment, the method comprises the steps of obtaining mammalian stem cells (such as iPSCs or HSCs) having the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant, wherein in one embodiment, the stem cells (e.g., iPSCs or HSCs) express at least one homozygous HLA haplotype; and introducing into the stem cells (e.g., by transfection) one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen-recognizing moiety directed to an antigenic determinant, the antigen-recognizing moiety operably linked to a T cell activation moiety. In another embodiment, the method further comprises the step of introducing into the stem cells (e.g., by transfection) 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-recognizing moiety directed to an antigenic determinant. Further disclosed herein, the plurality of nucleic acids encoding receptors may be introduced by a single vector or by separate vectors.

[0046] In another embodiment, the method comprises the steps of: obtaining T cells or thymocytes (preferably CD8+ T cells or thymocytes) expressing a TCR directed to a first antigenic determinant, wherein 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, wherein each chimeric antigen receptor includes an antigen-recognizing portion directed to an antigenic determinant, the antigen-recognizing portion of which is operably linked to a T cell activation portion; and obtaining stem cells (e.g., iPSCs) from the T cells or thymocytes. In another embodiment, the method further comprises, 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), wherein each antigen-binding receptor includes an antigen-recognizing portion directed to an antigenic determinant.

[0047] In yet another embodiment, the method includes, in some embodiments, the steps of: obtaining HSCs (e.g., from bone marrow or blood) expressing at least one homozygous HLA haplotype; introducing into the HSCs (i) one or more nucleic acids encoding a TCR directed to a first antigenic determinant; (ii) one or more nucleic acid molecules encoding one or more chimeric antigen receptors, wherein each chimeric antigen receptor comprises an antigen-recognizing moiety directed to an antigenic determinant different from the first antigenic determinant, the antigen-recognizing moiety being operably linked to a T cell activation moiety; and optionally (iii) one or more nucleic acid molecules encoding one or more antigen-binding receptors (e.g., non-signaling antigen-binding receptors), wherein each antigen-binding receptor comprises an antigen-recognizing moiety directed to an antigenic determinant different from the first antigenic determinant and also different from the antigenic determinant to which the chimeric antigen receptor is directed. As disclosed herein, the multiple nucleic acids encoding receptors may be introduced by a single vector or separate vectors. Using such genetically modified HSCs, T cells specific to multiple antigenic determinants can be generated.

[0048] In a further embodiment, cells are provided that express a TCR directed to a first antigenic determinant and express one or more chimeric antigen receptors, wherein each receptor includes an antigen-recognizing moiety directed to an antigenic determinant, and the antigen-recognizing moiety is operably linked to a T cell activation moiety. In some embodiments, the T cells further express antigen-binding receptors including an antigen-recognizing moiety directed to an antigenic determinant. In some embodiments, the T cells provided express at least one homozygous HLA haplotype.

[0049] In another embodiment, a method is provided for producing T cells expressing a TCR directed to a first antigenic determinant and expressing one or more CARs, wherein each CAR includes an antigen-recognizing moiety directed to an antigenic determinant, the antigen-recognizing moiety being operably linked to a T cell activation moiety, and optionally expressing one or more non-signaling antigen-binding receptors, each each including an antigen-recognizing moiety directed to an antigenic determinant. In some embodiments, the method provided herein is intended to produce T cells expressing at least one homozygous HLA haplotype.

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

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

[0052] According to this embodiment, a method is provided for treating a neoplasm, comprising the steps of administering an effective number of stem cells or T cells to a mammal as defined above, wherein the TCR is directed to a first tumor antigen determinant and the CAR is directed to a second tumor antigen determinant.

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

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

[0055] Yet another aspect of the present invention relates to the use of stem cells or T cells in the manufacture of pharmaceuticals for treating conditions characterized by the presence of an unwanted population of cells in mammals, as defined above. In certain embodiments, for example, the following are provided: (Item 1) A genetically modified mammalian stem cell, wherein the cell has the ability to differentiate into a T cell expressing a T cell receptor (TCR) directed to a first antigenic determinant, and comprises a nucleic acid molecule encoding a chimeric antigen receptor including an antigen-recognizing portion and a T cell-activating portion, wherein the antigen-recognizing portion is directed to a second antigenic determinant and operably linked to the T cell-activating portion. (Item 2) Genetically modified stem cells that express at least one homozygous HLA haplotype. (Item 3) Cells as described in item 1 or 2, which are induced pluripotent stem cells (iPSCs) or hematopoietic stem cells (HSCs). (Item 4) The cells described in item 3, wherein the iPSC or HSC has the ability to differentiate into CD4+ T cells or CD8+ T cells. (Item 5) The cells described in item 3, wherein the T cells express αβTCR or γδTCR. (Item 6) The iPSCs are cells as described in item 4, which are derived from T cells or thymocytes. (Item 7) The cells according to item 6, wherein the T cells or thymocytes derived from the iPSC are CD8+ or CD4+. (Item 8) The cells according to item 6 or 7, wherein the T cells or thymocytes derived from the iPSC express a TCR directed to the first antigenic determinant. (Item 9) The cell according to item 8, wherein the TCR expressed in the T cell or thymocyte derived from the iPSC is αβTCR or γδTCR. (Item 10) The cell according to item 1, wherein the first and second antigenic determinants are selected from the group consisting of tumor antigens, microbial antigens, or autoreactive immune cell antigens. (Item 11) The cell described in item 10, wherein the first antigenic determinant is selected from a tumor antigen, e.g., WT-1. (Item 12) The cells described in item 10 or 11, wherein the second antigenic determinant is selected from tumor antigens, such as TAG72, CD19, MAGE, and CD47. (Item 13) The cell according to any one of the preceding items, wherein the antigen recognition portion includes scFv. (Item 14) The cell according to 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) The cell according to item 14, wherein the hinge region is derived from the hinge region of IgG1, the hinge region of CD8, or the hinge region of CD28. (Item 16) The cell according to item 14, wherein the hinge region contains cysteine ​​that promotes the dimerization of the chimeric antigen receptor. (Item 17) The cell described in item 14, wherein the transmembrane domain is derived from the transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, or from an immunoglobulin such as CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or IgG4. (Item 18) The cell according to any one of the preceding items, wherein the T cell activation portion includes 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) The cell according to item 18, wherein the T cell activation portion further comprises an intracellular signaling sequence of a costimulatory molecule selected from the group consisting of ligands that specifically bind to CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, TIM-3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. (Item 20) A cell according to any one of the preceding items, further comprising an additional nucleic acid encoding an additional chimeric antigen receptor including an antigen-recognition portion and a T-cell-activating portion, wherein the antigen-recognition portion of the additional chimeric antigen receptor is directed to an additional antigenic determinant different from the first and second antigenic determinants. (Item 21) A cell according to any one of the preceding items, further comprising a nucleic acid encoding a non-signaling antigen-binding receptor, which includes the first antigenic determinant and an antigen recognition portion that is directed to an antigenic determinant different from the antigenic determinant to which the chimeric antigen receptor is directed. (Item 22) The cell according to item 21, wherein the antigen-recognizing portion is operably linked to the transmembrane domain by a hinge region. (Item 23) The cell according to item 21 or 22, wherein the antigen-binding receptor is directed to CD47. (Item 24) The cell according to item 23, comprising an scFv whose antigen-binding receptor is directed to CD47. (Item 25) The cell described in item 24, wherein the hinge and transmembrane region of the antigen-binding receptor are the hinge and transmembrane region of CD28. (Item 26) The cell according to 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) The cell according to item 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) A method for producing genetically modified mammalian stem cells, The steps include obtaining mammalian stem cells that have the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant; The steps include introducing one or more nucleic acid molecules into the stem cells, each comprising: one or more nucleic acid molecules encoding one or more chimeric antigen receptors, wherein each chimeric antigen receptor includes an antigen recognition portion directed to an antigen determinant different from the first antigen determinant, and the antigen recognition portion is operably linked to a T cell activation portion; and optionally, one or more nucleic acid molecules encoding one or more antigen-binding receptors (e.g., non-signaling antigen-binding receptors), wherein each antigen-binding receptor includes an antigen recognition portion directed to an antigen determinant different from the first antigen determinant and the antigen determinant to which the chimeric antigen receptor is directed; A method that includes this. (Item 29) The method according to item 28, wherein the stem cells express at least one homozygous HLA haplotype. (Item 30) A method for producing genetically modified mammalian stem cells, A step of obtaining T cells or thymocytes expressing a TCR directed to a first antigenic determinant, wherein the T cells or thymocytes are optionally CD8+ or CD4+; The steps include introducing one or more nucleic acid molecules encoding one or more chimeric antigen receptors, wherein each chimeric antigen receptor includes an antigen-recognition portion directed to an antigen-determinant different from the first antigen-determinant, and the antigen-recognition portion is 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), wherein each antigen-binding receptor includes an antigen-recognition portion directed to an antigen-determinant different from the first antigen-determinant and the antigen-determinant to which the chimeric antigen receptor is directed; Steps to derive stem cells from T cells or thymocytes A method that includes this. (Item 31) The method according to item 30, wherein the T cells or thymocytes express at least one homozygous HLA haplotype. (Item 32) The method according to 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 to 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 to a second antigenic determinant and operably linked to the T cell activation portion. (Item 34) The T cells described in item 33, wherein the T cells express at least one homozygous HLA haplotype. (Item 35) The T cells described in item 33 or 34, wherein the T cells are CD4+ T cells or CD8+ T cells. (Item 36) The T cell according to item 33 or 34, wherein the TCR is an αβTCR or a γδTCR. (Item 37) T cells according to item 33 or 34, derived from stem cells expressing at least one homozygous HLA haplotype. (Item 38) The T cells described in item 37, wherein the stem cells are iPSCs or HSCs. (Item 39) The T cell according to item 38, wherein the iPSC is derived from a T cell or thymocyte expressing a TCR directed to the first antigenic determinant. (Item 40) The T cell according to item 39, wherein the TCR expressed in the T cell or thymocyte derived from the iPSC is αβTCR or γδTCR. (Item 41) The T cells or thymocytes described in item 39 are CD8+. (Item 42) The T cell according to item 38, wherein the iPSC or HSC comprises a nucleic acid molecule encoding the chimeric antigen receptor. (Item 43) The T cell described in item 33, wherein the first and second antigenic determinants are selected from the group consisting of tumor antigens, microbial antigens, or autoreactive immune cell antigens. (Item 44) The T cell described in item 43, wherein the first antigenic determinant is selected from tumor antigens, e.g., WT-1 and EBVLMP2. (Item 45) The T cell described in item 44, wherein the second antigenic determinant is selected from tumor antigens, e.g., TAG72, CD19, MAGE, and CD47. (Item 46) A T cell according to any one of items 33 to 45, wherein the antigen recognition portion includes scFv. (Item 47) The T cell according to 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) The T cell described in item 47, wherein the hinge region is derived from the hinge region of IgG1, the hinge region of CD8, or the hinge region of CD28. (Item 49) The T cell described in item 47, wherein the hinge region contains cysteine ​​that promotes the dimerization of the chimeric antigen receptor. (Item 50) The T cell described in item 47, wherein the transmembrane domain is derived from the transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, or from an immunoglobulin such as CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or IgG4. (Item 51) The T cell according to any one of items 33 to 50, wherein the T cell activation region 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) The T cell according to item 51, wherein the T cell activation portion further comprises an intracellular signaling sequence of a costimulatory molecule selected from the group consisting of ligands that specifically bind to CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. (Item 53) A T cell according to any one of items 33 to 52, further expressing an additional chimeric antigen receptor comprising an antigen-recognizing portion and a T cell-activating portion, wherein the antigen-recognizing portion of the additional chimeric antigen receptor is directed to an additional antigen-determinant different from the first and second antigen-determinants. (Item 54) A T cell according to any one of items 33 to 53, further expressing a non-signaling antigen-binding receptor comprising an antigen recognition moiety that is directed to an antigen determinant different from the antigen determinant to which the first antigen determinant and the chimeric antigen receptor are directed. (Item 55) The T cell described in item 54, wherein the antigen-recognizing portion is operably linked to the transmembrane domain by a hinge region. (Item 56) The T cell described in item 54 or 55, wherein the antigen-binding receptor is directed to CD47. (Item 57) The T cell described in item 56, comprising an scFv whose antigen-binding receptor is directed to CD47. (Item 58) The T cell described in item 57, wherein the hinge and transmembrane region of the antigen-binding receptor are the hinge and transmembrane region of CD28. (Item 59) The T cell described in item 53, wherein, upon initial translation, the chimeric receptor and the additional chimeric antigen receptor 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) The T cell described in item 54, wherein, upon initial translation, the chimeric receptor and the antigen-binding receptor 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) T cells derived from stem cells as described in any one of items 1 through 27. (Item 62) A method for producing T cells, The steps of providing genetically modified stem cells as described in any one of items 1 to 27, and differentiating the genetically modified stem cells into T cells. A method that includes this. (Item 63) A method for producing T cells, The steps include obtaining stem cells that have the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant; The step of differentiating the aforementioned stem cells into T cells; The steps include introducing one or more nucleic acids into the T cells, each of which is a nucleic acid encoding one or more chimeric antigen receptors, wherein each chimeric antigen receptor is directed to an antigen determinant different from the first antigen determinant, or optionally, one or more nucleic acids encoding one or more antigen-binding receptors, wherein each antigen-binding receptor is directed to an antigen determinant different from the first antigen determinant and the antigen determinant to which the chimeric antigen receptor is directed; A method that includes this. (Item 64) The method according to item 63, wherein the stem cells express at least one homozygous HLA haplotype. (Item 65) The stem cells are iPSCs or HSCs as described in any one of items 62 to 64. The method. (Item 66) The method according to item 65, wherein the iPSC is derived from a T cell or a thymocyte. (Item 67) The method according to item 66, wherein the T cells or thymocytes are CD8+ or CD4+. (Item 68) The method according to item 65, wherein the iPSC is derived from a T cell or thymocyte expressing the TCR which is directed to the same antigenic determinant to which the TCR expressed in the T cell derived from the iPSC is directed. (Item 69) A method for treating a condition in a mammal characterized by the presence of an unwanted population of cells, comprising the step of administering to the mammal an effective number of T cells as described in any one of items 33 to 61. (Item 70) The method according to item 69, wherein the condition is a neoplasm, a microbial infection (such as HIV, an STD, or antibiotic-resistant bacteria), or an autoimmune condition. (Item 71) The method according to item 69, wherein the state is a neoplastic state, the TCR is directed to a first tumor antigen determinant, and the CAR is directed to a second tumor antigen determinant. (Item 72) The method according to item 71, wherein the first tumor antigen determinant is WT1. (Item 73) The method according to item 71 or 72, wherein the second tumor antigen determinant is TAG72. (Item 74) The method according to any one of items 71 to 73, wherein the cells administered to the mammal contain a nucleic acid molecule encoding a non-signaling CD47-binding receptor. [Brief explanation of the drawing]

[0056] [Figure 1A-1O] Stimulation and enlargement of cytotoxic T cells expressing a TCR specific to Wilms' tumor 1 (WT-1) antigen. Cells were isolated from whole blood peripheral blood mononuclear cells (PBMCs). Cells were gated for single cells (A, F, K), then by CD3-positive cells (conjugated with APCCy7; C, H, M), then by CD8 (conjugated with PECy7) and CD4 (conjugated with PerCp; D, I, N), and finally by CD8 cells alone (E, J, O), and scatter plots are also shown (B, G, L). WT-1 staining was performed using HLA-A02 tetramer specific to the WT-137 peptide. The samples presented are from two separate patients (Patient 1 A-E; Patient 2 F-J) who are HLA-A02 positive and are compared to Fluorescence Minus One (FMO; this stain lacks WT-1 tetramer staining and shows specific staining for WT-137, K-O). The percentages shown are the percentage 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 technique herein). In other studies (e.g., Schmeid et al., (2015)), they were only 10⁻⁶ CD8+ cells (ranging from 3 × 10⁻⁷ to 3 × 10⁻⁶ cells).

