Multiplexed iPSCs and immune effector cells targeting solid tumors

Abstract

To provide a method and composition for generating induced non-pluripotent cells differentiated from single-cell induced iPSC (induced pluripotent stem cell) clone lines. [Solution] A method and composition are provided for obtaining functionally enhanced induced effector cells obtained from targeted differentiation of genome-manipulated iPSCs. The iPSC-induced cells provided herein have stable functional genome editing that results in improved or enhanced therapeutic effects. Therapeutic compositions and their use are also provided, comprising functionally enhanced induced effector cells alone or in combination therapy with antibodies or checkpoint inhibitors.

Description

[Technical Field]

[0001] (Related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 109,842, filed on 4 November 2020, and U.S. Provisional Patent Application No. 63 / 228,490, filed on 2 August 2021, the disclosures of which are incorporated herein by reference in their entirety.

[0002] (Field of invention) This disclosure broadly relates to the field of ready-made immune cell products. More specifically, this disclosure relates to strategies for developing multifunctional effector cells that can deliver therapeutically appropriate properties in vivo. Cell products developed under this disclosure address significant limitations of patient-derived cell therapies.

[0003] (Referencing electronically submitted sequence listings) This application incorporates, by reference, a Computer Readable Form (CRF) of an ASCII text sequence listing, titled 184143-633601_SequenceListing_ST25.txt, filed with this application on November 4, 2021, with a size of 37,739 bytes. [Background technology]

[0004] The field of adoptive cell therapy currently focuses on using patient-derived and donor-derived cells, making it particularly challenging to achieve consistent production of cancer immunotherapy and to deliver therapies to all potentially beneficial patients. There is also a need to improve the efficacy and persistence of adoptive lymphocytes to promote favorable patient outcomes. Lymphocytes such as T cells and natural killer (NK) cells are potent antitumor effectors that play a crucial role in innate and adaptive immunity. However, using these immune cells for adoptive cell therapy remains challenging, and there is an unmet need for improvement. Therefore, there is a significant opportunity to maximize the potential of T cells, NK cells, or other immune effector cells in adoptive immunotherapy. [Overview of the project]

[0005] Functionally improved effector cells are needed to address a variety of issues, including response rate, cell exhaustion, transplant cell loss (viability and / or persistence), tumor escape due to target loss or lineage change, accuracy of tumor targeting, extra-target toxicity, extratumor effects, and efficacy against solid tumors, i.e., the tumor microenvironment and associated immunosuppression, recruitment, transport, and invasion.

[0006] The object of the present invention is to provide a method and composition for generating induced non-pluripotent cells differentiated from a single-cell induced iPSC (induced pluripotent stem cell) clone line, wherein the iPSC contains one or more genetic modifications in its genome. These one or more genetic modifications include DNA insertions, deletions, and substitutions, and these modifications are retained and continue to function in subsequent induced cells after differentiation, proliferation, passage, and / or transplantation.

[0007] The iPSC-induced non-pluripotent cells of this application contain CD34 +This includes, but is not limited to, cells, hematopoietic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic pluripotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, B cells, and immunoeffector cells having one or more functional features not present in primary NK cells, T cells, and / or NKT cells. The iPSC-induced non-pluripotent cells of this application have one or more genetic modifications in their genome through differentiation from iPSCs containing the same genetic modification. In engineered clonal iPSC differentiation strategies for obtaining genetically modified induced cells, it is also necessary that the possibility of iPSC development in differentiation is not adversely affected by the engineered modality of the iPSC, and that the engineered modality functions as intended in the induced cells. Furthermore, this strategy overcomes the current barriers to manipulating primary lymphocytes such as T cells or NK cells obtained from peripheral blood, namely, the difficulty in manipulating such cells due to their often lack of reproducibility and homogeneity, resulting in cells with insufficient cellular persistence accompanied by high cell death and low cell proliferation. Moreover, this strategy avoids the generation of heterogeneous effector cell populations obtained by other methods using a primary cell source that is initially heterogeneous.

[0008] Some aspects of the present invention provide genome-engineered iPSCs obtained using methods comprising (I), (II), or (III), respectively, which reflect a genome engineering strategy, simultaneously with and prior to a reprogramming process. (I): Genetically engineer iPSCs by performing one or both of the following (i) and (ii) in any order: (i) introduce one or more constructs into the iPSCs to enable targeted integration at selected sites; (ii)(a) introduce one or more double-strand breaks(or more) at selected sites(or more) into the iPSCs using one or more endonucleases capable of recognizing selected sites; and (b) culture the iPSCs from step (I)(ii)(a) to enable endogenous DNA repair to simultaneously or sequentially generate targeted indels at selected sites(or more), thereby obtaining genomically engineered iPSCs that can differentiate into partially or fully differentiated cells. (II): Genetically engineered and reprogrammed non-pluripotent cells to obtain a genome-engineered iPSC comprising (i) contacting the non-pluripotent cells with one or more reprogramming factors and, optionally, a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate the reprogramming of the non-pluripotent cells, and (ii) introducing either or both of (a) and (b) of step (II)(i) below into the reprogrammed non-pluripotent cells in any order: (a) one or more constructs(may) that enable targeted incorporation at selected sites(may); (b) one or more double-strand breaks(may) at selected sites using at least one endonuclease capable of recognizing the selected sites. The cells from step (II)(ii)(b) are then cultured to allow endogenous DNA repair to generate targeted indels at selected sites(may). Therefore, the resulting genome-modified iPSCs contain at least one functionally targeted genome edit, and these genome-modified iPSCs can differentiate into partially or fully differentiated cells. (III): Genetically engineer non-pluripotent cells to reprogram them to obtain genome-engineered iPSCs comprising (i) and (ii) below: (i) Introduce one or both of (a) and (b) below into the non-pluripotent cells in any order: (a) One or more constructs(s) that enable targeted incorporation at selected sites(s); (b) One or more double-strand breaks(s) at selected sites using at least one endonuclease capable of recognizing the selected sites. Culture the cells from step (III)(i)(b) to allow endogenous DNA repair to generate targeted indels at the selected sites. (ii) Contact the cells from step (III)(i) with one or more reprogramming factors and optionally a small molecule composition comprising a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to obtain a genome-modified iPSC containing targeted editing at a selected site, thereby obtaining a genome-modified iPSC containing at least one functional targeted genome edit, the genome-modified iPSC being able to differentiate into partially differentiated or fully differentiated cells.

[0009] In one embodiment of the method described above, at least one targeted genome edit at one or more selected sites includes the insertion of one or more exogenous polynucleotides encoding a safety switch protein, a targeted modality, a receptor, a signaling molecule, a transcription factor, a pharmaceutically active protein and peptide, a candidate drug target, or a protein that promotes engraftment, transport, homing, viability, self-renewal, persistence and / or viability of genome-engineered iPSCs or cells derived therefrom. In some embodiments, the exogenous polynucleotide for insertion is operably linked to one or more exogenous promoters, including (1) CMV, EF1α, PGK, CAG, UBC, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, or (2) one or more endogenous promoters located at a selected site, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genome-safe harbor. In some embodiments, the genome-engineered iPSCs produced using the methods described above comprise one or more different exogenous polynucleotides encoding proteins including caspase, thymidine kinase, cytosine deaminase, modified EGFR, or B cell CD20, and if the genome-engineered iPSC comprises two or more suicide genes, the suicide genes are incorporated into different safe harbor loci including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR, or RUNX1. In one embodiment, the exogenous polynucleotides encode partial or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and / or their respective receptors. In some embodiments, the partial or complete peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their respective receptors, encoded by exogenous polynucleotides, are in the form of a fusion protein.

[0010] In some other embodiments, the genome-engineered iPSCs produced using the methods provided herein include indels in one or more endogenous genes related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response modulation and modification, or proteins that suppress engraftment, transport, homing, viability, self-renewal, persistence, and / or viability of iPSCs or cells derived therefrom. In some embodiments, the endogenous genes for disruption include at least one of the following genes: CD38, B2M, TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, RFXAP, RAG1, and any of the genes in the chromosome 6p21 region.

[0011] In some other embodiments, the genome-engineered iPSCs produced using the methods provided herein include a caspase encoding an exogenous polynucleotide at the AAVS1 locus and a thymidine kinase encoding an exogenous polynucleotide at the H11 locus.

[0012] In some other embodiments, approaches (I), (II), and / or (III) further include contacting the genetically engineered iPSCs with a small molecule composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to maintain the pluripotency of the genetically engineered iPSCs. In one embodiment, the obtained genetically engineered iPSCs containing at least one targeted genome edit are functional, differentiateable, and can differentiate into non-pluripotent cells containing the same functional genome edit.

[0013] Accordingly, in one aspect, the present invention provides cells or populations thereof, the cells being eukaryotic cells, animal cells, human cells, immune cells, induced pluripotent cells (iPSCs), cloned iPSCs or induced cells differentiated therefrom, the cells comprising (i) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), and (ii) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), wherein the tgTCRα chain and the tgTCRβ chain are exogenous TCR complexes (TCRs) that recognize a first tumor antigen. exoThe construct comprises a polynucleotide forming a tgTCRα chain and one or more additional exogenous polynucleotides, optionally including (iii) a polynucleotide encoding a chimeric antigen receptor (CAR) or an engager targeting at least a second tumor antigen. In some embodiments, (i) the polynucleotide encoding the tgTCRα chain and the polynucleotide encoding the tgTCRβ chain are contained in a bicistronic construct, optionally (a) the construct is inserted into the constant region (TRAC or TRBC) of TCRα or TCRβ, (b) the insertion of the construct disrupts the expression of endogenous TCRα or endogenous TCRβ at the insertion site and / or (c) the expression of the construct is driven by the endogenous or exogenous promoter of the TCR, or (ii) the polynucleotide encoding the CAR or engager Otide is inserted into TRAC or TRBC, and optionally (a) insertion of a polynucleotide encoding a CAR or engager disrupts the expression of endogenous TCRα or endogenous TCRβ at the insertion site and / or (b) expression of the CAR or engager is driven by the endogenous or exogenous promoter of the TCR, or (iii) tgTCRα and tgTCRβ chains, a polynucleotide encoding a CAR or engager, or one or more additional polynucleotides are inserted into one or more safe harbor loci or selected loci. In certain embodiments, the polynucleotides encoding the construct and the CAR or engager are inserted into the constant region (TRAC or TRBC) of TCRα or TCRβ, respectively, but not into the same constant region, thereby disrupting the expression of both endogenous TCRα and endogenous TCRβ, knocking out the endogenous TCR, and avoiding unpaired TCRs, including (a) transgenic TCRα and endogenous TCRβ, or (b) transgenic TCRβ and endogenous TCRα. In some embodiments, the polynucleotides encoding the construct and the CAR or engager are incorporated into loci, respectively, including safe harbor loci or selected loci.In some embodiments, (i) the safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1; (ii) the selected locus is one of B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD71, CD44, CD58, CD54, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and / or (iii) the incorporation of an exogenous polynucleotide knocks out the expression of the gene at the locus.

[0014] In some embodiments, the iPSC is a cloned iPSC, a single-cell dissociated iPSC, an iPSC cell line, or an iPSC master cell bank (MCB) cell, or the induced cell is an induced CD34 +The invention may include cells, induced hematopoietic stem cells and progenitor cells, induced pluripotent hematopoietic progenitor cells, induced T cell progenitor cells, induced NK cell progenitor cells, induced T lineage cells, induced NKT lineage cells, induced NK lineage cells, induced B lineage cells, or induced effector cells having one or more functional features not present in the corresponding primary T cells, NK cells, NKT cells, and / or B cells. In some embodiments, the induced effector cells are hematopoietic cells that have longer telomeres compared to their corresponding primary cells. In some embodiments, the cells include one of the genotypes listed in Table 1, or the cells include (i) (1) CD19-CAR at the TRAC locus, (2) TRAC knockout, and (3) MR1-TCR or NYESO1-TCR, with or without optional (4) TRBC knockout; (ii) (1) BCMA-CAR and hnCD16 insertion at the TRAC locus, (2) TRAC knockout, and (3) MR1-TCR or NYESO1-TCR, with or without optional (4) TRBC knockout; or (iii) (1) MICA / B B-CAR insertion at the TRAC locus, (2) TRAC knockout, (3) hnCD16 insertion at the CD38 locus, (4) CD38 knockout, and (5) MR1-TCR or NYESO1-TCR, with or without optional (6) TRBC knockout. In some embodiments, the cells have therapeutic properties, compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue that do not have the same gene editing(s), including (i) increased cytotoxicity, (ii) improved persistence and / or viability, (iii) enhanced ability to migrate and / or activate or mobilize bystander immune cells to tumor sites, (iv) improved tumor penetration, (v) enhanced ability to reduce tumor immunosuppression, (vi) improved ability to rescue tumor antigen escapes, (vii) controlled apoptosis, (viii) enhanced or acquired ADCC, and (ix) ability to avoid sibling killing.

[0015] In some embodiments, the first tumor antigen and the second tumor antigen are, respectively, (i) MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Muc1, Muc16, CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, GD2, MSLN, VEGF-R2, PSMA, and PDL1; or (ii) ADGRE2, B7H3, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigens of cytomegalovirus (CMV) infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (ACh R), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, MR1, mucin 1 (Muc- 1) comprising at least one of the following: mucin 16 (Muc-16), mesoserin (MSLN), NKCSI, NKG2D ligand, c-Met, NYESO1, oncoemetic antigen (h5T4), PDL1, PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), and pathogen antigens, wherein the first tumor antigen and the second tumor antigen are the same or different.

[0016] In some embodiments, the first tumor antigen comprises at least one of MR1, NYESO1, and MICA / B, or (i) tgTCRα comprises a TCRα constant fragment comprising a variable alpha (Vα) fragment having at least about 85% identity to SEQ ID NO: 7 and a sequence having at least about 85% identity to SEQ ID NO: 8, and / or (ii) tgTCRβ comprises a TCRβ constant fragment comprising a variable alpha (Vβ) fragment having at least about 85% identity to SEQ ID NO: 9 and a sequence having at least about 85% identity to SEQ ID NO: 10.

[0017] In some embodiments, the CAR is (i) T cell specific or NK cell specific, (ii) bispecific antigen-binding CAR, (iii) switchable CAR, (iv) dimerized CAR, (v) split CAR, (vi) multi-chain CAR, (vii) induceable CAR, (viii) inactivating CAR, (ix) co-expressed with a cell surface-expressed exogenous cytokine and / or a portion or complete peptide of its receptor, optionally in a separate construct or a bicistronic construct, or (x) co-expressed with a checkpoint inhibitor, optionally in a separate construct or a bicistronic construct.

[0018] In embodiments in which the cell comprises one or more exogenous polynucleotides encoding an engager, the engager may comprise (i) a first binding domain that recognizes the extracellular portion of CD3, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof on the cell or bystander immunoeffector cell, and (ii) a second binding domain that targets a second tumor antigen different from the first tumor antigen targeted by the exogenous TCR, wherein the second binding domain of the engager may comprise B7H3, CD It is specific to one of the following: 10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROR1, or VEGF-R2.

[0019] In some embodiments, the cells are (i) CD38 knockout, (ii) HLA-I deficiency and / or HLA-II deficiency, (iii) introduced HLA-G or uncleaved HLA-G, or knockout of one or both CD58 and CD54, (iv) exogenous CD16 or its variant, (v) chimeric fusion receptor (CFR), (vi) cell surface-expressed exogenous cytokines and / or signaling complexes including a portion or complete peptide of their receptor, (vii) genotypes listed in Table 1 (viii) deletion or destruction of at least one of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (ix) HLA-E, 4-1BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The present invention may further include the introduction or upregulation of at least one of the following: R, Fc receptors, antibodies or functional variants or fragments thereof, checkpoint inhibitors, and surface trigger receptors for coupling with agonists. In embodiments in which cells include exogenous CD16 or a variant thereof, the exogenous CD16 or a variant thereof may include at least one of the following: (a) high affinity uncleaved CD16 (hnCD16), (b) F176V and S197P of the external domain domains of CD16, (c) a complete or partial external domain derived from CD64, (d) a non-natural (or non-CD16) transmembrane domain, (e) a non-natural (or non-CD16) intracellular domain, (f) a non-natural (or non-CD16) signaling domain, (g) a non-natural stimulating domain, and (h) a transmembrane, signaling, and stimulating domain not derived from CD16 but derived from the same or a different polypeptide.

[0020] In embodiments in which cells include a CFR, the CFR may include an external domain fused to a transmembrane domain operably connected to an internal domain, wherein the external domain, transmembrane domain, and internal domain do not contain endoplasmic reticulum (ER) retention signals or endocytosis signals. In some embodiments, (i) the external domain of the CFR contains the full or partial length of the extracellular portion of a signaling protein comprising at least one of the following: CD3ε, CD3γ, CD3δ, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, any functional variant, and combinations or chimeras thereof; (ii) the external domain of the CFR initiates signaling upon binding to a selected agonist; or (iii) the internal domain of the CFR contains at least the full or partial length of a polypeptide of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. The cytotoxic domain is included, and the internal domain is optionally: (a) a costimulatory domain containing the full length or a portion of CD2, CD27, CD28, CD40L, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide or any combination thereof; (b) a costimulatory domain containing the full length or a portion of CD28, 4-1BB, CD27, CD40L, ICOS, CD2 or a combination thereof; (c) a sustained signaling domain containing the full length or a portion of the internal domain of a cytokine receptor containing IL7R, IL15R, IL18R, IL12R, IL23R or a combination thereof; and / or (d) receptor tyrosine kinase. The further comprising one or more of the following: tyrosine kinase (RTK), tumor necrosis factor receptor (TNFR), EGFR, or a complete or partial intracellular portion of the FAS receptor.In some embodiments, the selected agonist is (i) an antibody or a functional variant or fragment thereof, or (ii) an engager, the selected agonist may be encoded by a polynucleotide contained in a cell, or contained in a culture medium containing a cell or population thereof.

[0021] In embodiments in which cells include a signaling complex comprising a partial or complete peptide of a cell surface-expressed exogenous cytokine and / or its receptor, the cell surface-expressed exogenous cytokine or its receptor may include (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and their respective receptors(s), or (b) (i) co-expression of IL15 and IL15Rα using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is cleaved or excluded, (iv) a fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) a fusion protein of IL15 and IL15Rβ, or (vi) IL15 and its common receptor (b) A fusion protein with γC, wherein the common receptor γC is either native or modified, and (vii) a homodimer of IL15Rβ, wherein any one of (i) to (vii) may be co-expressed with CAR in a separate construct or in a bicistronic construct; or (c) A fusion protein of IL7 and IL7Rα, (ii) a fusion protein of IL7 and the common receptor γC, wherein the common receptor γC is either native or modified, and (iii) a homodimer of IL7Rβ, wherein any one of (i) to (iii) may be co-expressed with CAR in a separate construct or in a bicistronic construct, and optionally (d) transiently expressed.

[0022] In embodiments in which cells contain a checkpoint inhibitor, the checkpoint inhibitor is PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2A It may be an antagonist to one or more checkpoint molecules, including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR.

[0023] In another embodiment, the present invention provides compositions comprising cells or populations thereof disclosed herein. In some embodiments of the composition, the cells or populations thereof comprise iPSC-induced effector cells, and the composition further comprises one or more therapeutic agents. In some embodiments, one or more therapeutic agents comprise peptides, cytokines, checkpoint inhibitors, antibodies or functional variants or fragments thereof, engagers, mitogens, growth factors, small RNAs, dsRNAs (double-stranded RNAs), mononuclear hematopoiesis, feeder cells, feeder cell components or their replacement factors, vectors comprising one or more polynucleic acids of interest, chemotherapeutic agents or radioactive moieties, or immunomodulatory drugs (IMiDs). In embodiments in which the composition comprises checkpoint inhibitors, the checkpoint inhibitors are (i) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A 2A(ii) one or more antagonist checkpoint molecules including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR, or (ii) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents. In some embodiments of the composition, one or more therapeutic agents include one or more of venetoclax, azacitidine, and pomalidomide.

[0024] In embodiments of a composition in which one or more therapeutic agents are antibodies or functional variants or fragments thereof, the antibodies or functional variants or fragments thereof include (a) anti-CD20, anti-CD22, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibodies, and (b) rituximab, bertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab, ibritumomab, ocrelizumab, inotuzumab, moxetumomab, eprat (c) may include one or more of the following: zumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, as well as their functional equivalents and biosimilars, or (c) daratumumab, wherein the induced effector cells include CD38 knockout and optionally express CD16 or a variant thereof.In embodiments of a composition in which one or more therapeutic agents are engagers, the engagers may include (i) a bi-specific T cell engager (BiTE), (ii) a bi-specific killer cell engager (BiKE), or (iii) a tri-specific killer cell engager (TriKE), or the engager may include (a) a first binding domain that recognizes the extracellular portion of CD3, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof on a cell or bystander immunoeffector cell, and (b) B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, CD7 It may contain a second binding domain specific to an antigen containing one of the following: 9a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROR1, or VEGF-R2.

[0025] In yet another aspect, the present invention provides a therapeutic use of the compositions provided herein by introducing the compositions into a subject suitable for adoptive cell therapy, the subject having an autoimmune disorder, hematological malignancy, solid tumor, cancer, or viral infection.

[0026] In yet another aspect, the present invention provides a master cell bank (MCB) containing clonal iPSCs provided herein.

[0027] In yet another aspect, the present invention provides a method for producing the induced effector cells described herein, the method comprising differentiating genetically engineered iPSCs, the iPSCs comprising (a) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), (b) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), and optionally (c) a polynucleotide encoding an engager targeting a chimeric antigen receptor (CAR) or a second tumor antigen, optionally the iPSCs comprising (i) CD38 knockout, (ii) HLA-I deficiency and / or HLA-II deficiency, (iii) introduced HLA-G or uncleaved HLA-G, or knockout of one or both CD58 and CD54, or (iv) exogenous (v) CD16 or its variants, (vi) chimeric fusion receptors (CFRs), (vi) cell surface-expressed exogenous cytokines and / or signaling complexes including a portion or complete peptide of their receptors, (vii) at least one of the genotypes listed in Table 1, (viii) deletion or disruption of at least one of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (ix) HLA-E, 4-1BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A 2AThe invention further comprises the introduction or upregulation of at least one of the following: R, Fc receptors, antibodies or functional variants or fragments thereof, checkpoint inhibitors, and surface trigger receptors for coupling with agonists. In some embodiments, the method further comprises genomically manipulating a cloned iPSC to knock in (a) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), (b) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), and optionally (c) a polynucleotide encoding a chimeric antigen receptor (CAR) or an engager targeting a second tumor antigen; optionally, further genomically manipulating the cloned iPSC to (i) knock out CD38, (ii) knock out B2M and / or CIITA, (iii) knock out one or both CD58 and CD54, and / or (iv) introduce a signaling complex comprising HLA-G or uncleaved HLA-G, exogenous CD16 or its variants, CFR and / or cell surface-expressed exogenous cytokines and / or their receptors, in partial or complete peptides. In some embodiments, the genomic manipulation includes targeted editing. In certain embodiments, targeted editing includes deletions, insertions, or indels, and targeted editing is performed by CRISPR, ZFN, TALEN, homing nucleases, homologous recombination, or any other functional variation thereof.

[0028] In yet another aspect, the present invention provides a chimeric antigen receptor (CAR) specific to the tumor cell surface antigen MR1, wherein the MR1-CAR comprises: (i) an external domain comprising at least one antigen-recognizing domain, the antigen-recognizing domain comprising (a) a variable alpha (Vα) fragment (MR1Vα) having at least about 85% identity to SEQ ID NO: 7 and a variable beta (Vβ) fragment (MR1Vβ) having at least about 85% identity to SEQ ID NO: 8, or (b) an extracellular domain of MR1 TCRα having at least about 85% identity to SEQ ID NO: 15 and an extracellular domain of MR1 TCRβ having at least about 85% identity to SEQ ID NO: 16; (ii) a transmembrane domain; and (iii) an internal domain comprising at least a first signaling domain, the first signaling domain comprising an internal domain derived from the cytoplasmic domain of a signaling protein specific to the activation or function of T cells and / or NK cells, wherein the tumor cell surface antigen MR1 is non-polymorphic.In some embodiments, the signaling proteins are 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain-containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain), CD3ζ1XX (CD3ζ variant), DAP10 (hematopoietic cell signaling molecule), DAP12 (TYRO protein tyrosine kinase binding protein), DNAM1 (CD226 antigen), FcERIγ (high affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta), IL-2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D It contains one of the following: type II intrinsic membrane protein, NKp30 (native cytotoxicity trigger receptor 3), NKp44 (native cytotoxicity trigger receptor 2), NKp46 (native cytotoxicity trigger receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain).

[0029] In some embodiments of the CAR, the internal domain further comprises a second signaling domain and optionally a third signaling domain, wherein the first, second, and third signaling domains are distinct. In some embodiments, the second or third signaling domain comprises the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8. In some embodiments, the transmembrane domain comprises the amino acid sequence or a portion thereof of the transmembrane region of CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide. In some embodiments, the external domain further comprises (i) a signal peptide and / or (ii) a spacer / hinge / linker.In some embodiments, the CAR comprises a bicistronic construct co-expressing a portion or full-length peptide of a cell surface-expressed exogenous cytokine or its receptor, the exogenous cytokine or its receptor comprising (a) at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21 and their respective receptors(s), or (b) (i) co-expression of IL15 and IL15Rα using a self-cleaving peptide, (ii) a fusion protein of IL15 and IL15Rα, (iii) an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is cleaved or excluded, or (iv) the membrane-bound Sushi domains of IL15 and IL15Rα (b) comprising at least one of the following: (v) a fusion protein of IL15 and IL15Rβ, (vi) a fusion protein of IL15 and common receptor γC, wherein common receptor γC is either native or modified, and (vii) a homodimer of IL15Rβ, wherein any one of (i) to (vii) is optionally co-expressed with CAR in a separate construct or in a bicistronic construct, or (c) comprising at least one of the following: (i) a fusion protein of IL7 and IL7Rα, (ii) a fusion protein of IL7 and common receptor γC, wherein common receptor γC is either native or modified, and (iii) a homodimer of IL7Rβ. In certain embodiments, MR1-CAR is specific to one or more of the following: colorectal cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, cervical cancer, melanoma, bone cancer, breast cancer, ovarian cancer, or hematological cancer.

[0030] In yet another embodiment, the present invention provides cells or populations thereof, the cells being eukaryotic cells, animal cells, human cells, immune cells, induced pluripotent cells (iPSCs), cloned iPSCs, or induced cells differentiated therefrom, the cells comprising polynucleotides encoding at least the chimeric antigen receptor (CAR) described herein. In various embodiments of the cells or populations thereof, the cells comprising (i) a polynucleotide encoding a transgenic TCRα chain (tgTCRα) and (ii) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), wherein the tgTCRα and tgTCRβ chains are exogenous TCR complexes (TCRs) that recognize a first tumor antigen other than MR1. exoThe present invention further comprises a polynucleotide forming a tgTCRα chain and optionally one or more additional exogenous polynucleotides including (iii) a polynucleotide encoding an engager that targets at least a second tumor antigen. In various embodiments, (i) the polynucleotide encoding the tgTCRα chain and the polynucleotide encoding the tgTCRβ chain are contained in a bicistronic construct, and optionally (a) the construct is inserted into the constant region (TRAC or TRBC) of TCRα or TCRβ, (b) the insertion of the construct disrupts the expression of endogenous TCRα or endogenous TCRβ at the insertion site and / or (c) the expression of the construct is driven by the endogenous or exogenous promoter of the TCR, or (ii) the polynucleotide encoding the CAR or engager The TID is inserted into a TRAC or TRBC, and optionally (a) insertion of a polynucleotide encoding a CAR or engager disrupts the expression of endogenous TCRα or endogenous TCRβ at the insertion site, and / or (b) the expression of the CAR or engager is driven by the endogenous or exogenous promoter of the TCR, or (iii) the tgTCRα and tgTCRβ chains, a polynucleotide encoding a CAR or engager, or one or more additional polynucleotides are inserted into one or more safe harbor loci or selected loci. In certain embodiments, (I) the polynucleotides encoding the construct and the CAR or engager are inserted into the constant region (TRAC or TRBC) of TCRα or TCRβ, respectively, but not into the same constant region, thereby disrupting the expression of both endogenous TCRα and endogenous TCRβ, knocking out the endogenous TCR, and avoiding unpaired TCRs including (a) transgenic TCRα and endogenous TCRβ, or (b) transgenic TCRβ and endogenous TCRα, or (II) the polynucleotides encoding the construct and the CAR or engager are incorporated into loci including a safe harbor locus or a selected locus.

[0031] In yet another aspect, the present invention provides a composition comprising the cells or a population thereof described herein. In some embodiments, the cells or a population thereof comprise iPSC-derived effector cells, and the composition further comprises one or more therapeutic agents. Accordingly, embodiments of the present invention provide for the therapeutic use of the compositions described herein by introducing the compositions described herein into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, a hematologic malignancy, a solid tumor, cancer, or a viral infection. In some embodiments, the subject has colorectal cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, cervical cancer, gastric cancer, melanoma, bone cancer, breast cancer, ovarian cancer, or a blood cancer.

[0032] In yet another aspect, the present invention provides a method of enhancing the function of a CAR-T cell, the CAR-T cell having specificity for a first tumor antigen via a chimeric antigen receptor (CAR), the method comprising introducing into the CAR-T cell (i) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), and (ii) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), wherein the tgTCRα chain and the tgTCRβ chain form an exogenous TCR complex (TCR exo ) having specificity for a second tumor antigen, and the CAR-induced tumor killing efficacy of the CAR-T cell is enhanced by the expression of the TCR exo . In various embodiments of the method, (i) endogenous TCR knockout by disrupting the expression of both endogenous TCRα and endogenous TCRβ, or (ii) the CAR is inserted into the constant region of TCRα or TCRβ (TRAC or TRBC), thereby disrupting the expression of endogenous TCRα or TCRβ of the VAR-T cell. In various embodiments of the method, the method uses a second tumor antigen recognized by the TCR exo to TCR exoThe method further includes activating the first tumor antigen specificity and the second tumor antigen specificity, which are different. In various embodiments of the method, the cessation of polynucleotide introduction into CAR-T cells is differentiated a genetically engineered iPSC into a T cell, wherein the iPSC comprises (a) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), (b) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), and (c) a polynucleotide encoding a CAR having the first tumor antigen specificity, thereby enabling TCR exo The present invention further comprises obtaining CAR-T cells having expression of . In various embodiments of the present invention, the method further comprises genomically manipulating cloned iPSCs to knock in (a) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), (b) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), and (c) a polynucleotide encoding a CAR having first tumor antigen specificity, thereby obtaining engineered iPSCs for T cell differentiation.

[0033] The various purposes and advantages of the compositions and methods provided herein will become apparent from the following description, together with the accompanying drawings which illustrate and illustrate specific embodiments of the invention. [Brief explanation of the drawing]

[0034] [Figure 1A] This figure shows the ectopic expression of MR1-TCR in iPSC-induced effector cells. [Figure 1B]This figure shows the ectopic expression of MR1-TCR in iPSC-induced effector cells. Figure 1A shows the transduction of the MR1-TCR construct into iPSCs reprogrammed from T cells (TiPSCs) or fibroblasts (FiPSCs), and their subsequent differentiation into T lineage effector cells (TiP-iT or FiP-iT, respectively). Figure 1B shows the ectopic expression of MR1-TCR in iPSC-induced effector cells. This suggests that the presence of endogenous TCRβ in TiPSCs can lead to TCR mispairing in induced T lineage effector cells (TiP-iT) with endogenous TCR knockout due to TRAC disruption. [Figure 2] This figure shows that TCRαβ expression can stabilize the surface expression of the CD3 molecule. [Figure 3] This figure shows that MR1 TCR-expressing effector cells can specifically kill A549 tumor cells, and that this can be inhibited by an MR1 blocking antibody. [Figure 4] Figure A shows that FiPSC-induced MR1-TCR+iT cells exhibit MR1-dependent cytokine release and degranulation responses. Figure B shows that TipsSC-induced MR1-TCR+iT cells exhibit MR1-dependent cytokine release and degranulation responses. [Figure 5A] This figure shows the purity of FiP-iT cells expressing MR1-TCR within the input population, and demonstrates that MR1-mediated TCR signaling, blocked by the antibody, can be restored by the addition of BiTE. [Figure 5B] This figure shows that MR1 TCR+FiP-iT exhibits MR1-dependent cytotoxicity in A549 cells, and that MR1-mediated TCR signaling blocked by the antibody can be restored by the addition of BiTE. [Figure 6] The figure shows that MR1 blocking antibodies can prevent MR1-TCR-mediated killing of tumor cells (above), and that the addition of BiTE enables targeting of tumor cells via CD3 binding of MR1-TCR-reconstituted cells, even in the presence of antibodies. [Figure 7A]This figure shows that BiTE restored cell activation and response in MR1-TCR+FiP-iT cells in the presence of an MR1 blocking antibody. [Figure 7B] This figure shows that BiTE restored cell activation and response in MR1-TCR+FiP-iT cells in the presence of an MR1 blocking antibody. [Figure 8A] This figure shows that when TCR-mediated MR1 recognition was blocked, BiTE restored cell activation and response in MR1-TCR+TiP-iT cells. [Figure 8B] This figure shows that when TCR-mediated MR1 recognition was blocked, BiTE restored cell activation and response in MR1-TCR+TiP-iT cells. [Figure 9A] This figure shows that MR1-TCR+FiP-iT exhibited MR1-dependent cytotoxicity in Nalm6 cells, and that MR1-mediated TCR signaling and cytotoxicity blocked by antibodies can be restored by the addition of BiTE. [Figure 9B] This figure shows that MR1-TCR+FiP-iT exhibited MR1-dependent cytotoxicity in Nalm6 cells, and that MR1-mediated TCR signaling and cytotoxicity blocked by antibodies can be restored by the addition of BiTE. [Figure 10] This figure shows an exemplary MR1-CAR design in which the transmembrane (TM) domain and cytoplasmic signaling domain can be altered. [Figure 11A] This figure shows that TRBC knockout reduces the mispairing problem and restores TCR function. [Figure 11B] This figure shows that TRBC knockout reduces the mispairing problem and restores TCR function. [Figure 11C] This figure shows that TRBC knockout reduces the mispairing problem and restores TCR function. [Figure 11D] This figure shows that TRBC knockout reduces the mispairing problem and restores TCR function. [Figure 11E]This figure shows that TRBC knockout reduces the mispairing problem and restores TCR function. In Figure 11D, the x-axis of %TNFα is 0.0, 0.5, 1.0, 1.5, and 2.0 from left to right. [Figure 12A] This figure shows the synergistic relationship between the co-expression of CAR and TCR on TiP-iT cells. [Figure 12B] This figure shows the synergistic relationship between the co-expression of CAR and TCR on TiP-iT cells. [Figure 12C] This figure shows the synergistic relationship between the co-expression of CAR and TCR on TiP-iT cells. [Figure 12D] This figure shows the synergistic relationship between the co-expression of CAR and TCR on TiP-iT cells. [Figure 13A] This figure shows the in vitro cytotoxicity of TiP-iT cells with co-expression of CAR, TCR, and CD16 (trimodal). [Figure 13B] This figure shows the in vitro cytotoxicity of TiP-iT cells with co-expression of CAR, TCR, and CD16 (trimodal). [Figure 14A] This figure shows further data supporting the synergistic relationship between CAR and TCR co-expression on trimodal TiP-iT cells, demonstrating the in vitro cytotoxicity of trimodal cells. [Figure 14B] This figure shows further data supporting the synergistic relationship between CAR and TCR co-expression on trimodal TiP-iT cells, demonstrating the in vitro cytotoxicity of trimodal cells. [Figure 14C] This figure shows further data supporting the synergistic relationship between CAR and TCR co-expression on trimodal TiP-iT cells, demonstrating the in vitro cytotoxicity of trimodal cells. [Figure 14D] This figure shows further data supporting the synergistic relationship between CAR and TCR co-expression on trimodal TiP-iT cells, demonstrating the in vitro cytotoxicity of trimodal cells. [Figure 14E]This figure shows further data supporting the synergistic relationship between CAR and TCR co-expression on trimodal TiP-iT cells, demonstrating the in vitro cytotoxicity of trimodal cells. [Figure 14F] This figure shows further data supporting the synergistic relationship between CAR and TCR co-expression on trimodal TiP-iT cells, demonstrating the in vitro cytotoxicity of trimodal cells. [Figure 14G] This figure shows further data supporting the synergistic relationship between CAR and TCR co-expression on trimodal TiP-iT cells, demonstrating the in vitro cytotoxicity of trimodal cells. [Figure 15] This figure shows that exogenous TCRs expressed in iT effectors can induce TCR antigen-specific tumor killing. [Figure 16] This figure shows that exogenous expression of TCR enhances CAR-induced tumor-killing efficacy in iT effectors. [Figure 17] This figure shows that in the presence of both CAR and TCR tumor antigens, the CAR / TCR iT effector exhibits enhanced tumor-killing efficacy compared to CAR iT. [Figure 18] This figure shows that activation of both TCR and CAR results in faster tumor killing compared to CAR activation alone. [Figure 19] This figure shows that exogenous TCR expression in CAR iT cells correlates with a higher number of iT effectors in cell proliferation. [Figure 20A] This figure shows that mixed tumor cell lines on day 0 each induce activation of CAR, TCR, or ADCC mediated by IgG antibodies that bind to CD16. [Figure 20B] This is a scheme for in vivo validation of trimodal iT cells using the Nalm6 mouse model. [Figure 21A] This figure shows FACS analysis of bone marrow-derived ex vivo CD10+ cells for BCMA-mCherry signaling and NYESO1-GFP signaling. [Figure 21B]This figure shows the absolute number of tumors in each bone marrow sample for the indicated tumor strains in all the groups shown. [Figure 22] This figure shows that CAR / TCR iT cells eliminated most of the tumor in the bone marrow on days 7 and 10, but not in the lung and brain regions. [Figure 23A] This figure shows a representative FACS plot illustrating the ratio between CAR-targeted Nalm6 (BCMA+) and TCR-targeted Nalm6 (BCMA-) inputs, as well as the analysis of bone marrow cell suspensions at day 22 from the no-effector group, the BCMA-CAR-only group, the BCMA-CAR+NYESO1-TCR+ group, and the BCMA-CAR+MR1-TCR+ group. [Figure 23B] This figure shows the absolute number of Nalm6 targets for BCMA+MR1ko and NYESO1+MR1wt for each group as shown. [Figure 24A] This figure provides confirmation of improved tumor growth inhibition (TGI) in mice treated with CAR / TCR iT cells using BLI analysis. [Figure 24B] This figure provides confirmation of improved tumor growth inhibition (TGI) in mice treated with CAR / TCR iT cells using area under the curve (AUC) analysis. [Figure 25] This is a representative FACS plot showing the absence of checkpoint inhibitor receptors, LAG3, TIM3, and PD1 on ex vivo iT cells from bone marrow of each group shown on day 22. [Figure 26] This figure shows the cytokine production and degranulation capacity of ex vivo iT cells from the bone marrow of each group shown on day 22. [Modes for carrying out the invention]

[0035] Genome modification of iPSCs (induced pluripotent stem cells) includes polynucleotide insertions, deletions, and substitutions. Exogenous gene expression in genetically engineered iPSCs often encounters problems such as gene silencing or reduced gene expression after long-term clonal proliferation of the original genetically engineered iPSCs, after cell differentiation, and in dedifferentiated cell types from genetically engineered iPSC-induced cells. On the other hand, directly manipulating primary immune cells such as T cells or NK cells is difficult, hindering the preparation and delivery of engineered immune cells for adoptive cell therapy. In various embodiments, the present invention provides an efficient and reliable targeted approach for the stable incorporation of one or more exogenous genes, including suicide genes and other functional modalities, which brings improved therapeutic properties with respect to engraftment, transport, homing, migration, cytotoxicity, viability, maintenance, proliferation, lifespan, self-renewal, persistence, and / or survival rate to iPSC-inducing cells, including, but not limited to, HSCs (hematopoietic stem cells and progenitor cells), T cell progenitor cells, NK cell progenitor cells, T lineage cells, NKT lineage cells, NK lineages, and immune effector cells having one or more functional features not present in primary NK cells, T cells, and / or NKT cells.

[0036] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms.

[0037] It should be understood that the present invention is not limited to, and is therefore subject to change, the specific methodologies, protocols, and reagents described herein. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

[0038] As used herein, the articles “a,” “an,” and “the” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “an element” means one or more elements.

[0039] The use of alternatives (e.g., "or") should be understood to mean one of the alternatives, both, or any combination thereof.

[0040] The terms "and / or" should be understood to mean either one or both of the alternatives.

[0041] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference. In one embodiment, the terms “about” or “approximately” refer to a range of a level, value, number, frequency, percentage, dimension, size, volume, weight, or length of approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of reference.

[0042] As used herein, the terms “substantially” or “essentially” mean a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more, compared to the quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of reference. In one embodiment, the terms “substantially the same” or “essentially the same” mean a range of a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of reference that is substantially identical to the quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of reference.

[0043] As used herein, the terms “substantially absent” and “essentially absent” are interchangeable and, when used to describe compositions such as cell populations or culture media, refer to compositions that do not contain a particular substance or its source, for example, 95%, 96%, 97%, 98%, 99%, or are undetectable by conventional means. The terms “absent” or “essentially absent” of a particular component or substance in a composition also mean that such component or substance is (1) not present in the composition at any concentration, or (2) present in the composition at a functionally inert low concentration. A similar meaning may apply to the term “not present,” which refers to the absence of a particular substance or its source in a composition.

[0044] Throughout this specification, unless the context requires otherwise, “comprise,” “comprises,” and “comprising” mean to include the step or element, or group of steps or elements, described, but not to exclude any other step or element, or group of steps or elements. In certain embodiments, the terms “include,” “have,” “contain,” and “comprise” are used synonymously.

[0045] "Consisting of" means that everything that follows the phrase is included and limited to it. Therefore, the phrase "consisting of" indicates that the listed elements are necessary or essential, and that no other elements can exist.

[0046] "Consisting essentially of" means including any elements listed after the phrase, but limited to other elements that do not interfere with or contribute to the activity or operation specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that the other elements may or may not be present, not necessarily as a matter of necessity, depending on whether or not they affect the activity or operation of the listed elements.

[0047] Throughout this specification, any reference to “one embodiment,” “embodiment,” “specific embodiment,” “related embodiment,” “specific embodiment,” “additional embodiment,” or “further embodiment,” or any combination thereof, means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, occurrences of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments.

[0048] The term "ex vivo" generally refers to activities performed outside of a living organism, such as experiments or measurements conducted in or on living tissues within an artificial environment outside of a living organism, preferably with minimal changes to natural conditions. In certain embodiments, an "ex vivo" procedure involves living cells or tissues taken from a living organism and cultured in an experimental apparatus, usually under sterile conditions, typically for several hours or up to about 24 hours, but depending on the circumstances, up to 48 hours, 72 hours or more. In certain embodiments, such tissues or cells may be collected and frozen and subsequently thawed for ex vivo processing. Tissue culture experiments or procedures using living cells or tissues that last longer than several days are typically considered "in vitro," although in certain embodiments, this term may be used interchangeably with "ex vivo."

[0049] The term "in vivo" generally refers to activities that occur inside a living organism.

[0050] As used herein, the terms “reprogramming,” “dedifferentiation,” “increased cellular capacity,” and “increased developmental capacity” refer to methods of increasing the capacity of a cell or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cellular capacity has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell that has not been reprogrammed. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell that has not been reprogrammed.

[0051] As used herein, the term “differentiation” refers to the process by which unspecialized (“uncommitted”) or less specialized cells acquire the characteristics of specialized cells, such as blood cells or muscle cells. Differentiated or induced-differentiation cells are cells that occupy a more specialized (“committed”) position within a cell lineage. When applied to the process of differentiation, the term “committed” refers to cells that, under normal circumstances, continue to differentiate into a particular cell type or subset of cell types, and under normal circumstances cannot differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term “pluripotency” refers to the ability of cells (i.e., the embryo itself) to form all lineages of living or somatic cells. For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: the ectoderm, mesoderm, and endoderm. Pluripotency is a range of developmental capabilities, from incomplete or partial pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot produce a complete organism to more primitive and pluripotent cells (e.g., embryonic stem cells) that can produce a complete organism.

[0052] As used herein, the terms “induced pluripotent stem cells” or “iPSC” refer to stem cells produced in vitro from differentiated adult, neonatal, or fetal cells that have been induced or modified, i.e., reprogrammed, to differentiate into cells capable of differentiating into all three germ layers or dermis: mesoderm, endoderm, and ectoderm, using reprogramming factors and / or small molecular weight chemicals. Produced iPSCs do not refer to naturally occurring cells.

[0053] As used herein, the term “embryonic stem cells” refers to the naturally occurring pluripotent stem cells in the inner cell mass of a blastocyst. Embryonic stem cells are pluripotent and, during development, give rise to all three primary germ layers: the ectoderm, endoderm, and mesoderm. They do not contribute to the extraembryonic membrane or placenta; that is, they are not totipotent.

[0054] As used herein, the term “pluripotent stem cell” refers to a developmentally capable cell that can differentiate into cells of one or more germ layers (ectoderm, mesoderm, and endoderm), but not into all three. Thus, pluripotent cells can also be called “partially differentiated cells.” Pluripotent cells are well known in the art, and examples of pluripotent cells include adult stem cells such as hematopoietic stem cells and neural stem cells. “Pluripotency” indicates that a cell can form many types of cells in a given lineage, but not cells in other lineages. For example, pluripotent hematopoietic cells can form many different types of blood cells (red, white, platelet, etc.), but cannot form neurons. Thus, the term “multipotency” refers to a state of cell development with a lower degree of potential than totipotency and pluripotency.

[0055] Pluripotency can be partially determined by evaluating the pluripotent characteristics of cells. These characteristics include, but are not limited to, (i) pluripotent stem cell morphology, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation consisting of cells from the three somatic cell lineages, and (vi) embryoid body formation consisting of cells from the three somatic cell lineages.

[0056] Two types of pluripotency have been described so far: a “priming” or “metastable” state of pluripotency, similar to the epiblastic stem cells (EpiSCs) of a late blastocyst, and a “naive” or “basal” state, similar to the inner cell mass of an early / pre-implantation blastocyst. While both pluripotent states exhibit the characteristics described above, the naive or basal state further exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonality and viability in single-cell culture, (iii) overall reduction in DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) decreased expression of differentiation markers compared to priming pluripotent cells. Standard methodologies of cell reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or eliminated from the resulting pluripotent cells, generally appear to exhibit the characteristics of a pluripotent preparation state. Under standard pluripotent cell culture conditions, such cells remain in a ready state and exhibit basal state characteristics unless exogenous transgene expression is maintained.

[0057] As used herein, the term “pluripotent stem cell morphology” refers to the classic morphological features of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a high nucleus-to-cytoplasmic ratio, prominent nucleoli, typical intercellular spacing, and a round, small shape.

[0058] As used herein, the term “subject” refers to any animal, preferably a human patient, livestock, or other domesticated animal.

[0059] "Pluripotency factors" or "reprogramming factors" refer to agents that can increase the developmental potential of cells, either alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of cells. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0060] "Culture" or "cell culture" refers to the maintenance, proliferation, and / or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in each case the singular is "medium"), "supplementary components," and "culture medium supplementary components" refer to the nutritional composition used to culture cells.

[0061] "To culture" or "to maintain" refers to sustaining, propagating (growing), and / or differentiating tissue or extracorporeal cells, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Culture" or "maintaining" can utilize the culture medium as a source of nutrients, hormones, and / or other factors that aid in cell growth and / or maintenance.

[0062] As used herein, the term “mesoderm” refers to one of the three germ layers that appear during early embryonic development and give rise to a variety of specialized cell types, including circulatory blood cells, muscle, heart, dermis, skeleton, and other supporting and connective tissues.

[0063] As used herein, the terms “definitive hematopoietic endothelium” (HE) or “definitive hematopoietic endothelium derived from pluripotent stem cells” (iHE) refer to a subset of endothelial cells that give rise to hematopoietic stem cells and progenitor cells in a process called endothelial hematopoietic transition. Hematopoietic cell development in the embryo progresses sequentially from the lateral plate mesoderm through angioblasts to definitive hematopoietic endothelial cells and hematopoietic precursors.

[0064] The terms “hematopoietic stem cells and progenitor cells,” “hematopoietic stem cells,” “hematopoietic progenitor cells,” or “hematopoietic precursor cells” refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation, including pluripotent hematopoietic stem cells (blood cells), myeloid precursors, megakaryocyte precursors, erythrocyte precursors, and lymphocyte precursors. Hematopoietic stem cells and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). As used herein, the term “secondary hematopoietic stem cells” refers to CD34 cells that can give rise to both mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells. + This refers to hematopoietic cells. Hematopoietic cells also include various subsets of primitive hematopoietic cells that give rise to primitive erythrocytes, megakaryocytes, and macrophages.

[0065] As used herein, the terms “T lymphocyte” and “T cell” are interchangeable and refer to the primary type of leukocyte that completes maturation in the thymus and has various roles in the immune system, including the identification of specific foreign antigens in the body, as well as the activation and inactivation of other immune cells in an MHC class I-restricted manner. A T cell may be any T cell, e.g., cultured T cells, e.g., primary T cells, or T cells from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from mammals. T cells are CD3 + It can be a cell. T cells are CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD8 +T cells can be any type of T cell and at any developmental stage, including but not limited to T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, gamma delta T cells (γδT cells), etc. Additional types of helper T cells include cells such as Th3 (Treg), Th17, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR). T cells or T cell-like effector cells can also be differentiated from stem cells or progenitor cells. T cell-like induced effector cells may possess T cell lineage in some respects, but at the same time, they have one or more functional characteristics that are not present in primary T cells.

[0066] "CD4 + "T cells" refer to a subset of T cells that express CD4 on their surface and are involved in cellular immune responses. They are characterized by their post-stimulation secretion profiles, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4, and IL10. The "CD4" molecule is a 55kD glycoprotein originally defined as a differentiation antigen for T lymphocytes, but is also found in other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin supergene family and is involved as a relevant recognition element in MHC (major histocompatibility complex) class II-restricted immune responses. In T lymphocytes, it defines a subset of helper / inducer cells.

[0067] "CD8 +"T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen found in thymocytes and cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin supergene family and is a relevant recognition element of major histocompatibility complex class I restricted interactions.

[0068] As used herein, the terms “NK cells” or “natural killer cells” refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms “adaptive NK cells” and “memory NK cells” are interchangeable and phenotypically CD3 - and CD56 + This refers to a subset of NK cells that express at least one of NKG2C and CD57, and optionally CD16, but lack the expression of one or more of PLZF, SYK, FceRγ, and EAT-2. In some embodiments, CD56 + The isolated subpopulation of NK cells includes expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and repressive KIRs, NKG2A, and / or DNAM-1. + This can be expressed as weakly positive (dim) or strongly positive (bright). NK cells, or NK cell-like effector cells, can be differentiated from stem cells or progenitor cells. NK cell-like induced effector cells may have some NK cell lineage characteristics, but at the same time, they possess one or more functional features that are not present in primary NK cells.

[0069] As used herein, the terms “NKT cells” or “natural killer T cells” refer to CD1d-restricted T cells that express a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are recognized. Invariant or type I NKT cells express a very limited TCR repertoire—a regular α chain (Vα24-Jα18 in humans) associated with a limited range of β chains (Vβ11 in humans). A second population of NKT cells, called non-classical or non-invariant type II NKT cells, exhibits a more heterogeneous use of TCRαβ. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0070] The term “effector cell” generally applies to specific cells in the immune system that perform a particular activity in response to stimulation and / or activation, or cells in the nervous system that produce a particular function upon activation. As used herein, the term “effector cell” is interchangeable with “differentiated immune cell,” which refers to a cell that has been edited and / or modified to perform a particular activity in response to stimulation and / or activation. Non-limiting examples of effector cells include T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0071] As used herein, the term “isolated” refers to cells or populations of cells separated from their original environment; that is, the environment of isolated cells substantially does not contain at least one component found in the environment in which “unisolated” reference cells exist. This term includes, for example, cells isolated from tissue or biopsy specimens, taken from some or all components as they would be found in their natural environment. This term also includes cells taken from at least one, some or all components because cells are found in a non-natural environment, e.g., in an environment from which they are isolated from a cell culture or cell suspension. Thus, isolated cells, when found in nature or when grown, stored, or persist in a non-natural environment, are partially or completely separated from at least one component, including other substances, cells, or populations of cells. Specific examples of isolated cells include partially pure cell compositions, substantially pure cell compositions, and cells cultured in media that do not exist in nature. Isolated cells can be obtained by separating desired cells or populations thereof from other substances or cells in the environment, or by removing one or more other cell populations or subpopulations from the environment.

[0072] As used herein, terms such as “purify” mean increasing purity. For example, purity can be increased to at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0073] As used herein, the term “coding” refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that function as a template for the synthesis of other polymers and macromolecules in biological processes having defined sequences of nucleotides (i.e., rRNA, tRNA, and mRNA) or defined sequences of amino acids and the biological properties derived therefrom. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is usually listed in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0074] "Construct" refers to a complex of macromolecules or molecules containing polynucleotides that are delivered to a host cell either in vitro or in vivo. As used herein, "vector" refers to any nucleic acid construct that can induce the delivery or transfer of foreign genetic material to a target cell and can replicate and / or express in the target cell. As used herein, the term "vector" includes the delivered construct. A vector may be a linear or cyclic molecule. A vector may or may not be incorporated. The main types of vectors include, but are not limited to, plasmids, episomal vectors, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, and Sendai virus vectors.

[0075] "Integration" means that one or more nucleotides of a construct are stably inserted into the cellular genome, i.e., covalently bonded to a nucleic acid sequence within the cell's chromosomal DNA. "Targeted integration" means that one nucleotide of a construct is inserted into the cell's chromosome or mitochondrial DNA at a pre-selected site or "integration site." As used herein, the term "integration" further refers to a process involving the insertion of one or more exogenous sequences or nucleotides of a construct, with or without the deletion of an endogenous sequence or nucleotide at the integration site. If there is a deletion at the insertion site, "integration" may further include the substitution of the deleted nucleotide in the endogenous sequence or one or more inserted nucleotides.

[0076] As used herein, the term “exogenous” is intended to mean that the reference molecule or reference activity is introduced into the host cell or is unnatural to the host cell. The molecule can be introduced, for example, by introducing the coding nucleic acid into the host's genetic material, for example, by incorporating it into the host's chromosome, or as non-chromosomal genetic material, for example, as a plasmid. Thus, as used in relation to the expression of coding nucleic acids, the term refers to the introduction of the coding nucleic acid into the cell in an expressible form. The term “endogenous” refers to a reference molecule or activity present in the host cell. Similarly, as used in relation to the expression of coding nucleic acids, this term refers to the expression of coding nucleic acids that are contained within the cell and not introduced exogenously.

[0077] As used herein, “the gene of interest” or “the polynucleotide sequence of interest” is a DNA sequence that, under the control of an appropriate regulatory sequence, is transcribed into RNA and, in some cases, translated into a polypeptide in vivo. The gene of interest or polynucleotide may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, the gene of interest may encode miRNA, shRNA, natural polypeptides (i.e., naturally occurring polypeptides) or fragments thereof, variant polypeptides (i.e., variants of natural polypeptides having less than 100% sequence identity with the natural polypeptide) or fragments thereof, engineered polypeptides or peptide fragments, therapeutic peptides or polypeptides, contrast markers, selection markers, and the like.

[0078] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotide of any length, which is either a deoxyribonucleotide or a ribonucleotide, or an analogue thereof. The sequence of a polynucleotide consists of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), except when the polynucleotide is RNA, in which case thymine is replaced by uracil (U). Polynucleotides may include genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides also refer to both double-stranded and single-stranded molecules.

[0079] As used herein, the terms “peptide,” “polypeptide,” and “protein” are interchangeable and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and long chains, also commonly called polypeptides or proteins in the art. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0080] As used herein, the term “subunit” refers to each distinct polypeptide chain of a protein complex, each of which can form a stable folded structure on its own. Many protein molecules consist of one or more subunits, and the amino acid sequences of each subunit may be identical, similar, or completely different. For example, the CD3 complex consists of CD3α, CD3ε, CD3δ, CD3γ, and CD3ζ subunits, which form CD3ε / CD3γ, CD3ε / CD3δ, and CD3ζ / CD3ζ dimers. Within a single subunit, the continuous portion of the polypeptide chain often folds into a compact, local, semi-independent unit called a “domain.” Many protein domains may further contain independent “structural subunits,” also called subdomains, which contribute to the common function of the domain. Thus, as used herein, the term “subdomain” refers to a protein domain within a larger domain, for example, a binding domain within the external domain of a cell surface receptor, or a stimulating or signaling domain within the internal domain of a cell surface receptor.

[0081] "Operatively linked" or "operatably linked" is interchangeable with "operatably connected" or "operatably connected," and refers to the association of nucleic acid sequences (or amino acids in a polypeptide with multiple domains) on a single nucleic acid fragment such that the function of one is influenced by the other. For example, a promoter is operatably linked to a coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter) if it can influence the expression of that coding sequence or functional RNA. A coding sequence can be operatably linked to a regulatory sequence in the sense or antisense direction. As a further example, a receptor-binding domain can be operatably connected to an intracellular signaling domain such that the binding of the receptor to a ligand translates the signal in response to that binding.

[0082] As used herein, a “fusion protein” or “chimeric protein” is a protein produced by genetic engineering to link two or more partial or complete polynucleotides encoding sequences that encode separate proteins, the expression of which results in a single peptide or plurality of polypeptides having functional properties derived from each of the original proteins or fragments thereof. A linker (or spacer) peptide may be added between two adjacent polypeptides from different sources in a fusion protein. The chimeric fusion receptor (CFR) described herein is a fusion protein or chimeric protein.

[0083] As used herein, the term “genetic imprint” refers to genetic or epigenetic information that contributes to the preferred therapeutic attributes of a source cell or iPSC and can be retained in source cell-derived iPSCs and / or iPSC-derived hematopoietic lineage cells. As used herein, “source cell” is a non-pluripotent cell that can be used to generate an iPSC through reprogramming, and the source cell-derived iPSC may further differentiate into specific cell types, including any hematopoietic lineage cell. Source cell-derived iPSCs and cells differentiated therefrom may be collectively referred to as “derived” cells or “derivative” cells, depending on the context. For example, as used throughout this application, derived effector cells, or derived NK lineage cells or derived T lineage cells, are cells differentiated from an iPSC compared to their corresponding primary cells obtained from a natural / native source such as peripheral blood, umbilical cord blood, or other donor tissue. As used herein, a genetic imprint conferring a preferred therapeutic attribute is incorporated into an iPSC by reprogramming selected source cells that are specific to a donor, disease, or therapeutic response, or by introducing a recombinant modality into the iPSC using genome editing. In the case of source cells obtained from a specifically selected donor, disease, or therapeutic situation, the genetic imprint contributing to the preferred therapeutic attribute may include a context-specific genetic or epigenetic modification that represents a retainable phenotype, i.e., the preferred therapeutic attribute, which is passed on to the iPSC-derived cells of the selected source cell, regardless of whether the underlying molecular event has been identified. Source cells specific to a donor, disease, or therapeutic response may include a genetic imprint that can be retained in iPSCs and induced hematopoietic lineage cells, which may include, but are not limited to, a pre-configured single-specific TCR from virus-specific T cells or invariant natural killer T (iNKT) cells, a traceable and desirable genetic polymorphism, such as homozygosity of a point mutation encoding a high-affinity CD16 receptor in a selected donor, and a predetermined HLA requirement, i.e., selected HLA-matched donor cells exhibiting a haplotype in an increased population.As used herein, preferred therapeutic attributes include improved engraftment, transport, homing, viability, self-renewal, persistence, modulation and modification of immune responses, survival rate, and cytotoxicity of induced cells. Preferred therapeutic attributes may also be indicated by antigen-targeted receptor expression, HLA presentation or absence thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immunomodulation, improved on-target specificity with reduced extratumor effects, and resistance to treatments such as chemotherapy. When induced cells with one or more therapeutic attributes are obtained by differentiating iPSCs that incorporate a genetic imprint(s) conferring preferred therapeutic attributes, such induced cells are also called “synthetic cells.” Generally, synthetic cells have one or more non-native cellular functions when compared to their nearest corresponding primary cells, whether the synthetic cells are differentiated from engineered pluripotent cells or obtained by engineering primary cells from natural / native sources such as peripheral blood, umbilical cord blood, or other donor tissues.

[0084] As used herein, the term “enhanced therapeutic properties” refers to the therapeutic properties of a cell that are enhanced compared to a typical immune cell of the same common cell type. For example, NK cells with “enhanced therapeutic properties” have enhanced, improved, and / or increased therapeutic properties compared to typical unmodified and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell engraftment, transport, homing, viability, self-renewal, persistence, modulation and modification of the immune response, survival rate, and cytotoxicity. Therapeutic properties of immune cells are also indicated by antigen-targeting receptor expression, HLA presentation or absence thereof, resistance to the tumor microenvironment, induction of bystander immune cells and immunomodulation, improved on-target specificity with reduced extratumor effects, and resistance to treatments such as chemotherapy.

[0085] As used herein, the term “engager” refers to a molecule, such as a fusion polypeptide, that can form a link between immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils) and tumor cells, and can activate immune cells. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTE), bispecific killer cell engagers (BiKE), triplicate killer cell engagers (TriKE), or multispecific killer cell engagers, and universal engagers that can be adapted to multiple immune cell types.