[0057] [Figure 2A-2G]CD8+ cytotoxic T cells with a TCR specific to Wilms' tumor 1 (WT-1) antigen are functional. This function is represented 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 with WT-137 peptide-PE conjugated fluorescent dye from CD8+ and HLA-A02 tetramers. These cytotoxic T cells with a WT-1 specific TCR demonstrated intracellular cytokine staining of IFN-γ (conjugated with Pacific blue fluorescent dye) upon stimulation with WT-1. The results shown are from two separate HLA-A02-positive patients (WT-1 #1 and WT-1 #2) (Patient 1: A-B, Patient 2: C-D), and are compared to (G) (FMO; EF; this stain lacks WT-1 tetramer staining and shows specific staining for WT-137). The percentages shown are the percentage of WT1+CD8+ cells. Over 80% of WT-1 TCR T cells produced IFNγ.

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

[0059] [Figure 3]Production of iPSCs derived from cancer-specific (e.g., WT-1) TCR T cells. 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 in lymphoblast cell lines (LCL) using EBV) that act as antigen-presenting cells. Cancer antigen-specific T cells have been shown to be double-labeled with a tetramer for CD8 (against cytotoxic T cells) and HLA-WT-1 that binds to the TCR of these CD8+ cells. These cells were then converted into iPSCs using Yamanaka programming factors. A rearranged TCR gene specific to WT-1 was embedded in the TCR locus of the iPSCs.

[0060] [Figure 4] Morphological progression of iPSC colonies into hematopoietic cell lineages and lymphocyte progenitor cells after 1, 5, 9, and 13 days of culture in OP9-supported cells. Note the numerous single hematopoietic-like cells up to day 13.

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

[0062] [Figure 6]Flow cytometry of HSCs in iPSC-derived cells after 13 days of culture in OP9 cells followed by 9 days of culture in OP9 DL-L1 cells. Cells were gated for viability, CD45 expression, and single-cell analysis, and then stained for CD34 and CD43 to examine HSC content. Note the reduction in HSCs from >90% in preliminary OPDL-L1 culture (Figure 5) to approximately 60% after 9 days of culture in OP9DL-L1 cells.

[0063] [Figure 7] Flow cytometry of T cell development in iPSC-derived cells after 9 days of culture in OP9 DL-L1 cells following 13 days of culture in OP9 cells. There is clear evidence of commitment to the first stage of thymocyte development, including T cell lineages with CD5 and CD7 expression, as well as immature (i.e., lacking CD3; data not described) 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 in OP9 cells followed by 16 days of culture in OP9 DL-L1 cells. Immature T cells expressing CD4 and / or CD8 were still clearly present, and HSCs were further reduced from approximately 60% to approximately 25%. Most importantly, mature CD8+ cells were present, expressing CD3, αβTCR, and CD8β chain (in addition to CD8α - not described). [Figure 8-2] Flow cytometry of HSC and T cell differentiation in iPSC-derived cells after 13 days of culture in OP9 cells followed by 16 days of culture in OP9 DL-L1 cells. Immature T cells expressing CD4 and / or CD8 were still clearly present, and HSCs were further reduced from approximately 60% to approximately 25%. Most importantly, mature CD8+ cells were present, expressing CD3, αβTCR, and CD8β chain (in addition to CD8α - not described).

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

[0066] [Figure 10] In vitro enhancement of WT-1-specific TCRs (CD8αβ T cells) induced from iPSCs derived from T cells demonstrates that WT-1-specific TCRs retained complete function (e.g., cytotoxicity to WT-1 expression targets) comparable to that of innate T cells. The effector:target ratio was 3:1; stepwise concentrations of WT-1 peptide were examined.

[0067] [Figure 11]Schematic diagrams of chimeric antigen receptors and antigen-binding receptor constructs. A panel of chimeric antigen receptor (CAR) constructs having scFv for either TAG72 or CD19 (as a positive control) was developed. Constructs used either human CD8 or CD28 as the hinge and transmembrane region, as well as the CD28, CD3ζ chain or 4-1BB cytoplasmic activation signaling domain. P2A is a signal sequence directed towards 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), whose leader is processed to release anti-CD47scFv anchored on the surface by the hinge / TM, and the endodomain tail does not contain a signaling sequence. Any CD47-binding external domain can be used for the purpose of binding to CD47 on target cells, for example, SIRP-alpha. The hinge region may contain cysteine ​​residues to direct dimerization by disulfide bond formation between adjacent hinge domains, which is characteristic of the natural CD8 hinge, or it may have cysteine ​​residues substituted with other residues such as serine, which do not form disulfide bonds and do 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 described in SEQ ID NOs: 1-20.

[0068] [Figure 12] Schematic diagram of retroviral transformation. Schematic diagram of the process attempted to generate a CAR containing a retroviral construct. The CAR construct is cloned into a pSAMEN plasmid vector and ligated to a fluorescent reporter EGFP by a P2A self-cleaving polypeptide to separate the CAR from 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 diagram of lentiviral transformation. Schematic diagram of the process attempted to generate a CAR containing a lentiviral construct. The CAR construct is cloned into a pWP1 plasmid vector and ligated to a fluorescent reporter EGFP by a P2A self-cleaving polypeptide to separate the CAR from the reporter. Upon successful cell transduction, P2A is expressed and cleaved, and EGFP is identified by flow cytometry and immunofluorescence microscopy.

[0070] [Figure 14A-14B] Figure 14A. Schematic diagram of a typical second-generation CAR structure. scFv binding domain to the target antigen; hinge region (stalk) that enables the CAR to be incorporated into the plasma membrane (the length of the hinge can vary in its effect on scFv binding to the target cell); cytoplasmic signaling domain that induces T cell activation by 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 diagram of a non-signaling antigen-binding receptor, a cleaved CD47 "attached stalk". The structure exhibits an scFv domain or single V-domain for CD47 antigen binding, attached to the hinge and transmembrane region, but without a signaling domain 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 thus without damage to normal cells. The hinge region may contain cysteine ​​residues that promote dimerization through the formation of disulfide bonds between adjacent hinge domains, or it may have cysteine ​​residue substitutions with other residues such as serine, in which case it does not form disulfide bonds and does not form a covalently stabilized dimer.

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

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

[0073] [Figure 17] TAG72 CAR-T mediated death of ovarian cancer (TAG72+) target cells. The effector:target ratio was (E:T)=1:1. TAG72 CAR-T effector cells (GFP-positive cells) developed from CD3-activated normal blood T cells were isolated via FACS with purity >95% and then stimulated for 72 hours in the presence of immobilized αCD3 / αCD28 and IL-2 for enhancement of cytolytic activity before use. Changes in cellular impedance (expressed herein as cellular indicators in arbitrary units) were monitored over 40 hours and compared with stimulated non-transduced CD3+ VE cells isolated from PBMCs and stimulated vector-only CAR-T cells. TAG72 CAR-T cells showed the highest death rate, however CD3 / CD28-activated non-CAR-T cells also showed death, albeit to a much lower degree.

[0074] [Figure 18] Determination of the specificity of TAG72 CAR-T cell death. TAG72 and CD19 CAR-T cells were isolated via FACS, respectively, and immediately added to TAG72hi / CD19low target cells without in vitro stimulation (E:T = 5:1). Changes in cellular impedance (expressed herein as cellular indicators in arbitrary units) were monitored for 15 hours. TAG72 CAR-T cells showed strong cell line death. CD19 CAR-T cells were identical 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 produced from WT-1-specific T cells. Figure 19A. WT-1-specific TCR T cells were successfully transduced using the TAG72 lentiviral CAR construct (31.3% positive, compared to <0.1% in controls). Figure 19B. iPSC-derived WT-1-specific TCR T cells formed from WT-1-specific TCR T cells were successfully transduced using dual-specific CAR constructs for TAG72 and non-signal-cleaved CD47 (55% transduction); 32% transduction using TAG72 alone. These transduced T cells contained three anti-cancer specificities: WT-1 (TCR); TAG72 (CAR); and cleaved non-signal-cleaved CD47. [Figure 19B] Figures 19A-19B. Flow cytometry analysis of CAR transduction of iPSC-derived WT-1-specific TCR CD8+ T cells produced from WT-1-specific T cells. Figure 19A. WT-1-specific TCR T cells were successfully transduced using the TAG72 lentiviral CAR construct (31.3% positive, compared to <0.1% in controls). Figure 19B. iPSC-derived WT-1-specific TCR T cells formed from WT-1-specific TCR T cells were successfully transduced using dual-specific CAR constructs for TAG72 and non-signal-cleaved CD47 (55% transduction); 32% transduction using TAG72 alone. These transduced T cells contained three anti-cancer specificities: WT-1 (TCR); TAG72 (CAR); and cleaved non-signal-cleaved CD47.

[0076] [Figure 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 with the ovarian cancer cell line CAOV4 for 24 hours in monolayer culture to assess cytotoxicity. Low effector ratio of 2:1: Despite the target ratio (inevitably due to the poor effector yield), there was specific death in WT-1 TCR T cells, which was further increased by transduction with TAG72 CAR. The technique is based on AquaAmine, which stains intracellular amines. When cells are dead or dying, the susceptible cell membrane allows the dye to penetrate the cell, staining the amines more strongly. Thus, the cytotoxicity of cells is indicated by an increase in the staining intensity of cellular amines. Note: Since some amines are present on the cell surface, living cells also produce some (low but) 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: Bispecific 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. 40× magnification. WT-1 TCR T cells undergo approximately 10% death (above background); TAG72 CAR-T cells undergo an additional 10% death (i.e., approximately 20% above background). The dual anti-cancer death mechanism is additive.

[0077] [Figure 21A]Figures 21A-21B. CAR transduction of iPS cells. Growth on day 5 in the MEF feeder layer. 4 days after incubation with CAR lentivirus. CAR+ transduction with TAG72, CD19, and GFP viruses (green) is superimposed on bright-field images at 20× magnification. The non-transduction control showed no GFP signal. Images of iPSC colonies at 4× magnification demonstrate the presence of iPSC colonies in the MEF feeder layer. It is noteworthy that in each system, some iPSC colonies appear to have spontaneously begun to differentiate. Transduction fibroblast-derived iPSCs are shown in Figure 21A. Figure 21B demonstrates good transduction of WT-1 T cell-derived iPSCs with TAG72 CAR. Thus, these iPSCs were well imprinted for both WT-1 TCR and TAG72 specificity. [Figure 21B] Figures 21A-21B. CAR transduction of iPS cells. Growth on day 5 in the MEF feeder layer. 4 days after incubation with CAR lentivirus. CAR+ transduction with TAG72, CD19, and GFP viruses (green) is superimposed on bright-field images at 20× magnification. The non-transduction control showed no GFP signal. Images of iPSC colonies at 4× magnification demonstrate the presence of iPSC colonies in the MEF feeder layer. It is noteworthy that in each system, some iPSC colonies appear to have spontaneously begun to differentiate. Transduction fibroblast-derived iPSCs are shown in Figure 21A. Figure 21B demonstrates good transduction of WT-1 T cell-derived iPSCs with TAG72 CAR. Thus, these iPSCs were well imprinted for both WT-1 TCR and TAG72 specificity.

[0078] [Figure 22-1]Flow cytometry analysis of chimeric antigen receptor transduction of iPSCs. These iPSCs are derived from adult fibroblasts, but may be from any source, including unselected T cells, CD8+ T cells, or cancer antigen-specific (e.g., WT-1) T cells. There is a clear population of fluorescent iPSCs that are well transdone with TAG72 or CD19. Figure 23 shows the overlay of transdone cells compared to untransdone controls. [Figure 22-2] Flow cytometry analysis of chimeric antigen receptor transduction of iPSCs. These iPSCs are derived from adult fibroblasts, but may be from any source, including unselected T cells, CD8+ T cells, or cancer antigen-specific (e.g., WT-1) T cells. There is a clear population of fluorescent iPSCs that are well transdone with TAG72 or CD19. Figure 23 shows the overlay of transdone cells compared to untransdone controls.

[0079] [Figure 23-1] An overlay of a dot plot comparing untransduced control cells (blue) to transduced iPSC cultures (green). Events at the GFP+ gate demonstrate good transduction and are expressed as the percentage of non-debris events. [Figure 23-2] An overlay of a dot plot comparing untransduced control cells (blue) to transduced iPSC cultures (green). Events at the GFP+ gate demonstrate good transduction and are expressed as the percentage of non-debris events.

[0080] [Figure 24] Reformation of CAR-transduced iPSC colonies after FACS sorting. CAR-transduced iPSCs can be isolated by flow cytometry (GFP-positive fluorescence) and reseed to form stable colonies. [Modes for carrying out the invention]

[0081] Detailed description of the invention This invention is partly based on the finding that, for example, TCR / CAR dual-expressing T cells directed to two distinct antigenic determinants can be consistently and stably generated by transfecting iPSCs derived from T cells exhibiting TCR specificity directed to a target antigenic determinant with a CAR cassette. Through the action of epigenetic memory, it has been found that T cells differentiated from these iPSCs stably express both the TCR specificity and the distinct antigenic determinant-directed CAR of the somatic T cells from which the iPSC originated. Specificity to additional antigenic determinants can be achieved by introducing additional nucleic acids encoding molecules that bind to such additional antigenic determinants into the cells. Thus, such multi-specific cells provide more effective therapeutic outcomes than currently available cells. These findings have therefore enabled the development of a continuous supply of stably transformed dual antigenic-specific T cells, particularly cytotoxic CD8+αβTCR T cells, for use in association with any disease state characterized by undesirable cell populations, such as neoplasms, viral infections, bacterial infections, or autoimmune states. This discovery and the subsequent generation of cells have now facilitated improvements in therapeutic regimens aimed at treating such conditions, particularly neoplasms such as solid tumors or hematological cancers (e.g., leukemia), including metastatic diseases.

[0082] Accordingly, one aspect of the present invention relates to genetically modified mammalian stem cells, or T cells differentiated therefrom, which have the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant, and which include a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor includes an antigen-recognizing portion directed to a second antigenic determinant, and the antigen-recognizing portion is operably linked to a T cell activation portion. In some embodiments, the genetically modified mammalian stem cells express at least one homozygous HLA haplotype.

[0083] The reference to “T cells” should be understood as a reference to any cell containing a T cell receptor. In this regard, the T cell receptor may contain any one or more of the α, β, γ, or δ chains. As will be understood by those skilled in the art, NKT cells also express T cell receptors, and therefore the present invention can also generate bispecific NKT cells. The present invention is not intended to limit to any particular subclass of T cells, however, in preferred embodiments the T cells of interest express an α / β TCR dimer. More preferably, the T cells express a CD4 + Helper T cells, CD8 + Killer T cells or NKT cells. The present invention is not limited to any one theory or mechanism of action, but CD8 + T cells are also known as cytotoxic cells. CD8 is a major part of the adaptive immune system. + T cells scan the intracellular environment, primarily targeting and destroying infected cells. Small peptide fragments derived from intracellular contents are processed and transported to the cell surface, where they are presented in association with MHC class I molecules. However, this is not limited to the response to viral infection, CD8 + T cells also provide an additional level of immune surveillance by monitoring and eliminating damaged or abnormal cells, including cancer cells. The CD8 peptide presented on MHC I. + T cell recognition typically leads to the destruction of target cells by activating the apoptotic pathway through the release of cytotoxic granules or lymphokines or FAS / FASL interactions. On the other hand, CD4 + T cells generally recognize peptides presented by antigen-presenting cells in association with MHC class II, and B cells and / or CD8 + This results in the release of cytokines designed to regulate the T cell immune response. Therefore, unlike cytotoxic T cells, helper T cells do not directly kill unwanted cells such as cancer cells, but can amplify such responses as long as they are killed by cytotoxic T cells and / or antibody 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 surface antigens generally 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 either the invariant TCR alpha chain or a small number of 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 - Distinguishable subpopulations, including cells, were identified. Type 2 NKT cells (or non-invariant NKT cells) express a broader TCRα chain and do not recognize the alpha-GalCer antigen. NKT cells produce cytokines that have multiple, often opposing effects, including inducing immunosuppression, such as promoting inflammation or tolerance. As a result, they can contribute to antimicrobial 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, references to genetically modified T cells in this invention should be understood to include references to NKT cells.

[0085] Since thymic T cell production is characterized by the random generation of a T cell receptor (TCR) repertoire, thymic proliferation must also involve a very rigorous selection process that eliminates or functionally neutralizes developing thymic T cells that have the potential to attack self. Thus, this "self-tolerance" reduces the potential for autoimmune disease. However, if non-virally induced cancers are defined as "self" diseases, then inevitably, this process impairs immune surveillance against cancer. This means that many of the T cells generated in the thymus, which may potentially be reactive with tumor-associated antigens, can be eliminated before they enter the bloodstream. At the very least, they will be numerically insufficient and likely express low-affinity TCRs.

[0086] In one embodiment, genetically modified mammalian stem cells, or T cells differentiated therefrom, wherein the cells express a CD4 that is directed to a first antigenic determinant. + A cell is provided that has the ability to differentiate into a T cell and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen-recognizing portion directed to a second antigenic determinant, and the antigen-recognizing portion is operably linked to a T cell activating portion. In one embodiment, the genetically modified mammalian stem cell expresses at least one homozygous HLA haplotype.

[0087] In another embodiment, genetically modified mammalian stem cells, or T cells differentiated therefrom, wherein the cells express a CD8 that is directed to a first antigenic determinant. + A cell is provided that has the ability to differentiate into a T cell and comprises a nucleic acid molecule encoding a chimeric antigen receptor, wherein the receptor comprises an antigen-recognizing portion directed to a second antigenic determinant, and the antigen-recognizing portion is operably linked to a T cell activating portion. In one embodiment, the genetically modified mammalian stem cell expresses 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. While the present invention is not limited 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 peptide fragments derived from antigens and present them to T cells. The MHC gene family is divided into three subgroups: Class I, Class II, and Class III. Class I MHC molecules express the β2 subunit and can therefore only be recognized by the CD8 co-receptor. Class II MHC molecules do not express the β2 subunit at all and can therefore be recognized by the CD4 co-receptor. Thus, since different lymphocytes express different TCR co-receptors, MHC molecules regulate which type of lymphocyte can bind to a given antigen with high affinity. The diversity of antigen presentation mediated by MHC Class I and II is acquired in at least three ways: (1) The MHC repertoire of an organism is usually polygenic (composed of multiple interacting genes); (2) MHC expression is codominant (by both pairs of heritable alleles); (3) MHC gene variants are highly diverse (they differ greatly between organisms within a species).

[0089] MHC molecules bind to both the T cell receptor and the CD4 / CD8 coreceptor in T lymphocytes. Antigenous epitopes held in the peptide-binding grooves of MHC molecules interact with the variable Ig-like domain of the TCR to induce T cell activation. However, MHC molecules can also act as antigens themselves, triggering an immune response in recipient tissues or cells expressing foreign MHC, and thus potentially causing transplant rejection. Furthermore, transplantation of immunocompetent cells can actually result in host tissue rejection, 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, and one or two HLA-DR from each parent, as well as combinations thereof). MHC variability is high in the human population, with at least 350 alleles for HLA-A, 620 alleles for HLA-B, 400 alleles for DR, and 90 alleles for DQ. Any two non-identical twins will express different MHC molecules.

[0090] While all MHC molecules can mediate transplant rejection, HLA-C and HLA-DP, which exhibit low polymorphism, are less significant. Transplant rejection can be minimized by attempts to match as much of the cell surface HLA repertoire between donor and recipient as possible. Perfect matching is only possible between identical twins. However, it is highly desirable to select donors based on minimizing incompatibility in one or more of the ranges of HLA antigens expressed on cells, and this can significantly minimize the problem of rejection. The usual method of managing tissue / cell rejection is the administration of immunosuppressive treatment regimens, and this method is undesirable in relation to treatment regimens based on the administration of genetically modified immune cells that need to function at an optimal level of functionality; therefore, this is a particular problem that is addressed 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 derived from iPSCs, which are homozygous for one or more MHC haplotypes, and the HLA allele of interest is the primary transplant antigen and is preferably expressed in 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. If the homozygous HLA haplotype corresponds to the dominant MHC I or MHC II HLA type (in terms of tissue rejection), the use of such cells will significantly reduce the problem of tissue rejection in a broader population receiving the cells of the present invention in relation to the treatment regime. With respect to the present invention, the genetically modified cells may be homozygous for one cellular HLA antigen, or they may be homozygous for more than one HLA antigen, e.g., 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] Therefore, the term "HLA-type" should be understood to refer to the complement of HLA antigens present in an individual's cells.

[0092] Obtaining suitable homozygous HLA T cells for use in generating iPSCs can be achieved by any suitable method, for example, a method comprising the steps of screening a population (e.g., by a blood bank) to identify individuals expressing HLA homozygotes, and then screening T cells from individuals exhibiting the desired TCR specificity. These normally very rare T cells can be selectively stimulated by specific antigenic peptides recognized by their TCRs, and their frequency can be greatly increased (e.g., <0.0001 to 0.2).

[0093] Information significant in terms of minimizing donor-recipient HLA mismatch across a given target population, thereby enabling the creation of a donor bank, is widely available in published literature describing the identification and practicality of homozygous haplotypes, as will be acknowledged by those skilled in the art. See, for example, Pappas et al. (2015). In one example, Table 1 identifies the 15 homozygous HLA haplotypes ranked highest compared to the proportion of the UK population that yield the smallest mismatch. The first eight listed homozygous HLA haplotypes are compatible with 49% of the population. Further examples are outlined in Table 2, detailing the first 10 ranked haplotypes compatible with racially distinct California populations. Table 2 includes matching frequencies for subpopulations, including Black or African American, Asian and Pacific Islander, White, Hispanic and Native American and Alaskan 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. Those skilled in the art will understand that, using the data illustrated in Table 3, a set of homozygous haplotypes that provide the smallest mismatch for the North Chinese population can be defined.

[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 manipulated to express dual T cells and chimeric antigen receptors directed to multiple distinct antigens, thereby providing a continuous supply of T cells that are more therapeutically effective than those used in currently available therapeutic cell treatment regimens. In this regard, the reference to “stem cells” should be understood as a reference to any cell that develops in the direction of multiple lineages that give a particular genetic makeup and therefore exhibits the potential to form new organisms or regenerate tissues or cell populations of an organism. The stem cells utilized by the present invention can be any suitable type having 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). “Totipotent” means that the target stem cell is capable of autoregeneration. "Pluripotency" means that the target stem cells can differentiate and form any one of the three germ layers, in particular the ectoderm, endoderm, and mesoderm.

[0098] In one particular embodiment, the target stem cells are induced pluripotent stem cells (iPSCs). While the present invention is not limited to any one theory or mechanism of action, the augmentation of adult stem cells does not necessarily involve both stem cell regeneration and differentiation into specific somatic cell lineages based on the development of asymmetric stem cell division. In particular, pluripotent stem cells can be supplied from T cells induced to transition to a multi-lineage latent stage. The development of techniques enabling the dedifferentiation of adult cells is significantly important, especially given the difficulty in inducing stem cell regeneration and augmentation in vitro.

[0099] According to this embodiment, therefore, there is a genetically modified mammalian stem cell, or a T cell differentiated therefrom, wherein the stem cell is an iPSC, which has the ability 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-recognizing portion directed to a second antigenic determinant, the antigen-recognizing portion being operably linked to a T cell-activating portion. 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 to this. That is, the iPSCs in question can be generated from cells that have not undergone terminal differentiation; in fact, iPSCs can, in principle, be induced from any nucleated cell, including, for example, blood-derived mononuclear cells and skin cells. For example, in connection with one embodiment of the present invention, the iPSCs in question can be generated from well-differentiated T cells or from precursor T cells such as thymocytes. Insofar as the thymocytes in question undergo TCR rearrangement to exhibit the desired antigen specificity in connection with the present invention, iPSCs can be generated from these cells. This may be the case, for example, when the particular TCR rearrangement in question is expected to be selected during thymocyte proliferation. One of the complicating factors associated with immune responsiveness to tumor cells or autoreactive cells will be recognized by those skilled in the art that, in this situation, the immune system needs to direct its immune response towards self-cells, and therefore self-antigens. Such immune cells are typically selected during T lymphocyte differentiation in the thymus to minimize the likelihood of developing autoimmune diseases. In relation to neoplasms and autoimmune states, however, the unwanted cells are self cells, and therefore the cell surface antigens that can be targeted are self antigens. Without limiting the present invention, as will be discussed in more detail later, one advantage of using iPSCs to generate TCR / CAR-expressing T cells directed to multiple distinct antigenic determinants is that epigenetic memory can enhance the differentiation of iPSCs into functional T cells expressing TCRs directed to the same antigens as the T cells from which the iPSCs were derived. However, with respect to the selection of specific TCR-expressing cells from which iPSCs were derived, it can be difficult to identify well-differentiated and suitable T cells, since T cells expressing functional TCRs directed to self antigens may be selected during thymocyte proliferation. Therefore, 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, iPSCs are transfected with one or more nucleic acid molecules encoding a TCR (such as a rearranged TCR gene) directed to 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) are stem cells that give rise to all blood cells of the lymphoid and myeloid lineage through the process of hematopoietic development. HSCs originate from the mesoderm and can be found in adult bone marrow, peripheral blood, and umbilical cord blood. HSCs can be collected from bone marrow, peripheral blood, and umbilical cord blood by established techniques and are 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 and transfected, for example, with one or more nucleic acids encoding a TCR directed to a first antigenic determinant, and then directed to differentiate into T cells. One or more nucleic acids encoding a CAR, and optionally one or more nucleic acids encoding a docking antigen-binding receptor, can be introduced into HSCs before or after differentiation into T cells.

[0103] Therefore, references to the “T cell receptor” (TCR) should be understood as references to heterodimers 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 association with MHC class II, while CD8+ T cells recognize peptides presented in association with MHC class I. Although the present invention is not limited to any one theory or mechanism of action, in the vast majority of human T cells, the TCR contains α and β chains, while a small population of cells expresses a TCR containing a γδ heterodimer. The TCR is a disulfide-linked, membrane-anchored heterodimeric protein. The γ, δ, α, and β chains consist of two extracellular domains: a variable (V) region and a constant (C) region, both of which form parts of the immunoglobulin superfamily and fold to form an antiparallel β-sheet. The constant region is adjacent to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex.

[0104] The variable domains of the TCR α and β chains express three hypervariable or complementarity-determining regions (CDRs), respectively, while the variable region of the β chain has an additional area of ​​hypervariability (HV4) that does not normally come into contact with the 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 by either somatic V(D)J recombination using RAG1 and RAG2 recombinases or gene conversion using cytidine deaminase. Each recombinated TCR possesses distinct antigen specificity determined by the structure of the antigen-binding site formed by the α and β chains in the case of αβ T cells, or by the γ and δ chains in the case of γδ T cells. The TCR α chain is generated by VJ recombination, while the β chain is 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 crossover 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, which is crucial for peptide / MHC recognition. It is a distinct combination of segments within this region, and along with palindromes and random nucleotide additions, it is a major contributor to the greater diversity of T cell receptor specificity for processed antigenic peptides.

[0105] Therefore, references to TCRs that are "directed" to antigenic determinants should be understood as references to TCRs that have undergone reorganization and exhibit specificity for antigenic determinants, preferably autologous (especially autologous cancer) antigenic determinants.

[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 + Other forms of T cells, NKT cells, thymocytes, or precursor T cells exist, but are not limited to these. In another embodiment, the cells are rearranged γδ It expresses TCR.

[0107] Therefore, the present invention provides genetically modified mammalian iPSCs or HSCs or T cells differentiated therefrom, wherein the iPSCs or HSCs have the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant, the TCR gene is derived from or converted by the rearranged cell and comprises a nucleic acid molecule encoding a chimeric antigen receptor, the receptor comprises an antigen-recognizing portion directed to a second antigenic determinant, and the antigen-recognizing portion is operably linked to a T cell activation portion. In some embodiments, the genetically modified mammalian iPSCs or HSCs express at least one homozygous HLA haplotype.

[0108] In one embodiment, the iPSC is derived from a T cell or a thymocyte.

[0109] In another embodiment, the iPSC is derived from a T cell or thymocyte expressing αβTCR.

[0110] In yet another embodiment, the iPSC is derived from a T cell or thymocyte expressing γδTCR.

[0111] The target stem cells may be freshly isolated from the organism being treated, or they may be supplied from a non-fresh source such as cultured cells (e.g., cultured to increase cell number and / or to make the cells receptive to differentiation signals) or frozen stocks that were isolated at a somewhat earlier point in time from the organism or another source. It should also be understood that the target cells may undergo any form of treatment or manipulation before differentiation, but not limited to purification, modification of cell cycle state, or formation of cell lines such as embryonic stem cell lines. Thus, the target cells may be primary or secondary cells. Primary cells are those isolated from the organism. Secondary cells are those that have undergone some form of in vitro manipulation, such as the preparation of an embryonic stem cell line, after isolation and before the application of the method of the present invention.

[0112] As long as 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 originate from more mature cell types, such as somatic cells, and have been converted / dedifferentiated to the pluripotent stage.

[0113] While this invention is not limited to any one theory or mechanism of action, iPSCs can be derived by introducing a specific set of pluripotency-related genes or “reprogramming factors” into a somatic cell type. The most commonly used set of reprogramming factors (also known as the Yamanaka factors) is the genes Oct4(Pou5f1), Sox2, cMyc, and Klf4. Yamanaka demonstrated in 2006 that transfection of these four specific genes encoding transcription factors converts human adult cells into pluripotent cells. While this combination is the most common combination used to produce iPSCs, each of the factors can be functionally replaced by unrelated genes such as associated transcription factors, miRNAs, small molecules, or lineage-specific factors. For example, induction of iPSCs has been achieved after transfection of Oct3 / 4, Sox2, Klf4, and cMyc using a retroviral system, and similarly by transfection of Oct4, Sox2, Nanog, and Lin28 using a lentiviral system. The first set of transcription factors is known as the Yamanaka factor, and the latter is generally known as the Thomson factor. Extensive modifications have been made to the basic reprogramming factor expression vectors, as will be recognized by those skilled in the art, to design new delivery mechanisms to increase efficiency and to minimize or remove vector sequences that might otherwise be incorporated into the reprogrammed iPSC genome. These methods are well known to those skilled in the art: (i) A single cassette reprogramming vector containing transgene excision mediated by Cre-Lox; (ii) Reprogramming by non-integrated viruses such as adenovirus or Sendai virus, but not limited to these. Alternatively, the 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 creating iPSCs that do not undergo chromosomal integration of viral vectors. Warren et al. transcribed mRNA to efficiently express reprogramming factors (Warren et al. (2010)). The efficiency can be increased by adding Lin28 to the Yamanaka reprogramming factor protocol, culturing in 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 mimes of mature miR-302b and / or miR-372 + 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)). It was also discovered that certain miRNAs can efficiently reprogram cells without the Yamanaka factor. (iii) PiggyBac - PiggyBac is a mobile genetic element (transposon) that can be integrated into the chromosomal TTAA region in the presence of a transpose and subsequently excised from the genome by re-expression of the transpose. When cloned into a piggyBac vector and co-transfected into MEFs, the Yamanaka factor can reprogram cells 14 to 25 days after transfection (Kaji et al. (2009); Woltjen et al. (2009)). The piggyBac vector is trans The re-expression of porzes allows for excision from iPSCs. (iv) Microcyclic vectors - Microcyclic vectors are the smallest vectors that contain only a eukaryotic promoter and the cDNA to be expressed. Lin28, GFP, Nanog, Sox2, and Oct4 microcyclic vectors expressed in human adipocytes can reprogram cells (Narsinh et al. (2011)). (v) Episomal Plasmids - Transient expression of reprogramming factors as episomal plasmids enables the generation of iPSCs. For example, the oriP / EBNA vector can be constructed with Yamanaka factor + Lin28 in one cassette, and another oriP / EBNA vector contains the SV40 large T antigen (Chuo et al. (2011)). These vectors were shown to be expressed in CD34+ umbilical cord blood, peripheral blood, and bone mononuclear cells in sodium butyrate-supplemented medium, and iPSC colonies were obtained on day 14. The transfected plasmids are eventually lost. It includes.