[0086] As used herein, the term “surface trigger receptor” refers to a receptor capable of inducing or initiating an immune response, such as a cytotoxic response. Surface trigger receptors can be manipulated and expressed in effector cells, such as T cells, NK cells, NKT cells, B cells, macrophages, or neutrophils. In some embodiments, the surface trigger receptor facilitates the binding of bispecific or multispecific antibodies between effector cells and specific target cells (e.g., tumor cells), regardless of the effector cell’s innate receptor and cell type. Using this approach, iPSCs containing a universal surface trigger receptor can be generated and differentiated into populations of various effector cell types expressing the universal surface trigger receptor. “Universal” means that the surface trigger receptor can be expressed and activated in any effector cell regardless of cell type, and all effector cells expressing the universal receptor can bind to or ligate to engagers that can be recognized by the surface trigger receptor, regardless of the engager’s tumor-binding specificity. In some embodiments, engagers with the same tumor-targeting specificity are used to bind to the universal surface trigger receptor. In some embodiments, engagers with different tumor targeting specificities are used to bind to universal surface trigger receptors. Thus, one or more effector cell types may be used to kill one specific type of tumor cell, or two or more types of tumor cells. Surface trigger receptors generally include a costimulatory domain for activating effector cells and an epitope specific to the epitope-binding region of the engager. A bispecific engager has one end specific to the epitope of the surface trigger receptor and the other end specific to the tumor antigen.

[0087] As used herein, the term “safety switch protein” refers to an engineered protein designed to prevent potential toxicity or other adverse effects of cell therapy. In some examples, the expression of safety switch proteins is conditionally controlled to address safety concerns in transplanted engineered cells in which the gene encoding the safety switch protein is permanently incorporated into the genome. This conditional regulation may be variable and may include control by post-translational activation via small molecules and tissue-specific and / or transient transcriptional regulation. Safety switches may mediate the induction of apoptosis, inhibition of protein synthesis, arrest of DNA replication and proliferation, transcription and post-transcriptional gene regulation, and / or antibody-mediated depletion. In some examples, safety switch proteins are activated by exogenous molecules, such as prodrugs, and when activated, induce apoptosis and / or cell death in therapeutic cells. Examples of safety switch proteins include, but are not limited to, suicide genes such as caspase 9 (or caspase 3 or 7), thymidine kinase, cytosine deaminase, B cell CD20, modified EGFR, and any combination thereof. In this strategy, a prodrug administered when an adverse event occurs is activated by a suicide gene product, killing the transduced cells.

[0088] As used herein, the term “pharmaceutically active protein or peptide” refers to a protein or peptide capable of achieving biological and / or pharmaceutical effects on an organism. Pharmaceutically active proteins may have therapeutic or mitigating properties for a disease and may be administered to improve, relieve, alleviate, reverse, or reduce the severity of the disease. Pharmaceutically active proteins may also have preventive properties and may be used to prevent the onset of a disease or, if it appears, to reduce the severity of such a disease or pathological condition. Examples of pharmaceutically active proteins include whole proteins or peptides, pharmaceutically active fragments or variants of proteins or peptides, pharmaceutically active analogs of proteins or peptides, and analogs of protein or peptide fragments. The term “pharmaceutically active protein” may also refer to multiple proteins or peptides that act synergistically or cooperatively to produce a therapeutic effect. Examples of pharmaceutically active proteins or peptides include, but are not limited to, receptors, binding proteins, transcription and translation factors, tumor growth inhibitory proteins, antibodies or fragments thereof, growth factors, and / or cytokines.

[0089] As used herein, the term “signaling molecule” refers to any molecule that modifies, participates in, inhibits, activates, reduces, or increases cellular signaling. “Signaling” refers to the transmission of molecular signals in the form of chemical modification by the recruitment of protein complexes along pathways that ultimately lead to biochemical events within a cell. Signaling pathways are well known in the art and include, but are not limited to, G protein-coupled receptor signaling, tyrosine kinase receptor signaling, integrin signaling, Tollgate signaling, ligand-dependent ion channel signaling, ERK / MAPK signaling pathways, Wnt signaling pathways, cAMP-dependent pathways, and IP3 / DAG signaling pathways.

[0090] As used herein, the term “targeting modality” refers to molecules, such as polypeptides, that are genetically incorporated into cells and enhance the specificity of antigens and / or epitopes, including but not limited to: i) antigen specificity when associated with a specific chimeric antigen receptor (CAR) or T cell receptor (TCR); ii) engager specificity when associated with a monoclonal antibody or bispecific engager; iii) targeting of transformed cells; iv) targeting of cancer stem cells; and v) other targeting strategies in the absence of specific antigens or surface molecules.

[0091] As used herein, the terms “specific” or “singularity” can be used to refer to the ability of a molecule, such as a receptor or engager, to selectively bind to a target molecule, as opposed to nonspecific or nonselective binding.

[0092] As used herein, the term “adoptive cell therapy” refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, identified as T or B cells, that are proliferating ex vivo prior to infusion, whether genetically modified or not.

[0093] As used herein, “therapeutically sufficient amount” means, within its meaning, a non-toxic but sufficient and / or effective amount of the particular treatment and / or pharmaceutical composition it refers to that provides the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the patient’s overall health, the patient’s age, and the stage and severity of the condition. In certain embodiments, “therapeutically sufficient amount” is sufficient and / or effective to alleviate, reduce, and / or improve at least one symptom associated with the disease or condition of the subject being treated.

[0094] Differentiation of pluripotent stem cells requires changes in the culture system, such as stimulants in the culture medium and changes in the physical state of the cells. The most common strategy utilizes the formation of embryoid bodies (EBs) as a common and important intermediate to initiate lineage-specific differentiation. An "embryoid body" is a three-dimensional cluster that has been shown to mimic embryonic development and gives rise to multiple lineages within a three-dimensional region. Through a differentiation process that typically lasts from hours to days, simple EBs (e.g., differentiated aggregated pluripotent stem cells) continue to mature and grow into cystic EBs, which typically last from days to weeks at which point they are processed to continue further differentiation. EB formation is initiated by bringing pluripotent stem cells into close proximity to each other in a three-dimensional multilayer cluster of cells. Typically, this is achieved by one of several methods, including settling the pluripotent cells in droplets, settling the cells in a "U" bottom well plate, or by mechanical agitation. Aggregates maintained in pluripotent culture maintenance medium do not form proper EBs, so further differentiation cues are needed for pluripotent stem cell aggregates to promote EB growth. Therefore, aggregates of pluripotent stem cells need to be transferred to a differentiation medium that provides cue induction to the selected lineage. EB-based culture of pluripotent stem cells typically produces a moderately proliferating population of differentiated cells (i.e., ectoderm, mesoderm, and endoderm) within EB cell clusters. While EB has been shown to promote cell differentiation, it produces heterogeneous cells in various differentiation states due to the inconsistency of the exposure of three-dimensional cell structures to the differentiation cue from the environment. Furthermore, EB is cumbersome to create and maintain. Moreover, cell differentiation through EB formation is accompanied by moderate cell proliferation, leading to reduced differentiation efficiency.

[0095] In contrast, "aggregate formation," unlike "EB formation," can be used to proliferate populations of pluripotent stem cell-inducing cells. For example, during the proliferation of pluripotent stem cells based on aggregates, the culture medium is selected to maintain proliferation and pluripotency. Cell proliferation generally increases the size of aggregates, which form larger aggregates, and these aggregates can routinely dissociate into smaller aggregates mechanically or enzymatically to maintain cell proliferation in culture and increase cell number. Unlike EB culture, cells cultured within aggregates in maintenance culture retain markers of pluripotency. Pluripotent stem cell aggregates require further differentiation queues to induce differentiation.

[0096] As used herein, “monolayer differentiation” is a term that refers to a method of differentiation distinct from differentiation across three-dimensional multilayer clusters of cells, i.e., “EB formation.” Among other advantages disclosed herein, monolayer differentiation avoids the need for EB formation to initiate differentiation. Because monolayer culture does not mimic embryonic development such as EB formation, differentiation into a particular lineage is considered minimal compared to the differentiation of all three germ layers in EB.

[0097] As used herein, “dissociated cells” or “single dissociated cells” means cells that have been substantially separated or purified from other cells or from a surface (e.g., the surface of a culture plate). For example, cells may be dissociated from an animal or tissue by mechanical or enzymatic means. Alternatively, cells that aggregate in vitro may be dissociated from each other enzymatically or mechanically, for example, by dissociation into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells may be dissociated from a culture plate or other surface. Thus, dissociation may involve disrupting cell interactions with the extracellular matrix (ECM) and substrate (e.g., a culture surface), or disrupting the ECM between cells.

[0098] As used herein, “master cell bank” or “MCB” refers to a clone master operated iPSC line, which is a clonal population of iPSCs that has been operated, characterized, tested, qualified, and expanded to include one or more therapeutic attributes, and has been shown to function reliably as starting cell material for the manufacture of cell-based therapeutics through targeted differentiation in a manufacturing setting. In various embodiments, the MCB is maintained, stored, and / or cryopreserved in multiple containers to prevent genetic mutation and / or potential contamination by reducing and / or eliminating the total number of times the iPS cell line is passaged, thawed, or handled during the manufacturing process.

[0099] As used herein, “feeder cells” or “feeder” is a term that describes one type of cell that is co-cultured with a second type of cell, providing a stimuli, growth factors, and nutrients to support the second cell type, thereby providing an environment in which the second type of cell can grow, proliferate, or differentiate. Feeder cells may originate from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts or immortalized mouse embryonic fibroblasts. In another example, peripheral blood-derived cells or transformed leukemia cells support the proliferation and maturation of natural killer cells. Feeder cells can typically be inactivated by irradiation or treatment with an antagonistic mitotic agent such as mitomycin to prevent them from proliferating more than the cells they support when co-cultured with other cells. Feeder cells may include endothelial cells, stromal cells (e.g., epithelial cells or fibroblasts), and leukemia cells. Without limiting the scope of the foregoing, one specific feeder cell type may be a human feeder, such as human dermal fibroblasts. Another feeder cell type may be a mouse embryonic fibroblast (MEF). In general, various feeder cells can be used in part to maintain pluripotency, direct differentiation into specific strains, enhance proliferative capacity, and promote maturation into specialized cell types such as effector cells.

[0100] As used herein, a “feeder-free” (FF) environment refers to an environment such as culture conditions, cell cultures, or culture media that essentially does not contain feeder or stromal cells and / or has not been pretreated by the culture of feeder cells. A “pretreated” medium refers to a medium in which feeder cells have been cultured for a certain period of time, for example, at least one day, and then harvested, and therefore contains many mediator substances, including growth factors and cytokines secreted by the feeder cells cultured in the medium. In some embodiments, a feeder-free environment does not contain either feeder cells or stromal cells and has not been pretreated by the culture of feeder cells.

[0101] As used in relation to genome editing or modification of iPSCs and iPSC-induced non-pluripotent cells differentiated therefrom, or genome editing or modification of non-pluripotent cells and non-pluripotent cell-induced iPSCs reprogrammed therefrom, “functional” means (1) at the gene level – successful knock-in, knock-out, knock-down gene expression, transgenic or controlled gene expression, e.g., inducible or transient expression at a desired cell developmental stage, achieved directly through genome editing or modification, or through “passaging” via differentiation or reprogramming from the initially genome-engineered starting cells, or (2) at the cell level – (i) obtained in the cell through direct genome editing. This refers to the removal, addition, or modification of successful cellular functions / characteristics through: (ii) gene expression modification; (iii) gene expression modification maintained in the cell through “passaging” via differentiation or reprogramming from the initially genome-engineered starting cell; (iii) downstream gene regulation in the cell as a result of gene expression modification that appears only in an earlier developmental stage of the cell or only in the starting cell that gives rise to the cell via differentiation or reprogramming; or (iv) enhanced or newly achieved cellular functions or characteristics exhibited in the mature cell product initially induced from genome editing or modification performed on iPSCs, progenitor cells, or dedifferentiated cell origins.

[0102] "HLA deficiency," including HLA class I deficiency, HLA class II deficiency, or both, refers to cells in which the surface expression level of the complete MHC complex, including the HLA class I protein heterodimer and / or HLA class II heterodimer, is insufficient, no longer maintained, or reduced, and the reduced or diminished level is lower than the level naturally detectable by other cells or synthetic methods.

[0103] As used herein, “modified HLA-deficient iPSC” refers to an HLA-deficient iPSC that is further modified by introducing genes that express proteins related to, but not limited to, non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 4-1BB, DAP10, DAP12, CD24, CD3ζ, 4-1BBL, CD47, CD113, and PDL1, resulting in improved differentiation potential, antigen targeting, antigen presentation, antibody recognition, persistence, immune evasion, resistance to suppression, proliferation, co-stimulation, cytokine stimulation, cytokine production (autocrine or paracrine), chemotaxis, and cytotoxicity, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G), chimeric antigen receptors (CARs), T cell receptors (TCRs), CD16 Fc receptors, BCL11b, NOTCH, RUNX1, IL15, 4-1BB, DAP10, DAP12, CD24, CD3ζ, 4-1BBL, CD47, CD113, and PDL1. “Modified HLA-deficient” cells also include cells other than iPSCs.

[0104] The term "ligand" refers to a substance that forms a complex with a target molecule and generates a signal by binding to a site on the target. Ligands can be natural or artificial substances that can specifically bind to a target. Ligands can be proteins, peptides, antibodies, antibody complexes, conjugates, nucleic acids, lipids, polysaccharides, monosaccharides, small molecules, nanoparticles, ions, neurotransmitters, or any other molecular entity that can specifically bind to a target. The target to which a ligand binds can be a protein, nucleic acid, antigen, receptor, protein complex, or cell. Ligands that bind to a target and alter its function, thereby triggering a signal transduction response, are called "agonists." Ligands that bind to a target and block or reduce the signal transduction response are called "antagonists."

[0105] The term “antibody” encompasses antibodies and antibody fragments that contain at least one binding site that specifically binds to a particular target of interest, the target may be an antigen or a receptor that can interact with a particular antibody. The term “antibody” includes, but is not limited to, immunoglobulin molecules or their antigen-binding or receptor-binding portions. For example, NK cells may be activated by the binding of an antibody or the Fc region of an antibody to its Fc-gamma receptor (FcγR), thereby inducing ADCC (antibody-dependent cellular cytotoxicity)-mediated effector cell activation. The specific fragment or portion of an antigen or receptor to which an antibody binds, or the target in general, is known as an epitope or antigenic determinant. The term “antibody” also includes, but is not limited to, antibody mimetic compounds that mimic the structure and / or function of an antibody or a specific fragment or portion thereof, including native antibodies and their variants, fragments of native antibodies and their variants, peptide bodies and their variants, and single-chain antibodies and their fragments. The antibody may be a mouse antibody, human antibody, humanized antibody, camel IgG, single variable new antigen receptor (VNAR), shark heavy chain antibody (Ig-NAR), chimeric antibody, recombinant antibody, single-domain antibody (dAb), anti-idiotype antibody, bispecific antibody, multispecific antibody, or multimeric antibody, or fragments thereof. An anti-idiotype antibody is specific to the binding of another antibody to an idiotope, and the idiotope is the antigenic determinant of the antibody. A bispecific antibody may be a BiTE (bispecific T cell engager) or BiKE (bispecific killer cell engager), and a multispecific antibody may be a TriKE (triplespecific killer cell engager).Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, Fabc, pFc, Fd, single-strand variable fragment (scFv), tandem scFv (scFv)2, single-strand Fab (scFab), disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb), camel heavy chain IgG and Nanobody® fragments, recombinant heavy chain-only antibody (VHH), and other antibody fragments that maintain antibody binding specificity.

[0106] "Fc receptors," abbreviated as FcR, are classified based on the type of antibody they recognize. For example, those that bind to the most common class of antibody, IgG, are called Fc-gamma receptors (FcγR), those that bind to IgA are called Fc-alpha receptors (FcαR), and those that bind to IgE are called Fc-epsilon receptors (FcεR). Classes of FcRs are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T cells, and B cells) and the signaling characteristics of each receptor. Fc-gamma receptors (FcγR) include several members with different molecular structures and therefore different antibody affinities, such as FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0107] The term "chimeric receptor" is a general term used to describe engineered artificial or hybrid receptor protein molecules that are constructed to contain two or more portions of amino acid sequences derived from at least two different proteins. Chimeric receptor proteins are engineered to give cells the ability to initiate signaling and perform downstream functions upon binding of agonist ligands to the receptor. Exemplary "chimeric receptors" include, but are not limited to, chimeric antigen receptors (CARs), chimeric fusion receptors (CFRs), chimeric Fc receptors (CFcRs), fusions of two or more receptors, and recombinant TCRs (rTCRs or exogenous TCRs) with specificity for tumor-associated antigens (TAAs).

[0108] The term "chimeric Fc receptor," abbreviated as "CFcR," is used to describe engineered Fc receptors in which the native transmembrane domain and / or intracellular signaling domain has been modified or replaced with a non-native transmembrane domain and / or intracellular signaling domain. In some embodiments of chimeric Fc receptors, in addition to one or both of the transmembrane and / or signaling domains being non-native, one or more stimulating domains can be introduced into the intracellular portion of the engineered Fc receptor to enhance receptor-induced cell activation, proliferation, and function. Unlike chimeric antigen receptors (CARs) that contain an antigen-binding domain to a target antigen, chimeric Fc receptors bind to Fc fragments, or the Fc region of an antibody, or to the Fc region contained in an engager or binding molecule, thereby activating cell function by binding to the molecule, either by bringing the target cell closer or not. For example, the Fcγ receptor may be engineered to include a selected transmembrane domain, stimulating domain, and / or signaling domain in the intracellular region that responds to IgG binding in the extracellular domain, thereby generating a CFcR. In one example, CFcRs are produced by manipulating the Fcγ receptor CD16 by replacing its transmembrane and / or intracellular domains. To further improve the binding affinity of CD16-based CFcRs, the extracellular domain of CD64 or a high-affinity variant of CD16 (e.g., F176V) can be incorporated. In some embodiments of CFcRs containing the high-affinity CD16 extracellular domain, the proteolytic cleavage site containing serine at position 197 is eliminated, or the extracellular domain of the receptor is replaced to be non-cleavable, i.e., non-shedding, thereby obtaining an hnCD16-based CFcR.

[0109] The FcγR receptor CD16 has been identified as having two isoforms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and promoting antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, "high affinity CD16," "uncleaved CD16," or "high affinity uncleaved CD16" (abbreviated as hnCD16) refers to native or non-native variants of CD16. Wild-type CD16 has low affinity and, upon activation of NK cells, is subjected to external domain shedding, a proteolytic cleavage process that regulates the cell surface density of various cell surface molecules on leukocytes. F176V and F158V are exemplary CD16 polymorphic variants with high affinity. CD16 variants in which the cleavage site (positions 195-198) in the membrane proximal region (positions 189-212) is altered or eliminated do not shed. The cleavage site and membrane proximal region are described in detail in International Publication 2015 / 148926, the full disclosure of which is incorporated herein by reference. The S197P variant of CD16 is an uncleaved version of CD16. CD16 variants containing both F158V and S197P are highly affinity and uncleaved. Another exemplary high-affinity uncleaved CD16 (hnCD16) variant is an engineered CD16 containing an external domain derived from one or more of the three exons of the CD64 external domain.

[0110] The “T cell receptor,” abbreviated as “TCR,” generally refers to a protein complex found on the surface of T cells that is involved in the recognition of antigen peptide fragments bound to major histocompatibility complex (MHC) molecules. Binding of the TCR to the antigen peptide initiates intracellular activation of TCR-CD3, recruitment of numerous signaling molecules, and branching and integration of signaling pathways, resulting in gene expression and recruitment of transcription factors crucial for T cell proliferation and gain-of-function. A typical TCR comprises two highly variable protein chains (α and β), each containing a constant region adjacent to the cell membrane and a variable region (i.e., a binding domain) that binds to the peptide / MHC. Where specified, as provided herein, the TCR also has specificity for tumor-associated antigens (TAAs) and includes recombinant TCRs (rTCR or exogenous TCRs) comprising transgenic TCRα and transgenic TCRβ chains.

[0111] I. Cells and compositions useful for adoptive cell therapy with enhanced properties Provided herein are strategies for systematically manipulating the regulatory circuits of clonal iPSCs without affecting the differentiation potential of iPSCs or the cell developmental biology of iPSCs and their induced cells, while enhancing the therapeutic properties of induced cells differentiated from iPSCs. The iPSC-induced cells are functionally improved and suitable for adoptive cell therapy after selective modality combinations have been introduced into the cells at the iPSC level through genomic manipulation. Previously, it was unclear whether modified iPSCs, including one or more gene edits provided, still possessed the ability to enter cell development and / or mature to produce functionally differentiated cells while retaining the modified activity and / or properties. Unexpected failures during targeted cell differentiation from iPSCs are attributed to embodiments including, but not limited to, specific gene expression or absence at developmental stages, HLA complex presentation requirements, protein shedding of introduced surface expression modalities, and the need to reconfigure differentiation protocols to allow for changes in the cellular phenotype and / or function. This application demonstrates that one or more selected genomic modifications provided herein do not adversely affect the differentiation potential of iPSCs, and that functional effector cells derived from engineered iPSCs possess enhanced and / or acquired therapeutic properties resulting from the individual or combined genomic modifications, which are retained in the effector cells after iPSC differentiation. Furthermore, all genomic modifications and combinations thereof that can be described in relation to iPSCs and iPSC-induced effector cells are applicable to primary origin cells, including primary immune cells such as T cells, NK cells, or immunomodulatory cells, whether cultured or proliferated, and the modifications result in engineered immune cells useful for adoptive cell therapy.

[0112] 1. Exogenous TCRs specific to tumor-associated antigens The alpha-beta T cell receptor (TCRαβ) is an antigen-specific receptor essential for the immune response and is present on the cell surface of αβ T lymphocytes. Binding of TCRαβ to the peptide-major histocompatibility complex (pMHC) initiates intracellular activation of TCR-CD3, recruitment of numerous signaling molecules, and branching and integration of signaling pathways, leading to the recruitment of transcription factors crucial for gene expression, T cell proliferation, and gain-of-function. Disruption of the constant region (TRAC or TRBC) of TCR alpha or TCR beta, either by direct T cell editing or by genomic iPSC editing and differentiation as a source for obtaining modified induced T cell lines, is linked to TCR neg This is one approach to producing T cells. In this specification, the terms "TCR negative" or "TCR neg The term "TCR" refers to the absence of endogenous TCR expression resulting from disruption of TCR gene expression by editing the constant region of any TCR chain, or from the absence of TCR gene expression in nature despite the presence of a TCR locus in the genome. neg When used in allogeneic adoptive therapy, T cells do not require HLA matching, exhibit reduced alloreactivity, and can prevent GvHD (Graft-versus-Host Disease).

[0113] However, in the case of iPSCs (TiPSCs) reprogrammed from T cells, it has been found herein that disrupting only TRAC or only TRBC can lead to the formation of an unpaired TCR complex resulting from the expression of the remaining endogenous TCR chain (either TRAC or TRBC) by pairing the endogenous TCR chain with the inserted exogenous TCR chain. For example, transduction of transgenic TCRα chains (tgTCRα) and transgenic TCRβ chains (tgTCRβ) into cells having only TRAC knockout can lead to unpairing between the exogenous TCRα chain and the endogenous TCRβ chain in the transduced effector cells, resulting in a non-functional unpaired TCR.

[0114] TCR disruption also leads to the elimination of the CD3 signaling complex from the T cell surface, despite endogenous CD3 subunit gene expression within the cell. The lack of cell surface CD3 can alter cell proliferation and / or viability, and reduce the functional potential of cells, due to incompatibility with technologies requiring cell surface CD3 recognition and binding (including, but not limited to, CD3-based antibody and engager technologies, CD3 / CD28 T cell activation bead technologies, and CD3-CAR stimulation technologies). Furthermore, TCR neg When iPSCs are used for directed T cell differentiation, undesirable effects on T cell developmental biology and T cell functional maturation may also exist. However, overexpression of CD3 in TCR-negative cells does not appear to restore the cell surface presentation of the CD3 complex and / or CD3 signaling. With respect to cells that do not express CD3 and / or TCR despite the presence of the TCR gene, such as NK cells or NK progenitor cells, acquired CD3 surface expression enables specific signaling and cellular function in NK lineage cells via CD3-based antibodies, engagers, and CAR technologies that would not have been naturally compatible with these cells.

[0115] As provided herein, in various embodiments, both endogenous TCRα and endogenous TCRβ are knocked out in cells using genome editing tools (TCRα - / - and TCRβ - / - or TCRα neg and TCRβ neg ), TCR neg This results in cells. In some other embodiments, only endogenous TCRα is knocked out, which also results in TCR neg The cells yield. Simultaneously with or following TCR knockout, a first polynucleotide encoding TCRα, including a defined variable region and a complete or partial constant region (tgTCRα) of TCRα, and a second polynucleotide encoding TCRβ, including a defined variable region and a complete or partial constant region (tgTCRβ) of TCRβ, are produced. negThe defined TCRα or TCRβ variable region may have any given specificity to an antigen, such that its sequence has been identified or can be identified. The defined TCRα or TCRβ variable region may also be specifically selected to target a selected tumor-associated antigen (TAA). Tumor-associated antigens are presented as peptides in the major histocompatibility complex (MHC) on the cell surface and interact with T cell receptors (TCRs) on effector T cells to stimulate an antitumor response.

[0116] In some alternative embodiments, the tgTCRα and tgTCRβ chains are exogenous TCR complexes (TCRs) that include antigen recognition regions that recognize tumor-associated antigens (TAAs). exo ) form. In certain embodiments, the antigen recognition regions contained in exogenous TCRα and exogenous TCRβ are derived from sources including mouse antibodies, human antibodies, humanized antibodies, camel Ig, single variable novel antigen receptors (VNARs), shark heavy chain-only antibodies (Ig NARs), chimeric antibodies, recombinant antibodies, or fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single-chain antigen-binding fragments (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen-binding fragments (sdAb, nanobodies), recombinant heavy chain-only antibodies (VHH), and other antibody fragments that maintain the binding specificity of the entire antibody.

[0117] In some embodiments, one or both of the first and second polynucleotides encoding TCRα and TCRβ are driven by endogenous promoters for TCRα and TCRβ, respectively. In some other embodiments, one or both of the first and second polynucleotides are driven by an exogenous promoter. In some embodiments, the second polynucleotide is driven by the endogenous promoter for TCRβ, while in some other embodiments, the second polynucleotide is driven by an exogenous promoter. In some embodiments, the exogenous promoter includes a constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoter. In some embodiments, the exogenous promoter includes one of CMV, EF1α, PGK, CAG, or UBC. In one embodiment, the exogenous promoter includes at least CAG.

[0118] In some embodiments, the polynucleotide encoding the full or partial length of the TCRα constant region and a given defined variable region includes at least a sequence having identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, when compared to the exemplary sequence SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the full or partial length of the TCRβ constant region and a given defined variable region includes at least a sequence having identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, when compared to the exemplary sequence SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the sequence identity is at least 80%. In some embodiments, the sequence identity is at least 90%. In some embodiments, the sequence identity is at least 95%. In some embodiments, sequence identity is 100%. In some embodiments of polynucleotides encoding the full length of the constant region of TCRα or TCRβ, the polynucleotide further includes a poly-A tail at the C' terminus. In some embodiments of polynucleotides encoding a partial length of the constant region of TCRα or TCRβ, the incorporation of the polynucleotide is at a site within the endogenous constant region and is in-frame with the rest of the endogenous sequence of the constant region of TCRα or TCRβ downstream of the incorporation site, so that the full-length transgenic / chimeric TRAC or TRBC is formed such that part of its sequence is exogenous / transgenic and another part is endogenous. Examples of N-terminal signal peptides include MALPVTALLLPLALLLHA (SEQ ID NO: 4, CD8asp) or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 5, IgKsp), or any signal peptide sequence or functional variant thereof known in the art. Examples of linker peptides include DYKDDDDK (SEQ ID NO: 6, FLAG), or any linker peptide sequence or functional variant thereof known in the art.

[0119] Sequence ID 1: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (TRAC) Sequence ID 2: DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRNHFRCRVSATFWQNPQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (TRBC1) Sequence ID 3 DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (TRBC2)

[0120] As discussed herein, transgenic TCRα (tgTCRα) having a constant region and a defined variable region, optionally together with transgenic TCRβ (tgTCRβ) having a constant region and a defined variable region, associates with an endogenous CD3 subunit containing a CD3ζ chain to form an exogenous TCR complex (TCR exo ) can form such a TCR. exoThe complex may have defined (or selected or targeted) peptide-MHC bonds, or it may not have peptide-MHC bonds, due to the specificity of the selected variable regions of tgTCRα and tgTCRβ. In some embodiments, the tgTCRα and tgTCRβ chains are inserted into the constant region (TRAC or TRBC) of TCRα or TCRβ, thereby disrupting the expression of endogenous TCRα or endogenous TCRβ at the insertion site. In some other embodiments, the exogenous variable region of the TCR is inserted into the TCR locus so that a recombinant TCR is expressed, and the recombinant TCR includes an exogenous variable region operably linked to the endogenous constant region of the TCR.

[0121] In this application, MR1-TCR is provided for proof-of-concept purposes in providing engineered immune effector cells with enhanced functionality. Major histocompatibility complex class-associated protein 1 (MR1) is a non-classical MHC class I protein that is widely expressed with minimal variability among patients, enabling a unique prospect of universal cancer immunotherapy. Thus, in one example, TCR formed within cells... exo This is specific to MR1. TCR exo In embodiments in which tgTCRα is specific to MR1Vα1, tgTCRα includes a variable alpha (Vα) fragment containing a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, identity with the exemplary sequence SEQ ID NO: 7 (MR1Vα), and a TCRα constant fragment containing a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, identity with the exemplary sequence SEQ ID NO: 8. exoIn some embodiments, tgTCRβ includes a variable beta (Vβ) fragment containing a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, identity with the exemplary sequence SEQ ID NO: 9, and a TCRβ constant fragment containing a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage in between, identity with the exemplary sequence SEQ ID NO: 10. Another aspect of this specification provides genetically engineered iPSCs, derivative cells thereof, or populations thereof, the cells comprising exogenous polynucleotides encoding at least tgTCRα, comprising a variable alpha (Vα) fragment and a TCRα constant fragment, and / or a variable beta (Vβ) fragment and a TCRβ constant fragment. In some embodiments, the incorporation of exogenous TCR chain polynucleotides occurs at a site within the cell's endogenous constant region, thereby disrupting the expression of endogenous TCR chains and endogenous TCR complexes.

[0122] Sequence ID 7

[0123] [Table 1] (Underlined areas are variable)

[0124] Sequence ID 8 IQNDPAVYQLRDKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0125] Sequence ID 9

[0126] [Table 2] (Underlined areas are variable)

[0127] Sequence ID 10

[0128] [Table 3] (Underlined areas are variable)

[0129] Another exemplary antigen-specific TCR involves incorporating MICA / B recognition to generate recombinant / exogenous TCRs. MICA and MICB are expression family members of the human major histocompatibility complex class I-related genes (MICs). Members of the MIC family are highly polymorphic (over 100 human alleles) but possess structurally conserved motifs. As tumor-associated antigens, MICA / B is primarily expressed in GI epithelium, endothelial cells, and fibroblasts, its expression is induced by cytotoxic / genotoxic stress, and it has high expression in epithelial carcinoma and melanoma carcinoma. On the other hand, shedding of MICA / B on tumor cells leads to an increase in soluble MICA / B that is not recognized by NKG2D expressed on NK cell subsets and T cell subsets, potentially enabling tumor evasion / escape and inhibiting immune surveillance. Thus, in another example, TCRs formed in cells... exoThe drug targets the tumor antigens MICA and MICB. In some embodiments, the antigen recognition region is an scFv that specifically binds to the conserved α3 ​​domains of MICA and MICB. In one embodiment, the scFv includes a variable region of the heavy chain represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 11, and a variable region of the light chain represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 12. In one embodiment of the MICA / B scFv, the scFv is represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 13, and the linker and / or signal peptide are exemplary and substitutable. In another embodiment of MICA / B scFv, the scFv is represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 14, and the linker and / or signal peptides are exemplary, and their lengths and sequences may vary. Another aspect of this specification provides genetically engineered iPSCs and their derived cells, the cells comprising at least an exogenous polynucleotide encoding MICA / B scFv.

[0130] Sequence ID 11 QIQLVQSGPELKKPGETVKVSCKASGYMFTNYAMNWVKQAPEKGLKWMGWINTHTGDPTYADDFKGRIAFSLETSASTAYLQINNLKNEDTATYFCVRTYGNYAMDYWGQGTSVTVSS (118AA.MICA / B scFv heavy chain (HC))

[0131] Sequence ID 12 DIQMTQTTSSLSASLGDRVTISCSASQDISNYLNWYQQKPDGTVKLLIYDTSILHLGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKFPRTFGGGTTLEIK (107AA.MICA / B scFv light chain (LC))

[0132] Sequence ID 13 (HC-Linker-LC)

[0133] [Table 4] (Signal peptide - other signal peptides are also possible; linker - other linkers are also possible)

[0134] Sequence ID 14 (LC-Linker-HC)

[0135] [Table 5] (Signal peptide - other signal peptides are also possible; linker - other linkers are also possible)

[0136] As further provided, single cells or populations thereof containing polynucleotides encoding tgTCRα and tgTCRβ may further comprise one or more additional manipulated modalities as described herein. A master cell bank comprising single-cell sorting and proliferated clonal manipulated iPSCs having at least one phenotype listed in Table 1 is further provided herein, providing a platform for additional iPSC manipulations and a renewable source for manufacturing ready-made manipulated homogeneous cell therapy products.

[0137] 2. Chimeric antigen receptor (CAR) expression Any CAR design known in the art may be applicable to genetically engineered iPSCs and their induced effector cells. A CAR is generally a fusion protein comprising an external domain containing an antigen-recognition region, a transmembrane domain, and an internal domain. In some embodiments, the external domain may further include a signal peptide or a leader sequence and / or a spacer. In some embodiments, the internal domain may further include a signaling peptide that activates effector cells expressing the CAR. In some embodiments, the internal domain may include a signaling domain, which is derived from the cytoplasmic domain of a signaling protein specific to the activation or function of T cells and / or NK cells. In some embodiments, the antigen-recognition domain may specifically bind to an antigen. In some embodiments, the antigen-recognition domain may specifically bind to an antigen associated with a disease or pathogen. In some embodiments, the disease-associated antigen is a tumor antigen, and the tumor may be a humoral tumor or a solid tumor. In some embodiments, the CAR is suitable for activating either T-cells or NK-cells expressing the CAR. In some embodiments, the CAR is NK cell-specific, containing an NK-specific signaling component. In certain embodiments, T cells are induced from CAR-expressing iPSCs, and the induced T cell lineage may include T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβ T cells, γδ T cells, or a combination thereof. In certain embodiments, NK cells are induced from CAR-expressing iPSCs.

[0138] In certain embodiments, the antigen-recognition region / domain includes mouse antibodies, human antibodies, humanized antibodies, camel Ig, single variable novel antigen receptors (VNARs), shark heavy chain-only antibodies (Ig NARs), chimeric antibodies, recombinant antibodies, or fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single-chain antigen-binding fragments (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen-binding fragments (sdAb, nanobodies), recombinant heavy chain-only antibodies (VHH), and other antibody fragments that maintain the binding specificity of the entire antibody. In some embodiments, the antigen-recognition region of the CAR is derived from the binding domain of a T cell receptor (TCR) targeting tumor-associated antigens (TAAs).

[0139] Non-exclusive examples of antigens that can be targeted by CAR include ADGRE2, B7H3, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDDS, CLEC12A, and cytomegalovirus. Antigens of CMV-infected cells, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, MR1, mucin 1 (Muc-1), mucin 16 (Muc-16) Examples include mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, NYESO1, oncoemetic antigen (h5T4), PDL1, PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms oncoprotein (WT-1), and various pathogen antigens known in the art. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that may cause disease.In some further embodiments, the CAR-T cells comprising an exogenous TCR complex further comprise a CAR specific for one of MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Muc1, Muc16, CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, GD2, MSLN, VEGF-R2, PSMA, and PDL1.

[0140] Some aspects of the present invention provide a CAR binding domain derived from a T cell receptor for a tumor-associated antigen. In various embodiments, the CAR is specific for a tumor cell surface antigen presented by the MR1 protein (MR1-CAR). In some embodiments, the antigen recognition domain of the extracellular domain of the MR1-CAR comprises a variable alpha (Vα) fragment and a variable beta (Vβ) fragment. In some embodiments, the variable alpha (Vα) fragment comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween when compared to the exemplary sequence, SEQ ID NO: 7 (MR1Vα). In some embodiments, the variable beta (Vβ) fragment comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween when compared to the exemplary sequence, SEQ ID NO: 9 (MR1Vβ).

[0141] In some embodiments, the antigen recognition domain of the external domain of MR1-CAR comprises the extracellular domain of MR1 TCRα and the extracellular domain of MR1 TCRβ. In some embodiments, the extracellular domain of MR1 TCRα comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween when compared to the exemplary sequence of SEQ ID NO: 15. In some embodiments, the extracellular domain of MR1 TCRβ comprises at least a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage identity therebetween when compared to the exemplary sequence of SEQ ID NO: 16.

[0142] SEQ ID NO: 15 [Table 6] 09>

[0143] SEQ ID NO: 16

[0144] [Table 7] (Underline indicates variable region)

[0145] Another aspect of the present specification provides genetically engineered iPSCs and their derived cells, the cells comprising an exogenous polynucleotide encoding at least MR1-CAR. In some embodiments, the iPSC-derived effector cells comprising an exogenous polynucleotide encoding at least MR1-CAR are T cells. In some embodiments, the iPSC-derived effector cells comprising an exogenous polynucleotide encoding at least MR1-CAR are NK cells. In some other embodiments, the iPSC-derived effector cells comprising an exogenous polynucleotide encoding at least MR1-CAR are NKT cells. [[ID=​​In another example, this specification provides CARs comprising antigen-recognition regions that target the tumor antigens MICA and MICB. In some embodiments of the MICA / B-targeted CAR, the antigen-recognition region is an scFv that specifically binds to the conserved α3 ​​domains of MICA and MICB. In one embodiment, the scFv includes a variable region of the heavy chain represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 11, and a variable region of the light chain represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 12. In one embodiment of MICA / B scFv, the scFv is represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 13, and the linker and / or signal peptide are exemplary and substitutable. In another embodiment of MICA / B scFv, the scFv is represented by an amino acid sequence that is at least about 99%, about 98%, about 96%, about 95%, about 90%, about 85%, or at least about 80% identical to SEQ ID NO: 14, and the linker and / or signal peptide are exemplary, and their length and sequence may vary. Another aspect of this specification provides genetically engineered iPSCs and their induced cells, the cells comprising at least an exogenous polynucleotide encoding MICA / B-CAR. In some embodiments, the iPSC-induced effector cells comprising at least an exogenous polynucleotide encoding MICA / B-CAR are T cells. In some embodiments, iPSC-induced effector cells containing at least an exogenous polynucleotide encoding MICA / B-CAR are NK cells. In some other embodiments, iPSC-induced effector cells containing at least an exogenous polynucleotide encoding MICA / B-CAR are NKT cells.

[0147] In some embodiments, the transmembrane domain of the CAR includes the full length or at least a portion of the native or modified transmembrane region of CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a T cell receptor polypeptide.

[0148] In some embodiments, the signaling peptide in the internal domain (or intracellular domain) is 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain-containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain), CD3ζ1XX (CD3ζ variant), DAP10 (hematopoietic cell signaling molecule), DAP12 (TYRO protein tyrosine kinase binding protein), DNAM1 (CD226 antigen), FcERIγ (high affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta), IL-2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D It includes the full length or at least a portion of the polypeptides of type II intrinsic membrane proteins, NKp30 (native cytotoxicity trigger receptor 3), NKp44 (native cytotoxicity trigger receptor 2), NKp46 (native cytotoxicity trigger receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain).

[0149] In some embodiments, the internal domain of CAR further includes a second signaling domain and optionally a third signaling domain, each of which is distinct from the first, second, and third signaling domains. In certain embodiments, the second or third signaling domain includes the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8.

[0150] In certain embodiments, the internal domain further comprises at least one co-stimulatory signaling region. This co-stimulatory signaling region may include the full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.

[0151] In one embodiment, a cell-applicable CAR provided herein includes a co-stimulatory domain derived from CD28 and a signaling domain comprising native or modified ITAM1 of CD3ζ represented by an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with SEQ ID NO: 17. In a further embodiment, the CAR comprising the co-stimulatory domain derived from CD28 and native or modified ITAM1 of CD3ζ also comprises a hinge domain and a transmembrane domain derived from CD28, the scFv may be connected to the transmembrane domain via the hinge, and the CAR comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical with SEQ ID NO: 18. In some embodiments, the sequence identity is at least 80%. In some embodiments, the sequence identity is at least 90%. In some embodiments, the sequence identity is at least 95%. In some embodiments, sequence identity is 100%.

[0152] Sequence ID 17 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGE RRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (153a.a.CD28 co-stimulation + CD3ζITAM)

[0153] Sequence ID 18

[0154] [Table 8] (219a.a.CD28 Hinge + CD28™ + CD28 Co-stimulation + CD3ζITAM)

[0155] In another embodiment, the CAR applicable to the cells provided herein includes a transmembrane domain derived from NKG2D, a co-stimulatory domain derived from 2B4, and a signaling domain including a native or modified CD3ζ represented by an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 19. The CAR including the transmembrane domain derived from NKG2D, the co-stimulatory domain derived from 2B4, and the signaling domain including a native or modified CD3ζ may further include a CD8 hinge, and the amino acid sequence of such a structure is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 20. In some embodiments, the sequence identity is at least 80%. In some embodiments, the sequence identity is at least 90%. In some embodiments, the sequence identity is at least 95%. In some embodiments, the sequence identity is 100%.

[0156] SEQ ID NO: 19

[0157] [Table 9] (263 a.a. NKG2D TM + 2B4 + CD3ζ)

[0158] SEQ ID NO: 20

[0159] [Table 10] (308 a.a. CD8 hinge + NKG2D TM + 2B4 + CD3ζ)

[0160] Non-restrictive CAR strategies include heterodimer-conditionally activated CARs through dimerization of a pair of intracellular domains (see, e.g., U.S. Patent No. 9,587,020); split CARs (homologous recombination of antigen-binding, hinge, and internal domains to generate a CAR (see, e.g., U.S. Patent No. 2017 / 0183407)); multi-strand CARs enabling non-covalent bonding between two transmembrane domains linked to an antigen-binding domain and a signaling domain, respectively (see, e.g., U.S. Patent Publication No. 2014 / 0134142); CARs with bispecific antigen-binding domains (see, e.g., U.S. Patent No. 9,447,194); or CARs with a pair of antigen-binding domains that recognize the same or different antigens or epitopes (see, e.g., U.S. Patent No. 8,409,577); or tandem CARs (see, e.g., Hegde et al., J Clin). See Invest.2016;126(8):3036-3052); inductive CARs (see, for example, U.S. Patent Publication Nos. 2016 / 0046700, 2016 / 0058857, and 2017 / 0166877); switchable CARs (see, for example, U.S. Patent Publication No. 2014 / 0219975); and any other designs known in the art.

[0161] In further embodiments, CAR and TCR exoiPSCs and their induced T cells have a CAR inserted into the TCR constant region, resulting in TCR knockout and placing CAR expression under the control of the endogenous TCR promoter. In some other embodiments, the CAR inserted into the TCR constant region is specific to a tumor antigen, including at least one of MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Muc1, Muc16, CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, GD2, MSLN, VEGF-R2, PSMA, and PDL1. Additional CAR insertion sites include, but are not limited to, AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. In some embodiments, TCR exo iPSCs or derivatives thereof, including CARs and optionally CFRs or engagers, further comprise exogenous CD16 having native or CD64-derived external domains in CD16 (F176V and / or S197P), and native or non-native transmembrane, stimuli, and signaling domains. In another embodiment, iPSCs and their derived NK cells include TCRs. exo The gene includes a CAR, which is inserted into the NKG2A or NKG2D locus, resulting in NKG2A or NKG2D knockout, thereby placing CAR expression under the control of the endogenous NKG2A or NKG2D promoter.

[0162] Accordingly, in addition to genetically engineered immune cells comprising functional modalities provided herein, aspects of the present invention provide induced cells obtained from differentiating genomically engineered iPSCs, both of which comprise one or more CARs along with additional modification modalities, as discussed herein. Further provided herein are at least CARs and TCRs. exoThis is a master cell bank containing single-cell sorting and augmented clonally manipulated iPSCs, which provides a platform for additional iPSC manipulations and a renewable source for manufacturing ready-made manipulated homogeneous cell therapy products.

[0163] 3. CD16 knock-in CD16 has been identified as two isoforms, the Fc receptor FcγRIIIa (CD16a; NM_000569.6) and FcγRIIIb (CD16b; NM_000570.4). CD16a is a transmembrane protein expressed by NK cells that binds to monomeric IgG attached to target cells, activating NK cells and promoting antibody-dependent cell-mediated cytotoxicity (ADCC). CD16b is exclusively expressed by human neutrophils. As used herein, “high affinity CD16,” “uncleaved CD16,” or “high affinity uncleaved CD16” (abbreviated as hnCD16) refer to various CD16 variants. Wild-type CD16 has low affinity and, upon activation of NK cells, is subject to downregulation, including external domain shedding, a proteolytic cleavage process that modulates the cell surface density of various cell surface molecules on leukocytes. F176V (also referred to as F158V in some publications) is an exemplary high-affinity CD16 polymorphic allele / variant, while the S197P variant is an example of a genetically engineered, non-cleavable version of CD16. Engineered CD16 variants, including both F176V and S197P, are high-affinity and non-cleavable, as described in detail in International Publication 2015 / 148926, the full disclosure of which is incorporated herein by reference. Furthermore, chimeric CD16 receptors in which the external domain of CD16 is essentially replaced by at least a portion of the external domain of CD64 can also achieve the desired high-affinity and non-cleavable function of a CD16 receptor capable of performing ADCC. In some embodiments, the substituted external domain of the chimeric CD16 includes one or more of the EC1, EC2, and EC3 exons of CD64 (UniPRotKB_P12314 or its isoforms or polymorphic variants).

[0164] Therefore, various embodiments of exogenous CD16 introduced into cells include functional CD16 variants and their chimeric receptors. In some embodiments, the functional CD16 variant is a high-affinity non-cleaved CD16 receptor (hnCD16). In some embodiments, hnCD16 includes both F176V and S197P, and in some embodiments, it includes F176V with the cleavage region eliminated. In some other embodiments, hnCD16 includes at least a portion of the CD64 external domain.

[0165] Accordingly, as intended and described herein, genetically engineered cloned iPSCs are provided to include exogenous CD16 or a variant thereof introduced into the iPSC in other edits. In some embodiments, the exogenous CD16 is a high-affinity uncleaved CD16 receptor (hnCD16). In some embodiments, the exogenous CD16 includes at least a portion of the CD64 exogenous domain. In some embodiments, the exogenous CD16 is a form of CD16-based chimeric Fc receptor (CFcR) including a transmembrane domain, stimulant domain and / or signaling domain not derived from CD16.

[0166] In some embodiments, primary origin or induced effector cells containing exogenous CD16 or its variants are NK lineage cells. In some embodiments, primary origin or induced effector cells containing exogenous CD16 or its variants are T lineage cells. In some embodiments, exogenous CD16 includes hnCD16. In some embodiments, hnCD16 includes the full-length or partial-length extracellular domain of CD64. Exogenous CD16 or its functional variant contained in iPSCs or induced cells has high affinity for binding to ligands that induce downstream signaling upon binding. Ligands that bind to exogenous CD16 or its functional variant include not only ADCC antibodies or fragments thereof, but also bispecific, tripspecific, or multispecific engagers or binders that recognize the extracellular binding domains of CD16 or CD64 of the exogenous CD16. Thus, at least one aspect of the present application provides induced effector cells or a population of such cells that are pre-loaded with one or more pre-selected ADCC antibodies via exogenous CD16 expressed on effector cells in an amount sufficient for therapeutic use in the treatment of a condition, disease, or infection as further detailed in the present disclosure, wherein the exogenous CD16 comprises the extracellular binding domain of CD64, or of CD16 having F176V and S197P.

[0167] In some other embodiments, exogenous CD16 includes CD16 or a variant-based CFcR. Chimeric Fc receptors (CFcRs) are produced to include a non-natural transmembrane domain, a non-natural stimulating domain, and / or a non-natural signaling domain by modifying or substituting the native CD16 transmembrane domain and / or intracellular domain. As used herein, the term “non-natural” means that the transmembrane domain, stimulating domain, or signaling domain is derived from a different receptor other than the receptor that provides the extracellular domain. In the examples herein, CD16 or a variant-based CFcR does not have a transmembrane domain, stimulating domain, or signaling domain derived from CD16. In some embodiments, the exogenous CD16-based CFcR includes CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or a non-native transmembrane domain derived from a T cell receptor polypeptide. In some embodiments, the exogenous CD16-based CFcR includes a non-native stimulative / inhibitory domain derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide. In some embodiments, the exogenous CD16-based CFcR includes a non-native signaling domain derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D polypeptide. In one embodiment of the CD16-based CFcR, the provided chimeric Fc receptor comprises both a transmembrane domain and a signaling domain derived from one of the following polypeptides: IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D.One particular embodiment of a CD16-based chimeric Fc receptor comprises a transmembrane domain of NKG2D, a stimulating domain of 2B4, and a signaling domain of CD3ζ, wherein the extracellular domain of CFcR is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P. Another exemplary embodiment of a CD16-based chimeric Fc receptor comprises a transmembrane domain and a signaling domain of CD3ζ, wherein the extracellular domain of CFcR is derived from the full length or a partial sequence of the extracellular domain of CD64 or CD16, and the extracellular domain of CD16 comprises F176V and S197P.

[0168] Various embodiments of the CD16-based chimeric Fc receptor described above can bind with high affinity to the Fc region of an antibody or fragment thereof, or to a bispecific, triplicate, or multispecific engager or binder. Upon binding, the stimulating and / or signaling domains of the chimeric receptor enable activation of effector cells and cytokine secretion, as well as the killing of tumor cells targeted by the antibody or the bispecific, triplicate, or multispecific engager or binder having a tumor antigen-binding component and an Fc region. Without being constrained by theory, CFcRs of the CD16-based chimeric Fc receptor can contribute to the killing ability of effector cells and increase the proliferation and / or potential of effector cells, either through the non-native transmembrane domain, stimulating domain, and / or signaling domain, or through the binding of an engager to the external domain. Antibodies and engagers can bring tumor cells expressing the antigen and effector cells expressing CFcR into close proximity, which also contributes to enhanced tumor cell killing. Exemplary tumor antigens for bispecific, tripspecific, or multispecific engagers or binders include, but are not limited to, B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EPCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. Some non-limiting exemplary bispecific, tripspecific, or multispecific engagers or binders suitable for binding effector cells expressing CD16-based CFcRs to attack tumor cells include CD16(or CD64)-CD30, CD16(or CD64)-BCMA, CD16(or CD64)-IL15-EPCAM, and CD16(or CD64)-IL15-CD33.

[0169] Unlike endogenous CD16 expressed by primary NK cells, which is cleaved from the cell surface following NK cell activation, various non-cleaved versions of CD16 in induced NK cells avoid CD16 shedding and maintain constant expression. In induced NK cells, non-cleaved CD16 increases the expression of TNFα and CD107a, indicators of improved cellular function. Non-cleaved CD16 also enhances antibody-dependent cell-mediated cytotoxicity (ADCC) and the binding of bispecific, tripspecific, or multispecific engagers. ADCC is a mechanism of NK cell-mediated lysis via the binding of CD16 to antibody-coated target cells. The additional high-affinity properties of hnCD16 introduced into induced NK cells also enable in vitro loading of hnCD16-mediated ADCC antibodies into NK cells before administering cells to subjects requiring cell therapy. As presented herein, hnCD16 may, in some embodiments, comprise F176V and S197P, or a full-length or partial-length external domain derived from CD64, or further comprise at least one of a non-native transmembrane domain, stimulating domain, and signaling domain. As disclosed herein, the application also provides induced NK cells or a population of such cells, pre-loaded with one or more pre-selected ADCC antibodies in quantities sufficient for therapeutic use in the treatment of a condition, disease, or infection, as further detailed herein.

[0170] Unlike primary NK cells, mature T cells derived from primary sources (i.e., natural / natural sources such as peripheral blood, umbilical cord blood, or other donor tissues) do not express CD16. It was unexpected that iPSCs containing expressed exogenous, uncleaved CD16 could differentiate into functional induced T cell lineages that not only express exogenous CD16 without compromising the developmental biology of T cells, but also perform function through the acquired ADCC mechanism. This ADCC acquired in induced T cell lineages can be further used as an approach to rescue antigen escape, which often occurs in dual-targeting and / or CAR-T cell therapy, resulting in tumor recurrence accompanied by reduced or absent expression of CAR-T target antigens, or the expression of mutant antigens that evade recognition by CAR. If the induced T cell lineage contains ADCCs acquired via exogenous CD16 (including functional variants and CD16-based CFcRs) expression, and the antibody targets a tumor antigen different from the tumor antigen targeted by the CAR, the antibody can be used to rescue CAR-T antigen escapes, thereby mitigating or preventing recurrence or relapse of target tumors commonly seen in CAR-T therapy. Such strategies, which reduce and / or prevent antigen escapes while achieving dual targeting, can also be applied to NK cells expressing one or more CARs.

[0171] Therefore, embodiments of the present invention are provided herein for TCR exoThe present invention provides genetically engineered immune cells and induced cells, including induced T lineage cells, which optionally include exogenous CD16 or a variant thereof in addition to CAR. In some embodiments, the exogenous CD16 included in the induced T lineage cells is hnCD16, which includes CD16 exogenous domains including F176V and S197P. In some other embodiments, the hnCD16 included in the induced T lineage cells includes a full-length or partial-length exogenous domain derived from CD64, or may further include at least one of a non-natural transmembrane domain, a stimulating domain, and a signaling domain. As described herein, such induced T lineage cells have an acquired mechanism for targeting tumors with monoclonal antibodies mediated by ADCC to enhance the therapeutic effect of the antibodies. As disclosed, the present invention also provides genetically engineered immune cells and induced cells, including induced T lineage cells or populations thereof, which are pre-loaded with one or more pre-selected ADCC antibodies in sufficient quantities for therapeutic use in the treatment of a condition, disease, or infection, as will be described in more detail below.

[0172] Further provided in this application are, but are not limited to, TCR exo A master cell bank comprising sorted single cells and proliferated clones of engineered iPSCs having at least one phenotype as provided herein, comprising CAR and exogenous CD16 or its variants, the cell bank provides a platform for further iPSC manipulation and a renewable source for manufacturing ready-made engineered homogeneous cell therapy products, including but not limited to induced NK cells and T cells, which have a clear and uniform composition and can be mass-produced on a considerable scale in a cost-effective manner.

[0173] 4. Engager In some embodiments, TCR exo Furthermore, iPSCs and their induced effector cells containing CAR and / or exogenous CD16 or its variants, as selected, are subject to TCR exoThe introduced expression of an engager (i.e., a second tumor antigen) having tumor targeting specificity different from that of the CAR may further include the introduction of such an engager. The engager is a fusion protein comprising two or more single-chain variable fragments (scFv) or other functional variants of different antibodies or fragments thereof, having at least one scFv that binds to effector cell surface molecules or surface trigger receptors, and at least another scFv that binds to target cells via target cell-specific surface molecules. Examples of engagers include, but are not limited to, bispecific T-cell engagers (BiTE), bispecific killer-cell engagers (BiKE), triplicate-cell engagers (TriKE), multispecific killer-cell engagers, or universal engagers that can adapt to multiple immune cell types. Such bispecific or multispecific engagers can induce effector cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils) to tumor cells and activate immune effector cells, demonstrating great potential to maximize the benefits of CAR-T cell therapy. In some embodiments, an engager expressed by engineered iPSC-induced effector cells binds to bystander immune cells containing surface molecules recognized and bound by the engager. In some embodiments, an engager expressed by engineered iPSC-induced effector cells containing a CAR binds to the inducing effector cells and activates them upon binding to a tumor antigen different from the CAR antigen. In some embodiments, an engager expressed by engineered iPSC-induced effector cells containing a CAR binds to the inducing effector cells via endogenous surface molecules of the effector cells. In some embodiments, an engager expressed by engineered iPSC-induced effector cells containing a CAR binds to the inducing effector cells via exogenous surface trigger receptors of the effector cells. In some embodiments, the exogenous surface trigger receptor of the effector cell expressing the engager includes a CFR (chimeric fusion receptor) as further provided herein.

[0174] In some embodiments, surface trigger receptors facilitate the binding of bispecific or multispecific antibodies between effector cells and specific target cells, such as tumor cells, regardless of the effector cells' innate receptors and cell type. In some other embodiments, the methods and compositions provided herein can be used to manipulate iPSCs to create a master cell bank containing optionally sorted single cells and proliferated cloned iPSCs, and then to direct the differentiation of iPSCs into T cells, NK cells, or other effector cells containing the same genotype as the source iPSCs, thereby introducing one or more exogenous surface trigger receptors into the effector cells.

[0175] This approach can also be used to generate iPSCs containing the universal surface trigger receptor, and then differentiate such iPSCs into populations of various effector cell types expressing the universal surface trigger receptor. In some embodiments, engagers with the same tumor targeting specificity are used to bind to different universal surface trigger receptors. In some embodiments, engagers with different tumor targeting specificities are used to bind to the same universal surface trigger receptor. Thus, one or more effector cell types may be used to kill one specific type of tumor cell, or two or more types of tumor cells. Surface trigger receptors generally include a co-stimulatory domain for effector cell activation and an anti-epitope specific to the engager's epitope, or vice versa, and the surface trigger receptor contains an epitope that is recognizable or specific to the engager's anti-epitope. For example, a bispecific engager has one end specific to the anti-epitope of the surface trigger receptor and the other end specific to a tumor antigen.

[0176] Exemplary effector cell surface molecules or surface trigger receptors that can be used for bispecific or multispecific engager recognition, coupling, or binding include, but are not limited to, CD3, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant or chimeric Fc receptor form thereof disclosed herein. In some embodiments, CD16 expressed on the surface of effector cells for engager recognition is hnCD16, which includes the extracellular domain of CD16 (including F176V and optionally S197P) or CD64, and native or non-native transmembrane domains, stimulating domains, and / or signaling domains, as described herein. In some embodiments, CD16 expressed on the surface of effector cells for engager recognition is a CD16-based chimeric Fc receptor (CFcR). In some embodiments, the CD16-based CFcR comprises the transmembrane domain of NKG2D, the stimulatory domain of 2B4, and the signaling domain of CD3ζ, the extracellular domain of CD16 is derived from the full-length or partial sequence of the extracellular domain of CD64 or CD16, and optionally the extracellular domain of CD16 comprises F176V and optionally S197P.

[0177] Exemplary tumor cell surface molecules for bispecific or multispecific engager recognition include, but are not limited to, B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, and ROR1. In one embodiment, the bispecific engager is a bispecific antibody (CD3×CD19 or CD3-CD19) specific to CD3 and CD19, and in another embodiment, the bispecific antibody is CD3-CD33. In another embodiment, the bispecific antibody is CD3-EpCAM. To bind to CD16 on effector cells, the bispecific antibody comprises CD16-CD30 or CD64-CD30. In yet another embodiment, the bispecific antibody comprises CD16-BCMA or CD64-BCMA. In yet another embodiment, the bispecific antibody further comprises a linker between the effector cell and the tumor cell antigen-binding domain, for example, modified IL15 can be used as a linker for effector NK cells (referred to in some publications as TriKE, or triplicate killer enhancer) to promote effector cell proliferation / autonomy. In one embodiment, TriKE is CD16-IL15-EpCAM or CD64-IL15-EpCAM. In another embodiment, TriKE is CD16-IL15-B7H3 or CD64-IL15-B7H3. In yet another embodiment, TriKE is CD16-IL15-CD33 or CD64-IL15-CD33. In yet another embodiment, TriKE is NKG2C-IL15-CD33. IL15 in TriKE also includes, but is not limited to, IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL18, and IL21, and may be derived from other cytokines.

[0178] In some embodiments, the engagers include ADGRE2, B7H3, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD52, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD269 (BCMA), CDS, CLEC12A, and cytom Antigens of Gallovirus (CMV)-infected cells (e.g., cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinases erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epithelial growth factor HER2 receptor 2, human telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, MR1, mucin 1 (Muc-1), mucin 16 (Muc-16), mesocerin It contains a first binding domain that is specific to one of the following: MSLN, NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NYESO1, carcinoembryonic antigen (h5T4), PDL1, PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms oncoprotein (WT-1), and various pathogen antigens.

[0179] In some embodiments, the surface trigger receptor for a bispecific or multispecific engager may be endogenous to the effector cell, sometimes depending on the cell type. In some other embodiments, the methods and compositions provided herein can be used to further manipulate iPSCs containing the genotypes listed in Table 1, direct the differentiation of the iPSCs into effector cells containing the same genotype and surface trigger receptor as the source iPSC, and introduce one or more exogenous surface trigger receptors into the effector cells.

[0180] In some embodiments, as an alternative to genome-engineered effector cells expressing an engager as provided herein, the engager may be used as a therapeutic agent in combination therapy compositions targeting one or more antigens associated with a condition, disease, or indication (as described above). In some embodiments, the engager is a bispecific T cell engager (BiTE). In some embodiments, the engager is a bispecific killer cell engager (BiKE). In some embodiments, the engager is a triplicate killer cell engager (TriKE). In some embodiments, the engager is a multispecific killer cell engager. In some embodiments, the engager is a universal engager compatible with multiple immune cell types. In some embodiments, the engager in a combination therapy or composition used therein activates bystander immune cells for tumor killing in the recipient of the composition. In some embodiments, the engager in a combination therapy or composition used therein activates effector cells contained in the combination therapy composition. In some embodiments of combination therapy compositions useful for the treatment of humoral or solid tumors, the composition comprises iPSC-induced effector cells containing at least one CAR provided herein. In some embodiments, the iPSC-induced effector cells of the composition comprise hematopoietic lineage cells comprising the genotypes listed in Table 1. In some embodiments, the iPSC-induced effector cells of the composition comprise NK lineage cells comprising the genotypes listed in Table 1. In some embodiments, the iPSC-induced effector cells of the composition comprise T lineage cells comprising the genotypes listed in Table 1.