[0115] In another aspect, those skilled in the art are also familiar with auxiliary methods known to enhance the programming efficiency of cells. For example, even when using the same method, there can be variability 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 inhibit histone deacetylation (Mali et al. (2010); Huangfu et al. (2008)) and the TGFβ and MEK signaling pathways. Blockade (Lin et al. (2009); Ichida et al. (2009)), enhancement of epigenetic modifier function (Esteban et al. (2010)), inhibition of the ROCK pathway (Noggle et al. (2011)) and This makes it easier to reprogram the system, including the induction of glycolysis (Zhu et al. (2010)). This becomes possible. One of these small molecules, the histone deacetylase inhibitors valproic acid and sodium butyrate, is most commonly used in reprogramming protocols. It is also noteworthy that culturing cells in 5% oxygen during the reprogramming process may increase the efficiency of iPSC induction (Yoshida et al. (2009)). Reprogramming In particular, for difficult cells, the addition of small molecules and culturing under hypoxic conditions can yield improvements. Another option is to use embryonic stem cell conditioned medium (ESCM) to induce the expression of endogenous reprogramming factors (Balasubramanian et al. (2009)). Efficiency is... Further improvement can be achieved by adding luproic acid. Such strategies can also be used to enhance the capacity of exogenously introduced reprogramming factors, thereby increasing reprogramming efficiency.

[0118] As long as 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 excision from bone marrow or from blood after HSCs have been released from bone marrow following treatment with a specific molecule, such as GM-CSF. The HSCs can then be purified by flow cytometry after labeling with, for example, anti-CD34 coated magnetic beads or fluorescent anti-CD34, for plasma membrane expression of CD34. These thus purified HSCs can be induced into T cell differentiation using the OP9 / OP9 DL-L1 system outlined, including in Example 3 and Figures 3 to 10.

[0119] References to target stem cells, particularly iPSCs or HSCs, that “have the ability” to differentiate into T cells expressing an antigen-determinant-directed TCR should be understood as references to any cell that is capable of transcribing and translating the target TCR gene and then assembling a TCR heterodimer as a functional receptor on the cell surface. As will be recognized by those skilled in the art, in most situations, stem cells such as iPSCs will not express a TCR in their undifferentiated form. Once TCR expression is induced along the T cell lineage, it is generally expected that TCR expression will 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 cells to express a TCR of particular specificity may be made possible 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, will associate to form a TCR heterodimer. Alternatively, and in connection with preferred embodiments of the present invention, the stem cells of the present invention are generated from T cells, thymocytes, or other cells in which the TCR gene has been rearranged. iPSCs generated from such cells are CD4 under appropriate cell culture conditions. + or CD8 +It was determined that when iPSCs are directed to differentiate into T cells, they will express the same TCR antigen specificity as the somatic T cells from which they are derived. More importantly, and as will be discussed in more detail later, it was determined that, with or without transfection of iPSCs or HSCs with one or more CARs, or one or more nucleic acids encoding the α and β chains of antigen / MHC class I specific TCRs, the T cells differentiated therefrom 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 to two or more distinct antigenic determinants. Thus, assuming that appropriate differentiation signals are given to iPSCs or HSCs, such stem cells are considered to have the "ability" to differentiate into T cells and express the necessary TCRs. In this regard, since TCR gene rearrangement is an entirely independent genomic event, the selection of a T cell subpopulation for generating iPSCs does not necessarily have to be identical to the T cell subpopulation that will ultimately be produced by the differentiation-directed iPSCs. For example, CD4 exhibiting appropriate TCR specificity + It is possible to select T cells and generate iPSCs. However, once those iPSCs are generated, a person skilled in the art can use CD8 + It is possible to aim for the differentiation of iPSCs into T cells. In this case, newly generated CD8 by epigenetic memory + T cells are CD8 + Although it exhibits T cell functionality, TCR specificity is derived from CD4, which is the basis for iPSCs. + This would be the case for T cells. The reverse is also true.

[0120] References to inducing the "conversion" of somatic cells, such as T cells, to multiseries latent phenotypes, such as iPSCs, should be understood as referring to inducing the genetic, morphological, and / or functional changes necessary to change a somatic phenotype to a multiseries (pluripotent) phenotype of the type defined herein.

[0121] As long as it is possible to choose to provide iPSCs that have the ability to produce TCRs by transfection of cells with DNA encoding TCRs, this transfection may occur at any point in time, such as before or after the generation of the iPSCs of the present invention, or may 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 multiseries differentiation potential. Therefore, references to cells exhibiting “multiseries differentiation potential” or “multiseries latency” should be understood as references to cells that exhibit 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, and such cells are usually called pluripotent or multiplicative. These cells exhibit latency that is more constrained to a limited range of lineages than totipotent cells, the latter being cells that can develop in virtually any possible differentiation direction, including all somatic lineages and gametes.

[0123] Cells classically referred to as “progenitor” cells or “precursor” cells are included in the definition of “multiseries differentiation potential,” on the premise that they can produce more than one somatic cell lineage under appropriate stimulation conditions. Insofar as references to “stem cells” are made herein in relation to cells produced by the method of the present invention, this should be understood as a reference to cells exhibiting multiseries differentiation potential as defined herein.

[0124] With respect to the present invention, it should be understood that a key characteristic of the target stem cells is that the multi-series differentiation potential exhibited by the cells includes the ability to differentiate into T cells and express a TCR exhibiting specificity for the target antigen. It is irrelevant whether TCR specificity is induced before or after the generation of the stem cells (e.g., by transfection of the stem cells with DNA encoding the target TCR). It should be understood that the stem cells claimed herein encompass all stem cells that exhibit the required differentiation potential, regardless of when and how their potential was introduced. Furthermore, it should be understood that the target stem cells do not need to be totipotent. However, if they exhibit the ability to differentiate along more than one somatic cell lineage, and one of these lineages is a T cell lineage, then such cells are included 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 cells of interest are obtained as a result of some form of molecular manipulation compared to cells observed in relation to the corresponding unmodified cells. In relation to the present invention, the stem cells of interest contain a nucleic acid molecule encoding a chimeric antigen receptor, and optionally further contain a nucleic acid molecule encoding an antigen-binding receptor. As disclosed herein, the nucleic acid encoding a receptor, whether a chimeric antigen receptor or an antigen-binding receptor, may 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 the receptor-encoding nucleic acid are considered genetically modified stem cells as herein. T cells differentiated from genetically modified stem cells, and T cells manipulated to contain a nucleic acid encoding a genetically modified CAR or antigen-binding receptor, are also considered genetically modified T cells as herein.

[0126] References to “nucleic acid molecules” should be understood as references to deoxyribonucleic acid and its ribonucleic acid. The nucleic acid molecules in question can be any appropriate form of nucleic acid molecule, e.g., genome, cDNA, or ribonucleic acid molecule. Therefore, the term “expression” refers to the transcription and translation of DNA or RNA that results in the synthesis of peptides, polypeptides, or proteins. A DNA construct corresponds, for example, to a construct that can be transfected into a cell for subsequent expression, while an example of an RNA construct is an RNA molecule transcribed from a DNA construct, which requires only translation to produce the desired protein. References to “expression products” refer to the products produced from the transcription and translation of nucleic acid molecules.

[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 on which antigen-binding moieties are grafted 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, but not to limit the present invention, the most common of these molecules are fusions of monoclonal antibodies with a single-chain variable fragment (scFv) fused to the transmembrane CD3 zeta chain and endodomain. Such molecules transmit CD3 zeta chain signals in response to recognition of their target by the scFv. When a T cell expresses this chimeric molecule, the cell recognizes and kills target cells expressing the antigen to which the scFv is directed. For example, to target malignant B cells, T cell specificity was redirected using a chimeric immune receptor specific to CD19, a B-series molecule.

[0128] The variable regions of immunoglobulin heavy and light chains are generally fused by a mobile linker to form scFvs. These scFvs typically follow a signal peptide that directs the initial protein to the endoplasmic reticulum and then surface-expressed, which is ultimately cleaved. The mobile spacer orients the scFv in different directions to enable antigen binding. The transmembrane domain is generally a typical hydrophobic alpha-helix, usually derived from the original molecule of the signaling end domain that delivers the desired signal. Therefore, a reference to the “antigen-recognizing region” 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-recognizing domain is usually the scFv. However, many other options exist. For example, the antigen-recognizing region from the native T cell receptor (TCR) alpha and beta single-chain has also been used, as it possesses a simple extracellular domain (e.g., the CD4 extracellular domain for recognizing HIV-infected cells) and other recognition components such as linked cytokines (resulting in recognition of cells possessing cytokine receptors). In fact, any portion 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 a suitable molecule for use will also be well known to those skilled in the art. In designing chimeric antigen receptors, particularly the extracellular domain, those skilled in the art can include additional portions that are useful in carrying out effective expression or functioning. 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 portion. Although the present invention is not limited to any one theory or mechanism of action, the single peptide directs the initial protein to the endoplasmic reticulum. This is necessary if the receptor is glycosylated and immobilized on the cell membrane. Any eukaryotic single peptide sequence can be used. Generally, a single peptide that is naturally attached to the amino terminus is used (e.g., in scFv with a light-linker-heavy-chain configuration, the native signal of the light chain is used).In another example, the extracellular domain may also include a spacer region that can be used to link the antigen recognition domain to the transmembrane domain. The spacer region should be sufficiently mobile to orient the antigen recognition domain in different directions to facilitate antigen recognition and binding. The simplest form of the spacer region is the hinge region of IgG1. Alternatives include the CH2CH3 region and CD3 portion of immunoglobulins. In most 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 a 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 change its length, thereby achieving additional functional benefits. For example, in conventional CARs containing a CD8 or CD28 hinge, one cysteine ​​(Cys) can be left on 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), resulting in the inability to form a stabilizing disulfide bond, thereby preventing dimerization and thus premature activation. Cys can also be removed entirely. Another design involves displaying only the VH domain in one CAR and only the VL domain in the other, so that Cys pairing aligns the VH / VL to form a functional monovalent Fv that targets the desired antigen.

[0129] The antigen-recognition portion of the target chimeric antigen receptor is operably linked to the T cell activation portion. The “T cell activation portion” refers to the receptor subregion involved in sending signals into the T cell after antigen recognition and binding, enabling its activation and induction of effector mechanisms. The T cell activation portion of the CAR is generally located within the intracellular domain (or “endodomain”) of the CAR; therefore, the intracellular domain of the CAR molecule also generally contains, or is an intracellular signaling domain, an “intracellular signaling domain.” A commonly used endodomain component is the intracellular domain of CD3 zeta containing three ITAMs. After antigen binding, this domain sends an activation signal to the T cell. CD3 zeta alone may not provide a sufficiently capable activation signal, and additional co-stimulatory signaling is desirable. For example, chimeric CD28 and OX40 can be used with CD3 zeta to send proliferation / survival signals, or all three can be used together. It should be understood that this intracellular signaling domain of the CAR is involved in the activation of at least one normal effector function of immune cells, preferably T cells expressing the CAR. The term "intracellular signaling domain" refers to a portion of a protein that translates effector functional signals, directing the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire domain. When a shortened portion of an intracellular signaling domain is used, it can be used in place of the complete chain, as long as it translates the effector functional signal. Therefore, the term "intracellular signaling domain" is intended to include any shortened portion of an intracellular domain sufficient to translate the effector functional signal.

[0130] Preferred examples of intracellular signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act together after antigen receptor engagement to initiate signaling, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional potential.

[0131] It is well known that the signal generated by the TCR alone is insufficient for sufficient T cell activation, and secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: a class that initiates antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences) and a class that acts in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulating or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulating manner may contain signaling motifs known as immune receptor tyrosine-type activation motifs or ITAMs. Examples of ITAMs containing particularly useful primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In CAR, cytoplasmic signaling molecules are particularly preferably those that include cytoplasmic signaling sequences derived from CD3 zeta.

[0132] In preferred embodiments, the cytoplasmic domain of the CAR can be designed to contain a CD3 zeta signaling domain by itself or can be combined with any other desired cytoplasmic domain useful in relation to the CAR of the present invention. For example, the cytoplasmic domain of the CAR may include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that contains 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 an effective response of lymphocytes to an antigen. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, TIM3, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention may be linked to each other randomly or in a specified order. Optionally, short oligo or polypeptide linkers, preferably amino acids between 2 and 10 in length, can form the linkage. Glycine-serine doublets provide particularly suitable linkers. In one embodiment, the cytoplasmic domain is designed to include a CD3 zeta signaling domain and a CD28 signaling domain.

[0133] As detailed above, the antigen recognition portion is operably linked to the T cell activation portion. "Operatably linked" means that the antigen recognition portion is linked, bound, or associated with the T cell activation portion, so that the signal can be induced by the T cell activation portion to activate the target T cell and activate its effector function. This is achieved, for example, by designing 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 avoid such domain binding to the transmembrane domain of the same or different surface membrane proteins, 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-bound or transmembrane protein. For example, the transmembrane region can be derived from the alpha, beta, or zeta chains of the T cell receptor, or from immunoglobulins such as CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or IgG4 (i.e., including at least their cell transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case the domain will mainly consist of 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 an amino acid between 2 and 10 in length, can form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. The glycine-serine doublet provides a particularly suitable linker. Generally, the transmembrane domain is a hydrophobic alpha-helix spanning the membrane. Generally, the transmembrane domain from the component most proximal to the membrane of the endodomain is used.

[0135] References to “antigen-binding receptors” should be understood as references to engineered receptors that are immobilized on the cell surface and bind to antigens. Similar to the chimeric antigen receptors disclosed herein, the antigen-binding receptors disclosed herein also include an antigen-recognizing moiety directed to an antigenic determinant. The antigen-recognizing moiety in the antigen-binding receptor can take the same form as the antigen-recognizing moiety of the chimeric antigen receptors described herein and is designed in the same manner. Also similar to the chimeric antigen receptors disclosed herein, the antigen-recognizing moiety 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 immobilized on 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 the transmembrane domain of the chimeric antigen receptor described above. However, unlike chimeric antigen receptors, antigen-binding receptors as defined herein are generally non-signaling and may include intracellular sequences lacking a T-cell activation domain. Such non-signaling antigen-binding receptors can bind to antigens but do not induce any signaling in T cells and are therefore also called “docking receptors” or “anchoring receptors.” Certain embodiments of antigen-binding receptors, such as non-signaling CD47-binding receptors, are described further below in this specification.