[0181] Therefore, in addition to genetically engineered immune cells including the functional modalities provided herein, embodiments of the present invention also include TCRs provided herein. exo And, optionally, CAR, and optionally, exogenous CD16 or its variant, TCR. exoThe present invention provides iPSC-inducing effector cells comprising, or one or more of the engagers. Further provided in this application is a master cell bank comprising sorted single cells and proliferated clones of engineered iPSCs having at least one phenotype as provided herein, including but not limited to engagers, the cell bank providing a platform for further iPSC manipulation and a renewable source for manufacturing ready-made engineered homogeneous cell therapy products, including but not limited to induced NK cells and T cells, which have a clear and uniform composition and can be mass-produced on a considerable scale in a cost-effective manner.

[0182] 5. Cell surface CFRs (Chimeric Fusion Receptors) The implementation of CFRs allows effector cells to initiate appropriate signaling cascades via CFR binding with selected agonists to enhance the therapeutic properties of effector cells expressing CFRs. Such enhanced effector cell therapeutic properties include, but are not limited to, increased activation and cytotoxicity; acquisition of dual-targeting ability; extended persistence; improved transport and tumor penetration; enhanced ability to prime, activate or mobilize bystander immune cells to tumor sites; enhanced resistance to immunosuppression; improved ability to rescue tumor antigen escapes; and / or regulation of cellular signaling feedback, metabolism, and apoptosis.

[0183] Therefore, in various embodiments, TCR exoiPSCs and induced cells comprising, optionally, one or more of CARs, exogenous CD16 or its variants and engagers, may further comprise a CFR, which generally comprises an external domain fused to a transmembrane domain operably connected to an internal domain, wherein the CFR lacks ER (endoplasmic reticulum) retention signals or endocytosis signals in either the external domain, transmembrane domain, or internal domain. The external domain of the CFR is for initiating signaling upon binding to an engager, the transmembrane domain is for membrane fixation of the CFR, and the internal domain comprises at least one signaling domain that modulates (i.e., activates or inactivates) a signaling pathway selected to enhance effector cell therapeutic properties (including, but not limited to, tumor killing, persistence, mobility, differentiation, TME neutralization, and / or controlled apoptosis). Elimination of ER retention signals from the CFR enables cell surface presentation of the CFR when expressed, while elimination of endocytosis signals from the CFR reduces CFR internalization and down-surface regulation. It is important to select a domain component that does not possess either an ER retention signal or an endocytosis signal, or to remove either an ER retention signal or an endocytosis signal from a selected component of the CFR using molecular engineering tools. In addition, the domains of the CFRs provided herein are modular, meaning that for a given internal domain of the CFR, the external domain of the CFR is switchable depending on the binding specificity of a selected agonist used with the CFR, such as an antibody, BiTE, TRiKE, or any other type of engager; and for a given external domain and specificity-matching agonist, the internal domain is switchable depending on the desired signaling pathway to be activated.

[0184] In some embodiments, the CFR external domain described herein includes the full or partial length of the extracellular portion of a protein involved in cell-cell signaling or interaction. In some embodiments, the CFR external domain includes the full or partial length of the extracellular portion of CD3ε, CD3γ, CD3δ, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof or combination thereof, and chimeric portions. In some embodiments, the CFR external domain is recognized by at least an agonist, for example, an antibody or engager (e.g., BiTE, BiKE, or TriKE) containing an epitope-specific binding domain in the CFR external domain. In some embodiments, an antibody or engager used as an agonist with CFR-expressing cells binds to at least one extracellular epitope of the CFR, the CFR comprising the full or partial length of the extracellular portion of CD3ε, CD3γ, CD3δ, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof, or a combination / chimeric form thereof. In some embodiments, the engager recognizes at least one tumor antigen, including B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, PSMA, PAMA, P-cadherin, or ROR1. In certain embodiments, both the ER retention signal and the endocytosis signal are absent or excluded from the CFR extradomain using genetic engineering techniques.

[0185] In some embodiments, the external domain of the CFR includes the full or partial length of the extracellular portion of CD3ε, CD3γ, CD3δ, or any functional variant or combination / chimeric form thereof, for the utilization of a CD3-based agonist. Non-limiting exemplary CD3-based agonists, including but not limited to antibodies or engagers, include CD3×CD19, CD3×CD20, CD3×CD33, CD3×EpCAM, CD3×B7H3, blinatumomab, catumakisomab, erzumakisomab, RO6958688, AFM11, MT110 / AMG110, MT111 / AMG211 / MEDI-565, AMG330, MT112 / BAY2010112, MOR209 / ES414, MGD006 / S80880, MGD007, and / or FBTA05. In some embodiments, the external domain of the CFR includes the full or partial length of the extracellular portion of NKG2C or any functional variant thereof to utilize an NKG2C-based agonist. Exemplary NKG2C-based agonists, including but not limited to antibodies or engagers, include NKG2C-IL15-CD33, NKG2C-IL15-CD19, and / or NKG2C-IL15-CD20. In some other embodiments, the external domain of the CFR includes the full or partial length of the extracellular portion of CD28 or any functional variant thereof to utilize a CD28-based agonist. Non-limiting exemplary CD28-based agonists, including but not limited to antibodies or engagers, include at least one of 15E8, CD28.2, CD28.6, YTH913.12, 37.51, 9D7(TGN1412), 5.11A1, ANC28.1 / 5D10, and / or 37407.

[0186] In some embodiments, the external domain of the CFR includes the full or partial length of the extracellular portion of CD16, CD64, or any functional variant or combination / chimeric form thereof, for the use of a CD16 or CD64-based agonist. Exemplary CD16 or CD64-based agonists, including but not limited to antibodies or engagers, include IgG antibodies or CD16 or CD64-based engagers. When the Fc portion of an IgG antibody binds to a CD16 or CD64-based CFR, it activates antibody-dependent cell-mediated cytotoxicity (ADCC) in CFR-expressing cells, along with other enhanced therapeutic properties conferred by the signaling domain contained within the internal domain of the CFR. Non-limiting, exemplary CD16 or CD64-based agonists, including antibodies or engagers, include at least one of CD16×CD30, CD64×CD30, CD16×BCMA, CD64×BCMA, CD16-IL-B7H3, CD64-IL-B7H3, CD16-IL-EpCAM or CD64-IL-EpCAM, CD16-IL-CD33 or CD64-IL-CD33, and “IL” in TriKE includes all or part of at least one cytokine, including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, or any functional variant or combination / chimeric form thereof.

[0187] Generally, a transmembrane domain is a three-dimensional protein structure that is thermodynamically stable in a membrane, such as a phospholipid bilayer of a biological membrane (e.g., the membrane of a cell or cell vesicle). Therefore, in some embodiments, the transmembrane domain of the CFR of the present invention includes a single α-helix, a stable complex of several transmembrane α-helices, a transmembrane β-barrel, a β-helix of gramicidin A, or any combination thereof. In various embodiments, the transmembrane domain of the CFR includes all or part of a “transmembrane protein” or “membrane protein” located within the membrane. As used herein, “transmembrane protein” or “membrane protein” is a protein located in and / or within the membrane. Examples of transmembrane proteins suitable for providing the transmembrane domain included in the CFR of the present invention include, but are not limited to, receptors, ligands, immunoglobulins, glycophorins, or combinations thereof. In some embodiments, the transmembrane domains included in the CFR include all or part of the transmembrane domains of 2B4, 4-1BB, BTLA, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, CS1, CTLA-4, DNAM1, DAP10, DAP12, FcERIγ, ICOS, ICAM-1, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, LAG3, PD1, NKp30, NKp44, NKp46, NKG2C, NKG2D, OX40, T cell receptor polypeptides (such as TCRα and / or TCRβ), nicotinic acetylcholine receptors, GABA receptors, or any combination thereof.

[0188] In some embodiments, the transmembrane domain includes all or part of the transmembrane domains of IgG, IgA, IgM, IgE, IgD, or any combination thereof. In some embodiments, the transmembrane domain includes all or part of the transmembrane domains of glycophorin A, glycophorin D, or any combination thereof. In certain embodiments of the CFR transmembrane domain, both the ER retention signal and the endocytosis signal are absent or eliminated using genetic engineering. In various embodiments, both the ER retention signal and the endocytosis signal are absent or eliminated from the CFR transmembrane domain using genetic engineering methods. In some embodiments, the transmembrane domain includes all or part of the transmembrane domains of CD28, CD8, or CD4.

[0189] In some embodiments, the internal domain of the CFR includes at least one signaling domain that activates a selected intracellular signaling pathway. In various embodiments of the CFR internal domain, both ER retention signaling and endocytosis signaling are absent or eliminated from them using genetic engineering techniques. In some embodiments, the internal domain includes at least one cytotoxic domain. In some other embodiments, the internal domain may optionally include, in addition to the cytotoxic domain, one or more of the following: a co-stimulatory domain, a sustained signaling domain, a death-inducing signaling domain, a tumor cell regulatory signaling domain, or any combination thereof. In some embodiments, the signaling peptide in the internal domain (or intracellular domain) includes the full length or at least a portion of the polypeptide of 2B4, CD2, CD3ζ, CD3ζ1XX, CD8, CD28, CD28H, CD137(4-1BB), CS1, DAP10, DAP12, DNAM1, FcERIγ, IL2Rγ, IL7R, IL21R, IL2Rβ(IL15Rβ), IL21, IL7, IL12, IL15, IL21, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In some embodiments, the cytotoxic domain of CFR comprises the full length or at least a portion of the polypeptide CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137(4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D. In one embodiment, the cytotoxic domain of CFR comprises an amino acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with at least one ITAM (immune receptor tyrosine-based activation motif) of CD3ζ. In one embodiment, the cytotoxic domain of CFR comprises modified CD3ζ.

[0190] In some embodiments, the CFR includes an internal domain containing a co-stimulatory domain in addition to a cytotoxic signaling domain. Suitable co-stimulatory domains for use in the CFR may include the full length or at least a portion of the polypeptides CD2, CD27, CD28, CD40L, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof. In some embodiments, the co-stimulatory domain of the CFR includes the full length or at least a portion of the polypeptides CD28, 4-1BB, CD27, CD40L, ICOS, CD2, or any combination thereof. In some embodiments, the CFR includes an internal domain containing a co-stimulatory domain of CD28 and a cytotoxic domain of CD3ζ (also referred to as "28ζ").

[0191] In some embodiments, the CFR includes an internal domain containing a sustained signaling domain in addition to a cytotoxic signaling domain and / or a co-stimulatory domain. Suitable sustained signaling domains for use in the CFR include, but are not limited to, all or part of the internal domains of cytokine receptors such as IL7R, IL15R, IL18R, IL12R, IL23R, or combinations thereof. In further embodiments, the internal domain of the CFR may include a receptor tyrosine kinase (RTK) such as EGFR to provide tumor cell control, or a complete or partial intracellular portion of a tumor necrosis factor receptor (TNFR) such as FAS to provide controlled cell death capability.

[0192] In various embodiments, the exemplary CFR comprises an extracellular portion of the CD3 subunit CD3ε, CD3δ, or CD3γ, or CD28; a transmembrane domain of CD28, CD8, or CD4; and at least one internal domain of CD3ε, CD3γ, CD3δ, or CD28, wherein the ER retention motif and / or endocytosis motif in the external domain, transmembrane domain, and / or internal domain are excluded. In various embodiments, the CFR provided herein further comprises a signal peptide at the N-terminus of the CFR external domain.

[0193] In some exemplary embodiments, the CFR comprises an external domain of one CD3 subunit, and in some other embodiments, the CFR comprises a single-chain heterodimer external domain comprising an external domain of CD3ε linked to an external domain of CD3δ or CD3γ. The type and length of the linker in the single-chain heterodimer external domain may vary.

[0194] Cell surface-expressed CFRs (including CD3-based CFRs, also referred to as cs-CD3 in some contexts) in the various constructs described herein can function as cell surface trigger receptors for binding to molecules with selected binding specificity, which include antibodies, engagers, and / or CARs. Cell surface-expressed CFRs in effector cells can also function in conjunction with engagers expressed by effector cells. exo Cells comprising, optionally, a polynucleotide encoding a CAR, exogenous CD16 or its variants, an engager, and / or one or more CFRs of the present invention may be any type of cell, including human and non-human cells, pluripotent or non-pluripotent cells, immune cells or immunomodulatory cells, APCs (antigen-presenting cells) or feeder cells, cells derived from primary sources (e.g., PMBCs), or cells derived from cultured or manipulated cells (e.g., cell lines, cells, and / or induced cells differentiated from iPSCs). In some embodiments, TCR exoFurthermore, cells containing polynucleotides encoding one or more of the following, optionally CAR, exogenous CD16 or its variants, engagers, and / or one or more CFRs, are primary or induced CD34 + The cell comprises hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T lineage cells, NKT lineage cells, NK lineage cells, or B lineage cells. In some embodiments, TCR exo Furthermore, induced cells containing polynucleotides encoding one or more of the following (optionally selected): CAR, exogenous CD16 or its variants, engagers, and / or one or more CFRs, are classified as TCR. exo , as well as effector cells obtained by differentiating iPSCs containing, optionally, polynucleotides encoding one or more of the following: CAR, exogenous CD16 or its variants, engagers, and / or one or more CFRs. In some embodiments, TCR exo Inducible effector cells, which optionally contain polynucleotides encoding one or more of the following: CAR, exogenous CD16 or its variants, engagers, and / or one or more CFRs, are obtained by generating inducible effector cells from iPSCs and then manipulating the inducible effector cells to incorporate one or more of the following: CAR, exogenous CD16 or its variants, engagers, and / or one or more CFRs.

[0195] Furthermore, TCR exoCells or populations thereof comprising, optionally, CARs, optionally, engagers, and / or polynucleotides encoding one or more CFRs may further comprise one or more further manipulated modalities as described herein and / or shown in Table 1. Further provided herein is a master cell bank comprising sorted single cells and proliferated clones of manipulated iPSCs having at least one phenotype as provided herein, the cell bank providing a platform for further iPSC manipulation and a renewable source for producing ready-made manipulated homogeneous effector cells that are clearly and uniformly composed and can be mass-produced on considerable scale in a cost-effective manner.

[0196] 6. Exogenously introduced cytokines and / or cytokine signaling By avoiding clinically appropriate systemic high-dose administration of cytokines, the risk of dose-limiting toxicity from such actions is reduced, and cytokine-mediated cell autonomy is established. To achieve lymphocyte autonomy without the need for additional soluble cytokine administration, a signaling complex comprising one or more partial-length or full-length peptides of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and / or their corresponding receptors may be introduced into cells to enable cytokine signaling with or without the expression of the cytokines themselves, thereby reducing the risk of cytokine toxicity and maintaining or improving cell growth, proliferation, expansion, and / or effector function. In some embodiments, the introduced cytokines and / or their respective native or modified receptors (signaling complexes) for cytokine signaling are expressed on the cell surface. In some embodiments, cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, the activation of cytokine signaling is transient and / or temporary.

[0197] Various construct designs for introducing protein complexes for cytokine signaling into cells are provided herein, including but not limited to IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In embodiments where the signaling complex is for IL15, the transmembrane (TM) domain may be native to the IL15 receptor or may be modified or substituted with the transmembrane domain of any other membrane-bound protein. In some embodiments, IL15 and IL15Rα are co-expressed using a self-cleaving peptide that mimics the trans presentation of IL15 without excluding the cis presentation of IL15. In other embodiments, IL15Rα is fused to IL15 at its C-terminus via a linker, mimicking the trans presentation of IL15 without excluding the cis presentation of IL15 and ensuring that IL15 is membrane-bound. In other embodiments, IL15Rα with a cleaved intracellular domain is fused to IL15 at its C-terminus via a linker, mimicking the trans-presentation of IL15, maintaining the membrane binding of IL15, and, in addition, excluding cis-presentation and / or other potential signaling pathways mediated by normal IL15R via its intracellular domain. In yet another embodiment, the cytoplasmic domain of IL15Rα can be omitted without adversely affecting the autonomous characteristics of effector cells possessing IL15. In other embodiments, the entire IL15Rα is essentially deleted, with the exception of the Sushi domain, which fuses with IL15 at one end and with a transmembrane domain at the other (mb-Sushi), and optionally has a linker between the Suchi domain and the transmembrane domain. The fused IL15 / mb-Sushi is expressed on the cell surface via the transmembrane domain of the membrane-bound protein. Thus, if only the desired trans-presentation of IL15 is retained, unwanted signaling mediated by IL15Rα, including cis-presentation, is eliminated.

[0198] In other embodiments, native or modified IL15Rβ is fused to IL15 at its C-terminus via a linker to enable constitutive signaling and maintain IL15 membrane binding and trans presentation. In other embodiments, native or modified common receptor γC is fused to IL15 at its C-terminus via a linker for cytokine constitutive signaling and membrane binding trans presentation. Common receptor γC is also called common gamma chain or CD132 and is also known as IL2 receptor subunit gamma or IL2RG. γC is a cytokine receptor subunit common to many interleukin receptor complexes, including but not limited to IL2, IL4, IL7, IL9, IL15, and IL21 receptors. In other embodiments, engineered IL15Rβ, which forms homodimers in the absence of IL15, is useful for generating cytokine constitutive signaling.

[0199] In embodiments where the signaling complex is directed toward IL7, any transmembrane (TM) domain of any of the various designs may be native to the IL15 receptor or may be modified or substituted with a transmembrane domain of any other membrane-bound protein. In some embodiments, native (or wild-type) or modified IL7R may be fused to IL7 at the C-terminus via a linker, enabling constitutive signaling and maintaining membrane binding and membrane-bound IL7.

[0200] In one embodiment, the native or modified common receptor γC described above is fused to IL7 at its C-terminus via a linker for constitutive and membrane-bound cytokine signaling complexes. In another embodiment, an engineered IL7R that forms a homodimer in the absence of IL7 is equally useful for generating constitutive cytokine signaling.

[0201] Accordingly, in various embodiments, the cytokines IL15 or IL7 and / or their receptors may be introduced into iPSCs using one or more of the construct designs described above, or into the induced cells during iPSC differentiation. In addition to induced pluripotent cells (iPSCs), cloned iPSCs, cloned iPS cell lines, or iPSC-induced cells comprising at least one of the engineered modalities disclosed herein are provided. Further provided is a master cell bank comprising sorted single cells and proliferated cloned engineered iPSCs having at least a signaling complex comprising a partial or complete peptide of the cell surface-expressed exogenous cytokines and / or their receptors described herein, the cell bank providing a platform for further iPSC manipulation and a renewable source for producing ready-made engineered homogeneous cell therapy products that are clearly and uniformly composed and can be mass-produced on a considerable scale in a cost-effective manner.

[0202] In iPSCs and cells derived therefrom that contain both CAR and exogenous cytokine and / or cytokine receptor signaling (signaling complex, or "IL"), CAR and IL may be expressed in separate constructs or co-expressed in a bicistronic construct that contains both CAR and IL. In some further embodiments, the signaling complex may be linked to either the 5' or 3' end of the CAR expression construct via a self-cleaving 2A coding sequence. Thus, the IL signaling complex (e.g., the IL7 signaling complex) and CAR may reside within a single open reading frame (ORF). In one embodiment, the signaling complex is contained within a CAR-2A-IL or IL-2A-CAR construct. When CAR-2A-IL or IL-2A-CAR is expressed, the self-cleaving 2A peptide allows the expressed CAR and IL to dissociate, and the dissociated IL may then be presented on the cell surface with its transmembrane domain fixed to the cell membrane. Bi-cistronic design of CAR-2A-IL or IL-2A-CAR enables coordinated expression of the CAR-IL signaling complex under the same regulatory mechanism, which can be selected to incorporate, for example, an inducible promoter for the expression of a single ORF or a promoter with temporal or spatial specificity, in terms of both timing and quantity. Self-cleaving peptides are found in members of the Picornaviridae family, including aftoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thosea asigna virus (TaV), and porcine tesiovirus-1 (PTV-1) (Donnelly, ML, et al., J. Gen. Virol, 82, 1027-101 (2001); Ryan, MD, et al., J. Gen. Virol., 72, 2727-2732 (2001)), as well as cardioviruses such as tyloviruses (e.g., Tyler mouse encephalomyelitis virus) and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-I, and TaV are sometimes referred to as "F2A," "E2A," "P2A," and "T2A," respectively.

[0203] Embodiments of the bicistronic CAR-2A-IL or IL-2A-CAR disclosed herein are also intended for the expression of any other cytokine or cytokine signaling complex provided herein, such as IL2, IL4, IL6, IL9, IL10, IL11, IL12, IL18, and IL21. In some embodiments, the bicistronic CAR-2A-IL or IL-2A-CAR is for the expression of one or more of IL2, IL4, IL7, IL9, IL15, and IL21.

[0204] TCR exo iPSCs and cells derived therefrom comprising a signaling complex comprising both CARs (optionally selected), an engager (optionally selected), and a cell surface-expressed exogenous cytokine and / or a partial or complete peptide of its cytokine receptor, wherein the iPSCs and derived cells may further comprise one or more of the following: CFR, exogenous CD16 or its variant, CD38 negativity, HLA-I and / or HLA-II deficiency, and / or HLA-G.

[0205] In some embodiments, iPSCs and their induced effector cells containing any one of the genotypes in Table 1 are characterized by deletion or disruption of at least one of the following genes within the chromosome 6p21 region: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, or HLA-E, 4-1BBL, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The system may further include the introduction or upregulation of at least one of the following: R, TCR, Fc receptor, antibody, and surface trigger receptor for bispecific, multispecific, or universal engager binding.

[0206] 7. Deficiency of HLA-I and HLA-II To avoid allogeneic rejection, multiple HLA class I and class II proteins must match for histocompatibility in allogeneic recipients. Provided herein are iPSC cell lines in which the expression of both HLA class I and HLA class II proteins is eliminated or substantially reduced, and induced cells differentiated therefrom. HLA class I deficiency can be achieved by functional deletion of any region of the HLA class I locus (chromosome 6p21), or by deletion or disruption of HLA class I-related genes, including but not limited to the beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene, and tapasin. For example, the B2M gene encodes a common subunit essential for the cell surface expression of all HLA class I heterodimers. B2M-negative cells are HLA-I deficient. HLA class II deficiency can be achieved by functional deletion or disruption of HLA class II-related genes, including but not limited to RFXANK, CIITA, RFX5, and RFXAP. CIITA is a transcriptional coactivator that functions through the activation of the transcription factor RFX5, which is required for the expression of class II proteins. CIITA-negative cells are HLA-II deficient. Provided herein are iPSC lines and their derived cells that have deficiencies in both HLA-I and HLA-II, for example, those lacking expression of both B2M and CIITA, and the resulting derived effector cells enable allogeneic cell therapy by eliminating the need for MHC (major histocompatibility complex) matching and avoiding recognition and killing by host (allogeneic) T cells.

[0207] In some cell types, the absence of HLA class I expression leads to lysis by NK cells. To overcome this "self-loss" response, HLA-G can be optionally knocked in to evade recognition and killing of NK cells by HLA-I-deficient effector cells derived from engineered iPSCs. In one embodiment, HLA-I-deficient iPSCs and their derived cells further include HLA-G knock-in. In some embodiments, the provided HLA-I-deficient iPSCs and their derived cells further include one or both of CD58 knockout and CD54 knockout. CD58 (or LFA-3) and CD54 (or ICAM-1) are adhesion proteins that initiate signal-dependent cell interactions and promote cell migration, including immune cells. CD58 knockout has been shown to be more efficient than CD54 knockout in reducing allogeneic NK cell activation, while dual knockout of both CD58 and CD54 has been shown to have the most enhanced reduction of NK cell activation. Some observations suggest that CD58 and CD54 dual knockout is more effective than HLA-G overexpression in HLA-I-deficient cells in overcoming the "self-loss" effect.

[0208] As provided herein, in some embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells have an exogenous polynucleotide encoding HLA-G. In some embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD58 negative. In some other embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are CD54 negative. In some yet other embodiments, HLA-I and HLA-II deficient iPSCs and their derived cells are both CD58 negative and CD54 negative.

[0209] Furthermore, TCR exoIn some embodiments of genetically engineered immune cells, iPSCs, and their derived cells, which optionally include one or more signaling complexes comprising CAR, exogenous CD16 or its variants, one or more CFRs, engagers, and / or cell surface-expressed exogenous cytokines and / or partial or complete peptides of their cytokine receptors, the cells are HLA-I and HLA-II deficient and have exogenous polynucleotides encoding HLA-G. exo In some embodiments of genetically engineered immune cells, iPSCs, and their derived cells, which optionally include one or more signaling complexes comprising CAR, exogenous CD16 or its variants, one or more CFRs, engagers, and / or cell surface-expressed exogenous cytokines and / or partial or complete peptides of their cytokine receptors, the cells are HLA-I and HLA-II deficient and CD58 negative. exo In some embodiments of genetically engineered immune cells, iPSCs, and their derived cells, which optionally include one or more signaling complexes comprising CAR, exogenous CD16 or its variants, one or more CFRs, engagers, and / or cell surface-expressed exogenous cytokines and / or partial or complete peptides of their cytokine receptors, the cells are HLA-I and HLA-II deficient and CD54 negative. exo In several other embodiments of genetically engineered immune cells, iPSCs, and their derived cells, which optionally include one or more signaling complexes comprising CAR, exogenous CD16 or its variants, one or more CFRs, engagers, and / or cell surface-expressed exogenous cytokines and / or partial or complete peptides of their cytokine receptors, the cells are HLA-I and HLA-II deficient and both CD58-negative and CD54-negative.

[0210] Further provided in this application is a master cell bank comprising sorted single cells and proliferated clones of engineered iPSCs having at least one phenotype as provided herein, including but not limited to HLA-I and / or HLA-II deficiency, the cell bank providing a platform for further iPSC manipulation and a renewable source for manufacturing ready-made engineered homogeneous cell therapy products, including but not limited to induced NK and T cells, which have a clear and uniform composition and can be mass-produced on a considerable scale in a cost-effective manner.

[0211] 8. CD38 Knockout The cell surface molecule CD38 is highly upregulated in multiple hematological malignancies derived from both the lymphoid and myeloid systems, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapies aimed at depleting cancer cells. Antibody-mediated depletion of cancer cells typically results from a combination of direct induction of apoptosis and activation of immune effector mechanisms such as ADCC (antibody-dependent cell-mediated cytotoxicity). In addition to ADCC, immune effector mechanisms linked to therapeutic antibodies may also include antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

[0212] CD38 is also highly expressed in plasma cells, as well as NK cells, activated T cells, and B cells, in addition to being highly expressed in malignant cells. During hematopoiesis, CD38 is expressed in conjunction with CD34 + It is expressed in stem cells, as well as in progenitor cells committed to lymphoid, erythroid, and myeloid lineages, and in the final stages of maturation, which continue to the plasma cell stage. CD38, a type II transmembrane glycoprotein, performs cellular functions as both a receptor and a multifunctional enzyme involved in the production of nucleotide metabolites. As an enzyme, CD38 is involved in NAD +CD38 catalyzes the synthesis of ADP-ribose from CD31 and the hydrolysis of the reaction, thereby producing the secondary messengers CADPR and NAADP, which stimulate the release of calcium from the endoplasmic reticulum and lysosomes, a calcium-dependent process crucial for cell adhesion. CD38 recognizes CD31 as a receptor and regulates cytokine release and cytotoxicity of activated NK cells. CD38 associates with cell surface proteins of lipid rafts and releases cytoplasmic Ca 2+ It has also been reported that it modulates flux and mediates signaling in lymphoid and myeloid cells.

[0213] In the treatment of malignant tumors, systemic use of T cells transduced with the CD38 antigen-binding receptor is effective. + CD38 in hematopoietic progenitor cells, monocytes, NK cells, T cells, and B cells + Because the fraction lyses and the function of recipient immune effector cells is impaired, the therapeutic response becomes incomplete, and efficacy is reduced or eliminated. Furthermore, in multiple myeloma patients treated with daratumumab, a CD38-specific antibody, a decrease in NK cells was observed in both bone marrow and peripheral blood, while other immune cell types such as T cells and B cells were unaffected despite CD38 expression (Casneuf et al., Blood Advances. 2017;1(23):2105-2114). While not bound by theory, this application offers a strategy to maximize the potential of CD38-targeted cancer therapy by overcoming the depletion or reduction of effector cells through sibling killing induced by CD38-specific antibodies and / or CD38 antigen-binding domains. Furthermore, since CD38 is upregulated by activated lymphocytes such as T cells and B cells, suppressing the activation of these recipient lymphocytes using CD38-specific antibodies such as daratumumab in recipients of allogeneic effector cells reduces and / or prevents allogeneic rejection of these effector cells, thereby increasing the viability and persistence of effector cells.

[0214] Thus, this application also provides strategies to enhance the persistence and / or viability of effector cells by reducing or preventing allogeneic rejection through the use of CD38-specific antibodies, secreted CD38-specific engagers, or CD38 CARs (chimeric antigen receptors) against the activation of recipient T and B cells (i.e., often the depletion of activated T and B lymphocytes prior to adoptive cell transplantation). Specifically, the strategies provided herein involve creating a master cell bank containing iPSC lines including CD38 knockout, sorted single cells, and proliferated clonal CD38-negative iPSCs, and achieving CD38-negative (CD38) cells through targeted differentiation of the manipulated iPSC lines. neg This includes obtaining induced effector cells, which, when the CD38-targeted therapeutic portion is used in conjunction with the effector cells, are protected from sibling killing and allogeneic rejection, among other advantages. In addition, anti-CD38 monoclonal antibody therapy significantly depletes the patient's activated immune system without adversely affecting the patient's hematopoietic stem cell compartment. CD38-negative induced cells have the ability to resist CD38 antibody-mediated depletion and can be effectively administered in combination with anti-CD38 antibodies or CD38-CARs without the use of toxic conditioning agents, thus reducing and / or replacing chemotherapy-based lymphocyte depletion.

[0215] In one embodiment provided herein, CD38 knockout in an iPSC strain is a biallelic knockout. As disclosed herein, the provided CD38-negative iPSC strain has at least TCR exo The iPSC may further optionally include CAR and, optionally optionally, one or more of CFR, exogenous CD16, or variants thereof, and may further include one or more additional manipulated modalities as described herein and shown in Table 1, wherein the iPSC is not limited to, but includes mesodermal cells with definitive hematopoietic endothelial (HE) capacity, definitive HE, CD34 +Hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells (MPPs), T cell progenitor cells, NK cell progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, erythrocytes, myeloid cells, neutrophil progenitor cells, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, macrophages, and immune effector cells having one or more functional features not present in primary NK cells, T cells, and / or NKT cells, can be targeted differentiation to produce functionally induced hematopoiesis. In some embodiments, when ADCC is induced using an anti-CD38 antibody or when an anti-CD38 CAR is used for targeted cell killing, CD38 neg iPSCs and / or induced effector cells are not eliminated by anti-CD38 antibodies, anti-CD38 CARs, or recipient-activated T or B cells, thereby increasing the persistence and / or viability of iPSCs and their effector cells in the presence of and / or after exposure to such therapeutic moieties. In some embodiments, effector cells increase in vivo persistence and / or viability in the presence of and / or after exposure to such therapeutic moieties.

[0216] 9. Genetically modified iPSC lines and iPSC-induced cells provided herein. Based on the above, this application provides iPSCs, iPS cell lines, or populations thereof, and induced effector cells obtained by differentiating iPSCs, each cell having at least TCR exoThe cells include polynucleotides encoding and optionally CARs, and the cells are eukaryotic cells, animal cells, human cells, induced pluripotent cells (iPSCs), iPSC-induced effector cells, immune cells, or feeder cells. Further provided is a master cell bank containing sorted single cells and proliferated clones of engineered iPSCs having the phenotypes described herein, which provides a renewable source for producing ready-made engineered homogeneous cell therapy products that have a clear and uniform composition and can be mass-produced on a considerable scale in a cost-effective manner. In some embodiments, the iPSC-induced cells are mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34, etc., but are not limited to these. + The hematopoietic cells include hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent precursors (MPPs), T cell precursors, NK cell precursors, bone marrow cells, and neutrophil precursors, and / or hematopoietic cells that share characteristics with T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In some embodiments, iPSC-induced hematopoiesis includes at least TCR exo and optionally include immunoeffector cells expressing CAR. Further provided herein are iPSCs, iPS cell lines, or clonal populations thereof, and induced functional cells obtained from differentiating iPSCs, each of which expresses TCR exoThe iPSCs can be selectively differentiated to produce functionally induced hematopoietic cells, comprising polynucleotides encoding one or more of the following: CAR, and optionally CFR; exogenous CD16 or its variants; cytokine signaling complexes comprising cytokines and / or their receptors or variants; HLA-I deficiency and / or HLA-II deficiency; introduction of HLA-G or uncleaved HLA-G, or knockout of one or both CD58 and CD54; and CD38 knockout. In some embodiments, the functionally induced hematopoietic cells are immune effector cells. In some embodiments, the functionally induced immune effector cells share features with NK cells and / or T cells. In some embodiments, the functionally induced immune effector cells that share features with NK cells and / or T cells are neither NK cells nor T cells.

[0217] In some embodiments of iPSCs, iPS cell lines, or clonal populations thereof, and induced functional cells obtained from differentiating iPSCs, each cell is a TCR. exo and optionally containing at least one polynucleotide encoding a CAR, the cell has endogenous TCR neg This is the case. When used herein, "TCR neg " means TCR negative, TCR - / - Also referred to as "TCR null" or TCR knockout, it includes cells that lack endogenous TCR expression, obtained naturally (e.g., NK cells or iPSC-induced NK cells), iPSC cells (e.g., iPSCs, iPSCs reprogrammed from T cells (TiPSCs)), or by regulating T cell gene expression or genome editing to knock out endogenous TCR or one or more of its subunits, or by obtaining TCR-negative induced cells differentiated from iPSCs having TCR knockout. Therefore, the TCR knocked out in the cells disclosed herein is the endogenous TCR complex. Disrupting the expression of either the constant region of TCRα or TCRβ in a cell is one of many ways to knock out the endogenous TCR complex in a cell. negThe cell is a TCR neg Despite the expression of all CD3 subunits in the cell, it cannot present the CD3 complex on the cell surface, which has an adverse effect on cell functions that require cell surface CD3 recognition, binding, and / or signaling. Therefore, a TCR containing a polynucleotide encoding CFR neg In some embodiments of the cell, CFR is a CD3 system. In some embodiments, the TCR exo A TCR containing a polynucleotide encoding CAR optionally neg The cell also includes, upon expression, a cell surface CD3 complex, or one or more of its subunits or subdomains (cs-CD3).

[0218] In some embodiments, the TCR exo The cell, optionally including a CAR and optionally an engager, also includes a CAR inserted into the constant region of the TCR. In some embodiments, the TCR exo The cell, optionally including a CAR and optionally an engager, is a TCR neg and includes a CAR inserted into the constant region of the TCR, and the expression of the CAR is driven by an endogenous TCR promoter. In some embodiments, the TCR exoCells containing, optionally, a CAR and optionally, an engager, also include exogenous cytokine signaling of IL2, IL4, IL7, IL9, IL15, IL21, or any combination thereof. In some embodiments, the exogenous cytokine signaling is bound to the cell membrane. In some embodiments, the exogenous cytokine signaling includes cytokines and / or their respective receptors or partially or completely introduced variants thereof. In some embodiments, the cytokine signaling is constitutively activated. In some embodiments, activation of cytokine signaling is inducible. In some embodiments, activation of cytokine signaling is transient and / or transient. In some embodiments, transient / transient expression of cell surface cytokine signaling is mediated by retroviruses, Sendai viruses, adenoviruses, episomes, minicircles, or RNA, including mRNA. In some embodiments, the exogenous cell surface cytokine signaling enables IL2 signaling. In some embodiments, the exogenous cell surface cytokine signaling enables IL4 signaling. In some embodiments, the exogenous cell surface cytokine signaling enables IL7 signaling. In some embodiments, exogenous cell surface cytokine signaling enables IL-9 signaling. In some embodiments, exogenous cell surface cytokine signaling enables IL-15 signaling. In some embodiments, exogenous cell surface cytokine signaling enables IL-21 signaling. In some embodiments, cells containing a CAR and optionally an engager further contain exogenous CD16 or its functional variant or chimeric receptor. In some embodiments, exogenous CD16 contains an external domain including F176V and S197P. In some embodiments, exogenous CD16 contains the full length or partial length of the external domain of CD64. In some other embodiments, exogenous CD16 contains a chimeric Fc receptor.Exogenous CD16 enables cell killing by ADCC, thereby providing a dual targeting mechanism to effector cells expressing, for example, a CAR.

[0219] In some embodiments, cells comprising a TCR exo , optionally a CAR, and optionally an engager further comprise a CD38 knockout. The cell surface molecule CD38 is highly upregulated in multiple hematologic malignancies derived from both lymphoid and myeloid lineages, including multiple myeloma and CD20-negative B-cell malignancies, making it an attractive target for antibody therapies that deplete cancer cells. CD38 is also expressed on plasmacytoid cells as well as NK cells, activated T cells, and B cells, in addition to being highly expressed on malignant cells. In some embodiments, effector cells that are CD38 - / - can avoid CD38-induced fratricide. In some embodiments, when using a CD3 engager comprising an anti-CD38 antibody, a CD38-binding CAR, or an anti-CD38 scFV to induce ADCC and / or tumor cell targeting, CD38 - / - iPSCs and / or their derived effector cells can target CD38-expressing (tumor) cells without causing elimination of effector cells, i.e., a decrease or depletion of CD38-expressing effector cells, thereby increasing the persistence and / or viability of the iPSCs and their effector cells.

[0220] TCR exo In some embodiments of cells comprising a polynucleotide encoding a TCR, optionally a polynucleotide encoding a CAR, and optionally a polynucleotide encoding an engager, the cells further comprise an HLA-I and / or HLA-II deficiency (e.g., a B2M knockout and / or a CIITA knockout), and optionally a polynucleotide encoding HLA-G or HLA-E. TCR exoIn some embodiments of cells comprising a polynucleotide encoding HLA-I, optionally a CAR, and optionally a polynucleotide encoding an engager, the cells further comprise HLA-I and / or HLA-II deficiencies (e.g., B2M knockout and / or CIITA knockout), and optionally one or both of CD58 and CD54 knockouts.

[0221] Considering the above, what is provided herein is TCR exo A polynucleotide encoding a CAR, optionally a polynucleotide encoding an engager, and optionally a TCR. neg iPSCs comprising one, two, three, or more, or all of the following: exogenous CD16 or its variants, CFR, cell surface-expressed exogenous IL-containing signaling complex, CD38 knockout, and B2M / CIITA knockout, wherein if B2M is knocked out, a polynucleotide encoding HLA-G, or one or both of CD58 knockout and CD54 knockout, is optionally introduced, and the iPSCs can be selectively differentiated to produce functionally induced hematopoietic cells.

[0222] Therefore, this application provides iPSCs and functionally induced hematopoietic cells thereof comprising any one of the following genotypes in Table 1. Also provided herein is a TCR comprising any one of the following genotypes in Table 1 without adversely affecting the differentiation ability of iPSCs and the function of induced effector cells. exoThe master cell bank contains sorted single cells and proliferated clones of engineered iPSCs, optionally having CAR, one or both of engagers ("Eg" in Table 1) and CFR, and optionally one or more of exogenous CD16 or its variants, cell surface-expressed exogenous IL-containing signaling complexes, CD38 knockout, and HLA-I and / or HLA-II deficiency. The cell bank provides a platform for further iPSC manipulation and a renewable source for producing ready-made engineered homogeneous cell therapy products. Depending on the insertion site of any one of the exogenous polynucleotides, the engineered effector cells may be negative for endogenous TCR expression. Furthermore, if the engineered effector cells are of an NK cell lineage, the cells are also TCR negative.

[0223] The "IL" provided in Table 1 represents one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21, depending on which specific cytokine / receptor or combination expression is selected. If IL7 is selected, IL represents IL7, including IL7Rα and IL7Rβ. Similarly, if IL15 is selected, IL represents IL15, including IL15Rα and IL15Rβ. In some embodiments, the cell surface-expressed exogenous cytokine and / or its receptor includes at least one of the following: co-expression of IL15 and IL15Rα using a self-cleaving peptide; a fusion protein of IL15 and IL15Rα; an IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα is cleaved or excluded; a fusion protein of IL15 and IL15Rβ; a fusion protein of IL15 and common receptor γC (where the common receptor γC is native or modified); and a homodimer of IL15Rβ.

[0224] In some embodiments, the cell surface-expressed exogenous cytokine and / or its receptor includes at least one of the following: co-expression of IL7 and IL7Rα using a self-cleaving peptide; a fusion protein of IL7 and IL7Rα; an IL7 / IL7Rα fusion protein in which the intracellular domain of IL7Rα is cleaved or excluded; a fusion protein of IL7 and IL7Rβ; a fusion protein of IL7 and common receptor γC (where the common receptor γC is native or modified); and a homodimer of IL7Rβ.

[0225] Furthermore, if the iPSC and its functionally induced hematopoietic cells have a genotype containing both CAR and IL, CAR and IL may be included in a bicistronic expression cassette containing a 2A sequence, at the option of selection. In contrast, in some other embodiments, CAR and IL are in separate expression cassettes included in the iPSC and its functionally induced hematopoietic cells.

[0226] [Table 11-1]

[0227] [Table 11-2]

[0228] [Table 11-3]

[0229] [Table 11-4]

[0230] [Table 11-5]

[0231] [Table 11-6]

[0232] [Table 11-7]

[0233] [Table 11-8]

[0234] [Table 11-9]

[0235] [Table 11-10]

[0236] 10. Antibodies for immunotherapy In some embodiments, in addition to the genome-modified effector cells provided herein, an additional therapeutic agent comprising an antibody or antibody fragment targeting an antigen associated with a condition, disease, or indication may be used in combination therapy with these effector cells, compared to expression on the genome-modified effector cells. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody, a humanized monoclonal antibody, or a chimeric antibody. In some embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen. In some embodiments, the tumor or virus-specific antigen activates the administered iPSC-induced effector cells to enhance their killing ability. In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents for administered iPSC-induced effector cells include anti-CD20 (rituximab, bertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab, ibritumomab, ocrelizumab), anti-CD22 (inotuzumab, moxetumomab, epratuzumab), anti-HER2 (trastuzumab, pertuzumab), and anti-CD5 Examples include, but are not limited to, 2 (aremutuzumab), anti-EGFR (cetuximab), anti-GD2 (dinutuximab), anti-PDL1 (avelumab), anti-CD38 (daratumumab, isatuximab, MOR202), anti-CD123 (7G3, CSL362), anti-SLAMF7 (elotuzumab), and their humanized or Fc-modified variants or fragments, or their functional equivalents and biosimilars.

[0237] In some embodiments, antibodies suitable for combination therapy as additional therapeutic agents to administered iPSC-induced effector cells include bispecific or multispecific antibodies that target two or more antigens or epitopes on target cells, or that target target cells while recruiting effector cells (e.g., T cells, NK cells, or macrophages) toward the target cells. Such bispecific or multispecific antibodies act as engagers that can direct effector cells toward tumor cells and activate them upon binding to tumor antigens, whether bystander immune cells (e.g., T cells, NK cells, NKT cells, B cells, macrophages, and / or neutrophils in the recipient of treatment) or engineered effector cells in the therapeutic composition, thus demonstrating great potential to maximize the benefits of antibody therapy. The engagers are specific to at least one tumor antigen and specific to at least one surface trigger receptor on the immune effector cells, which can provide a multitargeting approach for engineered cells disclosed herein to address tumor antigen escape and tumor heterogeneity. Examples of engagers include, but are not limited to, bispecific T cell engagers (BiTE), bispecific killer cell engagers (BiKE), triplicate killer cell engagers (TriKE), or multispecific killer cell engagers, or universal engagers that can engage multiple immune cell types.

[0238] In some embodiments, the iPSC-induced effector cells include hematopoietic lineage cells containing the genotypes listed in Table 1. In some embodiments of combinations useful for treating humoral or solid tumors, the combination includes at least TCRs provided herein. exo The combination includes, optionally, iPSC-induced effector cells containing CAR and optionally, CFR. In some other embodiments of combinations useful for treating humoral or solid tumors, the combination includes a pre-selected monoclonal antibody and at least TCRexo The combination comprises, optionally, CAR, and optionally, iPSC-induced effector cells containing one or more of CFR and exogenous CD16 or its variants. In some embodiments of combinations useful for treating humoral or solid tumors, the combination comprises a monoclonal antibody and at least TCR exo , optionally CAR, and optionally TCR neg iPSC-induced effector cells comprising one or more of the following: exogenous CD16 or its variants; CFRs; additional cytokine signaling complexes including cytokines and / or their receptors or variants; and CD38 knockout. In various embodiments, exogenous CD16 is hnCD16. While not bound by theory, hnCD16 provides an enhanced ADCC for monoclonal antibodies, but CAR not only targets specific tumor antigens but also prevents tumor antigen escape using a dual-targeting strategy combined with monoclonal antibodies that target different tumor antigens.

[0239] In some further embodiments, iPSC-induced NK cells included in the combination with daratumumab are TCR exo The molecule comprises, optionally, a CAR and optionally, one or more of the following: CD16, IL7, or IL15, wherein the CAR targets at least one of the following: B7H3, MICA / B, CD19, BCMA, CD20, CD22, CD123, HER2, CD52, EGFR, GD2, MSLN, VEGF-R2, PSMA, and PDL1, and the IL7 or IL15 signaling complex is co-expressed with the CAR or expressed separately.

[0240] 11. Checkpoint inhibitors Checkpoints are cellular molecules, often cell surface molecules, that can suppress or downregulate the immune response if not inhibited. Tumors have been shown to select specific immune checkpoint pathways, particularly as a primary mechanism of immune resistance against tumor antigen-specific T cells. Checkpoint inhibitors (CIs) are antagonists that can block inhibitory checkpoints and restore immune system function by reducing the expression or gene product of checkpoint genes or by decreasing the activity of checkpoint molecules. The development of checkpoint inhibitors targeting PD1 / PDL1 or CTLA4 has changed the oncological landscape, with these drugs resulting in long-term remission in multiple indications. However, many tumor subtypes are resistant to checkpoint blockade therapy, and relapse remains a significant concern. Therefore, one aspect of this application provides a therapeutic approach to overcome CI resistance by including genomically engineered functional iPSC-induced cells, as provided herein, in combination therapy with CIs. In one embodiment of the combination therapy, the iPSC-induced cells are NK cells. In another embodiment of the combination therapy, the iPSC-induced cells are T cells. In addition to exhibiting direct antitumor activity, the induced NK cells provided herein have been shown to be resistant to PDL1-PD1-mediated inhibition, enhance T cell migration, recruit T cells to the tumor microenvironment, and enhance T cell activation at tumor sites. Thus, T cell tumor infiltration facilitated by functionally potent genome-engineered induced NK cells suggests that NK cells can synergistically interact with T cell-targeted immunotherapy, including checkpoint inhibitors, to alleviate local immunosuppression and reduce tumor burden.

[0241] In one embodiment, iPSC-induced effector cells for checkpoint inhibitor combination therapy include CAR and, optionally, one, two, three, four, five or more of the following: engager expression, exogenous CD16 expression, CFR expression, HLA-I and / or HLA-II deficiency, CD38 knockout, and exogenous cell surface cytokine and / or receptor expression, and optionally, if B2M is knocked out, a polynucleotide encoding HLA-G, or knockout of one or both CD58 and CD54. In some embodiments, induced NK cells include one of the genotypes listed in Table 1. In some embodiments, the above-mentioned induced effector cells have a deletion or disruption of at least one of the following genes: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, and any gene in the chromosome 6p21 region; or HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention further includes the introduction or upregulation of at least one of the following: R, CAR, Fc receptors, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers.

[0242] In various embodiments, induced effector cells are directed to the TCR. exoThe iPSC clones are obtained by differentiating iPSC clones containing one, two, three, four, five or more of the following: expression, CAR expression, engager expression, exogenous CD16 expression, HLA-I and / or HLA-II deficiency, CD38 knockout, and exogenous cell surface cytokine expression, wherein if B2M is knocked out, a polynucleotide encoding HLA-G, or knockout of one or both of CD58 and CD54 is optionally introduced. In some embodiments, the above iPSC clones are deletions or disruptions of at least one of the following genes in the chromosome 6p21 region: TAP1, TAP2, tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, RFX5, RFXAP, RAG1, or HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The invention further includes the introduction or upregulation of at least one of the following: R, CAR, Fc receptors, and surface trigger receptors for binding to bispecific, multispecific, or universal engagers.

[0243] Checkpoint inhibitors suitable for combination therapy with induced NK cell or T cell lines provided herein include PD-1 (Pdcdl, CD279), PDL-1 (CD274), TIM-3 (Havcr2), TIGIT (WUCAM and Vstm3), LAG-3 (Lag3, CD223), CTLA-4 (Ctla4, CD152), 2B4 (CD244), 4-1BB (CD137), 4-1BBL (CD137L), A 2AAntagonists include, but are not limited to, R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIRs (e.g., 2DL1, 2DL2, 2DL3, 3DL1, and 3DL2).

[0244] In some embodiments, the antagonist that inhibits any of the checkpoint molecules described above is an antibody. In some embodiments, the checkpoint inhibitor antibody may be a mouse antibody, a human antibody, a humanized antibody, camel Ig, a single variable novel antigen receptor (VNAR), a shark heavy chain only antibody (Ig NAR), a chimeric antibody, a recombinant antibody, or a fragment of these antibodies. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, F(ab')3, Fv, single-chain antigen-binding fragment (scFv), (scFv)2, disulfide-stabilized Fv (dsFv), minibody, diabody, triabody, tetrabody, single-domain antigen-binding fragment (sdAb, nanobody), recombinant heavy chain only antibody (VHH), and other antibody fragments that maintain the binding specificity of the whole antibody, which may be more cost-effective to manufacture, easier to use, or more sensitive than the whole antibody. In some embodiments, one, two, three, or more checkpoint inhibitors include atezolizumab (anti-PDL1 mAb), avelumab (anti-PDL1 mAb), durvalumab (anti-PDL1 mAb), tremelimumab (anti-CTLA4 mAb), ipilimumab (anti-CTLA4 mAb), IPH4102 (anti-KIR), IPH43 (anti-MICA), IPH33 (anti-TLR3), lilimumab (anti-KIR), monalizumab (anti-NKG2A), nivolumab (anti-PD1 mAb), pembrolizumab (anti-PD1 mAb), and at least one of their derivatives, functional equivalents, or biosimilars.

[0245] In some embodiments, many miRNAs are found to be regulators that control the expression of immune checkpoints, so antagonists that inhibit any of the above-mentioned checkpoint molecules are microRNA-based (Dragomir et al., Cancer Biol Med. 2018, 15(2):103-115). In some embodiments, checkpoint antagonist miRNAs include, but are not limited to, miR-28, miR-15 / 16, miR-138, miR-342, miR-20b, miR-21, miR-130b, miR-34a, miR-197, miR-200c, miR-200, miR-17-5p, miR-570, miR-424, miR-155, miR-574-3p, miR-513, and miR-29c.

[0246] Some embodiments of the combination therapy with the provided iPSC-inducing effector cells include at least one checkpoint inhibitor that targets at least one checkpoint molecule, and the iPSC-inducing cells have the genotypes listed in Table 1. Some other embodiments of the combination therapy with the provided effector cells include two, three or more checkpoint inhibitors so that two, three or more checkpoint molecules are targeted. In some embodiments of the combination therapy including at least one checkpoint inhibitor and iPSC-inducing cells having the genotypes listed in Table 1, the checkpoint inhibitor is an antibody, or a humanized or Fc-modified variant or fragment, or a functional equivalent or biosimilar thereof, and the checkpoint inhibitor is produced by the iPSC-inducing cells by expressing an exogenous polynucleotide sequence encoding the antibody, or the fragment or variant thereof. In some embodiments, the exogenous polynucleotide sequence encoding the antibody, or the fragment or variant thereof that inhibits the checkpoint, is co-expressed with the CAR in either a separate construct or a bicistronic construct containing both the CAR and the sequence encoding the antibody or the fragment thereof. In some further embodiments, the sequence encoding the antibody or a fragment thereof may be ligated to either the 5' or 3' end of the CAR expression construct via an autocleaved 2A coding sequence, exemplified by, for example, CAR-2A-CI or CI-2A-CAR. Thus, the coding sequences of the checkpoint inhibitor and the CAR may be in a single open reading frame (ORF). Once the checkpoint inhibitor is delivered and expressed and secreted as a payload by induced effector cells capable of infiltrating the tumor microenvironment (TME), it activates the effector cells by binding to the TME, counteracting inhibitory checkpoint molecules, activating modalities such as CAR, or activating their receptors. In some embodiments, the checkpoint inhibitor co-expressed with the CAR may be the checkpoint molecule, PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2A It inhibits at least one of the following: R, BATE, BTLA, CD39 (Entpdl), CD47, CD73 (NT5E), CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2 (Pou2f2), retinoic acid receptor alpha (Rara), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR. In some embodiments, the checkpoint inhibitors co-expressed with CAR in induced cells having the genotypes listed in Table 1 are selected from the group including atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments, the checkpoint inhibitor co-expressed with CAR is atezolizumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is nivolumab, or its humanized or Fc-modified variant, fragment, or their functional equivalent or biosimilar. In some other embodiments, the checkpoint inhibitor co-expressed with CAR is pembrolizumab, or its humanized or Fc-modified variant, fragment, or functional equivalent or biosimilar thereof.

[0247] In some other embodiments of the combination therapies provided herein, which include iPSC-inducible cells and at least one antibody inhibiting a checkpoint molecule, the antibody is not produced by or within the iPSC-inducible cells and is administered before, concurrently with, or subsequently to the administration of iPSC-inducible cells having the genotypes listed in Table 1. In some embodiments, the administration of one, two, three, or more checkpoint inhibitors in the combination therapy with the provided induced NK cells or induced T cells is concurrent or sequential. In one embodiment of a combination therapy comprising iPSC-induced NK cells or iPSC-induced T cells having the genotypes listed in Table 1, the checkpoint inhibitors included in the therapy are one or more of the following: atezolizumab, avelumab, durvalumab, tremelimumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In several embodiments of a combination therapy comprising iPSC-induced NK cells or iPSC-induced T cells having the genotypes listed in Table 1, the checkpoint inhibitors included in the therapy are atezolizumab, or its humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars. In some embodiments of combination therapy involving iPSC-induced NK cells or iPSC-induced T cells having the genotypes listed in Table 1, the checkpoint inhibitor included in the therapy is nivolumab, or its humanized or Fc-modified variants, fragments, and their functional equivalents or biosimilars.

[0248] II. Methods for targeted genome editing at selected loci in iPSCs As used interchangeably herein, genome editing, genomic editing, or gene editing refers to a type of genetic engineering in which DNA is inserted, deleted, and / or replaced in the genome of a target cell. Targeted genome editing (interchangeable as “targeted genome editing” or “targeted gene editing”) allows for insertion, deletion, and / or replacement at pre-selected sites within the genome. If an endogenous sequence is deleted at the insertion site during targeted editing, the endogenous gene containing the affected sequence may be knocked out or knocked down by the sequence deletion. Thus, targeted editing can also be used to precisely disrupt the expression of endogenous genes. The term “targeted integration,” as used interchangeably herein, refers to a process involving the insertion of one or more exogenous sequences, with or without the deletion of an endogenous sequence at the insertion site. In contrast, randomly integrated genes are susceptible to positional effects and silencing, and their expression is unreliable and unpredictable. For example, centromere and subtelomere regions are particularly prone to transgene silencing. Newly integrated genes can influence surrounding endogenous genes and chromatin, potentially altering cellular behavior or promoting cell transformation. Therefore, inserting exogenous DNA into pre-selected loci, such as safe harbor loci or genome-safe harbors (GSH), is crucial for safe, efficient, copy number control, and reliable gene response regulation.

[0249] Targeted editing can be achieved through either a nuclease-independent or nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by a homologous sequence adjacent to the inserted exogenous polynucleotide via an enzymatic mechanism in the host cell.

[0250] Alternatively, targeted editing can be achieved more frequently by the specific introduction of double-strand breaks (DSBs) using specific rare-cut end nucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms, including non-homologous end joining (NHEJ), which occurs in response to DSBs. In the absence of a donor vector containing exogenous genetic material, NHEJ often results in random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast, when a donor vector containing a pair of homology arms and adjacent exogenous genetic material is present, the exogenous genetic material can be introduced into the genome during homologous recombination-directed repair (HDR), resulting in "targeted integration." In some situations, the targeted integration site is intended to be within the coding region of a selected gene, and therefore, targeted integration can disrupt gene expression, resulting in simultaneous knock-in and knock-out (KI / KO) in a single editing step.

[0251] It is possible to insert one or more transgenes at a selected site in a target gene locus (GOI) and simultaneously knock out the gene. Suitable gene loci for simultaneous knock-in and knockout (KI / KO) include, but are not limited to, B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT. Each site-specific targeted homology arm for site-selective insertion allows the transgene(s) to be expressed either under the site's endogenous promoter or under the exogenous promoter included in the construct. When two or more transgenes are inserted at a selected site (e.g., the CD38 locus), a linker sequence, such as a 2A linker or IRES, is placed between any two transgenes. The 2A linker encodes self-cleaving peptides derived from FMDV, ERAV, PTV-I, or TaV (referred to as "F2A," "E2A," "P2A," and "T2A," respectively), allowing for the production of distinct proteins from a single translation. In some embodiments, an insulator is included in the construct to reduce the risk of transgene and / or exogenous promoter silencing. The exogenous promoter may be CAG, or other constitutive, inducible, time-specific, tissue-specific, or cell-type-specific promoters, including but not limited to CMV, EF1α, PGK, and UBC.

[0252] Available endonucleases capable of introducing specific, targeted DSBs include, but are not limited to, zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), and RNA-induced CRISPR (clustered, equally spaced short repeat) systems. Furthermore, DICE (dual integrase cassette exchange) systems utilizing phiC31 and Bxb1 integrases are also promising tools for targeted incorporation.

[0253] ZFNs are targeted nucleases containing a nuclease fused to a zinc finger DNA-binding domain. “Zinc finger DNA-binding domain” or “ZFBD” refers to a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger is a domain of approximately 30 amino acids within a zinc finger-binding domain whose structure is stabilized by the coordination of a zinc ion. Examples of zinc fingers include, but are not limited to, C2H2, C3H, and C4 zinc fingers. “Engineered” zinc finger domains are domains that do not exist naturally, and their design / construction is primarily due to reasonable criteria, e.g., the application of substitution rules and computerized algorithms to process information in databases storing information on existing ZFP designs and binding data. See, for example, U.S. Patents 6,140,081, 6,453,242, and 6,534,261. See also International Publications 98 / 53058, 98 / 53059, 98 / 53060, 02 / 016536, and 03 / 016496. A “selected” zinc finger domain is a non-naturally occurring domain whose production arises primarily from empirical processes such as phage display, interaction trapping, or hybrid selection. ZFNs are described in detail in U.S. Patents 7,888,121 and 7,972,854, the full disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is the fusion of a FokI nuclease with a zinc finger DNA-binding domain.

[0254] TALEN is a targeted nuclease containing a nuclease fused to the TAL effector DNA-binding domain. “Transcription activator-like effector DNA-binding domain,” “TAL effector DNA-binding domain,” or “TALE DNA-binding domain” refers to the polypeptide domain of the TAL effector protein involved in the binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of plant cells and bind to effector-specific DNA sequences via their DNA-binding domains, activating gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA-binding domain depends on the effector variable number of incomplete 34-amino acid repeats containing polymorphisms at selected repeat positions, known as repeat variable residues (RVDs). TALEN is described in detail in U.S. Patent Publication 2011 / 0145940, which is incorporated herein by reference. The most well-known example of a TALEN in this field is a fusion polypeptide of FokI nuclease to a TAL effector DNA-binding domain.

[0255] Another example of a targeted nuclease used in the method of the present invention is a targeted Spo11 nuclease, a polypeptide comprising a Spo11 polypeptide having nuclease activity fused to a DNA-binding domain specific to the target DNA sequence, such as a zinc finger DNA-binding domain or a TAL effector DNA-binding domain.

[0256] Further examples of targeted nucleases suitable for the present invention include, but are not limited to, Bxb1, phiC31, R4, PhiBT1, and Wβ / SPBc / TP901-1, whether used individually or in combination.

[0257] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases, CRISPR-related nucleases from families including Cas, CPF, CSE, CSY, CSN, CSD, CST, CSH, CSA, CSM, and CMR, as well as restriction endonucleases, meganucleases, and homing endonucleases.

[0258] As an exemplary example, CRISPR / Cas9 requires two main components: (1) Cas9 endonuclease and (2) a crRNA-tracrRNA complex. When co-expressed, these two components form a complex and are recruited to a target DNA sequence containing the PAM and the seeding region proximal to the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) that guides Cas9 to target a selected sequence. These two components can then be delivered to mammalian cells via transfection or transduction. When using the CRISPR / Cpf system, it requires (1) a Cpf endonuclease (Cpf1, MAD7, and many more known in the art) and (2) a gNA (which often does not require tracrRNA) to guide the Cpf endonuclease to target a selected sequence.

[0259] DICE-mediated insertion provides unidirectional integration of exogenous DNA, strictly limited to small attB and attP recognition sites within each enzyme itself, using, for example, a pair of recombinases such as phiC31 and Bxb1. Since these targeted att sites are not naturally present in the mammalian genome, they must first be introduced into the genome at the desired integration site. See, for example, U.S. Patent Publication 2015 / 0140665, whose disclosure is incorporated herein by reference.