[0136] Examples of nucleic acid constructs encoding CARs and / or antigen-binding receptors are shown in Figure 11, and typical 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 Sequence IDs 1 to 20.

[0137] Those skilled in the art will acknowledge that the mechanisms by which these genetic modifications are introduced into cells can take any appropriate form that is well known and understood by those skilled in the art. For example, genetic material is generally conveniently introduced into cells by the use of expression constructs.

[0138] In one embodiment, cells expressing a TCR (i.e., stem cells such as iPSCs or HSCs) that have the ability to differentiate into T cells expressing a TCR, or cells expressing a TCR that can lead to stem cells such as iPSCs, are transfected with an expression construct encoding a CAR. The expression construct may include one or more DNA regions containing a promoter operably linked to the nucleotide sequence encoding the CAR, a second DNA region encoding an optionally selectable marker, and optionally a third DNA region encoding a suicide protein. In this regard, it should be understood that, as a matter of common practice, constructs may be designed with one or more optional additional components, such as suicide genes, which those skilled in the art may consider useful. In relation to the cells of the present invention proposed for use in vivo to treat patients, the ability to control the death of the genetically modified cells of the present invention and thus carry out their removal from the in vivo environment is highly desirable. While the present invention is not limited to any one theory or mechanism of action, adoptive transfer of cells of the present invention is not without risk, in particular, insofar as they may be directed to “auto” 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, outcomes similar to graft-versus-host disease may occur. In the overall therapeutic scheme, these side effects may still be more desirable than the nonspecific systemic death of healthy tissue characteristic of treatments such as chemotherapy, or the uncontrolled death of healthy tissue in autoimmune disorders. However, while killing cancer cells is paramount, the ability to control the elimination of cells according to the present invention is highly desirable and can be routinely achieved by the very well-known and widely used technique of incorporating induceable suicide genes into gene constructs introduced into the stem / T cells of the present invention.

[0139] The target promoter may be constitutive or inductive. If the target construct expresses more than one target protein, these may be under the control of separate promoters, or they may be under the control of a single promoter, as occurs in association with a bicistronic vector using an IRES sequence that promotes translation of more than one protein product in an unfused form from a single RNA transcript. The target construct may be additionally designed to facilitate the use of Cre recombinase-mediated splicing-inducible gene expression systems.

[0140] References to nucleic acid "expression constructs" should be understood as references to nucleic acid molecules designed to be transmissible to cells and to be transcribed. RNA molecules are then transcribed from them. Generally, expression constructs are also referred to by a number of widely used, interchangeable alternative terms, including "expression cassettes" and "vectors."

[0141] For the purpose of introducing nucleic acids encoding multiple receptors, the nucleic acids encoding multiple receptors, whether the receptors are CARs, antigen-binding receptors, or a combination thereof, may be placed in a single construct to be transfected into cells. In one embodiment, the nucleic acids encoding multiple receptors may be contained in a polycistronic vector that uses IRES sequences to facilitate the translation of multiple receptor proteins. In another embodiment, the nucleic acids encoding multiple receptors may be linked together within a single expression unit and reading frame, for example, by utilizing a self-cleaving peptide (e.g., P2A) such that one single polypeptide containing multiple receptor sequences is produced first, followed by processing to produce multiple receptors. In yet 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 preferred method, including recombinant or synthetic techniques. For this purpose, the target constructs may be constructed using the first principle considered when a complete synthetic approach is employed, or by appropriately modifying existing vectors. If the latter approach is adopted, the range of vectors that can be used as a starting point is broad, but not limited to: (i) Plasmids: Plasmids are small, independently replicating fragments of cytoplasmic DNA, commonly found in prokaryotic cells, and possess the ability to replicate autonomously. Plasmids are commonly used in connection with 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 in the episome, or they may be integrated into the host genome. Examples of available plasmids include bacterial pBR322 and pUC. (ii) Bacteriophages: Bacteriophages are viruses that infect and replicate in bacteria. Generally, they consist of a nucleic acid core enclosed within a protein coat (called a capsid). Depending on the type of phage, the nucleic acid may be either DNA (single-stranded or double-stranded) or RNA (single-stranded), and they may be linear or circular. Phages may be filamentous, polyhedron, or polyhedron with tails, with one or more tubular tail fibers attached to a tubular tail. Generally, phages can accommodate longer fragments of foreign DNA than plasmids, for example. Examples of phages, but not limited to these, include E. coli lambda phage, P1 bacteriophage, and T even phage (e.g., T4). (iii) Baculoviruses: A group of various DNA viruses that replicate only in invertebrates, generally classified under the family Baculoviridae. Their genomes consist 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 fasmids: Generally, cosmids are plasmid-derived but also contain a cos site for lambda phage, while phagemids represent chimeric phage plasmid vectors. Fasmids generally also represent plasmid-phage chimeras, but are defined by the fact that they contain the origins of both functional replications. Fasmids can therefore be grown in suitable host strains as either plasmids or phages. (vii) Commercially available vectors that are entirely synthesized 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 depends on numerous factors, including its final use in the genetically modified cells in which it will be placed, insofar as this is chosen over synthetically generating the construct. For example, if the cells are to be administered to humans in vivo, the use of certain types of vectors, such as viral vectors, may be less desirable. Furthermore, the amount of DNA to be introduced into the construct also needs to be considered. It is generally understood that certain vectors are more readily transfected by 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. Yet another example is that larger DNA insertions that need to be inserted further limit the selection of vectors from which the expression construct of the present invention is generated. For this purpose, 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 attempted to express, the number of selection markers utilized, and the incorporation of properties 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, “circular” nucleotide sequences should be understood as referring to the circular nucleotide sequence portion of any nucleotide molecule. For example, a nucleotide sequence may be completely circular, such as a plasmid, or partially circular, such as the circular portion of a nucleotide molecule produced during rolling circle replication (this may be relevant, for example, when the construct is first 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, “circular” nucleotide sequences correspond to the circular portion of this molecule. “Linear” nucleotide sequences, which are essentially linear in form, should be understood as referring to any nucleotide sequence. A linear sequence may be a linear nucleotide molecule, or it may be the linear portion of a nucleotide molecule that also includes non-linear portions, such as a circular portion. Examples of linear nucleotide sequences, but not limited to, include plasmid-derived constructs linearized to facilitate integration into host cell chromosomes, or constructs synthetically produced in linear form. For this purpose, it should be understood that the stereochemistry of the constructs of the present invention may or may not remain constant. For example, a circular plasmid-derived construct may be transfected into cells as a stable circular episome that undergoes replication and transcription in this form. However, in another example, the construct of interest may be transfected into cells in a circular form but undergo linearization within the cell before integration into the chromosome. This is not necessarily an ideal situation, as such linearization may occur in a random manner, cleaving the construct in important regions and thereby rendering it ineffective.

[0145] The nucleic acid molecules used in the methods of the present invention are derived from any human or non-human source. Non-human sources intended by the present invention include primates, domestic animals (e.g., sheep, pigs, cattle, 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), captured wild or domesticated animals (e.g., bulls, kangaroos, dingoes), reptiles, fish, insects, prokaryotes, or synthetic nucleic acids.

[0146] It should be understood that constructs encoding the receptors of the present invention may comprise nucleic acid material from more than one source. For example, while a construct may be derived from a specific microorganism, nucleic acid material from other microplastic sources may be introduced in modifying the construct to introduce the properties defined herein. These sources include, for example, viral or bacterial DNA (e.g., IRES DNA), mammalian DNA (e.g., CAR-coding DNA), or synthetic DNA (e.g., for introducing specific restriction endonuclease sites). Furthermore, the cell types to which the construct of interest is proposed to be expressed may be even more different, not corresponding to the same organism in whole or in part, of the nucleic acid material of the construct. For example, a construct essentially consisting of bacterial and viral DNA may nevertheless be expressed in mammalian stem cells as intended herein.

[0147] Without limiting the present invention in any way, the present invention preferably uses a DNA construct comprising a CAR sequence, the sequence comprising a nucleic acid sequence of an antigen-binding portion operably linked to a nucleic acid sequence of an intracellular domain. For example, the intracellular domain that may be used in a target CAR is, 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] Retroviral vectors, such as lentiviruses, are examples of suitable vectors for achieving long-term gene transfer because they enable the long-term stable integration of the transgene and its proliferation 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 promoters useful for controlling the expression of transcription and translation termination factors, start sequences, and desired nucleic acid sequences. Viral vector technology is well known in the art and is described, for example, by Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally preferred vectors contain a replication origin, promoter sequence, convenient restriction endonuclease site, 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] Numerous virus-based systems have been developed for gene transfer into mammalian cells. Retroviruses, for example, provide a favorable platform for gene delivery systems. Selected genes can be inserted into vectors using techniques known in the art and packaged into retroviral particles. Recombinant viruses can then be isolated and delivered to target stem cells. Numerous 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 initiation site, although recent studies have shown that many promoters also contain functional elements downstream of the initiation site. Spacing between promoter elements is often mobile, so promoter function is conserved even if the elements are inverted or moved relative to one another. In thymidine kinase (TK) promoters, 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] A suitable promoter is the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably ligated to it. Another suitable promoter is elongation growth factor la (EF-la). However, other constitutive promoter sequences, though not limited to these, may be used, including the monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Roussarcoma virus promoter, and, though not limited to these, human gene promoters such as actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, constructs should not be limited to the use of constitutive promoters. Inducible promoters are also intended to be used. The use of inductive promoters provides a molecular switch that has the ability to turn on the expression of the CAR polynucleotide sequence it operably ligated to when its expression is desired and turn it off when its expression is undesirable. Examples of inductive promoters, but not limited to these, include metallothionine promoters, glucocorticoid promoters, and progesterone promoters. Examples include sterone promoters and tetracycline promoters.

[0152] To evaluate the expression of a CAR polypeptide or a portion thereof, an expression vector introduced into cells may contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells attempted to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be carried on another small piece of DNA and may be used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as Neo. Epitope tags may 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 by 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 evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that encode polypeptides that are not present or expressed in the recipient organism or tissue, and whose expression manifests through several readily detectable characteristics, such as enzymatic activity. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into recipient cells. Suitable reporter genes include those encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000). FEBS Letters Vol. 479: pp. 79-82). Suitable expression systems are well known and known techniques. These can be prepared using techniques or commercially available. Generally, a construct having the minimum 5' adjacent region exhibiting the highest expression level of the reporter gene is identified as a promoter. Such promoter regions may be ligated to the reporter gene and used to evaluate drugs for their ability to regulate promoter-driven transcription. Reporters such as eGFP (enhanced green fluorescent protein) may be isolated by a self-cleaving peptide such as P2A and incorporated into the CAR as a C-terminal polypeptide elongation, and it is understood by those skilled in the art that reporters such as eGFP can be released into cells.

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

[0155] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, microparticle guns, microinjection, and electroporation. 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] The use of DNA and RNA vectors is a biological method for introducing a target polynucleotide into host cells. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, for example. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0157] Chemical means for introducing polynucleotides into host cells include colloidal dispersant systems such as macromolecular complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle is liposomes (e.g., artificial membrane vesicles).

[0158] When nonviral delivery systems are attempted, the exemplary delivery vehicle is a liposome. The use of lipid formulations is intended for the introduction of nucleic acids into host cells. In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via linking molecules that associate with both the liposome and the oligonucleotide, captured within a liposome, complexed with a liposome, dispersed in a lipid-containing solution and mixed with the lipid, combined with the lipid, contained as a suspension in a lipid, contained in or complexed in 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 may exist in bilayer structures such as micelles, or in "disintegrated" structures. They may be simply dispersed in solution and 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 naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing fatty acids, alcohols, amines, amino alcohols, aldehydes, and other long-chain aliphatic hydrocarbons and their derivatives.

[0159] Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("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; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Storage solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the only solvent that evaporates more readily than methanol. "Liposomes" is a general term encompassing various single and multi-membrane lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization 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 that of normal vesicles. Compositions are also included. For example, lipids can exist as micelle structures or simply as heterogeneous aggregates of lipid molecules. Similarly, lipofectamine nucleic acid complexes are also considered.

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

[0161] In some embodiments, the TCR and CAR and antigen-binding receptors of these cells are directed to antigenic determinants, respectively. The reference to “antigenic determinant” should be understood as a reference to any proteinaceous or non-proteinaceous molecule expressed by a cell that is attempted to be targeted by the receptor-expressing T cells of the present invention. It is understood that these molecules may be “self” molecules that are normally expressed in a patient’s body (e.g., expressed in some tumor cells or autoreactive cells) or non-self molecules (e.g., viral proteins) that are expected when a cell is 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, the reference to “antigen” or “antigenic determinant” refers to any proteinaceous or non-proteinaceous molecule that is attempted 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. Nevertheless, targeting this antigen may be desirable (in terms of incidental damage) to minimize the possibility of more severe side effects that may be observed in highly nonspecific and systemic treatments such as chemotherapy or immunosuppression, or to reduce the duration of treatment through highly targeted treatment, and / or to maximize the possibility of killing all unwanted cells. Preferably, the molecule is expressed on the cell surface.

[0162] It is understood by those skilled in the art that, at the TCR binding site, the target antigen determinant takes the form of an antigen-derived peptide expressed at either the MHC I or MHC II site. In relation to 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 native form of the antigen. The target epitope may be linear or conformational. It should be understood that the target antigen determinant may be any molecule expressed by the cell being attempted to target; that is, the targeted molecule may be exclusively expressed by the target cell or similarly expressed by non-target cells. Preferably, the target antigen determinant is a non-self antigen determinant or an antigen determinant that is otherwise exclusively expressed by the cell being attempted to target, or expressed at significantly higher levels than by normal cells. However, as already discussed herein, depending on the disease state being treated, it is not always possible to identify and target non-self antigen determinants.

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

[0164] Thus, in one embodiment, there is provided a genetically modified mammalian stem cell or a T cell differentiated therefrom, the cell expressing at least one homozygous HLA haplotype and having the ability to differentiate into a T cell expressing a TCR directed to a first antigen 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 portion directed to a second antigen determinant, said antigen recognition portion being operably linked to a T cell activation portion and optionally further comprising a nucleic acid encoding an antigen-binding receptor directed to a third antigen determinant, said antigen determinant being selected from a tumor antigen, a microbial antigen, or an autoreactive immune cell antigen.

[0165] In one embodiment, said stem cell is an iPSC. In another embodiment, the stem cell is an HSC.

[0166] In yet another embodiment, said stem cell, such as an iPSC, + has the ability to differentiate into a CD4 + T cell or a CD8

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

[0168] In yet another embodiment, said stem cells, such as iPSCs, are derived from T cells or thymocytes, preferably CD8 + T cells or thymocytes.

[0169] As those skilled in the art will understand, the identification of tumor-specific antigens is an important area of ​​research, but progress in this area has been limited. Since tumor cells are usually autologous cells (as opposed to tumors arising from transplanted tissue, for example), the antigens they express are not exclusively autoantigens, but are likely to also be expressed by non-neoplastic cells in the tissue from which the tumor originates. This is clearly not ideal due to the potential and unavoidable side effects (in terms of the destruction of non-neoplastic tissue) that can occur when antineoplastic 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, even if they are not exclusively expressed by tumor cells.

[0170] The selection of the antigen-binding moiety of the present invention depends on the specific type of cancer being 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, and mut. Examples include hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-LA, p53, prostain, PSMA, Her2 / neu, survivor 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 ("phagocytic rejection" receptor) is also a tumor target because it is often highly expressed in cancer cells compared to normal cells, specifically scavenger macrophages, which prevent these cancer cells from being attacked by immune system cells.

[0171] In one embodiment, a 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 antigens (CEAs). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute true tumor-specific immunoglobulin antigens that are distinct to individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B-cell lymphoma.

[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 multiseries 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 arising from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as the Epstein-Barr virus antigen (EBVA) and 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, B Examples 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, cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0173] The cells of the present invention are designed to be directed to multiple, i.e., two or more, antigenic determinants. As detailed herein, the multiple antigenic determinants may be, or comprise, multiple epitopes of a single molecule in some embodiments, or multiple epitopes of completely different molecules in other embodiments. The selection of which of the multiple antigenic determinants should be targeted, and furthermore, whether they should be targeted by TCRs or CARs, is well within the scope of the art. In one embodiment, the cells of the present invention are designed to eliminate tumor cells, and the TCR / CAR is directed to 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 to idiotype T cell or B cell receptors.

[0174] Accordingly, in one embodiment, genetically modified mammalian stem cells or T cells differentiated therefrom are provided, the cells having the ability to differentiate into T cells expressing a TCR directed to a first tumor antigenic determinant, comprising one or more nucleic acid molecules encoding one or more chimeric antigen receptors, each chimeric antigen receptor comprising an antigen-recognizing portion directed to a tumor antigenic determinant, the antigen-recognizing portion being operably linked to a T cell activation portion, the antigenic determinant being selected from TAG72, CD47, CD19, WT-1, MAGE, and EBVLMP2.

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

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

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

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

[0179] In yet another embodiment, the stem cells (such as iPSCs) are T cells or thymocytes, preferably CD8 + They are derived from T cells or thymocytes.

[0180] In one embodiment, a broad range of CARs were developed to target known tumor antigens, within the scope in which the cells of the present invention are directed to treat neoplasms. A non-limiting summary illustrating some of these CARs, along with their receptor structures, is provided in Table 5 below: [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 to CD19 or TAG72, a hinge (stalk) region and a transmembrane region both derived from CD28 or CD8, and a cytoplasmic endodomain also derived from CD28 or CD8 containing a T cell activation moiety. The CAR may include a reporter protein (such as EGFP) as a C-terminal polypeptide elongation, linked together by a P2A self-cleaving polypeptide for post-translational EGFP release. See, for example, Figures 11 and 14.

[0182] In related aspects, 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 signaling. Expression of the CD47-binding molecule on the cell surface anchors the cells of the present invention to the neoplastic cells they are targeted to, thereby facilitating improved interaction of their respective ligands with the TCR and CAR. With respect to the treatment of solid tumors, specifically, the increased stability and binding affinity of the interaction of the target cells enables an improved functional outcome with respect to neoplastic cell death as compared to cells that do not express the target CD47-binding molecule.

[0183] Thus, in related aspects of the present invention, there are provided genetically modified mammalian stem cells, or T cells differentiated therefrom, the cells having the ability to differentiate into T cells expressing a TCR directed to a first antigen determinant, and comprising (i) a nucleic acid molecule encoding a chimeric antigen receptor, said receptor comprising an antigen recognition portion directed to a second antigen determinant, said antigen recognition portion being 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 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 angiogenesis responses. CD47 is ubiquitously expressed in human cells and has been found to be overexpressed in a number of 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 the cytoplasmic tail. 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 primarily expressed in the brain and peripheral nervous system. Only keratinocytes express significant amounts of form 1. These isoforms are highly conserved between mouse and human, suggesting an important role for the cytoplasmic domain in CD47 function.

[0186] CD47 is a receptor for thrombospondin-1 (TSP-1), a secreted glycoprotein that plays a role in vascular development and angiogenesis. TSP-1 binding to CD47 influences several fundamental cellular functions, including cell migration and adhesion, cell proliferation, and apoptosis, playing a role in regulating 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 signaling, leading to various intercellular responses, including inhibition of phagocytosis (promoting cancer cell escape), stimulation of intercellular fusion, and T cell activation. Furthermore, CD47 interacts with several membrane integrins, most commonly integrin avb3. These interactions create CD47 / integrin complexes that influence a wide range of cellular functions, including adhesion, diffusion, and migration.

[0187] However, although CD47 is expressed eccentrically, it was determined that an increase in CD47 expression levels in neoplastic cells is sufficient to promote and eliminate the responsiveness of neoplastic cells by CD47-targeting molecules, prior to any inherent adverse effects on non-neoplastic cells.

[0188] Referring to a "binding receptor" directed towards CD47 should be understood as referring to any receptor that interacts with CD47. This can take the form of a CD47-binding receptor, such as a surface-presented antibody fragment, and preferably lacks signaling function.

[0189] This embodiment provides genetically modified mammalian stem cells, or T cells differentiated therefrom, the cells having the ability 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, wherein the receptor comprises an antigen-recognizing portion directed to a second antigenic determinant, the antigen-recognizing portion being operably linked to a T cell activating portion; and (ii) a nucleic acid molecule encoding a non-signaling antigen-binding receptor, wherein the receptor comprises an antigen-recognizing 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-signaling receptor. "Non-signaling" means that following the binding of the target receptor to CD47 on the target cell, there is no signaling that would alter the function of the cell in this invention. Rather, the purpose of the CD47-binding receptor is to provide improved anchoring of the target cell to the target cell, thereby improving the effectiveness of binding of TCRs and CARs directed to target antigen moieties such as tumor antigen moieties.

[0191] For example, in one design of a non-signaling antigen-binding receptor, the extracellular domain of the receptor includes an antigen-recognition moiety with binding specificity to CD47, a hinge (Stork) domain, a transmembrane domain, and an intracellular domain that completely lacks cytoplasmic signaling function. Such a non-signaling CD47-binding receptor can be used simply for adhesion rather than for signaling, thereby driving the docking of T cells to cancer cells via CD47 binding without the undesirable activation and death that would occur if it were engaged with normal CD47-expressing cells.

[0192] In some embodiments, the antigen-recognizing moiety of the non-signaling CD47-binding receptor may include antibody-like domains such as scFv, Fv, and Fab, as well as any CD47-targeting V-domains including single human and mammalian V-domains and their equivalents (VhH or vNAR) domains, or may include, but not limited to, dalpin, anticarin, notchin, ImmE7s, aphibodies, and Fn3 fibronectin domains, which are well known in the art. The antigen-recognizing moiety may also include one or more V-like domains (natural ligands for CD47) of SIRPα. In one embodiment, the antigen-recognizing moiety may include the natural V-like domains of SIRPα. In another embodiment, the antigen-recognizing moiety may include all three natural V-like domains of SIRPα. In other embodiments, a suitable molecule for use in providing an antigen-recognizing moiety 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 using three different versions of VH (1,2,3) and three different versions of VL (11,12,13), as shown in Figures 12A and 12B, published as U.S. Patent Application No. 14 / 656,431, U.S. No. 20150183874 A1. Liu et al. (PLOS One (2015) Sept. 21; 10(9):e0137345) described Hu5F9-G4, in which the selected V-domain was a heavy chain VH-2 containing four distinct residue changes in the framework (differentiating VH-2 from VH-1,3) and a light chain VL-12 containing two distinct residue changes in the framework (differentiating VL-12 from VL-11,13).

[0193] In some embodiments, the hinge region of a non-signaling CD47-binding receptor may be a native SIRPα hinge sequence, or a CD8 or CD28 hinge typically used in CARs, or an alternative hinge known in the art, such as a CD4 domain or mucin peptide hinge. The hinge region may be designed to include one or more cysteine ​​(Cys) residues to enable receptor dimerization. CD28 is a native 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 it can provide additional stabilization to the dimer.

[0194] It is understood by those skilled in the art that the introduction of nucleic acids encoding CARs and non-signaling antigen-binding receptors (such as non-signaling CD47-binding receptors) into cells (e.g., T cells or iPSCs) can be achieved using two separate transfection vectors, a single bisistronic vector, or a single gene encoding an internal cleavage signal for separating the CAR from the antigen-binding receptor. In one embodiment, the internal cleavage signal is P2A, a peptide sequence that directs self-cleavage for separating 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 post-translation by the P2A self-cleaving peptide for separating the CAR from the CD47-binding receptor.

[0195] The means for modifying stem cells of the present invention to also express non-signaling CD47-binding molecules are described in great detail above herein in terms of their effect on the expression of chimeric antigen receptors directed to tumor antigen moieties. Transfection and other methods for achieving receptor expression described herein will be understood by those skilled in the art as being equally applicable in relation to the target CD47-binding molecules.

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

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

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

[0199] In yet another embodiment, the stem cells, such as iPSCs, are T cells or thymocytes, preferably CD8 cells. + CD8 cells derived from T cells or thymocytes, having an endogenous TCR that is directed towards tumor antigens in some embodiments. + They are derived from T cells or thymocytes.

[0200] In yet another embodiment, the stem cells are directed towards TAG72 and WT1. Even more preferably, the CAR is directed towards TAG72 and the TCR is directed towards WT1.

[0201] In a further embodiment, a method for producing genetically modified mammalian stem cells is provided. Various means for producing genetically modified mammalian stem cells, specifically iPSCs, are described herein.

[0202] In further embodiments, T cells expressing a TCR directed to a first antigenic determinant and a chimeric antigen receptor are provided, the receptor comprising an antigen-recognizing portion directed to a second antigenic determinant, the antigen-recognizing portion being operably linked to a T cell activation portion. In some embodiments, the T cells express at least one homozygous HLA haplotype.

[0203] In one embodiment, T cells express multiple chimeric antigen receptors, each chimeric antigen receptor containing an antigen-recognizing portion directed to an antigenic determinant, and the antigen-recognizing portion is operably linked to the T cell activation portion.

[0204] In one embodiment, the multiple antigenic determinants targeted by the multiple chimeric antigen receptors are each different from the first antigenic determinant targeted by the TCR expressed on the target T cell. In another embodiment, the multiple antigenic determinants targeted by the multiple chimeric antigen receptors are each different and are also different from the first antigenic determinant targeted by the TCR expressed on the target T cell.

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

[0206] In another embodiment, 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 to a third antigenic determinant.

[0207] In one embodiment, the antigen-binding receptor is a non-signaling antigen-binding receptor; that is, the receptor anchors to the cell surface of a target T cell and binds to a third antigenic determinant, but does not translate the signal to the cytoplasmic portion of the T cell that affects T cell function (for this reason, it is also called a non-T cell signaling antigen-binding receptor). In one embodiment, the antigen-binding receptor includes an antigen-recognition moiety that is directed to the third antigenic determinant and operably linked to a transmembrane domain, but lacks a T cell activation moiety.

[0208] In specific embodiments, the antigen-binding receptor is a non-signaling antigen-binding receptor directed to CD47. For example, 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 the αβTCR. In other embodiments, the T cells provided herein express the γδTCR.

[0211] In some embodiments, the antigenic determinants to which the target T cell is directed, namely, a first antigenic determinant to which the TCR is directed, a single or multiple antigenic determinants to which a chimeric antigen receptor(s) is directed, and a single or multiple antigen-binding receptor(s), may be selected from tumor antigens, microbial antigens, or autoreactive immune cell antigens, if such antigen-binding receptor(s) are present. In a particular embodiment, the antigenic determinants are selected from tumor antigens. In a specific embodiment, the antigenic determinants to which the TCR is directed are selected from TCR-recognizing peptides such as WT-1 or EbvLMP2. In other specific embodiments, the antigenic determinants to which the chimeric antigen receptor and antigen-binding receptor are directed may be selected from, for example, TAG72, CD19, MAGE, or CD47.

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

[0213] In one embodiment, the iPSC or HSC derived from the target T cell is a genetically modified iPSC or HSC that expresses a TCR directed to the first antigenic determinant, comprises one or more nucleic acids encoding one or more chimeric antigen receptors, and optionally comprises one or more nucleic acids encoding antigen-binding receptors. In another embodiment, the iPSC or HSC derived from the target T cell has the ability to differentiate into a T cell expressing a TCR directed to the first antigenic determinant; one or more nucleic acids encoding one or more chimeric antigen receptors and optionally one or more nucleic acids encoding antigen-binding receptors are introduced after the iPSC or HSC has differentiated into a T cell. In some embodiments, the iPSC or HSC derived from the target T cell expresses 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 iPSC from which the target T cell originates is itself derived from a T cell or thymocyte. In one embodiment, the iPSC is derived from a CD8+ T cell or thymocyte. In one embodiment, the iPSC is derived from a T cell or thymocyte expressing a TCR directed to a first antigenic determinant, i.e., the TCR of the target T cell derived from the iPSC is directed to the same antigenic determinant.

[0215] The value of the cells of this invention is CD4 + or CD8 +This is based on directing the differentiation of target stem cells to T cells. In this context, "directing" the differentiation of stem cells to T cells should be understood as meaning that a cell culture system is applied that commits the stem cells to the T cell lineage and induces differentiation into mature T cells along that cell lineage. Means for bringing about directed differentiation of stem cells along the T cell lineage are well known to those skilled in the art. For example, as illustrated herein, it is known that introducing Notch-dependent signaling into a culture system results in directed differentiation of stem cells along the T cell lineage. Furthermore, particularly efficient differentiation is achieved when this signaling is provided to stem cells in the context of their co-culture in 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 context, OP-9 cells are engineered to express Delta-like 1 (OP9-DL1), thereby providing a very convenient means of generating T cells from stem cells. In another example, and as illustrated herein, target stem cells are initially cultured under feeder-free conditions that generate mesoderm, followed by co-culture in an OP9-DL1 cell line. CD8 + Particularly preferred methods for achieving T cell-directed differentiation are illustrated herein.

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

[0217] In one embodiment, the method comprises obtaining genetically modified stem cells (such as genetically modified iPSCs or HSCs) having the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant, each comprising one or more nucleic acids encoding one or more chimeric antigen receptors, each directed to an antigenic determinant (preferably different from the first antigenic determinant), and optionally one or more nucleic acids encoding one or more antigen-binding receptors, each directed to an antigenic determinant (preferably different from the first antigenic determinant); and differentiating such genetically modified stem 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) having the ability to differentiate into T cells expressing a TCR directed to a first antigenic determinant; differentiating the stem cells into T cells; and introducing into the T cells one or more nucleic acids (one or more) encoding one or more chimeric antigen receptors, each directed to an antigenic determinant (preferably different from the first antigenic determinant), and optionally one or more nucleic acids encoding one or more antigen-binding receptors (one or more) each directed to 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-coding nucleic acid is introduced into stem cells before differentiation into T cells or after differentiation from stem cells, the stem cells (such as iPSCs) may themselves be of T cell or thymocyte origin. Such T cells and thymocytes may have TCRs specific to nominal antigens, such as tumor antigens. In one embodiment, the stem cells are iPSCs. In one embodiment, the iPSCs are of CD8+ T cell or thymocyte origin. In another embodiment, the iPSCs are of T cell or thymocyte origin expressing a TCR that is directed to the same antigenic determinant as the TCR expressed on iPSC-derived T cells.

[0220] It should be understood that referring to "mammals" is not limited to these, but includes, but encompasses, humans, primates, domestic animals (e.g., sheep, cattle, horses, donkeys, pigs), companion animals (e.g., dogs, coats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captured wild animals (e.g., foxes, deer). Preferably, the mammal is human or primate. Most preferably, the mammal is human.

[0221] The development of the present invention is currently accelerating the development of means for treating disease conditions characterized by the presence of unwanted cell populations, such as neoplastic populations of cells, virus-infected cells, autoreactive immune cells, or infections by microorganisms such as antibiotic-resistant bacteria. More specifically, the cells of the present invention provide 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 diseases, or immunosuppression for managing autoimmunity. In this regard, referring to a disease condition “characterized by the presence of unwanted cell populations” should be understood as referring to any condition in which 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, is the symptom or cause. Treatment of the condition in question is achieved by administering T cells differentiated from the stem cells of the present invention, the dual TCR / CAR of the T cells being directed to two or more antigenic determinants expressed by the cells to be eliminated.