[0260] One aspect of the present invention provides a construct comprising one or more exogenous polynucleotides for targeted genomic integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the targeted integration method comprises introducing the construct into a cell to enable site-directed homologous recombination by a cell-host enzyme mechanism. In another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site into the cell to enable ZFN-mediated insertion. In yet another embodiment, the targeted integration method in a cell comprises introducing a construct comprising one or more exogenous polynucleotides into a cell and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site into the cell to enable TALEN-mediated insertion. In another embodiment, a method for targeted integration in cells includes introducing a construct containing one or more exogenous polynucleotides into cells and introducing a gRNA containing a Cas9 expression cassette and a guide sequence specific to the desired integration site into cells to enable Cas9-mediated insertion. In yet another embodiment, a method for targeted integration in cells includes introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in cells and introducing a construct containing one or more exogenous polynucleotides into cells and introducing an expression cassette for DICE recombinases to enable DICE-mediated targeted integration.

[0261] Promising sites for targeted integration include, but are not limited to, intragenetic or extragenetic regions of the human genome that, theoretically, can accommodate the predictable expression of newly integrated DNA without adversely affecting the host cell or organism, safe harbor loci or genome-safe harbors (GSHs). A useful safe harbor must enable sufficient transgene expression to produce the desired level of the protein or non-coding RNA encoded by the vector. The safe harbor must also not make cells more susceptible to malignant transformation or alter cellular function. For an insertion site to be a potential safe harbor locus, it should ideally meet criteria including, but not limited to, the following: the absence of disruption of regulatory elements or genes as determined by sequence annotation; being an intergeneric region within a gene-dense region, or a convergence site between two genes transcribed in opposite directions; maintaining a distance that minimizes the possibility of long-range interactions between the vector-encoded transcription activator and the promoters of adjacent genes, particularly oncological genes and microRNA genes; and having clearly ubiquitous transcriptional activity, as reflected by a broad spatial and temporal expression pattern of sequence tags (ESTs) exhibiting ubiquitous transcriptional activity. This latter characteristic is particularly important in stem cells, where chromatin remodeling during differentiation typically results in the silencing of some loci and the potential activation of others. Within a region suitable for exogenous insertion, the precise locus selected for insertion should lack repeating elements and conserved sequences, and primers for homology arm amplification should be easily designed.

[0262] Suitable sites for human genome editing, or more specifically, targeted incorporation, include, but are not limited to, human orthologues of the adeno-associated virus site 1 (AAVS1), the chemokine (CC motif) receptor 5 (CCR5) locus, and the mouse ROSA26 locus. Furthermore, the human orthologue of the mouse H11 locus may also be a suitable site for insertion using the targeted incorporation compositions and methods disclosed herein. In addition, collagen and HTRP loci can also be used as safe harbors for targeted incorporation. However, validation of each selected site has been shown to be necessary, particularly in stem cells, for specific incorporation events, and optimization of the insertion strategy, including promoter selection, exogenous gene sequencing and placement, and construct design, is often required.

[0263] For targeted indels, the editing site is often located in an endogenous gene whose expression and / or function is intended to be disrupted. In one embodiment, the endogenous gene containing the targeted indel is related to the modulation and modification of the immune response. In several other embodiments, the endogenous gene containing the targeted indel is related to targeted modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response modulation and modification, or proteins that suppress the engraftment, transport, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells, and cells derived from them.

[0264] Accordingly, another aspect of the present invention provides a method for targeted integration at a selected locus containing a genome-safe harbor, or at a pre-selected locus that is known or proven to be safely and adequately regulated for continuous or transient gene expression, such as the B2M, TAP1, TAP2, Tapasin, TRAC, or CD38 locus, as provided herein. In one embodiment, the genome-safe harbor for the targeted integration method comprises one or more desirable integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genome-safe harbor. In some embodiments, targeted integration is located at one or more loci where knockdown or knockout of the gene as a result of integration is desired, and such loci include, but are not limited to, B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.

[0265] In one embodiment, a method for targeted integration in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a construct containing a pair of homology arms specific to a desired integration site and one or more exogenous sequences to enable site-directed homologous recombination by a cell-host enzyme mechanism, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0266] In another embodiment, a method for targeted insertion in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a ZFN expression cassette containing a DNA-binding domain specific to a desired insertion site into cells to enable ZFN-mediated insertion, wherein the desired insertion site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method for targeted insertion in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a TALEN expression cassette containing a DNA-binding domain specific to a desired insertion site into cells to enable TALEN-mediated insertion, wherein the desired insertion site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.In another embodiment, a method for targeted insertion in cells comprises introducing a construct containing one or more exogenous polynucleotides into cells, and introducing a gRNA containing a Cas9 expression cassette and a guide sequence specific to a desired insertion site into the cells to enable Cas9-mediated insertion, wherein the desired insertion site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In yet another embodiment, a method for targeted integration in cells comprises introducing a construct containing one or more att sites of a pair of DICE recombinases into a desired integration site in a cell, introducing a construct containing one or more exogenous polynucleotides into the cell, and introducing a DICE recombinase expression cassette to enable DICE-mediated targeted integration, wherein the desired integration site includes AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT.

[0267] Furthermore, as provided herein, the above-described method for targeted incorporation in a safe harbor is used to insert polynucleotides of interest, such as safety switch proteins, targeted modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, and polynucleotides encoding proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival rates of stem cells and / or progenitor cells. In some other embodiments, a construct comprising one or more exogenous polynucleotides further comprises one or more marker genes. In one embodiment, the exogenous polynucleotide in the construct of the present invention is a suicide gene encoding a safety switch protein. Suitable suicide gene systems for induced cell death include, but are not limited to, caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV); cytosine deaminase (CD) and 5-fluorocytosine (5-FC). Furthermore, some suicide gene systems are cell type specific; for example, genetic modification of T lymphocytes by the B cell molecule CD20 can be eliminated upon administration of the mAb rituximab. Moreover, modified EGFR containing an epitope recognized by cetuximab can be used to deplete genetically modified cells when the cells are exposed to cetuximab. Thus, one aspect of the present invention provides a method for targeted incorporation of one or more suicide genes encoding safety switch proteins selected from caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR, and B cell CD20.

[0268] In some embodiments, one or more exogenous polynucleotides incorporated by the methods herein are driven by an operablely linked exogenous promoter contained in a construct for targeted incorporation. The promoter may be inductive or constitutive, and may be time-specific, tissue-specific, or cell-type-specific. Suitable constitutive promoters for the methods of the present invention include, but are not limited to, cytomegalovirus (CMV), elongation factor 1α (EF1α), phosphoglycerate kinase (PGK), hybrid CMV enhancer / chicken β-actin (CAG), and ubiquitin C (UBC) promoters. In one embodiment, the exogenous promoter is CAG.

[0269] Exogenous polynucleotides incorporated by the methods provided herein may be driven at the integration site by an endogenous promoter in the host genome. In one embodiment, the method of the present invention is used for targeted incorporation of one or more exogenous polynucleotides at the AAVS1 locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous AAVS1 promoter. In another embodiment, the method of the present invention is used for targeted incorporation at the ROSA26 locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous ROSA26 promoter. In yet another embodiment, the method of the present invention is used for targeted incorporation at the H11 locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous H11 promoter. In yet another embodiment, the method of the present invention is used for targeted incorporation at the collagen locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by the endogenous collagen promoter. In yet another embodiment, the method of the present invention is used for targeted incorporation at the HTRP locus in the cellular genome. In one embodiment, at least one incorporated polynucleotide is driven by an endogenous HTRP promoter. Theoretically, only correct insertion at the desired site would enable gene expression of the exogenous gene driven by the endogenous promoter.

[0270] In some embodiments, one or more exogenous polynucleotides included in the construct for a targeted incorporation method are driven by a single promoter. In some embodiments, the construct includes one or more linker sequences between two adjacent polynucleotides driven by the same promoter to enhance physical separation between the parts and maximize access to the enzymatic mechanism. The linker peptide of the linker sequence may consist of amino acids selected to make the physical separation between the parts (exogenous polynucleotides and / or proteins or peptides encoded therefrom) more flexible or more rigid, depending on the relevant function. The linker sequence may be protease-cleavable or chemically cleavable, resulting in distinct parts. Examples of enzymatic cleavage sites in the linker include sites for cleavage by proteolytic enzymes such as enterokinase, factor Xa, trypsin, collagenase, and thrombin. In some embodiments, the protease may be naturally produced by the host or introduced exogenously. Alternatively, the cleavage sites in the linker may be sites that can be cleaved upon exposure to a selected chemical or condition, such as cyanide bromide, hydroxylamine, or low pH. Any linker sequence may serve purposes other than providing cleavage sites. The linker sequence should allow for the effective positioning of a part relative to another adjacent part for the part to function properly. The linker may also be a simple amino acid sequence of sufficient length to prevent any steric hindrance between parts. Furthermore, the linker sequence can provide post-translational modifications including, but not limited to, phosphorylation sites, biotinylation sites, sulfated sites, and γ-carboxylation sites. In some embodiments, the linker sequence is flexible so as not to hold a biologically active peptide in a single undesirable conformation. To provide flexibility, the linker may be predominantly composed of amino acids with small side chains, such as glycine, alanine, and serine. In some embodiments, about 80 or 90 percent or more of the linker sequence consists of glycine, alanine, or serine residues, particularly glycine and serine residues.In some embodiments, the G4S linker peptide separates the terminal processing and endonuclease domains of the fusion protein. In other embodiments, the 2A linker sequence allows two distinct proteins to be produced from a single translation. Preferred linker sequences can be readily identified empirically. Furthermore, preferred sizes and sequences of linker sequences can also be determined by conventional computer modeling techniques. In one embodiment, the linker sequence encodes a self-cleaving peptide. In one embodiment, the self-cleaving peptide is 2A. In some other embodiments, the linker sequence provides an intra-sequence ribosome entry site (IRES). In some embodiments, any two consecutive linker sequences are distinct.

[0271] Methods for introducing constructs containing exogenous polynucleotides for targeted integration into cells can be achieved using methods of gene transfer into cells that are known in themselves. In one embodiment, the construct comprises a viral vector skeleton such as an adenovirus vector, adeno-associated virus vector, retrovirus vector, lentiviral vector, or Sendai virus vector. In some embodiments, plasmid vectors are used to deliver and / or express exogenous polynucleotides to target cells (e.g., pA1-11, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo). In some other embodiments, episomal vectors are used to deliver exogenous polynucleotides to target cells. In some embodiments, recombinant adeno-associated virus (rAAV) can be used for genetic engineering to introduce insertions, deletions, or substitutions via homologous recombination. Unlike lentiviruses, rAAV is not integrated into the host genome. Furthermore, episomal rAAV vectors mediate homologous gene targeting at a much higher rate compared to transfection with conventional targeted plasmids. In some embodiments, AAV6 or AAV2 vectors are used to introduce insertions, deletions, or substitutions at target sites in the iPSC genome. In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions herein contain at least one genotype listed in Table 1.

[0272] III. Methods for obtaining and maintaining genetically engineered iPSCs In one embodiment, the present invention provides a method for obtaining and maintaining a genome-engineered iPSC comprising one or more targeted edits at one or more desired sites, wherein the targeted edits remain intact and functional at each selected editing site(s) in the proliferated genome-engineered iPSC or iPSC-induced non-pluripotent cells. The targeted edits introduce insertions, deletions, and / or substitutions, i.e., targeted integrations and / or indels at selected sites, into the genome of the iPSC and / or cells derived therefrom. Compared to direct manipulation of patient-derived peripheral blood primary effector cells, many advantages of obtaining genomically engineered iPSC-induced effector cells through editing and differentiation of iPSCs provided herein include: unlimited supply of engineered effector cells; no need for repeated manipulation of effector cells, especially when multiple engineered modalities are involved; the resulting effector cells are rejuvenated due to elongated telomeres and less depletion; and the effector cell population is homogeneous with respect to editing sites, copy number, and the absence of allele mutations, random mutations, and expression variability, which is mainly due to the possibility of clonal selection in the engineered iPSCs provided herein.

[0273] In certain embodiments, genome-engineered iPSCs containing one or more targeted edits at one or more selected sites are maintained, passaged, and grown as single cells for extended periods in cell culture media shown in Table 2 as Fate Maintenance Medium (FMM), and the iPSCs retain the targeted edits and functional modifications at the selected sites(s). The components of the medium may be present in the medium in amounts within the optimal range shown in Table 2. iPSCs cultured in FMM have been shown to readily generate all three somatic cell lineages in in vitro differentiation via embryoid bodies or monolayers (without embryoid body formation) and in vivo differentiation via teratoma formation. See, for example, International Publication 2015 / 134652, whose disclosure is incorporated herein by reference.

[0274] [Table 12]

[0275] In some embodiments, genome-engineered iPSCs containing one or more targeted inclusions and / or indels are maintained, passaged, and grown in a medium containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and are not, or essentially not, TGFβ receptor / ALK5 inhibitors, and the iPSCs retain intact and functional targeted edits at selected sites.

[0276] Another aspect of the present invention provides a method for generating genome-engineered iPSCs, either through targeted editing of iPSCs, or by first generating genome-engineered non-pluripotent cells by targeted editing, and then reprogramming the selected / isolated genome-engineered non-pluripotent cells to obtain iPSCs containing the same targeted editing as the non-pluripotent cells. A further aspect of the present invention provides genome-engineered non-pluripotent cells that are simultaneously reprogrammed by introducing targeted inclusions and / or targeted indels into the cells, wherein the contacted non-pluripotent cells are under conditions sufficient for reprogramming, and the conditions for reprogramming include contacting the non-pluripotent cells with one or more reprogramming factors and small molecules. In various embodiments of the method for simultaneous genome engineering and reprogramming, targeted inclusions and / or targeted indels can be introduced into non-targeted pluripotent cells before or essentially simultaneously with initiating reprogramming by contacting the non-pluripotent cells with one or more reprogramming factors and optionally small molecules.

[0277] In some embodiments, to simultaneously manipulate and reprogram non-pluripotent cells genomes, targeted incorporations and / or indels may also be introduced into non-pluripotent cells after a multi-day process of reprogramming has been initiated by contacting the non-pluripotent cells with one or more reprogramming factors and small molecules, and the construct-carrying vector is introduced before the reprogrammed cells exhibit stable expression of one or more endogenous pluripotency genes, including but not limited to SSEA4, Tra181, and CD30.

[0278] In some embodiments, reprogramming is initiated by contacting non-pluripotent cells with at least one reprogramming factor and, optionally, a combination of a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FRM; Table 2). In some embodiments, genomically engineered iPSCs by any of the above methods are further maintained and proliferated using a mixture containing a combination of a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor (FMM; Table 2).

[0279] In some embodiments of a method for generating genetically engineered iPSCs, the method comprises genomically engineering an iPSC by introducing one or more targeted embeddings and / or indels into the iPSC to obtain a genetically engineered iPSC having at least one genotype listed in Table 1. Alternatively, a method for generating genetically engineered iPSCs comprises (a) introducing one or more targeted edits into non-pluripotent cells to obtain genetically engineered non-pluripotent cells containing targeted embeddings and / or indels at selected sites, and (b) contacting the genetically engineered non-pluripotent cells with one or more reprogramming factors and, optionally, a small molecule composition containing a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor and / or a ROCK inhibitor to obtain a genetically engineered iPSC containing targeted embeddings and / or indels at selected sites. Alternatively, a method for producing a genome-engineered iPSC comprises (a) contacting non-pluripotent cells with a small molecule composition comprising one or more reprogramming factors and, optionally, a TGFβ receptor / ALK inhibitor, a MEK inhibitor, a GSK3 inhibitor, and / or a ROCK inhibitor to initiate reprogramming of the non-pluripotent cells; (b) introducing one or more targeted inclusions and / or indels into the reprogrammed non-pluripotent cells for genome engineering; and (c) obtaining a genome-engineered iPSC containing the targeted inclusions and / or indels at a selected site. Any of the above methods may further comprise single-cell sorting of genome-engineered iPSCs to obtain cloned iPSCs. Through clonal proliferation of these genome-engineered iPSCs, a master cell bank is generated to contain single-cell sorting and proliferated cloned iPSCs having at least one phenotype as provided in Table 1 herein. The master cell bank is subsequently cryopreserved, providing a platform for further iPSC manipulation and a renewable source for manufacturing ready-made, manipulated, homogeneous cell therapy products, which have a clear and uniform composition and can be mass-produced on a considerable scale in a cost-effective manner.

[0280] Reprogramming factors are selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, SV40LT, HESRG, CDH1, TDGF1, DPPA4, DNMT3B, ZIC3, L1TD1 and any combination thereof, as disclosed in International Publication Nos. 2015 / 134652 and 2017 / 066634, the disclosures of which are incorporated herein by reference. One or more reprogramming factors may be in the form of polypeptides. Reprogramming factors may also be in the form of polynucleotides and are therefore introduced into non-pluripotent cells by vectors such as retroviruses, Sendai viruses, adenoviruses, episomes, plasmids, and minicircles. In certain embodiments, one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, one or more polynucleotides are introduced by an episomal vector. In various other embodiments, one or more polynucleotides are introduced by a Sendai virus vector. In some embodiments, one or more polynucleotides are introduced by a plasmid combination. See, for example, International Publication 2019 / 075057, the disclosure of which is incorporated herein by reference.

[0281] In some embodiments, non-pluripotent cells are transfected with multiple constructs containing different exogenous polynucleotides and / or different promoters by multiple vectors for targeted incorporation at the same or different selected sites. These exogenous polynucleotides may include suicide genes, or genes encoding proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, and / or survival of iPSCs or cells derived therefrom. In some embodiments, the exogenous polynucleotides encode RNA, including but not limited to siRNA, shRNA, miRNA, and antisense nucleic acids. These exogenous polynucleotides may be driven by one or more promoters selected from the group consisting of constitutive promoters, inducible promoters, time-specific promoters, and tissue-specific or cell-type-specific promoters. Thus, the polynucleotides can be expressed under conditions that activate the promoters, for example, in the presence of an inducer, or in a particular differentiated cell type. In some embodiments, the polynucleotides are expressed in iPSCs and / or cells differentiated from iPSCs. In one embodiment, one or more suicide genes are driven by a constitutive promoter, for example, capase-9 driven by CAG. These constructs, comprising different exogenous polynucleotides and / or different promoters, can be transfected into non-pluripotent cells simultaneously or sequentially. Non-pluripotent cells subjected to targeted incorporation of multiple constructs can be simultaneously exposed to one or more reprogramming factors to initiate reprogramming concurrently with genomic manipulation, thereby obtaining genomically engineered iPSCs with multiple targeted incorporations in the same cell pool. Thus, this robust method allows for the induction of clonally engineered iPSCs with multiple modalities integrated into one or more selected target sites through a simultaneous reprogramming and manipulation strategy.In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions provided herein include at least one genotype listed in Table 1.

[0282] IV. Method for obtaining genetically modified effector cells by differentiating genome-modified iPSCs. Further embodiments of the present invention provide a method for in vivo differentiation of genomically engineered iPSCs by teratoma formation, wherein differentiated cells induced in vivo from genomically engineered iPSCs retain intact and functional targeted editing, including targeted incorporation and / or indels, at desired sites(s). In some embodiments, differentiated cells induced in vivo from genomically engineered iPSCs via teratoma contain one or more inducible suicide genes incorporated at one or more desired sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, beta-2 microglobulin, CD38, GAPDH, TCR, or RUNX1, or other loci that meet the criteria for a genome-safe harbor. In some other embodiments, differentiated cells induced in vivo from genomically engineered iPSCs via teratoma contain polynucleotides encoding a targeted modality, or polynucleotides encoding proteins that promote the transport, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells. In some embodiments, differentiated cells induced in vivo from genomically engineered iPSCs via teratomas containing one or more inducible suicide genes further include one or more indels of endogenous genes associated with the regulation and mediation of immune responses. In some embodiments, the indels include one or more endogenous checkpoint genes. In some embodiments, the indels include one or more endogenous T cell receptor genes. In some embodiments, the indels include one or more endogenous MHC class I suppressor genes. In some embodiments, the indels include one or more endogenous genes associated with the major histocompatibility complex.In some embodiments, the indel is contained in one or more endogenous genes, including but not limited to AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT. In one embodiment, a genome-engineered iPSC containing one or more exogenous polynucleotides at selected sites further includes targeted editing in the gene encoding B2M (beta-2-microglobulin).

[0283] In certain embodiments, genome-engineered iPSCs containing one or more genetic modifications provided herein are used to induce a hematopoietic cell lineage or other specific cell type in vitro, and the induced non-pluripotent cells retain functional genetic modifications, including targeted editing, at selected sites(s). In some embodiments, the genome-engineered iPSCs used to induce a hematopoietic cell lineage or other specific cell type in vitro are master cell bank cells that are cryopreserved and thawed immediately before use. In one embodiment, genome-engineered iPSC-induced cells are mesodermal cells with definitive hematopoietic endothelial (HE) potential, definitive HE, CD34 + Hematopoietic cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotency precursors (MPPs), T cell precursors, NK cell precursors, bone marrow cells, neutrophil precursors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages, among others, derived from genome-engineered iPSCs, retain functional genetic modifications, including targeted editing, at desired sites(s).

[0284] For applicable differentiation methods and compositions for obtaining iPSC-induced hematopoietic cell lines, see, for example, International Publication No. 2017 / 078807, the disclosure of which is incorporated herein by reference. As provided, the methods and compositions for generating hematopoietic cell lines involve definitive hematopoietic endothelium (HE) induced from pluripotent stem cells, including iPSCs, in a scalable, monolayer EB-free culture platform under serum-free, feeder-free, and / or stroma-free conditions. Cells that can be differentiated according to the provided methods range from pluripotent stem cells to progenitor cells committed to specific terminally differentiated and transdifferentiated cells, and to cells of various lineages that have directly transitioned to hematopoietic fate without passing through pluripotent intermediates. Similarly, cells produced by differentiating stem cells range from pluripotent stem cells or progenitor cells to terminally differentiated cells, and to all intervening hematopoietic cell lineages.

[0285] A method for differentiating and proliferating hematopoietic lineage cells from pluripotent stem cells in monolayer culture comprises contacting pluripotent stem cells with a BMP pathway activator and, optionally, bFGF. As provided, pluripotent stem cell-induced mesodermal cells are obtained and proliferated from pluripotent stem cells without embryoid body formation. The mesodermal cells are then subjected to contact with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain proliferating mesodermal cells with definitive hematopoietic endothelial (HE) potential without embryoid body formation from pluripotent stem cells. Subsequent contact with bFGF, and optionally with a ROCK inhibitor and / or a WNT pathway activator, differentiates the mesodermal cells with definitive HE potential into definitive HE cells, which are similarly proliferated during differentiation.

[0286] The method for obtaining hematopoietic cells provided herein is superior to EB-mediated pluripotent stem cell differentiation because EB formation results in moderate to minimal cell proliferation, failing to enable monolayer culture and uniform differentiation of cells within a population, which are crucial for many applications requiring uniform proliferation, making it cumbersome and inefficient.

[0287] The provided monolayer differentiation platform facilitates differentiation into definitive hematopoietic endothelium, resulting in the induction of differentiated offspring such as hematopoietic stem cells and T cells, B cells, NKT cells, and NK cells. The monolayer differentiation strategy combines enhanced differentiation efficiency with large-scale proliferation, enabling the delivery of therapeutically appropriate numbers of pluripotent stem cell-induced hematopoietic cells for various therapeutic applications. Furthermore, monolayer culture using the methods provided herein results in functional hematopoietic lineage cells that enable the full range of in vitro differentiation, ex vivo modification, and in vivo long-term hematopoietic self-renewal, reconstitution, and engraftment. As provided, iPSC-induced hematopoietic lineage cells include, but are not limited to, definitive hematopoietic endothelium, hematopoietic pluripotent progenitor cells, hematopoietic stem cells and progenitor cells, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, B cells, macrophages, and neutrophils.

[0288] In some embodiments, the present invention provides a method for directing the differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage, the method comprising: (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate the differentiation and proliferation of mesodermal cells from the pluripotent stem cells; and (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF and a GSK3 inhibitor to initiate the differentiation and proliferation of mesodermal cells having definitive hematopoietic potential from the mesodermal cells, the composition optionally comprising a TGFβ receptor / ALK inhibitor (iii) Initiating a definitive hematopoietic endothelial differentiation and proliferation from pluripotent stem cell-induced mesoderm cells having definitive hematopoietic endothelial potential by contacting mesoderm cells having definitive HE potential with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11, and optionally a Wnt pathway activator, wherein the composition optionally does not contain a TGFβ receptor / ALK inhibitor.

[0289] In some embodiments, the method further comprises contacting pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but not a TGFβ receptor / ALK inhibitor, and seeding and growing the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs, or naive iPSCs, or iPSCs containing one or more genetic imprints, the one or more genetic imprints contained in the iPSCs being retained in hematopoietic cells differentiated therefrom. In some embodiments of the method for directing the differentiation of pluripotent stem cells into hematopoietic lineage cells, the differentiation of pluripotent stem cells into hematopoietic lineage cells is in a monolayer culture form without embryoid body formation.

[0290] In some embodiments of the method described above, the definitive hematopoietic endothelial cells obtained through pluripotent stem cell induction are CD34 + In some embodiments, the definitive hematopoietic endothelial cells obtained are CD34. + CD43 - In some embodiments, the definitive hematopoietic endothelial cell is CD34. + CD43 - CXCR4 - CD73 - In some embodiments, the definitive hematopoietic endothelial cell is CD34. + CXCR4 - CD73 - In some embodiments, the definitive hematopoietic endothelial cell is CD34. + CD43 - CD93 - In some embodiments, the definitive hematopoietic endothelial cell is CD34. + CD93 - That is the case.

[0291] In some embodiments of the method described above, the method further comprises (i) contacting definitive hematopoietic endothelium for pluripotent stem cell induction with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, and optionally a BMP activator, to initiate the differentiation of the definitive hematopoietic endothelium into pre-T cell precursors, and optionally (ii) contacting the pre-T cell precursors with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, but not one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate the differentiation of the pre-T cell precursors into T cell precursors or T cells. In some embodiments of the method, the pluripotent stem cell induction T cell precursors are CD34 + CD45 + CD7 + In some embodiments of this method, the pluripotent stem cell-inducing T cell precursor is CD45 + CD7 + That is the case.

[0292] In some further embodiments of the above-described method for directing the differentiation of pluripotent stem cells into hematopoietic cell lineage cells, the method further comprises (i) contacting definitive hematopoietic endothelium for pluripotent stem cell induction with a composition comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator, to initiate the differentiation of the definitive hematopoietic endothelium into pre-NK cell precursors; and optionally (ii) contacting the pre-NK cell precursor for pluripotent stem cell induction with a composition comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, but not comprising one or more of VEGF, bFGF, TPO, BMP activator, and ROCK inhibitor, to initiate the differentiation of the pre-NK cell precursor into NK cell precursors or NK cells. In some embodiments, the pluripotent stem cell induction NK precursor is CD3 - CD45 + CD56 +CD7 + In some embodiments, pluripotent stem cell-induced NK cells are CD3 - CD45 + CD56 + And, optionally, NKp46 + CD57 + and CD16 + It is further defined by this fact.

[0293] Therefore, using the above differentiation method, one or more populations of iPSC-induced hematopoietic cells can be obtained: (i) CD34 using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2. + (ii) Definitive hematopoietic endothelial cells (iHE) using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (ii) Definitive HSCs using one or more culture media selected from iMPP-A, iTC-A2, iTC-B2, iNK-A2, and iNK-B2; (iv) Pluripotent progenitor cells (iMPP) using iMPP-A; (v) T cell precursors (ipro-T) using one or more culture media selected from iTC-A2 and iTC-B2; (vi) T cells (iTC) using iTC-B2; (vii) NK cell precursors (ipro-NK) using one or more culture media selected from iNK-A2 and iNK-B2; and / or (viii) NK cells (iNK) and iNK-B2. In some embodiments, the media are: a.iCD34-C comprises a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of bFGF, VEGF, SCF, IL6, IL11, IGF, and EPO, and optionally a Wnt pathway activator, but does not contain a TGFβ receptor / ALK inhibitor. b.iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, Flt3L, and IL11. c.iTC-A2 comprises a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, TPO, and IL7, and optionally a BMP activator. d.iTC-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7. e.iNK-A2 comprises a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, TPO, IL3, IL7, and IL15, and optionally a BMP activator. f.iNK-B2 contains one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, and IL15.

[0294] In some embodiments, the genome-modified iPSC-induced cells obtained by the above method contain one or more inducible suicide genes integrated at one or more desired integration sites, including AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, RUNX1, B2M, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCRα or β constant region, NKG2A, NKG2D, CD25, CD38, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, or other loci that meet the criteria for a genome-safe harbor. In some other embodiments, genome-engineered iPSC-inducible cells include polynucleotides encoding safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins that promote the transport, homing, viability, self-renewal, persistence, and / or survival rate of stem cells and / or progenitor cells. In some embodiments, genome-engineered iPSC-inducible cells containing one or more suicide genes further include one or more indels contained in one or more endogenous genes associated with the regulation and mediation of immune responses, including but not limited to checkpoint genes, endogenous T cell receptor genes, and MHC class I suppressor genes. In one embodiment, genome-engineered iPSC-inducible cells containing one or more suicide genes include an indel in the B2M gene, and B2M is knocked out.

[0295] Furthermore, applicable dedifferentiation methods and compositions for obtaining second-fate genome-modified hematopoietic cells from first-fate genome-modified hematopoietic cells include, for example, those shown in International Publication No. 2011 / 159726, the disclosure of which is incorporated herein by reference. The methods and compositions provided herein enable partial reprogramming of initiating non-pluripotent cells into non-pluripotent intermediate cells by restricting the expression of the endogenous Nanog gene during reprogramming, and subjecting the non-pluripotent intermediate cells to conditions for differentiation of intermediate cells into desired cell types. In some embodiments, genome-modified iPSCs and their derived cells obtained using the methods and compositions herein include at least one genotype listed in Table 1.

[0296] V. Therapeutic use of functionally modalized inducible immune cells differentiated from genetically engineered iPSCs In some embodiments, the present invention provides compositions comprising an isolated population or subpopulation of functionally enhanced induced immune cells differentiated from genomically engineered iPSCs using the methods and compositions disclosed herein. In some embodiments, the iPSCs of the composition comprise one or more disclosed targeted gene edits that can be retained in iPSC-induced immune cells, and the genetically engineered iPSCs and cells derived therefrom are suitable for cell-based adoptive therapy. In one embodiment, the isolated population or subpopulation of genetically engineered effector cells of the composition comprises iPSC-induced CD34 +The composition includes cells. In one embodiment, an isolated population or subpopulation of genetically engineered effector cells of the composition includes iPSC-induced HSC cells. In one embodiment, an isolated population or subpopulation of genetically engineered effector cells of the composition includes iPSC-induced pro-T cells or T cells. In one embodiment, an isolated population or subpopulation of genetically engineered effector cells of the composition includes iPSC-induced pro-NK cells or NK cells. In one embodiment, an isolated population or subpopulation of genetically engineered effector cells of the composition includes iPSC-induced immunomodulatory cells or bone marrow-derived suppressor cells (MDSCs). In some embodiments, the iPSC-induced genetically engineered effector cells of the composition are further modified ex vivo for improved therapeutic potential. In one embodiment of the composition, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increase in the number or ratio of naive T cells, stem cell memory T cells and / or central memory T cells. In one embodiment of this composition, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of type I NKT cells. In another embodiment of this composition, an isolated population or subpopulation of genetically engineered immune cells derived from iPSCs includes an increased number or proportion of adaptive NK cells. In some embodiments of this composition, genetically engineered CD34 cells derived from iPSCs are included. + The isolated population or subpopulation of cells, HSC cells, T cells, NK cells, or bone marrow-derived suppressor cells are allogeneic. In some other embodiments of this composition, genetically engineered CD34 derived from iPSCs + An isolated population or subpopulation of cells, HSC cells, T cells, NK cells, or MDSCs is autologous.