[0222] It should be understood that the “cells” to be eliminated by the T cells of the present invention may be any cells, whether self or non-self. For example, to the extent that the T cells of the present invention are designed to treat disease conditions such as neoplasms, viral infections, or autoimmune diseases, the target population of cells to be eliminated is self-cells. However, to the extent that the condition to be treated is, for example, an infection by a microorganism such as antibiotic-resistant bacteria or a parasite, the “cells” to be eliminated are foreign cells. In this regard, the cells may be in a suspension (e.g., leukemia cells present in circulation) or as part of a tumor (e.g., a tumor or tissue). To the extent that the condition to be treated is a microbial infection, the cells may correspond to single-celled microorganisms (e.g., many bacteria) or as 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] Therefore, another aspect of the present invention is directed toward a method for treating a condition characterized by the presence of an unwanted population of cells in a mammal, as defined above, the method comprising administering an effective amount of stem cells or T cells differentiated therefrom to the mammal.

[0224] In one embodiment, the state is a neoplasm, a microbial infection (such as HIV, an STD, or antibiotic-resistant bacteria), or an autoimmune state.

[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 + It has the ability to differentiate into T cells.

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

[0228] In yet another embodiment, the 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, the receptor comprising an antigen-recognizing moiety directed to CD47.

[0230] In one specific aspect of these embodiments, a method is provided for treating a neoplasm, the method comprising administering an effective number of stem cells or T cells differentiated therefrom to the mammal, as defined above, wherein the TCR is directed to a first tumor antigen determinant and the CAR is directed to one or more additional tumor antigen determinants.

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

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

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

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

[0235] When referring to a “neoplastic state,” it should be understood as referring to a condition characterized by the presence or development of encapsulated or unencapsulated growth or aggregation of neoplastic cells. When referring to “neoplastic cells,” it should be understood as referring to cells exhibiting abnormal growth. The term “growth” should be understood in a broad sense, including the 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 nucleus / cytoplasmic ratio of individual cells, an increase in the rate of cell division, an increase in the number of cell divisions, a decrease in the length of the cell division period, an increase in the frequency of cell division periods or uncontrolled proliferation, and avoidance of apoptosis. Without limiting the invention in any way, the general medical meaning of the term “neoplasm” refers to “new cell growth,” e.g., neoplastic cell growth, that arises as a lack of responsiveness to normal growth management. 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 unencapsulated mass or other growth form or cellular aggregate containing neoplastic cells.

[0237] In relation to the present invention, the term “neoplasm” should be understood to include all types of cancerous growth or carcinogenic processes, metastatic tissue or malignant transformed cells, tissues or organs, regardless of their histopathological type or invasiveness.

[0238] Those skilled in the art will recognize that the term "cancer" refers to malignant tumors of epithelial or endocrine tissue, including respiratory cancers, gastrointestinal cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. The term includes carcinosarcoma, which includes, for example, malignant tumors consisting of cancerous and sarcomatous tissues. "Adenocarcinoma" refers to cancer or tumor cells of glandular origin that form recognizable glandular structures.

[0239] Neoplastic cells, including neoplasms, may be any cell type derived from any tissue, such as epithelial or non-epithelial cells. The terms “malignant neoplasm,” “cancer,” and “carcinoma” are to be understood as interchangeable in this specification.

[0240] The term “neoplasm” should be understood to refer to a lesion, tumor or other encapsulated or unencapsulated mass or other growth form or cellular aggregate containing neoplastic cells. Neoplastic cells, including the neoplasm, may be 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, neuroblastic carcinoma, pediatric tumors, head and neck cancers (e.g., squamous cell carcinoma), breast and prostate cancers, lung cancers (both small cell and non-small cell lung cancers), kidney cancers (e.g., renal cell adenocarcinoma), esophageal and gastric cancers, hepatocellular carcinoma, pancreatic and biliary duct neoplasms (e.g., adenocarcinoma and islet cell tumors), colorectal cancers, cervical and anal cancers, uterine and other reproductive system cancers, urinary tract cancers (e.g., ureters and bladder), germ cell tumors (e.g., testicular germ cell tumors or ovarian germ cell tumors), ovarian cancers (e.g., ovarian epithelial carcinoma), cancers of unknown primary origin, human immunodeficiency-associated malignancies (e.g., Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumors (e.g., thyroid), mesotheliomas and other pleural or peritoneal tumors, neuroendocrine tumors and carcinoid tumors.

[0241] In one specific embodiment, the neoplasm is leukemia or lymphoma.

[0242] In another embodiment, the neoplasm is transmissible.

[0243] The subject receiving treatment or prevention may be any human or animal in need of therapeutic or preventive treatment. In this regard, “treatment” and “prevention” as used herein may be considered in their broadest sense. The term “treatment” does not have to mean that a mammal is treated until complete recovery. Similarly, “prevention” does not have to mean that the subject will never contract the disease condition. Therefore, treatment and prevention include recovery from the symptoms of a particular condition, or preventing or otherwise reducing the risk of developing a particular condition. The term “prevention” may be considered to reduce the severity of developing a particular condition. “Treatment” may also reduce the severity of an existing condition.

[0244] Therefore, the present invention should be understood to encompass reducing or otherwise improving a condition in mammals. This should be understood as referring to the reduction or improvement of any one or more symptoms of a disease. While achieving a cure for a disease is always the most desirable outcome, slowing the progression of the disease also has significant clinical value. For example, in the context of viral infections such as HIV or STDs, even if a complete cure cannot be achieved, reducing the degree of viral load and spread can provide a means of managing the infection such 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 that characterize the antiviral drug cocktails that patients may need to 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. While "growth" of a cell or neoplasm should be understood as referring to the proliferation, differentiation, and / or maintenance of the viability of the cell in question, "downregulation of growth" of a cell or neoplasm refers to reducing, preventing, or suppressing the senescence process of the cell or the proliferation, differentiation, and / or maintenance of the viability of the cell in question. In a preferred embodiment, the growth of the subject is proliferation, and the downward control of the subject is CD8 +This is T cell-mediated death. In this regard, death can be demonstrated by a reduction in the size of the tumor mass, or by the suppression of further tumor growth, or by the delay in tumor growth. In this regard, without limiting the present invention to any theory or mode of action, neoplastic cells are directly lysed or apoptotically induced or CD4 + Or CD8 + They can be killed by any preferred mechanism, such as several other mechanisms that may be facilitated by T cells or T cells lacking these CD4 and CD8 markers. Therefore, the present invention should be understood to encompass reducing or otherwise restoring neoplasms in mammals. This should be understood as referring to the prevention, reduction, or restoration of any one or more symptoms of a neoplasm. Symptoms include, but are not limited to, pain at the site of tumor growth or metabolic or physiological impairment due to the neoplasm. It should be understood that the methods 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 methods of the present invention also extend to the prevention of the onset of any one or more symptoms.

[0245] Therefore, the methods of the present invention are useful from both therapeutic and palliative standpoints. For this purpose, the term “treatment” should be understood to encompass both therapeutic and palliative care. As will be understood by those skilled in the art, while the most desirable outcome is always the cure of a neoplasm, being able to slow or halt the progression of a neoplasm is significantly beneficial, even if it does not result in a complete cure. Without limiting the present invention in any way, there are certain neoplasms that, under conditions where they are sufficiently downregulated with respect to cell division, are not lethal to the patient and the patient can still have a reasonable quality of life. Furthermore, it should be understood that the methods provide useful alternatives to existing treatment regimens. For example, in some situations, the therapeutic outcomes of the methods 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 more well tolerated by the patient, is in the patient’s interest. As detailed above, it should be understood that the term “treatment” does not necessarily mean that the subject is treated until complete recovery. Therefore, as detailed above, treatment includes reducing the severity of an existing condition or inducing recovery or relief of the symptoms of a particular condition. In this regard, when the treatment of the present invention is applied when a primary tumor is treated, it can effectively function as a preventive measure to prevent the development 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 completely 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 preventive measure to prevent metastatic spread.

[0246] In this aspect of the present invention, the target cells are preferably autologous cells that have been isolated, genetically modified ex vivo, and transplanted back into the original organism from which they were collected. However, it should be understood that the present invention also extends to the use of cells from any other suitable source if the target cells exhibit a histocompatibility profile similar to that of the organism being treated, thereby enabling them to perform their function of removing unwanted cells before the transplanted cells are subjected to immune rejection by the host. Thus, such cells are practically autologous and do not raise the histocompatibility issues typically associated with the transplantation of cells exhibiting exogenous MHC profiles. Such cells should be understood as falling within the definition of histocompatibility. For example, under certain circumstances, it may be desirable, necessary, or actually important that the target cells are isolated from genetically identical twins or from embryos produced using gametes derived from the target organism, or cloned from the target organism (in which case the cells may correspond to stem cells directed toward differentiation into appropriate somatic cell types). The cells may be manipulated to exhibit a desired major histocompatibility profile. The use of such cells overcomes the inherent difficulties encountered in the context of tissue and organ transplantation.

[0247] However, when it is possible or not 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 exhibiting a different MHC profile. While the use of such cells in a therapeutic context may result in transplant-versus-host problems or host rejection of the graft, this problem can nevertheless be minimized by using cell populations that exhibit an MHC profile similar to that of the subject being treated, isolated / generated from relatives such as siblings, parents or children, or otherwise generated by methods illustrated herein.

[0248] In preferred embodiments, it is understood that the cells used are autologous. However, due to the circumstances of a given situation, it may not always be possible to generate an autologous stem cell population. This may be due to challenges such as the urgency of initiating treatment or the effectiveness of facilitating transformation and differentiation. In this case, and as detailed above, it may be desirable or necessary to use syngeneic or homogeneous cells, such as cells that have already been transfected and are available as cryopreserved products in a cell bank. Homogeneous but 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 illustrated herein.

[0249] To refer to the “effective number” means the number of cells necessary to at least partially achieve the desired effect, or to delay the onset, inhibit the progression, or completely halt the onset or progression of the specific condition being treated. Such a quantity naturally depends on the specific condition being treated, the severity of the condition, and the parameters of the individual patient, including age, general condition, size, weight, physiological status, concomitant therapies, 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 constituting the effective dose and the optimal mode of administration without excessive experimentation, the latter of which will be further discussed below. These factors are well known to those skilled in the art and can be addressed by routine experimentation alone. It is generally preferable to use the maximum number of cells, i.e., the most safe number based on sound medical judgment. However, it will be understood by those skilled in the art that a smaller number of cells may be administered for medical, psychological, or any other reason.

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

[0251] In relation to this aspect of the present invention, the target cells require introduction into a target organism. For this purpose, the cells may be introduced by any preferred method. For example, cell suspensions may be introduced by direct injection or in a blood clot, thereby immobilizing the cells within the clot and facilitating transplantation. Cells may also be introduced by surgical transplantation. This may be necessary, for example, when the cells exist in the form of a tissue graft. The transplantation site may be any preferred site, such as subcutaneously. Without limiting the present invention to any theory or mode of action, when cells are administered as encapsulated cell suspensions, the cells are aggregated into a mass. It should be understood that cells may continue to divide after transplantation. In this regard, as already described herein, the introduction of suicide genes provides a convenient means for managing ongoing division.

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

[0253] In the method of the present invention, other proteinaceous or non-proteinaceous molecules may be co-administered with the introduction of transformed cells. "Co-administration" means simultaneous administration via the same or different routes in the same or different formulations, or sequential administration via the same or different routes. "Sequential" administration means a time difference of several 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 with drug therapy to alleviate the symptoms of the condition until the transplanted cells have integrated and become fully functional (e.g., administration of antiviral drugs in the case of HIV patients). Alternatively, it may be necessary to initiate long-term drug therapy at the time the condition is treated to prevent relapse of the condition. For example, if the damage to the target is caused by an autoimmune condition, continuous use of low levels of immunosuppressants may be necessary from the time the autoreactive cells are destroyed.

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

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

[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 + It has the ability to differentiate into T cells.

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

[0259] In yet another embodiment, the stem cells, such as iPSCs, are T cells or thymocytes, preferably CD8 cells. + They are derived from T cells or thymocytes.

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

[0261] It should be understood that in this specification, when “cells” refers to isolated cells, or a population of isolated or substantially purified cells. When referring to a cell population, “substantially pure” means that the relevant cell type constitutes at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more percent of all cells in the cell population. For example, a cell population is substantially pure with respect to the relevant T cells if such T cells constitute at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more percent of all cells in the cell population.

[0262] The present invention is further described with reference to the following non-limiting embodiments. [Examples]

[0263] This specification is further presented by subsequent examples demonstrating the development of certain embodiments of the invention, including iPSC cells or HSC-derived dual anti-cancer-specific T cells. These examples should not be construed as limiting in any way.

[0264] (Example 1) Concentration of blood-derived cancer peptide antigen-specific T cells WT-1-specific TCR T cell stimulation and enhancement WT-1 specific T cells are extremely rare in normal human blood, but can be augmented and enriched for detection. In this regard, 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 L-glutamine and CD28 monoclonal antibodies, which act as T cell co-stimulators in the presence of WT-1, were added; anti-CD28 alone does not activate T cells. The PBMCs were then converted to Wilms tumor 1 (WT-1) peptide, four WT-1 peptides representing the main HLA class I binding motif: WT-1 37 (VLDFAPPGA, Sequence ID 22), WT-1 126 (RMFPNAPYL, Sequence ID 23), WT-1 187 (SLGEQQYSV, Sequence ID 24) and WT-1 235 Each of the cells (CMTWNQMNL, SEQ ID NO: 25) was stimulated overnight with 0.6 nmol / ml. The data shown in the examples of this application used WT-1 peptides 1-37 as representatives of this family of WT-1 peptides. WT-1 specific T cells can be identified using the HLA WT-1 specific tetramer or by early induction of the surface molecule CD137 on stimulated but non-quiescent 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 are WT-1 stimulated T cells) were magnetically separated using a magnetic cell separator. CD137-positive (WT-1 specific TCR) cells were cultured in T cell amplification 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 treated with mitomycin C and used as WT-1 peptide-loaded antigen-presenting feeder cells to induce a CD137-positive population, while the remaining CD3-positive cells (non-WT-1 specific) were cultured to act as control T cell types for downstream functional assays. Recombinant cytokine-containing culture medium was added every other day.

[0265] For flow cytometry analysis, cells were resuspended in FAC buffer: Cell 10 6 30 μl per cell. 10 μl of FcR blocking reagent was added to the cells at room temperature for 5 minutes. 10 μl of HLA-A02 WT-1 tetramer was added, and the cells were incubated at 4°C for 20 minutes, protected from light. 50 μl of the "T cell activation" cocktail was added, and the cells were incubated at 4°C for 20 minutes, protected from light. 100 μl of FAC buffer was added with 2 μl of AquaAmine, and the cells were incubated for 5 minutes, then centrifuged at 150xg for 5 minutes. The supernatant was aspirated or decanted, and the precipitate was resuspended in 100 μl of BD Cytofix / Cytoperm solution per sample. The cells were incubated at 4°C for 20 minutes. 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 in the dark at 4°C for 30 minutes. Cells were washed with BD / Perm wash and resuspended in FAC buffer before flow cytometry analysis. FACS data were acquired using a Miltenyi Quant cytometer.

[0266] T cells with a TCR specific to the WT-1 peptide are usually very rare (e.g., Schmeid et al., (2015)), and they make up only about 10% of CD8+ cells. -6 in It has been shown that (cells 3 × 10 -7 From 3 x 10 -6 (within the range). Following the stimulation protocol described above, WT-1 TCR-specific T cells increased approximately 100-fold, or about 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 augmented T cells were further stimulated using autoantigen-presenting cells (B cells transformed with EBV), and a wide range of WT-1 peptides were observed: WT-1 37 (VLDFAPPGA), WT-1 126(RMFPNAPYL), WT-1 187 (SLGEQQYSV) and WT1 235 Cells were pre-stimulated using (CMTWNQMNL). Interferon-gamma (IFNγ) production of T cells was examined by flow cytometry using a fluorescence-based assay. Cells were double-labeled for WT-1 peptide specificity via binding to the WT-1 peptide-HLA tetramer (see Figure 2).