[0297] In some embodiments of this composition, the iPSCs for differentiation contain selected gene imprints to convey desired therapeutic attributes in effector cells, provided that the cell developmental biology during differentiation is not disrupted and the gene imprints are retained and functional in the iPSC-induced differentiated hematopoietic cells.

[0298] In some embodiments of the composition, the genetic imprint of pluripotent stem cells includes (i) one or more recombinant modalities obtained through genomic insertions, deletions, or substitutions in the genome of pluripotent cells during or after reprogramming non-pluripotent cells into iPSCs, or (ii) one or more retainable therapeutic attributes of source-specific immune cells that are specific to a donor, disease, or therapeutic response, wherein the pluripotent cells are reprogrammed from source-specific immune cells, the iPSCs retain the source therapeutic attributes, and the source therapeutic attributes are also included in iPSC-induced hematopoietic lineage cells.

[0299] In some embodiments of this composition, the genetically modified modality comprises one or more of the following: safety switch proteins, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates; or proteins that promote engraftment, transport, homing, viability, self-renewal, persistence, immune response regulation and modification, and / or viability of iPSCs or cells derived therefrom. In some embodiments of this composition, the genetically modified iPSCs and cells derived therefrom comprise the genotypes listed in Table 1. In some other embodiments of this composition, genetically modified iPSCs and cells derived therefrom containing the genotypes listed in Table 1 are (1) deletions or disruptions of one or more genes of any of the following: TAP1, TAP2, Tapasin, NLRC5, PD1, LAG3, TIM3, RFXANK, CIITA, RFX5, or RFXAP, RAG1 and the chromosome 6p21 region, and (2) HLA-E, 4-1BBL, CD3, CD4, CD8, CD47, CD113, CD131, CD137, CD80, PDL1, A 2A The modality further includes additional genetic modification modalities, such as the introduction or upregulation of surface trigger receptors for coupling with R, CAR, Fc receptors, or bispecific, multispecific, or universal engagers.

[0300] In some other embodiments of the composition, the hematopoietic lineage cells include therapeutic attributes of source-specific immune cells relating to at least two combinations of the following: (i) expression of one or more antigen-targeting receptors, (ii) modified HLA, (iii) resistance to the tumor microenvironment, (iv) recruitment of bystander immune cells and immunomodulation, (iv) improved on-target specificity with reduced extratumor effects, and (v) improved homing, persistence, cytotoxicity, or antigen escape rescue.

[0301] In some embodiments of this composition, iPSC-induced hematopoietic cells, including the genotypes listed in Table 1, express at least one cytokine and / or its receptor, including IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, or IL21, or any modified protein thereof, and express at least CAR. In some embodiments of this composition, the manipulated expression of cytokines and CARs is NK cell-specific. In some other embodiments of this composition, the manipulated expression of cytokines and CARs is T cell-specific. In one embodiment, the CAR i...

Claims

1. A cell or a group thereof, wherein the cell is a eukaryotic cell, an animal cell, a human cell, an immune cell, an induced pluripotent cell (iPSC), a cloned iPSC, or an induced cell differentiated from the same, and the cell is (i) A polynucleotide encoding a transgenic TCRα chain (tgTCRα), (ii) A polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), wherein the tgTCRα chain and the tgTCRβ chain are exogenous TCR complexes (TCR) that recognize a first tumor antigen. ｅｘｏ ) form polynucleotides, and optionally, (iii) A cell or population thereof comprising one or more additional exogenous polynucleotides, each containing a polynucleotide encoding a chimeric antigen receptor (CAR) or engager that targets at least a second tumor antigen.

2. (i) The polynucleotide encoding the tgTCRα chain and the polynucleotide encoding the tgTCRβ chain are included in a bicistronic construct and optionally, (a) The construct is inserted into the steady region (TRAC or TRBC) of TCRα or TCRβ, (b) Insertion of the construct disrupts the expression of endogenous TCRα or endogenous TCRβ at the insertion site, and / or (c) The expression of the construct is driven by the endogenous or exogenous promoter of the TCR, or (ii) The polynucleotide encoding the CAR or the engager is inserted into the TRAC or TRBC, optionally, (a) Insertion of the polynucleotide encoding the CAR or the engager disrupts the expression of the endogenous TCRα or endogenous TCRβ at the insertion site, and / or (b) The expression of the CAR or the engager is driven by the endogenous or exogenous promoter of the TCR, or (iii) The cells or population thereof according to claim 1, wherein the tgTCRα chain and the tgTCRβ chain, the polynucleotide encoding the CAR or the engager, or the one or more additional polynucleotides are inserted into one or more safe harbor loci or selected loci.

3. (I) The construct and the polynucleotide encoding the CAR or the engager are each inserted into the constant region (TRAC or TRBC) of TCRα or TCRβ, but not into the same constant region, thereby disrupting the expression of both endogenous TCRα and endogenous TCRβ and knocking out the endogenous TCR. (a) the transgenic TCRα and the endogenous TCRβ, or (b) Avoid unpaired TCRs, including the transgenic TCRβ and the endogenous TCRα, Or (II) the cell or population thereof according to claim 2, wherein the construct and the polynucleotide encoding the CAR or the engager are each incorporated into a locus including a safe harbor locus or a selected locus.

4. (i) The safe harbor locus comprises at least one of AAVS1, CCR5, ROSA26, collagen, HTRP, H11, GAPDH, or RUNX1, (ii) The selected locus is one of B2M, TAP1, TAP2, Tapasin, NLRC5, CIITA, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD38, CD25, CD69, CD44, CD58, CD54, CD56, CD69, CD71, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT, and / or (iii) The cell or population thereof according to claim 3, wherein the incorporation of the exogenous polynucleotide knocks out the expression of the gene at the gene locus.

5. The first tumor antigen and the second tumor antigen are, (i) MR1, NYESO1, MICA / B, EpCAM, EGFR, B7H3, Muc1, Muc16, CD19, BCMA, CD20, CD22, CD38, CD123, HER2, CD52, GD2, MSLN, VEGF-R2, PSMA and PDL1, or (ii) ADGRE2, B7H3, carbonic anhydrase IX (CAIX), CCR1, CCR4, carcinoembryonic antigen (CEA), CD3, CD5, CD7, CD8, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CDS, CLEC12A, antigens of cytomegalovirus (CMV) infected cells, epithelium Glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2,3,4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER2), human Telomerase reverse transcriptase (hTERT), ICAM-1, integrin B7, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, melanoma antigen family A1 (MAGE-A1), MICA / B, MR1, mucin 1 (Muc-1), mucin 16 (Muc-16), It comprises at least one of the following: mesoserin (MSLN), NKCSI, NKG2D ligand, c-Met, NYESO1, oncoemetic antigen (h5T4), PDL1, PRAME, prostate stem cell antigen (PSCA), PRAME prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBC1, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms oncoprotein (WT-1), and pathogen antigens. The cells or population thereof according to claim 1, wherein the first tumor antigen and the second tumor antigen are the same or different.

6. The first tumor antigen comprises at least one of MR1, NYESO1, and MICA / B, or (i) The tgTCRα comprises a variable alpha (Vα) fragment having at least about 85% identity with SEQ ID NO: 7, and / or a TCRα constant fragment containing a sequence having at least about 85% identity with SEQ ID NO: 8, and / or (ii) The cell or population thereof according to claim 1, wherein the tgTCRβ comprises a variable alpha (Vβ) fragment having at least about 85% identity with SEQ ID NO: 9 and a TCRβ constant fragment having at least about 85% identity with SEQ ID NO:

10.

7. The aforementioned CAR is, (i) T cell-specific or NK cell-specific, (ii) bispecific antigen binding CAR; (iii) Switchable CAR, (iv) Dimerized CAR, (v) Split CAR, (vi) multi-chain CAR; (vii) Inductionable CAR, (viii) inactivated CAR; (ix) Optionally co-expressed in a separate construct or in a bicistronic construct with a partial or full-length peptide of an exogenous cytokine and / or its receptor expressed on the cell surface, (x) Cells of the population according to claim 1, which are optionally co-expressed with a checkpoint inhibitor in a separate construct or in a bicistronic construct.

8. The aforementioned Engager, (i) A first binding domain that recognizes the extracellular portion of CD3, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof of the cell or bystander immunoeffector cell, (ii) A second binding domain that targets a second tumor antigen different from the first tumor antigen targeted by the exogenous TCR, wherein the second binding domain of the engager is B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A The cells or population thereof according to claim 1, comprising a second binding domain that is specific to any one of CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROR1, or VEGF-R2.

9. The aforementioned cells are (i) CD38 knockout, (ii) HLA-I deficiency and / or HLA-II deficiency, (iii) Introduced HLA-G or non-cleavable HLA-G, or knockout of one or both CD58 and CD54, (iv) CD16 or its variant, (v) Chimeric fusion receptor (CFR), (vi) A signaling complex comprising a cell surface-expressed exogenous cytokine and / or a portion or complete peptide of its receptor, (vii) At least one of the genotypes listed in Table 1, (viiii) Deletion or destruction of at least one of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (ix) HLA-E, 4-1BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ A cell or population thereof according to any one of claims 1 to 8, further comprising the introduction or upregulation of at least one of the following: R, Fc receptor, antibody or functional variant or fragment thereof, checkpoint inhibitor, and surface trigger receptor for coupling with an agonist.

10. The CD16 or its variant is (a) high affinity non-cleavable CD16 (hnCD16), (b) External domains of CD16 F176V and S197P, (c) All or partial external domains derived from CD64, (d) Non-natural (or non-CD16) transmembrane domains, (e) Non-natural (or non-CD16) intracellular domains, (f) Non-natural (or non-CD16) signaling domains, (g) unnatural irritant domains, and (h) The cell or population thereof according to claim 9, comprising at least one of transmembrane, signaling, and stimulating domains derived from the same or a different polypeptide, but not from CD16.

11. The cell or population thereof according to claim 9, wherein the CFR comprises an external domain fused to a transmembrane domain operably connected to an internal domain, and the external domain, the transmembrane domain, and the internal domain do not contain endoplasmic reticulum (ER) retention signals or endocytosis signals.

12. (i) The external domain of the CFR includes the full length or partial length of the extracellular portion of a signaling protein comprising at least one of CD3ε, CD3γ, CD3δ, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, any functional variant, and combinations or chimeras thereof. (ii) The external domain of the CFR initiates signal transduction when it binds to a selected agonist, or (iii) The internal domain of the CFR includes a cytotoxic domain comprising at least the full length or a portion of the following polypeptides: CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (4-1BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, or NKG2D; the internal domain is optionally comprised of: (a) Costimulatory domains comprising the full length or a portion thereof of CD2, CD27, CD28, CD40L, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D polypeptide, or any combination thereof. (b) Costimulatory domains including the full length or a portion thereof of CD28, 4-1BB, CD27, CD40L, ICOS, CD2, or combinations thereof (c) A sustained signaling domain comprising the full length or a portion of the internal domain of a cytokine receptor including IL7R, IL15R, IL18R, IL12R, IL23R, or a combination thereof, and / or (d) The cell or population thereof according to claim 11, further comprising one or more complete or partial intracellular portions of receptor tyrosine kinase (RTK), tumor necrosis factor receptor (TNFR), EGFR, or FAS receptor.

13. The selected agonist is (i) an antibody, or a functional variant or fragment thereof, or (ii) an engager. The cell or population thereof according to claim 12, wherein the selected agonist is encoded by a polynucleotide contained in the cell or is contained in a culture medium containing the cell or population thereof.

14. The exogenous cytokine expressed on the cell surface or its receptor is, (a) comprising at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors(s), or (b) (i) Co-expression of IL15 and IL15Rα using self-cleaving peptides, (ii) Fusion protein of IL15 and IL15Rα (iii) IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα has been cleaved or excluded. (iv) A fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) Fusion protein of IL15 and IL15Rβ, (vi) A fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either natural or modified, and (vii) containing at least one of the homodimers of IL15Rβ, (b) Any one of (i) to (vii) may be co-expressed with CAR in a separate construct or a bicistronic construct, (c) (i) A fusion protein of IL7 and IL7Rα, (ii) A fusion protein of IL7 and the common receptor γC, wherein the common receptor γC is either native or modified, and (iii) comprising at least one homodimer of IL7Rβ, (c) any one of (i) to (vii) may be co-expressed with CAR in a separate construct or a bicistronic construct, Optional, (d) The cells or population thereof according to claim 9, which are transiently expressed.

15. The aforementioned checkpoint inhibitors are PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A ２Ａ The cells or population thereof according to claim 9, which are antagonists to one or more checkpoint molecules, including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, and inhibitory KIR.

16. The aforementioned cells, compared to their corresponding primary cells obtained from peripheral blood, umbilical cord blood, or any other donor tissue that does not have the same gene editing(s), (i) Increased cytotoxicity, (ii) Improved persistence and / or survival rate, (iii) Enhanced ability to migrate bystander immune cells to tumor sites and / or activate or mobilize them. (iv) Improved tumor penetration, (v) Enhanced ability to reduce tumor immunosuppression, (vi) Improved ability to rescue tumor antigen escapes, (vii) controlled apoptosis, (viiii) ADCC enhanced or acquired, and (ix) The ability to avoid fratricide, A cell or population thereof according to any one of claims 1 to 15, having therapeutic properties including one or more of the following.

17. The aforementioned induced cells are induced CD34 ＋ A cell or population thereof according to any one of claims 1 to 16, comprising cells, induced hematopoietic stem cells and progenitor cells, induced pluripotent hematopoietic progenitor cells, induced T cell progenitor cells, induced NK cell progenitor cells, induced T cell lineage cells, induced NKT cell lineage cells, induced NK cell lineage cells, induced B cell lineage cells, or induced effector cells having one or more functional features not present in the corresponding primary T cells, NK cells, NKT cells, and / or B cells.

18. The cell or population thereof according to claim 17, wherein the induced effector cell is a hematopoietic cell and contains longer telomeres compared to its corresponding primary cell.

19. The cells contain one of the genotypes listed in Table 1, or the cells are (i) (1) CD19-CAR at the TRAC locus, (2) TRAC knockout, and (3) MR1-TCR or NYESO1-TCR, and optionally (4) TRBC knockout; (ii) (1) BCMA-CAR and hnCD16 insertion at the TRAC locus, (2) TRAC knockout, and (3) MR1-TCR or NYESO1-TCR, and optionally (4) TRBC knockout; or (iii) a cell or population thereof according to any one of claims 1 to 18, comprising (1) a MICA / B-CAR insertion at the TRAC locus, (2) a TRAC knockout, (3) an hnCD16 insertion at the CD38 locus, (4) a CD38 knockout, and (5) an MR1-TCR or NYESO1-TCR, and optionally (6) a TRBC knockout.

20. A composition comprising cells or a population thereof as described in any one of claims 1 to 19.

21. The composition according to claim 20, wherein the cells or population thereof comprises the iPSC-induced effector cells, and the composition further comprises one or more therapeutic agents.

22. The composition according to claim 21, wherein the one or more therapeutic agents include a peptide, a cytokine, a checkpoint inhibitor, an antibody or a functional variant or fragment thereof, an engager, a mitogen, a growth factor, a small RNA, dsRNA (double-stranded RNA), a mononuclear blood cell, a feeder cell, a feeder cell component or a replacement factor thereof, a vector containing one or more target polynucleic acids, a chemotherapeutic agent or radioactive moiety, or an immunomodulator (IMiD).

23. (a) The checkpoint inhibitor is (i) PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A ２Ａ One or more antagonist checkpoint molecules including R, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E, or inhibitory KIR, (ii) one or more of the following: atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lilimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents. (iii) comprising at least one of atezolizumab, nivolumab, and pembrolizumab, or (b) The composition according to claim 22, wherein the one or more therapeutic agents comprises one or more of venetoclax, azacitidine, and pomalidomide.

24. The aforementioned antibody, or its functional variant or fragment, (a) anti-CD20, anti-CD22, anti-HER2, anti-CD52, anti-EGFR, anti-CD123, anti-GD2, anti-PDL1, and / or anti-CD38 antibody, (b) Rituximab, bertuzumab, ofatumumab, ubrituximab, okalatuzumab, obinutuzumab, ibritumomab, ocrelizumab, inotuzumab, moxetumomab, epratuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuzimab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and humanized or Fc-modified variants or fragments thereof, as well as one or more of their functional equivalents and biosimilars, or (c) The composition according to claim 22, comprising daratumumab, wherein the induced effector cells comprise CD38 knockout and optionally express CD16 or a variant thereof.

25. The aforementioned Engager, (i) Bispecific T cell engagers (BiTEs), (ii) Bispecific killer cell engagers (BiKE), or (iii) containing a triple-specific killer cell engager (TriKE), or The aforementioned Engager, (a) A first binding domain that recognizes the extracellular portion of CD3, CD28, CD5, CD16, CD64, CD32, CD33, CD89, NKG2C, NKG2D, or any functional variant thereof of the cell or bystander immunoeffector cell, (b) The composition according to claim 22, comprising an antigen-specific second binding domain containing any one of B7H3, CD10, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD38, CD44, CD52, CD79a, CD79b, CD123, CD138, CD179b, CEA, CLEC12A, CS-1, DLL3, EGFR, EGFRvIII, EpCAM, FLT-3, FOLR1, FOLR3, GD2, gpA33, HER2, HM1.24, LGR5, MSLN, MCSP, MICA / B, Muc1, Muc16, PDL1, PSMA, PAMA, P-cadherin, ROP1, or VEGF-R2.

26. A therapeutic use of a therapeutic composition according to any one of claims 20 to 25, comprising introducing the therapeutic composition into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, hematological malignancy, solid tumor, cancer, or viral infection.

27. A master cell bank (MCB) comprising cloned iPSCs according to any one of claims 1 to 19.

28. A method for producing induced effector cells according to any one of claims 1 to 19, wherein the method is: The method comprises differentiating a genetically modified iPSC into a T cell, wherein the iPSC comprises (a) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), (b) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), and optionally (c) a polynucleotide encoding an engager targeting a chimeric antigen receptor (CAR) or a second tumor antigen, and optionally the iPSC comprises (i) CD38 knockout, (ii) HLA-I deficiency and / or HLA-II deficiency, (iii) Introduced HLA-G or non-cleavable HLA-G, or knockout of one or both CD58 and CD54, (iv) CD16 or its variant, (v) Chimeric fusion receptor (CFR), (vi) A signaling complex comprising a cell surface-expressed exogenous cytokine and / or a portion or complete peptide of its receptor, (vii) At least one of the genotypes listed in Table 1, (viiii) Deletion or destruction of at least one of B2M, CIITA, TAP1, TAP2, Tapasin, NLRC5, RFXANK, RFX5, RFXAP, TCR, NKG2A, NKG2D, CD25, CD69, CD44, CD56, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT, or (ix) HLA-E, 4-1BBL, CD3, CD4, CD8, CD16, CD47, CD113, CD131, CD137, CD80, PDL1, A ２Ａ A method further comprising the introduction or upregulation of at least one of the following: R, Fc receptor, antibody or functional variant or fragment thereof, checkpoint inhibitor, and surface trigger receptor for coupling with an agonist.

29. The genomically engineered clone iPSC further comprises: (a) the polynucleotide encoding the transgenic TCRα chain (tgTCRα); (b) the polynucleotide encoding the transgenic TCRβ chain (tgTCRβ); and optionally (c) the polynucleotide encoding the engager targeting the chimeric antigen receptor (CAR) or the second tumor antigen, wherein the genomically engineered clone iPSC further comprises: (i) Knock out CD38, (ii) To knock out B2M and / or CIITA, (iii) Knock out one or both of CD58 and CD54, and / or The method according to claim 28, further comprising introducing a signaling complex comprising (iv) HLA-G or non-cleaved HLA-G, the CD16 or a variant thereof, the CFR and / or a partial or complete peptide of a cell surface-expressed exogenous cytokine and / or its receptor.

30. The method according to claim 29, wherein the genome manipulation includes targeted editing.

31. The method according to claim 30, wherein the targeted editing comprises deletion, insertion, or indel, and the targeted editing is performed by CRISPR, ZFN, TALEN, homing nuclease, homologous recombination, or any other functional variation thereof.

32. A chimeric antigen receptor (CAR) specific to the tumor cell surface antigen MR1, wherein the MR1-CAR is (i) an external domain comprising at least one antigen recognition domain, wherein the antigen recognition domain is (a) A variable alpha (Vα) fragment having at least about 85% identity with SEQ ID NO: 7 (MR1Vα) and a variable beta (Vβ) fragment having at least about 85% identity with SEQ ID NO: 8 (MR1Vβ), or (b) An external domain comprising the extracellular domain of MR1 TCRα having at least approximately 85% identity with SEQ ID NO: 15 and the extracellular domain of MR1 TCRβ having at least approximately 85% identity with SEQ ID NO: 16, (ii) Transmembrane domain and (iii) an internal domain comprising at least a first signaling domain, wherein the first signaling domain is derived from the cytoplasmic domain of a signaling protein specific to the activation or function of T cells and / or NK cells, The aforementioned tumor cell surface antigen MR1 is a non-polymorphic, chimeric antigen receptor.

33. The aforementioned signaling proteins are 2B4 (natural killer cell receptor 2B4), 4-1BB (tumor necrosis factor receptor superfamily member 9), and CD16 (IgG Fc region receptor III-A), CD2 (T cell surface antigen CD2), CD28 (T cell specific surface glycoprotein CD28), CD28H (transmembrane and immunoglobulin domain-containing protein 2), CD3ζ (T cell surface glycoprotein CD3 zeta chain), CD3ζ1XX (CD3ζ variant), DAP10 (hematopoietic cell signaling molecule), DAP12 (TYRO protein tyrosine kinase-binding protein), DNAM1 (CD226 antigen), FcERIγ (high affinity immunoglobulin epsilon receptor subunit gamma), IL21R (interleukin-21 receptor), IL-2Rβ / IL-15RB (interleukin-2 receptor subunit beta), IL-2Rγ (cytokine receptor common subunit gamma), IL-7R (interleukin-7 receptor subunit alpha), KIR2DS2 (killer cell immunoglobulin-like receptor 2DS2), NKG2D (NKG2-D The chimeric antigen receptor according to claim 32, comprising one of the following: type II intrinsic membrane protein, NKp30 (native cytotoxicity trigger receptor 3), NKp44 (native cytotoxicity trigger receptor 2), NKp46 (native cytotoxicity trigger receptor 1), CS1 (SLAM family member 7), and CD8 (T cell surface glycoprotein CD8 alpha chain).

34. The chimeric antigen receptor according to claim 32, wherein the internal domain further comprises a second signaling domain and optionally a third signaling domain, the first signaling domain, the second signaling domain and the third signaling domain being different.

35. The chimeric antigen receptor according to claim 34, wherein the second signaling domain or the third signaling domain comprises the cytoplasmic domain or a portion thereof of 2B4, 4-1BB, CD16, CD2, CD28, CD28H, CD3ζ, DAP10, DAP12, DNAM1, FcERIγ IL21R, IL-2Rβ (IL-15Rβ), IL-2Rγ, IL-7R, KIR2DS2, NKG2D, NKp30, NKp44, NKp46, CD3ζ1XX, CS1, or CD8.

36. The chimeric antigen receptor according to claim 32, wherein the transmembrane domain comprises the amino acid sequence of CD2, CD3D, CD3E, CD3G, CD3ζ, CD4, CD8, CD8a, CD8b, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, NKp46, NKG2C, NKG2D, CS1, or a part thereof of the transmembrane region of a T cell receptor polypeptide.

37. The aforementioned external domain is, (i) signal peptides, and / or (ii) The chimeric antigen receptor according to claim 32, further comprising a spacer / hinge / linker.

38. The CAR is contained in a bicistronic construct that co-expresses a portion or full-length peptide of an exogenous cytokine or its receptor expressed on the cell surface, and the exogenous cytokine or its receptor is (a) comprising at least one of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and their respective receptors(s), or (b) (i) Co-expression of IL15 and IL15Rα using self-cleaving peptides, (ii) Fusion protein of IL15 and IL15Rα (iii) IL15 / IL15Rα fusion protein in which the intracellular domain of IL15Rα has been cleaved or excluded. (iv) A fusion protein of IL15 and the membrane-bound Sushi domain of IL15Rα, (v) Fusion protein of IL15 and IL15Rβ, (vi) A fusion protein of IL15 and the common receptor γC, wherein the common receptor γC is either natural or modified, and (vii) containing at least one of the homodimers of IL15Rβ, (b) Any one of (i) to (vii) may be co-expressed with CAR in a separate construct or a bicistronic construct, (c) (i) A fusion protein of IL7 and IL7Rα, (ii) A fusion protein of IL7 and the common receptor γC, wherein the common receptor γC is either native or modified, and (iii) The chimeric antigen receptor according to claim 32, comprising at least one of the homodimers of IL7Rβ.

39. The chimeric antigen receptor according to claim 32, wherein the MR1-CAR is specific to one or more of the following: colorectal cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, cervical cancer, melanoma, bone cancer, breast cancer, ovarian cancer, or hematological cancer.

40. A cell or a group thereof, wherein the cell is a eukaryotic cell, an animal cell, a human cell, an immune cell, an induced pluripotent cell (iPSC), a cloned iPSC, or an induced cell differentiated therefrom, and the cell contains at least one polynucleotide encoding a chimeric antigen receptor (CAR) as described in any one of claims 32 to 39.

41. The aforementioned cells are (i) A polynucleotide encoding a transgenic TCRα chain (tgTCRα), (ii) A polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), wherein the tgTCRα chain and the tgTCRβ chain are exogenous TCR complexes (TCR) that recognize a first tumor antigen other than MR1. ｅｘｏ ) form polynucleotides, and optionally, (iii) a cell or population thereof according to claim 40, further comprising one or more additional exogenous polynucleotides, each containing a polynucleotide encoding an engager that targets at least a second tumor antigen.

42. (i) The polynucleotide encoding the tgTCRα chain and the polynucleotide encoding the tgTCRβ chain are included in a bicistronic construct and optionally, (a) The construct is inserted into the steady region (TRAC or TRBC) of TCRα or TCRβ, (b) Insertion of the construct disrupts the expression of endogenous TCRα or endogenous TCRβ at the insertion site, and / or (c) The expression of the construct is driven by the endogenous or exogenous promoter of the TCR, or (ii) The polynucleotide encoding the CAR or the engager is inserted into the TRAC or TRBC, optionally, (a) Insertion of the polynucleotide encoding the CAR or the engager disrupts the expression of endogenous TCRα or endogenous TCRβ at the insertion site, and / or (b) The expression of the CAR or the engager is driven by the endogenous or exogenous promoter of the TCR, or (iii) The cells or population thereof according to claim 41, wherein the tgTCRα chain and the tgTCRβ chain, the polynucleotide encoding the CAR or the engager, or the one or more additional polynucleotides are inserted into one or more safe harbor loci or selected loci.

43. (I) The construct and the polynucleotide encoding the CAR or the engager are each inserted into the constant region (TRAC or TRBC) of TCRα or TCRβ, but not into the same constant region, thereby disrupting the expression of both endogenous TCRα and endogenous TCRβ and knocking out the endogenous TCR. (a) the transgenic TCRα and the endogenous TCRβ, or (b) Avoid unpaired TCRs, including the transgenic TCRβ and the endogenous TCRα, Or (II) the cell or population thereof according to claim 42, wherein the construct and the polynucleotide encoding the CAR or the engager are each incorporated into a locus including a safe harbor locus or a selected locus.

44. A composition comprising cells or a population thereof as described in any one of claims 40 to 43.

45. The composition according to claim 44, wherein the cells or population thereof comprises the iPSC-induced effector cells, and the composition further comprises one or more therapeutic agents.

46. A therapeutic use of the therapeutic composition according to claim 44 or 45, comprising introducing the therapeutic composition into a subject suitable for adoptive cell therapy, wherein the subject has an autoimmune disorder, hematological malignancy, solid tumor, cancer, or viral infection.

47. The therapeutic use of the composition according to claim 46, wherein the subject is colorectal cancer, lung cancer, kidney cancer, prostate cancer, bladder cancer, cervical cancer, gastric cancer, melanoma, bone cancer, breast cancer, ovarian cancer, or hematological cancer.

48. A method for enhancing the function of CAR-T cells, wherein the CAR-T cells have a first tumor antigen specificity mediated by CAR, and the method involves the CAR-T cells, (i) A polynucleotide encoding a transgenic TCRα chain (tgTCRα), (ii) A polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), wherein the tgTCRα chain and the tgTCRβ chain are a second tumor antigen specific exogenous TCR complex (TCR ｅｘｏ This includes introducing a polynucleotide that forms a ) The CAR-induced tumor killing efficacy of the CAR-T cells is enhanced by the expression of TCR ｅｘｏ , method.

49. The aforementioned CAR-T cells are (i) Endogenous TCR knockout by disrupting the expression of both endogenous TCRα and endogenous TCRβ, or (ii) The CAR is inserted into the steady-state region (TRAC or TRBC) of TCRα or TCRβ, The method according to claim 48, wherein the expression of endogenous TCRα or endogenous TCRβ in the CAR-T cells is thereby disrupted.

50. The above method is TCR ｅｘｏ Using the second tumor antigen recognized by the above, TCR ｅｘｏ The method according to claim 48 or 49, further comprising activating the first tumor antigen specificity and the second tumor antigen specificity being different.

51. The step of introducing polynucleotides into the CAR-T cells is, The method involves differentiating genetically modified iPSCs into T cells, wherein the iPSCs contain (a) a polynucleotide encoding a transgenic TCRα chain (tgTCRα), (b) a polynucleotide encoding a transgenic TCRβ chain (tgTCRβ), and (c) a polynucleotide encoding a CAR having first tumor antigen specificity, and thereby the TCR ｅｘｏ The method according to claim 48, further comprising obtaining CAR-T cells having the expression of .

52. The method according to claim 51, further comprising genomically manipulating a cloned iPSC to knock in (a) the polynucleotide encoding the transgenic TCRα chain (tgTCRα), (b) the polynucleotide encoding the transgenic TCRβ chain (tgTCRβ), and (c) the polynucleotide encoding a CAR having a first tumor antigen specificity, thereby obtaining an engineered iPSC for T cell differentiation.

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