[0268] WT-1 stimulated T cells clearly expressed interferon-gamma (IFNγ) in well-established T cell function measurements (80-90%) (Figure 2) (e.g., Ghanekar et al., (2001)). Potentially increasing the level of CD8 T cell activation (WT-1) (T cell targeting) The LAG3 inhibitor IMP321 was used. LAG3 is typically a "checkpoint blockade" that inhibits the stimulating function of dendritic cells (DCs) and the response of CD8 T cells to DCs as antigen-presenting cells. When added to a WT-1 specific T cell activation assay, IMP321 had no effect after 24 hours, but rare CD8+WT-1 specific TCR T cells doubled after 4 days (Figure 2H).

[0269] (Example 2) Generation of iPSCs derived from human blood T cells Numerous approaches exist for inducing iPSCs from human blood T cells, each maintaining the characteristics of the original T cells at various levels. iPSCs were produced from a broad repertoire of peripheral blood T lymphocyte pools (T-iPSCs) derived from healthy humans. T cells were pre-activated, for example, using mitogen PHA or anti-CD3 and anti-CD28 antibodies. Multiple T-iPSC clones were generated using dual retroviral vector cassettes containing two Yamanakari programming factors each (Oct4, Sox2, KLF, cMyc), 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 showing the reconstituted TCR gene.

[0270] The production of WT-1-specific blood T cell-derived iPSCs is summarized in Figure 3.

[0271] (Example 3) Induction of iPSC-derived human T cells This example demonstrates the generation of genuine T cells derived from iPSCs. These T cells were shown to express key characteristics of typical T cells, such as those normally produced by the thymus. They were shown to express mainstream T cell αβTCRs as well as CD8 having both β and α chains.

[0272] T cells were induced from adult whole blood T cells or pre-selected CD8+ T cells, or antigen-specific T cells (e.g., those specific to WT-1) (T-iPSCs), or iPSCs derived from adult fibroblasts. Two basic differentiation stages were performed: culture in OP9 cells to hematopoietic (hematopoietic stem cells or "HSCs") and partially lymphoid; and these cultured cells were transferred to OP cell lines genetically modified to express the Notch signaling molecule delta-like ligand 1 (OP9-DL-L1) for induction of subsequent T cell differentiation.

[0273] Phase 1 preparation of OP9-supporting cells and iPSC colonies - Day 8: Mitomycin-treated mouse embryonic fibroblasts were placed in a feeder layer in 3 mL of MEF medium (DMEM + 15% FCS + 1% pen / strep L-glutamine) on a 0.1% gelatin-coated TC plate, with 0.3 × 10⁶ cells. 6 (14,250 cells / cm 2 ) were seeded and incubated overnight. 0.25 × 10¹⁶ cells were placed in 11 mL of OP9 medium (αMEM + 20% FCS + 1% pen / strep) in a 0.1% gelatin-coated 10 cm TC plate. 6 Pre-preparation was done by sowing the seeds individually.

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

[0275] Phase 2 iPSC conversion to hematopoietic cells Day 0: Initiation of hematopoietic special differentiation. iPS colonies were isolated and seeded on OP9 for HSC differentiation. Colony suspensions were added dropwise to ensure uniform distribution to the OP9 plate. Fresh differentiation medium was added on days 1, 5, and 9.

[0276] Day 13: Collection 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 hours), and the colonies were further broken down into single cells with trypsin / EDTA 0.05% at 37°C for 30 minutes. The cells were gently washed and examined by phase-contrast microscopy (Figure 4) and flow cytometry (Figure 5). The hematopoietic properties of the cells were confirmed by flow cytometry (Figure 5).

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

[0279] In a preferred embodiment, OP9-adapted cells are CD34 to enhance the efficiency of contact with OP9DL-L1 cells. + CD43 + The (HSCs) were purified and then seeded into OP9 DLL-1 cells for the first stage of T cell differentiation. A critical component of the process disclosed herein was the initial harvesting of cells grown under OP9 DL-L1 cells.

[0280] Cells harvested 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). The suspension was added dropwise to OP9 DLL-1 cells and incubated at 37°C. Cells were harvested at 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 the expression of CD7 and CD9, early markers of T cell development with CD4 and CD8 expression, as well as the expression of the following marker (Figure 7). Even at this early stage, approximately 10% of cells already expressed both CD4+ and CD8+; these CD4+CD8+ cells are characteristic of T cells that normally develop in the thymic cortex (Heng et al., (2010)).

[0282] Flow cytometry demonstrated progressive T cell development, starting with the expression of CD5, then CD7+, and then CD8+. Most importantly, the induced T cells expressed the "optimal thymic-producing" CD8 T cell phenotype. They expressed CD8β chains in addition to CD8α chains (other reported T cell induction systems do not induce optimal signaling CD8β chains; e.g., Themeli et al., (2013)). As an indicator of function, they also expressed CD3 with αβTCRs. Expression occurred. 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] Seven days later (13 days on OP9 cells followed by 16 days on OP DL-L1 cells), these developing T cells underwent a decisive transition to express T cell receptor complexes, including CD8+ T cells that were clearly positive for CD3 and αβTCR; in addition, these cells expressed the crucial CD8β—these are the desirable cells for CAR-T. There was a corresponding further reduction in CD34+CD43+HSCs (Figure 8).

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

[0286] Using the process described above, T cells expressing a WT-1 specific TCR were produced from iPSCs that themselves originated from WT-1 TCR CD8+ T cells (Figure 9). These iPSC-derived WT-1 T cells exhibited cytotoxic function equivalent to that of the original T cells from which the iPSCs originated (Figure 10).

[0287] (Example 4) Development of CAR structures The components of chimeric antigen receptors (CARs) in T cells include the antigen-recognition component of the CAR, mediated by the scFv external domain, which is represented by a single-stranded Fv (scFv) containing a transmembrane (TM) region and anchored by a CD8 or CD28 hinge, and the signaling of the CAR via the cytoplasmic endodomain represented by CD28, 4-1BB, and CD3 zeta (CD3ζ) chains. Two preferred viral delivery systems—retroviruses and lentiviruses—are also present. Exemplary CAR and CD47-binding receptor constructs are 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 Sequence IDs 1-20.

[0289] (Example 6) Transduction of T cells into chimeric antigen receptors lentivirus production 293T cells coated with poly-L-lysine (Sigma) at 175 cm² 2 The cells were seeded in a flask. Two hours before transfection, the medium was replaced with DMEM supplemented with 10% FCS. The lentivirus transfer vector DNA was combined with the packaging and envelope plasmid DNA and mixed with lipofectamine 2000. The solution was briefly vortexed and incubated at room temperature for 30 minutes. The solution was then mixed again and 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 cleared by centrifugation at 1500 rpm for 5 minutes at 4°C, and 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 polyalomer conical tube (Beckman). Centrifugation was performed for 90 minutes at 20,000 g. The supernatant was completely removed, and the virus precipitate was resuspended in 300 μL of PBS and stored at -80°C until use.

[0290] Generation of CAR-T cells Figure 11 and Sequence IDs 1-6 show a panel of the developed chimeric antigen receptor (CAR) and CD47-binding receptor constructs, along with scFvs specific to either TAG72 or CD19 (as a positive control). These constructs use either human CD8 or CD28 and the CD28, CD3ζ chain or 4-1BB cytoplasmic activation signaling domain as hinge regions. The CAR and CD47-binding receptor constructs are cloned into lentiviral vectors as described in the previous chapter.

[0291] Optimal lentiviral transduction of T cells involves their activation at the TCR and costimulatory receptors. Therefore, on day 0, fresh PBMCs were harvested from healthy donors by apheresis and enriched for activated T cells using anti-CD3 and anti-CD28 antibodies conjugated to paramagnetic beads (Dynabeads ClinExVivo CD3 / CD28, Invitrogen, Camarillo, CA, USA) in a 3:1 (beads:cells) ratio. Cells and beads were co-incubated at room temperature for 1 hour, and CD3+ cell enrichment was performed using a magnet (Invitrogen). Cells in the CD3+ fraction were placed in starting medium, 1 × 10⁶ cells. 6 The cells were resuspended in T cell amplification medium containing 100 IU / ml IL-2 at a concentration of cells / ml. On day 1, RetroNectin was used to coat the cell culture dishes overnight at 4°C at a concentration of 2 mg / cm² in a 10 mg / mL solution in PBS. On day 2, the RetroNectin solution was aspirated and a blocking solution consisting of 0.5% human serum albumin in the same volume of PBS was added to each bag, and the mixture was incubated at room temperature for 30 minutes. The blocking solution was aspirated and each bag was washed with PBS. The lentivirus supernatant was rapidly thawed and added to each dish containing T cell amplification medium containing 300 IU / ml IL-2. The cultures were returned to the incubator and left for at least 24 hours. Transduction was stopped on day 4; cells were transferred to fresh T cell amplification medium in 0.5-1 × 10⁶ cells. 6 The culture was resuspended at a concentration of cells / mL. The culture was maintained until day 14, and the cell concentration was increased to 1 × 10⁶ cells. 6 Fresh augmentation medium was added every other day to maintain a cell / mL concentration.

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

[0293] Evaluation of CAR-T cell function We investigated the ability of TAG72 CAR-T cells (derived from normal PBMCs) to kill TAG72-expressing target cancer cells in vitro. A real-time cell monitoring system (xCELLigence) was used to determine the in vitro CAR-T cell death efficiency. Target cells (e.g., TAG72+ ovarian cancer cell line CaOV4) 10,000~2×10 6 Cells / 100 μL were placed in RTCA plates. In some cases, tethering of target cells with anti-hCD40 or by pre-coating the plates with human fibronectin may be necessary. Target cells were maintained at 37°C, 5% CO2 for 3–12 hours to allow cell adhesion. Following target cell adhesion, CAR-T effector cells were added in various effector:target ratios (ranging from 1:1 to 10:1). In some experiments, CAR-T effector cells were isolated via FACS before use based on GFP expression of 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 an indicator of cell detachment and ultimately cell death.

[0294] Figure 17 shows the results from this experiment, which was monitored for 40 hours. The ovarian cancer cell line CaOV4 grew consistently during this period (blue line). In contrast, cultures supplemented with TAG72-specific CAR T cells showed a significantly less early growth phase than those of target cells alone, followed by a stepwise elimination of target cells over time (purple line). To eliminate nonspecific death due to CD3 / CD28 activation, TAG72 CAR-T cells were isolated by flow cytometry and compared with 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 in the first 24 hours of culture with TAG72-expressing cancer cells, as negative controls of T cells transfected with the vector alone and non-transfected T cells did not show cancer cell death during this period.

[0295] The above study was performed using peripheral blood-derived polyclonal T cells. To demonstrate CAR-transduction of monospecific T cells expressing nominal cancer peptide antigen-specific TCRs, iPSC-derived WT-1 TCR-specific T cells formed from WT-1-specific TCRs were transduced with the TAG72 CAR lentivirus. Figure 22A shows good CAR transduction of these iPSC-derived WT-1-specific TCR CD8+ T cells themselves, produced from WT-1-specific T cells. The CAR exhibited specificity for TAG72. Most importantly, Figure 22B shows good transduction of iPSC-derived WT-1-specific TCR CD8+ T cells themselves, produced from WT-1-specific T cells, with CAR constructs for both TAG72 and CD47. This indicates that T cells can be produced with three specificities for cancer: WT-1 (TCR), TAG72 (CAR), and CD47 (cleaved form, CD47-binding receptor).

[0296] The results demonstrated the development of iPSC-derived bispecific CAR-transduced cancer-specific TCR (WT-1) that originated from WT-1-specific TCR T cells derived 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 death. After adjusting for spontaneous cell death, even with a low effector-target ratio (here, 2 effectors to 1 target cell), WT-1 cells resulted in approximately 10% cell death, followed by transduction with the addition of TAG72 CAR, resulting in an additional 10% death.

[0298] (Example 7) Transduction of iPSCs into chimeric antigen receptors The production of multispecific CAR-T cells can be achieved through multiple approaches, including CAR transduction of existing blood T cells (Figure 15) or transduction of iPSCs (expressing cancer-specific TCRs and CARs) that are subsequently induced into T cells (e.g., SEQ ID NOs: 1-6). Several iPSC lines have been used to advance CAR-T transduction. These iPSCs may be non-T cell-derived or derived from cancer antigen-specific T cells (e.g., WT-1) that retain TCR gene rearrangements. These iPSCs were either derived from adult fibroblasts or from T cells with endogenous TCRs specific to a specific cancer antigen (WT-1 peptide).

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

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

[0301] Transduced iPSCs can also be isolated by fluorescence-based cell sorting. Positive cells can be collected and seeded to form healthy (CAR-transduced) iPSC colonies (Figure 24).

[0302] Those skilled in the art will understand that the invention as described herein allows for variations and modifications other than those specifically described herein. It will be understood that the invention includes all such variations and modifications. The invention also includes all steps, properties, compositions and compounds that are individually or collectively referred to or indicated herein, as well as any and all combinations of any two or more of the 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, wherein the stem cell has the ability to differentiate into a T cell expressing a T cell receptor (TCR) directed to a first antigenic determinant, and the stem cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR) including an antigen-recognizing portion directed to a second antigenic determinant, wherein the antigen-recognizing portion is operably linked to a T cell activation portion by a hinge region and a transmembrane domain. (1) The antigen recognition portion includes 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) Genetically modified mammalian stem cells in which the T cell activation region comprises (a) a 4-1BB signaling domain or a CD28 signaling domain, and (b) a TCR zeta signaling domain.

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

20.

3. The stem cells according to claim 1 or 2, wherein the stem cells express at least one homozygous HLA haplotype.

4. The stem cell according to any one of claims 1 to 3, wherein the stem cell is selected from induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), or lymphocyte progenitor cells.

5. The stem cell according to any one of claims 1 to 4, wherein the hinge region contains cysteine ​​that promotes the dimerization of the CAR.

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

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

8. The stem cell according to claim 6 or 7, wherein the non-signaling antigen-binding receptor includes an antigen-recognizing portion directed to CD47.

9. The stem cell according to any one of claims 6 to 8, wherein the antigen recognition portion directed to the third antigen determinant is operably linked to the transmembrane domain by a hinge region.

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

11. T cells derived from stem cells according to any one of claims 1 to 10.

12. A method for producing genetically modified mammalian stem cells, wherein the method is (i) Introducing one or more nucleic acids encoding one or more chimeric antigen receptors into a T cell or thymocyte, wherein each chimeric antigen receptor includes an antigen-recognizing portion directed to an antigen-determinant different from the first antigen-determinant, the antigen-recognizing portion is operably linked to a T cell-activating portion, at least one of the nucleic acids is a nucleic acid encoding the chimeric antigen receptor according to claim 1 or 2, and the T cell or thymocyte expresses a TCR directed to the first antigen-determinant; (ii) A method comprising the step of deriving stem cells from the T cells or thymocytes.

13. The method according to claim 12, further comprising step (i) introducing one or more nucleic acids encoding at least one non-signaling antigen-binding receptor having an antigen-recognizing moiety directed to an additional antigenic determinant into the T cell or thymocyte.

14. A composition for use in the treatment of a condition in mammals, comprising stem cells according to any one of claims 1 to 10 or T cells according to claim 11.

15. The composition according to claim 14, wherein the state is a neobiotic state.

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